cytochrome-c-t and Colorectal-Neoplasms

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Calystegia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; HT29 Cells; Humans; Membrane Potential, Mitochondrial; Methylene Chloride; Plant Extracts; S Phase Cell Cycle Checkpoints

Apoptin is a small molecular weight protein encoded by the VP3 gene of chicken anemia virus (CAV). It can induce apoptosis of tumor cells and play anti-tumorigenic functions. In this study, we identified a time-dependent inhibitory role of apoptin on the viability of HCT116 cells. We also demonstrated that apoptin induces pyroptosis through cleaved caspase 3, and with a concomitant cleavage of gasdermin E (GSDME) rather than GSDMD. GSDME knockdown switched the apoptin-induced cell death from pyroptosis to apoptosis

Topics: Animals; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Colorectal Neoplasms; Cytochromes c; Female; HCT116 Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Pore Forming Cytotoxic Proteins; Pyroptosis; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Compounds isolated from marine animals have different pharmacological effects. In this study, we investigated the effects of sea nettle (Chrysaora quinquecirrha) crude venom on human colon cancer mitochondria.. First, mitochondria were isolated from healthy colon tissue and cancerous colon tissue, and then mitochondrial function (SDH activity), reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release were measured.. The results showed that crude venom of Chrysaora quinquecirrha (180, 360 and 720 µg/ml) can significantly impair mitochondrial function (**P<0.01 and ***P<0.001) and consequently increase the level of ROS (*P<0.05 and ****P<0.0001), collapse in MMP (*P<0.05 and ****P<0.0001), mitochondrial swelling (**** P<0.0001) and release of cytochrome c (* P<0.05 and *** P<0.001) only in mitochondria isolated from human colon cancer tissue.. The results concluded that crude venom of Chrysaora quinquecirrha (180, 360 and 720 µg/ml) has no side effects on normal mitochondria and only selectively affects cancerous mitochondria. It seems that after further research, Chrysaora quinquecirrha can be considered as a drug candidate for the treatment of patients with colon cancer.

Topics: Animals; Antineoplastic Agents; Colon; Colorectal Neoplasms; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Rectum; Sea Nettle, East Coast; Venoms

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

The anti-cancer effects of Aloe vera barbadensis extract C (AVBEC) have been demonstrated in a previous study. However, the specific functional ingredient and mechanism remain undefined. This study aimed to evaluate the function and associated mechanisms of purified polysaccharide (ABPA1) from AVBEC on colorectal cancer. Here, we identify that ABPA1 can induce colorectal cancer apoptosis. In vivo, ABPA1 significantly suppressed tumor growth in an orthotopic colon cancer model. Mechanistically, ABPA1 alters mitochondrial membrane permeability by promoting Bax translocation while causing cytochrome-c release, which initiates the caspase cascade reaction. Additionally, we found that ABPA1 exerted distinct impacts on the mitochondrial metabolism of colorectal cancer cells. Our study elucidated the mechanism by which the polysaccharide ABPA1 induces apoptosis in colorectal cancer cells through the regulation of Bax and cytochrome-c mediated mitochondrial pathway, indicating that ABPA1 may be developed as a mitochondrial-targeting anti-cancer drug.

Topics: Aloe; Apoptosis; bcl-2-Associated X Protein; Colorectal Neoplasms; Cytochromes c; Humans; Mannans

A large body of research has demonstrated a synergistic anticancer effect between docosahexaenoic acid (DHA) and standard chemotherapy regimens against colorectal cancer (CRC). In this study, we investigated the chemotherapeutic potential of cotreatment with DHA and isoliquiritigenin (ISL) against CRC HCT-116 cells. Apoptosis was confirmed by Annexin V/PI staining and expression of apoptosis-associated proteins. The synergistic effect of DHA and ISL combination on apoptosis was detected using combination index approaches. Flow cytometry was carried out using fluorescent probes to measure the production of reactive oxygen species (ROS). DHA and ISL in combination synergistically enhanced the decrease in cell viability versus the compounds used alone. Moreover, we demonstrated that the synergistic anti-CRC activity of cotreatment with these two compounds was achieved by inducing the apoptosis caspase-dependently mediated through augmented ROS generation followed by increased Fas ligand mRNA expression and cytochrome c release. Our data also demonstrated that cotreating with DHA and ISL strongly upregulated the phosphorylation of ERK and JNK, which are functionally associated with ROS induced by the two compounds in combination. Interestingly, further study revealed that inhibiting ERK phosphorylation strongly enhanced Fas ligand mRNA expression and the combination of the two compounds induced stronger cytotoxicity, whereas inhibiting JNK phosphorylation significantly reduced the apoptotic signals mediated by cotreatment with these two compounds. Excessive ROS-induced JNK activation and cytochrome c release from mitochondria played a key role in the synergistic anticancer activity of CRC cells by cotreating with DHA and ISL.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Chalcones; Colorectal Neoplasms; Cytochromes c; Docosahexaenoic Acids; Drug Synergism; Humans; MAP Kinase Kinase 4; Reactive Oxygen Species

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

Topics: Apoptosis; Caspase 3; Cell Survival; Colorectal Neoplasms; Cytochromes c; Deferoxamine; Down-Regulation; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Models, Biological; Tumor Hypoxia; Tumor Suppressor Protein p53

Topics: Antineoplastic Agents; Antiprotozoal Agents; Apoptosis; Caspases; Cell Cycle; Cell Survival; Colorectal Neoplasms; Cytochromes c; Fluorouracil; HCT116 Cells; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Interleukin-6; Janus Kinase 2; Molecular Docking Simulation; Nitro Compounds; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Thiazoles; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

Scoulerine, an isoquinoline alkaloid isolated from Corydalis plants, has been reported to possess potent anti-proliferative and pro-apoptotic function in cancer cells. However, the effects and underlying mechanisms of scoulerine on colorectal cancer (CRC) progression remain elusive. CCK-8 and LDH assays were used to evaluate cell viability. Apoptosis was assessed by flow cytometry analysis, caspase-3/7 activity assay, and Western blot analysis of Bax, Bcl-2 and cytochrome c (Cyt C) expression. Oxidative stress level was examined by measuring reactive oxygen species (ROS) and glutathione (GSH) contents and superoxide dismutase (SOD) activity. Endoplasmic reticulum (ER) stress activation was detected by Western blot analysis of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) expression. Results showed that scoulerine dose-dependently suppressed CRC cell viability. Scoulerine induced apoptosis and increased caspase-3/7 activity in CRC cells. Bax and cytosolic Cyt C expression was enhanced while Bcl-2 and mitochondrial Cyt C expression was reduced in scoulerine-treated CRC cells. Additionally, scoulerine induced oxidative damage in CRC cells by increasing ROS generation and reducing GSH content and SOD activity. Scoulerine activated ER stress, as evidenced by the increased GRP78 and CHOP expression in CRC cells. Interestingly, blocking ROS production by ROS scavenger N-acetyl-cysteine (NAC) attenuated scoulerine-induced ER stress. Inhibition of ER stress by 4-phenyl butyric acid (4-PBA) abolished scoulerine-induced ROS generation in CRC cells. Blockage of ROS and ER stress attenuated scoulerine-induced cell viability reduction and apoptosis in CRC cells. In conclusion, scoulerine promoted cell viability reduction and apoptosis by activating ROS-dependent ER stress in CRC cells.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Berberine Alkaloids; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Humans; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Colorectal carcinoma is one of the utmost diagnosed cancer with a steep increase in mortality rate. The incidence has been increasing in developing countries like India due to a westernization life style. Flavonoids have been explored widely for its various pharmacological activity including antitumor activity. Orientin, an analogue of luteolin (citrus flavonoid) isolated from rooibos and tulsi leaves is also expected to deliver significant antitumor activity similar to that of luteolin. The present study anticipates exploring the antitumor activity of orientin against colorectal carcinoma cells (HT29). Orientin exhibited remarkable cytotoxicity and antiproliferative activity against HT29 cells, which is clearly evident from tetrazolium based cytotoxicity and lactate dehydrogenase release assays. Orientin induce G0/G1 cell cycle arrest and regulates cyclin and cyclin-dependent protein kinases in order to prevent the entry of the cell cycle to the S phase. Annexin V-FITC (V-Fluorescein Isothiocyanate) dual staining reveals the apoptotic induction ability of orientin. The Bcl-2 family proteins along with the inhibitor of apoptotic proteins were regulated and the tumor suppressor p-53 expression have been decreased. In conclusion, our results proposed that orientin could be a potent chemotherapeutic agent against colorectal cancer after ascertaining their molecular mechanisms.

