cytochrome-c-t and Liver-Neoplasms

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Cytochromes c; Galectin 3; Humans; Liver Neoplasms; Membrane Proteins; NF-kappa B; Parkinson Disease; Signal Transduction

Sorafenib resistance limits its survival benefit for treatment of hepatocellular carcinoma (HCC). Cholesterol metabolism is dysregulated in HCC, and its role in sorafenib resistance of HCC has not been fully elucidated. Aiming to elucidate this, in vitro and in vivo sorafenib resistant models were established. Sterol regulatory element binding transcription factor 2 (SREBF2), the key regulator of cholesterol metabolism, was activated in sorafenib resistant HepG2 and Huh7 cells. Knockdown of SREBF2 resensitized sorafenib resistant cells and xenografts tumors to sorafenib. Further study showed that SREBF2 positively correlated with StAR related lipid transfer domain containing 4 (STARD4) in our sorafenib resistant models and publicly available datasets. STARD4, mediating cholesterol trafficking, not only promoted proliferation and migration of HepG2 and Huh7 cells, but also increased sorafenib resistance in liver cancer. Mechanically, SREBF2 promoted expression of STARD4 by directly binding to its promoter region, leading to increased mitochondrial cholesterol levels and inhibition of mitochondrial cytochrome c release. Importantly, knockdown of SREBF2 or STARD4 decreased mitochondrial cholesterol levels and increased mitochondrial cytochrome c release, respectively. Moreover, overexpression of STARD4 reversed the effect of SREBF2 knockdown on mitochondrial cytochrome c release and sorafenib resistance. In conclusion, SREBF2 promotes STARD4 transcription, which in turn contributes to mitochondrial cholesterol transport and sorafenib resistance in HCC. Therefore, targeting the SREBF2-STARD4 axis would be beneficial to a subset of HCC patients with sorafenib resistance.

Topics: Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cholesterol; Cytochromes c; Drug Resistance, Neoplasm; Homeostasis; Humans; Liver Neoplasms; Membrane Transport Proteins; Sorafenib; Sterol Regulatory Element Binding Protein 2

Rosmarinic acid (RA) is a well-known phenolic acid widely present in over 160 species of herbal plants and known to exhibit anti-tumor effects on breast, prostate, and colon cancers in vitro. However, its effect and mechanism in gastric cancer and liver cancer are unclear. Moreover, there is no RA report yet in the chemical constituents of Rubi Fructus (RF). In this study, RA was isolated from RF for the first time, and the effect and mechanism of RA on gastric and liver cancers were evaluated using SGC-7901 and HepG2 cells models. The cells were treated with different concentrations of RA (50, 75, and 100 μg/mL) for 48 h, and the effect of RA on cell proliferation was evaluated by the CCK-8 assay. The effect of RA on cell morphology and mobility was observed by inverted fluorescence microscopy, cell apoptosis and cell cycle were determined by flow cytometry, and the expression of apoptosis-related proteins cytochrome C, cleaved caspase-3, Bax, and Bcl-2 was detected by western blotting. The results revealed that, with an increase in the RA concentration, the cell viability, mobility, and Bcl-2 expression decreased, while the apoptosis rate, Bax, cytochrome C, and cleaved caspase-3 expression increased, and SGC-7901 and HepG2 cells could be induced to arrest their cell cycle in the G0/G1 and S phases, respectively. These results together indicate that RA can induce apoptosis of SGC-7901 and HepG2 cells through the mitochondrial pathway. Thus, this study supplements the material basis of the anti-tumor activity of RF and provides an insight into the potential mechanism of RA-inducing apoptosis of gastric cancer SGC-7901 cells and liver cancer HepG2 cells, thereby facilitating further developmental studies on and the utilization of the anti-tumor activity of RF.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; Male; Proto-Oncogene Proteins c-bcl-2; Rosmarinic Acid; Stomach Neoplasms

This study explored the anti-tumor effect of ginkgetin, an extract from ginkgo biloba, on human hepatocellular carcinoma cell lines and the underlying mechanisms. Cell viability was measured by MTT assay. Apoptotic cell morphology was observed under an inverted microscope after Hoechst 33,258 staining, and the ratio of apoptotic and necrotic cells was examined by flow cytometry after FITC/PI staining. Cell cycle changes were analyzed using flow cytometry. Cytochrome c release and caspase 3 and 8 activities were monitored using the relevant reagent kits. The levels of cell cycle-related proteins were detected by Western blot. MTT results indicated that ginkgetin significantly reduced HepG2 cell viability in a dose-dependent manner. Cellular morphology observation revealed that ginkgetin induced typical apoptotic morphological features of HepG2 cells, such as increased apoptotic bodies and cell shrinkage. Cell cycle analysis showed that ginkgetin increased the proportion of cells in the S phase. S-phase cell accumulation could be attributed to the decreased expression of cell cycle regulatory factors. Similarly, ginkgetin also induced the apoptosis and S phase cell accumulation of another human HCC cell line SK-HEP-1. Furthermore, ginkgetin treatment increased caspase-3 activity and cytochrome c release but not caspase-8 activity, implying that ginkgetin might mediate cell apoptosis through the mitochondrial pathway. In addition, the tumor formation experiment in nude mice showed that ginkgetin administration inhibited tumor growth. These results suggest that ginkgetin could be a cell apoptosis stimulator by affecting the balance between cell proliferation and apoptosis, suggesting that ginkgetin might be suitable for human HCC treatment.

Topics: Animals; Apoptosis; Biflavonoids; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Nude

Liver cancers, such as hepatocellular carcinoma (HCC), are a highly prevalent cause of cancer-related deaths. Current treatments to combat liver cancer are limited. (-)-Agelasidine A, a compound isolated from the methanol extract of

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Cytochromes c; Endoplasmic Reticulum Stress; Guanidines; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Porifera; Sulfones

Traditional Chinese medicine formula (TCMF) possesses unique advantages in the prevention and treatment of malignant tumors such as hepatocellular carcinoma (HCC) and colorectal cancer (CRC). However, the unclear chemical composition and mechanism lead to its unstable efficacy and adverse reactions occurring frequently, especially injection. We previously proposed the research idea and strategy for compound-composed Chinese medicine formula (CCMF).. A demonstration study was performed through screening of the compound-composed optimal formula (COF) from Aidi injection, confirmation of the synergistic effect, and exploration of the related mechanism in the treatment of HCC and CRC.. The feedback system control (FSC) technique was applied to screening of COF. CCK-8 and calcein-AM/PI assays were performed to evaluate cell proliferation. Cell apoptosis was assessed using flow cytometry and DAPI staining. JC-1 probe and mitochondrial staining were employed to detect mitochondrial membrane potential (MMP) and the release of cytochrome c into cytoplasm, respective. Quantitative proteomics, drug affinity responsive target stability (DARTS) assay, bioinformatics, and molecular docking were carried out to explore the targets of the compounds and the synergistic mechanism involved.. COF was obtained from Aidi injection, which comprises cantharidin (CAN): calycosin-7-O-β-D-glucoside (CAG): ginsenoside Rc: ginsenoside Rd = 1:12:12:8 (molar ratio). The monarch drug CAN in combination with minister medicines consisting of CAG, Rc and Rd (abbr. TD) displayed evidently synergistic effect, which inhibited cell viability, increased dead cell number, induced apoptosis, reduced MMP, promoted cytochrome c leakage of HCC and CRC cells, and suppressed the increases of tumor volume and weight in HCC and CRC bearing nude mice. TD probably antagonized CAN enhanced activity of the ubiquitin proteasome system (UPS) to depress the degradation of cytotoxic proteins through binding to ubiquitin proteasome, thus exerting the synergistic effect with CAN activated protein phosphatase 2A (PP2A) to activate the mitochondrial apoptosis pathway. In addition, the CAN enhanced protein expression of UPS was also observed for the first time.. CAN and TD exert synergism through activation of PP2A and inhibition of UPS. It makes sense to elucidate the scientific nature of the compatibility theory of TCMF based on CCMF, which will be an important research direction of the modernization of traditional Chinese medicines.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cytochromes c; Liver Neoplasms; Mice; Mice, Nude; Molecular Docking Simulation; Proteasome Endopeptidase Complex; Ubiquitins; Xenograft Model Antitumor Assays

Raptinal is a novel antineoplastic agent that induces an expeditious intrinsic apoptotic pathway, in addition to the shutdown of mitochondrial function for cancerous cells, because of silver nanoparticles (AgNPs) that have been shown to provide a worthy approach to overcome tumors. In this study, Both Raptinal and Raptinal-loaded silver nanoparticles (AgNPs) were tested as the first time in hepatocellular carcinoma-induced mice to evaluate its efficacy and targeting to HCC. Seventy-two albino male mice of comparable age were classified into six groups; early stage of HCC was induced using diethyl nitrosamine (DEN)/carbon tetrachloride (CCL4). Liver function was assessed in all groups using ALT, AST, total bilirubin, and alpha-fetoprotein (AFP) as well as histopathological examination. Quantitative gene expression of key apoptotic gene markers p53, cytochrome c, and caspase 3 was assessed in all liver homogenates. The results showed that Raptinal-loaded AgNPs group had significant increase in both apoptotic genes of cytochrome c and Caspase 3 at P = 0.0001 compared with Raptinal-free drug group. AFP levels were significantly decreased in Raptinal-loaded AgNPs group compared with both Raptinal-free drug and HCC groups at P = 0.0001. Degenerative changes in the hepatocytes with focal necrosis and inflammatory cell infiltration in histopathology confirm the biochemical analysis. Our study is considered one of the first studies using Raptinal in vivo. Moreover, it showed that Raptinal and/or the combination between Raptinal and AgNPs showed a promising therapeutic agent in treating early HCC.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Caspase 3; Cyclopentanes; Cytochromes c; Disease Models, Animal; Fluorenes; Gene Expression Regulation, Neoplastic; Hepatocytes; Liver; Liver Neoplasms; Male; Metal Nanoparticles; Mice; Silver; Tumor Suppressor Protein p53

Hypoxia, as a form of stress, plays a critical role in oncogenesis, including metabolic reprogramming. Mitochondrial, the centers of energy production, re-balance mitochondria dynamic to maintain cell survival during high levels of environmental stresses. NDRG1 is a hypoxia-inducible protein that is involved in various human cancers, including HCC. However, little is known about whether NDRG1 participants in the quality control of mitochondrial in times of stress. Here, we firstly showed that how NDRG1 exerted its role through mediating mitochondrial dynamic in HCC cells under hypoxia. Initially, we identified that NDRG1 expression varies with oxygen content. NDRG1 silencing notably induced cell apoptosis under hypoxia, while no obviously change of wildtype cells in hypoxia compared with that in normoxia. Further analysis revealed that NDRG1 silencing in HCC cells led to increase of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx, which meant mitochondrial damage were induced. In the analysis of mitochondria, we found that more released cytochrome c located in cytosolic with NDRG1 knockdown in hypoxia, which may be due to mitochondria division. And the following experiment proved that more fragmented mitochondria were presented in NDRG1 silencing cells, as well as destroyed mitochondrial membrane potential with evidence by JC-1 was verified. Moreover, these trends could be reversed by Mdivi1. Further research showed that NDRG1 silencing disrupt hypoxia-enhanced aerobic glycolysis through effectively decreased glucose uptake, lactate output and ECAR value. In sum, we provide the first direct evidence that NDRG1-driven change in mitochondrial dynamics and aerobic glycolysis maintain cells survival in HCC during hypoxia.

Topics: Apoptosis; bcl-X Protein; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Reprogramming; Cytochromes c; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondrial Dynamics; Proto-Oncogene Proteins c-bcl-2; Tumor Hypoxia

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bacillus pumilus; Callyspongia; Carcinoma, Hepatocellular; Cell Survival; Cytochromes c; Dipeptides; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Phosphatidylinositol 3-Kinases; Reactive Oxygen Species; Signal Transduction

Discovering the utmost effective and targeted chemotherapy for hepatocellular carcinoma is still a significant challenge. In the present study, diethylnitrosamine was used as a liver carcinogen and boldine a compound of boldo. We anticipated the hypothesis that boldine endow antiproliferative and promote apoptosis on hepatocarcinoma rats. We analyzed that boldine alters the tumor biomarkers and liver markers enzyme levels. Also, we determined boldine modulate the enzymatic and nonenzymatic antioxidant activities, as well as messenger RNA and protein expressions of Bcl2, Bax, and cleaved caspase 3 by reverse transcription polymerase chain reaction and Western blot analysis, respectively. It was also manifested by histopathology studies in liver tissues of HCC rats. Our finding suggested that boldine has antioxidant activity, and moreover, also contributes apoptotic nature by upregulating the protein expression of Bax, and cleaved caspase 3. Our data accomplishes that boldine a candidate drug has dynamic therapeutic activity and suitable for the treatment of HCC.

Topics: alpha-Fetoproteins; Animals; Antioxidants; Apoptosis; Aporphines; bcl-2-Associated X Protein; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Cytochromes c; Diethylnitrosamine; Liver; Liver Neoplasms; Male; Oxidative Stress; Peumus; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; RNA, Messenger; Weight Gain

Ivalin, a natural compound isolated from

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Asteraceae; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cytochromes c; DNA-Binding Proteins; Humans; Lactones; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; NF-kappa B; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction

Flavonoids are natural compounds that show various biological effects, such as the anti-cancer effect. Chrysin is a flavonoid compound found in honey and propolis. Studies have shown that chrysin has anti-cancer activity due to induction of apoptosis signaling. In the present study, we examined the cytotoxic effect of chrysin against liver mitochondria obtained from the hepatocellular carcinoma (HCC) rat model. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) was used for induction of HCC. Mitochondria were isolated from liver hepatocytes using differential centrifugation. Then, hepatocytes and mitochondria markers related to apoptosis signaling were investigated. Our finding indicated an increase in mitochondrial reactive oxygen species (ROS) generation, collapse in the mitochondrial membrane potential (MMP), swelling in mitochondria, and cytochrome c release (about 1.6 fold) after exposure of mitochondria obtained from the HCC rats group with chrysin (10, 20, and 40 µM) compared to the normal rats group. Furthermore, Chrysin was able to increase caspase-3 activity in the HCC rats group (about 2.4 fold) compared to the normal rats group. According to the results, we proposed that chrysin could be considered as a promising complementary therapeutic candidate for the treatment of HCC, but it requires a further in vivo and clinical studies.

Topics: Animals; Carcinoma, Hepatocellular; Cytochromes c; Dose-Response Relationship, Drug; Flavonoids; Gene Expression Regulation; Liver Neoplasms; Male; Mitochondria, Liver; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Succinate Dehydrogenase

Evidence suggests that auranofin (AF) exhibits anticancer activity by inhibiting thioredoxin reductase (TrxR). Here, in this study, we have investigated the synergistic effects of AF and morin and their mechanism for the anticancer effects focusing on apoptosis in Hep3B human hepatocellular carcinoma cells. We assessed the anticancer activities by annexin V/PI double staining, caspase, and TrxR activity assay. Morin enhances the inhibitory effects on TrxR activity of AF as well as reducing cell viability. Annexin V/PI double staining revealed that morin/AF cotreatment induced apoptotic cell death. Morin enhances AF-induced mitochondrial membrane potential (ΔΨm) loss and cytochrome c release. Further, morin/AF cotreatment upregulated death receptor DR4/DR5, modulated Bcl-2 family members (upregulation of Bax and downregulation of Bcl-2), and activated caspase-3, -8, and -9. Morin also enhances AF-induced reactive oxygen species (ROS) generation. The anticancer effects results from caspase-dependent apoptosis, which was triggered via extrinsic pathway by upregulating TRAIL receptors (DR4/DR5) and enhanced via intrinsic pathway by modulating Bcl-2 and inhibitor of apoptosis protein family members. These are related to ROS generation. In conclusion, this study provides evidence that morin can enhance the anticancer activity of AF in Hep3B human hepatocellular carcinoma cells, indicating that its combination could be an alternative treatment strategy for the hepatocellular carcinoma.

Topics: Animals; Apoptosis; Auranofin; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Down-Regulation; Flavonoids; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Up-Regulation

The prevalent human hepatocellular carcinoma (HCC) is a leading cause of global cancer-related mortality. The small molecular weight triepoxide derivative, 1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl) (teroxirone), has been proved effective against the proliferation of lung cancer cells. The purpose is to further examine if teroxirone regulate growth and metastatic potential of HCC cells with aims at disclosing more of the reaction mechanisms.. Measurements of cell viability and flow cytometry were conducted to test sensitivities of teroxirone against HCC cells. The signaling pathway leading to apoptotic death was unraveled by Western blotting analysis. The metastatic progression was evaluated by cell-based phenotype assay that included migration, invasion, gelatin zymography and wound assay. The in vivo drug efficiency was done in immune-deficient mice with the established xenograft tumors.. Teroxirone inhibited growth of HCC cells, but not hepatic cells. The drug induced apoptosis in HCC cells bearing mutant p53. Pretreatment of caspase-3 inhibitor restored cell viabilities by suppressing extrinsic pathway-mediated cell death. More experiments suggested that sub-apoptotic concentrations of teroxirone mitigated migration, invasion and wound healing of HCC cells. The drug reduced growth of the xenograft tumors as established in animal models by activating apoptotic death.. The findings asserted that teroxirone is an eligible addition to the existing options as an anticancer agent to eliminate HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cytochromes c; Female; Humans; Liver Neoplasms; Matrix Metalloproteinases; Mice, Nude; Neoplasm Invasiveness; Signal Transduction; Triazines; Wound Healing; Xenograft Model Antitumor Assays

RBFOX3, an RNA-binding fox protein, plays an important role in the differentiation of neuronal development, but its role in the chemosensitivity of hepatocellular carcinoma (HCC) to 5-FU is unknown.. In this study, we examined the biological functions of RBFOX3 and its effect on the chemosensitivity of HCC cells to 5-FU in vitro and in a mouse xenograft model.. RBFOX3 was found to have elevated expression in HCC cell lines and tissue samples, and its knockdown inhibited HCC cell proliferation. Moreover, knockdown of RBFOX3 improved the inhibitory effect of 5-fluorouracil (5-FU) on cell proliferation, migration and invasion, and enhanced the apoptosis induced by 5-FU. However, overexpression of RBFOX3 reduced the inhibitory effect of 5-fluorouracil (5-FU) on cell proliferation, migration and invasion, and decreased the apoptosis induced by 5-FU. We further elucidated that RBFOX3 knockdown synergized with 5-FU to inhibit the growth and invasion of HCC cells through PI3K/AKT and epithelial-mesenchymal transition (EMT) signaling, and promote apoptosis by activating the cytochrome-c/caspase signaling pathway. Finally, we validated that RBFOX3 regulated 5-FU-mediated cytotoxicity in HCC in mouse xenograft models.. The findings from this study indicate that RBFOX3 regulates the chemosensitivity of HCC to 5-FU in vitro and in vivo. Therefore, targeting RBFOX3 may improve the inhibition of HCC growth and progression by 5-FU, and provide a novel potential therapeutic strategy for HCC.

Topics: Animals; Antigens, Nuclear; Apoptosis; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytochromes c; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Fluorouracil; Humans; Liver Neoplasms; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasm Metastasis; Nerve Tissue Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

Overexpression of renin angiotensin system (RAS) components and nuclear factor-kappa B (NF-kB) has a key role in various cancers. Blockade of RAS and NF-kB pathway has been suggested to reduce cancer cell proliferation. This study aimed to investigate the role of angiotensin II and NF-kB pathway in liver hepatocellular carcinoma cell line (HepG2) proliferation by using azilsartan (as a novel Ag II antagonist) and Bay 11-7082 (as NF-kB inhibitor). HepG2 cells were treated with different concentrations of azilsartan and Bay 11-7082. Cytotoxicity was determined after 24, 48, and 72?h by MTT assay. Reactive oxygen spices (ROS) generation and cytochrome c release were measured following azilsartan and Bay11- 7082 treatment. Apoptosis was analyzed qualitatively by DAPI staining and quantitatively through flow cytometry methodologies and Bax and Bcl-2 mRNA and protein levels were assessed by real time PCR and ELISA methods, respectively. The cytotoxic effects of different concentration of azilsartan and Bay11- 7082 on HepG2 cells were observed as a reduction in cell viability, increased ROS formation, cytochrome c release and apoptosis induction. These effects were found to correlate with a shift in Bax level and a downward trend in the expression of Bcl-2. These findings suggest that azilsartan and Bay11- 7082 in combination or alone have strong potential as an agent for prevention or treatment of liver cancer after further studies.

