cytochrome-c-t and Adenocarcinoma

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Benzoates; Breast Neoplasms; Cell Cycle Checkpoints; Cytochromes c; Female; Gold; Humans; MCF-7 Cells; Phosphines; Reactive Oxygen Species; Sulfhydryl Compounds

Topics: Adenocarcinoma; Apoptosis; Caspase 3; Caspase 9; Cell Survival; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Flax; Humans; Mitochondria; Peptides, Cyclic; Proto-Oncogene Proteins c-bcl-2

Benzyl isothiocyanate (BITC) is known to inhibit the metastasis of gastric cancer cells but further studies are needed to confirm its chemotherapeutic potential against gastric cancer. In this study, we observed cell shrinkage and morphological changes in one of the gastric adenocarcinoma cell lines, the AGS cells, after BITC treatment. We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, a cell viability assay, and found that BITC decreased AGS cell viability. Reactive oxygen species (ROS) analyses using 2',7'-dichlorofluorescin diacetate (DCFDA) revealed that BITC-induced cell death involved intracellular ROS production, which resulted in mitochondrial dysfunction. Additionally, cell viability was partially restored when BITC-treated AGS cells were preincubated with glutathione (GSH). Western blotting indicated that BITC regulated the expressions of the mitochondria-mediated apoptosis signaling molecules, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cytochrome c (Cyt c). In addition, BITC increased death receptor DR5 expression, and activated the cysteine-aspartic proteases (caspases) cascade. Overall, our results showed that BITC triggers apoptosis in AGS cells via the apoptotic pathways involved in ROS-promoted mitochondrial dysfunction and death receptor activation.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Biological Products; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Isothiocyanates; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Stomach Neoplasms

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Topics: Adenocarcinoma; ADP Ribose Transferases; Animals; Ankyrin Repeat; Bacterial Toxins; Breast Neoplasms; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cytochromes c; Exotoxins; Female; Heme; Humans; Liposomes; Luminescent Proteins; Ovarian Neoplasms; Particle Size; Pseudomonas aeruginosa Exotoxin A; Receptor, ErbB-2; Red Fluorescent Protein; Virulence Factors

Ginger (Zingiber officinale Roscoe), a monocotyledonous herb, is widely used as an herbal medicine owing to the phytoconstituents it possesses. In the current study, the quantity of [6]-gingerol, the major phenolic ketone, in the fresh ginger and dried ginger rhizome was found to be 6.11 µg/mg and 0.407 µg/mg. Furthermore, [6]-gingerol was assessed for its antiapoptotic effects in human gastric adenocarcinoma (AGS) cells evidenced by acridine orange/ethidium bromide staining technique and Annexin-V assay. An increase in reactive oxygen species (ROS) generation led to a decrease in mitochondrial membrane potential (MMP) and subsequent induction of apoptosis. Results disclose that perturbations in MMP are associated with deregulation of Bax/Bcl-2 ratio at protein level, which leads to upregulation of cytochrome-c triggering the caspase cascade. These enduringly suggest that [6]-gingerol can be effectively used for targeting the mitochondrial energy metabolism to manage gastric cancer cells.

Topics: Acridine Orange; Adenocarcinoma; Annexin A5; Apoptosis; bcl-2-Associated X Protein; Caspases; Catechols; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Cytochromes c; Ethidium; Fatty Alcohols; Humans; Membrane Potential, Mitochondrial; Plant Extracts; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Stomach Neoplasms; Up-Regulation; Zingiber officinale

CBP (CREB-binding protein) is a transcriptional co-activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase-dependent signaling pathways. Co-immunoprecipitation and immunofluorescence analyses revealed the co-localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways.

Topics: Acetylation; Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cleavage And Polyadenylation Specificity Factor; CREB-Binding Protein; Cytochromes c; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Histone Acetyltransferases; Humans; Lung Neoplasms; MAP Kinase Signaling System; Prognosis; RNA, Small Interfering; Telomerase

Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human lung adenocarcinoma A549 cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an upregulation of pro-apoptotic Bax and Bak genes and downregulation of anti-apoptotic Bcl-2 and Bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase-3 and -9, and morphological characteristics of apoptosis, including plasma membrane blebbing and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartment (VAC) and suppressions of nuclear transcription factors nuclear factor-κB (NF-κB) and both topoisomerase I and II activities. Further study reveals that the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against A549 cells is accompanied by downregulations of NF-κB binding activity and proliferative control involving apoptosis and both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

Topics: Acrolein; Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cinnamomum zeylanicum; Cytochromes c; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Humans; Lung Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; NF-kappa B; Topoisomerase Inhibitors; Xenograft Model Antitumor Assays

Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Caco-2 Cells; Caspase 3; Caspase 8; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Drug Screening Assays, Antitumor; Flow Cytometry; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Mutation; Receptors, Death Domain; Treatment Outcome; Triterpenes; Tumor Suppressor Protein p53

Barrett's esophagus follows the classic step-wise progression of metaplasia-dysplasia-adenocarcinoma. While Barrett's esophagus is a leading known risk factor for esophageal adenocarcinoma, the pathogenesis of this disease sequence is poorly understood. Mitochondria are highly susceptible to mutations due to high levels of reactive oxygen species (ROS) coupled with low levels of DNA repair. The timing and levels of mitochondria instability and dysfunction across the Barrett's disease progression is under studied.. Using an in-vitro model representing the Barrett's esophagus disease sequence of normal squamous epithelium (HET1A), metaplasia (QH), dysplasia (Go), and esophageal adenocarcinoma (OE33), random mitochondrial mutations, deletions and surrogate markers of mitochondrial function were assessed. In-vivo and ex-vivo tissues were also assessed for instability profiles.. Barrett's metaplastic cells demonstrated increased levels of ROS (p < 0.005) and increased levels of random mitochondrial mutations (p < 0.05) compared with all other stages of the Barrett's disease sequence in-vitro. Using patient in-vivo samples, Barrett's metaplasia tissue demonstrated significantly increased levels of random mitochondrial deletions (p = 0.043) compared with esophageal adenocarcinoma tissue, along with increased expression of cytoglobin (CYGB) (p < 0.05), a gene linked to oxidative stress, compared with all other points across the disease sequence. Using ex-vivo Barrett's metaplastic and matched normal patient tissue explants, higher levels of cytochrome c (p = 0.003), SMAC/Diablo (p = 0.008) and four inflammatory cytokines (all p values <0.05) were secreted from Barrett's metaplastic tissue compared with matched normal squamous epithelium.. We have demonstrated that increased mitochondrial instability and markers of cellular and mitochondrial stress are early events in the Barrett's disease sequence.

Topics: Adenocarcinoma; Barrett Esophagus; Cell Line; Cell Line, Tumor; Cytochromes c; Cytoglobin; Cytokines; Disease Progression; Enzyme-Linked Immunosorbent Assay; Esophagus; Gene Expression Regulation, Neoplastic; Globins; Humans; Inflammation Mediators; Metaplasia; Mitochondria; Mitochondrial Proteins; Mutation; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors

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

The aim of this study was to construct an expression vector carrying the hypoxia/radiation dual‑sensitive chimeric hypoxia response element (HRE)/early growth response 1 (Egr‑1) promoter in order to overexpress the therapeutic second mitochondria‑derived activator of caspases (Smac). Using this expression vector, the present study aimed to explore the molecular mechanism underlying radiotherapy‑induced A549 human lung adenocarcinoma cell death and apoptosis under hypoxia. The plasmids, pcDNA3.1‑Egr1‑Smac (pE‑Smac) and pcDNA3.1‑HRE/Egr-1‑Smac (pH/E‑Smac), were constructed and transfected into A549 human lung adenocarcinoma cells using the liposome method. CoCl2 was used to chemically simulate hypoxia, followed by the administration of 2 Gy X‑ray irradiation. An MTT assay was performed to detect cell proliferation and an Annexin V‑fluorescein isothiocyanate apoptosis detection kit was used to detect apoptosis. Quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression, respectively. Infection with the pE‑Smac and pH/E‑Smac plasmids in combination with radiation and/or hypoxia was observed to enhance the expression of Smac. Furthermore, Smac overexpression was found to enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis. The cytochrome c/caspase‑9/caspase‑3 pathway was identified to be involved in this regulation of apoptosis. Plasmid infection in combination with X‑ray irradiation was found to markedly induce cell death under hypoxia. In conclusion, the hypoxia/radiation dual‑sensitive chimeric HRE/Egr‑1 promoter was observed to enhance the expression of the therapeutic Smac, as well as enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis under hypoxia. This apoptosis was found to involve the mitochondrial pathway.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Early Growth Response Protein 1; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mitochondrial Proteins; Promoter Regions, Genetic; Radiation, Ionizing; Response Elements; RNA, Messenger; Sequence Analysis, DNA

Curcumin, a polyphenol compound derived from the rhizome of the plant Curcuma longa L. has been verified as an anticancer compound against several types of cancer. However, understanding of the molecular mechanisms by which it induces apoptosis is limited. In this study, the anticancer efficacy of curcumin was investigated in human gastric adenocarcinoma SGC-7901 cells. The results demonstrated that curcumin induced morphological changes and decreased cell viability. Apoptosis triggered by curcumin was visualized using Annexin V-FITC/7- AAD staining. Curcumin-induced apoptosis of SGC-7901 cells was associated with the dissipation of mitochondrial membrane potential (MMP) and the release of cytochrome c into the cytosol. Furthermore, the down-regulation of Bcl-2 and up-regulation of Bax that led to the cleavage of caspase-3 and increased cleaved PARP was observed in SGC-7901 cells treated with curcumin. Therefore, curcumin-induced apoptosis of SGC-7901 cells might be mediated through the mitochondria pathway, which gives the rationale for in vivo studies on the utilization of curcumin as a potential cancer therapeutic compound.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Curcuma; Curcumin; Cytochromes c; Down-Regulation; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Permeability; Plant Preparations; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Stomach Neoplasms; Up-Regulation

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.

Topics: Adenocarcinoma; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Docosahexaenoic Acids; Drug Synergism; eIF-2 Kinase; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Mitochondria; Signal Transduction; Sphingolipids; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; X-Linked Inhibitor of Apoptosis Protein

Phloroglucinol is a polyphenolic compound that is used to treat and prevent several human diseases, as it exerts beneficial biological activities, including anti-oxidant, anti‑inflammatory and anticancer properties. The aim of the present study was to investigate the effects of phloroglucinol on apoptotic signaling pathways in HT-29 colon cancer cells. The results indicated that phloroglucinol suppressed cell viability and induced apoptosis in HT-29 cells in a concentration-dependent manner. Phloroglucinol treatment of HT-29 cells resulted in characteristic apoptosis-related changes: altered Bcl-2 family proteins, cytochrome c release, and activation of caspase-3 and caspase-8. This study also showed that proteins involved in apoptosis were stimulated by treatment with phloroglucinol. These findings demonstrated that phloroglucinol exerts anticancer activity in HT-29 colon cancer cells through induction of apoptosis.