Topics: Antineoplastic Agents; Apoptosis; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; DNA Damage; Flavonoids; G1 Phase Cell Cycle Checkpoints; Glucosides; HT29 Cells; Humans; Mitochondria; Oligopeptides; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Deregulation of glycolysis is a common phenomenon in human colorectal cancer (CRC). In the present study, we reported that Hexokinase 2 (HK2) is overexpressed in human CRC tissues and cell lines, knockout of HK2 inhibited cell proliferation, colony formation, and xenograft tumor growth. We demonstrated that the natural compound, xanthohumol, has a profound anti-tumor effect on CRC via down-regulation of HK2 and glycolysis. Xanthohumol suppressed CRC cell growth both

Topics: Animals; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Female; Flavonoids; Glycolysis; Hexokinase; Humans; Immunohistochemistry; Mice; Mice, Nude; Propiophenones; Signal Transduction; Xenograft Model Antitumor Assays

Cordycepin, or 3'‑deoxyadenosine, is a derivative of the nucleoside adenosine. Initially extracted from the fungus Cordyceps militaris, cordycepin exhibits antitumor activity against certain cancer cell lines; however, the mechanism by which cordycepin counteracts colorectal cancer (CRC) remains poorly understood. The aim of the present study was to explore the underlying mechanisms of cordycepin against human CRC. To investigate the molecular mechanisms of cordycepin against colon cancer and in driving apoptosis, p53 and Bcl‑2‑like protein 4‑null (Bax‑/‑) colon cancer HCT116 cell lines were used. Cell viability and growth were repressed in a dose‑dependent manner in cells treated with cordycepin. Treatment with cordycepin resulted in increased apoptosis in HCT116 cells; however, flow cytometic analysis demonstrated that apoptosis was notably decreased in the Bax‑/‑ HCT116 cell lines, but not in the p53‑/‑ HCT116 cell lines. Furthermore, cordycepin exposure resulted in the translocation of Bax from the cytosol to the mitochondria and the subsequent release of cytochrome c from the mitochondria. Results from the present study demonstrated that cordycepin inhibited colon cancer cell growth in vitro and this appears to be through the endogenous Bax‑dependent mitochondrial apoptosis pathway, which suggested a molecular mechanism for cordycepin against human colon cancer. These results indicated the possibility of cordycepin as a novel drug for the prevention of colon cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cytochromes c; Deoxyadenosines; HCT116 Cells; Humans; Mitochondria; Tumor Suppressor Protein p53

Thiosemicarbazones (TSCs), and their copper derivatives, have been extensively studied mainly due to the potential applications as antitumor compounds. A part of the biological activity of the TSC-Cu

Topics: Cell Line, Tumor; Colorectal Neoplasms; Copper; Crystallography, X-Ray; Cytochromes c; Drug Screening Assays, Antitumor; Glutathione; Humans; Molecular Structure; Myoglobin; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; Thiosemicarbazones

Colorectal cancer (CRC) is a common and life‑threatening type of malignant cancer, which is associated with a high mortality rate. Cisplatin (CDDP) is a commonly used chemotherapy drug with significant side effects. Brusatol (BR) is one of the principal chemical compounds isolated from the Chinese herb Bruceae Fructus, which has been reported to markedly inhibit the proliferation of numerous cancer cell lines. The present study aimed to investigate the possible synergistic anticancer effects of CDDP combined with BR on CT‑26 cells, and to evaluate the underlying mechanisms of action. The growth inhibitory effects of BR, CDDP, and BR and CDDP cotreatment on CT‑26 cells were assessed by MTT assay. Cell apoptosis were determined by flow cytometry and western blot analysis. The results indicated that compared with single‑agent treatment, cotreatment of CT‑26 cells with CDDP and BR synergistically inhibited cell proliferation and increased cellular apoptosis. Furthermore, treatment of CT‑26 cells with CDDP and BR resulted in a marked increase in the release of cytosolic cytochrome c, decreased expression of procaspase‑3 and procaspase‑9, and upregulation of the B‑cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio compared with treatment with BR or CDDP alone. These results strongly suggested that the combination of CDDP and BR was able to produce a synergistic antitumor effect in CRC cells, thus providing a solid foundation for further development of this combination regimen into an effective therapeutic method for CRC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Shape; Cisplatin; Colorectal Neoplasms; Cytochromes c; Drug Synergism; Gene Expression Regulation, Neoplastic; Inhibitory Concentration 50; Mice; Quassins

Colorectal cancer is the third leading cause of cancer-related deaths in the word. Coptisine (COP), an isoquinoline alkaloid derived from Coptis chinensis Franch, possesses a wide variety of pharmacological effects. However, its anti-proliferative effect on colon cancer is not fully elucidated. In the present study, we aimed to ascertain whether COP inhibits HCT-116 cell growth and to further explore the molecular mechanism in vitro and in vivo.. Cell viability was determined by MTT assay. Cell migration was detected using wound healing assay. Apoptosis, mitochondrial membrane potential (Δψ. The results demonstrated that COP induces apoptosis in HCT-116 cells through PI3K/Akt and mitochondrial-associated apoptotic pathway. Our findings suggest that COP has potential to be a therapeutic candidate for colon cancer patients.

Topics: Animals; Apoptosis; Berberine; Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cytochromes c; HCT116 Cells; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

The present study aimed to demonstrate the antiproliferative effect of hyperoside from Zanthoxylum bungeanum leaves (HZL) and explain the underlying molecular mechanisms in the SW620 human colorectal cancer cell line. The cytotoxic effects of HZL were determined using a3‑(4,5‑dimethylthiazol‑2‑yl)2,5‑diphenyltetrazolium bromide assay. Apoptosis and cell cycle were detected using flow cytometry. Reactive oxygen species (ROS) levels and mitochondrial membrane potential (∆Ψm) were assessed using 2',7'‑dichlorofluorescin diacetate and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide fluorescence spectrophotometry, respectively. Western blot analysis was used to quantify the expression levels of apoptosis‑associated proteins. Reverse transcription‑quantitative polymerase chain reaction analysis was used to determine the mRNA expression of glutathione peroxidase (GSH‑Px) and catalase (CAT). HZL had a marked anti‑proliferative effect on the SW620 human colorectal cancer cells by inducing cell cycle G2/M phase arrest and apoptosis, which was associated with an increase in the expression of p53 and p21. Further mechanistic investigations revealed that the induction of apoptosis was associated with increased generation of ROS, reduced ∆Ψm, and upregulation of B‑cell lymphoma 2‑associated X protein, cytochrome c, caspase‑9, apoptotic protease activating factor 1 and caspase‑3. The antitumor potency of HZL was also attributed to inhibition of the mRNA expression levels of GSH‑Px and CAT. These data indicated that HZL may be involved in the pro‑apoptotic signaling of SW620 human colorectal cancer cells via induction of the caspase‑dependent apoptosis and p53 signaling pathways.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Catalase; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; G2 Phase Cell Cycle Checkpoints; Glutathione Peroxidase; Humans; M Phase Cell Cycle Checkpoints; Membrane Potential, Mitochondrial; Plant Leaves; Quercetin; Reactive Oxygen Species; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation; Zanthoxylum

The alkynyl gold(I) derivative [Au(C≡CPh)(PTA)] (PTA=1,3,5-triaza-7-phosphaadamantane) induces apoptosis in colorectal carcinoma tumour cells (Caco-2) without affecting to normal enterocytes. [Au(C≡CPh)(PTA)] is a slight lipophilic drug, stable in PBS (Phosphate Buffered Saline) and able to bind BSA (Bovin Serum Albumin) by hydrophobic interactions. Once inside the cell, [Au(C≡CPh)(PTA)] targets seleno proteins such as Thioredoxin Reductase 1, increasing ROS (Reactive Oxygen Species) levels, reducing cell viability and proliferation and inducing mitochondrial apoptotic pathway, pro-apoptotic and anti-apoptotic protein imbalance, loss of mitochondrial membrane potential, cytochrome c release and activation of caspases 9 and 3. Moreover, unlike other metal-based drugs such as cisplatin, [Au(C≡CPh)(PTA)] does not target nucleic acid, reducing the risk of side mutation in the DNA. In consequence, our results predict a promising future for [Au(C≡CPh)(PTA)] as a chemotherapeutic agent for colorectal carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caco-2 Cells; Caspase 3; Caspase 9; Cattle; Colorectal Neoplasms; Coordination Complexes; Cytochromes c; Gold; Humans; Membrane Potential, Mitochondrial; Neoplasm Proteins; Oxidation-Reduction; Serum Albumin, Bovine; Thioredoxin Reductase 1

Colorectal cancer (CRC) is the 3. 3c-induced inhibition of proliferation was measured in the absence and presence NAC using MTT in HT-29 and SW620 cells and xCELLigence RTCA DP instrument. 3c-induced apoptotic studies were performed using flow cytometry. 3c-induced redox alterations were measured by ROS production using fluorescence plate reader and flow cytometry and mitochondrial membrane potential by flow cytometry; NADPH and GSH levels were determined by colorimetric assays. Bcl2 family protein expression and cytochrome c release and PARP activation was done by western blotting. Caspase activation was measured by ELISA. Cell migration assay was done using the real time xCELLigence RTCA DP system in SW620 cells and wound healing assay in HT-29.. Many anticancer therapeutics exert their effects by inducing reactive oxygen species (ROS). In this study, we demonstrate that 3c-induced inhibition of cell proliferation is reversed by the antioxidant, N-acetylcysteine, suggesting that 3c acts via increased production of ROS in HT-29 cells. This was confirmed by the direct measurement of ROS in 3c-treated colorectal cancer cells. Additionally, treatment with 3c resulted in decreased NADPH and glutathione levels in HT-29 cells. Further, investigation of the apoptotic pathway showed increased release of cytochrome c resulting in the activation of caspase-9, which in turn activated caspase-3 and -6. 3c also (i) increased p53 and Bax expression, (ii) decreased Bcl2 and BclxL expression and (iii) induced PARP cleavage in human colorectal cancer cells. Confirming our observations, NAC significantly inhibited induction of apoptosis, ROS production, cytochrome c release and PARP cleavage. The results further demonstrate that 3c inhibits cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3.. Our findings thus demonstrate that 3c disrupts redox balance in colorectal cancer cells and support the notion that this agent may be effective for the treatment of colorectal cancer.