Topics: Angiotensin II Type 1 Receptor Blockers; Antineoplastic Agents; Apoptosis; Benzimidazoles; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; NF-kappa B; Nitriles; Oxadiazoles; Oxidative Stress; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Sulfones

Spiders and spider venoms have been used in traditional Chinese medicine to treat various ailments for more than 1000 years. For instance, several large spiders have been utilized by the Li People, who mainly live in Hainan Island of China, in their own unique traditional Chinese medicine therapy. Recent studies have indicated that spider venoms may be an important source of bioactive compounds for anti-tumor treatments. However, the specific mechanisms underlying these activities are not yet completely understood.. The present study investigated how the venom of the spider Haplopelma hainanum regulate proliferation and apoptosis in HepG2 cells via the underlying molecular mechanisms.. We treated HepG2 cells with various concentrations of the spider venom (0, 10, 50, 100 and 200 μg/mL) for 48 h, and then analyzed anti-proliferation activity, apoptosis-inducing effects, mitochondrial membrane potential (Δψm) and changes in the pro-apoptotic pathway. The anti-proliferation activity was detected by the MTT assay and Western blotting. Flow cytometry was used to analyze both apoptosis and mitochondrial membrane potential. The key pro-apoptotic molecules in the caspase-3 and -9 dependent mitochondrial pathway, including Bcl2 family, were assessed through realtime PCR, Western blotting and enzymatic test.. The results indicated that the venom of H. hainanum exhibited potent inhibition effects in HepG2 cells through suppressing proliferation, reducing the mitochondrial membrane potential, activating caspase-3 and caspase-9, and inducing the apoptosis through a mitochondrial-dependent pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Proliferation; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Poly(ADP-ribose) Polymerases; Spider Venoms; Spiders

Hepatocellular carcinoma (HCC) is the one of most common and deadly cancers, and is also highly resistant to conventional chemotherapy treatments. Mitochondrial phosphoglycerate mutase/protein phosphatase (PGAM5) regulates mitochondrial homeostasis and cell death, however, little is known about its roles in cancer. The aim of this study was to explore the clinical significance and potential biological functions of PGAM5 in hepatocellular carcinoma. For the first time, our results show that PGAM5 is significantly upregulated in HCC compared with corresponding adjacent noncancerous hepatic tissues and high PGAM5 expression is an independent predictor of reduced survival times in both univariate and multivariate analyses. Additionally, in vivo and in vitro studies showed that depleting PGAM5 expression inhibited tumor growth and increased the 5-fluorouracil sensitivity of HCC cells. Conversely, restoring PGAM5 expression in PGAM5-knockdown cells dramatically enhanced HCC cell resistance to 5-fluorouracil. Importantly, we demonstrated that the mechanism of 5-fluorouracil resistance conferred to HCC cells by PGAM5 was via inhibiting BAX- and cytochrome C-mediated apoptotic signaling by interacting and stabilizing Bcl-xL. Consistently, in the same cohorts of HCC patient tissues, Bcl-xL expression was positively correlated with PGAM5, and together predicted poor prognoses. In Conclusion, Our data highlight the molecular etiology and clinical significance of PGAM5 in HCC. Targeting the novel signaling pathway mediated by PGAM5/Bcl-xL may represent a new therapeutic strategy to improve the survival outcomes of HCC patients.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Hepatocellular; Cohort Studies; Cytochromes c; Drug Resistance, Neoplasm; Female; Fluorouracil; Gene Silencing; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Mitochondria; Mitochondrial Proteins; Phosphoprotein Phosphatases; Prognosis; Xenograft Model Antitumor Assays

Evidence has been provided of the anti-proliferative activity of citalopram against some cancer cells.. The apoptotic impact of citalopram, an antidepressant, against liver hepatocellular carcinoma cell line HepG2 was investigated in relation to the oxidative pathway and nuclear factor (NF)κB activation.. The cytotoxic effects of citalopram on HepG2 cells were determined by MTT assay. Reactive oxygen species (ROS) formation and cytochrome c release were measured following treatment with citalopram. Apoptosis analysis and Bax and Bcl--2 mRNA and protein levels were also determined.. The cytotoxic effects of different concentrations of citalopram on HepG2 cells were observed as a reduction in cell viability and an increase in ROS formation. Citalopram caused an increase in mitochondrial Bax levels and a decrease in Bcl2 levels and also caused cytochrome c release. Moreover, DAPI staining and flow cytometry assays revealed citalopram-induced apoptosis in HepG2 cells. Oxidant scavengers and Bay 11-7082 (an irreversible inhibitor of NFκB activation) prevented the citalopram-associated cell death, increased BAX and decreased Bcl2.. Outcomes from current study suggest that citalopram might exhibit apoptotic effect against hepatocellular carcinoma cell line by induction of cell death through cytochrome c release and ROS-dependent activation of NFκB.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Hep G2 Cells; Humans; Liver Neoplasms; Molecular Structure; NF-kappa B; Reactive Oxygen Species; Structure-Activity Relationship

Curcumin (CUR), a widely used food additive, is derived mainly from Curcuma species that has been applied traditionally for the treatment of various diseases, including hepatocellular carcinoma (HCC). However, its poor systemic bioavailability hampers its clinical application, which may be related to its wide metabolism. Tetrahydrocurcumin (THC) is a major metabolite of CUR and has been reported to have multiple biologic activities. We investigated, for the first time, the efficacy and associated mechanism of action of THC and CUR in a H22 ascites tumor-bearing model in mice. THC evoked a significant dose-dependent promotion of survival and was significantly more effective than CUR in inhibiting tumor growth, including increased body weight, abdominal circumference, ascites volume, and the viability of cancer cells. Experiments on essential immune organs indicated that THC had a more favorable margin of safety than the reference drug cyclophosphamide. THC induced the apoptosis of H22 cells obviously by increasing the level of p53 and decreasing the level of murine double minute 2. THC also decreased the expression of Bcl-2 significantly and increased the expression of Bcl2-associated X, resulting in the release of cytochrome C. THC significantly activated and induced cleavage of caspase-3 and caspase-9 to induce the apoptosis of H22 cells. Taken together, these results indicate that THC was more effective than CUR in inhibiting the apoptosis of H22-induced ascites tumor cells and achieved it via regulation of a mitochondrial apoptosis pathway. THC might be a bioactive anti-tumor form of CUR and represented a potentially effective agent for HCC treatment.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Ascites; Cell Line, Tumor; Curcumin; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; Mice; Mitochondria

Cinobufacini, a traditional Chinese medicine, has been used widely for cancer treatment, such as hepatocellular carcinoma (HCC), sarcoma, and leukemia. Previous studies done by our lab indicated that cinobufacini could suppress HCC cells through mitochondria-mediated and Fas-mediated apoptotic pathways. Here, we use a combination of cinobufacini and doxorubicin to inhibit the growth of HCC cells. The combination group induced more significant apoptosis by affecting proteins and RNA of apoptosis-related elements, such as Bcl-2, Bax, Bid, and cytochrome c. Furthermore, cinobufacini, as a mixture of a number of components, had stronger apoptosis-inducing activity than particular individual components or a simple mixture of a few components. Overall, these results suggested that the combination of cinobufacini and doxorubicin may provide a new strategy for inhibiting the proliferation of HCC cells.

Topics: Amphibian Venoms; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cytochromes c; Doxorubicin; Drug Combinations; Drugs, Chinese Herbal; fas Receptor; Hep G2 Cells; Humans; Liver Neoplasms; Mitochondria; Mitochondrial Membranes; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Hepatocellular carcinoma (HCC), also named cancerous hepatoma, is the most common type of malignant neoplasia of the liver. In this research, we screened the Persian Gulf sea cucumber

Topics: 2-Acetylaminofluorene; Acrylonitrile; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Chromatography, Thin Layer; Cytochromes c; Diethylnitrosamine; Holothuria; Humans; Indian Ocean; Liver; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Membrane Potential, Mitochondrial; Mitochondria, Liver; Rats; Rats, Wistar; Reactive Oxygen Species; Stilbenes

In the present study, a flavonoid was isolated from Origanum vulgare and identified as didymin. The effect and mechanism of O. vulgare didymin (OVD) on human HepG2 liver carcinoma cell was then assessed. Our results showed that OVD strongly inhibited the viability, clonogenicity and migration of HepG2 cells. OVD significantly induced apoptosis and induced cell cycle arrest at G2/M phase by regulating cyclin B1, cyclin D1 and CDK4. The anti-proliferative and pro-apoptotic effects were associated with changes in the Bcl-2/Bax ratio and induction of caspase-mediated apoptosis. Moreover, OVD attenuated the mitochondrial membrane potential, accompanied by the release of cytochrome c. In addition, OVD inhibited the ERK/MAPK and PI3K/Akt pathways by increasing the level of Raf kinase inhibitor protein (RKIP). Our study indicates that OVD induces apoptosis against of HepG2 cells through mitochondrial dysfunction and inactivation of the ERK/MAPK and PI3K/Akt pathways by up-regulating RKIP.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Cycle; Cell Cycle Proteins; Cell Movement; Cell Survival; Clone Cells; Cytochromes c; Flavonoids; Glycosides; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Phosphatidylethanolamine Binding Protein; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Up-Regulation

This study was designed to examine the potential antitumor effect of some macrolides: clarithromycin, azithromycin, and erythromycin on chemically induced hepatocellular carcinoma (HCC) in rats and on human hepatoma cells (HepG2) as well. The possible underlying antiapoptotic mechanisms were investigated. Antiproliferative activity was assessed in HepG2 using Sulforhodamine-B staining method. In vivo, HCC was induced in rats by initiation-selection-promotion protocol using diethylnitrosamine (200 mg/kg, single i.p. injection)/2-acetylaminofluorene (0.03% w/w supplemented-diet for 2 weeks)/carbon tetrachloride (2 ml/kg diluted in corn oil 1:1, single intra-gastric dose)/phenobarbitone sodium (0.05% w/w supplemented-diet for 28 weeks). Macrolides were administered once daily starting from the 3rd week until the 17th week at a dose of 100 mg/kg in the current 33-week study period. Clarithromycin showed a higher efficacy in the suppression of HepG2 proliferation with lower IC50 value than doxorubicin. In vivo, chemically-induced HCC rat model proved that clarithromycin suppressed HCC via induction of apoptosis through up-regulation of both extrinsic/intrinsic apoptotic pathways' proteins (TNFR1, cleaved caspase-3, and Bax with an increased Bax/Bcl-2 ratio) along with MMP-9 normalization. Similarly, azithromycin demonstrated antitumorigenic effect through both apoptotic pathways, however, to a lesser extent compared to clarithromycin. Moreover, azithromycin suppressed the proliferation of HepG2, however, at a higher IC50 than doxorubicin. Surprisingly, erythromycin increased HepG2 proliferation in vitro, along with worsened tumorigenic effect of the carcinogenic agents in the in vivo study with ineffective apoptotic outcome. Some macrolides represent potential antitumor agents; however, this evident anticancer activity is an individual effect rather than a group effect and involves modulation of both intrinsic and extrinsic apoptotic pathways.

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Cell Survival; Cytochromes c; Disease Models, Animal; Hep G2 Cells; Humans; Ki-67 Antigen; Liver; Liver Neoplasms; Macrolides; Male; Matrix Metalloproteinase 9; Proto-Oncogene Proteins c-bcl-2; Rats, Wistar; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha

Quercetin is a potent cancer therapeutic agent and dietary antioxidant present in fruit and vegetables. Quercetin prevents tumor proliferation by inducing cell cycle arrest and is a well known cancer therapeutic agent and autophagy mediator. Recent studies showed that drug delivery by nanoparticles have enhanced efficacy with reduced side effects. In this regard, gold-quercetin into poly(DL-lactide-co-glycolide) nanoparticles was examined. In this study, we explored the role and possible underlying mechanisms of quercetin nanoparticle in regulation of antitumor activity in liver cancer cells. Treatment with quercetin nanoparticle effectively inhibited the liver cancer cell proliferation, cell migration and colony formation, thus suppressing liver cancer progression. Quercetin nanoparticle also upregulated apoptosis markedly. Further study suggested that quercetin nanoparticle accelerated the cleavage of caspase-9, caspase-3, and induced the up-releasing of cytochrome c (Cyto-c), contributing to apoptosis in liver cancer cells. Quercetin nanoparticles also promoted telomerase reverse transcriptase (hTERT) inhibition through reducing AP-2β expression and decreasing its binding to hTERT promoter. In addition, quercetin nanoparticle had an inhibitory role in cyclooxygenase 2 (COX-2) via suppressing the NF-κB nuclear translocation and its binding to COX-2 promoter. Quercetin nanoparticle also inactivated Akt and ERK1/2 signaling pathway. Taken together, our results suggested that quercetin nanoparticle had an antitumor effect by inactivating caspase/Cyto-c pathway, suppressing AP-2β/hTERT, inhibiting NF-κB/COX-2 and impeding Akt/ERK1/2 signaling pathways. Our results provided new mechanistic basis for further investigation of quercetin nanoparticles to find potential therapeutic strategies and possible targets for liver cancer inhibition.

Topics: Antioxidants; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclooxygenase 2; Cytochromes c; Drug Delivery Systems; Gold; Humans; Liver Neoplasms; MAP Kinase Signaling System; Nanoparticles; NF-kappa B; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Quercetin; Telomerase; Transcription Factor AP-2

To investigate the mechanism of human liver cancer HepG-2 cells apoptosis induced by total saponins of Ornithogalum caudatum( OCA-TS).. The anti-proliferative effects of OCA-TS on HepG-2 cells was detected by MTT assay; the inverted microscope was used to observe cell morphology; transmission electron microscope( TEM) was used to observe the cellular ultrastructure changes; flow cytometry method was used to detect the apoptosis rate, mitochondrial membrane potential, and protein expression level of Cyt-C; Caspase-3 activity was measured by ELISA.. OCA-TS can inhibit the proliferation of HepG-2 cells and the IC50 was 79. 80 ± 0. 18 μg / m L. After treated by OCA-TS, cells became round, and the refractivity of cells receded, the number of suspension cells increased. By TEM method, the cells presented typical apoptosis characteristics. With the increasing of concentration of OCA-TS, cell apoptosis rate, the protein expression level of Cyt-C and the activity of Caspase-3 were increased markedly( P < 0. 05 or P < 0. 01).. OCA-TS can effectively inhibit the proliferation of human liver cancer HepG-2 cells by inducing apoptosis of HepG-2 cells through mitochondrial pathway.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Ornithogalum; Saponins

DYC-279 is a newly synthesized compound. In this study, we revealed that DYC-279 could inhibit the proliferation of HepG2 cells in a dose- and time-dependent manner using the CCK-8 test. FACS showed that DYC-279 induced a G2/M arrest and apoptosis in a dose-dependent manner. Western blot demonstrated that DYC-279-induced G2/M arrest effect was correlated with the inhibition of cyclin-dependent kinase 1 activity, including a concomitant downregulation of cyclinD1 and cdc2 and upregulation of cyclinB1 in HepG2 cells. DYC-279 also significantly increased the ratio of Bax/Bcl-2, and stimulated the released of cytochrome c into cytosol and also activated caspase-9 and caspase-3, suggesting DYC-279 induced apoptosis via mitochondrial apoptotic pathway. These data support that DYC-279 has anticancer properties in HepG2 cells and may be used as a novel effective reagent in the treatment of human liver cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; CDC2 Protein Kinase; Cell Proliferation; Cell Survival; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinases; Cytochromes c; Hep G2 Cells; Humans; Liver Neoplasms; Piperazines; Proto-Oncogene Proteins c-bcl-2; Triazoles

The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers.. This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development.. We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis-associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors.. This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348-60. ©2016 AACR.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disease Progression; Exosomes; Female; GTP Phosphohydrolases; Humans; Liver Neoplasms; Lung Neoplasms; MCF-7 Cells; Mitochondria; Mitochondrial Proteins; Superoxides; Tumor Suppressor Proteins

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death. In patients for whom HCC could not be detected early, current treatments show poor tolerance and low efficacy. So, alternative therapies with good efficacy are urgently needed. The aim of this research was to evaluate the selective apoptotic effects of myricetin (MYR), a flavonoid compound, on hepatocytes and mitochondria obtained from the liver of HCC rats. In this study, HCC induced by diethylnitrosamine (DEN), as an initiator, and 2-acetylaminofluorene (2-AAF), as a promoter. To confirm the HCC induction, serum levels of alpha-fetoprotein (AFP), AST, AST and ALP and histopathological changes in the liver tissue were evaluated. Rat liver hepatocytes and mitochondria for evaluation of the selective cytotoxic effects of MYR were isolated, and mitochondrial and cellular parameters related to apoptosis signalling were then determined. Our results showed that MYR was able to induce cytotoxicity only in hepatocytes from the HCC but not from the untreated control group. Besides, MYR (12.5, 25 and 50 μM) induced a considerable increase in reactive oxygen species (ROS) level, mitochondrial swelling, mitochondrial membrane permeabilization (MMP) and cytochrome c release only in cancerous but not in untreated normal hepatocyte mitochondria. MYR selectively increased caspase-3 activation and apoptotic phenotypes in HCC, but not untreated normal hepatocytes. Finally, our finding underlines MYR as a promising therapeutic candidate against HCC and recommends the compound for further studies.

Topics: 2-Acetylaminofluorene; Alanine Transaminase; Alkaline Phosphatase; alpha-Fetoproteins; Animals; Apoptosis; Aspartate Aminotransferases; Carcinoma, Hepatocellular; Caspase 3; Cytochromes c; Diethylnitrosamine; Disease Models, Animal; Flavonoids; Hepatocytes; Liver; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Swelling; Organ Size; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

To study the therapeutic effect of norcantharidin (NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism.. Two hepatocellular carcinoma (HCC) cell lines, HepG2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. HepG2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.. As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant (P < 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partially inhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant (P > 0.05). The effect on inducing apoptosis in HepG2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant (P < 0.05).. NCTD combined with ABT-737 has a positive role in the treatment of HCC, and it has great value in clinical research.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Liver Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Sulfonamides; Time Factors

Tetramethylpyrazine (TMP) was originally isolated from a traditional Chinese herbal medicine, Ligusticum chuanxiong In the present study, TMP exhibits potent antitumor activities in vitro. However, the molecular mechanisms remain to be defined. Hence, this study aims to investigate the antiproliferative and apoptotic effects of TMP on HepG2 and elucidate the underlying mechanisms. Analyses using Cell Counting Kit-8 and real-time cell analyzer indicated that TMP significantly inhibited HepG2 cell proliferation. We also observed that TMP induced cell cycle arrest at the G0/G1 checkpoint and apoptosis, using flow cytometry and high-content screening. Furthermore, our results predicted that TMP could directly decrease mitochondrial membrane potential (Δψm), increase the release of cytochrome c, and increase caspase activation, indicating that mitochondrial pathway apoptosis could be the mechanism for TMP within HepG2 cells. Moreover, TMP altered expression of p53 and the Bcl-2/Bax protein ratio, which revealed that TMP induced cell cycle arrest and caspase-dependent mitochondrial apoptosis in HepG2 cells in vitro. These studies provided mechanistic insights into the antitumor properties of TMP, which may be explored as a potential option for treatment of hepatocellular carcinoma.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Resting Phase, Cell Cycle; Signal Transduction; Tumor Suppressor Protein p53

Celastrol, an active ingredient of Tripterygium Wilfordii, is a traditional Chinese medicinal herb, which has attracted interests for its potential anti-inflammatory and anti-cancer activities. The aim of this study was to evaluate the anti-tumor effect of Celastrol against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats and furthermore, to explore the underlying mechanism. Sprague-Dawley rats were intragastrically administered with DEN (10mg/kg) for 6 days every week and persisting 16 weeks. The number of nodules was calculated. Hematoxylin-Eosin (HE) staining was used to evaluate the hepatic pathological lesions. The levels of serum alanine aminotransferase (ALT), glutamic oxalacetic transaminase (AST), alkaline phosphatase (ALP) and alpha fetoprotein (AFP) were analyzed by Elisa kits, and the protein levels of p53, Murine double minute (MDM) 2, Bax, Bcl-2, Bcl-xl, cytochrome C, Caspase-3, Caspase-9 and Poly (ADP-ribose) polymerase (PARP) were analyzed by western blot. The results showed that Celastrol could significantly decrease the mortality, the number of tumor nodules and the index of liver in the Celastrol groups compared with DEN-treated group. Moreover, Celastrol obviously improved the hepatic pathological lesions and decreased the elevated levels of ALT, AST, ALP and AFP. Meanwhile, Celastrol suppressed the expression of the protein MDM2, activated the intrinsic mitochondrial apoptosis pathway induced by p53, inhibited anti-apoptotic Bcl-2 and Bcl-xl, induced the pro-apoptotic Bax, cytochrome C, PARP and caspases. These results suggested that Celastrol had a good therapeutic action in reversing DEN-induced HCC rats, which may be associated with the apoptosis of hepatoma cells induced by Celastrol.

Topics: Animals; Antineoplastic Agents; bcl-2-Associated X Protein; bcl-X Protein; Carcinogenesis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cytochromes c; Diethylnitrosamine; Gene Expression Regulation, Neoplastic; Liver; Liver Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mitochondria; Pentacyclic Triterpenes; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-mdm2; Rats; Rats, Sprague-Dawley; Triterpenes; Tumor Suppressor Protein p53

5,6-Dihydroxy-3,7,4'-trimethoxyflavonol (AH5), 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavonol (AH22), artemetin, and oroxylin A are four flavonoids with the same 2-phenyl-chromone skeleton isolated from the Chinese herb Aster himalaicus. The aim of this study was to evaluate the structure-activity relationship of these four analogs and the mediation of AH5 cytotoxicity via G2/M arrest and apoptosis in human hepatocellular carcinoma (HCC) cells. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated AH5 showed the better potency to inhibit proliferation in human HCC cells, which suggested hydroxyl binding to C6 is necessary to anticancer properties, whereas binding to C3' attenuated the activities and increased toxicity in tested cells. Flow cytometry analysis revealed that AH5-induced G2/M arrest and significantly apoptosis in these cell lines. HepG-2 cells were used to further evaluate the antitumor effects and mechanisms of AH5. AH5-induced apoptosis was further confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining and the increased ratio of Bax/Bcl-2. Moreover, AH5 induced the release of cytochrome C and the activation of caspase-9 and caspase-3, thus suggesting mitochondria activation might be involved. Western blot showed that AH5 induced the phosphorylation of Cdc2 and decreased the level of Cyclin B1. These results demonstrated that AH5 could be a proapoptotic leading compound for developing novel anticancer drugs.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cyclin B1; Cytochromes c; Drug Design; Flavonoids; Flavonols; G2 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Indoles; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship

Timosaponin A-III (TSA-III), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. However, the molecular mechanism underlying the anticancer activity of TSA-III has not been fully elucidated. In this study, the apoptotic effects of TSA-III were investigated in HepG2 cells. Treatment with TSA-III significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis in HepG2 cells. This induction was associated with increased fluorescence intensity of Annexin V-FITC, activation of caspases, and altered expression of inhibitor of apoptosis protein (IAP) family members. In addition, TSA-III mediated mitochondrial dysfunction with the release of HtrA2/Omi, Smac/Diablo, and cytochrome c. These findings suggest that TSA-III induces mitochondria-mediated and caspase-dependent apoptosis in HepG2 cells by altering expression of the IAP family. Thus, TSA-III could possibly be used to treat other types of cancer with similar pathologic mechanisms.

Topics: Anemarrhena; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Caspases; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Survival; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Liver Neoplasms; Mitochondria, Liver; Protein Transport; Rhizome; Saponins; Steroids

The therapeutic strategy for advanced nasopharyngeal carcinoma (NPC) is still challenging. It is an urgent need to uncover novel treatment targets for NPC. Therefore, understanding the mechanisms underlying NPC tumorigenesis and progression is essential for the development of new therapeutic strategies. Here, we showed that TP53-regulated inhibitor of apoptosis (TRIAP1) was aberrantly overexpressed and associated with poor survival in NPC patients. TRIAP1 overexpression promoted NPC cell proliferation and suppressed cell death in vitro and in vivo, whereas TRIAP1 knockdown inhibited cell tumorigenesis and enhanced apoptosis through the induction of mitochondrial fragmentation, membrane potential alteration and release of cytochrome c from mitochondria into the cytosol. Intersecting with our previous miRNA data and available bioinformatic algorithms, miR-320b was identified and validated as a negative regulator of TRIAP1. Further studies showed that overexpression of miR-320b suppressed NPC cell proliferation and enhanced mitochondrial fragmentation and apoptosis both in vitro and in vivo, while silencing of miR-320b promoted tumor growth and suppressed apoptosis. Additionally, TRIAP1 restoration abrogated the proliferation inhibition and apoptosis induced by miR-320b. Moreover, the loss of miR-320b expression was inversely correlated with TRIAP1 overexpression in NPC patients. This newly identified miR-320b/TRIAP1 pathway provides insights into the mechanisms leading to NPC tumorigenesis and unfavorable clinical outcomes, which may represent prognostic markers and potential therapeutic targets for NPC treatment.

Topics: Adult; Aged; Animals; Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cytochromes c; Cytosol; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Male; Membrane Potentials; Mice; Mice, Inbred BALB C; MicroRNAs; Middle Aged; Mitochondria; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Proportional Hazards Models; Signal Transduction

Human liver cancer is one of the most frequently diagnosed cancers worldwide. The development of resistance to therapy limits the application against the disease. To improve treatment, new effective anticancer agents are constantly pursued. Previously, we reported that an indolylquinoline, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), is effective in suppressing the growth of human lung cancer by impairing mitochondria functions. The present study revealed that EMMQ inhibited cell growth and induced apoptosis in liver cancer cells, but not in normal cells. This study demonstrated that EMMQ induced DNA damage by activating p53 and γ-H2AX and cell arrest by suppressing cyclin D1 and CDK2. Damaged DNA injured mitochondrial functions by lowering the membrane potential and producing reactive oxygen species. The subsequent mitochondrial cytochrome c release attenuated pro-survival signals and increased apoptotic characteristics. Introduction of p53 shRNA abrogated drug effects by reducing DNA damage while maintaining mitochondria integrity. In brief, the study demonstrates that the effectiveness of EMMQ accentuated apoptosis of hepatocarcinoma cells by activating p53. Based on these collective findings, the study offered a new perspective of EMMQ that was shown to be a promising candidate to treat liver cancer.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Cytochromes c; DNA Damage; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Histones; Humans; Indoles; Liver; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Quinaldines; Quinolines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

Cisplatin is a potent chemotherapeutic drug widely used for the treatment of human cancer. However, its efficacy against hepatocellular carcinoma (HCC) is poor for reasons that remain unclear. We show here that prothymosin-alpha (PTMA) is overexpressed in HCC cell lines. Silencing PTMA using short-hairpin RNA sensitizes HCC cells to cisplatin, while ectopic expression of PTMA induces cell resistance to the drug. Cisplatin inhibits both the JNK pathway and PTMA in a dose-dependent manner. Treatment with a JNK inhibitor also reduces PTMA protein stability and sensitizes HCC cells to cisplatin. Notably, the effects of PTMA silencing and JNK inhibition can be reversed by ectopic expression of PTMA. We show that PTMA silencing induces translocation of proapoptotic Bax to mitochondria and enhances cisplatin-induced cytochrome c release and caspase-9 activation. Conversely, ectopic expression of PTMA reverses these effects. Our results indicate that PTMA is positively regulated by JNK and protects HCC cells against cisplatin-induced cell death. The JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC.