Topics: Adenocarcinoma; Animals; Apoptosis; Caspase 3; Caspase 8; Cell Line; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; HT29 Cells; Humans; Intestine, Small; Phloroglucinol; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction

Interferon-gamma (IFN-γ) has been used to treat various malignant tumors. However, the molecular mechanisms underlying the direct anti-proliferative activity of IFN-γ are poorly understood. In the present study, we examined the in vitro antitumor activity of IFN-γ on two human non-small-cell lung carcinoma (NSCLC) cell lines, H322M and H226. Our findings indicated that IFN-γ treatment caused a time-dependent reduction in cell viability and induced apoptosis through a FADD-mediated caspase-8/tBid/mitochondria-dependent pathway in both cell lines. Notably, we also postulated that IFN-γ increased indoleamine 2,3-dioxygenase (IDO) expression and enzymatic activity in H322M and H226 cells. In addition, inhibition of IDO activity by the IDO inhibitor 1-MT or tryptophan significantly reduced IFN-γ-induced apoptosis and death receptor 5 (DR5) expression, which suggests that IDO enzymatic activity plays an important role in the anti-NSCLC cancer effect of IFN-γ. These results provide new mechanistic insights into interferon-γ antitumor activity and further support IFN-γ as a potential therapeutic adjuvant for the treatment of NCSLC.

Topics: Adenocarcinoma; Antiviral Agents; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Cytochromes c; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-gamma; Lung Neoplasms; Membrane Potential, Mitochondrial; Receptors, TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched.. The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded.. All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident.. This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Survival; Central Nervous System Neoplasms; Chromans; Cytochromes c; DNA Fragmentation; Glioblastoma; Humans; Isomerism; Lung Neoplasms; Mitochondria; Tocotrienols; Vitamin E

To evaluate the effects of bufalin in A549 human lung adenocarcinoma epithelial cells in vitro and assess the underlying mechanisms.. Human A549 non-small cell lung cancer (NSCLC) cells were treated with various concentrations of bufalin. Cell proliferation was measured by CCK-8 assay, apoptotic cell percentage was calculated by flow cytometry and morphological change was observed by inverted phase contrast microscopy/transmission electron microscopy. In addition, the membrane potential of mitochondria was detected by JC-1 fluorescence microscopy assay, and the related protein expression of cytochrome C and caspase-3 was analyzed by Western blotting.. Bufalin could inhibit the proliferation of A549 cells via induction of apoptosis, with the evidence of characteristic morphological changes in the nucleus and mitochondria. Furthermore, bufalin decreased the mitochondrial membrane potential with up-regulation of cytochrome C in the cytosol, and activation of caspase-3.. Bufalin inhibits the proliferation of A549 cells and triggers mitochondria-dependent apoptosis, pointing to therapeutic application for NSCLC.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Bufanolides; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Up-Regulation

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

2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1-25 μM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 μM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.

Topics: 2-Methoxyestradiol; Adenocarcinoma; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Shape; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Activation; Estradiol; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Microtubules; Protein Multimerization; Sulfonamides; Tubulin; Tubulin Modulators

Geraniin has previously been reported to possess extensive biological activity. In this study, we reported that geraniin is an inhibitor of tumor activity in vitro and in vivo. Geraniin suppressed the proliferation of A549 cells in a dose- and time-dependent manner. Geraniin arrested the cell cycle in the S phase and induced a significant accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. Western blot analysis showed that geraniin inhibited Bcl-2 expression and induced Bax expression to disintegrate the outer mitochondrial membrane and cause cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascades. Additionally, geraniin resulted in tumor growth inhibition in A549 xenografts. Our results indicate cytotoxic activity of geraniin towards cancer cells in vitro and in vivo.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Death; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Glucosides; Humans; Hydrolyzable Tannins; Lung Neoplasms; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; S Phase

Tautomycetin (TMC), originally isolated from Streptomyces griseochromogenes, has been suggested as a potential drug retaining specificity toward colorectal cancer. However, we found that TMC exhibited inhibitory effects on cell proliferation of many cancer cell lines including adriamycin-resistant human breast adenocarcinoma. We investigated its anti-tumor activity and mechanisms in human breast cancer cells for the first time. In this study, we showed that TMC effectively inhibited breast cancer cell proliferation, migration, and invasion. TMC also induced apoptosis in MCF-7 cells. This apoptotic response was in part mediated by Bcl-2 cleavage, leading to the release of cytochrome c, which facilitates binding of Apaf-1 to caspase-9 in its presence and subsequent activation of caspase-7 in apoptosis induction signaling pathways. Furthermore, we identified that TMC induced apoptosis by suppressing Akt signaling pathway activation, which is independent of protein phosphatase PP1 inhibition. The levels of downstream targets of Akt, including phospho-forkhead transcription factor and Bad, were also reduced after TMC treatment. Overall, our results indicate that TMC could be used as a potential drug candidate for breast cancer therapy. More importantly, our study provides new mechanisms for the anticancer effects of TMC.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Breast Neoplasms; Caspase 7; Caspase 9; Cell Movement; Cell Proliferation; Cytochromes c; Female; Furans; Humans; Lipids; MCF-7 Cells; Oncogene Protein v-akt; Proto-Oncogene Proteins c-bcl-2; Receptors, Neuropeptide Y; Signal Transduction; Tumor Cells, Cultured

Anacardic acid (AA) is a constituent of the cashew nut shell and is known as an inhibitor of nuclear factor-κB (NF-κB). We investigated the cytotoxicity of AA on cancer cells and more experiments to reveal the cell death mechanism focused on A549 lung adenocarcinoma cells for our interest in lung cancer. To examine the molecular mechanism of cell death in AA treated A549 cells, we performed experiments such as transmission electron microscopy (TEM), western blot analysis, fluorescence-activated cell sorting (FACS), genomic DNA extraction and staining with 4',6-diamidino-2-phenylindole (DAPI). For the first time we revealed that AA induces caspase-independent apoptosis with no inhibition of cytotoxicity by pan-caspase inhibitor, Z-VAD-fmk, in A549 cells. Our results showed the possibility of mitochondrial-mediated apoptosis through the activation of apoptosis-inducing factor (AIF) and an intrinsic pathway executioner such as cytochrome c. This study will be helpful in revealing the cell death mechanisms and in developing potential drugs for lung cancer using AA.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Amino Acid Chloromethyl Ketones; Anacardic Acids; Apoptosis; Apoptosis Inducing Factor; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; HEK293 Cells; Hep G2 Cells; Humans; Lung Neoplasms; Mitochondria; NF-kappa B; Signal Transduction

The aim of this study was to analyze the immunoexpression of (Smac) DIABLO, AIF, cytochrome c, Ki-67 and cleaved caspase-3 in gastric cancer. A tissue microarray (TMA) paraffin block was constructed using gastric adenocarcinoma tissue and adjacent normal adjacent mucosa from 87 patients who had not previously undergone radiotherapy or chemotherapy. Immunohistochemistry was used to evaluate the protein levels. Samples were positive for (Smac) DIABLO in 37 (45.6%) and 37 (46.8%), for AIF in 31 (36.9%) and 36 (45.6%), for cytochrome c in 60 (68.9%) and 44 (54.4%), for Ki-67 in 63 (72.4%) and 52 (61.9%) and for cleaved caspase-3 in 21 (24.1%) and 3 (3.4%) cases of tumor and adjacent normal tissues, respectively. Our results suggest that increased expression of Ki-67 and cleaved caspase-3 could contribute to carcinogenesis. The expression of these proteins indicates an attempt of cells to maintain tissue homeostasis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Biomarkers, Tumor; Cytochromes c; Female; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Male; Middle Aged; Mitochondrial Proteins; Stomach Neoplasms; Tissue Array Analysis

Strobilanthes crispus has been traditionally used as antidiabetic, anticancer, diuretic, antilytic and laxative agent. However, cytotoxicity and antiproliferative effect of S. crispus is still unclear.. Strobilanthes cripus was able to reduce cell viability and proliferation in MTT and BrdU assays. Both cell cycle progression and Tunel assay suggested that IC50 of S. crispus ethanol extract induced sub-G1 cell cycle phase, and DNA fragmentation. On the other hand, translocation of mitochondria cytochrome c release, induction of caspase 3/7 and p53 while suppress XIAP on treated MCF-7 cell were also observed in this study.. Our findings suggest that S. crispus ethanol extract induced apoptosis and DNA fragmentation on hormone dependent breast cancer cell line MCF-7 via mitochondria dependent p53 apoptosis pathway.