Topics: Amides; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Sulfanilic Acids; Tumor Cells, Cultured; Wound Healing

The stress-upregulated catecholamines-activated β1- and β2-adrenergic receptors (β1/2-ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of β1/2-ARs signaling for the treatment of CRC and elucidated the significance of β2-AR expression in CRC in vitro and in clinical samples. The impacts of β1/2-AR antagonists in CRC in vitro and CRC-xenograft in vivo were examined. We found that repression of β2-AR but not β1-AR signaling selectively suppressed cell viability, induced G1-phase cell cycle arrest, caused both intrinsic and extrinsic pathways-mediated apoptosis of specific CRC cells and inhibited CRC-xenograft growth in vivo. Moreover, the expression of β2-AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidence that the expression profiles, signaling, and blockage of β2-AR have a unique pattern in CRC comparing to other cancers. β2-AR antagonism selectively suppresses the growth of CRC accompanying active β2-AR signaling, which potentially carries wild-type KRAS, in vitro and in vivo via the inhibition of β2-AR transactivated EFGR-Akt/ERK1/2 signaling pathway. Thus, β2-AR blockage might be a potential therapeutic strategy for combating the progressions of β2-AR-dependent CRC.

Topics: Adrenergic beta-1 Receptor Antagonists; Adrenergic beta-2 Receptor Antagonists; Animals; Apoptosis; Atenolol; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; ErbB Receptors; G1 Phase Cell Cycle Checkpoints; Gene Expression; HCT116 Cells; HT29 Cells; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Nude; Propanolamines; Propranolol; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptors, Adrenergic, beta; Signal Transduction; Xenograft Model Antitumor Assays

Trichosanthes kirilowii exhibits various biological functions including anti-inflammatory, antidiabetic and anticancer activities. In this study, a novel protein with anti-cancer activity, named as TKP, was purified from Trichosanthes kirilowii fruit by cell-base screening. Mass spectrometry and protease assays revealed that TKP is a serine protease. TKP decreased cell viability in a concentration-and time-dependent manner in human colorectal adenocarcinoma cells. Notably, TKP presented very little effect on human colon epithelial cell line NCM460. Furthermore, TKP inhibited colorectal adenocarcinoma cells to aggregate into viable colony clusters and induced cell apoptosis. The loss of mitochondrial membrane potential, upregulation of cytochrome c and Bax, downregulation of Bcl-2, and activation of caspase-9 and -3 were observed, indicating mitochondria-dependent apoptosis induced by TKP. We found that TKP activated MAPK/ERK and suppressed PI3K/AKT signaling. Inhibition of MAPK/ERK signaling by employing inhibitor PD98059 did not antagonize the effects of TKP. Treatment with PI3K inhibitor LY294003 increased the impact of TKP on mitochondria-related apoptosis proteins (cytochrome c, Bcl-2, Bax, caspase-9 and caspase-3) and cell apoptosis. On the contrary, PI3K/AKT activator insulin-like growth factor-1 reversed the effects of TKP. Taken together, our results demonstrated that TKP has potential anti-colorectal cancer activity by inducing apoptosis, which was regulated by the PI3K/AKT-mediated mitochondria-dependent pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Down-Regulation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Phosphatidylinositol 3-Kinases; Plant Proteins; Proto-Oncogene Proteins c-akt; Serine Proteases; Signal Transduction; Trichosanthes; Up-Regulation

Store-operated Ca(2+) entry (SOCE) inhibitors are emerging as an attractive new generation of anti-cancer drugs. Here, we report that SKF-96365, an SOCE inhibitor, exhibits potent anti-neoplastic activity by inducing cell-cycle arrest and apoptosis in colorectal cancer cells. In the meantime, SKF-96365 also induces cytoprotective autophagy to delay apoptosis by preventing the release of cytochrome c (cyt c) from the mitochondria into the cytoplasm. Mechanistically, SKF-96365 treatment inhibited the calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)/AKT signaling cascade in vitro and in vivo. Overexpression of CaMKIIγ or AKT abolished the effects of SKF-96365 on cancer cells, suggesting a critical role of the CaMKIIγ/AKT signaling pathway in SFK-96365-induced biological effects. Moreover, Hydroxychloroquine (HCQ), an FDA-approved drug used to inhibit autophagy, could significantly augment the anti-cancer effect of SFK-96365 in a mouse xenograft model. To our best knowledge, this is the first report to demonstrate that calcium/CaMKIIγ/AKT signaling can regulate apoptosis and autophagy simultaneously in cancer cells, and the combination of the SOCE inhibitor SKF-96365 with autophagy inhibitors represents a promising strategy for treating patients with colorectal cancer.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Calcium Channel Blockers; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Proliferation; Colorectal Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Female; HCT116 Cells; HT29 Cells; Humans; Hydroxychloroquine; Imidazoles; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-akt; Time Factors; TOR Serine-Threonine Kinases; Transfection; Xenograft Model Antitumor Assays

Hwang-Heuk-San (HHS) is a polyherbal formulation that has been used in traditional Korean medicine for hundreds of years to treat gastrointestinal malignancy. However, to date, the mechanisms responsible for the anticancer effects remain unclear. In the present study, we investigated the anticancer effects of HHS using HCT116 human colorectal cancer (CRC) cells. Our results showed that HHS treatment significantly reduced cell survival and increased apoptotic cell death in a concentration-dependent manner. The treatment of HCT116 cells with HHS also significantly elevated the accumulation of reactive oxygen species (ROS), which was followed by the attenuation of the mitochondrial membrane potential through the upregulation of Bax and the downregulation of Bcl-2, which was accompanied by the release of cytochrome c to the cytosol. In addition, HHS treatment caused the truncation of Bid and activated the caspases (caspase-8, -9 and -3), which was associated with the induction of the Fas ligand, the death receptors (DRs), DR4 and DR5, downregulation of the inhibitors of protein expression in the apoptosis protein family, and the degradation of poly(ADP-ribose)-polymerase. However, a pan-caspase inhibitor reversed the HHS-induced apoptosis and growth suppression, indicating that HHS induces apoptosis though a caspase-dependent intrinsic and extrinsic apoptotic pathway in HCT116 cells. Moreover, HHS treatment inhibited the activation of phosphatidylinositol-3-kinase (PI3K)/Akt signaling, and a pharmacological inhibitor of PI3K significantly potentiated the apoptotic effects of HHS when employed in combination in HCT116 cells. Furthermore, the blocking of ROS generation by antioxidant N-acetyl cysteine attenuated the HHS-induced release of cytochrome c, caspase activation and PI3K/Akt inactivation, thereby preventing HHS-induced apoptosis and reduction in cell viability. These findings suggest that HHS-induced ROS generation is required for caspase-dependent apoptotic cell death involving inhibition of the PI3K/Akt signaling pathway in HCT116 cells. Overall, our findings suggest that HHS may be an effective treatment for CRC cancer, and further studies are required to identify the active compounds in HHS.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cytochromes c; Down-Regulation; HCT116 Cells; Humans; Medicine, Korean Traditional; Membrane Potential, Mitochondrial; Mitochondria; Phosphatidylinositol 3-Kinase; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Up-Regulation

Colorectal cancer is the most commonly diagnosed malignancy with high mortality rates worldwide. Improved therapeutic strategies with minimal adverse side effects are urgently needed. In this study, the anti-tumor effects of EF24, a novel analog of the natural compound curcumin, were evaluated in colorectal cancer cells.. The anti-tumor activity of EF24 on human colon cancer lines (HCT-116, SW-620, and HT-29) was determined by measures of cell cycle arrest, apoptosis, and mitochondrial function. The contribution of ROS in the EF24-induced anti-tumor activity was evaluated by measures of H. The findings indicated that EF24 treatment dose-dependently inhibited cell viability and caused cell cycle arrest at G2/M phase in all the tested colon cancer cell lines. Furthermore, we demonstrated that EF24 treatment induced apoptosis effectively via enhancing intracellular accumulation of ROS in both HCT-116 and SW-620 cells, but with moderate effects in HT-29 cells. We found that EF24 treatment decreased the mitochondrial membrane potential in the colon cancer cells, leading to the release of mitochondrial cytochrome c. Also, EF24 induced activation of caspases 9 and 3, causing decreased Bcl-2 protein expression and Bcl-2/Bax ratio. Pretreatment with NAC, a ROS scavenger, abrogated the EF24-induced cell death, apoptosis, cell cycle arrest, and mitochondrial dysfunction, suggesting an upstream ROS generation which was responsible for the anticancer effects of EF24.. Our findings support an anticancer mechanism by which EF24 enhanced ROS accumulation in colon cancer cells, thereby resulting in mitochondrial membrane collapse and activated intrinsic apoptotic signaling. Thus, EF24 could be a potential candidate for therapeutic application of colon cancer.