Topics: Antineoplastic Agents; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 9; Cell Line, Tumor; Cisplatin; Cytochromes c; Gene Silencing; Humans; Liver Neoplasms; MAP Kinase Kinase 4; Mitochondria; Protein Precursors; Protein Transport; Thymosin

Celastrol is a natural terpenoid isolated from Tripterygium wilfordii, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Here, we investigated whether celastrol induces apoptosis on hepatocellular carcinoma Bel-7402 cells and further explored the underlying molecular mechanisms. Celastrol caused a dose- and time-dependent growth inhibition and apoptosis of Bel-7402 cells. It increased apoptosis through the up-regulation of Bax and the down-regulation of Bcl-2 in Bel-7402 cells. Moreover, celastrol induced the release of cytochrome c and increased the activation of caspase-3 and caspase-9, suggesting that celastrol-induced apoptosis was related to the mitochondrial pathway. These results indicated that celastrol could induce apoptosis in Bel-7402 cells, which may be associated with the activation of the mitochondria-mediated pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cytochromes c; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Mitochondria, Liver; Pentacyclic Triterpenes; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tripterygium; Triterpenes; Tumor Cells, Cultured

Recently, we isolated HY253, a novel decahydrofluorene analog with a molecular structure of 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol from the roots of Aralia continentalis, which is known as Dokwhal, a traditional medicinal herb. Moreover, we previously reported its cytotoxic activity on cancer cell proliferation in human lung cancer A549 and cervical cancer HeLa cells. The current study aimed to evaluate its detailed molecular mechanisms in cell cycle arrest and apoptotic induction in human hepatocellular carcinoma HepG2 cells. Flow cytometric analysis of HepG2 cells treated with 60 micrometer HY253 revealed appreciable cell cycle arrest at the G1 phase via inhibition of Rb phosphorylation and down-regulation of cyclin D1. Furthermore, using western blots, we found that up-regulation of cyclin-dependent kinase inhibitors, such as p21(CIP1) and p27(KIP1), was associated with this G1 phase arrest. Moreover, TUNEL assay and immunoblottings revealed apoptotic induction in HepG2 cells treated with 100 micrometer HY253 for 24 h, which is associated with cytochrome c release from mitochondria, via down-regulation of anti-apoptotic Bcl-2 protein, which in turn resulted in activation of caspase-9 and -3, and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Accordingly, we suggest that HY253 may be a potent chemotherapeutic hit compound for treating human liver cancer cells via up-regulation and activation of the p53 gene.

Topics: Apoptosis; Aralia; Blotting, Western; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cytochromes c; Flow Cytometry; Fluorenes; G1 Phase; Hep G2 Cells; Humans; Liver Neoplasms; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Native buckwheat, a common component of food products and medicine, has been observed to inhibit cancer cell proliferation in vitro. The aim of the present study was to evaluate the in vitro and in vivo anti-tumoral effects of recombinant buckwheat trypsin inhibitor (rBTI) on hepatic cancer cells and the mechanism of apoptosis involved. Apoptosis in the H22 cell line induced by rBTI was identified using MTT assays, DNA electrophoresis, flow cytometry, morphological observation of the nuclei, measurement of cytochrome C and assessment of caspase activation. It was identified that rBTI decreases cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation. rBTI-induced apoptosis occurred in association with mitochondrial dysfunction, leading to the release of cytochrome C from the mitochondria to the cytosol, as well as the activation of caspase-3, -8 and -9. In conclusion, the results of the present study suggested that rBTI specifically inhibited the growth of the H22 hepatic carcinoma cell line in vitro and in vivo in a concentration-dependent and time-dependent manner, while there were minimal effects on the 7702 normal liver cell line. In addition, rBTI‑induced apoptosis in H22 cells was, at least in part, mediated by a mitochondrial pathway via caspase-9.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Caspase 9; Cell Line; Cell Proliferation; Cytochromes c; DNA Fragmentation; Fagopyrum; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mitochondria; Transplantation, Heterologous; Trypsin Inhibitors

Jiedu Xiaozheng Yin decoction (JXY) is a type of Chinese traditional medicine, which has been used to treat various types of cancer. The present study explored the mechanisms underlying the anticancer activity of JXY. The effects of ethyl acetate extraction of JXY (EE-JXY) were evaluated on the HepG2 human hepatoma cell line in vitro and in vivo. Following treatment of the HepG2 cells with EE-JXY for 24 h, cell viability, apoptosis, mitochondrial membrane potential, caspase enzyme activity and the expression levels of apoptotic-associated proteins (Bcl-2 and Bax) were detected by MTT, flow cytometry, ELISA and western blotting respectively. In addition, HepG2 cells were subcutaneously transplanted into BALB/c nude mice, and the tumor bearing mice were treated with either EE-JXY (0.06 g/kg) or normal saline for 21 days. Tumor volume and weight were measured and recorded. The apoptotic index, and the expression levels of Bax and cytochrome c were determined with immunohistochemical staining. Treatment with EE-JXY inhibited the proliferation of HepG2 cells, and reduced cell viability in a dose-and time-dependent manner. Furthermore, EE-JXY induced HepG2 cell apoptosis, as demonstrated by a loss of plasma membrane asymmetry and externalization of phosphatidylserine, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Furthermore, EE-JXY inhibited tumor growth and increased the apoptotic index of tumors in tumor-bearing mice. In conclusion, the results of the present study suggest that JXY inhibits HepG2 cell proliferation through mitochondrion-mediated apoptosis, which may partially explain its anticancer activity.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Male; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2

8-Methoxypsoralen (8-MOP), a formerly considered photosensitizing agent, induces apoptosis when used alone. On this basis, the present study was designed to explore the effects and mechanisms of 8-MOP-induced apoptosis in human hepatocellular carcinoma HepG2 cells, independent of its photoactivation.. We analyzed the cell viability with MTT assay. Flow cytometry was used to examine the apoptosis rate, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation after specific staining. The expression and location of apoptosis-associated protein as well as the activation status of cell signaling pathway were determined by Western blot analysis.. 8-MOP significantly decreased cell viability and induced cell apoptosis through mitochondrial apoptotic pathway, as demonstrated by increased Bax/Bcl-2 ratio, collapsed MMP, and induced cytochrome c release (Cyt c) and apoptosis-inducing factor (AIF) transposition. ROS generation was significantly increased by 8-MOP and the eradication of ROS significantly abolished 8-MOP-induced apoptosis. In addition, the activation of ERK1/2 was drastically decreased by 8-MOP as ERK inhibitor PD98059, indicating a role of ERK1/2 signaling pathway in 8-MOP-induced cell apoptosis.. 8-MOP induces intrinsic apoptosis by increasing ROS generation and inhibiting ERK1/2 pathway in HepG2 cells. The findings are important in substantiating the anti-tumor role of 8-MOP in cancer therapy.

Topics: Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Cytochromes c; HCT116 Cells; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; MCF-7 Cells; Membrane Potential, Mitochondrial; Methoxsalen; Mitochondria; Reactive Oxygen Species; Signal Transduction

Annexin A7 caused an anti-apoptosis phenotype in mouse hepatocarcinoma cells. Yet, the underlying mechanism remains unclear. In this study, we found that Annexin A7 had an inhibitory effect on the mitochondrial Cytochrome C release and Caspase-3 cleavage. Furthermore, Annexin A7 was identified to form a complex with BAG4 which is a negative regulator of apoptosis. Moreover, Annexin A7 was positively correlated with the protein levels of BAG4, Hsp70 and Bcl-2. Both Hsp70 and Bcl-2 bind BAG4 and inhibit Cytochrom C dependent activation of Caspase-3. Taken together, our study indicated Annexin A7 modulated BAG4 and BAG4-binding proteins in mitochondrial apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Annexin A7; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cytochromes c; Liver Neoplasms; Mice; Mitochondria

Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC‑7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC‑7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC‑7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase‑8 and caspase‑9 were activated in MUC1 gene‑silenced SMMC‑7721 cells. The pro‑apoptotic protein Bcl‑2‑associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti‑apoptotic protein B‑cell lymphoma 2 was decreased in MUC1 gene‑silenced cells. In addition, results from the co‑immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase‑8 and these interactions were reduced upon MUC1 gene silencing in SMMC‑7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC‑7721 cells through Bax‑mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Mucin-1; Phenotype; Protein Binding; Receptors, Death Domain; RNA Interference; Signal Transduction

Cadmium (Cd) is a well known environmental and industrial toxicant causing damaging effects in numerous organs. In this study, we examined the role of heme oxygenase-1 (HO-1) in modulating the Cd-induced apoptosis in human hepatoma (HepG2) cells after 24 h exposure.. HepG2 cells were exposed to 5 and 10 μM Cd as CdCl2 for 24 h while other sets of cells were pre-treated with either 10 μM Cobalt protoporphyrin (CoPPIX) or 10 μM Tin protoporphyrin (SnPPIX) for 24 h, or 50 μM Z-DEVD-FMK for 1 h before exposure to 5 and 10 μM CdCl2 for 24 h. Expressions of caspase 3, cytosolic cytochrome c, mitochondrial Bax and anti-apoptotic BCL-xl proteins were assessed by western blot. Intracellular reactive oxygen species (ROS) production was determined using the dihydrofluorescein diacetate (H2DFA) method. Cell viability was assessed by MTT assay, while a flow cytometry method was used to assess the level of apoptosis in the cell populations.. Our results show that there were a significant increase in the expression of cytosolic cytochrome c, mitochondrial Bax protein, and caspase 3 at 5 and 10 μM compared to the control, but these increases were attenuated by the presence of CoPPIX. The presence of SnPPIX significantly enhanced Cd-induced caspase 3 activities. CoPPIX significantly decreased the level of ROS production by 24.6 and 22.2 % in 5 and 10 μM CdCl2, respectively, but SnPPIX caused a significant increase in ROS production in the presence of CdCl2. HepG2 cell viability was also significantly impaired by 13.89 and 32.53 % in the presence of 5 and 10 μM CdCl2, respectively, but the presence of CoPPIX and Z-DEVD-FMK significantly enhanced cell survival, while SnPPIX enhanced Cd-impaired cell viability. The presence of CoPPIX and Z-DEVD-FMK also significantly decreased the population of apoptotic and necrotic cells compared with Cd.. In summary, the present study showed that HO-1 attenuates the Cd-induced caspase 3 dependent pathway of apoptosis in HepG2 cells, probably by modulating Cd-induced oxidative stress.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cadmium; Carcinoma, Hepatocellular; Caspase 3; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Heme Oxygenase-1; Hep G2 Cells; Humans; Liver Neoplasms; Metalloporphyrins; Mitochondria; Oligopeptides; Protoporphyrins; Reactive Oxygen Species

Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN+CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated.. ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1-pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry.. CytochromeP4502E1 mediated bioactivation of DEN+CCl4 induced Akt dependent pMdm2-HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2-HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure.. SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN+CCl4 induced hepatocarcinoma.

Topics: Alkylating Agents; Animals; Antineoplastic Agents; Apoptosis; Berberine; Blotting, Western; Carbon Tetrachloride; Carcinoma, Hepatocellular; Caspase 3; Cells, Cultured; Cysteine; Cytochrome P-450 CYP2E1; Cytochromes c; Diethylnitrosamine; Flow Cytometry; Hepatocytes; Histone Deacetylase 1; Immunoenzyme Techniques; Immunoprecipitation; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Reactive Oxygen Species

Zinc deficiency and excess influence cellular homeostasis and are believed to modulate apoptosis. Zinc also regulates cell growth and proliferation. Understanding of the role of zinc in the mechanisms associated with these changes is limited because of its diverse, complex, and cell-specific effects. Therefore, we investigated the oxidative stress responses and the underlying molecular mechanisms associated with the disruption of intracellular zinc homeostasis in H4IIE rat hepatoma cells. We found that zinc excess (100 μM) and DTPA (diethylenetriaminepentaacetic acid; 50-100 μM) induced zinc deficiency both generate reactive oxygen species (ROS) and decrease viability in H4IIE cells. However, cotreatment with the antioxidant, N-acetyl-L-cysteine (NAC) both reduced ROS production and protected cells from death. We additionally observed an increase in Bax mRNA and cytochrome c release from the mitochondria in DTPA-treated cells and an elevated expression of Fas/Fas ligand mRNA with zinc treatment. Both treatments increased p53 and MdM2 protein concentrations along with caspase 3/7 activity. These results suggest that zinc deficiency stimulates mitochondrial-dependent apoptosis whereas zinc activates the extrinsic-apoptotic pathway. Both decreasing and increasing cellular zinc concentrations modulate ROS mediated apoptosis and warrant further research on zinc mediated cancer chemoprevention in this and other cancer cell lines.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cytochromes c; Liver Neoplasms; Mitochondria; Oxidative Stress; Proto-Oncogene Proteins c-mdm2; Rats; Reactive Oxygen Species; Tumor Suppressor Protein p53; Zinc

In some cancer cells, translocation of cytochrome c (Cyt c) from mitochondria to the cytoplasma is inhibited. This inhibition prevents cells from undergoing apoptotic cell death and can lead to uncontrolled cell growth. Increasing cytoplasmic concentration of Cyt c can induce apoptosis in cancer cells as a strategy of cancer therapy. Here we proposed a galactosylated albumin based carrier for intracellular delivery of Cyt c to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. Cyt c was chemically conjugated to galactosylated albumin with a reducible disulfide linker in order to release Cyt c from the carrier inside cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types resulting in cytotoxicity in a dose dependent manner through the induction of apoptosis. The lack of ASGPR specific uptake might be due to other carbohydrate-recognizing receptors expressed on tumor cells. In general, our work has shown that intracellular delivery of Cyt c to tumor cells can be an alternative therapeutic approach and galactosylated albumin can be a protein drug carrier for intracellular delivery.

Topics: Albumins; Apoptosis; Cell Line, Tumor; Cytochromes c; Galactose; Humans; Liver Neoplasms; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Saccharina japonica is a family member of Phaeophyceae (brown macro-alga) and extensively cultivated in China, Japan and Korea. Here, the potential anti-cancer effect of n-hexane fraction of S. japonica was evaluated in SK-Hep1 human hepatocellular carcinoma cells. The N-hexane fraction reduced cell viability and increased the numbers of apoptotic cells in a both dose- and time-dependent manner. Apoptosis was activated by both caspase-dependent and independent pathways. The caspase-dependent cell death pathway is mediated by cell surface death receptors and activated caspase-8 amplified the apoptotic signal either through direct activation of downstream caspase-3 or pro-apoptotic proteins (Bad, Bax and Bak) subsequently leading to the release of cytochrome c. On the other hand, caspase-independent apoptosis appeared mediated by disruption of mitochondrial membrane potential and translocation of AIF to the nucleus where they induced chromatin condensation and/or large-scale DNA fragmentation. In addition, the n-hexane fraction induced endoplasmic reticulum (ER)-stress and cell cycle arrest. The results suggested that potential anti-cancer effects of n-hexane extract from S. japonica on SK-Hep1 cells.

Topics: Active Transport, Cell Nucleus; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Hepatocellular; Caspase 3; Caspase 8; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Chromatin; Cytochromes c; DNA Fragmentation; Endoplasmic Reticulum Stress; HEK293 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Phaeophyceae; Plant Extracts

Green tea polyphenol (GTP) is one of the most promising chemopreventive agent for cancer; it can inhibit cancer cell proliferation and induce apoptosis through p53-dependent cell signaling pathways. Unfortunately, many tumor cells lack the functional p53, and little is known about the effect of GTP on the p53-deficient/mutant cancer cells. To understand the p53-independent mechanisms in GTP-treated p53-dificient/mutant cancer cells, we have now examined GTP-induced cytotoxicity in human hepatoma Hep3B cells (p53-deficient). The results showed that GTP could induce Bax and Bak activation, cytochrome c release, caspase activation, and necroptosis of Hep3B cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore (MPTP), were interdependently activated by GTP, with translocation and homo-oligomerization on the mitochondria. Bax and Bak induce cytochrome c release. Importantly, cytochrome c release and necroptosis were diminished in Hep3B cells (Bax(-/-)) and Hep3B cells (Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate GTP-induced cytochrome c release and necroptosis. Together, the findings suggested that GTP-induced necroptosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Liver Neoplasms; Polyphenols; Tea; Tumor Suppressor Protein p53

Apoptotic liver cancer cells have important roles in liver tumorigenesis and liver cancer progression. Recent studies have shown that δ‑opioid receptors are highly expressed in human liver and liver cancer cells. The present study aimed to investigate the role of activated δ‑opioid receptors on human liver cancer cell apoptosis and its interrelation with the mitochondria and the protein kinase C/extracellular‑signal‑regulated kinase (PKC/ERK) signaling pathway. H2O2 was used to induce apoptosis in human liver cancer cells. During apoptosis, mitochondrial transmembrane potentials were observed to decrease, cytochrome c expression was found to increase and B cell lymphoma 2 (Bcl‑2) expression decreased. These findings suggested that H2O2‑induced apoptosis was mediated through the mitochondrial pathway. Of note, activated δ‑opioid receptors were observed to inhibit H2O2‑induced apoptosis in human liver cancer cells. Following δ‑opioid receptor activation, the number of apoptotic liver cancer cells decreased, mitochondrial transmembrane potentials were restored, cytoplasmic cytochrome c and Bcl‑2‑associated X protein expression decreased and Bcl‑2 expression increased. These data suggested that δ‑opioid receptor activation inhibited mitochondria‑mediated apoptosis. In addition, activation of δ‑opioid receptors was observed to increase the expression of PKC and ERK in human liver cancer cells. Furthermore, upon inhibition of the PKC/ERK signaling pathway, the protective effect associated with the δ‑opioid receptor on liver cancer cell apoptosis was inhibited, which was not associated with the status of δ‑opioid receptor activation. These findings suggested that the PKC/ERK signaling pathway has an important role in δ‑opioid receptor‑mediated inhibition of apoptosis in human liver cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Humans; Hydrogen Peroxide; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Receptors, Opioid, delta; Signal Transduction

Glycoborinine (GB), a natural carbazole alkaloid isolated from Glycosmis pentaphylla, has been shown to be a potential molecule against cancer cells. In this study, the cell-signaling pathway of its anti-tumor activity was investigated. MTT assay result showed that GB inhibited HepG2 cell proliferation in a dose- and time-dependent manner and 50% inhibiting concentration (IC50) of GB-induced cell death was 39.7 μM for a period of 48 h. GB-induced HepG2 apoptosis was confirmed by Hochest 33258 staining and PI staining. The level of reactive oxygen species (ROS) was measured with H2DCF-DA staining and the change of mitochondrial membrane potential (△Ψ(m)) was analyzed with tetrechloro-tetraethylbenzimidazolcarbocyanine iodide (JC-1) probe. Results showed that GB at 12.5, 25, and 50 μM promoted ROS production. GB induced HepG2 apoptosis through a mitochondrial apoptotic pathway, which was demonstrated by GB-induced increase in the ratio of Bax/Bcl-2, cytochrome C release, the ratio of cleaved caspase-3/procaspase-3, and the ratio of cleaved poly ADP-ribose polymerase (cleaved PARP)/poly ADP-ribose polymerase (PARP). To summarize, this study demonstrated that GB could induce HepG2 apoptosis through the mitochondrial-dependent pathway, which might provide a promising approach to cure liver cancer with GB.