Topics: Acanthaceae; Adenocarcinoma; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Female; G1 Phase; Hormones; Humans; Inhibitory Concentration 50; Mitochondria; Phytotherapy; Plant Extracts; Tumor Suppressor Protein p53; X-Linked Inhibitor of Apoptosis Protein

The human WWOX gene, known as WW domain-containing oxidoreductase, is located on 16q23.3-24.1, a chromosome region that spans the common fragile site, FRA16D. Abnormal transcripts or even loss of expression are frequently found in a number of cancer cell types, including breast, ovarian, prostate and lung cancer cells. It has therefore been proposed that the WWOX gene encodes a candidate tumor suppressor, possibly a pro-apoptotic protein. However, the mechanism behind this is not entirely clear. In the present study, we examined the pro-apoptotic action of WWOX using transient expression in A549 cells. We observed that the ectopic expression of WWOX caused apoptosis in A549 cells. We further observed procaspase-3 and procaspase-9 activation and the release of cytochrome C from the mitochondria in A549 cells transfected with pcDNA3.0-WWOX. These data indicate that WWOX induces apoptosis in A549 cells via the mitochondrial pathway.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; Humans; Lung Neoplasms; Mitochondria; Oxidoreductases; Signal Transduction; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase

Baohuoside I (also known as Icariside II) is a flavonoid isolated from Epimedium koreanum Nakai. Although Baohuoside I exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human lung cancer cells are poorly understood. Therefore, in the present study, we investigated the usefulness of Baohuoside I as a potential apoptosis-inducing cytotoxic agent using human adenocarcinoma alveolar basal epithelial A549 cells as in vitro model. The apoptosis induced by Baohuoside I in A549 cells was confirmed by annexin V/propidium iodide double staining, cell cycle analysis and dUTP nick end labeling. Further research revealed that Baohuoside I accelerated apoptosis through the mitochondrial apoptotic pathway, involving the increment of BAX/Bcl-2 ratio, dissipation of mitochondrial membrane potential, transposition of cytochrome c, caspase 3 and caspase 9 activation, degradation of poly (ADP-ribose) polymerase and the over-production of reactive oxygen species (ROS). A pan-caspase inhibitor, Z-VAD-FMK, only partially prevented apoptosis induced by Baohuoside I, while NAC, a scavenger of ROS, diminished its effect more potently. In addition, the apoptotic effect of Baohuoside I was dependent on the activation of ROS downstream effectors, JNK and p38(MAPK), which could be almost abrogated by using inhibitors SB203580 (an inhibitor of p38(MAPK)) and SP600125 (an inhibitor of JNK). These findings suggested that Baohuoside I might exert its cytotoxic effect via the ROS/MAPK pathway.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Flavonoids; Humans; Lung Neoplasms; MAP Kinase Kinase 4; Membrane Potential, Mitochondrial; Mitochondria; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species

Colorectal cancer is one of the leading causes of death in the world. Plant-derived products have proven to be valuable sources for discovery and development of unique anticancer drugs. In this study, the inhibitory effects of ethanolic extract of Melia toosendan fruit (EMTF), a traditional medicine in the Chinese Pharmacopeia were evaluated in vitro and in vivo against colon cancer. Human colon cancer cells SW480 and murine colorectal adenocarcinoma cells CT26 were used to investigate cell proliferation. The results showed that EMTF inhibited cell proliferation of SW480 and CT26 by promoting apoptosis as indicated by nuclear chromatin condensation and DNA fragmentation. Through increasing mitochondrial membrane permeability and cytochrome c release from mitochondria, EMTF induced caspase-9 activity which further activated caspase-3 and poly(ADP-ribose) polymerase cleavage, leading the tumor cells to apoptosis. The in vivo results confirmed reduction of tumor volume and apoptotic effects and the side effects were not induced by EMTF. Therefore, EMTF may be an effective chemotherapeutic agent for colon cancer treatment.

Topics: Adenocarcinoma; Animals; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Female; Fruit; Humans; Melia; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mitochondria; Plant Extracts; Poly(ADP-ribose) Polymerases; Xenograft Model Antitumor Assays

The present study was designed to investigate the abnormalities in the expression of apoptosis-associated proteins that lead to the progression of breast cancer. Sixty breast cancer patients histologically categorized as grade I, II and III, and as pre- and post-menopausal were chosen for the study. We analyzed the expression of the anti-apoptotic and pro-apoptotic Bcl-2 family proteins as well as cytochrome C, Apaf-1 and caspases in tumour and adjacent tissues by immunohistochemical and Western blot analyses. The breast tumours analyzed in the present study were characterized by increased expression of Bcl-2, Bcl-xL and Mcl-1, associated with downregulation in the expression of Bax, cytosolic cytochrome C, Apaf-1 and caspases. The magnitude of the changes was however more pronounced in premenopausal patients and in grade III tumours. The results of the present study confirm that differential expression patterns of Bcl-2 family proteins and caspases are involved in evasion of apoptosis and in the progression of breast cancer.

Topics: Adenocarcinoma; Adult; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; bcl-X Protein; Breast; Breast Neoplasms; Caspase 3; Caspase 8; Caspase 9; Cytochromes c; Disease Progression; Female; Humans; Middle Aged; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Postmenopause; Premenopause; Proto-Oncogene Proteins c-bcl-2

Deregulation of apoptosis will influence the balance of cell proliferation and cell death, resulting in various fatal diseases that can include cancer. In prior research reports related to cancer therapy, phytohemagglutinin, a lectin extracted from red kidney beans, demonstrated the ability to inhibit the growth of human cancer cells. However, one of its isoforms, erythroagglutinating, has yet to be evaluated on its anticancer effects.. PHA-E was used to induce apoptosis of A-549 lung cancer cells and the possible signal transduction pathway was elucidated, as measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, G6PD release assay, flow cytometry, and Western blot analysis.. PHA-E treatment caused a dose-dependent increase of cell growth inhibition and cytotoxicity on A-549 cells. In annexin V/propidium iodide [i.e., PI] and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)/PI assay, we found that the rate of apoptotic cells was raised as the concentration of PHA-E increased. Treatment of A-549 cells with PHA-E resulted in enhancing the release of cytochrome c, which thus activated an increase in caspase 9 and caspase 3, the upregulation of Bax and Bad, the downregulation of Bcl-2 and phosphorylated Bad, and finally the inhibition of the epidermal growth factor receptor and its downstream signal pathway PI3K/Akt and MEK/ERK.. PHA-E can induce growth inhibition and cytotoxicity of lung cancer cells, which is mediated through an activation of the mitochondria apoptosis pathway. These results suggest that PHA-E can be developed into a new therapeutic treatment that can be applied as an effective anti-lung cancer drug in the near future.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cytochromes c; Glucosephosphate Dehydrogenase; Humans; Lung Neoplasms; Mitochondria; Phytohemagglutinins; Signal Transduction

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol, which is present in cruciferous vegetables and has been reported to possess anti-carcinogenic properties. In the present study, we examined whether DIM inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen and proliferating cell nuclear antigen, and increased the number of terminal dUTP nick-end labeling-positive cells in the dorsolateral lobes of the prostate. Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate cancer cells were cultured with various concentrations of DIM. DIM induced a substantial reduction in the numbers of viable cells and induced apoptosis in LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly (ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability and the translocation of cytochrome c and Smac/Diablo from the mitochondria. Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid. These results indicate that DIM inhibits prostate carcinogenesis via induction of apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

Topics: Adenocarcinoma; Animals; Anticarcinogenic Agents; Antigens, Polyomavirus Transforming; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Carrier Proteins; Caspases; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin A; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Disease Models, Animal; Humans; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondrial Membranes; Mitochondrial Proteins; Oligopeptides; Peptides; Permeability; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Transport

The current study was an attempt to elucidate the mechanism of human colon cancer cell proliferation inhibition by limonin and limonin glucoside (LG) isolated from seeds of Citrus reticulata. The structures of purified compounds were confirmed by NMR and quantified using HPLC. These compounds of more than 95% purity were subjected to proliferation inhibition assay using human colon adenocarcinoma (SW480) cells. The IC50 value of 54.74 and 37.39 μM was observed for limonin and LG, respectively at 72 h. Following confirmation of proliferation inhibition, pattern of DNA fragmentation and activation of caspase-3 of the cells treated with limonoids suggest involvement of apoptosis. Furthermore, reduction in the transcription ratio of bcl2/bax and induction of cytochrome c release from mitochondria to cytosol with treatment of limonoids confirm the activation of intrinsic apoptosis pathway. The activity of Bax and Bcl2 was confirmed through analysis of mitochondrial membrane potential and intracellular calcium in the cells treated with limonin and LG; the net content of caspase-8 was not affected by limonoids. Results of the current study provide compelling evidence on the induction of mitochondria mediated intrinsic apoptosis by both limonin and LG in cultured SW480 cells for the first time.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Citrus; Colonic Neoplasms; Cytochromes c; Gene Expression Regulation, Neoplastic; Glucosides; Humans; Limonins; Mitochondria; Plant Extracts

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Ascorbic Acid; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cathepsins; Cell Line, Tumor; Cytochromes c; Fluorescent Antibody Technique; Humans; Lung Neoplasms; Lysosomes; Microscopy, Confocal; Mitochondria; Photosensitizing Agents; Porphyrins; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering

Aerosolized cyclosporine A (CsA) increases the local concentration of CsA in lung tissue and has proven to be an effective therapy for refractory rejection in lung transplant patients. However, the safety of high concentrations of CsA on tumour progression remains controversial.. Human lung adenocarcinoma A549 cells were cultured with or without 1-3 μg/ml of CsA. The percentage of apoptotic cells was evaluated by Annexin V staining. The expressions of caspase-3, -9, -8 and cytochrome c were determined by Western blotting.. CsA therapy suppressed the growth of human lung cancer cells and increased the percentage of apoptotic cells compared with control cells. Western blot analysis revealed that CsA increased the levels of cytosolic cytochrome c and cleaved caspase-3 and -9, but not of cleaved caspase-8 in the lung cancer cells, suggesting that CsA-induced apoptosis is associated with the activation of caspase-3 and -9.. Our findings indicate that a high concentration of CsA has cytocidal effects through the caspase-3- and -9-dependent apoptotic pathway. This result shows that local administration of CsA does not increase the risk of secondary lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cyclosporine; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Isoenzymes; Lung Neoplasms; Mitochondria

Pancreatic adenocarcinoma is one of the most common malignancies worldwide. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine can induce activation of Akt and nuclear factor-κB (NF-κB), which is associated with its chemoresistance. It has been reported that gemcitabine combination therapies result in improved survival outcomes in pancreatic cancer. Therefore, agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Emodin is an active component of Chinese medicinal herbs and can inhibit the activation of Akt and NF-κB. In this study, we investigated whether emodin could enhance the anticancer effect of gemcitabine on pancreatic cancer in vivo. We demonstrated that treatment of gemcitabine combined with emodin efficiently suppressed tumor growth in mice inoculated with pancreatic tumor cells. This treatment paradigm promoted apoptotic cell death and mitochondrial fragmentation. Furthermore, it reduced phosphorylated-Akt (p-Akt) level, NF-κB activation and Bcl-2/Bax ratio, increased caspase-9 and -3 activation, Cytochrome C (CytC) release occurred in combination therapy. Collectively, emodin enhanced the activity of gemcitabine in tumor growth suppression via inhibition of Akt and NF-κB activation, thus promoting the mitochondrial-dependent apoptotic pathway. Therefore, our findings may provide new insights into understanding the pharmacological regulation of emodin on gemcitabine-mediated proapoptosis in pancreatic cancer and may aid in the design of new therapeutic strategies for the intervention of human pancreatic cancers.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Body Weight; Caspase 3; Cell Line, Tumor; Cytochromes c; Emodin; Female; Humans; Mice; NF-kappa B; Pancreatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rheum; Tumor Burden; Xenograft Model Antitumor Assays