Topics: Antineoplastic Agents; Apoptosis; Benzylidene Compounds; Caspases; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Curcumin; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Piperidones; Reactive Oxygen Species

Small molecule BH3 mimetics comprise a promising new chemotherapeutic strategy for treating relapsed or chemoresistant cancer. In this study, we investigated the cellular mechanism of action by which BM-1197, a Bcl-xL/Bcl-2 dual inhibitor, triggers apoptosis in a panel of colorectal cancer (CRC) lines. Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, we determined that BM-1197 inhibited CRC cell growth in a concentration- and time-dependent manner. The 50 % inhibitory concentration (IC50) values for the most sensitive cell lines, SW620 and SW480, ranged from 0.07 to 1.10 μM in response to a 72-h treatment. In CRC cells, BM-1197 induced apoptotic death without affecting the expression of Bcl-2 family proteins. However, BM-1197 effectively triggered a conformational change in Bax, releasing Bim from Bcl-xL by disrupting the interaction between Bcl-xL and Bak/Bax. Compared with the control group, BM-1197 treatment significantly increased the fraction of SW480 cells in the sub-G1 phase, the apoptosis rate, and cellular internucleosomal DNA fragmentation. The proapoptotic activity was associated with cytochrome c release, caspase-3 activation, and PARP-1 cleavage. Collectively, BM-1197 effectively suppressed the growth of the human CRC cell line SW480 by inducing mitochondria-dependent apoptotic cell death. These data have specific implications for the in vivo analysis and clinical evaluation of BM-1197 in CRC.

Topics: Aniline Compounds; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Humans; Protein Conformation; Protein Multimerization; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been found to exhibit various anti-tumor effects. In this work, to investigate its pharmacological effects on human colorectal carcinoma HCT-116 and LoVo cells, cell proliferation and apoptosis were respectively evaluated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC, and propidium iodide (PI) staining. Western blotting was used to detect the expression levels of Bim, Bax, Bcl-2, cytosolic cytochrome c, procaspase-9, cleaved caspase-9, procaspase-3, and caspase-3 proteins. Caspase-Glo-9 and Caspase-Glo-3 assays were applied to determine caspase-9 and caspase-3 activity. MicroRNA-32 (miR-32) expression level was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The in vivo anti-tumor effects of oridonin were evaluated using cell lines HCT-116 and LoVo xenograft model. The results indicated that oridonin effectively inhibited cell proliferation and induced apoptosis in HCT-116 and LoVo cells in a concentration-dependent manner. Oridonin treatment upregulated the expression levels of Bim, Bax, cytosolic cytochrome c, cleaved caspase-9 and cleaved caspase-3 proteins, downregulated the expression levels of Bcl-2, procaspase-9 and procaspase-3 proteins, and meanwhile obviously activated caspase-9 and caspase-3 in a dose-dependent manner in HCT-116 and LoVo cells. The results of qRT-PCR demonstrated that oridonin treatment significantly decreased miR-32 expression, and furthermore, suppression of miR-32 expression by miR-32 inhibitors augmented oridonin-mediated inhibitory and apoptotic effects in HCT-116 and LoVo cells. In vivo results indicated that oridonin administration through intraperitoneal injection suppressed tumor growth in nude mice. Therefore, these findings suggest that oridonin maybe is a potential candidate for colorectal cancer treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cytochromes c; Diterpenes, Kaurane; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; MicroRNAs; Proto-Oncogene Proteins; Xenograft Model Antitumor Assays

The electric field and the concomitant heat (electrohyperthermia) can synergistically induce cell death in tumor tissue, due to elevated glycolysis, ion concentration, and permittivity in malignant compared with nonmalignant tissues. Here we studied the mechanism and time course of tumor destruction caused by electrohyperthermia.. Bilateral implants of HT29 colorectal cancer in the femoral regions of Balb/c (nu/nu) mice were treated with a single 30-min shot of modulated, 13.56-MHz, radiofrequency-generated electrohyperthermia (mEHT). Tumors at 0, 1, 4, 8, 14, 24, 48, and 72 h posttreatment were studied for morphology, DNA fragmentation, and cell death response-related protein expression using tissue microarrays, immunohistochemistry, Western immunoblots, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.. Modulated EHT treatment induced significant tumor destruction in HT29 xenografts with a peak of a sevenfold increase compared with the untreated controls. The significant treatment-related elevation of DNA fragmentation--detected with TUNEL assay--and apoptotic bodies between 24 and 72 h posttreatment was proof of a programmed cell death response. This was associated with significant mitochondrial accumulation of bax and mitochondrial-to-cytoplasmic release of cytochrome c proteins between 8 and 14 h. Cleaved caspase-3 levels were low and mainly localized to inflammatory cells. The substantial cytoplasmic-to-nuclear translocation of apoptosis-inducing factor (AIF) and its 57-kDa activated fragment detected between 14 and 24 h after treatment indicated AIF as an effector for DNA fragmentation.. Modulated EHT treatment can induce programmed cell death-related tumor destruction in HT29 colorectal adenocarcinoma xenografts, which dominantly follows a caspase-independent subroutine.

Topics: Adenocarcinoma; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 2; Colorectal Neoplasms; Cytochromes c; DNA Fragmentation; Gene Expression Regulation, Neoplastic; Heterografts; HT29 Cells; Humans; Hyperthermia, Induced; In Situ Nick-End Labeling; Magnetic Field Therapy; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasm Transplantation; Rats

Colorectal cancer continues to be one of the most common causes of cancer death, and the poor survival rates and liver metastases at the time of diagnosis urgently need more effective strategy for colorectal cancer treatment.. The activities of N-(5-bromopyridin-2-yl)-2-((6-(2-chloroacetamido)benzo[d]thiazol-2-yl)thio)acetamide (YLT205), which is a novel small molecule compound synthesized by us, were investigated using MTT assay, flow cytometry, western blotting and mice tumor xenograft models.. YLT205 induced apoptosis of human colorectal cell lines in a dose-dependent manner. The occurrence of apoptosis was associated with activation of caspases-9 and -3, down-regulation of Bcl-2 and up-regulation of Bax in HCT116 cells. Moreover, YLT205 disrupted mitochondrial membranes and induced the release of cytochrome c into cytosol. Impaired phosphorylation of p44/42 mitogen-activated protein kinase was also observed while the expression of phosphorylated protein kinase B (Akt) was not affected. Furthermore, in HCT116 and SW620 tumor-bearing nude mice models, YLT205 dose-dependently inhibited tumor growth without obvious adverse effects. Immunohistochemistry analyses revealed YLT205 also induced apoptosis and inhibited tumor cell proliferation in vivo.. These studies suggested that YLT205 might be a potential drug candidate for human colorectal cancer therapy.

Topics: Acetamides; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Down-Regulation; Flavonoids; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Phosphorylation; Transplantation, Heterologous

To investigate the molecular mechanism of colorectal cancer (CRC) cell apoptosis induced by the Jumonji domain containing 2B (JMJD2B) silencing.. Both HCT116 and LoVo CRC cell lines were used for analyses. Cell apoptosis after JMJD2B silencing was determined by flow cytometry. JC-1 fluorescence probe was applied to measure the mitochondrial outer membrane permeabilization by flow cytometry and fluorescence microscopy. Immunofluorescence was used to detect cytochrome C translocation from mitochondria to cytosol after JMJD2B silencing. The efficacy of JMJD2B silencing on the protein levels of Bcl-2 family, caspase proteins, CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were detected by Western blot.. JMJD2B silencing induced CRC cell apoptosis via a decrease of the anti-apoptotic gene Bcl-2 family expression, leading to the translocation of Bak and Bax proteins and the promotion of mitochondrial membrane disruption, resulting in the release of cytochrome C from mitochondria and subsequent caspase-9 and caspase-3 cleavage. It also increased the amount of cleaved caspase-8 involved in the death receptor-related apoptotic pathway. Bcl-2 homology 3 interacting-domain death agonist (Bid), a specific caspase-8 substrate involved in the Fas signaling pathway, subsequently induced cleavage via caspase-8 activation. However, levels of CHOP and GRP78 remained unchanged after JMJD2B silencing.. JMJD2B silencing induced CRC cell apoptosis via both mitochondria-related and death receptor-related pathways. The cleavage of Bid activated by caspase-8 might serve as a crosstalk mediator between these two pathways in CRC.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspases; Colorectal Neoplasms; Cytochromes c; Down-Regulation; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Silencing; Humans; Jumonji Domain-Containing Histone Demethylases; Mitochondria; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Death Domain; RNA Interference; Tumor Cells, Cultured

α-Methyl-n-butylshikonin (MBS), one of the active components in the root extracts of Lithospermum erythrorhizon, posses antitumor activity. In this study, we assess the molecular mechanisms of MBS in causing apoptosis of SW620 cells. MBS reduced the cell viability of SW620 cells in a dose-and time-dependent manner and induced cell apoptosis. Treatment of SW620 cells with MBS down-regulated the expression of Bcl-2 and up-regulated the expression of Bak and caused the loss of mitochondrial membrane potential. Additionally, MBS treatment led to activation of caspase-9, caspase-8 and caspase-3, and cleavage of PARP, which was abolished by pretreatment with the pan-caspase inhibitor Z-VAD-FMK. MBS also induced significant elevation in the phosphorylation of JNK and p38. Pretreatment of SW620 cells with specific inhibitors of JNK (SP600125) and p38 (SB203580) abrogated MBS-induced apoptosis. Our results demonstrated that MBS inhibited growth of colorectal cancer SW620 cells by inducing JNK and p38 signaling pathway, and provided a clue for preclinical and clinical evaluation of MBS for colorectal cancer therapy.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Proliferation; Colorectal Neoplasms; Cytochromes c; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Naphthoquinones; p38 Mitogen-Activated Protein Kinases; Tumor Cells, Cultured

Recently, we found that lovastatin, a HMG-CoA reductase inhibitor, and gamma-tocopherol, one of the significant types of vitamin E in diet, additively induced apoptosis in a colorectal carcinoma cell line. In this study we mechanistically monitored the loss of mitochondrial membrane potential, amount of cytosolic cytochrome c and caspase 3 activity after treatment by lovastatin and gamma-tocopherol. HT29 cells were treated with different doses of lovastatin and gamma-tocopherol for 48 and 72 h. Lovastatin and gamma-tocopherol in combination induced the release of cytochrome c, caspase 3 activation, and loss of mitochondrial membrane potential more significantly compared to their controls. Our data showed that lovastatin plus gamma tocopherol potently induced mitochondrial membrane potential collapse, cytochrome c release along with caspase 3 activation that reveals the importance of targeting programmed cell death signaling at different points of its signaling pathway for cancer therapy.