Topics: Apoptosis; Benzimidazoles; Carbazoles; Carbocyanines; Caspase 3; Cytochromes c; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Inhibitory Concentration 50; Liver Neoplasms; Membrane Potential, Mitochondrial; Molecular Structure; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

To develop carriers for efficient anti-cancer drug delivery with reduced side effects, a biocompatible and redox-responsive nanocontainer based on mesoporous silica nanoparticles (MSNs) for tumor-targeted triplex therapy was reported in this study. The nanocontainer was fabricated by immobilizing cytochrome c (CytC) onto the MSNs as sealing agent via intermediate linkers of disulfide bonds for redox-responsive intracellular drug delivery. AS1411 aptamer was further tailored onto MSNs for cell/tumor targeting. The successful construction of redox- responsive MSNs was confirmed by BET/BJH analysis, transmission electron microscopy, Fourier transform infrared spectroscopy, fluorescence spectroscopy and thermogravimetric analysis (TGA), respectively. Detailed investigations demonstrated that anticancer drug of doxorubicin (DOX) loaded nanocontainer could be triggered by reductant (e.g. glutathione) within cellular microenvironment and release DOX to induce tumor cell apoptosis in vitro. More importantly, the nanocontainer displayed great potential for tumor targeting and achieved triplex therapy effects on the tumor inhibition in vivo through the loading DOX, gatekeeper of CytC and AS1411 aptamer, which were reflected by the change of tumor size, TUNEL staining and HE staining assays.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aptamers, Nucleotide; Cytochromes c; Delayed-Action Preparations; Doxorubicin; Drug Delivery Systems; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Oxidation-Reduction; Porosity; Silicon Dioxide

To explore inhibition effects of veliparib as PARP inhibitor combined doxorubicin for BEL-7404 proliferation of human liver cancer cell line.. BEL-7404 was taken as the object of study and conventional culture was performed. It was treated by doxorubicin and (or) veliparib after 24 h. Cell proliferation rate was detected by four methyl thiazolyl tetrazolium (MTT) assay, cell apoptosis was measured with annexin V-FITC/PI double staining method by flow cytometry, DNA damage degree evaluation by single cell gel electrophoresis assay, and cytosolic C levels of the mitochondrial and cytosol by polyacrylamide gel electrophoresis (Western blotting).. Cell proliferation rate of doxorubicin combined veliparib group was lower than that of the control group and doxorubicin alone treated group significantly (P<0.01), the apoptosis rate was significantly higher than that of the control group and doxorubicin alone treated group (P<0.05). At the same time, DNA damage level of doxorubicin combined with veliparib group was significantly higher than doxorubicin alone treatment group and the control group (P<0.01), and cytochrome C in the cytosol was significantly higher than that of control group and doxorubicin alone treated group (P<0.01).. Veliparib, PARP inhibitor could inhibit PARP activity, block tumor cell DNA repair, and have significant sensitizing effect for hepatocellular carcinoma cell line BEL-7404 treated with doxorubicin. This might provide a new target for clinical treatment of hepatic carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Damage; Doxorubicin; Humans; Liver Neoplasms

Celecoxib is a potent nonsteroidal anti-inflammatory drug that has demonstrated promise in cancer chemoprevention and treatment. The present study was conducted to gain insight into the molecular mechanism by which celecoxib induces apoptosis in the H22 mouse hepatoma cell line. The effect of celecoxib on the viability of H22 mouse hepatoma cells was assessed with sulforhodamine B assay. Apoptosis and mitochondrial membrane potential were detected by a flow cytometric assay. The protein expression levels of Bax, Bcl‑2, cytochrome c, caspase-3, caspase-9, apoptosis-inducing factor (AIF), peroxisome proliferator-activated receptor (PPAR)γ and nuclear factor (NF)-κB were determined by western blot analysis. The data demonstrated that celecoxib reduced the percentage of viable H22 cells in a dose- and time-dependent manner, which was associated with cell apoptosis. Furthermore, celecoxib induced apoptosis via the loss of the mitochondrial transmembrane potential (ΔΨm), the release of cytochrome c and AIF, and the activation of caspase-9 and caspase-3. Celecoxib also increased the abundance of the pro-apoptotic protein Bax and reduced the levels of the anti-apoptotic protein Bcl-2. The data demonstrated that celecoxib induced apoptosis in mouse liver cancer cells via the mitochondria-dependent pathway rather than the PPARγ/NF-κB signaling pathway, which indicates that celecoxib may be an effective agent in the clinical management of hepatocellular carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Celecoxib; Cell Line, Tumor; Cytochromes c; Liver Neoplasms; Membrane Potential, Mitochondrial; Mice; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Signal Transduction; Sulfonamides

To investigate the roles of Golgi protein (GP) 73 in the regulation of cell proliferation and apoptosis.. Stealth RNAi targeting GP73 gene sequence was used to silence its expression in Hep G2 cells and Bel7402 cells. Stealth RNAi effects were assessed by reverse transcriptase polymerase chain reaction and ELISA. Cell proliferation assay and cell cycle analysis were assessed by MTT assay and flow cytometry. Apoptosis was assessed by flow cytometry and transmission electron microscopy. Apoptosis-related proteins were assessed by western immunoblot analysis.. Stealth RNAi targeting GP73 gene sequence markedly reduced the expression of GP73 gene. The reduction of GP73 in Hep G2 cells and Bel7402 cells inhibited cell proliferation and induced apoptosis, however, terminal apoptosis occurred in Hep G2 cells, but early apoptosis occurred in Bel7402 cells. Reduced expression of GP73 gene might lead to a reduction in Bcl-2/Bax ratio, an increase in cytochrome c, but a reduction in capase-3.. GP73 might play an important role in proliferation and apoptosis in hepatocellular carcinoma cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; Cytochromes c; Down-Regulation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Signal Transduction; Transfection

Incidence of hepatocellular carcinoma (HCC) is dramatically increasing and is the third cause of cancer death worldwide. One key approach to control HCC is chemoprevention by naturally occurring agents. This study aims at investigating the antitumor effect of oleanolic acid (OA) and the molecular mechanisms. BALB/c mice were injected subcutaneously with HepG2 cells to establish transplanted tumors. Apoptosis and cell cycle arrest-related markers and signaling cascades were determined by western blot, immunofluorescence, reverse transcriptase-polymerase chain reaction and flow cytometric analysis. OA exhibited inhibitory effect on HCC through induction of apoptosis and cell cycle arrest both in transplanted tumors and in HepG2 cells. OA induced apoptosis through mitochondrial pathway, evidenced by inhibition of Akt/mammalian target of rapamycin pathway, mitochondrial dysfunction, transient increase of adenosine triphosphate, increase of Bax/Bcl-2 ratio, increased release of cytochrome c and activation of caspase/poly (ADP-ribose) polymerase. Activation of mitochondrial apoptotic pathway may be due to reactive oxygen species generated by mitochondrial fatty acid oxidation, resulted from enhancement of lipolysis regulated by cyclic adenosine 3',5'-monophosphate response element-binding protein-hormone-sensitive lipase/peroxisome proliferator-activated receptor γ signaling. OA induced G2/M cell cycle arrest through p21-mediated downregulation of cyclin B1/cdc2. Cyclooxygenase-2 (COX-2) and p53 were involved in OA-exerted effect, and extracellular signal-regulated kinase-p53 signaling played a central role in OA-activated cascades responsible for apoptosis and cell cycle arrest. OA demonstrated significant antitumor activities in HCC in vivo and in vitro models. These data provide new insights into the mechanisms underlying the antitumor effect of OA.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin B1; Cyclooxygenase 2; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; G2 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mitochondria; Neoplasm Transplantation; Oleanolic Acid; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; TOR Serine-Threonine Kinases; Transplantation, Heterologous; Tumor Suppressor Protein p53

The anticancer activities of gambogic acid (GA) on two hepatocellular carcinoma cells with either p53 deletion (Hep3B) or p53 mutation (Huh7) were investigated in the present study. GA inhibited the growth of Hep3B and Huh7 through similar apoptotic pathways. After treatment of Hep3B and Huh7 with GA for 24 h, the IC₅₀ was determined for both cell lines at 1.8 and 2.2 µM, respectively. The results showed that both cancer cells underwent morphological changes and DNA fragmentation. GA induced apoptosis in the two cell lines through caspases-3/7, -8 and -9 in the mitochondrial pathway. The results suggest that both the caspases in the extrinsic death receptor pathway and the mitochondrial-dependent pathway are involved in the GA-induced cell apoptosis. The inhibitory effects of GA on Hep3B and Huh7 are independent of p53-associated pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Garcinia; Humans; Liver Neoplasms; Mitochondria; Plant Extracts; Signal Transduction; Tumor Suppressor Protein p53; Xanthones

The mechanisms of toxicity related to human hepatocellular carcinoma HepG2 cell exposures to cadmium telluride quantum dots (CdTe-QDs) were investigated. CdTe-QDs caused cytotoxicity in HepG2 cells in a dose- and time-dependent manner. Treated cells showed an increase in reactive oxygen species (ROS). Altered antioxidant levels were demonstrated by depletion of reduced glutathione (GSH), a decreased ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) and an increased NF-E2-related Factor 2 (Nrf2) activation. Enzyme assays showed that superoxide dismutase (SOD) activity was elevated whereas catalase (CAT) and glutathione-S-transferase (GST) activities were depressed. Further analyses revealed that CdTe-QD exposure resulted in apoptosis, indicated by changes in levels of caspase-3 activity, poly ADP-ribose polymerase (PARP) cleavage and phosphatidylserine externalization. Extrinsic apoptotic pathway markers such as Fas levels and caspase-8 activity increased as a result of CdTe-QD exposure. Involvement of the intrinsic/mitochondrial apoptotic pathway was indicated by decreased levels of B-cell lymphoma 2 (Bcl2) protein and mitochondrial cytochrome c, and by increased levels of mitochondrial Bcl-2-associated X protein (Bax) and cytosolic cytochrome c. Further, mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (Erk1/2), and p38 were all activated. Our findings reveal that CdTe-QDs cause oxidative stress, interfere with antioxidant defenses and activate protein kinases, leading to apoptosis via both extrinsic and intrinsic pathways. Since the effects of CdTe-QDs on selected biomarkers were similar or greater compared to those of CdCl2 at equivalent concentrations of cadmium, the study suggests that the toxicity of CdTe-QDs arises from a combination of the effects of cadmium and ROS generated from the NPs.

Topics: Apoptosis; Cadmium Compounds; Carcinoma, Hepatocellular; Catalase; Cell Survival; Cytochromes c; Glutathione; Glutathione Disulfide; Hep G2 Cells; Humans; Liver Neoplasms; Microscopy, Confocal; Mitogen-Activated Protein Kinases; NF-E2-Related Factor 2; Oxidative Stress; Quantum Dots; Signal Transduction; Superoxide Dismutase; Tellurium

Small-molecule Bcl-2/Bcl-xL inhibitor Navitoclax represents a promising cancer therapeutic since preclinical and clinical studies with Navitoclax have demonstrated strong anticancer activity in several types of cancers. However, because Navitoclax has a low binding affinity to Mcl-1, anticancer activity by Navitoclax is often attenuated by the elevated expression of Mcl-1 in hepatocellular carcinoma (HCC) and other cancers, posing a serious problem for its potential clinical utilities. Therefore, approaches that suppress the expression of Mcl-1 are urgently needed to overcome Navitoclax-resistance in these cancers. Here, we reported that aspirin markedly suppressed Mcl-1 expression, and significantly enhanced Navitoclax-mediated cell viability inhibition and apoptosis induction in HCC cells. We further showed that aspirin robustly enhanced Navitoclax-triggered cytosolic cytochrome c release, activation of initiator caspase-9 and effector caspase-3, and cleavage of PARP. Importantly, the cell death induction by the combination could be rescued by a cell-permeable caspase-9 inhibitor Z-LEHD-FMK, indicative of an indispensable role of mitochondrial apoptosis pathway during the combination effect. Taken together, our study suggests that aspirin can be used to enhance Navitoclax-mediated anticancer activity via suppression of Mcl-1. Since aspirin is one of the most commonly used medicines, our findings therefore have translational impacts on Navitoclax-based therapy for HCC.

Topics: Aniline Compounds; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Aspirin; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Hep G2 Cells; Humans; Liver Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides

α-Mangostin is a dietary xanthone that has been shown to have anti-cancer and anti-proliferative properties in various types of human cancer cells. This study investigates the molecular mechanism of the apoptosis-inducing effects of α-mangostin on human hepatocellular carcinoma (HCC) cells. We observed that α-mangostin reduces the viability of HCC cells in a dose- and time-dependent manner. α-Mangostin mediated apoptosis of SK-Hep-1 cells is accompanied by nuclear chromatin condensation and cell cycle arrest in the sub-G1 phases as well as phosphatidylserine exposure. Furthermore, α-mangostin triggered the mitochondrial caspase apoptotic pathway, as indicated by the loss of mitochondrial membrane potential, the release of cytochrome c from mitochondria, and the regulation of B cell lymphoma 2 family member expression. Moreover, α-mangostin inhibited a sustained activation of p38 mitogen-activated protein kinase (MAPK) phosphorylation, and treatment with a p38 MAPK inhibitor enhanced α-mangostin-induced caspase activation and apoptosis in SK-Hep-1 cells. In vivo xenograft mice experiments revealed that α-mangostin significantly reduced tumor growth and weight in mice inoculated with SK-Hep-1 cells. These findings demonstrate that α-mangostin induces mitochondria-mediated apoptosis through inactivation of the p38 MAPK signaling pathway and that α-mangostin inhibits the in vivo tumor growth of SK-Hep-1 xenograft mice.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cytochromes c; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mitochondria; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Xanthones

Daidzein, which belongs to the group of isoflavones from soybeans, has been extensively researched prostate, cervix, brain, breast, and colon cancer cell lines. However, daidzein has not been thoroughly investigated in human hepatic cancer cells; therefore, we investigated whether it inhibits hepatic cancer cell growth. Decreased cell proliferation was measured in daidzein-treated hepatic cancer cells (SK-HEP-1) upon real-time cell electronic sensing analysis however, it was not affected on normal human hepatocytes (Chang). Daidzein-induced apoptosis was demonstrated by comet and TUNEL assay. Moreover, we conducted two-dimensional electrophoresis to study the mechanism of daidzein-induced apoptosis in daidzein-treated SK-HEP-1 cells. Expression of peroxiredoxin-3 (Prdx-3), which modulates redox homeostasis of cells, was increased in protein analysis. Additionally, we measured the levels of reactive oxygen species and it was decreased in daidzein-treated SKHEP-1 cells. Daidzein-induced apoptosis in SK-HEP-1 cells was also associated with the up-regulation of Bak and down-regulation of Bcl-2 and Bcl-xL proteins. Moreover, daidzein treatment increased in the release of mitochondrial cytochrome c and activation of APAF-1, caspase 9 and caspase 3. Overall, these result indicate that daidzein is a potent inducer of apoptosis in hepatic cancer cells via mitochondrial pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Comet Assay; Cytochromes c; Down-Regulation; Humans; In Situ Nick-End Labeling; Isoflavones; Liver Neoplasms; Mitochondria; Peroxiredoxin III; Reactive Oxygen Species; Signal Transduction; Up-Regulation

Extract of Myrica cerifera bark has long been fruitfully used as a hepato-protective and anti-cancer drug in various complementary and alternative systems of medicine. Myricanone, its principal bioactive compound, had also been reported to have apoptosis-promoting ability. We evaluated its anti-cancer potential in vitro in HepG2 liver cancer cells and tried to understand the signal cascades involved in accomplishing apoptosis. Further, we ascertained by using a (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay (MTT) assay if it had cytotoxic effects on normal noncancerous liver cells (WRL-68). We deployed various tools and protocols, like phase contrast, scanning electron and fluorescence microscopies, performed an annexinV-FITC/PI assay and cell cycle analysis, and estimated the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization through flow cytometry. Further, analyses of cytochrome-c translocation and of HSP70 and caspase expressions were also done by using immunoblota and Enzyme linked immunosorbent assay (ELISA). Results revealed that myricanone induced apoptosis in HepG2 cells through generation of ROS, depolarization of the mitochondrial membrane, early release of cytochrome-c, down-regulation of HSP70 and activation of a caspase cascade; it had no, or insignificant, cytotoxic effects in WRL-68 cells in vitro and in mice in vivo. Thus, myricanone has great potential for use in formulating an effective drug against both hepatotoxicity and hepatocellular cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Cell Cycle; Cytochromes c; Diarylheptanoids; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mitochondria; Myrica; Plant Extracts; Reactive Oxygen Species; Signal Transduction

Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established; however, the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca(2+) were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase 3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein.

Topics: Adenosine Triphosphate; Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cyclosporine; Cytochromes c; Hep G2 Cells; Humans; In Situ Nick-End Labeling; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Permeability; Phytotherapy; Plant Extracts; Rheum

Novel liposomes composed of L-α-dimyristoylphosphatidylcholine (DMPC) and trehalose surfactant (DMTreCn) were produced by the method of sonication in buffer solution. The thickness of fixed aqueous layer of DMTreCn was larger than that of DMPC liposomes and increased in a dose-dependent manner. The remarkable inhibitory effects of DMTreCn on the growth of human hepatocellular carcinoma (HCC) (Hep-G2 and HuH-7) cells were obtained along with apoptosis, without affecting the growth of normal cells. DMTreCn induced apoptosis of Hep-G2 and HuH-7 cells through the activation of caspase-3, 8 and 9. Release of cytochrome c from mitochondria and activation of Bcl-2 family protein (BAX) were recorded, indicating that DMTreCn induced apoptosis of Hep-G2 and HuH-7 cells through mitochondrial pathway via BAX. It is noteworthy that the remarkable inhibitory effects of DMTreCn on the growth of human HCC cells were obtained along with apoptosis for the first time.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cell Membrane; Cytochromes c; Flow Cytometry; Humans; Liposomes; Liver Neoplasms; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; Surface-Active Agents; Trehalose; Tumor Cells, Cultured

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths reported worldwide. The incidence is higher in Asia and Africa, where there is greater endemic prevalence of hepatitis B and C. The devastating outcome of cancer can be minimized only by the use of potent therapeutic agents. Tridham (TD) has been acknowledged since olden days for its wide spectrum of biological properties and was used by traditional practitioners of Siddha and other indigenous systems of medicine. The present study aims at investigating the mechanistic action of TD by assessing the antiproliferative and pro-apoptotic effects on human hepatocellular carcinoma cell line (Huh7).. Cell viability and apoptosis assay using MTT analysis and trypan blue staining, DAPI staining, DNA fragmentation, cell cycle analysis, mitochondrial membrane potential, real-time reverse transcription-polymerase chain reaction, western blotting and immunofluorescence staining were determined in Huh7 cells.. Viability studies of TD treated Huh7 cells showed an inhibition in cell growth in time and dose dependent manner. Chromatin condensation, DNA fragmentation and apoptotic bodies, which are structural changes characteristic of apoptosis, were found following TD treatment of Huh7 cells. DAPI staining and agarose gel electrophoresis confirmed the induction of apoptosis by TD. Cell cycle analysis of Huh7 cells treated with TD exhibited a marked accumulation of cells in the sub-G1 phase of the cell cycle in a dose dependent manner. Immunofluorescent staining for Ki-67 showed a higher level of expression in untreated cells as compared to TD treated cells. We observed a significant loss in the mitochondrial membrane potential and the release of cytochrome c into the cytosol in TD treated cells. Down regulation of Bcl-2, up regulation of Bax and Bad as well as activation of caspases-3 and 9 were also observed. The p53 gene expression was found to be unaltered in TD treated cells.. These results suggest that TD induces apoptosis of Huh7 cells through activation of Bax and triggered caspase cascade, independent of p53 function. This study throws light on the mechanistic action of TD in triggering apoptosis in Huh 7 cells.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Humans; Liver Neoplasms; Medicine, Ayurvedic; Membrane Potential, Mitochondrial; Plant Extracts; Tumor Suppressor Protein p53

Phlorotannins have been reported to demonstrate several biological properties, including antioxidant activity, and activities useful in the treatment of diabetic complications and in chemoprevention of several vascular diseases. In this study, we focused on the apoptosis induced by dieckol, a marine algal phlorotannin isolated from Ecklonia stolonifera, on human hepatocellular carcinoma (HCC) Hep3B cells. Dieckol reduced the numbers of viable cells and increased the numbers of apoptotic cells in a dose-dependent manner. Immunoblotting analysis revealed that dieckol increased the expression levels of cleaved caspases-3, 7, 8, and 9, and cleaved poly(ADP-ribose) polymerase. Dieckol increased the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with apoptosis-inducing factor. In addition, dieckol induced increased expression of truncated Bid and Bim. The results indicate that dieckol induces apoptosis via the activation of both death receptor and mitochondrial-dependent pathways in HCC Hep3B cells.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Benzofurans; BH3 Interacting Domain Death Agonist Protein; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; HEK293 Cells; Humans; Liver Neoplasms; Membrane Proteins; Mitochondrial Membranes; Permeability; Phaeophyceae; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins

Sorafenib has been used to treat advanced hepatocellular carcinoma (HCC), but the underlying molecular mechanisms remain controversial and why some patients do not respond to this therapy is poorly understood. In this study, we show that sorafenib triggers cell growth inhibition and apoptosis in HCC cells by directly targeting the mitochondria. Treatment with sorafenib induces rapid mitochondrial fragmentation, which is associated with the deregulation of mitochondria fusion-related protein optic atrophy 1 (OPA1). Exposure of cells or isolated mitochondria to sorafenib substantially induces cytochrome c release. Our data indicate that siRNA-mediated OPA1 knockdown significantly sensitizes HCC cells to sorafenib-induced apoptosis. Furthermore, sorafenib has no apparent apoptotic toxicity to normal human primary hepatocytes. Sorafenib inhibits HCC xenograft tumor growth in vivo and murine xenograft tumor tissue analysis reveals mitochondria fusion protein. OPA1 expression levels are strongly downregulated by sorafenib treatment. Western blotting evaluation of patient HCC with matched non-tumor tissue samples demonstrates that OPA1 expression is decreased in up to 40% of HCC patients. Taken together, we have shown that sorafenib suppresses the tumorigenesis of HCC through the induction of mitochondrial injury via OPA1. Our results provide new insights into the pathogenesis of HCC and suggest that OPA1 is a novel therapeutic target in patients with HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; Down-Regulation; Gene Knockdown Techniques; GTP Phosphohydrolases; Humans; Liver; Liver Neoplasms; Mice; Mice, SCID; Mitochondria; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; ras Proteins; RNA, Small Interfering; Signal Transduction; Sorafenib; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, therapies against HCC to date have not been completely effective. Sinomenine hydrochloride (SH), an anti‑arthritis drug applied in clinical practice, has been reported to have in vitro anti‑neoplastic activity in various cancer cells. Whether SH inhibits HCC remains unknown. For this purpose, in this study, MTT assay was used to determine cell growth. Flow cytometry, Hoechst staining, DNA fragmentation, western blot analysis, immunohistochemisty and TUNEL staining were performed to investigate the mechanisms involved. The in vivo activity of SH was determined using a mouse xenograft model. SH inhibited the growth of various types of human HCC cells in vitro. We found that SH promoted cell cycle arrest in the G1 phase and sub‑G1 formation, associated with the increased p21/WAF1/Cip1 expression. Additionally, SH induced caspase‑dependent apoptosis, which involved the disruption of mitochondrial membrane potential, the increased release of cytochrome c and Omi/HtrA2 from the mitochondria into the cytoplasm, the downregulation of Bcl‑2 and the upregulation of Bax, the activation of a caspase cascade (caspase‑8, -10, -9 and -3) and PARP, as well as the decreased expression of survivin. The SH‑suppressed growth of human HCC xenografts in vivo occurred due to the decrease in proliferation and the induction of apoptosis, implicating the activation of caspase‑3, the upregulation of p21 and the downregulation of survivin. These findings suggest that SH exhibits anticancer efficacy in vitro and in vivo involving cell cycle and caspase‑dependent apoptosis and may serve as a potential drug candidate against HCC.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Hepatocellular; Caspases; Cell Cycle Checkpoints; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Flow Cytometry; Humans; In Vitro Techniques; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Morphinans; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

In the present study, in order to investigate the anticancer effects of O-desmethylangolensin (O-DMA) on human hepatocellular carcinoma Hep3B cells, we first examined the antiproliferative effect of O-DMA. When Hep3B cells were treated with O-DMA at various concentrations (5-200 µM) for 24, 48 or 72 h, cell proliferation decreased significantly in a dose- and time-dependent manner. Moreover, O-DMA exposure at the IC50 concentration for 72 h arrested cells at the G2/M phase, which was accompanied by a reduction in CDK1, and an increase in cyclin A and B. Under the same conditions, O-DMA significantly increased the number of sub-G1 phase cells. Additionally, an Annexin V assay revealed that exposure to O-DMA affected the rate of cell apoptosis. O-DMA caused the downregulation of Bcl-2 and upregulation of Bax, which led to cytochrome c release from the mitochondria and activation of caspase-3. Taken together, these data suggest that O-DMA exhibits anticancer activity by arresting the cell cycle at G2/M phase and causing mitochondrial-dependent apoptosis in Hep3B cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B; Cytochromes c; G2 Phase Cell Cycle Checkpoints; Humans; Isoflavones; Liver Neoplasms; Mitochondria; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2

To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus (C. orbiculatus) extract in human hepatocellular carcinoma cells.. Human hepatocellular carcinoma cells (HCCLM6) were treated with C. orbiculatus extract (COE) at different nontoxic concentrations (10, 20, 40, 80, and 160 microg/mL). The effect of COE on HCCLM6 viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis.. COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner. Apoptosis was accompanied by increased Bax expression and decreased Bcl-2 expression. In addition, COE treatment led to the release of cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Furthermore, activation of extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulation of Akt phosphorylation was observed.. COE induces mitochondrial-mediated, caspase-dependent apoptosis in HCCLM6 cells, which might be attributed to the activation of mitogen-activated protein kinase (MAPK) and inhibition of Akt signaling pathways. These data suggest that COE may be a potential treatment for human hepatocellular carcinoma.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Celastrus; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitochondria; Plant Extracts; Proto-Oncogene Proteins c-bcl-2

Ginsenoside Rh2 (G-Rh2) has been shown to induce apoptotic cell death in a variety of cancer cells. However, the details of the signal transduction cascade involved in G-Rh2-induced cell death is unclear. In this manuscript we elucidate the molecular mechanism of G-Rh2-induced apoptosis in human hepatoma SK-HEP-1 cells by demonstrating that G-Rh2 causes rapid and dramatic translocation of both Bak and Bax, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Interestingly, siRNA-based gene inactivation of caspase-8 effectively delays caspase-9 activation and apoptosis induced by G-Rh2, indicating that caspase-8 also plays an important role in the G-Rh2-induced apoptosis program. Taken together, our results indicate that G-Rh2 employs a multi pro-apoptotic pathway to execute cancer cell death, suggesting a potential role for G-Rh2 as a powerful chemotherapeutic agent.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Ginsenosides; Humans; Liver Neoplasms

Hepatic inflammation and degeneration induced by lipid depositions may be the major cause of nonalcoholic fatty liver disease. In this study, we tried to investigate the effects of saturated and unsaturated fatty acids on hepatoma cell apoptosis.. H4IIE liver cells were treated with palmitic acid, linoleic acid, or both with or without the calcium-specific chelator BAPTA-AM after which the expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis, caspase-3 levels, and calcium flux were measured.. Palmitic or linoleic acid (250 μM) induced H4IIE cell apoptosis, which required calcium flux but not caspase-3. Apoptosis was not observed when cells were co-treated with linoleic acid (125 μM) and palmitic acid (250 μM). Importantly, the release of cytochrome C from mitochondria into cytoplasm during cell apoptosis was specifically detected only when linoleic acid (125 μM), but not palmitic acid (250 μM), was added to the cells. Depletion of intracellular calcium flux by the calcium-specific chelator, BAPTA-AM, abolished linoleic acid-induced apoptosis. Moreover, in the presence of BAPTA-AM, expression of the unfolded protein response (UPR)-associated genes, CHOP, GRP78, and GRP94, was induced by linoleic acid, but not palmitic acid.. The results suggest that linoleic acid promotes cell apoptosis through the release of cytochrome C, only if the intracellular calcium flux is unperturbed and intact. These results confirm that ER stress contributes to fatty acid-induced liver cell apoptosis.