Higd-1a (hypoxia induced gene domain family-1a) is a mitochondrial inner membrane protein with a conformation of N-terminal outside-C-terminal outside and loop inside. There are four Higd genes, Higd-1a, -1b, -1c and -2a, in the mouse. Higd-1a and -2a are expressed primarily in the brain, heart, kidney and leukocytes. HIF (hypoxia-inducible factor) overexpression induced the endogenous expression and promoter activity of Higd-1a. Mutation of the HRE (hypoxia-response element) site at -32bp in the Higd-1a promoter reduced the promoter activity, suggesting that transcription of Higd-1a is regulated by binding of the transcription factor HIF to the HRE. Higd-1a promoted cell survival under hypoxia. RAW264.7 cells stably transfected with Higd-1a underwent less apoptosis than control cells in a hypoxic condition, and hypoxia-induced apoptosis was strongly enhanced when endogenous Higd-1a was silenced by siRNA. The survival effect of Higd-1a was completely abolished by deletion of the 26 N-terminal amino acids, and we showed that Higd-1a increased survival by inhibiting cytochrome C release and reducing the activities of caspases. However, expression of Bcl-2, Bax, Bad, and BNIP3 and translocation of AIF were unaffected under the same conditions. Higd-2a also enhanced cell survival under hypoxia. Cells transfected with Higd-2a underwent less apoptosis than control cells in hypoxic conditions, and hypoxia-induced apoptosis increased when endogenous Higd-2a was depleted. Together these observations indicate that Higd-1a is induced by hypoxia in a HIF-dependent manner and its anti-apoptotic effect results from inhibiting cytochrome C release and reducing caspase activities.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Apoptosis; Blotting, Western; Caspases; Cell Hypoxia; Cells, Cultured; Cytochromes c; DNA, Mitochondrial; Enzyme Activation; Flow Cytometry; HeLa Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Macrophages; Membrane Proteins; Mice; Mitochondria; Mitochondrial Proteins; Molecular Sequence Data; Pancreatic Neoplasms; Real-Time Polymerase Chain Reaction; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid

In the present study, the anticancer activity of chamaejasmine towards A549 human lung adenocarcinoma cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of chamaejasmine, cell cycle distribution, ROS generation, mitochondrial membrane potential (Δψ(m)) disruption, and expression of cytochrome c, Bax, Bcl-2, caspase-3, caspase-9 and PARP were measured in A549 cells. Chamaejasmine inhibited the growth of A549 cells in a time and dose-dependent manner. The IC₅₀ value was 7.72 µM after 72 h treatment. Chamaejasmine arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that chamaejasmine inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of chamaejasmine towards A549 in vitro.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Biflavonoids; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cytochromes c; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Plant Preparations; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Radix Stephaniae tetrandrae, which contains tetrandrine (Tet) and fangchinoline, is traditionally used as an analgesic, antirheumatic, and antihypertensive drug in China. In this study, we investigated its effect on breast cancer cell proliferation and its potential mechanism of action in vitro. Treatment of cells with fangchinoline significantly inhibited MDA-MB-231 cell proliferation in a concentration- and time-dependent manner. To define the mechanism underlying the antiproliferative effects of fangchinoline, we studied its effects on critical molecular events known to regulate the apoptotic machinery. Specifically, we addressed the potential of fangchinoline to induce apoptosis of breast cancer cells. Fangchinoline induced internucleosomal DNA fragmentation, chromatin condensation, activation of caspases-3, -8, and -9, and cleavage of poly(ADP ribose) polymerase, as well as enhanced mitochondrial cytochrome c release. Furthermore, fangchinoline increased the expression of the proapoptotic protein B cell lymphoma-2 associated X (Bax) and decreased the expression of the antiapoptotic protein B cell lymphoma-2 (Bcl-2). In addition, the proliferation-inhibitory effect of fangchinoline was associated with decreased levels of phosphorylated Akt. Our results indicate that fangchinoline can inhibit breast cancer cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and decreasing phosphorylated Akt. Thus fangchinoline may be a novel agent that can potentially be developed clinically to target human malignancies.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Benzylisoquinolines; Breast Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Drugs, Chinese Herbal; Female; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins c-bcl-2; Stephania tetrandra

Rottlerin is a polyphenolic compound derived from Mallotus philipinensis. In the present study, we show that rottlerin decreased tumor size and stimulated apoptosis in an orthotopic model of pancreatic cancer with no effect on normal tissues in vivo. Rottlerin also induced apoptosis in pancreatic cancer (PaCa) cell lines by interacting with mitochondria and stimulating cytochrome c release. Immunoprecipitation results indicated that rottlerin disrupts complexes of prosurvival Bcl-xL with Bim and Puma. Furthermore, siRNA knockdown showed that Bim and Puma are necessary for rottlerin to stimulate apoptosis. We also showed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2' stimulate apoptosis through a common mechanism. They both directly interact with mitochondria, causing increased cytochrome c release and mitochondrial depolarization, and both decrease sequestration of BH3-only proteins by Bcl-xL. However, the effects of rottlerin and BH3I-2' on the complex formation between Bcl-xL and BH3-only proteins are different. BH3I-2' disrupts complexes of Bcl-xL with Bad but not with Bim or Puma, whereas rottlerin had no effect on the Bcl-xL interaction with Bad. Also BH3I-2', but not rottlerin, required Bad to stimulate apoptosis. In conclusion, our results demonstrate that rottlerin has a potent proapoptotic and antitumor activity in pancreatic cancer, which is mediated by disrupting the interaction between prosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thus rottlerin represents a promising novel agent for pancreatic cancer treatment.

Topics: Acetophenones; Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-Associated Death Protein; bcl-X Protein; Benzamides; Benzopyrans; Cell Line, Tumor; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Humans; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasm Transplantation; Pancreatic Neoplasms; Protein Kinase C-delta; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

NF-kappaB activation is known to reduce the efficiency of chemotherapy in cancer treatment. Ursolic acid, a minimally toxic compound, has shown the capability to inhibit NF-kappaB activation in living cells. Here, for the first time, we investigated the effects and mechanisms of NF-kappaB inhibition by ursolic acid on chemotherapy treatment (Taxol or cisplatin) of cancer. ASTC-a-1 (human lung adenocarcinoma), Hela (human cervical cancer) cells, primary normal mouse cells of lung and liver and mouse in vivo model were used. Activity of signal factors (NF-kappaB, Akt, Fas/FasL, BID, Bcl-2, cytochrome c and caspase-8, 3) was used to analyze the mechanisms of ursolic acid-chemo treatment. Ursolic acid-mediated suppression of NF-kappaB drastically reduced the required dosage of the chemotherapeutic agents to achieve identical biological endpoints and enhanced the chemotherapeutic agent-induced cancer cells apoptosis. Chemosensitization by ursolic acid in cancer cells was dependent on the amplified activation of intrinsic pathway (caspase-8-BID-mitochondria-cytochrome c-caspase-3) by augmentation of BID cleavage and activation of Fas/FasL-caspase-8 pathway. Prolonged treatment with relatively low doses of ursolic acid also sensitized cancer cells to the chemotherapeutic agents through suppression of NF-kappaB. Chemosensitization by ursolic acid was observed only in cancer cells, but not in primary normal cells. The inhibitive effect of ursolic acid on NF-kappaB was reversible, and the reversal was not accompanied by a loss in cells viability. By supplementing chemotherapy with minimally toxic ursolic acid, it is possible to improve the efficacy of cancer treatment by significantly reducing the necessary drug dose without sacrificing the treatment results.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Caspase 8; Cell Proliferation; Cells, Cultured; Cisplatin; Cytochromes c; Drug Synergism; Fas Ligand Protein; fas Receptor; Female; Flow Cytometry; Humans; Liver; Luciferases; Lung; Lung Neoplasms; Mice; NF-kappa B; Protein Transport; RNA, Small Interfering; Signal Transduction; Triterpenes; Ursolic Acid; Uterine Cervical Neoplasms

We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3).. LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined.. LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect.. Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Docetaxel; Down-Regulation; Humans; Male; Neoplasm Metastasis; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Androgen; RNA, Small Interfering; Signal Transduction; Taxoids

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. This study was performed to elucidate whether EGFR and PI3K signaling pathways are involved in NFD-induced apoptosis of human lung adenocarcinoma A549 cells.. The effect of NFD on cell viability and apoptosis was measured by the MTT assay and flow cytometry. The phosphorylation levels of EGFR and its regulatory molecules by NFD treatment were studied by immunoblots.. Immunoblot showed that NFD inhibited EGFR phosphorylation and the activation of PI3K/Akt, downstream molecules of EGFR pathway, in A549 cells. The levels of downstream targets of Akt, including phospho-glycogen synthase kinase-3beta (p-GSK-3beta), GSK-3beta, forkhead transcription factor (FKHR), and cyclin D1, were also reduced after NFD treatment. Moreover, inactivation of nuclear factor-kappaB (NFkappaB), modulation of IkappaKalpha/beta and IkappaBalpha, up-regulation of Bad and Bax, and down-regulation of anti-apoptotic proteins including phospho-Bad, Bcl-2, survivin, and XIAP were also found in NFD-treated cells. In addition, NFD treatment disrupted mitochondrial membrane potential (DeltaPsim) and resulted in release of mitochondrial cytochrome c and activation of both caspases-9 and caspase-3.. These findings indicate that EGFR and PI3K/Akt signaling pathways play important roles in NFD-induced apoptosis of A549 cells.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; ErbB Receptors; Flow Cytometry; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Naphthoquinones; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

In the present study, the antiproliferative mechanism of action of 1 microM 2-methoxyestradiol (2ME) was investigated in the MCF-7 cell line. Measurement of intracellular cyclin B and cytochrome c protein levels, reactive oxygen species formation, cell cycle progression and apoptosis induction were conducted by means of flow cytometry. Morphological changes were evaluated using transmission electron microscopy and fluorescent microscopy by employing Hoechst 33342 and acridine orange. Gene expression changes were conducted by means of microarrays. 2ME-treated cells demonstrated an increase in cyclin B protein levels, hydrogen peroxide formation, intracellular levels of cytochrome c, as well as an increase in early and late stages of apoptosis. In addition, morphological data revealed the presence of autophagic processes. Fluorescent microscopy showed an increase in acridine orange staining and electron microscopy revealed an increase in vacuolar formation in 2ME-treated cells. The gene expression of several genes associated with mRNA translation, autophagy-related processes and genes involved in microtubule dynamics were affected. The study contributes to the mechanistic understanding of 2ME's growth inhibition in MCF-7 cells and highlights the possibility of both apoptotic and autophagic processes being activated in response to 2ME treatment in this cell line.