Topics: Apoptosis; Caspase 3; Colorectal Neoplasms; Cytochromes c; Drug Synergism; gamma-Tocopherol; HEK293 Cells; HT29 Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Signal Transduction

Parthenolide (PT), a NF-κB inhibitor, has recently been demonstrated as a promising anticancer agent that promotes apoptosis of cancer cells. 5-fluorouracil (5-FU) has been a drug of choice for treatment of colorectal cancer (CRC). Unfortunately, many of the therapies that use 5-FU alone or in combination with other agents are likely to become ineffective due to drug resistance. In the present study, we investigated the antitumor effect of PT combined with 5-FU on a human CRC cell line, SW620. The results demonstrated that combination of PT and 5-FU induced apoptosis which was determined using MTT, cell cycle analysis, annexin-V assay, and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome C release, and activation of caspase 3 and 9. Moreover, intra-peritoneal injection of PT and 5-FU showed significant inhibition of tumor growth in the xenograft model. These results demonstrate that PT exhibits anticancer activity in human colorectal cancer in vitro and in vivo. These findings provide an efficacious strategy to overcome 5-FU resistance in certain CRC.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antimetabolites, Antineoplastic; Apoptosis; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Drug Combinations; Drug Resistance, Neoplasm; Drug Synergism; Fluorouracil; Humans; Mice; Mice, Nude; Mitochondria; Neoplasm Transplantation; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Xenograft Model Antitumor Assays

RU486 (mifepristone) exerts an anticancer effect on cancer cells via induction of apoptosis. However, the molecular mechanisms are not fully understood. Here, we investigated the effect of RU486 on the apoptosis of U937 human leukemia cells. RU486 markedly increased apoptosis in U937 cells as well as in MDA231 human breast carcinoma, A549 human lung adenocarcinoma epithelial and HCT116 human colorectal carcinoma cells. RU486 increased dose-dependent release of mitochondrial cytochrome c, and reduced the mitochondrial membrane potential (MMP, Δψm) in RU486-treated U937 cells. We also found that overexpression of Bcl-2 completely blocked RU486-mediated apoptosis. However, reactive oxygen species signaling had no effect on RU486-induced apoptosis. RU486 increased the phosphorylation of p38 MAPK and JNK, but p38 MAPK only was associated with RU486-mediated apoptosis. Taken together, RU486 induces apoptosis through reduction in the mitochondrial membrane potential and activation of p38 MAPK in U937 human leukemia cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Female; HCT116 Cells; Hormone Antagonists; Humans; Leukemia; Lung Neoplasms; Lymphoma; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mifepristone; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Glucocorticoid; U937 Cells

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.

Topics: Animals; Apoptosis; Caspases; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Cytosol; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Models, Biological; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Xenograft Model Antitumor Assays

Polyamine metabolism is an intriguing tumor therapeutic target. The present study was designed to assess the synergistic antitumor effects of NPC-16, a novel polyamine naphthalimide conjugate, with celecoxib and to elucidate the mechanism of these effects on human colorectal cancer cells.. Cell proliferation was assessed by the MTT assay. Cell apoptosis and mitochondria membrane potential were evaluated by high content screening analysis. Intracellular polyamine content was detected by HPLC. Protein expression was detected by western blot analysis.. The co-treatment with celecoxib enhanced NPC-16-induced apoptosis in HCT116 (COX-2 no expression), HT29 (COX-2 higher expression) and Caco-2 (COX-2 higher expression) colorectal cancer cells, which was mediated by the elevated NPC-16 uptake via the effect of celecoxib on polyamine metabolism, including the up-regulated spermidine/spermine N(1)-acetyltransferase (SSAT) activity and reduced intracellular polyamine levels. The presence of celecoxib does not result in obviously different effect on the NPC-16-triggered apoptosis in diverse COX-2 expressed colorectal cell lines, suggesting that COX-2 was not one vital factor in the apoptotic mechanism. Furthermore, this synergistic apoptosis was involved in the PKB/AKT signal pathway, Bcl-2 and caspase family members. Z-VAD-FMK, a cell permeable pan caspase inhibitor, almost completely inhibited celecoxib and NPC-16 co-induced apoptosis, indicating that this apoptosis was caspase dependent.. Co-treatment of celecoxib and NPC-16 could induce colorectal cancer cell apoptosis via COX-2-independent and caspase-dependent mechanisms. The combination therapy with these agents might provide a novel therapeutic model for colorectal cancer.

Topics: Acetyltransferases; Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Celecoxib; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclooxygenase 2; Cytochromes c; Drug Synergism; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Naphthalimides; Polyamines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Sulfonamides; Up-Regulation

Sessile marine animals such as sponges, ascidians, and bryozoans are a rich source of bioactive natural products, many of which exhibit potent anticancer activity.. We extracted and purified a polypeptide with potent antitumor activity from Ciona savignyi by acetone fractionation, ultrafiltration, ion exchange chromatography, gel chromatography, and high-performance liquid chromatography. An MTT assay was used to study the cytotoxicity of the isolated fraction and the purified polypeptide. Cell cycle and Western blot analysis were performed to study the mode of action of the purified polypeptide.. A novel polypeptide with potent antitumor activity was purified. The molecular weight of the polypeptide, designated CS5931, was 5931 Da, and use of the genome basic local alignment search tool (BLAST) revealed that the N-terminal sequence of CS5931 is identical to that of granulin A from C savignyi. CS5931 exhibited significant cytotoxicity for several cancer cell types and induced apoptotic death in HCT-8 cells in a dose- and time-dependent manner. Cell cycle analysis demonstrated that CS5931 caused cell cycle arrest at the G(2)/M phase, and a sub-G(1) peak appeared after treating the cells with CS5931 for 12 hours. The mitochondrial-mediated pathway was implicated in CS5931-induced apoptosis.. Our observations clearly demonstrate the antiproliferative and proapoptotic activities of the polypeptide CS5931 from C savignyi and the mitochondrial-mediated pathway involved in the polypeptide-induced cell death.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; G2 Phase Cell Cycle Checkpoints; Granulins; Humans; Intercellular Signaling Peptides and Proteins; Mitochondria; Peptides; Proto-Oncogene Proteins c-bcl-2; Urochordata

Parthenolide (PT), a principal active component in medicinal plants, has been used conventionally to treat migraine and inflammation. This component has recently been reported to induce apoptosis in cancer cells, through mitochondrial dysfunction. In the present study, we investigated PT-mediated cell death signaling pathway by focusing on the involvement of Bcl-2 family members in human colorectal cancer cells. We also investigated the inhibitory effect of PT on tumor growth in xenografts. Using the human colorectal cancer cell lines HT-29, SW620 and LS174T, we demonstrated that treatment of these cancer cells with PT induces apoptosis using MTT, Annexin V assay and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome c release and caspase activation. Moreover, intraperitoneal injection of PT showed significant inhibition of tumor growth, angiogenesis in the xenograft model. These results demonstrate that PT exhibits anti-cancer activity in human colorectal cancer in vitro and in vivo. These findings may also provide a novel approach for the treatment of colorectal cancer.

Topics: Apoptosis; Carcinogenesis; Colorectal Neoplasms; Cytochromes c; Genes, bcl-2; HT29 Cells; Humans; Mitochondria; Neovascularization, Pathologic; Sesquiterpenes; Signal Transduction; Xenograft Model Antitumor Assays

The antitumor activity of 7-piperazinethylchrysin (7-PEC) was investigated in HCT-116 human colon cancer cells. MTT assay revealed that the IC50 of 7-PEC in HCT-116 cells was 1.5 µM after 72 h of treatment, much lower than that of chrysin (>100 µM). The data showed that 7-PEC was able to inhibit the growth of HCT-116 cells in a concentration- and time-dependent manner. Topical morphological changes of apoptotic body formation after 7-PEC treatment were observed by Hoechst 33258 staining. 7-PEC reduced mitochondrial membrane potential (∆Ψm) of cells in a concentration-dependent manner and increased the production of intracellular reactive oxygen species (ROS). After treatment with 7-PEC, a significant increase of Bax protein expression and decrease of Bcl-2 protein expression were observed at the same time. These events paralleled with activation of p53, caspase-3 and -9 and the release of cytochrome c (cyt‑c), as well as poly(ADP-ribose) polymerase-1 (PARP1) cleavage and downregulation of p-Akt. However, the apoptosis induced by 7-PEC was blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. These results demonstrate that 7-PEC-induced mitochondrial dysfunction in HCT-116 human colon cancer cells triggers events responsible for caspase-dependent apoptosis pathways, and the elevated ratio of Bax/Bcl-2 is likely involved in this effect.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Survival; Colorectal Neoplasms; Cytochromes c; Down-Regulation; Flavonoids; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Piperazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Tumor Suppressor Protein p53