Topics: Animals; Apoptosis; Calcium Signaling; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cytochromes c; Endoplasmic Reticulum Stress; Fatty Liver; Gene Expression; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Linoleic Acids; Liver Neoplasms; Membrane Proteins; Non-alcoholic Fatty Liver Disease; Palmitic Acids; Rats; Transcription Factor CHOP; Unfolded Protein Response

Radiation-induced bystander effect (RIBE) has important implication in tumour radiotherapy, but the bystander signals are still not well known.. The role of cytochrome-c (cyt-c) and free radicals in RIBE on human hepatoma cells HepG2 was investigated by detecting the formation of bystander micronuclei (MN) and the generation of endogenous cyt-c, inducible nitric oxide (NO) synthase (iNOS), NO, and reactive oxygen species (ROS) molecules.. When HepG2 cells were cocultured with an equal number of irradiated HepG2 cells, the yield of MN in the nonirradiated bystander cells was increased in a manner depended on radiation dose and cell coculture time, but it was diminished when the cells were treated with cyclosporin A (CsA), an inhibitor of cyt-c release. Meanwhile the CsA treatment inhibited radiation-induced NO but not ROS. Both of the depressed bystander effect and NO generation in the CsA-treated cells were reversed when 5 μM cyt-c was added in the cell coculture medium. But these exogenous cyt-c-mediated overproductions of NO and bystander MN were abolished when the cells were pretreated with s-methylisothiourea sulphate, an iNOS inhibitor.. Radiation-induced cyt-c has a profound role in regulating bystander response through an iNOS-triggered NO signal but not ROS in HepG2 cells.

Topics: Bystander Effect; Carcinoma, Hepatocellular; Cell Line, Tumor; Coculture Techniques; Cyclosporine; Cytochromes c; Gamma Rays; Hep G2 Cells; Humans; Isothiuronium; Liver Neoplasms; Micronucleus Tests; Nitric Oxide; Nitric Oxide Synthase Type II; Reactive Oxygen Species; Signal Transduction

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has been shown to exhibit inhibitory effects on human cancer cells. However, its antitumor ability toward hepatocellular carcinoma (HCC) has not been studied. In this study, we demonstrated that DHA significantly inhibited HCC cell growth in vitro and in vivo via inducing G2/M cell cycle arrest and apoptosis. The induction of p21 and the inhibition of cyclin B and CDC25C contributed to DHA-induced G2/M arrest. DHA-induced apoptosis was associated with mitochondrial membrane depolarization, release of cytochrome c, activation of caspases, and DNA fragmentation. Activation of caspase 9 and caspase 3, but not caspase 8, was detected in DHA-treated cells. Attenuation of apoptosis in cells pretreated with Z-VAD-FMK suggested the involvement of caspase cascade. Furthermore, p53 facilitated apoptosis caused by DHA. Bcl-2 family proteins were also responsible for DHA-induced apoptosis. DHA exposure decreased Mcl-1 expression but increased the levels of Noxa and active Bak. Bak was released from the Mcl-1/Bak complex due to the decline of Mcl-1. Further study revealed that Mcl-1 was rapidly degraded in DHA-treated cells and that DHA-induced apoptosis was largely inhibited by overexpression of Mcl-1 or RNAi-mediated decrease of Bak and Noxa. In a HCC-xenograft mouse model, the intraperitoneal injection of DHA resulted in significant inhibition of HCC xenograft tumors. Taken together, our data, for the first time, demonstrate the potential antitumor activity of DHA in HCC.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Artemisinins; bcl-2 Homologous Antagonist-Killer Protein; Carcinoma, Hepatocellular; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Genes, p53; Humans; Liver Neoplasms; Mice; Mitochondrial Membranes; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most common internal malignant tumors. Glypican-3 (GPC3) is involved in the biological and molecular events in the tumorigenesis of HCC. We used RNA interference to evaluate the molecular effects of GPC3 suppression at the translational level and demonstrated for the first time that GPC3 silencing results in a significant elevation of the Bax/Bcl-2 ratio, the release of cytochrome c from mitochondria and the activation of caspase-3. The results suggest that GPC3 regulates cell proliferation by enhancing the resistance to apoptosis through the dysfunction of the Bax/Bcl-2/cytochrome c/caspase-3 signaling pathway and therefore plays a critical role in the tumorigenesis of HCC. Thus, the knockdown of GPC3 should be further investigated as an attractive novel approach for the targeted gene therapy of HCC.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 3; Cytochromes c; Gene Silencing; Genetic Therapy; Glypicans; Hep G2 Cells; Humans; Liver Neoplasms; RNA Interference

6-Gingerol, a major phenolic compound derived from ginger, has been known to possess anticarcinogenic activities. However, the mechanisms are not well understood. In our previous study, it was demonstrated that lysosome and mitochondria may be the primary targets for 6-gingerol in HepG2 cells. Therefore, the aim was to evaluate lysosome-mitochondria cross-signaling in 6-gingerol-induced apoptosis. Apoptosis was detected by Hoechst 33342 and TUNEL assay after 24 h treatment, and the destabilization of lysosome and mitochondria were early upstream initiating events. This study showed that cathepsin D played a crucial role in the process of apoptosis. The release of cathepsin D to the cytosol appeared to be an early event that preceded the release of cytochrome c from mitochondria. Moreover, inhibition of cathepsin D activity resulted in suppressed release of cytochrome c. To further determine the involvement of oxidative stress in 6-gingerol-induced apoptosis, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined. Taken together, these results suggest that cathepsin D may be a positive mediator of 6-gingerol induced apoptosis in HepG2 cells, acting upstream of cytochrome c release, and the apoptosis may be associated with oxidative stress.

Topics: Apoptosis; Catechols; Cathepsin D; Cytochromes c; Fatty Alcohols; Glutathione; Hep G2 Cells; Humans; Liver Neoplasms; Lysosomes; Mitochondria; Oxidative Stress; Reactive Oxygen Species

Shochu distillation remnants (SDR) are by-products in the manufacturing process of the Japanese liquor Shochu and include various useful organic compounds derived from the fermentation of grains. We have obtained valuable powder (PSDR) from freeze-dried SDR by the treatment with ethanol. In this study, we examined the anticancer effects of barley-, rice-, and sweet potato-PSDR against HepG2 and HuH-7 cells of human hepatocellular carcinoma (HCC) in vitro. All PSDR inhibited the growth of both these HCC cells through the induction of apoptosis. Especially, barley-PSDR was the most effective for the growth inhibition and apoptosis induction of HCC cells of all PSDR. We next examined the apoptotic mechanisms induced by barley-PSDR. Decrease in mitochondrial membrane potential and release of cytochrome c from mitochondria were observed in HCC cells after the treatment with barley-PSDR. Furthermore, barley-PSDR induced the nuclear translocation of apoptosis-inducing factor (AIF) from mitochondria, while it did not significantly affect the activities of caspase-3, -8, and -9. The results suggested that barley-PSDR induced apoptosis against HCC cells via the caspase-independent mitochondrial pathway. The findings in this study suggest that PSDR has the possibility of therapeutic and/or preventive agents of HCC.

Topics: Active Transport, Cell Nucleus; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cytochromes c; Distillation; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Powders

Alantolactone, a sesquiterpene lactone, possesses anti-inflammatory property. In this study, we provide evidence that it could be developed as a novel agent against human liver cancer. We observed that alantolactone treatment to HepG2, Bel-7402 and SMMC-7721 cells, human liver cancer cell lines resulted in a dose-dependent inhibition of cell growth. We selected HepG2 cell line as a test model system. Alantolactone treatment of HepG2 cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G2-M phase. This induction of apoptosis seems to be mediated via modulating the protein levels of Bcl-2 family and activation of caspases. Moreover, caspase-8 and Bid activation, loss of mitochondrial transmembrane potential and cytochrome c release suggest the existence of a cross-talk between the death receptor and the mitochondrial pathways. We also observed that alantolactone treatment of cells resulted in a dose-dependent decrease in NF- κB/p65. In addition, a significant and progressive increase in the level of p53 protein in alantolactone-treated cells was observed. Taken together, our data suggest that alantolactone could be developed as an agent against human liver cancer.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Carcinoma, Hepatocellular; Caspase 8; Cytochromes c; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Lactones; Liver Neoplasms; Poly(ADP-ribose) Polymerases; Sesquiterpenes, Eudesmane

Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.

Topics: Animals; Apoptosis; Artemisinins; Cell Line, Tumor; Cytochromes c; Drug Synergism; Enzyme Activation; Flow Cytometry; Histone Deacetylase Inhibitors; In Situ Nick-End Labeling; Liver Neoplasms; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Phosphorylation

The mechanisms underlying the antiproliferative and antitumor activities of aromatic turmerone (ar-turmerone), a volatile turmeric oil isolated from Curcuma longa Linn., have been largely unknown. In this study, 86% pure ar-turmerone was extracted by supercritical carbon dioxide and liquid-solid chromatography and its potential effects and molecular mechanisms on cell proliferation studied in human hepatocellular carcinoma cell lines. Ar-turmerone exhibited significant antiproliferative activity, with 50% inhibitory concentrations of 64.8 ± 7.1, 102.5 ± 11.5, and 122.2 ± 7.6 μg/mL against HepG2, Huh-7, and Hep3B cells, respectively. Ar-turmerone-induced apoptosis, confirmed by increased annexin V binding and DNA fragmentation, was accompanied by reactive oxygen species (ROS) production, mitochondrial membrane potential dissipation, increased Bax and p53 up-regulated modulator of apoptosis (PUMA) levels, Bax mitochondrial translocation, cytochrome c release, Fas and death receptor 4 (DR4) augmentation, and caspase-3, -8, and -9 activation. Exposure to caspase inhibitors, Fas-antagonistic antibody, DR4 antagonist, and furosemide (a blocker of Bax translocation) effectively abolished ar-turmerone-triggered apoptosis. Moreover, ar-turmerone stimulated c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) phosphorylation and activation; treatment with JNK and ERK inhibitors markedly reduced PUMA, Bax, Fas, and DR4 levels and reduced apoptosis but not ROS generation. Furthermore, antioxidants attenuated ar-turmerone-mediated ROS production; mitochondrial dysfunction; JNK and ERK activation; PUMA, Bax, Fas, and DR4 expression; and apoptosis. Taken together, these results suggest that ar-turmerone-induced apoptosis in HepG2 cells is through ROS-mediated activation of ERK and JNK kinases and triggers both intrinsic and extrinsic caspase activation, leading to apoptosis. On the basis of these observations, ar-turmerone deserves further investigation as a natural anticancer and cancer-preventive agent.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Carbon Dioxide; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Chromatography, Supercritical Fluid; Curcuma; Cytochromes c; Drug Screening Assays, Antitumor; Extracellular Signal-Regulated MAP Kinases; Fas Ligand Protein; Hep G2 Cells; Humans; Ketones; Liver Neoplasms; MAP Kinase Kinase 4; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction

Researchers have proposed that VAA-I, a specific plant lectin found in Viscum album, has therapeutic effects on cancer and autoimmune diseases. VAA-I has shown some promising treatment results in some types of tumor cell lines, especially SMMC-7721 cells (human hepatocellular carcinoma cells). However, few details are known about the mechanism and process of cell death induced by VAA-I in tumor cells. In this study, the cell morphology results showed that SMMC-7721 cells treated with VAA-I exhibited several features typical of apoptotic cell death, which was confirmed by the Caspase inhibition assay. Fluo-3-acetoxymethyl ester (AM) fluorescence imaging techniques showed that rVAA-I significantly elevated the intracellular calcium level ([Ca2+]i) in SMMC-7721 cells. These findings suggest that apoptosis may play the most important role in SMMC-7721 cell death induced by rVAA-I. Finally, in the SMMC-7721 cells treated with rVAA-I, a series of genes in the p38 mitogen-activated protein kinase (MAPK) signaling pathway were expressed differentially, and further found that PI 3-kinase pathway is involved in rVAA-I signal transduction in SMMC-7721 cells.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cytochromes c; Humans; Liver Neoplasms; Mitochondria; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Pichia; Protein Kinase Inhibitors; Recombinant Proteins; Ribosome Inactivating Proteins; Ribosome Inactivating Proteins, Type 2; Signal Transduction; Time Factors; Toxins, Biological; Viscum album

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.

Topics: Antimetabolites, Antineoplastic; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cytochromes c; Fluorouracil; Humans; In Situ Nick-End Labeling; Liver Neoplasms; Recombinant Proteins; Tumor Cells, Cultured

To study the effects of cadmium chloride (CdCl(2)) combined with hSmac on the proliferation and apoptosis of hepatocellular carcinoma cells, i.e. SMMC-7721.. SMMC-7721 cells were transfected with pcDNA3.1(+)-hSmac using a lipofectamine-mediated method, and then cell viability was detected by MTT assay after exposure to 10, 20, and 30 μmol/l CdCl(2). Apoptosis was determined by both acridine orange-ethidium bromide staining and flow cytometry, and expressions of caspase-3, caspase-9, and cytochrome c by Western blot.. CdCl(2) had cytotoxicity to SMMC-7721 cells, and it could inhibit proliferation in a dose-dependent manner and induce apoptosis; hSmac could inhibit proliferation and induce apoptosis independently in SMMC-7721 cells. Furthermore, cotreatment with CdCl(2) and hSmac could enhance antiproliferative and proapoptotic effects in SMMC-7721 cells.. hSmac could enhance the cytotoxicity of CdCl(2).

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cadmium Chloride; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Combined Modality Therapy; Cytochromes c; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mitochondrial Proteins

Coumarins are natural compounds found in many plants that possess medical value by itself and its modified derivatives.. Six novel coumarin derivatives were synthesized and examined for their potential anticancer cytotoxicity.. Among the 6 derivatives, 3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) presented the strongest cytotoxicity against human hepatoma HepG2 cells in vitro with an IC(50) value of 8.46 ± 0.28 μM in a 48-hour treatment. Further experiments revealed that DMFC induced apoptosis in HepG2 cells through both extrinsic and intrinsic apoptotic pathways in a p53-dependent manner. Mechanistically, DMFC activated caspases 3, 8 and 9, depolarized mitochondrial membrane potential and induced cytochrome c and apoptosis-inducing factor release. DMFC-induced apoptosis was also characterized by DNA fragmentation, phosphatidylserine externalization and sub-G1 peak in DNA histograms. Moreover, both caspase 8 and 9 inhibitors suppressed the apoptosis induced by DMFC. Western blot analyses revealed that DMFC also significantly increased the expression levels of p53, Fas death receptor, Fas-associated death domain protein and proapoptotic Bcl-2 family members such as Bax, Bad and tBid, as well as decreased the levels of pro-survival members such as Bcl-2 and Bcl-xl.. DMFC is potentially an effective therapeutic agent in liver cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-X Protein; Benzofurans; Carcinoma, Hepatocellular; Caspases; Coumarins; Cytochromes c; DNA Fragmentation; fas Receptor; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Many clinical reports have proven that the combination therapy of interferon-alpha plus 5-fluorouracil is remarkably effective for advanced hepatocellular carcinoma (HCC). However, the mechanism of this therapy is not well understood. Here, we demonstrated that the combination therapy synergistically inhibited the growth of Fas-negative HCC cells, arrested cell-cycle progression and induced apoptosis. Moreover, the combination therapy significantly increased the protein expression of caspase-8, activated Bid and cytochrome c. Meanwhile, the expression of anti-apoptotic gene Bcl-xL was reduced and intracellular calcium elevated obviously during the early stage of treatment. Therefore, mitochondrial pathway was involved in the apoptosis of Fas-negative HCC cells induced by IFN-α/5-FU and Ca(2+) partially promoted the beneficial effect against HCC.

Topics: Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Calcium; Carcinoma, Hepatocellular; Caspase 8; Cell Line; Cell Proliferation; Cytochromes c; Disease Progression; Fluorouracil; Humans; In Vitro Techniques; Interferon-alpha; Liver Neoplasms; Mitochondria

To evaluate the pro-apoptotic and anti-tumorigenic properties of Thapring - a Traditional Tibetan Medicine - in hepatoma cells and in a transgenic mouse model of hepatocellular carcinoma.. The pro-apoptotic action and growth inhibition property of Thapring were assessed in Huh7, HepG2 and A549 cell lines using flow cytometry and MTT assay, respectively. Confocal microscopy for colocalization of cytochrome c and mitochondria was done using dsRed mitotracker in Huh7 cells. The activation of p38 MAP kinase and p53 pathway was evaluated by Western blotting. Serological studies for liver function, vascular endothelial growth factor and superoxide dismutase were assessed in the serum of X15-myc transgenic mice. Immuno-histochemical studies for Bcl2 and p21(Waf1) expression were also carried out in the liver section of the above mice.. Treatment with Thapring inhibited proliferation and accumulation of hepatoma cells in G1 phase. There was increased cytochrome c release from mitochondria and decreased Bcl2 levels - the key markers of apoptotic cell death. Besides activation of p38 MAP kinase and increased p53 expression were also observed. Oral administration of Thapring in transgenic mice lowered serum VEGF levels and conferred hepatoprotection as evident from normal serum ALT levels. Further, immunohistochemical analysis of the liver samples revealed reduced expression of anti-apoptotic protein Bcl2 and over-expression of cell cycle regulator p21(Waf1).. The ability of Thapring to impose growth arrest and trigger pro-apoptotic death in cell culture as well as ameliorative effects in vivo provides scientific basis for its usefulness as traditional medicine and its clinical application in adjunct/combination therapy along with other known anticancer drugs.

Topics: Alanine Transaminase; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Enzyme Activation; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Immunohistochemistry; Liver Neoplasms; Medicine, Tibetan Traditional; Mice; Mice, Transgenic; Microscopy, Confocal; p38 Mitogen-Activated Protein Kinases; Plant Extracts; Plant Preparations; Plants, Medicinal; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

We recently established that GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component. In this study, we demonstrated that GL-V9 inhibited cells growth via inducing apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells. Following the treatment of HepG2 cells with GL-V9, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and caspase-9, while caspase-8 remained unchanged. The expression ratio of Bcl-2/Bax was also decreased in GL-V9-treated cells. Meanwhile, the cell cycle-related proteins, such as cyclin B1, CDK1 and cdc25 were down-regulated in GL-V9-induced G2/M cell cycle arrest. Furthermore, we showed that GL-V9-induced apoptosis in HepG2 cells was achieved through mitochondrial pathway. It also regulated changes of mitochondrial membrane potential and increased the production of intracellular reactive oxygen species. Besides, the growth inhibitory effect of GL-V9 was examined in vivo using murine implanted tumor model. These studies indicate that GL-V9 shows promise as a therapeutic agent against human hepatoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cytochromes c; Female; Flavonoids; G2 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Liver Neoplasms; M Phase Cell Cycle Checkpoints; Membrane Potential, Mitochondrial; Mice; Mitochondria; Protein Transport; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Sorafenib is a newly established cancer drug found to be an effective systemic treatment for advanced hepatocellular carcinoma (HCC). However, little is known about any potential effectors that modify tumor cell sensitivity towards sorafenib. Here, we present the first evidence that glucose-regulated protein 78 (GRP78) is intimately associated with acquisition of resistance towards sorafenib.. The role of GRP78 in acquisition of resistance towards sorafenib was determined using HepJ5 (a GRP78-overexpressing subline) and HepG2 as its pair-matched control. RNA interference in cancer cells was applied to determine the influence of GRP78 expression on sensitivity to sorafenib treatment.. We found that HepG2 cells exhibited higher sensitivity toward sorafenib, with 50% inhibition concentration (IC(50)) >20 microMu for HepJ5 and 4.8 microM for HepG2. Specifically, when HepG2 cells received 20 microM sorafenib treatment for 24 h, over 80% of cells underwent apoptosis compared with only 32% of HepJ5 cells under similar experimental conditions. Similarly, GRP78 knockdown in HepJ5 cells by small interfering RNA (siRNA) technique enhanced the efficacy of sorafenib-mediated cell death. This was reflected by a shift of IC(50) values from >20 microM to 4.8 microM.. GRP78 is a positive modifier for sorafenib resistance acquisition in HCC and represents a prime target for overcoming sorafenib resistance.

Topics: Antineoplastic Agents; Apoptosis; Benzenesulfonates; Blotting, Western; Carcinoma, Hepatocellular; Caspases; Cell Cycle; Cell Proliferation; Cytochromes c; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Flow Cytometry; Heat-Shock Proteins; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Niacinamide; Phenylurea Compounds; Pyridines; RNA, Small Interfering; Sorafenib; Tumor Cells, Cultured

Anthraquinone compounds have been shown to induce apoptosis in different cancer cell types. Effects of chrysophanol, an anthraquinone compound, on cancer cell death have not been well studied. The goal of this study was to examine if chrysophanol had cytotoxic effects and if such effects involved apoptosis or necrosis in J5 human liver cancer cells. Chrysophanol induced necrosis in J5 cells in a dose- and time-dependent manner. Non-apoptotic cell death was induced by chrysophanol in J5 cells and was characterized by caspase independence, delayed externalization of phosphatidylserine and plasma membrane disruption. Blockage of apoptotic induction by a general caspase inhibitor (z-VAD-fmk) failed to protect cells against chrysophanol-induced cell death. The levels of reactive oxygen species production and loss of mitochondrial membrane potential (DeltaPsi(m)) were also determined to assess the effects of chrysophanol. However, reductions in adenosine triphosphate levels and increases in lactate dehydrogenase activity indicated that chrysophanol stimulated necrotic cell death. In summary, human liver cancer cells treated with chrysophanol exhibited a cellular pattern associated with necrosis and not apoptosis.