Topics: 2-Methoxyestradiol; Adenocarcinoma; Antineoplastic Agents; Apoptosis; Autophagy; Breast Neoplasms; Caspase 3; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Shape; Cyclin B1; Cytochromes c; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Oligonucleotide Array Sequence Analysis; Reactive Oxygen Species; Receptors, Estrogen; Vacuoles

Several studies have shown that curcumin can induce apoptosis and inhibit growth in human tumor cell lines. However, the mechanism is not completely understood yet. The present studies were designed to investigate the effects of curcumin on human A549 lung adenocarcinoma cells lines to better understand its effect on apoptosis and apoptosis-related genes in vitro. Apoptosis induction, mitochondria membrane potential, mitochondria structure, and apoptotic associated gene expression were examined by flow cytometric assay, confocal microscopy, Western blotting and electron microscopy. After treatment with curcumin, percentage of apoptotic cells increased dose- and time-dependently, and morphology observation revealed typical apoptotic features. Our data further indicated that the expression of Bax proteins in A549 cells was increased in a dose-dependent manner, whereas the expression of Bcl-2 was significantly decreased, thus the ratio of Bax/Bcl-2 was increased. The apoptotic process was accompanied by the change of mitochondrial function and structure which led to release of the cytochrome c, and activation of caspase-9 and caspase-3. Furthermore, curcumin also induced a dose-dependent cleavage of PARP. Caspases activation during the course of curcumin-induced apoptosis was additionally confirmed by using a broad-spectrum caspases inhibitor, Z-VAD-fmk. As expected, the inhibitor was able to decrease curcumin-induced apoptosis on A549 cells. These results suggested that mitochondria played an important role in the curcumin-induced apoptosis, and mitochondria membrane potential loss initiated apoptosis via the activation of caspases.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Shape; Curcumin; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Time Factors

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.. The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.. We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.. Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.. Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Ceramides; Cytochromes c; Cytoplasm; Female; Glutathione; Humans; Lipid Peroxidation; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction

Fucoidan is a sulfated polysaccharide found in brown algae; it has been shown to exhibit a number of biological effects, including anti-tumor effects. In this study, we evaluated the effects of fucoidan on apoptosis in HT-29 and HCT116 human colon cancer cells.. HT-29 and HCT116 cells were cultured with various concentrations of fucoidan (0 - 20 microg/mL). Apoptosis was assayed via Hoechst staining and Annexin V staining followed by flow cytometric analysis. Western blot analyses and JC-1 staining were conducted to determine the levels of apoptosis-regulating proteins and mitochondrial membrane permeability, respectively.. Fucoidan induced substantial reductions in viable cell numbers and apoptosis of HT-29 and HCT116 cells in a dose-dependent manner. In HT-29 cells, fucoidan also increased the levels of cleaved caspases-8, -9, -7, and -3, and cleaved poly (ADP-ribose) polymerase (PARP) levels. The levels of the X-linked inhibitor of apoptosis protein and survivin were attenuated in the fucoidan-treated cells. Fucoidan was also shown to enhance mitochondrial membrane permeability, as well as the cytochrome c and Smac/Diablo release from the mitochondria. Fucoidan increased the levels of the Bak and truncated Bid proteins, but reduced the levels of Mcl-1. Additionally, fucoidan increased the levels of the tumor necrosis factor-related apoptosis-inducing ligand, Fas and death receptor 5 proteins. The caspase-8 and -9 inhibitors Z-IETD-FMK and Z-LEHD-FMK induced a reduction in fucoidan-mediated apoptosis. Caspase-8 inhibitor inhibited the fucoidan-induced cleavage of Bid, caspases-9 and -3, and PARP.. The findings of this study indicate that fucoidan induces apoptosis in HT-29 and HCT116 human colon cancer cells, and that this phenomenon is mediated via both the death receptor-mediated and mitochondria-mediated apoptotic pathways. These results suggest that fucoidan may prove useful in the development of a colon cancer-preventive protocol.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; HT29 Cells; Humans; Phaeophyceae; Phytotherapy; Plant Extracts; Polysaccharides; Proto-Oncogene Proteins c-bcl-2

Allicin is an active compound derived from garlic that has been shown to have antitumor properties in vitro. The current study was designed to explore the effects and the underlying mechanism of allicin on gastric cancer cells. The MTT assay was used to detect cell viability. Transmission electron microscopy, Rh123 and propidium iodide staining, annexin V/FITC assay and the mitochondrial membrane potential were used to assess for the presence of apoptosis. Immunocytochemistry, western blot analysis, and Q-RT-PCR were used to detect gene expression. We found that allicin reduced cell viability in a dose- and time-dependent manner, partly through induction of apoptosis in gastric cancer cells. At the molecular level, allicin induced cytochrome c release from the mitochondria and increased caspase-3, -8, and -9 activation, with concomitant upregulation of bax and fas expression in the tumor cells. Allicin treatment inhibited proliferation and induced apoptosis in SGC-7901 cancer cells. Both intrinsic mitochondrial and extrinsic Fas/FasL-mediated pathways of apoptosis occur simultaneously in SGC-7901 cells following allicin treatment. Data from the current study demonstrated that allicin should be further investigated as a novel cancer preventive or therapeutic agent in control of gastric cancer, with potential uses in other tumor types.

Topics: Adenocarcinoma; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Survival; Cytochromes c; Disulfides; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Models, Biological; Signal Transduction; Stomach Neoplasms; Sulfinic Acids; Tumor Cells, Cultured

Pyrogallol (PG) is a polyphenol compound and a known O2- generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (DeltaPsi(m)). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G1 Phase; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Mitochondria; Oligopeptides; Pyrogallol

Inhibitor of growth 4 (ING4) is considered to be a tumor suppressor implicated in several human malignancies by tumor growth inhibition and apoptosis enhancement. In present study, the effects of ING4 on apoptosis and its mechanisms were investigated through the transduction of ING4 cDNA into lung adenocarcinoma cell line A549.. The effects of ING4 on A549 apoptosis were observed by FCM analysis, TUNEL assay, and electron microscopy. Simultaneously, the effects of ING4 on the expression of several apoptosis-related proteins in cell line A549 were evaluated by Western blot analysis.. Both Annexin-V FITC analysis by FCM and TUNEL assay revealed more apoptotic cells in A549 cells with exogenous ING4 gene. For electron microscopy, A549 cells with exogenous ING4 gene showed typical morphological changes of apoptosis. The deregulation of Bcl-2 family proteins (Bcl-2, Bcl-xl, Bax, Bak, Bid) and the major apoptotic executioners of mitochondria pathway (Cyt-c, caspase3, PARP) were also observed.. Our findings suggest that exogenous ING4 can enhance A549 apoptosis via regulating the expression of Bcl-2 family proteins and the activation of mitochondrial apoptotic pathway.

Topics: Adenocarcinoma; Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Homeodomain Proteins; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Electron; Mitochondria; Neoplasm Transplantation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Transfection; Transplantation, Heterologous; Tumor Suppressor Proteins

Coumarin (1,2-benzopyrone) is a naturally occurring fragrant compound found in a variety of plants and spices. Coumarins have attracted intense interest in recent years because of their diverse pharmacological activities. This study examines the antioxidant coumarin 7,8-diacetoxy-4-methylcoumarin (DAMC) and its thiocoumarin derivative 7,8-diacetoxy-4-methylthiocoumarin (DAMTC) for their effect on human non-small cell lung cancer A549 cells. Here we show that both DAMC and DAMTC not only inhibited cell proliferation, but also induced apoptosis with an IC(50) of 160 microg/ml as confirmed by morphological examination, annexin-V assay and flow cytometric analysis. Interestingly, it was observed that these two coumarin compounds exhibited little cytotoxicity towards peripheral blood mononuclear cells but induced apoptosis in malignant cells. DAMC/DAMTC treatment also resulted in pronounced release of apoptogenic cytochrome c from mitochondria to cytosol, alteration of mitochondrial membrane potential (DeltaPsi(m)), and activation of caspase-9 and caspase-3. Although an increase in the levels of reactive oxygen species (ROS) was observed, pre-treatment with antioxidant showed no protective effect against DAMC/DAMTC-induced apoptosis. Results of present study suggest that downregulation of Bcl-xl, Cox-2 and mitogen activated protein kinase pathway and upregulation of p53, Akt and NF-kappaB pathway are involved in the underlying molecular mechanism of apoptosis induction by DAMC and DAMTC in A549 cells.

Topics: Adenocarcinoma; Apoptosis; Caspases; Cell Proliferation; Coumarins; Cytochromes c; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Lung Neoplasms; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

We explored the in vitro and in vivo mechanism of antitumor action of the synthetic flavonoid 2'-nitroflavone on LM3 murine mammary adenocarcinoma cells. In vitro assays showed that 2'-nitroflavone increased the population of LM3 hypodiploid cells and produced a typical ladder of DNA fragmentation. Apoptotic cell death was also characterized by the activation of caspase-8, -9 and -3, by an increment in the expression levels of the proapoptotic protein Bax and by the release of cytochrome c to cytosol. The in vivo effect of 2'-nitroflavone on tumor growth was studied in BALB/c mice injected subcutaneously with LM3 cells. Results showed that tumor volume and weight were significantly reduced at doses of 10 and 40 mg/kg of 2'-nitroflavone, respectively. Apoptotic cells were identified by TUNEL assay in tumor slices from mice treated with 10 mg/kg of 2'-nitroflavone. Western blot analysis of tumor lysate supernatants from treated mice revealed an upregulation of the total levels of Bax and Fas receptor. In addition, administration of 40 mg/kg of 2'-nitroflavone to nontumor-bearing mice showed no histopathological effects on different organ tissues. This is the first report of the in vivo growth inhibitory effect of 2'-nitroflavone as an apoptotic agent likely useful for mammary adenocarcinoma treatment.