Apoptosis is a highly regulated mechanism of cell death where pro-apoptotic proteins and caspases play an important role. Activation of pro-caspases at a definite time is essential to control the whole caspase cascade. Mitochondrion contains some pro-apoptotic proteins, which need to come out in cytoplasm for apoptotic function such as Cytochrome c (Cyt c), while the Bcl-2 protein family works as the guard of mitochondrial membrane and prevents the escape of Cyt c. Once Cyt c is out in cytoplasm, it binds with Apaf-1 (another pro-apoptotic protein also essential for proper cell differentiation) and pro-caspase-9, forming the Apoptosome complex. In this study, the role of two non-steroidal anti-inflammatory drugs (NSAIDs), Diclofenac and Celecoxib, in experimentally induced early neoplasm of colon via apoptosome mechanism had been studied. It has been recognized that the prolonged use of NSAIDs has its effect on reducing the risk of colorectal cancer through apoptotic pathways. However, the role of NSAIDs in respect of apoptosome is not clear.. Western blotting and immunohistochemistry were performed, along with morphological and histological analysis.. According to the expression levels of Cytochrome c, Apaf-1, Caspases, and Bcl-2, it was observed that NSAIDs do follow the mitochondrial or intrinsic pathway of apoptosis.. The effects of Diclofenac and Celecoxib on the expression of pro- and anti-apoptotic proteins have been observed, which may constitute the mechanism by which the NSAIDs are efficient in controlling the proliferation of neoplasm in the colon.

Topics: 1,2-Dimethylhydrazine; Aberrant Crypt Foci; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosomes; Blotting, Western; Carcinogens; Celecoxib; Colorectal Neoplasms; Cyclooxygenase 2 Inhibitors; Cytochromes c; Diclofenac; Immunoenzyme Techniques; Male; Mitochondria; Pyrazoles; Rats; Rats, Sprague-Dawley; Sulfonamides

Apoptosis has a vital role in maintaining tissue homeostasis, and dysregulation of the apoptotic pathway is now widely recognized as a key step in tumourigenesis. Increasingly, evidence has demonstrated that microRNA (miRNA) can exert various biological functions in tumours by targeting oncogenes or tumour suppressors. Nevertheless, the role of miRNA in apoptosis remains unclear. Here we show that ectopical expression of miR-148a can induce apoptosis in colorectal cancer cells. In addition, MYB can inhibit miR-148a by directly acting on the transcription factor binding site in miR-148a gene and miR-148a can posttranscriptionally silence Bcl-2. Subsequently, the intrinsic apoptosis pathway is activated by releasing cytochrome c, cleaving caspase 9, caspase 3 and PARP, which eventually induce cancer-cell apoptosis. These findings are part of a hitherto undocumented apoptotic regulatory pathway in which a pleiotropic transcription factor controls the expression of a miRNA and the miRNA inhibits the target, leading to activation of an intrinsic mitochondrial pathway and tumour apoptosis.

Topics: 3' Untranslated Regions; Apoptosis; Binding Sites; Caspase 3; Caspase 9; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Humans; MicroRNAs; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Δψ(m)) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase 7; Caspase 9; Cell Survival; Colorectal Neoplasms; Cytochromes c; Fluorouracil; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; X-Linked Inhibitor of Apoptosis Protein

Hepatoma-derived growth factor (HDGF) is a novel multifunctional growth factor that elicits pleiotropic effects on biological processes such as lung remodeling and renal development. Recent studies demonstrated that HDGF is related to tumor proliferation, invasion, angiogenesis, and apoptosis. However, the molecular mechanism of HDGF's involvement in apoptosis remains to be clarified. In this study, we first analyze the role of HDGF in colorectal carcinoma (CRC) progression by immunohistochemistry. Then we used small interference RNA (HDGF-siRNA) to block HDGF and assessed its effect on inducing apoptosis of CRC loVo cells. Apoptosis was detected using flow cytometry (FCM), DNA ladder analysis, and Hoechst 33258 staining. In addition, the expression levels of some apoptosis-related proteins were examined by western blot. The result showed that HDGF expression gradually increased in the colorectal carcinogenesis process. Further studies demonstrated that knock-down of HDGF can down-regulate the survivin, activate the mitochondrial pathway, and induce apoptosis in loVo cells. These findings suggest that HDGF is involved in colorectal carcinogenesis process. Further blocking HDGF exhibits potent pro-apoptotic properties in colon cancer cells. Thus, HDGF might be a potential therapeutic target for human colorectal cancer. These findings may have major implications in the treatment of colorectal cancer.

Topics: Adenoma; Apoptosis; Blotting, Western; Cell Proliferation; Colitis; Colon; Colorectal Neoplasms; Cytochromes c; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Precancerous Conditions; Rectum; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering

Norcantharidin exhibits cytotoxicity in many cancer cell lines, including colorectal cancer (CRC) cells. Its cytotoxic potency on primary CRC cells and other normal constituent cells of the human body remains elusive. This study investigates whether norcantharidin differentially exhibits cytotoxicity on primarily isolated CRC cells and dermal fibroblasts. The in vitro chemosensitivity of norcantharidin was measured using a MTT tetrazolium assay and compared with 73 primary tumor cells from surgically excised colorectal tumors, six human CRC cell lines and dermal fibroblasts. Observations of cytotoxicity to primary tumor cells reveal significant differences among genders and histological types; however, drug-induced chemosensitivity was not correlated with age or clinical stages of CRC patients. Norcantharidin had a similar cytotoxic effect on primary tumor cells and CRC cell lines in a dose-dependent manner. In contrast, normal fibroblasts were more resistant to norcantharidin-induced cytotoxicity than CRC cells. DAPI staining results demonstrated that norcantharidin caused CRC cell apoptosis by nuclear fragmentation and chromatin condensation. The release of cytochrome c and the triggering of caspase-3, -8 and -9 activation mediated apoptotic induction. Conversely, pretreatment with caspases or mitogen-activated protein kinase (MAPK) inhibitors significantly suppressed norcantharidin-induced CRC cytotoxicity. These in vitro results suggest that norcantharidin may be a safe and effective anti-cancer drug for CRC.

Topics: Aged; Antineoplastic Agents; Apoptosis; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Caspases; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Fluorescent Dyes; Histones; Humans; Indoles; Male; Middle Aged; Mitogen-Activated Protein Kinases

Hepatoma-derived growth factor (HDGF) is correlated with aggressive tumor behaviors such as invasion and angiogenesis. Nevertheless, the potential involvement of HDGF in cell resistance to natural plant phenols-based chemotherapy is still unclear. This study demonstrated that over-expression of HDGF could confer the resistance of human colorectal cancer (CRC) cells to nordihydroguaiaretic acid (NDGA) toxicity. Enforced expression of HDGF could inhibit NDGA-induced apoptosis through the mitochondrial pathway in CRC cells, and attenuate the inhibitory effect of NDGA on tumor growth. Therefore, our results suggest that HDGF represents a potential molecule associated with chemotherapy resistance, which may have major implications in improving chemotherapy of colorectal cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Humans; Intercellular Signaling Peptides and Proteins; Male; Masoprocol; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

Cancer is one of the leading causes of death in the world. The triterpenoid compound asiatic acid derived from the tropical medicinal plant Centella asiatica displays cytotoxic activity on fibroblast cells and several other kinds of cells. The present work studies asiatic acid-mediated growth inhibition of cancer cells and the underlying mechanism. Asiatic acid markedly inhibited cancer cell proliferation. Apoptosis of SW480 human colon cancer cells was induced by asiatic acid as shown by flow cytometry, DNA fragmentation and nuclear chromatin condensation experiments. Through increasing mitochondrial membrane permeability and cytochrome c release from mitochondria into cytosol, asiatic acid induced caspase-9 activity, which further activated caspase-3 and poly(ADP-ribose) polymerase cleavage resulting in irreversible apoptotic death in the tumor cells. Taken together, these results suggest that mitochondrial death apoptosis cascade plays very important roles in asiatic acid-induced cancer apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Centella; Chromatin; Colonic Neoplasms; Colorectal Neoplasms; Cytochromes c; DNA Fragmentation; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Molecular Structure; Pentacyclic Triterpenes; Poly Adenosine Diphosphate Ribose; Stomach Neoplasms; Triterpenes

Agents that inhibit histone deacetylases (HDAC inhibitors) have been shown to enhance radiation response. The aim of this study was to evaluate the effects of low, minimally cytotoxic concentrations of the HDAC inhibitor, valproic acid (VPA), on radiation response of colorectal cancer cells. Cell lines LS174T and an isogenic pair of HCT116, which differed only for the presence of wild-type p53, were exposed to ionizing radiation (IR) alone, VPA alone, or the combination. Clonogenic survival, gamma-H2AX induction, apoptosis, changes in mitochondrial membrane potential, and mitochondrial levels of p53 and Bcl-2 family proteins were assessed. In vivo studies monitored tumor growth suppression after therapy in mice bearing HCT116/p53(+/+) and HCT116/p53(-/-) tumor xenografts. VPA led to radiosensitization, which was dependent on p53 status. A decrease in clonogenic survival, an increase in apoptosis, and an increase in levels of gamma-H2AX were observed after VPA+IR, compared to IR alone, in wild-type p53 cells (LS174T and HCT116/p53(+/+)), as opposed to p53 null cells (HCT116/p53(-/-)). Exposure to VPA resulted in enhancement of IR-induced mitochondrial localizations of Bax and Bcl-xL, mitochondrial membrane potential, and cytochrome c release only in wild-type p53 cell lines. VPA also enhanced tumor growth suppression after IR only in wild-type p53 xenografts. These data suggest that VPA may have an important role in enhancing radiotherapy response in colorectal cancer, particularly in tumors with the wild-type p53 genotype.