Topics: Adenosine Triphosphate; Anthraquinones; Antineoplastic Agents, Phytogenic; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Membrane; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Damage; Free Radical Scavengers; Humans; Lactate Dehydrogenases; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria, Liver; Necrosis; Osmolar Concentration; Phosphatidylserines; Reactive Oxygen Species; Time Factors

Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402.. The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.. Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).. The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.

Topics: Adenosine Diphosphate Ribose; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Bufo bufo; Carcinoma, Hepatocellular; Caspase 9; Cell Line; Cell Proliferation; China; Cytochrome c Group; Cytochromes c; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms; Poly(ADP-ribose) Polymerases; Signal Transduction

Loss of 16q is one of the most frequent alterations in many malignancies including hepatocellular carcinomas (HCC), suggesting the existence of a tumor suppressor gene (TSG) within the frequently deleted region. In this report we describe the identification and characterization of one candidate TSG, tyrosine aminotransferase gene (TAT), at 16q22.1. Loss of one TAT allele was detected in 27/50 (54%) of primary HCCs by quantitative real-time polymerase chain reaction. In addition, homo-deletion of TAT alleles was detected in two cases. Down-regulation of TAT was detected in 28/50 (56%) of HCCs, which was significantly associated with the loss of TAT allele and hypermethylation of TAT 5' CpG island (CGI) region (P < 0.001). Functional studies found that TAT has a strong tumor suppressive ability. Introduction of the TAT gene into HCC cell lines could effectively inhibit colony formation in soft agar, foci formation, and tumor formation in nude mice. Further study found that the tumor suppressive mechanism of TAT was associated with its proapoptotic role in a mitochondrial-dependent manner by promoting cytochrome-c release and activating caspase-9 and PARP.. Taken together, our findings suggest that TAT plays an important suppressive role in the development and progression of HCC.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 9; Chromosomes, Human, Pair 16; CpG Islands; Cytochromes c; DNA Methylation; Down-Regulation; Enzyme Activation; Gene Deletion; Humans; Liver Neoplasms; Mice; Mice, Nude; RNA, Small Interfering; Tyrosine Transaminase

We investigated the effects of antcin A, antcin C, and methyl antcinate A (MAA) isolated from Antrodia camphorata on the proliferation of human liver cancer cell lines Huh7, HepG2, and Hep3B and the normal cell rat hepatocytes. The three compounds selectively inhibit the proliferation of tumor cells rather than normal cells, with IC(50) values ranging from 30.2 to 286.4 microM. The compound MAA was a more potent cytotoxic agent than antcins A and C with IC(50) values of 52.2, 78.0, and 30.2 microM against HepG2, Hep3B, and Huh7 cells, respectively. To elucidate the molecular mechanism, treatment of Huh7 cells with 100 microM MAA induced an apoptotic cell death, which was characterized by the appearance of sub-G1 population, DNA fragmentation, TUNEL positive cells, and caspase activation. MAA triggered the mitochondrial apoptotic pathway, as indicated by an increase in the protein expression of Bax, Bak, and PUMA, as well as a decrease in Bcl-(XL) and Bcl-2 and disruption of mitochondrial membrane potential and promotion of mitochondrial cytochrome c release, as well as activation of caspases-2, -3, and -9. We also found that pretreatment with inhibitors of caspases-2, -3, and -9 noticeably blocked MAA-triggered apoptosis. Furthermore, intracellular reactive oxygen species (ROS) generation and NADPH oxidase activation were observed in MAA-stimulated Huh7 cells. Mechanistic studies showed that MAA induces mitochondrial translocation of cofilin. When Huh7 cells were treated with cyclosporine A and bongkrekic acid, an inhibitor of the mitochondria permeability transition pore, the levels of cell death induced by MAA were significantly attenuated. Additionally, pretreatment of Huh7 cells with antioxidants ascorbic acid and N-acetyl cysteine markedly attenuated the MAA-induced apoptosis by upregulation of Bax, Bak, and PUMA, mitochondrial translocation of cofilin, activation of caspase-3, and cell death. Taken together, our results provide the first evidence of the activation of the ROS-dependent cofilin- and Bax-triggered mitochondrial pathway as a critical mechanism of MAA-induced cell death in liver cancer cells.

Topics: Animals; Antineoplastic Agents; Antrodia; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 2; Caspase 3; Caspase 9; Cell Line, Tumor; Cofilin 1; Cytochromes c; Hepatocytes; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; NADPH Oxidases; Oxidants; Proto-Oncogene Proteins; Rats; Reactive Oxygen Species; Triterpenes

In this work, direct electron transfer of cytochrome c (cyt. c)--a model for studying the electron transfer of enzymes is achieved at hexagonal ZnO nanosheets by one-step electrodeposition. UV-vis spectra and electrochemical data demonstrate that such ZnO nanosheets can supply a bio-compatible surface to keep the bioactivity of cyt. c. The redox formal potential (E(0)') of cyt. c is estimated to be 338.2+/-4.3 mV (vs. AgAgCl) at the nanostructured ZnO surface. This value is much more positive than those of enzymes previously obtained at other metal oxides and zeolite surfaces. Experiment data show, under the optimized potential of 0.0 V (vs. AgAgCl), the electrochemical determination of H(2)O(2) is free from not only anodic interferences like ascorbic acid (AA) and dopamine (DA), but also a cathodic interference-O(2). Such an excellent selectivity enable the present H(2)O(2) biosensor determine the extracellular H(2)O(2) released from living human hepatoma cells.

Topics: Adsorption; Animals; Biocatalysis; Biosensing Techniques; Cell Line, Tumor; Cytochromes c; Electrochemistry; Electron Transport; Horses; Humans; Hydrogen Peroxide; Liver Neoplasms; Nanotubes; Oxidation-Reduction; Particle Size; Surface Properties; Zinc Oxide

Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti-tumor properties. To better understand berberine-induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97-L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine-induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3-methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97-L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97-L cells through activation of Beclin-1 and inhibition of the mTOR-signaling pathway by suppressing the activity of Akt and up-regulating P38 MAPK signaling. This is the first study to describe the role of Beclin-1 activation and mTOR inhibition in berberine-induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Berberine; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Hep G2 Cells; Humans; Immunoblotting; Liver Neoplasms; Membrane Potential, Mitochondrial; Membrane Proteins; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; RNA Interference

To investigate the mechanism of norcantharidin (NCTD)-induced SMMC-7721 hepatoma cell apoptosis.. SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin V/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression.. NCTD inhibited SMMC-7721 cell growth in a time- and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD.. NCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.

Topics: Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potentials; Mitochondria

Norcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells.. The cytotoxicity was measured by MTT assay for cellular viability and by flow cytometry. The mitochondrial membrane potential and reactive oxygen species production was evaluated by flow cytometry analysis. The role of caspase activities were assayed using caspase apoptosis detection kit . Western blot analysis was used to evaluate the level of Cyto-C, Bcl-2, Bax, Bid, caspase 3, -9, -8 and PARP expression. After treatment with NCTD, a decrease in the viability of HepG2 cells and increase in apoptosis were observed. NCTD-induced apoptosis was accompanied by an increase in ROS production, loss of mitochondrial membrane potential and release of cytochrome c(cyto-c) from the mitochondria to the cytosol and down-regulation of anti-apoptotic protein Bcl-2 levels with concurrent up-regulation in pro-apoptotic protein Bax levels. However, another pro-apoptotic molecule, Bid, showed no change in such same treatment. NCTD-increased activity of caspase 9,caspase 3 and the subsequent cleavage caspase substrate PARP were also observed. The expression levels of pro-caspase-8 were not changed after NCTD treatment.. These results indicate that NCTD induced cytotoxicity in HepG2 cells by apoptosis, which is mediated through ROS generation and mitochondrial pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Caspases; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Flow Cytometry; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria, Liver; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Time Factors

Fluvastatin, a lipophilic statin, was known to inhibit proliferation and induce apoptosis in many cancer cells. Its potential anticancer was evaluated in three hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and MHCC-97H). Cells were treated with fluvastatin in vitro and its effect on cell proliferation, cell cycle, invasion and apoptosis was determined. Mechanism of apoptosis induced by fluvastatin on HCC cell lines was also investigated through western blotting and mitochondrial membrane potential (MMP) analysis. It was observed that fluvastatin inhibited proliferation of HCC cells by inducing apoptosis and G2/M phase arrest in a dose-dependent manner. The results of cell invasion assay revealed that fluvastatin significantly decreased the invasion potency of HCC cells. A mitochondria-operated mechanism for fluvastatin induced apoptosis might be involved and was supported by Western blotting and MMP analysis. After fluvastatin treatment, expression of Bcl-2 and procaspase-9 were downregulated, cytochrome c (cytosolic extract), Bax and cleaved-caspase-3 protein expression were increased. Furthermore, a breakdown of MMP in HCC cells was observed. To conclude, these results have provided a rationale for clinical investigations of fluvastatin in future as a potential anticancer reagent for growth control of HCC.

Topics: Anticholesteremic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Cytochromes c; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Indoles; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Tumor Cells, Cultured

Garcinol, derived from Garcinia indica and other related species, has been found to modulate several cell signalling pathways involved in apoptosis and cancer development. Growth arrest and DNA damage-inducible gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors; it is expressed at low levels under normal conditions but strongly induced upon growth arrest, DNA damage, and endoplasmic reticulum (ER) stress. This study investigated the effect of garcinol on Hep3B cells, a human hepatocellular cancer cell line lacking functional p53, with the goal of elucidating the molecular mechanisms of p53-independent apoptosis in hepatocellular cancer. Overall, garcinol activated not only the death receptor and the mitochondrial apoptosis pathways but also the ER stress modulator GADD153. Garcinol treatment led to the accumulation of reactive oxygen species (ROS), increased GADD153 expression, and reduced mitochondrial membrane potential. An increase in the Bax/Bcl-2 ratio resulted in enhanced apoptosis. Caspase-8 and tBid (truncated Bid) expression also increased in a time-dependent manner. The enzymatic activities of caspase-3 and caspase-9 increased approximately 13-fold and 7.8-fold, respectively. In addition, the proteolytic cleavage of poly-(ADP-ribose)-polymerase (PARP) and DNA fragmentation factor-45 (DFF-45) increased in dose- and time-dependent manners. Our data suggest a promising therapeutic application of garcinol in p53-independent apoptosis in cancers.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Arabidopsis Proteins; Breast Neoplasms; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Female; Hep G2 Cells; Humans; Intramolecular Transferases; Liver Neoplasms; Mitochondrial Diseases; Plant Extracts; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Reactive Oxygen Species; Terpenes; Transcription Factor CHOP

Despite recent significant advances in the treatment of human carcinoma (HCC), the results of chemotherapy to date remain unsatisfactory. 5-Fluorouracil (5-FU) still represents the cornerstone of treatment of carcinoma, and resistance to the actions of 5-FU is a major obstacle to successful chemotherapy. More effective treatment strategies may involve combinations of agents with activity against HCC. Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone isolated from Andrographis paniculata, has been shown to suppress the growth of HCC cells and trigger apoptosis in vitro. To assess the suitability of ANDRO as a chemotherapeutic agent in HCC, its cytotoxic effects have been evaluated both as a single agent and in combination with 5-FU. ANDRO potentiates the cytotoxic effect of 5-FU in HCC cell line SMMC-7721 through apoptosis. ANDRO alone induces SMMC-7721 apoptosis with p53 expression, Bax conformation and caspase-3,8,9 activation. Surprisingly, the addition of ANDRO to 5-FU induces synergistic apoptosis, which could be corroborated to the increased caspase-8, p53 activity and the significant changes of Bax conformation in these cells, resulting in increased losses of mitochondrial membrane potential, increased release of cytochrome c, and activation of caspase-9 and caspase-3. Suppression of caspase-8 with the specific inhibitor z-IETD-fmk abrogates largely ANDRO/5-FU biological activity by preventing mitochondrial membrane potential disappearance, caspase-3,9 activation and subsequent apoptosis. The results suggest that ANDRO may be effective in combination with 5-FU for the treatment of HCC cells SMMC-7721.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Diterpenes; Drug Synergism; Fluorouracil; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Tumor Suppressor Protein p53

Extracellularly added recombinant granulysin was reported to kill mammalian target cells. The sites of actions and molecular mechanisms of granulysin in target cell killing, however, are presently unclear. In order to provide new insights into its potential mechanism of target cell damage, we here constructed recombinant plasmids carrying 9 kDa granulysin cDNA and examined effects of intracellularly expressed granulysin on the target hepatoma SMMC-7721 cells. The localization of intracellularly expressed granulysin was examined by fluorescence microscopy and confocal microscopy. Effects of granulysin on cell proliferation and apoptosis were measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, flow cytometry and fluorescence microscopy, respectively. Changes of mitochondrial membrane potential were monitored by fluorescence microscopy. On the other hand, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF) was evaluated by Western blot and confocal microscopy. Intracellularly expressed granulysin was preferentially localized in cytoplasm, noticeably inhibited cell proliferation and induced cell death accompanied by reduced mitochondrial membrane potential, release of AIF and cytochrome c from mitochondria. Taken together, our findings demonstrate for the first time that localization and effect of intracellularly expressed granulysin on non-native cancer cells and indicate its potential utility in gene therapy for cancer.

Topics: Antigens, Differentiation, T-Lymphocyte; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Plasmids

The high conductive TiO(2) nanoneedles film is first employed as a support matrix for immobilizing model enzyme, cytochrome c (cyt c) to facilitate the electron transfer between redox enzymes and electrodes. Reversible and direct electron transfer of cyt c is successfully achieved at the nanostructured TiO(2) surface with the redox formal potential (E(0)') of 108.0 +/- 1.9 mV versus Ag|AgCl and heterogeneous electron transfer rate constant (k(s)) of 13.8 +/- 2.1 s(-1). Experimental data indicate that cyt c is stably immobilized onto the TiO(2) nanoneedles film and maintains inherent enzymatic activity toward H(2)O(2). On the basis of these results, the cyt c-TiO(2) nanocomposits film is capable of sensing H(2)O(2) at a suitable potential, 0.0 V (vs Ag|AgCl), where not only common anodic interferences like ascorbic acid, uric acid, 3,4-dihydroxyphenylacetic acid but also a cathodic interference, O(2), are effectively avoided. Besides high selectivity, the present biosensor for H(2)O(2) shows broad dynamic range and low detection limit. These remarkable analytical advantages, as well as the characteristic of TiO(2) nanoneedles film such as high conductivity, biocompatibility, and facile ability to miniaturize establishes a novel approach to detection of extracellular H(2)O(2) released from human liver cancer cells.

Topics: Biosensing Techniques; Cell Line, Tumor; Cytochromes c; Electrochemistry; Electrodes; Electron Transport; Extracellular Space; Humans; Hydrogen Peroxide; Linear Models; Liver Neoplasms; Nanostructures; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Surface Properties; Titanium

The aim of this study was to investigate mechanisms involved in the growth inhibitory effect of silymarin, in humanhepatocellular carcinoma.. The human hepatocellular carcinoma cell line HepG2 was utilized and the MTT assay was performed to study the antiproliferative effect of silymarin. Dual staining was undertaken for ethidium bromide/acridine orange, propidium iodide staining and DNA fragmentation studies were executed to confirm the presence of apoptosis. Cell-cycle analysis was revealed by flow cytometry and mitochondrial transmembrane potential was measured by uptake of the mitochondrial-specific lipophilic cationic dye rhodamine 123. Western blotting analysis for cytochrome c, p53, Bax, Bcl-2, APAF-1, caspase-3, survivin, beta-catenin, cyclin D1, c-Myc and PCNA was carried out.. Silymarin inhibited population growth of the hepatocellular carcinoma cells in a dose-dependent manner, and the percentage of apoptotic cells was increased after treatment with 50 and 75 microg/ml silymarin for 24 h. Silymarin treatment increased the proportion of cells with reduced DNA content (sub-G(0)/G(1) or A(0) peak), indicative of apoptosis with loss of cells in the G(1) phase. Silymarin also decreased mitochondrial transmembrane potential of the cells, thereby increasing levels of cytosolic cytochrome c while up-regulating expression of pro-apoptotic proteins (such as p53, Bax, APAF-1 and caspase-3) with concomitant decrease in anti-apoptotic proteins (Bcl-2 and survivin) and proliferation-associated proteins (beta-catenin, cyclin D1, c-Myc and PCNA).. Our results demonstrate that silymarin treatment inhibited proliferation and induced apoptosis in the human hepatocellular carcinoma cell line HepG2.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; beta Catenin; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Flavonoids; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Mitochondria; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Silymarin; Survivin; Tumor Suppressor Protein p53

Capsaicin, a pungent ingredient of red pepper, has been reported to possess antitumor activities. In this study, the effects of capsaicin on human HepG2 cells were investigated. Capsaicin reduced viability by PI incorporation in HepG2 cells in a dose and time dependent manner. Capsaicin promoted intracellular Ca2+ production and reactive oxygen species (ROS). The alpha psi(m) significantly decreased after capsaicin treatment for 24 h. Co-treatment of HepG2 cells with capsaicin and BAPTA (an intracellular Ca2+ chelator) significantly reduced intracellular Ca2+ levels, prevented alpha psi(m) disruption and inhibited apoptosis induction. The protein levels of Bcl-2 decreased and Bax increased in the mitochondrial fraction while the Bax protein decreased, and p53 and cytochrome c protein levels increased in the cytosolic fraction in HepG2 cells after capsaicin treatment for 24 h by Western blot. Immunostaining and confocal microscopic analysis also showed that capsaicin promoted cytoplasmic GADD153 expression and GRP78 nuclear translocation. The caspase-3 activity significantly increased after capsaicin treatment for 24 h. Our results indicated that the capsaicin-induced apoptosis in HepG2 cells may result from the elevation of intracellular Ca2+ production, ROS, disruption of alpha psi(m), regulation of Bcl-2 family protein expression and caspase-3 activity.

Topics: Apoptosis; Calcium; Capsaicin; Carcinoma, Hepatocellular; Caspase 3; Cell Survival; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria, Liver; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Activation of the nuclear transcription factor-kappaB (NF-kappaB) has been implicated in liver tumorigenesis. We evaluated the effects of a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in two human liver cancer cell lines HA22T/VGH and HuH-6. DHMEQ treatment dose dependently decreased the DNA-binding capacity of the NF-kappaB p65 subunit, inhibited cell growth and proliferation, and increased apoptosis as shown by caspase activation, release of cytochrome c, poly(ADP-ribose) polymerase cleavage, and down-regulation of survivin. DHMEQ also induced a dose-dependent activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling, and inhibition of this pathway significantly reduced cell growth. It is noteworthy that we observed that DHMEQ stimulated reactive oxygen species (ROS) production in a dose-dependent manner and that pretreatment of the cells with the antioxidant N-acetyl-L-cysteine (NAC) significantly reduced DHMEQ-induced ROS generation. Accordingly, NAC completely reversed the DHMEQ-induced growth inhibition, caspase activation, and cell death. DHMEQ-treated cells exhibited DNA damage, as evaluated by accumulation in nuclear foci of phospho-H2AX, which was completely reversed by NAC. Moreover, DHMEQ induced the expression of genes involved in the endoplasmic reticulum stress response (GRP78, CHOP, TRB3) and promoted the splicing of XBP1 mRNA in a dose-dependent fashion in both cell lines, which was reversed in the presence of NAC. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased DHMEQ-induced cell growth inhibition. These data suggest that DHMEQ antitumor effects are primarily mediated through ROS generation. Thereby, considering that cancer cells are under increased ER stress and oxidative stress conditions, DHMEQ may greatly improve various anticancer strategies.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclohexanones; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Drug Evaluation, Preclinical; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinases; NF-kappa B; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species

It is reported that gambogic acid (GA), the main active compound of gamboge which is a dry resin extracted from Garcinia hanburyi tree, has potent antitumor activity both in vivo and in vitro. Activation of mitochondrial apoptotic pathway in cancer cells is one effective therapy for cancer treatment. In the present study, we focus on the effect of GA on induction of reactive oxygen species (ROS) accumulation and triggering the mitochondrial signaling pathway in human hepatoma SMMC-7721 cells. The results indicated that GA induced ROS accumulation and collapse of mitochondrial membrane potential in SMMC-7721 cells in a concentration-dependent manner and subsequently induced that release of Cytochrome c and apoptosis-inducing factor from mitochondria to cytosol, which inhibited ATP generation and induced apoptosis in the cells. Moreover, GA elevated the phosphorylation of c-Jun-N-terminal protein kinase (JNK) and p38, which was the downstream effect of ROS accumulation. Furthermore, N-acetylcysteine, a ROS production inhibitor, partly reversed the activation of JNK and p38 and the induction of apoptosis in GA-treated cells. Collectively, our study demonstrated that accumulation of ROS played an important role in GA-induced mitochondrial signaling pathway, which provided further theoretical support for the application of GA as a promising anticancer agent.

Topics: Acetylcysteine; Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytochromes c; Flow Cytometry; Free Radical Scavengers; Glutathione; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Confocal; Mitochondria, Liver; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Xanthones

The role of reactive oxygen species (ROS) and p38 mitogen-activated protein kinases (MAPK) in tanshinones-induced apoptosis was investigated in HepG2 cells in this study. The major tanshinones (cryptotanshinone, dihydrotanshinone, tanshinone I, tanshinone IIA), isolated from Salvia miltiorrhiza, inhibit cell growth and induce caspase-dependent apoptosis concentration-dependently, with dihydrotanshinone being the most potent. All four tanshinones were found to induce ROS generation, but only dihydrotanshinone can induce activation of p38 MAPK. The p38 MAPK activation by dihydrotanshinone was inhibited by N-acetyl cysteine pretreatment. It is thus concluded that ROS-mediated p38 MAPK activation plays a vital role in dihydrotanshinone-induced apoptosis in HepG2 cells.

Topics: Abietanes; Acetylcysteine; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Furans; Humans; Imidazoles; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; L-Lactate Dehydrogenase; Liver Neoplasms; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phenanthrenes; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Transport; Pyridines; Quinones; Reactive Oxygen Species; Time Factors

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on the induction of apoptosis in hepatocarcinoma cell lines in vitro and further examine the molecular mechanisms of EGCG-induced apoptosis. In the present study, it was observed that EGCG rapidly induced apoptosis in hepatocarcinoma SMMC7721 cells. EGCG-induced apoptosis was in association with the attenuation of mitochondrial transmembrane potentials (Deltapsi(m)), the alteration of Bcl-2 family proteins, the release of cytochrome c from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during EGCG-induced apoptosis of hepatocarcinoma SMMC7721 cells. The antioxidant N-acetyl-l-cysteine (NAC) significantly reduced ROS production and EGCG-induced apoptosis, suggesting that ROS plays a key role in EGCG-induced apoptosis in hepatocarcinoma SMMC7721 cells. In summary, EGCG-induced apoptosis through mitochondrial pathways, and ROS affected EGCG-induced apoptosis in hepatocarcinoma SMMC7721 cells.