Topics: Adenocarcinoma; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cytochromes c; Female; Flavones; Flow Cytometry; In Situ Nick-End Labeling; In Vitro Techniques; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Proto-Oncogene Proteins c-bcl-2; Xenograft Model Antitumor Assays

Gene therapy and virotherapy are among the approaches currently being used to treat lung cancer. The success of cancer gene therapy depends on treatments where different types of tumors can be selectively targeted and destroyed without affecting normal cells and tissue. Previously, we described a promoter system (TTS) that we designed that is specifically targeted to lung cancer cells but which does not affect other types of cells including stem cells. In our study, we have enhanced the utility of the TTS system by inserting the pro-apoptotic gene BH3 domain interacting death agonist (Bid) into the TTS promoter system (TTS/Bid) to create a drug regulatable lung cancer-specific gene therapy. A recombinant adenoviral vector was used to introduce TTS/Bid (Ad-TTS/Bid) into lung cancer cells. BID expression and apoptosis occurred in A549 pulmonary adenocarcinoma cells but little Bid expression or apoptosis occurred in MCF7 breast cancer cells or in normal human lung fibroblasts. The use of cisplatin enhanced the processing of full length BID to t-BID which significantly increased lung cancer-specific cell death. In in vivo experiments, intraperitonal injection of cisplatin enhanced the antitumor effects of the vector in a lung cancer xeno-graft mouse model. Moreover, dexamethasone effectively suppressed exogenous BID expression and the antitumor effect of Ad-TTS/Bid both in vitro and in vivo. Here, we describe the efficacy of the use of cisplatin and dexamethasone with the anti lung cancer promoter system (Ad-TTS/Bid) for a safe and effective gene therapy against advanced lung cancer.

Topics: Adenocarcinoma; Adenoviridae; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Caspases; Cisplatin; Colony-Forming Units Assay; Cytochromes c; Dexamethasone; Female; Fibroblasts; Flow Cytometry; Humans; Immunoblotting; Lung; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Promoter Regions, Genetic; Xenograft Model Antitumor Assays

To determine the effect of ellagic acid on apoptosis and proliferation in pancreatic cancer cells and to determine the mechanism of the pro-survival effects of ellagic acid.. The effect of ellagic acid on apoptosis was assessed by measuring phosphatidylserine externalization, caspase activity, mitochondrial membrane potential and DNA fragmentation; and proliferation by measuring DNA thymidine incorporation. Mitochondrial membrane potential was measured in permeabilized cells, and in isolated mitochondria. Nuclear factor kappa B (NF-kappa B) activity was measured by electromobility shift assay (EMSA).. We show that ellagic acid, a polyphenolic compound in fruits and berries, at concentrations 10 to 50 mmol/L stimulates apoptosis in human pancreatic adenocarcinoma cells. Further, ellagic acid decreases proliferation by up to 20-fold at 50 mmol/L. Ellagic acid stimulates the mitochondrial pathway of apoptosis associated with mitochondrial depolarization, cytochrome C release, and the downstream caspase activation. Ellagic acid does not directly affect mitochondria. Ellagic acid dose-dependently decreased NF-kappa B binding activity. Furthermore, inhibition of NF-kappa B activity using IkB wild type plasmid prevented the effect of ellagic acid on apoptosis.. Our data indicate that ellagic acid stimulates apoptosis through inhibition of the prosurvival transcription factor NF-kappa B.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Ellagic Acid; Humans; I-kappa B Proteins; Membrane Potential, Mitochondrial; Mitochondria; NF-kappa B; Pancreatic Neoplasms

Duchesnea indica (Andr.) Focke has been commonly used to treat cancer in Asian countries of centuries, and more recently, has been shown to possess anticancer properties in vivo and in vitro. However, little is known about the underlying mechanism of its anticancer action. In the present study, we investigated the effect of Duchesnea phenolic fraction (DPF) on SKOV-3 ovarian cancer cells to provide insights into the mechanisms of growth suppression involved in DPF-mediated apoptosis and cell cycle arrest.. Cytotoxic activity of DPF on SKOV-3 cells was determined using MTT assay, apoptosis (AO/EB staining, DNA fragmentation, FACS), caspase-3 activation and cell cycle analysis studies. The role of the molecules in apoptosis and cell cycle regulation was analyzed by Western blot and RT-PCR.. DPF significantly inhibited SKOV-3 cell proliferation in a dose-dependent manner and markedly induced apoptosis evidenced by characteristic apoptotic morphological changes, nuclear DNA fragmentation and sub-G1 peak. DPF suppressed Bcl-2 levels, enhanced Bax levels and Bax/Bcl-2 ratio, and simultaneously translocated Bax to mitochondria followed by mitochondrial release of cytochrome c into the cytosol and activation of effector caspase-3. Furthermore, DPF provoked S phase arrest in SKOV-3 cells with down-regulation of cyclin A, E, D1 and CDK2.. DPF exhibits cytotoxicity towards human ovarian cancer SKOV-3 cells through induction of apoptosis via mitochondrial pathway and arresting cell cycle progression in S phase. All together, these data sustain our contention that DPF has anticancer properties and merits further investigation as a potential therapeutic agent.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Female; HeLa Cells; Humans; Mitochondria; Ovarian Neoplasms; Phenol; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rosaceae

Cisplatin is an efficient anticancer agent. Cisplatin-based chemotherapy is believed to involve different signal transduction pathways, among which calpain activation has been proposed as an important factor in the induced apoptosis. In our study, based on real-time single cell analysis, we investigated the molecular involvement of calpain in cisplatin-induced apoptosis in living human lung adenocarcinoma cells. After cisplatin treatment, calpain was activated, resulting in Bid cleavage at 4-5 hr, followed by Bid translocation and cytochrome c release, leading to cell death. Calpeptin and PD150606, specific inhibitors of calpain, blocked Bid activation completely; however, cytochrome c release was delayed by more than 2 hr, which was associated with the delay of caspase-3 activation and cell death. Remarkably, calpain-mediated release of cytochrome c and cell death was significantly compromised in the Bid knockdown cells. Z-IETD-fmk and Z-VDVAD-fmk were used to block the activation of caspase-8 and caspase-2, respectively; however, the progression of apoptosis were not affected, suggesting that caspase-8 and caspase-2 were not involved in this experimental model. Taken together, the data demonstrate that calpain mediated cisplatin-induced apoptosis in human lung adenocarcinoma cells through activating Bid, which then regulated the mitochondrial apoptotic pathway. The delays of cytochrome c release, caspase-3 activation and subsequent cell death by inactivating calpain or silencing Bid exclude other earlier or parallel pathways, strongly suggesting that the calpain-mediated pathway is the kinetically earliest one, which dominates the cisplatin-induced apoptosis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Calpain; Caspases; Cisplatin; Cytochromes c; Humans; Lung Neoplasms; Mitochondria; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

Acrolein is a toxic combustion product that elicits apoptotic and/or necrotic cell death depending on the conditions under which exposure occurs. As a strong electrophile, side-reactions with nucleophilic media constituents seem likely to accompany study of its toxicity in vitro, but these reactions are poorly characterized. We have thus examined the effect of media composition on the toxicity of acrolein in A549 cells. Cells were exposed to acrolein in either Dulbecco's buffered saline (DBS) or F12 supplemented with various concentrations of fetal bovine serum. Cell viability was assessed using the MTT assay, while heme oxygenase-1 (HO-1) and cytoplasmic cytochrome c were measured as respective markers of transcriptional response and apoptosis. Protein damage was evaluated using the protein carbonyl assay. Compared to F12 media (with or without serum), maximal cell death as evaluated using the MTT assay, as well as adduction of intracellular proteins, occurred when cells were exposed to acrolein in DBS. In contrast, cytochrome c release was maximal in cells exposed to acrolein in serum-containing F12, conditions which inhibited protein modification and overt cell death. These findings highlight the need for careful attention to experimental conditions when conducting in vitro toxicological studies of reactive substances.

Topics: Acrolein; Adenocarcinoma; Alkylation; Animals; Apoptosis; Cattle; Cell Line, Tumor; Cell Survival; Culture Media; Cytochromes c; Cytoplasm; Heme Oxygenase-1; Humans; Protein Carbonylation; Serum; Transcription, Genetic

Upper gastrointestinal adenocarcinomas are a common cause of cancer-related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas.. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining on tumor tissue microarrays (TMA) were used to study the expression of AURKA in upper gastrointestinal adenocarcinomas. To investigate the biological and signaling impact of AURKA, the authors used multiple in vitro assays that included 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling), cytochrome C release, flow cytometry, luciferase reporter, and Western blot analysis.. Frequent overexpression of AURKA transcript in upper gastrointestinal adenocarcinomas was detected compared with normal samples (47%; P= .001). The immunohistochemical analysis of 130 tumors demonstrated moderate-to-strong immunostaining of AURKA in >50% of upper gastrointestinal adenocarcinomas. By using camptothecin as a drug-induced apoptosis in vitro model, the authors demonstrated that the expression of AURKA provided protection against apoptosis to gastrointestinal cancer cells (AGS and RKO) (P= .006) and RIE-1 primary intestinal epithelial cells (P= .001). The AURKA overexpression mediated an increase in phosphorylation of AKT(Ser473) with an increase in HDM2 level. The shRNA-knockdown of AKT in AURKA-overexpressing cells reversed this effect and showed a significant increase in the p53 protein level, indicating a possible nexus of AURKA/AKT/p53. Indeed, overexpression of AURKA led to a remarkable reduction in the transcription activity of p53, with subsequent reductions in transcript and protein levels of its downstream proapoptotic transcription targets (p21, BAX, NOXA, and PUMA).. Study results indicated that AURKA provides potent antiapoptotic properties to gastrointestinal cells by regulating levels of p53 through the AKT/HDM2 axis.

Topics: Adenocarcinoma; Apoptosis; Aurora Kinase A; Aurora Kinases; Biomarkers, Tumor; Blotting, Western; Camptothecin; Coloring Agents; Cytochromes c; Enzyme Inhibitors; Esophageal Neoplasms; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Luciferases; Luminescent Agents; Polymerase Chain Reaction; Protein Array Analysis; Protein Serine-Threonine Kinases; Stomach Neoplasms; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p53

mAtNOS1 is a novel gene recently reported in mammalian genome with functions that are not fully understood. The present study shows that in human mammary adenocarcinoma MCF-7 cells, mAtNOS1 expression increases mitochondrial nitric oxide and calcium. Our study further shows that overexpression of mAtNOS1 induces apoptosis in MCF-7 cells by increasing mitochondrial protein tyrosine nitration and cytochrome c release. The present study suggests a novel function for mAtNOS1 in regulating mitochondrial nitric oxide and calcium and inducing apoptosis of MCF-7 cells.