Topics: Animals; bcl-2-Associated X Protein; Cell Cycle; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Flow Cytometry; Genotype; Histone Deacetylase Inhibitors; Humans; Membrane Potentials; Mice; Mitochondria; Mitochondrial Membranes; Radiation-Sensitizing Agents; T-Lymphocytes; Tumor Suppressor Protein p53; Valproic Acid

The mitochondrial enzyme manganese superoxide dismutase (MnSOD) has been shown to have two faces with regard to its role in tumor development. On the one side, it is well documented that overexpression of MnSOD slows down cancer cell growth, whereas on the other side MnSOD also has a metastasis-promoting activity. We set out to examine the role of MnSOD in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, thought to be a first-line tumor surveillance mechanism and failure to undergo apoptosis might contribute to metastasis formation. We show that overexpression of MnSOD at moderate levels is able to protect cells from TRAIL-induced apoptosis. While caspase-8 activation and Bid cleavage were not affected by MnSOD, we detected a marked decrease in caspase-3 activation pointing to a mitochondrial resistance mechanism. Indeed, we found that MnSOD-overexpressing cells showed reduced cytochrome c and no Smac/DIABLO release into the cytosol. The resulting lack of X-linked inhibitor of apoptosis (XIAP) inhibition by cytosolic Smac/DIABLO most likely caused the TRAIL resistance as RNAi against XIAP-rescued caspase-3 activity and TRAIL sensitivity. Our results show that reactive oxygen species are involved in TRAIL-induced Smac/DIABLO release and in TRAIL-triggered apoptosis. Hence, high levels of MnSOD, which decompose and neutralize these reactive oxygen species, might contribute to metastasis formation by allowing disseminated tumor cells to escape from TRAIL-mediated tumor surveillance. As part of TRAIL regimens, adjuvant treatment with XIAP inhibitors in the form of Smac/DIABLO mimetics or MnSOD inhibitors might be able to break TRAIL resistance of malignant tumor cells.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Humans; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Phenotype; Reactive Oxygen Species; RNA Interference; Superoxide Dismutase; TNF-Related Apoptosis-Inducing Ligand; Tumor Stem Cell Assay; X-Linked Inhibitor of Apoptosis Protein

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.

Topics: Animals; Apoptosis; Caspases; Cell Proliferation; Cells, Cultured; Ceramides; Colorectal Neoplasms; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Humans; Mice; Mice, Inbred BALB C; Mitochondria; Molecular Structure; Reverse Transcriptase Polymerase Chain Reaction; Sphingolipids; Sphingosine; Survival Rate; Tumor Cells, Cultured

Bidens alba has been used for healing cuts, injuries, swellings, hypertension, jaundice, and diabetes in some countries. However, the effect of B. alba on human cancer remains poorly understood. The goal of this study was to investigate whether B. alba protein-extract could have an anticancer property against human colorectal cancer. The human colorectal cancer SW 480 cells treated with the protein-extract of B. alba would cause marked DNA damages and apoptosis-related cellular morphologies. Treatment with 225 microg/ml B. alba protein-extract also led to the SW480 cells to produce readily intracellular reactive oxygen species (ROS) after 1h of treatment and last to 24 h. The intracellular glutathione (GSH) depletion occurred after 12-24h of treatment. The treatment of the protein-extract would also caused mitochondrial transmembrane potential (DeltaPsi(m)) to decrease and cytosolic cytochrome c to increase. The caspase 3/7 activities were activated from 3 to 6 h after the treatment. The percentages of apoptosis induced by the protein-extract of B. alba decreased 26.4%, 10.1%, and 29.4% when the SW 480 cells were pretreated with Vitamin C, N-acetylcysteine, and Boc-Asp(OMe)-fmk, respectively. Taken together, we demonstrated for the first time that the protein-extract of B. alba could induce apoptosis that was related to the ROS production and GSH depletion in human colorectal cancer. The protein-extract of B. alba might have therapeutic value against the human colorectal cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bidens; Caspases; Cell Count; Cell Cycle; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Cytosol; Flow Cytometry; Glutathione; Humans; In Situ Nick-End Labeling; Indicators and Reagents; Mitochondria; Plant Extracts; Plant Proteins; Reactive Oxygen Species; Superoxide Dismutase; Tetrazolium Salts; Thiazoles

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL shows strong cytotoxicity to many cancer cells but minimal cytotoxicity to most normal cells. Interestingly, our recent studies have demonstrated that pretreatment with TRAIL induces acquired resistance to TRAIL (Song et al. 2007 J Biol Chem 282: 319). Acquired TRAIL resistance develops within 1 day and gradually decays within 5 days after TRAIL treatment. In our current study, we examined whether human colorectal carcinoma CX-1 cells with acquired TRAIL resistance are resistant to UV irradiation as well. CX-1 cells were treated with 200 ng/ml TRAIL for 6 h and incubated various times (0.25-5 days) and then challenged to UV irradiation. Unexpectedly, we observed an increase in apoptosis in acquired TRAIL resistant cells after UVC as well as UVB exposure. This was due to an increase in caspase activation which was mediated through cytochrome c release. These results suggest that cells with acquired TRAIL resistance are sensitive to UV irradiation.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Humans; TNF-Related Apoptosis-Inducing Ligand; Ultraviolet Rays; Ultraviolet Therapy

Chemically synthesized sugar-cholestanols with mono-, di-, and tri-saccharides attached to cholestanol showed strong inhibiting activity against the proliferation of colorectal and gastric cancer cells. In contrast, cholestanol without sugar moieties was totally ineffective. Furthermore, when cancer cells were exposed to GlcNAcRbetacholestanol (R=(-) or beta1-3Gal), the compound was rapidly taken up via the lipid rafts/microdomains on the cell surface. The uptake of sugar-cholestanol in mitochondria increased gradually and was followed by the release of cytochrome c from mitochondria and the activation of apoptotic signals through the mitochondrial pathway and the caspase cascade, leading to apoptotic cell death, characterized by DNA ladder formation and nuclear fragmentation. Additionally, the examination of GlcNAcRbetacholestanol in a mouse model of peritoneal dissemination showed a dramatic reduction of tumor growth (P < 0.003) and prolonged mouse survival time (P<0.0001). Based on these observations, we believe that the sugar-cholestanols described here have clinical potential as novel anticancer agents.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cholestanols; Chromatography, Liquid; Colorectal Neoplasms; Cytochromes c; DNA Fragmentation; Female; HT29 Cells; Humans; Injections, Intraperitoneal; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Oligosaccharides; Stomach Neoplasms; Tandem Mass Spectrometry

ST13 is a cofactor of heat shock protein 70 (Hsp70). To date, all data since the discovery of ST13 in 1993 until more recent studies in 2007 have proved that ST13 is downregulated in tumors and it was proposed to be a tumor suppressor gene, but no work reported its antitumor effect and apoptotic mechanism. In the work described in this paper, ST13 was inserted into ZD55, an oncolytic adenovirus with the E1B 55-kDa gene deleted, to form ZD55-ST13, which exerts an excellent antitumor effect in vitro and in an animal model of colorectal carcinoma SW620 xenograft. ZD55-ST13 inhibited tumor cells 100-fold more than Ad-ST13 and ZD55-EGFP in vitro. However, ZD55-ST13 showed no damage of normal fibroblast MRC5 cells. In exploring the mechanism of ZD55-ST13 in tumor cell killing, we found that ZD55-ST13-infected SW620 cells formed apoptotic bodies and presented obvious apoptosis phenomena. ZD55-ST13 induced the upregulation of Hsp70, the downregulation of antiapoptotic gene Bcl-2, and the release of cytochrome c. Cytochrome c triggered apoptosis by activating caspase-9 and caspase-3, which cleave the enzyme poly(ADP-ribose) polymerase in ZD55-ST13-infected SW620 cells. In summary, overexpressed ST13 as mediated by oncolytic adenovirus could exert potent antitumor activity via the intrinsic apoptotic pathway and has the potential to become a novel therapeutic for colorectal cancer gene therapy.

Topics: Adenoviridae; Animals; Apoptosis; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Down-Regulation; Female; Genes, bcl-2; Genetic Therapy; HSP70 Heat-Shock Proteins; Humans; Mice; Mice, Nude; Mitochondria; Oncolytic Virotherapy; Tumor Suppressor Proteins; Up-Regulation

In human colorectal cancer cells, the polyphenol resveratrol (RV) activated the caspase-dependent intrinsic pathway of apoptosis. This effect was not mediated via estrogen receptors. Pepstatin A, an inhibitor of lysosomal cathepsin D (CD), not (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, an inhibitor of cathepsins B and L, prevented RV cytotoxicity. Similar protection was attained by small interference RNA-mediated knockdown of CD protein expression. RV promoted the accumulation of mature CD, induced lysosome leakage and increased cytosolic immunoreactivity of CD. Inhibition of CD or its post-transcriptional down-regulation precluded Bax oligomerization, permeabilization of mitochondrial membrane, cytosolic translocation of cytochrome c, caspase 3 activation and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling positivity occurring in RV-treated cells. The present study identifies the lysosome as a novel target of RV activity and demonstrates a hierarchy of the proteolytic pathways involved in its cytotoxic mechanism in which the lysosomal CD acts upstream of the cytosolic caspase activation. Our data indicate that metabolic, pharmacologic or genetic conditions affecting CD expression and/or activity could reflect on the sensitivity of cancer cells to RV.

Topics: Caspase Inhibitors; Cathepsin D; Cathepsin L; Cathepsins; Cell Death; Cell Line; Colorectal Neoplasms; Cysteine Endopeptidases; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; HT29 Cells; Humans; Lysosomes; Resveratrol; Stilbenes; Time Factors

The human probiotic Propionibacterium freudenreichii kills colorectal adenocarcinoma cells through apoptosis in vitro via its metabolites, the short chain fatty acids (SCFA), acetate and propionate. However, the precise mechanisms, the kinetics of cellular events and the impact of environmental factors such as pH remained to be specified. For the first time, this study demonstrates a major impact of a shift in extracellular pH on the mode of propionibacterial SCFA-induced cell death of HT-29 cells, in the pH range 5.5 to 7.5 prevailing within the colon. Propionibacterial SCFA triggered apoptosis in the pH range 6.0 to 7.5, a lethal process lasting more than 96 h. Indeed at pH 7.5, SCFA induced cell cycle arrest in the G2/M phase, followed by a sequence of cellular events characteristic of apoptosis. By contrast, at pH 5.5, the same SCFA triggered a more rapid and drastic lethal process in less than 24 h. This was characterised by sudden mitochondrial depolarisation, inner membrane permeabilisation, drastic depletion in ATP levels and ROS accumulation, suggesting death by necrosis. Thus, in digestive cancer prophylaxis, the observed pH-mediated switch between apoptosis and necrosis has to be taken into account in strategies involving SCFA production by propionibacteria to kill colon cancer cells.

Topics: Acetic Acid; Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle; Cell Fractionation; Cell Line, Tumor; Cell Membrane; Cell Shape; Chromatin; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Fatty Acids, Volatile; Humans; Hydrogen-Ion Concentration; Mitochondria; Necrosis; Probiotics; Propionates; Propionibacterium

Among 13 different cell lines, gossypol (GOS) showed the most potent cytotoxic effect against human colorectal carcinoma cells including HT29, COLO205, COLO320HSR and COLO320DM cells according to an MTT assay. The cytotoxic effect of GOS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS-treated cells. Decreases in Bcl-xL and phosphorylated Bad proteins were found in GOS-treated cells. GOS induction of ROS production was detected by in vitro plasmid digestion, and an increase in the intracellular peroxide level was observed in GOS-treated COLO205 cells by the DCHF-DA assay. Antioxidants including N-acetyl-L-cysteine (NAC), catalase (CAT), tempol (TEM) and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS-induced Reactive oxygen species (ROS) production through blocking the occurrence of apoptosis. GOS induced mitochondrial dysfunction characterized by a loss of the mitochondria membrane potential via DiOC6 staining, and the release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from mitochondria to the cytoplasm was observed. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO205 cells. Furthermore, an intraperitoneal injection of GOS or gossypol acetic acid (GAA) significantly reduced the growth of colorectal carcinoma induced by a subcutaneous injection of COLO205 cells in nude mice. Results of the present study provide the first evidences demonstrating the in vitro and in vivo antitumor effects of GOS via an ROS-dependent mitochondrial apoptosis in colorectal carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-Associated Death Protein; bcl-X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cell Survival; Collagen Type XI; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Ethidium; Flow Cytometry; Gossypol; Humans; Mice; Mice, Nude; Mitochondria; Reactive Oxygen Species; Signal Transduction

Resistance to apoptosis is one of the important determinants of resistance to 5-fluorouracil (5-FU) in colorectal cancer cells. Human Ring-Finger homologous to Inhibitor of apoptosis protein type (hRFI) is a newly discovered gene that has been shown to inhibit death receptor-mediated apoptosis in colorectal cancer cells. However, the molecular mechanism of the inhibition of apoptosis is presently unknown. In order to investigate the molecular function of hRFI in the regulation of 5-FU-induced apoptosis in colorectal cancer cells, HCT116 cells were stably transfected with hRFI or LacZ as a control. hRFI overexpression resulted in cellular resistance to 5-FU through an inhibition of the mitochondrial apoptotic pathway and specific upregulation of Bcl-2 and Bcl-XL. Futhermore, hRFI overexpression resulted in the activation of nuclear factor-kappaB (NF-kappaB). Inhibition of NF-kappaB effectively reversed the resistance to apoptosis as well as the upregulation of Bcl-2 and Bcl-XL in the hRFI transfectant, indicating that the activation of NF-kappaB is the key mechanism for all these findings. Overexpression of hRFI in SW480 and COLO320 colorectal cancer cells similarly resulted in resistance to 5-FU with the activation of NF-kappaB and upregulation of Bcl-2 and Bcl-XL. hRFI might be a novel therapeutic target for gene therapy in colorectal cancer.

Topics: Antimetabolites, Antineoplastic; Apoptosis; bcl-X Protein; Carrier Proteins; Colorectal Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Fluorouracil; HCT116 Cells; Humans; Mitochondria; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Up-Regulation

Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2. Cardiotoxin III-induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub-G1 formation) with an IC(50) of 4 mg/mL at 48 h. 3. Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase-9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl-2, protein in the cells. Moreover, the CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. 4. However, CTX III did not generate the formation of reactive oxygen species and anti-oxidants, including N-acetylcysteine, and catalase could not block CTX III-induced apoptosis in the Colo205 cells. 5. Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial- and caspase-dependent mechanism and alteration of Bax/Bcl-2 ratio in human colorectal Colo205 cancer cells.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Cobra Cardiotoxin Proteins; Colorectal Neoplasms; Cytochromes c; Cytosol; DNA Fragmentation; Enzyme Inhibitors; Flow Cytometry; Humans; Mitochondria; Reactive Oxygen Species; Up-Regulation

No published data are available about the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and the role of PPARgamma in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARgamma is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARgamma, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARgamma mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARgamma down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.

Topics: Apoptosis; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Colorectal Neoplasms; Cyclooxygenase 2; Cytochromes c; Enzyme Activation; Humans; Hypoglycemic Agents; Ligands; Pioglitazone; Poly(ADP-ribose) Polymerases; PPAR gamma; Retinoblastoma Protein; Thiazolidinediones

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L), a member of the tumor necrosis factor (TNF) gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively kill tumor cells. Although microenvironments of solid tumors (hypoxia, nutrient deprivation, and low pH) often affect the effectiveness of chemotherapy, few studies have been reported on the relationship between tumor microenvironments and TRAIL. In this study, we investigated whether low extracellular pH affects TRAIL-induced apoptotic death. When human prostate carcinoma DU145 cells were treated with 200 ng/ml His-tagged TRAIL for 4 h, the survival was approximately 10% at pH 6.3-6.6 and 61.3% at pH 7.4. Similar results were observed in human colorectal carcinoma CX-1 cell line. The TRAIL-mediated activation of caspase, cytochrome c release, and poly (ADP-ribose) polymerase (PARP) cleavage was promoted at low extracellular pH. Immunoprecipitation followed by western blot analysis shows that low extracellular pH enhances the association of truncated Bid with Bax during treatment with TRAIL. Western blot analysis also shows that the low extracellular pH-enhanced TRAIL cytotoxicity does not involve modulation of the levels of TRAIL receptors (DR4, DR5, and DcR2), FLIP, inhibitor of apoptosis (IAP), and Bcl-2. Overexpression of Bcl-2 effectively prevented low extracellular pH-augmented TRAIL cytotoxicity. Taken together, we propose that TRAIL-mediated cytotoxicity is greatly enhanced in low pH environments by promoting caspase activation.

Topics: Adenocarcinoma; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Male; Membrane Glycoproteins; Mitochondria; Models, Biological; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Shikonin is a main constituent of the roots of Lithospermum erythrorhizon that has antimutagenic activity. However, its other biological activities are not well-known. Shikonin displayed a strong inhibitory effect against human colorectal carcinoma COLO 205 cells and human leukemia HL-60 cells, with estimated IC(50) values of 3.12 and 5.5 microM, respectively, but were less effective against human colorectal carcinoma HT-29 cells, with an estimated IC(50) value of 14.8 microM. Induce apoptosis was confirmed in COLO 205 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by loss of mitochondrial membrane potential, reactive oxygen species (ROS) generation, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor (DFF-45) were accompanied by activation of caspase-9 and -3 triggered by shikonin in COLO 205 cells. Here, we found that shikonin-induced apoptotic cell death was accompanied by upregulation of p27, p53, and Bad and down-regulation of Bcl-2 and Bcl-X(L), while shikonin had little effect on the levels of Bax protein. Taken together, we suggested that shikonin-induced apoptosis is triggered by the release of cytochrome c into cytosol, procaspase-9 processing, activation of caspase-3, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by shikonin may provide a pivotal mechanism for its cancer chemopreventive action.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Caspases; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; Membrane Potentials; Mitochondria; Naphthoquinones; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Caspases; Cell Division; Colorectal Neoplasms; Cytochromes c; Cytosol; Drug Resistance, Neoplasm; Enzyme Activation; G2 Phase; Gene Expression Profiling; Humans; Mitochondria; Organoplatinum Compounds; Oxaliplatin; Predictive Value of Tests; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Protein p53