Topics: Apoptosis; Catechin; Cell Line, Tumor; Cytochromes c; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species

In evaluating the cytotoxic effects and the mechanisms of the apoptotic and necrotic actions of trimethyltin chloride (TMT) on human hepatoma G2 (HepG2) cells, the present study focused on the involvement of antiproliferation, DNA damage, cell death, apoptosis-related proteins, and p53-dependent transcriptional activity. Twenty-four hour TMT treatments (4-64 microM) induced apoptosis and necrosis in HepG2 cells. Thirty-two micromolar and higher concentration significantly increases cell death. DNA damage was observed at 8 microM. Additionally, TMT increased the activity of cellular caspase-3 and the release of mitochondrial cytochrome c in a concentration-dependent manner. Our data demonstrated that the Bcl-2 family of proteins was involved in the apoptotic process but that p53 expression level was not affected. The results of luciferase reporter assay indicated that TMT-induced apoptosis seemed to adopt a transcription-dependent route, by activating p53 target genes such as PUMA and p21.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Comet Assay; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Humans; Liver Neoplasms; Necrosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Trimethyltin Compounds; Tumor Suppressor Protein p53

Baicalein has been reported to induce growth-inhibitory activity in vitro in human cancer cells; however, the molecular mechanism of action is not completely understood. A pharmacological dose (10-100 microM) of baicalein exerted a cytotoxic effect on human hepatoma J5 cells resulting in G2/M arrest and apoptosis. In addition to cytotoxicity in J5 cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9 and -3 occurred. Baicalein induced AIF and Endo G release from mitochondria indicating that baicalein stimulates apoptosis through the caspase-independent pathway, while undergoing apoptosis, there was a remarkable accumulation of G2/M cells. Also, the ratio of Bax/Bcl-2 was increased leading to changes in mitochondria membrane potential (DeltaPsim) and release of cytochrome c, whereas the baicalein-induced apoptosis was partially abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G2/M cells remained. These results demonstrate that the cytotoxicity of baicalein in J5 cells is attributable to apoptosis mainly involving G2/M-arrest in an ER-dependent manner, via a mitochondria-dependent caspase pathway and as well as contributions of AIF and Endo G pathways.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Calcium; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cell Shape; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Damage; Dose-Response Relationship, Drug; Endodeoxyribonucleases; Enzyme Activation; Flavanones; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Time Factors

Sorafenib (Nexavar, BAY43-9006), a bi-arylurea, is a newly established anti-cancer drug and its functional attribute of cytotoxicity is based on the multi-kinase inhibitory action. Here, we report yet another novel pathway in which sorafenib can induce apoptotic cell death preferentially and efficaciously on an experimentally proven drug- and radio-resistant human Hep G2 cells via a mitochondria-dependent oxidative stress mechanism. A real time confocal imaging assay revealed that sorafenib could rapidly provoke the production of ROS plethorically, mainly concentrating in the mitochondria, albeit substantial amounts of ROS could also be detected in cytosol and nucleus. The rapid production of ROS could simultaneously induce intracellular glutathione (iGSH) depletion. A nearly 90% of iGSH was found to be depleted in 1h period after the cells received the drug treatment. Besides mitochondria, iGSH depletion could also be detected in other cellular compartment including cytoplasm and nucleus. Interestingly, we also demonstrated that sorafenib could trigger mitochondrial Ca(2+) overload. All these events compoundedly serve as the final arbitrator to initiate lethal apoptotic process through the release of cytochrome c and caspase 3/7 activation. Collectively, we provide first evidence here that sorafenib can provoke an alternative pathway for apoptosis induction of Hep G2 cells through a mitochondria-dependent oxidative stress mechanism which is independent of original kinase inhibitory attribute of the drug action. Most importantly, we also demonstrate that sorafenib can effectively eradicate a highly drug- and radio-resistant HCC cells. Thus, our data can provide the basis for a potential applicability of sorafenib in a combined treatment modality.

Topics: Antineoplastic Agents; Apoptosis; Benzenesulfonates; Calcium; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Glutathione; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Intracellular Space; Liver Neoplasms; Microscopy, Confocal; Mitochondria; Niacinamide; Oxidative Stress; Phenylurea Compounds; Pyridines; Reactive Oxygen Species; Sorafenib

To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line.. Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting.. CIE inhibited proliferation of MHCC97H cells in a time- and dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial cells. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression.. CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chrysanthemum; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Endothelial Cells; Hepatocytes; Humans; Liver Neoplasms; Phytotherapy; Plant Extracts; Rats; Rats, Sprague-Dawley

Hepatocellular carcinoma suppressor 1 (HCCS1) was discovered as a novel tumor suppressor gene. We recently observed that adenovirus-mediated gene transfer of HCCS1 leads to cytotoxicity to human hepatocarcinoma cells. Here, we have demonstrated that adenovirus-mediated overexpression of HCCS1 induces apoptosis in hepatocarcinoma cells and have further characterized the apoptotic cascade. The results showed that lysosomal cathepsin D is released into the cytosol in response to HCCS1 overexpression and consequently triggers Bax insertion into the mitochondrial membrane, which leads to the release of cytochrome c. In addition, HCCS1 overexpression can induce an increase in intracellular free Ca(2+) concentration, which also results in cytochrome c release. The released cytochrome c activates downstream caspases, leading to the occurrence of the late stages of apoptosis. Moreover, we demonstrated that the disruption of HCCS1 in mice leads to embryonic lethality, accompanied by abnormal labyrinth architecture resulting from the excessive proliferation of trophoblast cells in the placenta. These results suggest that HCCS1 plays a role in apoptosis regulation and development.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Calcium; Carcinoma, Hepatocellular; Caspases; Cathepsin D; Cell Line, Tumor; Cytochromes c; Female; Genes, Lethal; Humans; Liver Neoplasms; Lysosomes; Mice; Mice, Knockout; Placenta; Pregnancy; Tumor Suppressor Proteins; Vesicular Transport Proteins

We investigated whether HS-1200 has anti-proliferation effects on human hepatoma cells in vitro. Here, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) were observed after treatment of HS-1200, indicating the occurrence of apoptotic cell death, which was associated with up-regulation of Bax, cleaved-caspase-3 and cleaved-caspase-9. Inhibition of caspase-9 rescued HS-1200-induced apoptosis. Furthermore, cells treated with HS-1200 showed a reduction in mitochondrial membrane potential (Deltapsi(m)) and caused cytochrome c release into the cytosol. The results indicated that synthetic chenodeoxycholic acid HS-1200 could induce cell apoptosis in BEL7402 human hepatoma cell line, via a Bax/cytochrome c/caspase-9 independent pathway. This study suggested that HS-1200 is potentially useful as an apoptosis inducer for the treatment of hepatocellular carcinoma.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Proliferation; Chenodeoxycholic Acid; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2

To investigate the effects of the essential oil of Curcuma wenyujin (CWO) on growth inhibition and on the induction of apoptosis in human HepG2 cancer cells.. The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. DNA fragmentation was visualized by agarose gel electrophoresis. Cell cycle and mitochondrial transmembrane potential (Delta psi m) were determined by flow cytometry (FCM). Cytochrome C immunostaining was evaluated by fluorescence microscopy. Caspase-3 enzymatic activity was assayed by the cleavage of Ac-DEVD-R110. Cleaved PARP and active caspase-3 protein levels were measured by FCM using BD(TM) CBA Human Apoptosis Kit.. Treatment with CWO inhibited the growth of HepG2 cells in a dose-dependent manner, and the IC50 of CWO was approximately 70 mug/mL. CWO was found to inhibit the growth of HepG2 cells by inducing a cell cycle arrest at S/G(2). DNA fragmentation was evidently observed at 70 mug/mL after 72 h of treatment. During the process, cytosolic HepG2 cytochrome C staining showed a markedly stronger green fluorescence than in control cells in a dose-dependent fashion, and CWO also caused mitochondrial transmembrane depolarization. Furthermore, the results clearly demonstrated that both, activity of caspase-3 enzyme and protein levels of cleaved PARP, significantly increased in a dose-dependent manner after treatment with CWO.. CWO exhibits an antiproliferative effect in HepG2 cells by inducing apoptosis. This growth inhibition is associated with cell cycle arrest, cytochrome C translocation, caspase 3 activation, Poly-ADP-ribose polymerase (PARP) degradation, and loss of mitochondrial membrane potential. This process involves a mitochondria-caspase dependent apoptosis pathway. As apoptosis is an important anti-cancer therapeutic target, these results suggest a potential of CWO as a chemotherapeutic agent.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Line; Cell Line, Tumor; Curcuma; Cytochromes c; Humans; Liver Neoplasms; Membrane Potentials; Microscopy, Fluorescence; Mitochondrial Membranes; Oils, Volatile; Plant Extracts

Oridonin, a diterpenoid isolated from Rabdosia rubescences, could induce apoptosis through the generation of reactive oxygen species (ROS) in human hepatoma HepG2 cells. p53, a specific inhibitor of pifithrin alpha (PFT alpha), markedly inhibited ROS generation and apoptosis, showing that p53 was responsible for the cytotoxity of oridonin through mediation by ROS. Moreover, the ROS activated the p38 kinase, which in turn promoted the activation of p53, as verified by evidence showing that the ROS scavenger N-acetyl-cysteine (NAC) not only blocked the phosphorylation of p38 but also partially inhibited the activation of p53, and the p38 inhibitor SB203580 reduced the activation of p53 as well. Mitochondria were either the sources or the targets of ROS. This study showed that oridonin stimulated mitochondrial transmembrane permeabilization in a ROS-dependent manner because NAC almost thoroughly reversed the drop of mitochondrial transmembrane potential (Deltapsim) and the release of cytochrome c from the mitochondrial inter-membrane space into cytosol. Furthermore, as a result of mitochondrial permeability transition, procaspases-9 and -3 were cleaved into 37- and 17-kDa proteolytic products, respectively, which acted as executors of oridonin-induced apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Diterpenes, Kaurane; Humans; Isodon; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53

To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22.. Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry.. MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner.. ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Liver Neoplasms; Membrane Potential, Mitochondrial; Mice; Mitochondria, Liver; Plant Extracts; Scutellaria

This study was carried out to investigate the apoptotic effects of glycine- and proline-rich glycoprotein [Solanum nigrum Linne (SNL) glycoprotein, 150-kDa] isolated from SNL, which has been used as an antipyretic and anticancer agent in Korean herbal medicine. We found that SNL glycoprotein has obviously cytotoxic and apoptotic effects at 80 microg/ml of SNL glycoprotein for 4 h in Hep3B cells (hepatocellular carcinoma cells). In mitochondria-mediated apoptosis pathway, SNL glycoprotein has abilities to stimulate release of mitochondrial cytochrome c, activations of caspase-9 and caspase-3, cleavage of poly(ADP-ribose)polymerase and production of intracellular reactive oxygen species in Hep3B cells. In nuclear factor-kappa B (NF-kappaB)-mediated apoptosis pathway, the results showed that SNL glycoprotein dose-dependently blocked DNA binding activity of NF-kappaB, activity of inducible nitric oxide synthase (iNOS) and production of inducible nitric oxide (NO). Interestingly, pyrrolidine dithiocarbamate (for NF-kappaB inhibitor) and Nomega-nitro-l-arginine methylester hydrochloride (for NO inhibitor) effectively stimulated the caspase-3 activation and induced apoptosis in Hep3B cells. These results indicate that SNL glycoprotein transfers its cell death signal from cytochrome c to caspase 3 by inhibiting NF-kappaB and iNOS activation in Hep3B cells. Here, we speculate that SNL glycoprotein is one of the chemotherapeutic agents to modulate mitochondria-mediated apoptosis signals in Hep3B cells.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA; Enzyme Activation; Glycine; Glycoproteins; Humans; Liver Neoplasms; Mitochondria, Liver; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Plant Proteins; Poly(ADP-ribose) Polymerases; Proline; Reactive Oxygen Species; Signal Transduction

Podophyllotoxin is a well known anti-tumor chemical, but because of its strong side effects much effort has been paid to reduce cytotoxicity by modifying its structure. Here, we evaluate the anti-tumor activity of a new isolated derivative of podophyllotoxin, 4'-demethyl-4-dehydroxy-4-seleno-phenyl-beta-peltatin-epipodophyllotoxin (CPZ) and find that CPZ can suppress the proliferation of human hepatoma SMMC-7721 cells in a dose- and time-dependent manner. Phase-contrast microscope observation and flow cytometric analysis through PI stains showed that the reagents have strong inhibition of SMMC-7721 cell growth, as the cells were blocked in the G2/M period. Cell apoptosis induced by CPZ was further confirmed by staining with M30 Cytodeath antibody. Rh123 label testing revealed that the mitochondrial membrane potential had been decreased by CPZ treatment. Under the stress of CPZ, cytochrome c was secreted into the cytoplasm by mitochondria, and Bax in cytoplasm was translocated into the mitochondrial membrane. These results suggest that CPZ-induced apoptosis may work through a Bax-dependent pathway.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Shape; Cytochromes c; Etoposide; Humans; Liver Neoplasms; Membrane Potentials; Mitochondria; Molecular Structure; Podophyllotoxin

Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an inexpensive traditional Chinese medicine commonly used for the treatment of liver diseases, and a few studies have indicated that SGR has anti-hepatocarcinogenic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell growth by causing cell-cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation assay. SGRE-induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. The SGRE-mediated mitochondria-caspase dependent apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS. These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further development of SGR for liver disease therapy.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Flavonols; Gene Expression; Humans; Inhibitory Concentration 50; Liver Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Smilax; Spirostans

TIP30 (Tat-interacting protein 30), a newly found proapoptotic factor, appears to be involved in multiple functions including metabolic suppression, apoptosis induction, and diminishing angiogenic properties. In the present study, we reported that mitochondrial events were required for apoptosis induced by TIP30 in hepatocellular carcinoma cells (HCC cells). Translocation of Bax was essential for TIP30-induced apoptosis, whereas overexpression of the anti-apoptotic protein Bcl-xL delayed both second mitochondria-derived activator of caspases (Smac/DIABLO) release and onset of apoptosis. Furthermore, TIP30-induced apoptosis was dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (z-VAD-fmk) blocked DNA fragmentation. Release of Smac/DIABLO from the mitochondria through the TIP30-P53-Bax cascade was required to remove the inhibitory effect of XIAP (X-linked Inhibitor of Apoptosis) and allowed apoptosis to proceed. Our results showed for the first time that Bax-dependent release of Smac/DIABLO, cytochrome c and AIF from the mitochondria mediated the contribution of the mitochondrial pathway to TIP30-mediated apoptosis. Our data suggested that adenovirus-mediated overexpression of TIP30 was capable of inducing therapeutic programmed cell death in vitro by activating the mitochondrial pathway of apoptosis. On the basis of these studies, elucidating the mechanism by which TIP30 induces cell death might establish it as an anticancer approach.

Topics: Acetyltransferases; Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Cytochromes c; Down-Regulation; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Permeability; Protein Binding; Protein Transport; Transcription Factors; X-Linked Inhibitor of Apoptosis Protein

Intracellular redox homeostasis plays a critical role in determining tumor cells' sensitivity to drug-induced apoptosis. Here we investigated the role of thioredoxin-1 (TRX1), a key component of redox regulation, in arsenic trioxide (As(2)O(3))-induced apoptosis. Over-expression of wild-type TRX1 in HepG(2) cells led to the inhibition of As(2)O(3)-induced cytochrome c (cyto c) release, caspase activation and apoptosis, and down-regulation of TRX1 expression by RNAi sensitized HepG(2) cells to As(2)O(3)-induced apoptosis. Interestingly, mutation of the active site of TRX1 from Cys(32/35) to Ser(32/35) converted this molecule from an apoptotic protector to an apoptotic promoter. In an effort to understand the mechanisms of this conversion, we used isolated mitochondria from mouse liver and found that recombinant wild-type TRX1 could protect mitochondria from the apoptotic changes. In contrast, the mutant form of TRX1 alone elicited mitochondria-related apoptotic changes, including the mitochondrial permeability transition pore (mPTP) opening, loss of mitochondrial membrane potential, and cyto c release from mitochondria. These apoptotic effects were inhibited by cyclosporine A (CsA), indicating that mutant TRX1 targeted to mPTP. Alteration of TRX1 from its reduced form to oxidized form in vivo by 2,4-dinitrochlorobenzene (DNCB), a specific inhibitor of TRX reductase, also sensitized HepG(2) cells to As(2)O(3)-induced apoptosis. These data suggest that TRX1 plays a central role in regulating apoptosis by blocking cyto c release, and inactivation of TRX1 by either mutation or oxidization of the active site cysteines may sensitize tumor cells to As(2)O(3)-induced apoptosis.

Topics: Animals; Arsenic Trioxide; Arsenicals; Binding Sites; Carcinoma, Hepatocellular; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Dinitrochlorobenzene; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; Liver Neoplasms; Mice; Mitochondria; Mitochondrial Swelling; Mutant Proteins; NADP; Oxidation-Reduction; Oxides; Reactive Oxygen Species; Recombinant Proteins; RNA Interference; Thioredoxins

Sarsasapogenin is a steroidal sapogenin with antitumor properties. To explain the mechanism of its apoptotic effect, mitochondrial activity was assessed via a 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCM) was used to estimate the changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and cellular-reduced glutathione (GSH) level. Laser scanning confocal microscope (LSCM) recorded instantaneous ROS burst after application of sarsasapogenin. Western blotting was used to determine the expression level and intracellular distribution of cytochrome c (cyt c). It is demonstrated that during apoptosis, ROS burst acted as an early event followed by depolarization of MMP, prolonged ROS generation, and significantly declined GSH level. Cyt c was upregulated and released from mitochondria to cytosol during the process. These findings show that a mitochondrial ROS burst is an early upstream apoptotic signal which may trigger the mitochondrial apoptotic pathway and play a vital role in sarsasapogenin-induced HepG2 cell apoptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Glutathione; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Confocal; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Spirostans; Time Factors

Alpha-lipoic acid (alpha-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, alpha-LA induces apoptosis in different cancer cells types. However, whether alpha-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated whether alpha-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that alpha-LA inhibits the growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased levels of the cyclin/CDKs inhibitors, p27(Kip1) and p21(Cip1). Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that alpha-LA induces apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests that it may prove useful in liver cancer therapy.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; Ascorbic Acid; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; Liver Neoplasms; MAP Kinase Kinase 4; Proto-Oncogene Proteins c-akt; Rats; Reactive Oxygen Species; Thioctic Acid; Tumor Suppressor Protein p53

Solanum nigrum L. (SN) has been used in traditional folk medicine to treat different cancers. It is also used as a hepatoprotective and anti-inflammatory agent. In this study, we demonstrated that the extract of SN (SNE) induced a strong cytotoxic effect toward HepG2 cells but much less to Chang liver and WRL-68 cells. The mechanisms of the cytotoxic effect were concentration-dependent. High doses of SNE (2 and 5 mg/mL) induced apoptotic cell death in HepG2 cells, as evidenced by increases in the expressions of p-JNK and Bax, mitochodrial release of cytochrome c, and caspase activation. On the other hand, cells treated with low concentrations of SNE (50-1000 microg/mL) revealed morphological and ultrastructural changes of autophagocytic death under electron microscopic observation. Furthermore, these cells showed increased levels of autophagic vacuoles and LC3-I and LC3-II proteins, specific markers of autophagy. The levels of Bcl-2 and Akt that have been implicated in the down-regulation of autophagy were decreased upon SNE treatment. Taken together, these findings indicate that SNE induced cell death in hepatoma cells via two distinct antineoplastic activities of SNE, the ability to induce apoptosis and autophagocytosis, therefore suggesting that it may provide leverage to treat liver cancer.

Topics: Apoptosis; Autophagy; Caspase 3; Cell Line; Cell Line, Tumor; Cytochromes c; Fetus; Humans; Liver; Liver Neoplasms; Plant Extracts; Solanum nigrum; Stomach Neoplasms

To investigate the apoptosis effect of SARS coronavirus nucleocapsid protein on cultured cell lines and to explore the possible pathway of apoptosis. pCDNA3.1(-)/his-myc vector containing the SARS coronavirus nucleocapsid gene (N), matric gene (M), spike gene (S) were transfected into COS-1, Huh-7 and HepG2 cells. Apoptosis induced by SARS coronavirus N protein under starvation of serum of COS-1 cells was monitored by Annexin V and electron microscopy assays. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (DeltaPsim) were determined by flow cytometric assay. Cytochrome C, cleaved caspase (cysteine aspartic acid protease)-3, 9, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. After removal of serum in COS-1 cells, we observed the loss of DeltaPsim, the increase of ROS and cytochrome C release into cytosol and subsequent activation of caspase-3 and PARP cleavage. The pan-caspase inhibitor z-VAD-fmk can block the activation of caspase 3, 9 and PARP cleavage. In conclusion, SARS coronavirus N protein can induce apoptosis of COS-1 cells by activating mitochondrial pathway. SARS coronavirus M, S protein can not induce apoptosis in COS-1, HepG2 and Huh-7 and SARS coronavirus N protein can not induce apoptosis in HepG2 and Huh-7 by methods used in this study.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 9; Cell Line, Tumor; Chlorocebus aethiops; Coronavirus Nucleocapsid Proteins; COS Cells; Cytochromes c; Flow Cytometry; Genetic Vectors; Humans; Liver Neoplasms; Membrane Glycoproteins; Membrane Potential, Mitochondrial; Microscopy, Electron; Mitochondria; Nucleocapsid Proteins; Phosphatidylserines; Reactive Oxygen Species; Severe acute respiratory syndrome-related coronavirus; Spike Glycoprotein, Coronavirus; Transfection; Viral Envelope Proteins

In several neoplastic diseases, including hepatocellular carcinoma, the expression of P-glycoprotein and cyclooxygenase-2 (COX-2) are often increased and involved in drug resistance and poor prognosis. P-glycoprotein, in addition to drug resistance, blocks cytochrome c release, preventing apoptosis in tumor cells. Because COX-2 induces P-glycoprotein expression, we evaluated the effect of celecoxib, a specific inhibitor of COX-2 activity, on P-glycoprotein-mediated resistance to apoptosis in cell lines expressing multidrug resistant (MDR) phenotype. Experiments were done using MDR-positive and parental cell lines at basal conditions and after exposure to 10 or 50 micromol/L celecoxib. We found that 10 micromol/L celecoxib reduced P-glycoprotein, Bcl-x(L), and Bcl-2 expression, and induced translocation of Bax from cytosol to mitochondria and cytochrome c release into cytosol in MDR-positive hepatocellular carcinoma cells. This causes the activation of caspase-3 and increases the number of cells going into apoptosis. No effect was shown on parental drug-sensitive or on MDR-positive hepatocellular carcinoma cells after transfection with MDR1 small interfering RNA. Interestingly, although inhibiting COX-2 activity, 50 micromol/L celecoxib weakly increased the expression of COX-2 and P-glycoprotein and did not alter Bcl-x(L) and Bcl-2 expression. In conclusion, these results show that relatively low concentrations of celecoxib induce cell apoptosis in MDR cell lines. This effect is mediated by P-glycoprotein and suggests that the efficacy of celecoxib in the treatment of different types of cancer may depend on celecoxib concentration and P-glycoprotein expression.