Topics: Adenocarcinoma; Apoptosis; Arabidopsis Proteins; Breast Neoplasms; Calcium; Cell Line, Tumor; Cytochromes c; Female; Humans; Immunochemistry; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Transfection; Tyrosine

In this study, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, with other chemotherapeutic drugs including estrogen receptor-beta (ER-beta) antagonist (tamoxifen) and topoisomerase II inhibitor (etoposide) on some metastatic prostate cancer (PC) cell lines. Immunohistochemial analyses revealed that EGFR expression was enhanced in 38% of primary prostatic adenocarcinomas (Gleason scores 4-10) as compared to the corresponding normal tissues of the same prostate gland from 32 PC patients. The RT-PCR and Western blot data have also indicated the higher expression levels of EGFR and ER-beta transcripts and proteins in metastatic LNCaP, DU145 and PC3 cells relative to nonmalignant normal prostate cells. Moreover, the results from MTT and FACS analyses revealed that the drugs, alone or in combination at lower concentrations, inhibited the growth of 17beta-estradiol (E2) plus EGF and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145 and PC3 cells. Importantly, the combined gefitinib, tamoxifen and etoposide also caused a higher rate of apoptotic death of PC cells as compared to single agents. The cytotoxic effects induced by these drugs in PC3 cells appear to be mediated through the accumulation of cellular ceramide and activation of caspase cascades via a mitochondrial pathway. These findings indicate that the combined use of inhibitors of EGF-EGFR and E2-ER-beta signaling with etoposide, which act by increasing the cellular ceramide levels and caspase activity, represents a promising strategy for a more effective treatment of metastatic PC forms.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Caspases; Cell Proliferation; Cells, Cultured; Ceramides; Cytochromes c; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Etoposide; Flow Cytometry; Gefitinib; Humans; Immunoenzyme Techniques; Male; Membrane Potential, Mitochondrial; Neoplasms, Hormone-Dependent; Prostate; Prostatic Neoplasms; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tamoxifen

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

Gastroduodenal reflux is implicated in esophageal carcinogenesis. This effect is mediated by reactive oxygen species. We hypothesized that this is mediated by DNA mismatch lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), which is repaired by the Mut Y homologue (MYH). We tested the effect of reflux, either alone or in combination with the human dietary mutagen methyl-n-amyl nitrosamine (MNAN), on DNA damage in adenocarcinoma and squamous cell cancer of the esophagus in a rat model.. Reflux was promoted in male Sprague-Dawley rats by duodenoesophageal anastomosis (8 weeks) without gastric bypass. MNAN treatment (25 mg/kg per week intraperitoneally for four doses) commenced at 10 weeks age. Ten animals served as controls. Quantification of 8-oxoG was performed by using immunohistochemistry, and MYH was analyzed by Western blot. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL), cytochrome C, and caspase.. Tumors (adenocarcinoma) developed in 15 (50%) of 30 animals with reflux alone; this increased to 26 (86.6%) of 30 when reflux was combined with MNAN treatment, with tumor histology consistent with adenosquamous and squamous cell cancer. DNA damage, as reflected by positive 8-oxoG staining in reflux groups, was significantly increased compared with control (p < 0.01), and this was maximal in tissues with malignant transformation. Protein levels of the DNA repair enzyme MYH were significantly less in tissues subjected to reflux compared with controls (p < 0.05). TUNEL, cytochrome C, and caspase positivity confirmed increased apoptosis in cancer lesions.. Gastroduodenal reflux leads to increased DNA damage and downregulation of the DNA mismatch repair pathway. This pathway has an important role in esophageal carcinogenesis in rats.

Topics: Adenocarcinoma; Animals; Apoptosis; Carcinogens; Carcinoma, Squamous Cell; Caspases; Cytochromes c; DNA Damage; DNA Mismatch Repair; DNA Repair Enzymes; Down-Regulation; Duodenogastric Reflux; Esophageal Neoplasms; Guanosine; In Situ Nick-End Labeling; Male; Nitrosamines; Rats; Rats, Sprague-Dawley; Staining and Labeling

Chemotherapeutic drugs are usually designed to induce cancer cell death via cell cycle arrest and/or apoptosis pathways. In this study, we used the chemical drug 15,16-dihydrotanshinone I (DHTS) to inhibit breast cancer cell proliferation and tumor growth, and investigate the underlying molecular mechanisms. Human breast cancer cell lines MCF-7 and MDA-MB-231 were both used in this study, and DHTS was found to significantly decrease cell proliferation by a dose-dependent manner in both cells. Flow cytometry indicated that DHTS induced G1 phase arrest in synchronous MCF-7 and MDA-MB-231 cells. When analyzing the expression of cell cycle-related proteins, we found that DHTS reduced cyclin D1, cyclin D3, cyclin E, and CDK4 expression, and increased CDK inhibitor p27 expression in a dose-dependent manner. In addition, DHTS inhibited the kinase activities of CDK2 and CDK4 by an immunocomplex kinase assay. In addition, DHTS also induced apoptosis in both cells through mainly mitochondrial apoptosis pathways. We found that DHTS decreased the anti-apoptotic protein Bcl-xL level and increased the loss of mitochondria membrane potential and the amount of cytochrome c released. Moreover, DHTS activated caspase-9, caspase-3, and caspase-7 and caused cell apoptosis. The fact that DHTS-induced apoptosis could be blocked by pretreating cells with pan-caspase inhibitor confirmed that it is mediated through activation of the caspase-3-dependent pathway. In a nude mice xenograft experiment, DHTS significantly inhibited the tumor growth of MDA-MB-231 cells. Taken together, these results suggest that DHTS can inhibit human breast cancer cell proliferation and tumor growth, and might have potential chemotherapeutic applications.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Furans; G1 Phase; Humans; Male; Mammary Neoplasms, Experimental; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Phenanthrenes; Quinones; Xenograft Model Antitumor Assays

Cytokines such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in colon cancer cells through engagement of death receptors. Nevertheless, evading apoptosis induced by anticancer drugs characterizes many types of cancers. This results in the need for combination therapy. In this study, we have investigated whether the flavonoid quercetin could sensitize human colon adenocarcinoma cell lines to TRAIL-induced apoptosis. We report that quercetin enhanced TRAIL-induced apoptosis by causing the redistribution of DR4 and DR5 into lipid rafts. Nystatin, a cholesterol-sequestering agent, prevented quercetin-induced clustering of death receptors and sensitization to TRAIL-induced apoptosis in colon adenocarcinoma cells. In addition, our experiments show that quercetin, in combination with TRAIL, triggered the mitochondrial-dependent death pathway, as shown by Bid cleavage and the release of cytochrome c to the cytosol. Together, our findings propose that quercetin, through its ability to redistribute death receptors at the cell surface, facilitates death-inducing signaling complex formation and activation of caspases in response to death receptor stimulation. Based on these results, this study provides a challenging approach to enhance the efficiency of TRAIL-based therapies.

Topics: Adenocarcinoma; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspases; Colonic Neoplasms; Cytochromes c; Cytosol; Death Domain Receptor Signaling Adaptor Proteins; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immunoblotting; Immunoprecipitation; Membrane Microdomains; Mitochondria; Quercetin; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

The c-myc oncogene encodes for a transcriptional factor involved in many cellular processes such as proliferation, differentiation and apoptosis. According to these different functions, the role of c-Myc protein in cellular sensitivity to anti-cancer drugs is controversial. We defined the role of c-Myc in cancer cell sensitivity to vinblastine (VLB) using human colon cancer cells: LoVo wild-type or transfected with a plasmid containing the human c-myc gene in antisense orientation (LoVo-mycANS). Analysis of VLB cytotoxicity demonstrated a 3-fold increase in VLB sensitivity in LoVo-mycANS cells. Comparison between cells revealed different apoptosis kinetics: accumulation of cells in sub-G1 phase and poly(ADP-ribose) polymerase cleavage occurred earlier in LoVo-mycANS. Then, we demonstrated a mitochondrial membrane potential disruption followed by cytochrome c release that indicates the involvement of mitochondria in this apoptotic signaling pathway. This earlier apoptosis was accompanied by a Bcl-2 decrease and a p53 increase. In conclusion, the decrease in c-Myc expression enhanced the VLB sensitivity, triggering earlier apoptosis through induction of the intrinsic pathway. Thus, c-myc induction is a resistance factor and our findings suggest that tumors carrying low levels of c-Myc protein could be more responsive to vinca alkaloids treatment. Moreover, the downregulation of c-myc oncogene by an antisense strategy might represent a useful goal for improving the efficacy of this anti-neoplastic drug family.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Colonic Neoplasms; Cytochromes c; DNA, Antisense; Down-Regulation; Drug Tolerance; G1 Phase; Humans; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vinblastine

Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Ceramides; Cytochromes c; Enzyme Activation; Fatty Acids, Monounsaturated; Female; Gene Targeting; Humans; Mitochondria; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering; Sphingolipids

Lamellarin D is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cell lines and is a potent inhibitor of topoisomerase I. However, lamellarin D maintains a marked cytotoxicity toward cell lines resistant to the reference topoisomerase I poison camptothecin. We therefore hypothesized that topoisomerase I is not the only cellular target for the drug. Using complementary cell-based assays, we provide evidence that lamellarin D acts on cancer cell mitochondria to induce apoptosis. Lamellarin D, unlike camptothecin, induces early disruption of the inner mitochondrial transmembrane potential (Deltapsi(m)) in the P388 leukemia cell line. The functional alterations are largely prevented by cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), but not by the inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone. Deltapsi(m) disruption is associated with mitochondrial swelling and cytochrome c leakage. Using a reliable real-time flow cytometric monitoring of Deltapsi(m) and swelling of mitochondria isolated from leukemia cells, we show that lamellarin D has a direct MPT-inducing effect. Furthermore, mitochondria are required in a cell-free system to mediate lamellarin D-induced nuclear apoptosis. The direct mitochondrial effect of lamellarin D accounts for the sensitivity of topoisomerase I-mutated P388CPT5 cells resistant to camptothecin. Interestingly, a tumor-active analogue of lamellarin D, designated PM031379, also exerts a direct proapoptotic action on mitochondria, with a more pronounced activity toward mitochondria of tumor cell lines compared with nontumor cell lines. Altogether, this work reinforces the pharmacologic interest of the lamellarins and defines lamellarin D as a lead in the search for treatments against chemoresistant cancer cells.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Camptothecin; Cell Line, Tumor; Cell Membrane Permeability; Cell-Free System; Coumarins; Cytochromes c; Heterocyclic Compounds, 4 or More Rings; Humans; Isoquinolines; Leukemia P388; Lung Neoplasms; Membrane Potentials; Mice; Mitochondria; Mitochondrial Membranes; NIH 3T3 Cells; Rats

Inhibition of cyclooxygenase-2 has been proposed to be a potential mechanism for the chemoprevention of gastrointestinal tumors by nonsteroidal anti-inflammatory drugs. This study investigates the mechanisms by which the cyclooxygenase-2 inhibitor SC236 induces apoptosis of gastric cancer cell lines and its downstream signaling pathway.. Two gastric cancer cell lines, AGS and MKN28, were treated with SC236 and assessed for cell growth and apoptosis. The involvement of mitogen-activated protein kinase and Akt kinase/protein kinase B (Akt/PKB) pathways and their downstream signalings were studied in the AGS cell line.. SC236 treatment induced apoptosis in gastric cancer cells and caused activation of p38 and stress-activated protein kinase/jun kinase, but down-regulated Akt/PKB. The specific p38 inhibitor SB203580 and the dominant-negative stress-activated protein kinase/jun kinase both failed, while the constitutively active form of Akt/PKB was able to block SC236-induced apoptosis. SC236-induced apoptosis was coupled with release of cytochrome c and activation of caspases.. One of the pathways involved in SC-236-induced apoptosis in gastric cancer cells is through downregulation of Akt and then release of cytochrome c.