Topics: Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Hepatocellular; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2; Cytochromes c; Drug Resistance, Multiple; HT29 Cells; Humans; Liver Neoplasms; Mice; Mitochondria; NIH 3T3 Cells; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Sulfonamides

20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, is reported to induce apoptosis in a variety of cancer cells. We purified the compound and measured its in vitro anti-tumor activity. IH-901 inhibited cell growth of human hepatocellular carcinoma SMMC7721 cells in a dose- and time-dependent manner. We also found that IH-901 induced apoptotic cell death concurrent with cell cycle arrest in G0-G1 phase in SMMC7721 cells. At the molecular level, we show that IH-901 upregulates cytochrome c, p53, and Bax expression, and downregulates pro-caspase-3 and pro-caspase-9 expressions in a dose-dependent manner, while the levels of Bcl-2 and Bcl-X(L) were unchanged in IH-901-treated SMMC7721 cells. These results provide significant insight into the anticarcinogenic action of IH-901.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cell Proliferation; Cytochromes c; Flow Cytometry; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sapogenins; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Curcumin, a major pigment of turmeric, is a natural antioxidant possessing a variety of pharmacological activities and therapeutic properties. But its mechanisms are unknown. In our previous study, we found that a 2-h exposure to curcumin induced DNA damage to both the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA) in HepG2 cells and that mtDNA damage was more extensive than nDNA damage. Therefore, experiments were initiated to evaluate the role of mtDNA damage in curcumin-induced apoptosis. The results demonstrated that HepG2 cells challenged with curcumin for 1 h showed a transient elevation of the mitochondrial membrane potential (DeltaPsim), followed by cytochrome c release into the cytosol and disruption of DeltaPsim after 6 h exposure to curcumin. Apoptosis was detected by Hoechst 33342 and annexin V/PI assay after 10 h treatment. Interestingly, the expression of Bcl-2 remained unchanged. A resistance to apoptosis for the corresponding rho0 counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in HepG2 cells mediated by curcumin. The effects of PEG-SOD in protecting against curcumin-induced cytotoxicity suggest that curcumin-induced cytotoxicity is directly dependent on superoxide anion O2- production. These data suggest that mitochondrial hyperpolarization is a prerequisite for curcumin-induced apoptosis and that mtDNA damage is the initial event triggering a chain of events leading to apoptosis in HepG2 cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Nucleus; Curcumin; Cytochromes c; DNA Damage; DNA, Mitochondrial; Free Radical Scavengers; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; Superoxides

The molecular mechanisms behind the anti-neoplastic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are not completely understood and cannot be explained by the inhibition of the cyclooxygenase (COX) enzymes COX-1 and COX-2 alone. We previously reported that both the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitor CAY10404 exhibit anti-tumor effects in human hepatoma cells. NSAID inhibitors have many COX-independent actions and, among others, the mitogen-activated protein kinase (MAPK) pathways are targets for NSAIDs. Here, we examined the role of MEK/ERK1/2 signaling in the anti-neoplastic effects of both selective COX-1 and COX-2 inhibitors in two human hepatoma cell lines. Treatment of hepatoma cells with the selective COX-1 inhibitor SC-560, as well as with the selective COX-2 inhibitor CAY10404, was associated with activation of ERK1/2 in a time- and dose-dependent manner. Treatment with COX-1 and COX-2 inhibitors in the presence of the selective MEK1/2 inhibitor U0126 effectively suppressed ERK1/2 activation and combinations of either SC-560 or CAY10404 with U0126 resulted in synergistic effects on cell growth inhibition and induction of apoptosis. In HuH-6 hepatoma cells the combination-induced apoptosis was associated with caspase-9 and -3 activation, PARP cleavage, release of cytochrome c from the mitochondria into the cytosol and down-regulation of survivin and beta-catenin levels. In conclusion, our study showed that growth inhibitory concentrations of selective COX-1 and COX-2 inhibitors increased ERK1/2 phosphorylation in hepatoma cells, and that inhibition of the MEK/ERK signaling pathway potentiates the antitumor activity of both types of inhibitors. Therefore, our results provide preclinical support for a combined chemotherapeutic approach with selective NSAIDs and MEK inhibitors for the treatment of hepatocellular carcinoma.

Topics: Apoptosis; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Caspases; Cell Proliferation; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Enzyme Inhibitors; Flow Cytometry; Humans; Isoxazoles; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Pyrazoles; Sulfones; Time Factors; Tumor Cells, Cultured

Cepharanthine (CEP), a biscoclaurine alkaloid, has been reported to induce cell death, however, the molecular mechanism of this phenomenon remains unclear. We herein report that CEP induced apoptosis in HuH-7 cells through nuclear fragmentation, DNA ladder formation, cytochrome c release, caspase-3 activation and poly-(ADP-ribose)-polymerase cleavage. CEP triggered the generation of reactive oxygen intermediates, the activation of mitogen activated protein kinase (MAPK) p38, JNK1/2 and p44/42, and the downregulation of protein kinase B/Akt. Antioxidants and SP600125, an inhibitor of JNK1/2, but not inhibitors of p38 MAPK and MEK1/2, significantly prevented cell death, thus implying that reactive oxygen species and JNK1/2 play crucial roles in the CEP-induced apoptosis of HuH-7 cells.

Topics: Alkaloids; Animals; Anthracenes; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Benzylisoquinolines; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Line, Tumor; Collagen Type XI; Cytochromes c; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species

In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.

Topics: Apoptosis; Calcium; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Line, Tumor; Cyclic N-Oxides; Cytochromes c; Enzyme Activation; Humans; Liver Neoplasms; Mitochondria, Liver; Proto-Oncogene Proteins c-bcl-2; Seeds; Strychnine; Strychnos nux-vomica

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Carcinoma, Hepatocellular; Cathepsin B; Cytochromes c; Fatty Acids, Nonesterified; Fatty Liver; Flow Cytometry; Hepatocytes; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Proto-Oncogene Proteins; Rats; RNA, Small Interfering; Signal Transduction

Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calcium; Calpain; Carcinoma, Hepatocellular; Caspase 12; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Fruit; Humans; Liver Neoplasms; Membrane Potentials; Mitochondrial Membranes; Polyporales; Proto-Oncogene Proteins c-bcl-2

Solanum indicum L., an anti-inflammatory and wound-healing herb in traditional Chinese medicine, is enriched of unique dioscins, that is, indiosides. Our previous studies revealed that several synthetic indiosides from Solanum indicum L. have potent anticancer effects. This study was to investigate the anticancer mechanism of synthetic indiosides I from Solanum indicum L.. Human hepatocarcinoma Bel-7402 cells were treated with different concentrations of indiosides. The inhibitory rate of cell proliferation and 50% inhibitory concentration (IC50) of indiosides were detected by the acid phosphatase assay (APA). Cell morphology was observed under optical microscope with crystal violet staining. The expression of apoptosis-related proteins was detected by Western blot.. Indiosides significantly inhibited proliferation of Bel-7402 cells: when treated with indioside I for 72 h, the IC50 value was 4.2 microg/ml, cell density was decreased, and bubbles were observed in cytoplasm. Western blot showed that the cytoplasmic level of cytochrome c was increased significantly, Caspase-3 was activated, and poly (ADR-ribose) polymerase (PARP) was cleavaged after indioside I treatment.. Indiosides have dose-dependent inhibitory effect on proliferation of Bel-7402 cells, and can induce cell apoptosis through mitochondria-dependent pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Cytoplasm; Dose-Response Relationship, Drug; Glycosides; Humans; Liver Neoplasms; Molecular Structure; Poly(ADP-ribose) Polymerases; Solanum; Steroids

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Caspase 8; Caspases; Cell Proliferation; Cytochromes c; Dioxolanes; Down-Regulation; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Lignans; Liver Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Mitochondrial Proteins; Proto-Oncogene Proteins c-bcl-2; Up-Regulation; Xenograft Model Antitumor Assays

Hepatitis C virus (HCV) core protein is known to cause oxidative stress and alter apoptosis pathways. However, the apoptosis results are inconsistent, and the real significance of oxidative stress is not well known. The aim of this study was twofold. First, we wanted to confirm whether core-induced oxidative stress was really significant enough to cause DNA damage, and whether it induced cellular antioxidant responses. Second, we wanted to evaluate whether this core-induced oxidative stress and the antioxidant response to it was responsible for apoptosis changes.. HCV core protein was expressed under control of the Tet-Off promoter in Huh-7 cells and HeLa cells. We chose to use deoxycholic acid (DCA) as a model because it is known to produce both reactive oxygen species (ROS) and apoptosis.. Core expression uniformly increased ROS and 8-hydroxy-2'-deoxyguanosine (8-OHdG) under basal and DCA-stimulated conditions. Core protein expression also increased manganese superoxide dismutase levels. Core protein inhibited DCA-mediated mitochondrial membrane depolarization and DCA-mediated activation of caspase-9 and caspase-3, despite the increase in ROS by DCA. Core protein inhibited DCA-mediated apoptosis by increasing Bcl-x(L) protein and decreasing Bax protein, without affecting the proportion of Bax between mitochondria and cytosol, resulting in suppression of cytochrome c release from mitochondria into cytoplasm.. HCV core protein induces oxidative DNA damage, whereas it inhibits apoptosis that is accompanied by enhancement of ROS production. Thus, oxidative stress and apoptosis modulation by core protein are independent of each other.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; Cytosol; Dactinomycin; Deoxycholic Acid; Deoxyguanosine; Enzyme Activation; Humans; Intracellular Membranes; Liver Neoplasms; Mitochondria, Liver; Oxidative Stress; Protein Synthesis Inhibitors; Reactive Oxygen Species; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Viral Core Proteins

To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment.. Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, Bcl-X(L) and cytochrome c, and the cleavage of poly (ADP-ribose) polymerase (PARP) were assayed by using Western blots.. Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or anti-Fas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-X(L) expression.. Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.

Topics: Antibiotics, Antineoplastic; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Beclin-1; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; DNA, Neoplasm; Doxorubicin; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Liver Neoplasms; Membrane Proteins; Mitochondrial Membranes; Permeability; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transfection

Interleukin-12 (IL-12), a cytokine with antitumor activity, was examined for the suppressive effect on hepatocellular carcinoma (HCC) in mouse model, and its mechanism of antitumor activity was analyzed.. Mice implanted with MIH-2 HCC cells were treated with recombinant mouse IL-12 (500 ng/mouse). Involvement of CD4(+), CD8(+), NK cells and interferon (IFN)-gamma on tumor suppression by IL-12 was examined by treatment of mice with each antibody. Interferon-gamma (IFN-gamma) production by tumor infiltrating cells was analyzed by immunofluorescence microscopy and flow cytometric analysis. Signal transduction for apoptosis induction was examined by immunoblot analysis.. The growth of implanted MIH-2 tumors was significantly suppressed by IL-12 and the suppression was inhibited by depletion of CD8(+)T cells. IL-12 treatment caused numerous IFN-gamma-producing CD8(+)T cells to infiltrate into MIH-2 tumors. Antitumor activity of IL-12 was blocked by treating mice with anti-IFN-gamma mAb. CD8(+)T cells from IL-12-treated mice attached to MIH-2 cells and produced IFN-gamma in vitro. Cell attachment might be associated with intercellular adhesion molecule-1 induced by IFN-gamma. In vitro treatment with IFN-gamma induced apoptosis of MIH-2 cells via a mitochondria-dependent pathway.. IL-12 suppressed HCC growth in mouse model. IFN-gamma produced by IL-12-activated tumor-infiltrating CD8(+)T cells directly induced apoptosis of HCC cells.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Carcinoma, Hepatocellular; Caspase 9; CD8-Positive T-Lymphocytes; Cytochromes c; Interferon-gamma; Interleukin-12; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Models, Animal; Proto-Oncogene Proteins; Recombinant Proteins

We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.

Topics: Animals; Annexin A5; Apoptosis; Caspase 3; Caspase 8; Cells, Cultured; Cytochromes c; DNA Fragmentation; Hepatocytes; Interferon alpha-2; Interferon-alpha; Liver; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Precancerous Conditions; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reactive Oxygen Species; Recombinant Proteins; Transforming Growth Factor beta1

Several studies showed the effectiveness of combination therapy with IFN-alpha and 5-fluorouracil (5-FU) for advanced hepatocellular carcinoma. However, only little is known about the underlying mechanism of combination therapy. In the present study, we examined whether apoptosis through IFN-alpha/beta receptor (IFN-alpha/betaR) was associated with the effects of combination therapy.. HuH7, PLC/PRF/5, HLE, and HLF were treated with IFN- (500 units/mL), 5-FU (0.5 microg/mL), or their combination for 10 days. In addition, IFN-alpha/betaR gene transfer with combination therapy was done.. Ten-day treatment by combination therapy resulted in >80% cell growth inhibition. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis showed synergistic effects for combination therapy on PLC/PRF/5, HLE, and HLF. Concordant results were obtained with DNA fragmentation. Moreover, there was an evidence showing that changes in the expression of Bcl-2 family lead to apoptosis. On the other hand, the expression of IFN-alpha/betaR and up-regulation of alpha-phospho-signal transducer and activator of transcription 1, IFN regulatory factor-1 by combination therapy were observed in all cell lines. Furthermore, IFN-alpha/type 2 IFN receptor long form-transfected HuH7 cells treated with combination therapy showed strong DNA fragmentation compared with nontransfected or transfected with IFN-alpha- and 5-FU-treated HuH7.. Our results showed that combination of IFN-alpha plus 5-FU strongly induced cell growth inhibition of human hepatocellular carcinoma cells and indicated that one of the direct mechanisms of combination therapy may in part be attributable to alterations in induction of apoptosis through IFN-alpha/betaR.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Fluorouracil; Gene Expression; Humans; Interferon Regulatory Factor-1; Interferon-alpha; Liver Neoplasms; Phosphoproteins; Phosphorylation; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Receptors, Interferon; STAT1 Transcription Factor; Statistics as Topic; Time Factors; Trans-Activators; Up-Regulation

To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).. Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation, cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential (MMP) was determined by measuring the retention of the dye 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)) and the protein expression including cytochrome C and poly-(ADP-ribose) polymerase (PARP) were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.. We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3, and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cyclosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.. These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochromes c; Drugs, Chinese Herbal; Enzyme Inhibitors; Humans; Liver Neoplasms; Membrane Potentials; Mitochondria; Oligopeptides; Poly(ADP-ribose) Polymerases

We reported previously that jasmonates can kill human cancer cells. Many chemotherapeutic drugs induce mitochondrial membrane permeability transition, membrane depolarization, osmotic swelling, and release of cytochrome c, involving the opening of the permeability transition pore complex (PTPC). Because jasmonates exert their cytotoxic effects independent of transcription, translation, and p53 expression, we hypothesized that these compounds may act directly on mitochondria. Mitochondrial membrane depolarization was determined by flow cytometry, and cytochrome c release by Western blotting. Mitochondria were isolated by mechanical lysis and differential centrifugation. Cytotoxicity was measured by a tetrazolium-based assay, and mitochondrial swelling by spectrophotometry. Jasmonates induced membrane depolarization and cytochrome c release in intact human cancer cell lines. Jasmonates induced swelling in mitochondria isolated from Hep 3B hepatoma cells, but not in mitochondria isolated from 3T3 nontransformed cells or from normal lymphocytes, in a PTPC-mediated manner. Methyl jasmonate induced the release of cytochrome c from mitochondria isolated from cancer cell lines in a PTPC-mediated manner, but not from mitochondria isolated from normal lymphocytes. A correlation was found between cytotoxicity of methyl jasmonate and the percentage of leukemic cells in the blood of patients with chronic lymphocytic leukemia (CLL). Jasmonates induced membrane depolarization in CLL cells, and swelling and release of cytochrome c in mitochondria isolated from these cells. In conclusion, jasmonates act directly on mitochondria derived from cancer cells in a PTPC-mediated manner, and could therefore bypass premitochondrial apoptotic blocks. Jasmonates are promising candidates for the treatment of CLL and other types of cancer.

Topics: Acetates; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Calcium; Carcinoma, Hepatocellular; Cell Membrane Permeability; Cells, Cultured; Cyclopentanes; Cytochromes c; Fibroblasts; Flow Cytometry; Humans; Ion Channels; Leukemia, Lymphocytic, Chronic, B-Cell; Liver Neoplasms; Lymphocytes; Membrane Potentials; Mice; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Oxylipins; Plant Growth Regulators

Curcumin exhibits anti-inflammatory and antitumor activities. Although its functional mechanism has not been elucidated so far, numerous studies have shown that curcumin induces apoptosis in cancer cells. In the present study, we show that subtoxic concentrations of curcumin sensitize human renal cancer cells to the tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. This apoptosis induced by the combination of curcumin and TRAIL is not interrupted by Bcl-2 overexpression. We found that treatment with curcumin significantly induces death receptor 5 (DR5) expression both at its mRNA and protein levels, accompanying the generation of the reactive oxygen species (ROS). Not only the pretreatment with N-acetylcystine but also the ectopic expression of peroxiredoxin II, an antioxidative protein, dramatically inhibited the apoptosis induced by curcumin and TRAIL in combination, blocking the curcumin-mediated DR5 upregulation. Taken together, the present study demonstrates that curcumin enhances TRAIL-induced apoptosis by ROS-mediated DR5 upregulation.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Caspase Inhibitors; Caspases; Colonic Neoplasms; Curcumin; Cystine; Cytochromes c; Drug Combinations; Drug Resistance, Neoplasm; Enzyme Activation; Flow Cytometry; Humans; Liver Neoplasms; Luciferases; Membrane Glycoproteins; Peroxidases; Peroxiredoxins; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

Phenylethyl isothiocyanate (PEITC) is a well recognized potential chemopreventive compound against human cancers. In this study, the molecular mechanism of PEITC-induced apoptosis was examined with two antioxidants (N-acetyl-cysteine and vitamin E) and a caspase-3 inhibitor (z-DEVD-fmk). Results demonstrated that PEITC significantly induced human hepatoma PLC/PRF/5 (CD95-negative) cells undergoing apoptosis. Treatment with 0 approximately 10 microM PEITC-triggered cell apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and the subsequent appearance of sub-G1 population. Results also displayed that PEITC-induced apoptosis involves the up-regulation of p53 and Bax protein, down-regulation of the XIAP, Bcl-2, Bcl-(XL) and Mcl-1 proteins, cleavage of Bid, and the release of cytochrome c and Smac/Diablo, which were accompanied by the activation of caspases -9, -3 and -8. PEITC-induced the generation of reactive oxygen species and the decrease of mitochondrial membrane potential (Deltapsim) in a time-dependent pattern. N-acetyl-cysteine and vitamin E at 100 microM, and z-DEVD-fmk at 50 microM markedly blocked PEITC-induced apoptosis, which was demonstrated by a decline in the reactive oxygen species generation and the release of the cytochrome c and Smac/Diablo from mitochondria to the cytosol. N-acetyl-cysteine, vitamin E and z-DEVD-fmk also prevented the PEITC in inducing the loss of Deltapsim. They also affected the activity of XIAP and Bax proteins. Taken together, these studies suggest that PEITC is an apoptotic inducer that acts on the mitochondria and the feedback amplification loop of caspase-8/Bid pathways in PLC/PRF/5 cells.

Topics: Acetylcysteine; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carcinoma, Hepatocellular; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; fas Receptor; Flow Cytometry; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Isothiocyanates; Liver Neoplasms; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Oligopeptides; Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Time Factors; Tumor Suppressor Protein p53; Up-Regulation; Vitamin E; X-Linked Inhibitor of Apoptosis Protein

To investigate the effects of nitric oxide on mitochondrial permeability and cytochrome C (cyt C) of human hepatocellular carcinoma cell lines.. NO-mediated apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 was investigated by flow cytometry. The growth and proliferation of human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 were evaluted by MTT assay. Mitochondrial transmembrane potential was analyzed by flow cytometry with double staining of Rh123 and PI, and cytoplasmid cyt C was measured by Western blot. The cells were preincubate with cyclosporin A or GSH synthesis blocker BSO to explore their effect on the results of the above experiments.. NO donor sodium nitroprusside (SNP) induced apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 and resulted in the decrease of the mitochondrial transmembrane potential and the increase of the amount of cytoplasmid cyt C in time-dependent manner. Cyclosporin A (CsA) specific inhibitor of the mitochondrial permeability transition pore could partially prevent the decrease of delta psi m and the release of cyt C. In contrast, GSH synthesis blocker BSO promoted the decrease of delta psi m and the release of cyt C.. NO may induce apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 by decreasing delta psi m, opening the mitochondrial permeability transition pore, and releasing the cyt C.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; Humans; Intracellular Membranes; Liver Neoplasms; Membrane Potentials; Mitochondria; Nitric Oxide; Nitric Oxide Donors; Permeability

To investigate the effect of Tauroursodeoxycholic acid (TUDCA) on Taurodeoxycholic acid (TDCA)-induced HepG2 cell apoptosis and to clarify the molecular mechanism of its anti-apoptosis effect of TUDCA.. Morphologic evaluation of apoptotic cells was performed by Hoechst 33258 staining and electron microscope. DNA fragment was detected by electrophoresis on 1.5% agarose gels. Apoptosis rate was measured by flow cytometry using PI dye. Following incubation of HepG2 cells either with TDCA alone, or coincubation with TUDCA and TDCA, the releasing level of cytochrome c from mitochondria into cytosol was determined by western blot, also the activity of caspase-3, 8, 9.. Incubating the cells with 400 micromol/L TDCA for 12 h induced the cells apoptosis significantly. The apoptotic rate decreased from 50.35% +/- 2.20% to 13.78% +/- 0.84% after coincubation with TUDCA, and this anti-apoptotic effect of TUDCA was confirmed by morphological and DNA ladder detection. TUDCA significantly inhibited the release of cytochrome C from mitochondria into cytosol, and the activity of caspase-9, 3 (t > or = 13.00, P < 0.01), especially at 12 h, caspase-3 activity decreased by 54.9% (t = 16.88, P < 0.01) and 52.5%, however it had no obvious effect on the activity of caspase-8 (t = 1.94, P > 0.05).. TUDCA prevents HepG2 cells apoptosis induced by TDCA through modulating mitochondrial membrane stability, inhibiting the release of cytochrome c and the activation of procaspase-9 and 3. Anti-apoptotic mechanism of TUDCA may be considered to be one of the most important reasons that TUDCA exerts significant efficacy in the treatment of cholestatic liver diseases.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cytochromes c; Humans; Liver Neoplasms; Taurochenodeoxycholic Acid; Taurodeoxycholic Acid; Tumor Cells, Cultured

Acute liver failure (ALF), characterized by massive hepatocyte necrosis, is often caused by drug poisoning, particularly with acetaminophen (APAP). Hepatocyte necrosis is consecutive to glutathione depletion by NAPQI, a metabolite of APAP, and to mitochondrial damages caused by reactive oxygen species (ROS) overproduction. Considering the structure of Mangafodipir, a contrast agent currently used in magnetic resonance imaging of the liver, we hypothesized that this molecule could exert an antioxidant activity and be possibly used as a treatment of APAP-induced ALF.. Mangafodipir is endowed with superoxide dismutase, catalase, and glutathione reductase activities. It can inhibit ROS production by hepatocytes in culture, and protect those cells from oxidative stresses induced by exposure to xanthine oxidase, H(2)O(2), or UV light. Moreover, preventive or curative administration of Mangafodipir to mice with APAP-induced ALF significantly increases survival rates, and abrogates aspartate aminotransferase elevation and histological damage.. Those data point out the potential interest of Mangafodipir in the treatment of toxic ALF in humans.

Topics: Acetaminophen; Animals; Antibodies, Monoclonal; Antioxidants; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Edetic Acid; Enzymes; fas Receptor; Female; Glutathione; Humans; Liver; Liver Failure, Acute; Liver Neoplasms; Mice; Mice, Inbred BALB C; Oxidoreductases; Pyridoxal Phosphate; Reactive Oxygen Species; Superoxides; Survival Analysis

Topics: Blood; Cholecystitis; Cytochromes; Cytochromes c; Liver Function Tests; Liver Neoplasms; Peptic Ulcer