Topics: Acridine Orange; Adenocarcinoma; Apoptosis; Blotting, Western; Caspases; Cyclooxygenase 2 Inhibitors; Cytochromes c; Down-Regulation; Enzyme Activation; Humans; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Sulfonamides; Transfection; Tumor Cells, Cultured

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Mitochondria; Olea; Triterpenes; Tumor Suppressor Protein p53

Rotaviruses, which are the main cause of viral gastroenteritis in young children, induce structural and functional damages in infected mature enterocytes of the small intestine. To investigate a relationship between rotavirus infection and cell death by apoptosis, we used the human intestinal Caco-2 cell line. We demonstrated by several methods including TUNEL and ELISA detection of cytoplasmic histone-associated DNA fragments that the infection of fully differentiated Caco-2 cells by the RRV rotavirus strain induces apoptosis. Rotavirus infection leads to the loss of mitochondrial membrane potential and the release of cytochrome C from mitochondria. We showed that rotavirus-induced apoptosis was dependent of the multiplicity of infection and increased with time from 4 h to 24 h of infection. Flow cytometric analysis showed that DNA fragmentation occurs in productively infected cells, suggesting that rotavirus induces apoptosis by a direct mechanism. We also demonstrated that non-replicative RRV particles are not sufficient to induce apoptosis and viral gene expression seems required. Intracellular calcium plays a role in RRV-induced apoptosis because treatment with an intracellular calcium ion chelator (BAPTA-AM) partially inhibited apoptosis.

Topics: Adenocarcinoma; Annexin A5; Apoptosis; Cell Differentiation; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Flow Cytometry; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Rotavirus

To investigate the underlying mechanism of apoptosis-inducing effect of a specific COX-2 inhibitor SC236 in gastric cancer cells.. Western blot analysis was used to measure apoptosis-related proteins, cytochrome c, and caspase-3. The catalytic activity of the caspases was measured using a colorimetric assay.. Treatment of AGS gastric cancer cells with SC236 caused a significant elevation of the pro-apoptotic protein Bak, release of cytochrome c to the cytosol, and activation of caspase-3. A specific caspase-3 inhibitor, z-DEVD-fmk, blocked SC236-induced apoptosis.. SC236 inhibits cell growth and induces apoptosis in gastric cancer cells at least partly through the up-regulation of Bak, stimulation of cytochrome c release, and activation of caspase-3.

Topics: Adenocarcinoma; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspase 3; Caspases; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Cytochromes c; Humans; Oligopeptides; Poly(ADP-ribose) Polymerases; Pyrazoles; Stomach Neoplasms; Sulfonamides

We previously reported that the garlic-derived compound S-allylmercaptocysteine (SAMC) causes growth inhibition, mitotic arrest, and induction of apoptosis in SW480 human colon cancer cells by inducing microtubule depolymerization and c-Jun NH(2) terminus kinase-1 activation. In the present study, we compared the aforementioned effects of SAMC to those of a series of garlic-derived and other organosulfur compounds. Among the 10 compounds tested, only SAMC, diallyl disulfide (DADS), and S-trityl-L-cysteine (trityl-cys) cause significant inhibition of cell growth with IC(50) values of 150, 56, and 0.9 micromol/L, respectively. These three compounds also induce G(2)-M cell cycle arrest and apoptosis. Further studies reveal that, like SAMC, the garlic-derived compound DADS exerts antiproliferative effects by binding directly to tubulin and disrupting the microtubule assembly, thus arresting cells in mitosis and triggering mitochondria-mediated signaling pathways that lead to apoptosis. However, the synthetic compound trityl-cys exerts its effect on M-phase arrest and growth inhibition by mechanisms that involve spindle impairment but do not involve disruption of microtubule structure or dynamics. Furthermore, trityl-cys does not induce marked loss of mitochondrial membrane potential or release of cytochrome c, but it does induce caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Structure-function analysis suggests that both the allyl and the disulfide moieties are important features for the antiproliferative effects of SAMC and DADS. These findings may be useful in the identification, synthesis, and development of organosulfur compounds that have anticancer activity.

Topics: Adenocarcinoma; Allyl Compounds; Apoptosis; Caspase 3; Caspases; Cell Division; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Garlic; Humans; Membrane Potentials; Microtubules; Mitochondria; Mitosis; Poly(ADP-ribose) Polymerases; Sulfur Compounds; Tubulin; Tumor Cells, Cultured

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

Various human colon cancer cell lines tested in vitro differed significantly in susceptibility to growth inhibition of recombinant human interferon-beta (rHuIFN-beta). Two p53-mutant lines, COH and CC-M2, derived from high-grade colon adenocarcinoma, showed signs of apoptosis after treatment with 250 IU/ml of HuIFN- beta in the culture medium. The similarly p53-mutated HT-29 line from a grade I adenocarcinoma showed no apoptosis, however, and only cell cycle G1/G0 or S phase retardation with 1000 IU/ml HuIFN-beta. After HuIFN-beta exposure, COH and CC-M2 cells showed increased levels of Fas and FasL proteins, alteration of mitochondrial membrane potential, and activation of caspase-9, caspase-8, and caspase-3 in a time-dependent manner. Treatment of COH and CC-M2 cells with anti-FasL antibodies or rFas/Fc fusion protein, however, could not prevent the apoptosis induced by HuIFN-beta. In contrast, cell-permeable specific inhibitors of the three caspases could inhibit the DNA fragmentation and cell death but not the mitochondrial membrane potential changes. Treatment with mitochondria-stabilizing reagents could significantly abrogate the apoptosis and caspase activation induced by HuIFN-beta. These results suggest that in COH and CC-M2 colon cancer cell lines, HuIFN-beta induces apoptosis mainly through mitochondrial membrane alteration and subsequent activation of the caspase cascade pathway, but not by the Fas/FasL interaction or the p53-dependent apoptotic mechanism.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Fas Ligand Protein; fas Receptor; Genes, p53; Humans; Interferon Type I; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Mutation; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Recombinant Proteins

Most patients with pancreatic adenocarcinoma present with surgically incurable disease. Gemcitabine, the principal agent used to treat such patients, has little impact on outcome. Overexpression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, a feature of this malignancy, is associated with resistance to anoikis and increased metastasis. The purpose of this study was to determine the role of CEACAM6 in cellular chemoresistance to gemcitabine. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express very low levels of the protein. Suppression of CEACAM6 expression was achieved in BxPC3 cells, which inherently overexpress CEACAM6, by stable transfection of a CEACAM6 small interfering RNA-generating vector. The effects of modulating CEACAM6 expression on gemcitabine-induced cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay, flow cytometric apoptosis quantification, caspase profiling, and Western analysis of cytoplasmic cytochrome c release. The roles of Akt and c-Src kinases as downstream targets of CEACAM6 signaling were examined. Stable overexpression of CEACAM6 in Capan2 increased gemcitabine chemoresistance, whereas CEACAM6 gene silencing in BxPC3 markedly increased the sensitivity of these cells to gemcitabine. Differential expression of CEACAM6 modulates Akt activity in a c-Src-dependent manner, and CEACAM6 overexpression appears to protect cells from cytochrome c-induced caspase 3 activation and apoptosis.

Topics: Adenocarcinoma; Antigens, CD; Antigens, Neoplasm; Antimetabolites, Antineoplastic; Caspase 3; Caspases; Cell Adhesion Molecules; Cell Line, Tumor; CSK Tyrosine-Protein Kinase; Cytochromes c; Deoxycytidine; Drug Resistance, Viral; Enzyme Activation; Gemcitabine; Gene Silencing; GPI-Linked Proteins; Humans; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; src-Family Kinases; Transfection

Bilirubin is the principal end product of heme degradation. Prompted by epidemiologic analyses demonstrating an inverse correlation between serum bilirubin levels and cancer mortality, we examined the effect(s) of bilirubin on the growth and survival of colon adenocarcinoma cells. Adenocarcinoma cell monolayers were treated with bilirubin over a range of bilirubin:BSA molar ratios (0-0.6), and viability was assessed colorimetrically. Apoptosis was characterized by TUNEL assay, annexin V staining and caspase-3 activation. The mechanism(s) by which bilirubin induces apoptosis was investigated by Western blotting for cytochrome c release, assaying for caspase-8 and caspase-9 activation and for mitochondrial depolarization by JC-1 staining. The direct effect of bilirubin on the membrane potential of isolated mitochondria was evaluated using light-scattering and fluorescence techniques. Bilirubin decreased the viability of all colon cancer cell lines tested in a dose-dependent manner. Cells exhibited substantial apoptosis when exposed to bilirubin concentrations ranging 0-50 microM, as demonstrated by an 8- to 10-fold increase in TUNEL and annexin V staining and in caspase-3 activity. Bilirubin treatment evokes specific activation of caspase-9, enhances cytochrome c release into the cytoplasm and triggers the mitochondrial permeability transition in colon cancer monolayers. Additionally, bilirubin directly induces the depolarization of isolated rat liver mitochondria, an effect that is not inhibited by cyclosporin A. Bilirubin stimulates apoptosis of colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, apparently by directly dissipating mitochondrial membrane potential. As this effect is triggered at concentrations normally present in the intestinal lumen, we postulate a physiologic role for bilirubin in modulating colon tumorigenesis.

Topics: Adenocarcinoma; Annexin A5; Apoptosis; Bilirubin; Caspase 3; Caspases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Tumor Cells, Cultured

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that beta-Carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents beta-Carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. beta-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-kB activation is involved in beta-Carotene-induced caspase cascade. These results support a pharmacological role for beta-Carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells.

Topics: Adenocarcinoma; Apoptosis; beta Carotene; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; HL-60 Cells; Humans; Melanoma; Membrane Potentials; Mitochondria; Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured