cytochrome-c-t and Colonic-Neoplasms

Colorectal cancer is the third most lethal and fourth most commonly diagnosed cancer worldwide. Sinapic acid, a derivative of hydroxycinnamic acid, is a promising phytochemical exhibiting numerous pharmacological activities in various systems. It is a substantial chain-breaking antioxidant that operates as a radical scavenger. The aim of this research was to investigate the antiproliferative effect of sinapic acid on the HT-29 cell line besides the mechanisms underlying this activity. The effect of sinapic acid on the viability of HT-29 cell line was investigated using XTT assay. The levels of BCL-2, cleaved caspase 3, BAX, cleaved PARP, and 8-oxo-dG were measured using ELISA. Gamma-H2AX and cytochrome

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Proliferation; Colonic Neoplasms; Coumaric Acids; Cytochromes c; HT29 Cells; Humans; Poly(ADP-ribose) Polymerase Inhibitors

Colorectal cancer (CRC) can be either prevented or alleviated using conventional drugs combined with natural treatments. Andean berry (AB, Vaccinium meridionale Sw.) is an underutilized berry with promising anti-inflammatory and antiproliferative effects that could be used to alleviate CRC markers in combination with Aspirin, a well-known CRC preventive drug. This research aimed to evaluate the impact of Aspirin, AB juice (ABJ), and their mixture on colorectal cancer in vitro and in vivo. The treatments (ABJ: 0, 10, 20, and 30 % v/v; Aspirin: 0, 10, 15, and 20 mM; and their combination) were assessed on SW480 cells to test their antiproliferative and pro-apoptotic effect. To evaluate their chemopreventive and chemoprotective effect in vivo, azoxymethane (AOM, 15 mg/kg BW) was used as a chemical inductor of early-stage colon cancer. Balb/c mice (8 weeks' age) were randomly assigned to five groups (n = 6 mice/group): control (no treatment), positive control (AOM-treated mice), AOM + Aspirin (20 mM: 25 mg/kg BW), AOM + ABJ (30 % v/v), and AOM + Aspirin + ABJ (Aspirin: 25 mg/kg BW; ABJ: 30 % v/v). ABJ contained phenolic compounds such as 3,4-dihydroxybenzoic and gallic acids, morin, and rutin. The mixture showed a strongest antiproliferative effect than their counterparts (+10.39-46.23 %). Except for Aspirin (20 mM), the cells were not able to proliferate based on the cloning efficiency test. The mixture was the most effective treatment arresting the cell cycle and increasing G2/M cell population (p < 0.01). Aspirin and ABJ showed mainly intrinsic and extrinsic-mediated apoptotic processes, while the mixture decreased most pro-apoptotic (cytochrome C, DR4, DR5, TNFRSF1A, Bax, and Bad) and anti-apoptotic proteins (Hsp70, Hsp32, and XIAP) compared to the untreated cells. In silico simulations highlighted the interaction between rutin and catalase as the strongest affinity (-10.30 Kcal/mol). ABJ and the mixture decreased aberrant crypt foci in vivo compared to AOM-only treated mice and protected the colonic and liver architecture, this was latter used as a secondary indicator of AOM-metabolic activity. The chemopreventive approach was more effective, related to a prior regulation of cancer-protective mechanisms in vivo, alleviating the AOM-induced damage. The results indicated that Aspirin and ABJ mixtures exhibit antiproliferative and pro-apoptotic effects in SW480 cells inducing mechanisms linked to extrinsic (TNF and TRAIL-mediated apoptosis) and intrinsic (B

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; Aspirin; bcl-2-Associated X Protein; Colonic Neoplasms; Cytochromes c; Fruit; Mice; Rutin; Vaccinium

Colon cancer is one of the most common causes of deaths by cancer worldwide. Stem cells have immunosuppressive properties that promote tumor targeting and circumvent obstacles currently in gene therapy. Bone marrow stem cells are believed to have anticancer potential. The transplantation of mesenchymal stem cells (MSCs), a type of bone marrow stem cells, has been considered a potential therapy for patients with solid tumors due to their capability to enhance the immune response; MSC transplantation has received renewed interest in recent years. The present study aimed to evaluate the antiapoptotic effects of the MSCs on 1,2-dimethylhydrazine (DMH)-induced inflammation in the rat model of colorectal cancer. The rats were randomly allocated into four groups: control, treated with MSCs, induced by DMH, and induced by DMH and treated with MSCs. The MSCs were intra-rectally injected, and DMH was subcutaneously injected at 20 mg/kg body weight once a week for 15 weeks. The administration of MSCs into rats starting from day 0 of the DMH injection was found to enhance the histopathological picture. The MSC treatment resulted in fewer inflammatory cells than in the DMH group. Therefore, our findings suggest that BMCs have antitumor effects by modulating the cellular redox status and down-regulating the pro-inflammatory genes. Thus, BMCs may provide therapeutic value for colon cancer treatment.

Topics: 1,2-Dimethylhydrazine; Animals; Apoptosis; Carcinogens; Cells, Cultured; Colonic Neoplasms; Cytochromes c; Male; Mesenchymal Stem Cell Transplantation; Oxidative Stress; Rats; Rats, Wistar

To preserve bioactivity and achieve colon targeted release of phycocyanin (PC), the polysaccharides-based electrospun fiber mat (EFM) containing PC and prebiotics was prepared and characterized. In vitro release tests confirmed the colon targeting behavior of PC, in particular, faster release of PC was achieved due to the addition of prebiotics. Ritger-Peppas model confirmed that the release of PC in simulated colon fluids follows a mechanism of anomalous transport (non-Fickian). CCK-8 results showed that the combination of PC and prebiotics exerted a significant anti-proliferative effect on HCT116 cells with an IC50 values of 22.31, 17.12 and 11.63 mg/mL after 24, 48, and 72 h, respectively. Furthermore, the cell cycle and apoptosis analysis revealed that the inhibition activity on HCT116 cells was caused by arresting cell cycle at G0/G1 phase that is relevant to the inhibition of cyclin D1 and CDK4 and the up-regulation of p21 expression, and inducing cell apoptosis by mediating the mitochondrial pathway as well, in which the decrease of Bcl-2/Bax, activation of caspase 3 and release of cytochrome c were included. This study suggests that the PC-loaded EFM with GOS holds a great potential as an effective formulation for colon cancer prevention.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Colon; Colonic Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; HCT116 Cells; Humans; Phycocyanin; Polysaccharides; Prebiotics; Proto-Oncogene Proteins c-bcl-2; Sincalide

BACKGROUND In developed countries, colon cancer is a leading cause of cancer-associated mortality. Dietary changes have resulted in an increased incidence of colon cancer in Asia. This study aimed to investigate the effects of the structural analog of endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH₂) on human colon cancer cells in vitro. MATERIAL AND METHODS Human DLD-1 and RKO colon cancer cells and CCD-18Co normal human colonic fibroblasts were treated with increasing doses of the structural analog of endomorphin-2. Cells underwent the MTT assay, fluorescence confocal flow cytometry, and Hoechst 33258 staining to investigate cell proliferation, the cell cycle, and apoptosis. Western blot was used to measure the expression levels of poly(ADP-ribose) polymerase-1 (PARP-1), cytochrome c, caspase-3, and caspase-9. The 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence method measured reactive oxygen species (ROS). RESULTS Cell proliferation of DLD-1 and RKO cells was inhibited by the endomorphin-2 analog in a dose-dependent manner, and a 100 µM dose reduced DLD-1 and RKO cell proliferation by 28% and 23%, respectively, at 72 h. Endomorphin-2 analog induced cell apoptosis and the generation of ROS, activated caspase-3 and caspase-9, and increased the levels of p53 and cytochrome c release, and down-regulated of Akt activation in DLD-1 and RKO cells in a dose-dependent manner. Treatment of the DLD-1 and RKO cells with the endomorphin-2 analog increased the expression of Bax and reduced the expression of Bcl-2. CONCLUSIONS Endomorphin-2 analog inhibited colon cancer cell proliferation, activated apoptosis, and down-regulated Akt phosphorylation of human DLD-1 and RKO colon cancer cells in vitro in a dose-dependent manner.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colon; Colonic Neoplasms; Cytochromes c; Humans; Oligopeptides; Poly (ADP-Ribose) Polymerase-1; Reactive Oxygen Species

This study aimed at investigating the anticancer activity of an exopolysaccharide (EPS) isolated from Lactobacillus helveticus MB2-1. The crude EPS from L. helveticus MB2-1 (LHEPS) was fractionated into three fractions, namely LHEPS-1, LHEPS-2 and LHEPS-3. LHEPS-1 exhibited the most effective anti-proliferative activity, which was associated with a stronger inhibition rate and increased lactate dehydrogenase leakage of human colon cancer HT-29 cells. Flow cytometry analysis and colorimetric assay revealed that LHEPS-1 induced cell cycle arrest by preventing G1 to S transition and increased the apoptosis rate. Furthermore, LHEPS-1 enhanced the production of intracellular reactive oxygen species (ROS) and the activity of caspases-8/9/3, increased the levels of pro-apoptotic Bax and mitochondrial cytochrome c, while decreased the anti-apoptotic Bcl-2 level, indicating that LHEPS-1 might induce the apoptosis of HT-29 cells through a ROS-dependent pathway and a mitochondria-dependent pathway. These findings suggest that LHEPS-1 may be developed as an effective food and/or drug for the prevention and therapeutics of cancer, especially human colon cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Proliferation; Colonic Neoplasms; Cytochromes c; HT29 Cells; Humans; Lactobacillus helveticus; Mitochondria; Polysaccharides; Reactive Oxygen Species

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer cytokine with minimal toxicity towards normal cells. Nevertheless, most primary cancers are often intrinsically TRAIL-resistant or can acquire resistance after TRAIL therapy. This study aimed to investigate the inhibitory effect of co-treatment of 3-bromopyruvate (3-BP) as a potent anticancer agent with TRAIL on colon cancer cells (HT-29). The results of present study indicated that combined treatment with 3-BP and TRAIL inhibited the proliferation of HT-29 cells to a greater extent (88.4%) compared with 3-BP (54%) or TRAIL (11%) treatment alone. In contrast, the combination of 3-BP and TRAIL had no significant inhibitory effect on the proliferation of normal cells (HEK-293) (8.4%). At a cellular mechanistic level, the present study showed that 3-BP sensitized human colon cancer cells to TRAIL-induced apoptosis via reactive oxygen species generation, upregulation of Bax, downregulation of Bcl-2 and survivin, release of cytochrome c into the cytosol, and activation of caspase-3. In normal cells, 3-BP, TRAIL, or combination of both had no significant effect on the reactive oxygen species levels, release of cytochrome c, and caspase-3 activity. Therefore, the combination of 3-BP and TRAIL can be a promising therapeutic strategy for treatment of colon cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Colonic Neoplasms; Cytochromes c; Down-Regulation; Drug Synergism; Enzyme Activation; HEK293 Cells; HT29 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Pyruvates; Reactive Oxygen Species; Survivin; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

To search novel therapy for human colon cancer, scutellarein identified from

Topics: Apigenin; Apoptosis; Colonic Neoplasms; Cytochromes c; HCT116 Cells; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Scutellaria

Pyroptosis, a form of programmed cell death (PCD), has garnered increasing attention as it relates to innate immunity and diseases. However, the involvement of pyroptosis in the mechanism by which lobaplatin acts against colorectal cancer (CRC) is unclear. Our study revealed that treatment with lobaplatin reduced the viability of HT-29 and HCT116 cells in a dose-dependent manner. Morphologically, HT-29 and HCT116 cells treated with lobaplatin exhibited microscopic features of cell swelling and large bubbles emerging from the plasma membrane, and transmission electron microscopy (TEM) revealed multiple pores in the membrane. GSDME, rather than GSDMD, was cleaved in lobaplatin-induced pyroptosis in HT-29 and HCT116 cells due to caspase-3 activation. Knocking out GSDME switched lobaplatin-induced cell death from pyroptosis to apoptosis but did not affect lobaplatin-mediated inhibition of growth and tumour formation of HT-29 and HCT116 cells in vivo and in vitro. Further investigation indicates that lobaplatin induced reactive oxygen species (ROS) elevation and JNK phosphorylation. NAC, a ROS scavenger, completely reversed the pyroptosis of lobaplatin-treated HT-29 and HCT116 and JNK phosphorylation. Activated JNK recruited Bax to mitochondria, and thereby stimulated cytochrome c release to cytosol, followed by caspase-3/-9 cleavage and pyroptosis induction. Therefore, in colon cancer cells, GSDME mediates lobaplatin-induced pyroptosis downstream of the ROS/JNK/Bax-mitochondrial apoptotic pathway and caspase-3/-9 activation. Our study indicated that GSDME-dependent pyroptosis is an unrecognized mechanism by which lobaplatin eradicates neoplastic cells, which may have important implications for the clinical application of anticancer therapeutics.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caco-2 Cells; Caspase 3; Cell Survival; Colonic Neoplasms; Cyclobutanes; Cytochromes c; HCT116 Cells; HT29 Cells; Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase 4; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Organoplatinum Compounds; Phosphate-Binding Proteins; Pyroptosis; Reactive Oxygen Species; Receptors, Estrogen; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

The application of plant extracts for therapeutic purposes has been used in traditional medicine since the plants are a source of a great variety of chemical compounds that possess biological activity. Actually, the effect of these extracts on diseases such as cancer is being widely studied. Colorectal adenocarcinoma is one of the main causes of cancer related to death and the second most prevalent carcinoma in Western countries. The aim of this work is to study the possible effect of two fenugreek (Trigonella foenum graecum) protein hydrolysates on treatment and progression of colorectal cancer. Fenugreek proteins from seeds were hydrolysed by using two enzymes separately, which are named Purafect and Esperase, and were then tested on differentiated and undifferentiated human colonic adenocarcinoma Caco2/TC7 cells. Both hydrolysates did not affect the growth of differentiated cells, while they caused a decrease in undifferentiated cell proliferation by early apoptosis and cell cycle arrest in phase G1. This was triggered by a mitochondrial membrane permeabilization, cytochrome C release to cytoplasm, and caspase-3 activation. In addition, the hydrolysates of fenugreek proteins displayed antioxidant activity since they reduce the intracellular levels of ROS. These findings suggest that fenugreek protein hydrolysates could be used as nutraceutical molecules in colorectal cancer treatment.

Topics: Apoptosis; Caco-2 Cells; Caspase 3; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Dietary Supplements; Gene Expression Regulation, Neoplastic; Humans; Plant Proteins; Protein Hydrolysates; Reactive Oxygen Species; Thioredoxin Reductase 1; Trigonella

In this study, the prebiotic potential of soluble dietary fibre extracted from plantain inflorescence (PIF) was investigated. PIF demonstrated prebiotic potential by enhancing the growth of the probiotics under study and thereby hindered colon cancer development. The soluble dietary fibre from Musa paradisiaca inflorescence (PIF) was fermented using Lactobacillus casei and Bifidobacterium bifidum. The fermentation supernatants (LS and BS) were enriched with short chain fatty acids (SCFA) and were able to initiate apoptotic signalling in HT29 colon cancer cells leading to cell death. Both BS and LS exhibited cytotoxic effect; induced DNA damage and enhanced generation of reactive oxygen species in HT29 cells leading to apoptosis. The induction of apoptosis was facilitated by the reduction of membrane potential of mitochondria and ATP synthesis; enhanced delivery of cytochrome c and interference with the expression of pro/antiapoptotic proteins. BS, which exhibited better activity, was further analysed for the identification of differentially regulated proteins by performing two dimensional electrophoresis and MALDI-TOF mass spectrometry. Results emphasized on the fact that, the exposure to BSalteredthe HT29 proteins expression, particularly the upregulation of apoptosis- inducing factor-AIFM1 leading to apoptosis of HT29 cells.

Topics: Apoptosis; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Dietary Fiber; Fatty Acids, Volatile; Fermentation; HT29 Cells; Humans; Musa; Probiotics

Colorectal cancer (CRC) is one of the leading causes of cancer death, and diet plays an important role in the etiology of CRC. Traditional medical practitioners in many South Asian countries use plantain inflorescence to treat various gastro-intestinal ailments. The aim of the present study was to investigate the anticancer effects of extracts of inflorescence of Musa paradisiaca against HT29 human colon cancer cells and elucidate the mechanism of these effects by studying the modulation of cascades of transcriptional events. In vitro assays depicted that methanol extract of Musa paradisiaca inflorescence (PIMET) was cytotoxic to HT29 cells. PIMET induced DNA damage and arrested the cell cycle at the G2/M phase. Expression studies showed that PIMET pretreatment upregulates pro-apoptotic Bcl2 and downregulates anti-apoptotic Bax proteins. Different assays showed that the deregulation of pro/antiapoptotic proteins reduces the mitochondrial membrane potential and ATP production; moreover, it enhances cytochrome c release, which triggers the apoptotic pathway, and further cleaves caspase 3 and PARP proteins, resulting in apoptosis. Changes in the protein expression profile of HT29 cells after PIMET treatment were analyzed using mass-spectrometry-based proteomics. PIMET treatment significantly altered the expression of HT29 protein; interestingly, X-linked inhibitor of apoptosis protein was also downregulated. Alteration in the expression of this protein has significant effects, leading to HT29 cell death.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Inflorescence; Musa; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Transcription, Genetic

Epidemiology and evidence have demonstrated that colon carcinoma is one of the most common gastrointestinal tumors in the clinic. Reports have suggested that Tunicamycin significantly inhibits aggressiveness of colon carcinoma cells by promotion of apoptosis. In the present study, the inhibitory effect of tunicamycin on colon cancer cells and the potential underlying molecular mechanism was investigated. Western blotting, immunohistochemistry, apoptotic assays and immunofluorescence were used to analyze the therapeutic effects of tunicamycin on apoptosis, growth, aggressiveness and cell cycle of colon tumor cells, by downregulation of fibronectin, vimentin and E‑cadherin expression levels. In vitro experiments demonstrated that tunicamycin significantly inhibited growth, migration and invasion of colon carcinoma cells. In addition, tunicamycin administration promoted apoptosis of colon carcinoma cells via upregulation of apoptotic protease activating factor 1 and cytochrome c expression levels, which are proteins that have a role in mitochondrial apoptosis signaling. Cell cycle assays revealed that tunicamycin suppressed proliferation and arrested S phase entry of colon carcinoma cells. Mechanistic analysis demonstrated that tunicamycin reduced expression and phosphorylation levels of extracellular signal‑regulated kinase (ERK), c‑JUN N‑terminal kinase (JNK) and protein kinase B (AKT), and inhibited mammalian target of rapamycin (mTOR) expression levels in colon carcinoma cells. Endogenous overexpression of ERK inhibited tunicamycin‑mediated downregulation of JNK, AKT and mTOR expression, which further blocked tunicamycin‑mediated inhibition of growth and aggressiveness of colon carcinoma. In vivo assays revealed that tunicamycin treatment significantly inhibited tumor growth and promoted apoptosis, which led to long‑term survival of tumor‑bearing mice compared with the control group. In conclusion, these results suggested that tunicamycin may inhibit growth and aggressiveness of colon cancer via the ERK‑JNK‑mediated AKT/mTOR signaling pathway, and suggested that tunicamycin may be a potential anti‑cancer agent for colon carcinoma therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cadherins; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Kinase 4; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Receptor, PAR-1; Signal Transduction; Survival Analysis; TOR Serine-Threonine Kinases; Tumor Burden; Tunicamycin; Vimentin; Xenograft Model Antitumor Assays

As a natural compound, Ornithogalum caudatum Ait is primarily used as an anti-inflammatory and antitumor agent in Chinese folk medicine. In 1992, OSW-1 was isolated from this compound, which is a new member of cholestane saponin family. In numerous recent studies, OSW-1 has been shown to have powerful cytotoxic anticancer effects against various malignant cells. However, the therapeutic efficacy of OSW-1 on colon cancer and the underlying mechanism are not understood. To explore the mechanism underlying OSW-1 in antitumor therapy, a therapeutic function analysis of OSW-1 on colon cancer was performed in vitro and in vivo. It was shown that with low toxicity on normal colonic cells, OSW-1 suppresses colon cancer cells in vitro and this inhibition was via the intrinsic apoptotic pathway, which increased cellular calcium, changed mitochondrial membrane potential, disrupted mitochondrial morphology, and led to the release of cytochrome c and the activation of caspase-3. Furthermore, in a nude mouse model, OSW-1 had a powerful effect on suppressing colon tumor proliferation without significant side effects through the apoptosis pathway. Taken together, these results demonstrate that OSW-1 is a potential drug for colon cancer treatment.

Topics: Animals; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cholestenones; Colonic Neoplasms; Cytochromes c; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mice; Saponins; Xenograft Model Antitumor Assays

Resveratrol, a polyphenolic compound, is a naturally occurring phytochemical and is found in a variety of plants, including grapes, berries and peanuts. It has gained much attention for its potential anticancer activity against various types of human cancer. However, the usefulness of resveratrol as a chemotherapeutic agent is limited by its photosensitivity and metabolic instability. In this study the effects of a synthetic analogue of resveratrol, HS-1793, on the proliferation and apoptotic cell death were investigated using HCT116 human colon cancer cells. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of HS-1793 on human colon cancer and its mechanisms of action have not been extensively studied. HS-1793 inhibited cell growth and induced apoptotic cell death in a concentration-dependent fashion. Induction of apoptosis was determined by morphological changes, cleavage of poly(ADP-ribose) polymerase, alteration of Bax/Bcl-2 expression ratio, and caspase activations. Flow cytometric analysis revealed that HS-1793 induced G2/M arrest in the cell cycle progression in HCT116 cells. Furthermore, HS-1793 showed more potent anticancer effects in several aspects than resveratrol in HCT116 cells. In addition, HS-1793 suppressed Akt and the phosphatidylinositol-3 kinase/Akt inhibitor LY294002 was found to enhance its induction of apoptosis. Thus, these findings suggest that HS-1793 have potential as a candidate chemotherapeutic agent against human colon cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Proliferation; Chromones; Colonic Neoplasms; Cytochromes c; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; Humans; Morpholines; Naphthols; Phosphorylation; Proto-Oncogene Proteins c-akt; Resorcinols; Resveratrol; Stilbenes

Wasabia japonica (wasabi) has been shown to exhibit properties of detoxification, anti-inflammation and the induction of apoptosis in cancer cells. This study aimed to investigate the molecular mechanism of the cytotoxicity of wasabi extract (WE) in colon cancer cells to evaluate the potential of wasabi as a functional food for chemoprevention.. Colo 205 cells were treated with different doses of WE, and the cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Apoptosis and autophagy were detected by 4',6-diamidino-2-phenylindole, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-yanine iodide and staining for acidic vascular organelles (AVOs), along with Western blotting.. The results demonstrated that WE induced the extrinsic pathway and mitochondrial death machinery through the activation of TNF-α, Fas-L, caspases, truncated Bid and cytochrome C. WE also induced autophagy by decreasing the phosphorylation of Akt and mTOR and promoting the expression of microtubule-associated protein 1 light chain 3-II and AVO formation. An in vivo xenograft model verified that tumor growth was delayed by WE treatment.. Our studies revealed that WE exhibits anti-colon cancer properties through the induction of apoptosis and autophagy. These results provide support for the application of WE as a chemopreventive functional food and as a prospective treatment of colon cancer.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Autophagy; BH3 Interacting Domain Death Agonist Protein; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; Humans; Indoles; Mice; Mice, Inbred BALB C; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Phosphorylation; Plant Extracts; Proto-Oncogene Proteins c-akt; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Wasabia; Xenograft Model Antitumor Assays

The newly-synthesized naftopidil analog HUHS1015 suppresses tumor growth and induces apoptosis of cells from a variety of cancer types. The present study was conduced to assess the effect of HUHS1015 on human colonic cancer cells and to clarify the underlying mechanism.. HUHS1015 reduced cell viability of Caco-2 and CW2 human colonic cancer cell lines in a concentration (0.3-100 mM)-dependent manner. HUHS1015 increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in both cell lines. In flow cytometry using propidium iodide and annexin V, HUHS1015 significantly increased the populations of cells undergoing primary necrosis, early apoptosis, and late apoptosis/secondary necrosis in both cell lines. In the cell-cycle analysis, HUHS1015 increased the proportion of the sub-G1 phase of cell, which corresponds to apoptotic cells. HUHS1015 perturbed the mitochondrial membrane potential and reduced the intracellular ATP level. HUHS1015 activated caspases 3, -4, -8, and -9, particularly caspase-3. HUHS1015 promoted cytochrome c release from the mitochondria. HUHS1015 significantly inhibited tumor growth in mice inoculated with CW2 cells.. HUHS1015 induces necrosis by lowering the intracellular ATP level in association with mitochondrial damage and caspase-dependent apoptosis. This occurs in part by stimulating cytochrome c release from the mitochondria to activate caspase-9 followed by the effector caspase-3, responsible for suppression of colonic cancer proliferation in the mouse xenograft model.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Antineoplastic Agents; Apoptosis; Caco-2 Cells; Caspases; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Humans; Male; Membrane Potential, Mitochondrial; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Necrosis; Propanolamines; Time Factors; Tumor Burden; 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

Doxycycline (DOX) exhibits anti-inflammatory, anti-tumor, and pro-apoptotic activity and is being tested in clinical trials as a chemotherapeutic agent for several cancers, including colon cancer.. In the current study, the chemotherapeutic activity of doxycycline was tested in a rat model of colon carcinogenesis, induced by colon specific cancer promoter, 1,2, dimethylhydrazine (DMH) as well as study the effect of DOX-alone on a separate group of rats.. Doxycycline administration in DMH-treated rats (DMH-DOX) unexpectedly increased tumor multiplicity, stimulated progression of colonic tumor growth from adenomas to carcinomas and revealed metastasis in small intestine as determined by macroscopic and histopathological analysis. DOX-alone treatment showed markedly enhanced chronic inflammation and reactive hyperplasia, which was dependent upon the dose of doxycycline administered. Moreover, immunohistochemical analysis revealed evidence of inflammation and anti-apoptotic action of DOX by deregulation of various biomarkers.. These results suggest that doxycycline caused chronic inflammation in colon, small intestine injury, enhanced the efficacy of DMH in tumor progression and provided a mechanistic link between doxycycline-induced chronic inflammation and tumorigenesis. Ongoing studies thus may need to focus on the molecular mechanisms of doxycycline action, which lead to its inflammatory and tumorigenic effects.

Topics: 1,2-Dimethylhydrazine; Animals; Body Weight; Carcinogenesis; Caspase 3; Caspase 9; Cell Proliferation; Chronic Disease; Colonic Neoplasms; Cytochromes c; Down-Regulation; Doxycycline; Immunohistochemistry; Inflammation; Intestine, Small; Male; Matrix Metalloproteinase 9; Neoplasm Metastasis; NF-kappa B; Rats, Sprague-Dawley; Tumor Suppressor Protein p53; Up-Regulation; Vascular Endothelial Growth Factor A

Plants in the Meliaceae family are known to possess interesting biological activities, such as antimalaral, antihypertensive and antitumour activities. Previously, our group reported the plant-derived compound cycloart-24-ene-26-ol-3-one isolated from the hexane extracts of Aglaia exima leaves, which shows cytotoxicity towards various cancer cell lines, in particular, colon cancer cell lines. In this report, we further demonstrate that cycloart-24-ene-26-ol-3-one, from here forth known as cycloartane, reduces the viability of the colon cancer cell lines HT-29 and CaCO-2 in a dose- and time-dependent manner. Further elucidation of the compound's mechanism showed that it binds to tumour necrosis factor-receptor 1 (TNF-R1) leading to the initiation of caspase-8 and, through the activation of Bid, in the activation of caspase-9. This activity causes a reduction in mitochondrial membrane potential (MMP) and the release of cytochrome-C. The activation of caspase-8 and -9 both act to commit the cancer cells to apoptosis through downstream caspase-3/7 activation, PARP cleavage and the lack of NFkB translocation into the nucleus. A molecular docking study showed that the cycloartane binds to the receptor through a hydrophobic interaction with cysteine-96 and hydrogen bonds with lysine-75 and -132. The results show that further development of the cycloartane as an anti-cancer drug is worthwhile.

Topics: Aglaia; Antineoplastic Agents; Apoptosis; Caco-2 Cells; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; HT29 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Plant Leaves; Receptors, Tumor Necrosis Factor, Type I; Triterpenes

Recent findings revealed in cancer cells novel stress response pathways, which in response to many chemotherapeutic drugs causing nucleolar stress, will function independently from tumor protein p53 (p53) and still lead to cell cycle arrest and/or apoptosis. Since it is known that most cancers lack functional p53, it is of great interest to explore these emerging molecular mechanisms. Here, we demonstrate that nucleolar stress induced by 5-fluorouracil (5-FU) in colon cancer cells devoid of p53 leads to the activation of ribosomal protein L3 (rpL3) as proapoptotic factor. rpL3, as ribosome-free form, is a negative regulator of cystathionine-β-synthase (CBS) expression at transcriptional level through a molecular mechanism involving Sp1. The rpL3-CBS association affects CBS stability and, in addition, can trigger CBS translocation into mitochondria. Consequently apoptosis will be induced through the mitochondrial apoptotic cell death pathway characterized by an increased ratio of Bax to Bcl-2, cytochrome c release and subsequent caspase activation. It is noteworthy that silencing of CBS is associated to a strong increase of 5-FU-mediated inhibition of cell migration and proliferation. These data reveal a novel mechanism to accomplish p53-independent apoptosis and suggest a potential therapeutic approach aimed at upregulating rpL3 for treating cancers lacking p53.

Topics: Antimetabolites, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Nucleolus; Colonic Neoplasms; Cystathionine beta-Synthase; Cytochromes c; Enzyme Activation; Fluorouracil; Genes, p53; HCT116 Cells; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Ribosomal Protein L3; Ribosomal Proteins; Ribosomes; Sp1 Transcription Factor; Transcription, Genetic; Tumor Suppressor Protein p53

Here, we report the p53 dependent mitochondria-mediated apoptotic mechanism of plant derived silver-nanoparticle (PD-AgNPs) in colorectal cancer cells (CRCs). PD-AgNPs was synthesized by reduction of AgNO3 with leaf extract of a medicinal plant periwinkle and characterized. Uptake of PD-AgNPs (ξ - 2.52 ± 4.31 mV) in HCT116 cells was 3 fold higher in comparison to synthetic AgNPs (ξ +2.293 ± 5.1 mV). A dose dependent increase in ROS production, activated JNK and decreased mitochondrial membrane potential (MMP) were noted in HCT116 but not in HCT116 p53(-/-) cells after PD-AgNP exposure. PD-AgNP-mediated apoptosis in CRCs is a p53 dependent process involving ROS and JNK cascade.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; HCT116 Cells; Humans; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase Kinases; Nanoparticles; Plant Extracts; Reactive Oxygen Species; Silver; Treatment Outcome; Tumor Suppressor Protein p53

Curcuma purpurascens BI. (Zingiberaceae) commonly known as 'Koneng Tinggang' and 'Temu Tis' is a Javanese medicinal plant which has been used for numerous ailments and diseases in rural Javanese communities. In the present study, the apoptogenic activity of dichloromethane extract of Curcuma purpurascens BI. rhizome (DECPR) was investigated against HT-29 human colon cancer cells.. Acute toxicity study of DECPR was performed in Sprague-Dawley rats. Compounds of DECPR were analyzed by the gas chromatography-mass spectrometry-time of flight (GC-MS-TOF) analysis. Cytotoxic effect of DECPR on HT-29 cells was analyzed by MTT and lactate dehydrogenase (LDH) assays. Effects of DECPR on reactive oxygen species (ROS) formation and mitochondrial-initiated events were investigated using a high content screening system. The activities of the caspases were also measured using a fluorometric assay. The quantitative PCR analysis was carried out to examine the gene expression of Bax, Bcl-2 and Bcl-xl proteins.. The in vivo acute toxicity study of DECPR on rats showed the safety of this extract at the highest dose of 5 g/kg. The GC-MS-TOF analysis of DECPR detected turmerone as the major compound in dichloromethane extract. IC50 value of DECPR towards HT-29 cells after 24 h treatment was found to be 7.79 ± 0.54 μg/mL. In addition, DECPR induced LDH release and ROS generation in HT-29 cells through a mechanism involving nuclear fragmentation and cytoskeletal rearrangement. The mitochondrial-initiated events, including collapse in mitochondrial membrane potential and cytochrome c leakage was also triggered by DECPR treatment. Initiator caspase-9 and executioner caspase-3 was dose-dependently activated by DECPR. The quantitative PCR analysis on the mRNA expression of Bcl-2 family of proteins showed a significant up-regulation of Bax associated with down-regulation in Bcl-2 and Bcl-xl mRNA expression.. The findings presented in the current study showed that DECP suppressed the proliferation of HT-29 colon cancer cells and triggered the induction of apoptosis through mitochondrial-dependent pathway.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspases; Colonic Neoplasms; Curcuma; Cytochromes c; HT29 Cells; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Phytotherapy; Plant Extracts; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Rhizome; RNA, Messenger; Signal Transduction; Zingiberaceae

Standard chemotherapeutic protocols, based on maximum tolerated doses, do not prevent nor overcome chemoresistance caused by the efflux transporter P-glycoprotein (Pgp). We compared the effects of two consecutive low doses versus a single high dose of doxorubicin in drug-sensitive Pgp-negative and drug-resistant Pgp-positive human and murine cancer cells. Two consecutive low doses were significantly more cytotoxic in vitro and in vivo against drug-resistant tumors, while a single high dose failed to do so. The greater efficacy of two consecutive low doses of doxorubicin could be linked to increased levels of intracellular reactive oxygen species. These levels were produced by high electron flux from complex I to complex III of the mitochondrial respiratory chain, unrelated to the synthesis of ATP. This process induced mitochondrial oxidative damage, loss of mitochondrial potential and activation of the cytochrome c/caspase 9/caspase 3 pro-apoptotic axis in drug-resistant cells. Our work shows that the "apparent" ineffectiveness of doxorubicin against drug-resistant tumors overexpressing Pgp can be overcome by changing the timing of its administration and its doses.

Topics: Animals; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caspase 3; Caspase 9; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Drug Administration Schedule; Drug Resistance, Neoplasm; Female; HT29 Cells; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mitochondria; Oxidative Stress; Random Allocation; Xenograft Model Antitumor Assays

Besides other mechanism(s) 17β-estradiol (E2) facilitates neuronal survival by increasing, via estrogen receptor β (ERβ), the levels of neuroglobin (NGB) an anti-apoptotic protein. In contrast, E2 could exert protective effects in cancer cells by activating apoptosis when the ERβ level prevails on that of ERα as in colon cancer cell lines. These apparently contrasting results raise the possibility that E2-induced NGB up-regulation could regulate the ERβ activities shunning this receptor subtype to trigger an apoptotic cascade in neurons but not in non-neuronal cells. Here, human colorectal adenocarcinoma cell line (DLD-1) that only expresses ERβ and HeLa cells transiently transfected with ERβ encoding vector has been used to verify this hypothesis. In addition, neuroblastoma SK-N-BE cells were used as positive control. Surprisingly, E2 also induced NGB up-regulation, in a dose- and time-dependent manner, in DLD-1 cells. The ERβ-mediated activation of p38/MAPK was necessary for this E2 effect. E2 induced NGB re-allocation in mitochondria where, subsequently to an oxidative stress injury (i.e., 100μM H2O2), NGB interacted with cytochrome c preventing its release into the cytosol and the activation of an apoptotic cascade. As a whole, these results demonstrate that E2-induced NGB up-regulation could act as an oxidative stress sensor, which does not oppose to the pro-apoptotic E2 effect in ERβ-containing colon cancer cells unless a rise of oxidative stress occurs. These results support the concept that oxidative stress plays a critical role in E2-induced carcinogenesis and further open an important scenario to develop novel therapeutic strategies that target NGB against E2-related cancers.

Topics: Apoptosis; Cell Line, Tumor; Colon; Colonic Neoplasms; Cytochromes c; Estradiol; Estrogen Receptor beta; Gene Expression Regulation, Neoplastic; Globins; Humans; Mitochondria; Nerve Tissue Proteins; Neuroglobin; Oxidative Stress; RNA, Messenger; Up-Regulation

The development of metal-based agents has had a tremendous role in the present progress in cancer chemotherapy. One well-known example of metal-based agents is Schiff based metal complexes, which hold great promise for cancer therapy. Based on the potential of Schiff based complexes for the induction of apoptosis, this study aimed to examine the cytotoxic and apoptotic activity of a CdCl2(C14H21N3O2) complex on HT-29 cells. The complex exerted a potent suppressive effect on HT-29 cells with an IC50 value of 2.57 ± 0.39 after 72 h of treatment. The collapse of the mitochondrial membrane potential and the elevated release of cytochrome c from the mitochondria to the cytosol indicate the involvement of the intrinsic pathway in the induction of apoptosis. The role of the mitochondria-dependent apoptotic pathway was further proved by the significant activation of the initiator caspase-9 and the executioner caspases-3 and -7. In addition, the activation of caspase-8, which is associated with the suppression of NF-κB translocation to the nucleus, also revealed the involvement of the extrinsic pathway in the induced apoptosis. The results suggest that the CdCl2(C14H21N3O2) complex is able to induce the apoptosis of colon cancer cells and is a potential candidate for future cancer studies.

Topics: Apoptosis; Cadmium Chloride; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; HT29 Cells; Humans; L-Lactate Dehydrogenase; Matrix Metalloproteinases; Microscopy, Confocal; Mitochondria; NF-kappa B; Protein Transport; Reactive Oxygen Species; Schiff Bases; Signal Transduction

Direct antitumor effects of bisphosphonates (BPs) have been demonstrated in various cancer cells in vitro. However, the effective concentrations of BPs are typically much higher than their clinically relevant concentrations. Oral cancers frequently invade jawbone and may lead to the release of Ca(2+) in primary lesions. We investigated the effects of the combined application of zoledronic acid (ZA) and Ca(2+) on proliferation and apoptosis of oral cancer cells. Human oral cancer cells, breast cancer cells, and colon cancer cells were treated with ZA at a wide range of concentrations in different Ca(2+) concentration environments. Under a standard Ca(2+) concentration (0.6mM), micromolar concentrations of ZA were required to inhibit oral cancer cell proliferation. Increasing extracellular Ca(2+) concentrations greatly enhanced the potency of the ZA cytocidal effect. The ability of Ca(2+) to enhance the cytocidal effects of ZA was negated by the Ca(2+)-selective chelator EGTA. In contrast, the cytocidal effect of ZA was less pronounced in breast and colon cancer cells regardless of whether extracellular Ca(2+) was elevated. In oral cancer cells incubated with 1.6mM Ca(2+), ZA up-regulated mitochondrial Bax expression and increased mitochondrial Ca(2+) uptake. This was associated with decreased mitochondrial membrane potential and increased release of cytochrome c. We suggest that ZA can specifically produce potent cytocidal activity in oral cancer cells in an extracellular Ca(2+)-dependent manner, implying that BPs may be useful for treatment of oral squamous cell carcinoma with jawbone invasion leading to the hypercalcemic state.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Calcium; Calcium Chelating Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Diphosphonates; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Imidazoles; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; Zoledronic Acid

Cancer stem cells (CSCs) are shown to be responsible for initiation and progression of tumors in a variety of cancers. We previously showed that anthocyanin-containing baked purple-fleshed potato (PP) extracts (PA) suppressed early and advanced human colon cancer cell proliferation and induced apoptosis, but their effect on colon CSCs is not known. Considering the evidence of bioactive compounds, such as anthocyanins, against cancers, there is a critical need to study anticancer activity of PP, a global food crop, against colon CSCs. Thus, isolated colon CSCs (positive for CD44, CD133 and ALDH1b1 markers) with functioning p53 and shRNA-attenuated p53 were treated with PA at 5.0 μg/ml. Effects of baked PP (20% wt/wt) against colon CSCs were also tested in vivo in mice with azoxymethane-induced colon tumorigenesis. Effects of PA/PP were compared to positive control sulindac. In vitro, PA suppressed proliferation and elevated apoptosis in a p53-independent manner in colon CSCs. PA, but not sulindac, suppressed levels of Wnt pathway effector β-catenin (a critical regulator of CSC proliferation) and its downstream proteins (c-Myc and cyclin D1) and elevated Bax and cytochrome c, proteins-mediating mitochondrial apoptosis. In vivo, PP reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis and suppressed tumor incidence similar to that of sulindac. Combined, our data suggest that PP may contribute to reduced colon CSCs number and tumor incidence in vivo via suppression of Wnt/β-catenin signaling and elevation of mitochondria-mediated apoptosis.

Topics: Animals; Anthocyanins; Antineoplastic Agents; Apoptosis; Azoxymethane; bcl-2-Associated X Protein; beta Catenin; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Colonic Neoplasms; Cytochromes c; Food; Humans; In Situ Nick-End Labeling; Lentivirus; Male; Mice; Mitochondria; Neoplastic Stem Cells; RNA, Small Interfering; Solanum tuberosum; Sulindac; Tumor Suppressor Protein p53; Wnt Proteins

Monitoring the effects of treatment in malignant diseases is very important in study of the influence on the cell metabolism. Energy production in cancer cells is abnormally dependent on aerobic glycolysis. In addition to the dependency on glycolysis, cancer cells have other atypical metabolic characteristics. The purpose of the present study is to evaluation and analysis of the colon cancer cells under anti-angiogenic treatment, to establish the changes in the cellular energy metabolism and apoptotic potential. Anti-angiogenic drugs block the vascular endothelial growth factors, preventing the formation of new vessels.. We use immunohistochemical analysis of cytochrome c release and histoenzymatic analysis of adenosine triphosphatase (ATP-ase), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) enzymes. Colorectal tumor tissue samples were obtained by biopsy following the surgical procedures at the County Clinical Hospital of Oradea (Romania).. The obtained results show that the apoptotic potential of malignant cells increases during the anti-angiogenic treatment, in the same time the rate of glycolysis increases, due to installed hypoxia and reduced ATP synthesis. Our results have been confirmed by international studies too.. It was been demonstrated that the apoptotic potential of malignant cells increases significantly during anti-angiogenic treatment. There is growing evidence that cancer's "Achilles' heel" is tumor cell metabolism.

Topics: Adenosine Triphosphatases; Apoptosis; Colonic Neoplasms; Cytochromes c; Cytosol; Female; Humans; Immunohistochemistry; L-Lactate Dehydrogenase; Male; Middle Aged; Neoplasm Metastasis; Succinate Dehydrogenase

Cytotoxic activity of 5,7-dimethoxy-2-phenyl-N-propylquinolin-4-amine (DMPPQA) was investigated in human colon cancer cells HCT-116 and umbilical vein endothelial cell line HUVEC. The IC(50) of DMPPQA on HCT-116 and HUVEC cells were respectively 1.26 and 7.43 µM after 72 h treatment. DMPPQA inhibited the growth of HCT-116 and HUVEC cells in concentration- and time-dependent manners. Typical morphological changes of apoptotic body formation were seen after DMPPQA with Hoechst 33258 staining. FCM analysis showed that DMPPQA induced apoptosis, mitochondrial membrane potential loss (ΔΨm) and increase in the production of intracellular reactive oxygen species (ROS) of HCT-116 cells. After treating with DMPPQA, apoptosis-related protein expression of Bax, cytochrome c, caspase-9, caspase-3, PARP-1 and P53 increased and Bcl-2 protein expression decreased. DMPPQA treatment of HUVECs reduced cell migration and microcapillary tube formation in a Matrigel matrix. It also decreased VEGF protein expression. Thus DMPPQA acts as an angiogenesis inhibitor and induces cell apoptosis by a caspase-dependent mitochondrial pathway.

Topics: Aminoquinolines; Angiogenesis Inhibitors; Apoptosis; bcl-2-Associated X Protein; Caspases; Colonic Neoplasms; Cytochromes c; HCT116 Cells; Human Umbilical Vein Endothelial Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Tumor Suppressor Protein p53

We previously noted that kaempferol, a flavonol present in vegetables and fruits, reduced cell cycle progression of HT-29 cells. To examine whether kaempferol induces apoptosis of HT-29 cells and to explore the underlying molecular mechanisms, cells were treated with various concentrations (0-60 μmol/L) of kaempferol and analyzed by Hoechst staining, Annexin V staining, JC-1 labeling of the mitochondria, immunoprecipitation, in vitro kinase assays, Western blot analyses, and caspase-8 assays. Kaempferol increased chromatin condensation, DNA fragmentation and the number of early apoptotic cells in HT-29 cells in a dose-dependent manner. In addition, kaempferol increased the levels of cleaved caspase-9, caspase-3 and caspase-7 as well as those of cleaved poly (ADP-ribose) polymerase. Moreover, it increased mitochondrial membrane permeability and cytosolic cytochrome c concentrations. Further, kaempferol decreased the levels of Bcl-xL proteins, but increased those of Bik. It also induced a reduction in Akt activation and Akt activity and an increase in mitochondrial Bad. Additionally, kaempferol increased the levels of membrane-bound FAS ligand, decreased those of uncleaved caspase-8 and intact Bid and increased caspase-8 activity. These results indicate that kaempferol induces the apoptosis of HT-29 cells via events associated with the activation of cell surface death receptors and the mitochondrial pathway.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; HT29 Cells; Humans; Kaempferols; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt

Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC50 value of 2.87  μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G1 cell population. At a concentration of 6.25  μg/ml, the Cu(BrHAP)2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents.

Topics: Apoptosis; Caspase 3; Caspase 7; Caspase 9; Cell Proliferation; Colonic Neoplasms; Copper; Cytochromes c; HT29 Cells; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Organometallic Compounds; Reactive Oxygen Species; Signal Transduction

Shogaols, the major constituents of thermally processed ginger, have been proven to be highly effective anticancer agents. Our group has identified cysteine-conjugated shogaols (M2, M2', and M2″) as the major metabolites of [6]-, [8]-, and [10]-shogaol in human and found that M2 is a carrier of its parent molecule [6]-shogaol in cancer cells and in mice, while being less toxic to normal colon fibroblast cells. The objectives of this study are to determine whether M2' and M2″ behave in a similar manner to M2, in both metabolism and efficacy as anticancer agents, and to further explore the biological pro-apoptotic mechanisms of the cysteine-conjugated shogaols against human colon cancer cells HCT-116 and HT-29. Our results show that [8]- and [10]-shogaol have similar metabolic profiles to [6]-shogaol and exhibit similar toxicity toward human colon cancer cells. M2' and M2″ both show low toxicity against normal colon cells but retain potency against colon cancer cells, suggesting that they have similar activity to M2. We further demonstrate that the cysteine-conjugated shogaols can cause cancer cell death through the activation of the mitochondrial apoptotic pathway. Our results show that oxidative stress activates a p53 pathway that ultimately leads to p53 up-regulated modulator of apoptosis (PUMA) induction and down-regulation of B-cell lymphoma 2 (Bcl-2), followed by cytochrome c release, perturbation of inhibitory interactions of X-linked inhibitor of apoptosis protein (XIAP) with caspases, and finally caspase 9 and 3 activation and cleavage. A brief screen of the markers attenuated by the proapoptotic activity of M2 revealed similar results for [8]- and [10]-shogaol and their respective cysteine-conjugated metabolites M2' and M2″. This study highlights the cysteine-conjugated metabolites of shogaols as novel dietary colon cancer preventive agents.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Caspase 9; Catechols; Cell Cycle; Colonic Neoplasms; Cysteine; Cytochromes c; HCT116 Cells; HT29 Cells; Humans; Oxidative Stress; Proto-Oncogene Proteins; Signal Transduction; Tumor Suppressor Protein p53; Zingiber officinale

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

Ibulocydine (IB), a novel prodrug of CDK inhibitor, has been reported to have anti-cancer effect in human hepatoma cells. In order to address its feasibility as a radiosensitizer to improve radiotherapeutic efficacy for human cancers, this study was designed.. Human cancer cells of lung and colon were treated with IB and/or radiotherapy (RT). The cellular effects were assessed by CCK-8, clonogenic, flow cytometric, and western blotting assays. In vivo radiotherapeutic efficacy was evaluated using the xenograft mouse model.. Combined treatment of IB and RT significantly reduced viability and survival fraction of the cells. Apoptotic cell death accompanied with activation of caspases, decrease in Bcl-2/Bax expression, loss of mitochondrial membrane potential (MMP) leading to release of cytochrome c into cytosol was observed. Recovery of Bcl-2 expression level by introducing Bcl-2 expressing plasmid DNA compromised the loss of MMP and apoptosis induced by IB and RT. In vivo therapeutic efficacy of combined treatment was verified in the xenograft mouse model, in which tumor growth was markedly delayed by RT with IB.. IB demonstrated the property of sensitizing human cancer cells to RT by induction of mitochondria-mediated apoptosis, suggesting that IB deserves to be applied for chemoradiotherapy.

Topics: Animals; Apoptosis; Caspases; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Flow Cytometry; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Pyrimidine Nucleosides; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays

In this study, we attempted to develop a multimodality approach using chemotherapeutic agent mitomycin C, biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), and mild hyperthermia to treat colon cancer. For this study, human colon cancer LS174T, LS180, HCT116 and CX-1 cells were infected with secretory TRAIL-armed adenovirus (Ad.TRAIL) and treated with chemotherapeutic agent mitomycin C and hyperthermia. The combinatorial treatment caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, dissociation of Bak from Bcl-xL, oligomerization of Bak, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed the synergistic death effect. Interestingly, Beclin-1 was dissociated from Bcl-xL and overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed dissociation of Beclin-1 from Bcl-xL. A combinatorial treatment of mitomycin C, Ad.TRAIL and hyperthermia induced Beclin-1 cleavage, but the Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells, suppressing the apoptosis induced by the combination therapy. We believe that this study supports the application of the multimodality approach to colon cancer therapy.

Topics: Adenoviridae; Antibiotics, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Beclin-1; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Genetic Vectors; Humans; Hyperthermia, Induced; MAP Kinase Kinase 4; Membrane Proteins; Mitochondria; Mitomycin; Protein Multimerization; TNF-Related Apoptosis-Inducing Ligand

Annona muricata known as "the cancer killer" has been widely used in the traditional medicine for the treatment of cancer and tumors. The purpose of this study is to investigate the anticancer properties of ethyl acetate extract of Annona muricata leaves (EEAM) on HT-29 and HCT-116 colon cancer cells and the underlying mechanisms.. The effect of EEAM on the cell proliferation of HT-29 and HCT-116 cells was analyzed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. High content screening system (HCS) was applied to investigate the cell membrane permeability, mitochondrial membrane potential (MMP), nuclear condensation and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. Flow cytometric analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. The protein expression of Bax and Bcl-2 was determined using immunofluorescence analysis. In addition, the potential of EEAM to suppress the migration and invasion of colon cancer cells was also examined.. EEAM exerted significant cytotoxic effects on HCT-116 and HT-29 cells as determined by MTT and LDH assays. After 24 h treatment, EEAM exhibited the IC₅₀ value of 11.43 ± 1.87 µg/ml and 8.98 ± 1.24 µg/ml against HT-29 and HCT-116 cells, respectively. Flow cytometric analysis demonstrated the cell cycle arrest at G1 phase and phosphatidylserine externalization confirming the induction of apoptosis. EEAM treatment caused excessive accumulation of ROS followed by disruption of MMP, cytochrome c leakage and activation of the initiator and executioner caspases in both colon cancer cells. Immunofluorescence analysis depicted the up-regulation of Bax and down-regulation of Bcl-2 proteins while treated with EEAM. Furthermore, EEAM conspicuously blocked the migration and invasion of HT-29 and HCT-116 cells.. These findings provide a scientific basis for the use of A. muricata leaves in the treatment of cancer, although further in vivo studies are still required.

Topics: Annona; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Down-Regulation; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; HT29 Cells; Humans; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mitochondria; Plant Extracts; Plant Leaves; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Up-Regulation

Different plant parts of Flacourtia indica have long been used in Ayurvedic medicine. Previous studies have demonstrated that the methanolic extract of F. indica possess anti-inflammatory properties. The present study was aimed at investigating the anticancer effects of methanol extract of Flacourtia indica (FIM) aerial parts in human colon cancer (HCT116) cells. Treatment of cells with FIM at a concentration of 500 μg/ml for 24 hours significantly reduced cell viability and induced apoptosis, which was associated with the increased cytoplasmic expression of cytochrome c, activation of caspase-3, and the cleavage of poly-(ADP-ribose) polymerase. Incubation with FIM also inhibited the levels of Bcl-2, Bcl-xl and survivin, which are the markers of cell proliferation, whereas the expression of Bax remained unchanged. Treatment with FIM led to the generation of reactive oxygen species (ROS) in a concentration-dependent manner. Pharmacological inhibition of ROS generation by pretreatment of cells with N-acetyl cysteine abrogated FIM-induced apoptosis in HCT116 cells. Thus, these results demonstrate that FIM has anti-proliferative and pro-apoptotic effects in HCT116 cells and the effects are, at least in part, due to the ROS dependent activation of caspases.

Topics: Apoptosis; bcl-X Protein; Carcinoma; Caspase 3; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Inhibitor of Apoptosis Proteins; Methanol; Plant Components, Aerial; Plant Extracts; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Salicaceae; Survivin

Crude extract of Rheum palmatum L (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the effects of CERP on LS1034 human colorectal cancer cells in vitro and also examined possible mechanisms of cell death. Flow cytometric assays were used to measure the percentage of viable cells, cell cycle distribution including the sub-G1 phase (apoptosis), the activities of caspase-8, -9, and -3, reactive oxygen species (ROS) and Ca(2+) levels, and mitochondrial membrane potential (ΔΨm). DNA damage, nuclei condensation, protein expression, and translocation were examined by Comet assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, Western blotting, and confocal laser system microscope, respectively. CERP induced apoptosis as seen by DNA fragmentation and DAPI staining in a concentration- and time-dependent manner in cancer cells. CERP was associated with an increase in the Bax/Bcl-2 protein ratio and CERP promoted the activities of caspase-8, -9, and -3. Both ROS and Ca(2+) levels were increased by CERP but the compound decreased levels of ΔΨm in LS1034 cells. Laser confocal microscope also confirmed that CERP promoted the expressions of AIF, Endo G, cytochrome c, and GADD153 to induce apoptosis through mitochondrial-dependent pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Calcium; Caspases; Cell Cycle; Cell Line, Tumor; Colonic Neoplasms; Comet Assay; Cytochromes c; DNA Damage; Humans; Indoles; Membrane Potential, Mitochondrial; Plant Extracts; Protein Transport; Reactive Oxygen Species; Rheum

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid distributed in medical herbs, exerts antitumor effects and is emerging as a promising compound for cancer prevention and therapy, but its excise mechanisms of action in colon cancer cells remains largely unknown. Here, we identified the molecular mechanisms by which UA inhibited cell proliferation and induced apoptosis in human colon cancer SW480 and LoVo cells. Treatment with UA led to significant inhibitions in cell viability and clone formation and changes in cell morphology and spreading. UA also suppressed colon cancer cell migration by inhibiting MMP9 and upregulating CDH1 expression. Further studies showed that UA inhibited the phosphorylation of Akt and ERK proteins. Pretreatment with an Akt or ERK-specific inhibitor considerably abrogated the proliferation inhibition by UA. UA also significantly inhibited colon cancer cell COX-2 expression and PGE2 production. Pretreatment with a COX-2 inhibitor (celecoxib) abrogated the UA-induced cell proliferation. Moreover, we found that UA effectively promoted NF-κB and p300 translocation from cell nuclei to cytoplasm, and attenuated the p300-mediated acetylation of NF-κB and CREB2. Pretreatment with a p300 inhibitor (roscovitine) abrogated the UA-induced cell proliferation, which is reversed by p300 overexpression. Furthermore, UA treatment induced colon cancer cell apoptosis, increased the cleavage of PARP, caspase-3 and 9, and trigged the release of cytochrome c from mitochondrial inter-membrane space into cytosol. These results indicate that UA inhibits cell proliferation and induces apoptosis in colon cancer cells through simultaneous modulation of the multiple signaling pathways such as MMP9/CDH1, Akt/ERK, COX-2/PGE2, p300/NF-κB/CREB2, and cytochrome c/caspase pathways.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Cyclooxygenase 2; Cytochromes c; Dinoprostone; E1A-Associated p300 Protein; Humans; NF-kappa B; Protein Transport; Signal Transduction; Time Factors; Triterpenes; Ursolic Acid

A novel synthetic compound 6-acetyl-9-(3,4,5-trimetho-xybenzyl)-9H-pyrido[2,3-b]indole (HAC-Y6) demonstrated selective anticancer activity. In the present study, COLO 205 cells were treated with HAC-Y6 to investigate the molecular mechanisms underlying its effects. HAC-Y6 induced growth inhibition, G2/M arrest and apoptosis in COLO 205 cells with an IC50 of 0.52±0.035 µM. Annexin V/PI double staining demonstrated the presence of apoptotic cells. JC-1 staining analysis showed that HAC-Y6 decreased mitochondrial membrane potential in support of apoptosis. An immunostaining assay revealed that HAC-Y6 depolymerized microtubules. Treatment of COLO 205 cells with HAC-Y6 resulted in increased expression of BubR1 and cyclin B1 and decreased expression of aurora A, phospho-aurora A, aurora B, phospho-aurora B and phospho-H3. HAC-Y6 treatment increased protein levels of active caspase-3, caspase-9, Endo G, AIF, Apaf-1, cytochrome c and Bax, but treatment with the compound caused reduced levels of procaspase-3, procaspase-9, Bcl-xL and Bcl-2. Overall, our results suggest that HAC-Y6 exerts anticancer effects by disrupting microtubule assembly and inducing G2/M arrest, polyploidy and apoptosis via mitochondrial pathways in COLO 205 cells.

Topics: Apoptosis; Blotting, Western; Carbolines; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Flow Cytometry; Humans; Immunoenzyme Techniques; Indoles; Membrane Potential, Mitochondrial; Mitochondria; Mitosis; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Previous studies have suggested that soy sauce contains specific bioactive components and various biological activities of soy sauce have been observed. Soy soluble polysaccharide (SSPS), a predominant bioactive compound in soy sauce, has numerous pharmacological actions, including anti‑inflammatory and immunomodulating activities. In the current study, the apoptotic effects of SSPS were investigated in HCT‑116 human colon cancer cells. Treatment with SSPS significantly inhibited cell growth in a concentration‑dependent manner by inducing apoptosis but not necrosis. This induction was associated with the generation of reactive oxygen species (ROS), mitochondrial dysfunction, activation of caspases and cleavage of the poly (ADP‑ribose) polymerase protein. Induction of apoptotic cell death of HCT‑116 cells by SSPS showed a correlation with the downregulation of members of the inhibitor of apoptosis protein family, including X‑linked inhibitor of apoptosis protein and antiapoptotic Bcl‑2, and upregulation of Bax and Bad. Administration of N‑acetyl‑L‑cysteine, a scavenger of ROS, significantly decreased SSPS‑induced apoptosis. These results indicate a critical role of signaling cascades involving a ROS‑mediated caspase pathway in the anticancer effects of SSPS.

Topics: Apoptosis; Caspases; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Enzyme Activation; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

5-Fluorouracil (5-FU) is the most widely used chemo drug for the treatment of colon cancer. However, a sub-population of colon cancer patients do not respond to 5-FU and this treatment does not provide survival benefit due to chemo resistance. The mechanisms involved in 5-FU resistance are not fully understood and multiple factors have been involved in the sensitivity of cancer cells to 5-FU. We previously reported that β6-integrin plays an important role in invasion, metastasis and degradation of extracellular matrix of colon cancer. In this study, we investigated whether β6-integrin is associated with chemo resistance in colon cancer. We found that over-expression of β6-integrin protected SW480 and HT-29 colon cancer cells from 5-FU-induced growth inhibition and apoptosis, which were accompanied by changes in cytochrome C released from the mitochondria and activity of caspase-3 and caspase-9. Moreover, β6-integrin resulted in up-regulation of Bcl-2 and down-regulation of Bax. We also found that β6-integrin induced 5-FU resistance through the ERK/MAP kinase pathway and the β6-ERK2 direct binding. The results indicate β6-integrin might be a novel therapeutic target in colon cancer therapy.

Topics: Antimetabolites, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Fluorouracil; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Integrin beta Chains; MAP Kinase Signaling System; Mitochondria; Proto-Oncogene Proteins c-bcl-2

D-limonene is recognized as a potential chemotherapeutic agent, however, the details of this mechanism remain unclear. In this study, we investigated the effects of d-limonene on colon cancer cell viability and its potential mechanism of action in vitro. After 48 h of treatment, d-limonene suppressed the viability of LS174T cells in a dose-dependent manner and caused a dose-dependent apoptotic cell death. D-limonene activated caspase-3 and -9 and PARP cleavage in a dose-dependent manner. Moreover, an increase in Bax protein and cytosol cytochrome c from mitochondria and a decrease in bcl-2 protein were observed following treatment with d-limonene. In addition, d-limonene decreased the levels of p-Akt (Ser473), p-Akt (Thr308) and p-GSK-3β (Ser9), suggesting that d-limonene induced apoptosis via the mitochondrial death pathway and the suppression of the PI3K/Akt pathway.

Topics: Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Proliferation; Colonic Neoplasms; Cyclohexenes; Cytochromes c; Cytosol; Flow Cytometry; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Limonene; Mitochondria; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Terpenes; Tumor Cells, Cultured

The purpose of this study was to investigate the anti-tumor effect and explore the mechanisms of celecoxib (a selective cyclooxygenase-2 inhibitor) combined with 5-fluorouracil (5-FU) on the treatment of human colorectal cancer in a BALB/C nude mouse subcutaneous xenograft model.. Effects of celecoxib combined with 5-FU on the proliferation of xenograft carcinoma induced by HT-29 were investigated. The apoptotic cells were detected by electron microscope and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Immunohistochemistry and Western blot were used to estimate the expression of cytochrome C, caspase-3 and caspase-9.. Compared with the control group, treatment groups showed significant inhibition of tumor growth. More apoptotic cells existed after treatment with celecoxib combined with 5-FU. Cytochrome C, caspase-3 and caspase-9 were increased in treated groups, and more obviously in the drug combination group. Cyclooxygenase-2 (COX-2) were decreased after treatment with celecoxib only or combined with 5-FU. And the combined group showed a greater decrease.. Celecoxib combined with 5-FU could inhibit the growth of tumors in vivo by inducing apoptosis and activation of the cytochrome C dependency apoptosis signal pathway. A decrease of COX-2 and an increase of cytochrome C, caspase-3 and caspase-9 may be involved in this process.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Caspases; Celecoxib; Cell Proliferation; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytochromes c; Fluorouracil; HT29 Cells; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; Injections, Subcutaneous; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Pyrazoles; Sulfonamides; Xenograft Model Antitumor Assays

Deoxynivalenol (DON), known colloquially as "vomitoxin", is a pathogenic mycotoxin produced by Fusarium fungi. Human food poisoning outbreaks, with nausea, diarrhea, and vomiting as primary symptoms, have been associated with Fusarium-infected cereals. Therefore, this study was designed to determine the molecular aspects of DON in human colon cancer cells (HT-29). To this aim, we have monitored the effects of DON on (i) cellular morphological changes via optical and transmission electron microscopy, especially in regards to cell viability and mitochondria changes, and (ii) its effects on key regulators of cell apoptosis, including cytochrome c, caspase-9, caspase-3, Bcl-2, Bax, and Bid. Our results showed that DON treatment inhibited cell proliferation, induced significant morphological changes, and promoted the activation of cytochrome c and caspases. Furthermore, changes in Bcl-2, Bax, and Bid expression were detected. The relative expression profile of Bcl-2 was contrary to that of Bax and Bid, as Bcl-2 expression decreased as the concentrations DON increased, reaching a minimum at the highest concentration of DON. We concluded that DON-induced apoptosis was caused by mitochondrial dysfunction and subsequent release of cytochrome c into the cytoplasm and successive activation of caspases, and this was likely regulated by Bcl-2 family proteins.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Cytoplasm; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Microscopy, Electron, Transmission; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Trichothecenes

Apoptosis is a synchronized procedure of cell death that is regulated by caspases and proapoptotic proteins. During apoptosis, translocation of cytochrome c, an electron carrier, from mitochondria into the cytosol is regulated by Bcl-2 family members. Cytochrome c in association with an apoptotic protease activating factor (Apaf), a proapoptotic protein essential for cell differentiation and procaspase-9 form the apoptosome complex, which consecutively activates effector caspase, caspase-3, and coordinate the implementation of apoptosis. In the current study, an attempt has been made to gain insight into piroxicam, a traditional nonsteroidal antiinflammatory drug and c-phycocyanin, a biliprotein from Spirulina platensis (cyanobacterium) mediated apoptosis in DMH-induced colon cancer. Male Sprague-Dawley rats were segregated into 5 groups: control, DMH, DMH + piroxicam, DMH + c-phycocyanin, and DMH + piroxicam + c-phycocyanin. Results illustrated that piroxicam and c-phycocyanin treatments stimulate cytochrome c release by downregulating the Bcl-2 (an antiapoptotic protein) expression significantly, while promoting the level of Bax (a proapoptotic protein), thereby activating caspases (caspases-9 and -3) and Apaf-1. The outcomes of the present study clearly signify that piroxicam and c-phycocyanin may mediate mitochondrial-dependent apoptosis in DMH-induced colon cancer. Moreover, apoptosis induction was more apparent in the combination regimen of piroxicam and c-phycocyanin than the individual drugs alone.

Topics: 1,2-Dimethylhydrazine; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Carcinogens; Caspase 3; Caspase 9; Colonic Neoplasms; Cyclooxygenase 1; Cyclooxygenase 2; Cytochromes c; Down-Regulation; Male; Membrane Proteins; Mitochondria; Phycocyanin; Piroxicam; Rats; Rats, Sprague-Dawley; Spirulina

Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.

Topics: Apoptosis; Colonic Neoplasms; Cytochromes c; Dynamins; Gene Knockdown Techniques; GTP Phosphohydrolases; HCT116 Cells; Humans; Membrane Proteins; Microtubule-Associated Proteins; Mitochondrial Membranes; Mitochondrial Proteins; RNA, Small Interfering

Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Humans; Male; Mice; Mice, Inbred BALB C; Mitochondria; Noscapine; Xenograft Model Antitumor Assays

Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by γ-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), eIF-2α (eukaryotic initiation factor 2α), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1α by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer.

Topics: Acriflavine; Animals; Apoptosis; Blotting, Western; Caspase 3; Chemoradiotherapy; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Endoplasmic Reticulum Stress; Female; Gamma Rays; HCT116 Cells; HT29 Cells; Humans; Mice; Mice, Nude; Mitochondria; Molecular Structure; Phosphorylation; Radiation Tolerance; Radiation-Sensitizing Agents; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

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

Many scientific studies have shown that laminarin has anti-tumor effects, but the anti-tumor mechanism was unclear. The purpose of this study was to investigate the effect of laminarin on the induction of apoptosis in human colon cancer LOVO cells and the molecular mechanism involved. LOVO cells were treated with different concentrations of laminarin at different times. Morphology observations were performed to determine the effects of laminarin on apoptosis of LOVO cells. Flow cytometry (FCM) was used to detect the level of intracellular reactive oxygen species (ROS) and pH. Laser scanning confocal microscope (LSCM) was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP) and mitochondrial membrane potential (MMP). Western blotd were performed to analyze the expressions of Cyt-C, Caspase-9 and -3. The results showed the apoptosis morphology, which showed cell protuberance, concentrated cytoplasm and apoptotic bodies, was obvious after 72 h treatment. Laminarin treatment for 24 h increased the intracellular level of ROS and Ca²⁺; decreased pH value; activated intracellular MPTP and decreased MMP in dose-dependent manners. It also induced the release of Cyt-C and the activation of Caspase-9 and -3. In conclusion, laminarin induces LOVO cell apoptosis through a mitochondrial pathway, suggesting that it could be a potent agent for cancer prevention and treatment.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Caspase 3; Caspase 9; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Glucans; Humans; Hydrogen-Ion Concentration; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Polysaccharides; Reactive Oxygen Species; Signal Transduction

Colon cancer is the third most common malignancy around the world. Surgery, chemotherapy, and radiotherapy are generally used to treat colon cancer, but no effective therapy for advanced colon carcinoma is available. Therefore, there is a need to identify other therapeutic agents against this disease. Magnolol, a hydroxylated biphenyl compound present in Magnolia officinalis, exerts anticancer potential and low toxicity. Emerging evidence has suggested that activation of AMP-activated protein kinase (AMPK), a potential cancer therapeutic target is involved in apoptosis in colon cancer cells. However, the effects of magnolol on human colon cancer through activation of AMPK remain unexplored. In this study, we explored whether magnolol exerts an antiproliferative effect, and induces apoptosis in HCT-116 human colon cancer cells. Magnolol displayed several apoptotic features, including propidium iodide labeling, DNA fragmentation, and caspase-3 and poly(ADP-ribose) polymerase cleavages. We showed that magnolol induced the phosphorylation of AMPK in dose- and time-dependent manners. The selective AMPK inhibitor compound C abrogated the effect of magnolol on AMPK activation, suppression of proliferation, and caspase-3 cleavage. Magnolol downregulated expression of the antiapoptotic protein Bcl2, upregulated expression of pro-apoptotic protein p53 and Bax, and caused the release of mitochondrial cytochrome c. Magnolol-induced p53 and Bcl2 expression was abolished in the presence of compound C. Magnolol inhibited migration and invasion of HCT-116 cells through AMPK activation. These findings demonstrate that AMPK mediates the anticancer effects of magnolol through apoptosis in HCT-116 cells.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Caspase 3; Cell Movement; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; HCT116 Cells; Humans; Lignans; Magnolia; Mitochondria; Phosphorylation; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Propidium; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Suppressor Protein p53

Colorectal cancer is the third leading cause of cancer-related mortality in the world. The main cause of death because of colorectal cancer is hepatic metastases, which can be treated using isolated hepatic perfusion (IHP), allowing treatment of colorectal metastasis with various methods. In this study, we present a novel potent multimodality strategy comprising humanized death receptor 4 (DR4) antibody mapatumumab in combination with oxaliplatin and hyperthermia to treat human colon cancer cells. Oxaliplatin and hyperthermia sensitized colon cancer cells to mapatumumab in the mitochondrial-dependent apoptotic pathway and increased reactive oxygen species (ROS) production, leading to Bcl-xL phosphorylation at serine 62 in a c-jun-NH2-kinase (JNK)-dependent manner. Overexpression of Bcl-xL reduced the efficacy of the multimodality treatment, whereas phosphorylation of Bcl-xL decreased its antiapoptotic activity. The multimodality treatment dissociated Bcl-xL from Bax, allowing Bax oligomerization to induce cytochrome c release from mitochondria. In addition, the multimodality treatment significantly inhibited colorectal cancer xenografts' tumor growth. The successful outcome of this study will support the application of multimodality strategy to colorectal hepatic metastases.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cell Cycle Checkpoints; Cell Line, Tumor; Colonic Neoplasms; Combined Modality Therapy; Cytochromes c; Drug Synergism; HCT116 Cells; Humans; Hyperthermia, Induced; Male; MAP Kinase Kinase 4; Mice; Mice, Nude; Mitochondria; Organoplatinum Compounds; Oxaliplatin; Phosphorylation; Protein Transport; Reactive Oxygen Species

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.

Topics: Adipose Tissue; Aldehydes; Animals; Body Weight; Cachexia; Catalase; Colonic Neoplasms; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Gene Expression; Genes, APC; GTP Phosphohydrolases; Hindlimb; Interleukin-6; Ion Channels; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Muscle; Mitochondrial Proteins; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Oxidative Phosphorylation; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Sirtuin 1; STAT3 Transcription Factor; Succinate Dehydrogenase; Superoxide Dismutase; Trans-Activators; Transcription Factors; Uncoupling Protein 3

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

Colon cancer is the third most malignant neoplasm in the world and chemoprevention through dietary intervention is an emerging option to reduce its mortality. Ellagic acid (EA) a major component of berries possesses attractive biological deeds. This study is aimed to investigate the effect of ellagic acid in fostering apoptosis in 1,2-dimethyl hydrazine (DMH) mediated experimental colon carcinogenesis model. Wistar male rats were segregated into four groups: group I-control rats, group II-rats received ellagic acid (60 mg/kg body weight p.o. every day), rats in group III-induced with DMH (20 mg/kg body weight, s.c.) for 15 weeks, DMH-induced group IV rats were initiated with ellagic acid treatment. The present study is designed to explore the significance of phosphoinositide-3-kinase (PI3K)/Akt molecular pathway as well as ellagic acid's chemopreventive effect in colon cancer. DMH-induced rats exhibited elevated expressions of PI3K and Akt as confirmed by immunofluorescence, immunoblot and confocal microscopic analysis. Mechanistically, ellagic acid was found to prevent PI3K/Akt activation that in turn, results in modulation of its downstream Bcl-2 family proteins. Bax expression and caspase-3 activation was noted after ellagic acid supplementation leading to elevation of cytochrome c (cyt c) levels and finally cell death. These observations were supported by the DNA fragmentation results, which showed the occurrence of apoptosis. This study reveals the involvement of PI3K-Akt signaling through which ellagic acid induces apoptosis and subsequently suppresses colon cancer during DMH-induced rat colon carcinogenesis. In conclusion, our findings demonstrate that ellagic acid begets apoptosis in DMH-induced colon carcinoma.

Topics: 1,2-Dimethylhydrazine; Animals; Apoptosis; Carcinogens; Caspase 3; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Ellagic Acid; Enzyme Activation; Gene Expression Regulation, Neoplastic; Male; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Subunits; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction

Methyl jasmonate (MJ) is a plant stress hormone with selective cytotoxic anti-cancer activities. The TNF-related apoptosis-inducing ligand (TRAIL) death pathway is an attractive target for cancer therapy. Although TRAIL receptors are specifically expressed in primary cancer cells and cancer cell lines, many types of cancer cells remain resistant to TRAIL-induced cytotoxicity. Here we have assessed a possible synergy between MJ and TRAIL cytotoxicity in colorectal cancer (CRC) cell lines.. CRC cell lines were pre-incubated with sub-cytotoxic concentrations of MJ followed by TRAIL administration. Cell death was determined by XTT assay and microscopy. Cytochrome c release, caspase cleavage, TRAIL-associated factors, X-linked inhibitor of apoptosis (XIAP) and survivin protein levels were detected by immunoblotting. Survivin transcription was examined by RT-PCR.. Pre-treatment with MJ resulted in increased TRAIL-induced apoptotic cell death, increased cytochrome c release and caspase cleavage. TNFRSF10A, TNFRSF10B, TNFRSF10D, Fas-associated death domain and cellular FLICE-like inhibitory protein remained unchanged during MJ-induced TRAIL sensitization, whereas MJ induced a significant decrease in survivin protein levels. Overexpression of survivin prevented MJ-induced TRAIL cytotoxicity, implying a role for survivin in MJ-induced TRAIL sensitization. MJ decreased survivin mRNA indicating that MJ may affect survivin transcription. In a β-catenin/transcription factor (TCF)-dependent luciferase activity assay, MJ decreased TCF-dependent transcriptional activity.. MJ, at sub-cytotoxic levels, sensitized CRC cells to TRAIL-induced apoptosis. Thus, combinations of MJ and TRAIL, both selective anti-cancer agents, have potential as novel treatments for CRC.

Topics: Acetates; Antineoplastic Agents; Blotting, Western; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cyclopentanes; Cytochromes c; Down-Regulation; Drug Synergism; Flow Cytometry; Genes, Reporter; Humans; Inhibitor of Apoptosis Proteins; Luciferases; Oxylipins; Receptors, Death Domain; Reverse Transcriptase Polymerase Chain Reaction; Survivin; TNF-Related Apoptosis-Inducing Ligand; Transfection

Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism.. We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison.. HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling.. All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose a plausible sequential molecular mechanism for the expression of the different proteins responsible for the intrinsic mitochondrial apoptotic pathway. Further studies with other cell lines will be needed to confirm the general nature of these findings.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspases; Colonic Neoplasms; Comet Assay; Cytochromes c; HT29 Cells; Humans; MAP Kinase Kinase 4; Proto-Oncogene Proteins c-bcl-2; Triterpenes; Tumor Suppressor Protein p53

To investigate the effect of α-mangostin on the growth and apoptosis induction of human colon cancer cells.. The three colorectal adenocarcinoma cell lines tested (COLO 205, MIP-101 and SW 620) were treated with α-mangostin to determine the effect on cell proliferation by MTT assay, cell morphology, chromatin condensation, cell cycle analysis, DNA fragmentation, phosphatidylserine exposure and changing of mitochondrial membrane potential. The molecular mechanisms of α-mangostin mediated apoptosis were further investigated by Western blotting analysis including activation of caspase cascade, cytochrome c release, Bax, Bid, p53 and Bcl-2 modifying factor.. The highest inhibitory effect of α-mangostin on cell proliferation of COLO 205, MIP-101 and SW 620 were 9.74 ± 0.85 μg/mL, 11.35 ± 1.12 μg/mL and 19.6 ± 1.53 μg/mL, respectively. Further study showed that α-mangostin induced apoptotic cell death in COLO 205 cells as indicated by membrane blebbing, chromatin condensation, DNA fragmentation, cell cycle analysis, sub-G1 peak (P < 0.05) and phosphatidylserine exposure. The executioner caspase, caspase-3, the initiator caspase, caspase-8, and caspase-9 were expressed upon treatment with α-mangostin. Further studies of apoptotic proteins were determined by Western blotting analysis showing increased mitochondrial cytochrome c release, Bax, p53 and Bmf as well as reduced mitochondrial membrane potential (P < 0.05). In addition, up-regulation of tBid and Fas were evident upon treatment with α-mangostin (P < 0.01).. α-Mangostin may be effective as an anti-cancer agent that induced apoptotic cell death in COLO 205 via a link between extrinsic and intrinsic pathways.

Topics: Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; Protein Kinase Inhibitors; Xanthones

Methyl 3,5-dicaffeoyl quinate (MDQ) is a flavonoid glucoside found in several plants that scavenges 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and peroxynitrite, and inhibits the formation of cholesteryl ester hydroperoxide during the copper ion-induced oxidation of blood plasma in rats. In this study, MDQ inhibited proliferation and induced apoptosis in HT-29 cells in a dose-dependent manner as detected by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), trypan blue exclusion, and flow cytometric assays. Western blot analysis showed that apoptosis was dependent on caspase-3 activity. PARP cleavage and the cytosolic release of cytochrome c from mitochondria increased significantly. In addition, these events were accompanied by a collapse in the mitochondrial membrane potential and a decreased Bcl-2/Bax ratio. Furthermore, the MDQ-induced G(0)/G(1) arrest was correlated with an increase in p27 and a decrease in cyclin D1 and p53. MDQ also inhibited the phosphorylation of PI3K/Akt and ERK; significantly reduced NF-κB; and in general displayed a significant anti-proliferative effect via a cell cycle arrest and apoptotic induction in HT-29 cells. These results suggest that MDQ has therapeutic potential against human colon carcinoma.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Cycle; Chlorogenic Acid; Colonic Neoplasms; Cyclooxygenase 2; Cytochromes c; Dose-Response Relationship, Drug; HT29 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Tumor Cells, Cultured

Three-dimensional (3D) cell cultures have increased complexity compared to simple monolayer and suspension cultures, recapitulating the cellular architecture and molecular gradients in tissue. As such, they are popular for in vitro models in biological research. Classical imaging methodologies, like immunohistochemistry, are commonly used to examine the distribution of specific species within the spheroids. However, there is a need for an unbiased discovery-based methodology that would allow examination of protein/peptide distributions in 3D culture systems, without a need for prior knowledge of the analytes. We have developed a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based imaging approach to examine protein distributions in 3D cell culture models. Using colon carcinoma cell lines, we detect changes in the spatial distribution of proteins across 3D culture structures. To identify the protein species present, we are combining results from the MS/MS capabilities of MALDI-MS to sequence peptides in a de novo fashion and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) of homogenized cultures. As a proof-of-principle, we have identified cytochrome C and Histone H4 as two of the predominant protein species in the 3D colon carcinoma cultures.

Topics: Cell Culture Techniques; Colonic Neoplasms; Cytochromes c; Histones; Humans; Mass Spectrometry; Tumor Cells, Cultured

An unexpected and fascinating aspect of the neuropeptides orexins has recently emerged when it was shown that orexins acting at orexin receptors OX1R or OX2R induce dramatic apoptosis resulting in massive reduction in cell growth in various cancer cell lines. This mini-review will provide the reader with recent findings related to the proapoptotic actions of orexins and the entirely novel mechanism whereby the seven membrane-spanning G-protein-coupled receptor (GPCR) OX1R triggers apoptosis. Recent data show that orexins induce tyrosine phosphorylation of the tyrosine-based motifs - immunoreceptor tyrosine-based inhibitory motif and immunoreceptor tyrosine-based switch motif - in OX1R. These phosphorylations result in the recruitment and activation of the phosphotyrosine phosphatase SHP-2 and subsequent cytochrome c-mediated mitochondrial apoptosis. Finally, this mini-review will also speculate on: (1) the potential importance of tyrosine-based motifs in the large family of GPCRs; (2) the interest of orexin receptors as therapeutic targets in cancer therapy; (3) the possible role of orexin receptor-mediated apoptosis in physiology and pathophysiology in the brain (neurodevelopment, neurodegenerative diseases) and in the periphery.

Topics: Amino Acid Motifs; Animals; Apoptosis; Brain; Colonic Neoplasms; Cytochromes c; Humans; Mitochondria; Neurons; Orexin Receptors; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Signal Transduction; Tyrosine

Sulforaphane (SUL), an isothiocyanate naturally present in widely consumed vegetables, particularly broccoli, has recently attracted attention due to its inhibitory effects on tumor cell growth by inducing apoptosis. We investigated the ability of SUL to induce autophagy in human colon cancer cells and whether inhibition of autophagy could potentiate the proapoptotic effect of SUL.. The proliferation of cells treated with SUL was assessed by MTS assay and colony-forming assay. Apoptosis and caspases activity were investigated by flow cytometry. The formation of acidic vesicular organelles (AVOs) was detected in acridine-orange-stained cells by flow cytometry. Western blotting was used for the detection of light chain 3 (LC3). Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry.. The proapoptotic effect was observed by treatment of cells with relatively high concentrations of SUL for long periods of time. After 16 h of treatment, evident formation of AVOs and recruitment of LC3 to autophagosomes, features of autophagy, were observed. Treatment of cells with a specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL, which was dependent on the activation of caspases and the release of cytochrome c to the cytosol.. The present results demonstrate induction of autophagy in colon cancer cells as a protective reaction against the proapoptotic effect of SUL, and consequently, the potentiation of the proapoptotic effect by autophagy inhibition. These findings provide a premise for use of autophagy inhibitors in combination with chemotherapeutic agents for treatment of colorectal cancer.

Topics: Adenine; Anticarcinogenic Agents; Apoptosis; Autophagy; Blotting, Western; Caspases; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Colony-Forming Units Assay; Cytochromes c; Flow Cytometry; Humans; Isothiocyanates; Sulfoxides; Thiocyanates

Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Benzopyrans; Butyrates; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Down-Regulation; Drug Screening Assays, Antitumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Protein Kinase Inhibitors; Up-Regulation; Urinary Bladder Neoplasms

This study explored the role of intrinsic mitochondrial membrane potential (DeltaPsiM) in etoricoxib-mediated apoptosis in 1,2-dimethylhydrazine dihydrochloride (DMH) induced colon cancer. Male Sprague--Dawley rats were divided into four groups: control, DMH, DMH+etoricoxib and etoricoxib. After 6 weeks of treatment period, animals were killed and colons were analyzed for morphological and histopathological features. Well-characterized preneoplastic aberrations such as multiple plaque lesions, hyperplasia and dysplasia were found in the DMH-treated group whereas these features were reduced with coadministration of etoricoxib and DMH. DeltaPsiM was assessed by 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazol carbocyanine iodide (JC-1) fluorescent staining of the isolated colonocytes. DMH treatment led to a significant increase in DeltaPsiM which was found to be low in the DMH+etoricoxib group. The expression of important proapoptotic proteins, cytochrome C and Bax, were analyzed by western blot and immunohistochemistry. DMH treatment reduced the expression of Bax and cytochrome C whereas etoricoxib promoted the expression of the same. Protein expression of antiapoptotic protein Bcl-2 was also studied in colonic mucosa and was found high in the DMH-treated group and low in DMH+etoricoxib treated animals. Etoricoxib treatment may exert its chemopreventive action in colon carcinogenesis by modulating the DeltaPsiM.

Topics: Animals; Antineoplastic Agents; Apoptosis; Colonic Neoplasms; Cytochromes c; Etoricoxib; Genes, bcl-2; Intestinal Mucosa; Male; Membrane Potential, Mitochondrial; Mitochondria; Pyridines; Rats; Rats, Sprague-Dawley; Sulfones

beta-Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that beta-elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of beta-elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of beta-elemene on prostate cancer cell death was mediated through the induction of apoptosis.. The MTT assay was used to evaluate the ability of beta-elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA-based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl-2, caspases, cytochrome c and poly(ADP-ribose) polymerase (PARP) were analysed by Western blotting.. Here, we showed that beta-elemene had an antiproliferative effect on androgen-insensitive prostate carcinoma DU145 and PC-3 cells. Treatment with beta-elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of beta-elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 microg/ml (230-465 microm). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by beta-elemene in a dose- and time-dependent manner. Moreover, beta-elemene exposure resulted in a decreased Bcl-2 protein level, increased cytochrome c release, and activated PARP and caspase-3, -7, -9, and -10 in prostate cancer cells.. Overall, these findings suggest that beta-elemene exerts broad-spectrum antitumour activity against many types of solid carcinoma and supports a proposal of beta-elemene as a new potentially therapeutic drug for castration-resistant prostate cancer and other solid tumours.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Lung Neoplasms; Male; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sesquiterpenes; Time Factors; Uterine Cervical Neoplasms

The present study explored the role of intrinsic mitochondrial membrane potential (delta psi M) in NSAID-induced apoptosis in the early stages of colon cancer. 1,2-Dimethylhydrazine dihydrochloride (DMH) was used to induce colon cancer and its chemoprevention was studied by diclofenac in a rat model. After 6 weeks of treatment with DMH (early stage), morphological analysis revealed a marked occurrence of preneoplastic features [i.e., mucosal plaque lesions (MPLs) in the colonic tissue]. Coadministration of diclofenac with DMH resulted in a significant reduction of these lesions, thereby proving the chemopreventive efficacy of diclofenac at the chosen anti-inflammatory dose. DMH treatment also led to a significant increase in delta psi M in the isolated colonocytes as assessed by JC-1 fluorescent staining, measured both by fluorescence microscopy and spectrofluorometerically. Further, there was seen a reduction in the number of cells showing low delta psi M, and hence monomer intensity of JC-1 by DMH treatment. To study the mechanism of these alterations in delta psi M in the present work, we studied the protein expression of important proapoptotic proteins, cytochrome c and Bax, by Western blot analysis and immunohistochemistry. DMH treatment reduced the mitochondrial translocation of Bax whereas cytochrome c was found to be located prominently in the mitochondria. Protein expression of antiapoptotic Bcl-2 was also studied in the colonic mucosa, which was expectedly found to be overexpressed after DMH treatment. Diclofenac treatment ameliorated the elevated delta psi M and its associated events to exert its chemopreventive action against early stages of colon cancer.

Topics: 1,2-Dimethylhydrazine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Colonic Neoplasms; Cytochromes c; Diclofenac; Male; Membrane Potential, Mitochondrial; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley

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

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

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

We have investigated the mechanisms of maslinic acid with regard to its inhibitory effects on the growth of HT29 colon-cancer cells. High concentrations of maslinic acid are present in the protective wax-like coating of olives. Our results show that treatment with maslinic acid results in a significant inhibition of cell proliferation in a dose-dependent manner and causes apoptotic death in colon-cancer cells. We found that it inhibits considerably the expression of Bcl-2 whilst increasing that of Bax; it also stimulates the release of mitochondrial cytochrome-c and activates caspase-9 and caspase-3. All these results point clearly to the activation of the mitochondrial apoptotic pathway in response to the treatment of HT29 colon-cancer cells with maslinic acid. Our results suggest that maslinic acid has the potential to provide significant natural defence against colon-cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Proliferation; Colonic Neoplasms; Cytochromes c; HT29 Cells; Humans; Mitochondria; Olea; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Triterpenes

Dietary flavonols have been found to possess preventive and therapeutic potential against several kinds of cancers. This study is conducted to investigate the anti-proliferation effects of kaempferol, a major component of food flavonols, against colon cancer cells. In the human HCT116 colon cancer cell line, kaempferol induced p53-dependent growth inhibition and apoptosis. Furthermore, kaempferol was found to induce cytochrome c release from mitochondria and activate caspase-3 cleavage. The Bcl-2 family proteins including PUMA were involved in this process. Kaempferol also induced ATM and H2AX phosphorylation in HCT116 cells, inhibition of ATM by a chemical inhibitor resulted in abrogation of the downstream apoptotic cascades. These findings suggest kaempferol could be a potent candidate for colorectal cancer management.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Ataxia Telangiectasia Mutated Proteins; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Collagen Type XI; Colonic Neoplasms; Cytochromes c; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Kaempferols; Mitochondria; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Time Factors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

5-fluorouracil-(5-FU)-based adjuvant chemotherapy is widely used for the treatment of colorectal cancer. However, 5-FU resistance in the course of treatment has become more common. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored.. Two colon-cancer-derived cell lines, colon26 and HT29, were used to investigate the effect of 5-FU, 3-methyladenine (3-MA, an autophagy inhibitor), or their combination on apoptotic cell death and autophagy. MTT assay, Hochest plus propidium iodide (PI) staining, and DNA fragmentation assay were used to observe apoptosis. Meanwhile, monodansylcadaverine (MDC) was used to detect autophagy. Finally, immunoblotting assay was used to explore the molecular change that occurred.. We observed the apoptosis induced by 5-FU in colon cancer cells. Meanwhile, autophagy was also stimulated. The combination treatment of 3-MA and 5-FU significantly increased the apoptotic cell death. By isolating the subcellular fractions of mitochondria and cytosol, we observed that the release of cytochrome c was increased in combination-treated cells. Cytochrome c resulted in the activation of caspase-3, thus activating PARP. Moreover, the anti-apoptotic protein, Bcl-xL, was significantly downregulated by 3-MA.. Our results suggest that 5-FU-induced apoptosis in colon cancer cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-defense mechanism in 5-FU-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.

Topics: Adenine; Antimetabolites, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Drug Synergism; Drug Therapy, Combination; Fluorouracil; Humans

Fisetin, or 3,3',4',7-tetrahydroxyflavone, is present in fruits and vegetables and has been previously reported to inhibit the proliferation of a variety of cancer cells (Lu X, Jung J, Cho HJ, Lim do Y, Lee HS, Chun HS, Kwon DY, Park JH. J Nutr 135: 2884-2890, 2005). We have demonstrated in a previous work that 20-60 micromol/l fisetin inhibits cyclin-dependent kinase activities resulting in cell cycle arrest in HT-29 colon cancer cells. In the present study, we attempted to characterize the mechanisms by which fisetin induces apoptosis in HCT-116 cells. DNA condensations, cleavage of poly(ADP-ribose) polymerase (PARP), and cleavage of caspases 9, 7, and 3 were induced in HCT-116 cells treated with 5-20 micromol/l of fisetin. Fisetin induced a reduction in the protein levels of antiapoptotic Bcl-xL and Bcl-2 and an increase in the levels of proapoptotic Bak and Bim. Fisetin did not affect the Bax protein levels, but induced the mitochondrial translocation of this protein. Fisetin also enhanced the permeability of the mitochondrial membrane and induced the release of cytochrome c and Smac/Diablo. Additionally, fisetin caused an increase in the protein levels of cleaved caspase-8, Fas ligand, death receptor 5, and TNF-related apoptosis-inducing ligand, and the caspase-8 inhibitor Z-IETD-FMK suppressed fisetin-induced apoptosis and the activation of caspase-3. Furthermore, fisetin increases p53 protein levels, and the inhibition of p53 expression by small interference RNA resulted in a decrease in the fisetin-induced translocation of Bax to the mitochondria, release of mono- and oligonucleosome in the cytoplasm, and PARP cleavage. These results show that fisetin induces apoptosis in HCT-116 cells via the activation of the death receptor- and mitochondrial-dependent pathway and subsequent activation of the caspase cascade. The induction of p53 results in the translocation of Bax to the mitochondria, which contributes to fisetin-induced apoptosis in HCT-116 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; Bcl-2-Like Protein 11; bcl-X Protein; Caspase Inhibitors; Caspases; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Flavonoids; Flavonols; HCT116 Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Permeability; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Time Factors; Tumor Suppressor Protein p53; Up-Regulation

Despite the reported cytotoxicity and apoptosis-inducing properties of sulforaphane (SF) in colon cancer cells, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we investigated its mechanisms in metastatic SW620 cell line. Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and do not depend on generation of ROS, making SF an attractive chemopreventive agent whose antitumor properties should be further investigated in colon cancer.

Topics: Anticarcinogenic Agents; Apoptosis; Caspase 2; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Damage; Drug Screening Assays, Antitumor; Humans; Isothiocyanates; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Oxidative Stress; Sulfoxides; Thiocyanates; Tumor Suppressor Protein p53

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

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

Earlier we showed that RACK1 regulates growth of human colon cells by suppressing Src activity at G(1) and mitotic checkpoints. Here, we show that RACK1 also induces apoptosis of the cells, partly by inhibiting Src. In the intrinsic pathway, RACK1 inhibits expression of anti-apoptotic Bcl-2 and Bcl-X(L), induces expression of pro-apoptotic Bim, targets Bim and Bax to the mitochondria, induces oligomerization of Bax (which requires Bim and inhibition of Src), depolarizes mitochondria membranes, releases cytochrome c, and activates caspases-9 and -3 and death substrates. Bax and Bim are required for RACK1-mediated mitochondrial cell death. RACK1-induced oligomerization of Bax is required for staurosporine-mediated cell death. RACK1 also induces apoptosis by blocking Src activation of the Akt cell survival pathway. This leads to activation of the transcription factor FOXO3, a potent inducer of apoptosis and G(1) arrest. Collectively, our results show that RACK1, partly by inhibiting Src, promotes mitochondrial cell death and blocks Akt-mediated cell survival. Thus, RACK1 inhibits growth and induces death of colon cells. Exploitation of these dual functions could lead to novel colon cancer therapies that mimic RACK1 function.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-X Protein; Caspases; Cell Survival; Colonic Neoplasms; Cytochromes c; G1 Phase; GTP-Binding Proteins; HT29 Cells; Humans; Membrane Proteins; Mitochondria; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptors for Activated C Kinase; Receptors, Cell Surface; Signal Transduction; src-Family Kinases; Staurosporine

Although many studies have focused on anticarcinogenic properties of capsaicin and resveratrol, molecular mechanisms by which they selectively induce apoptosis are incompletely characterized. We examined the role of nitric oxide (NO) and influence of p53 status during apoptosis induced by these agents in two isogenic HCT116 human colon carcinomas, wild-type p53 (p53-WT) and complete knockout of p53 (p53-null) cells. Capsaicin and resveratrol, alone or in combination, inhibited cell growth and promoted apoptosis by the elevation of NO; combined treatment in p53-WT cells was most effective. Increased NO production after treatment uniformly stimulated p53 and Bax expression through Mdm2 down-regulation in p53-WT cells, whereas all were unaffected in p53-null cells. Both cell types underwent a reduction in the levels of anti-apoptotic Bcl-2 protein, cytochrome c loss from mitochondria and activation of caspase 9 together with caspase 3, independently of p53 status. Concomitantly, we observed DR4, Fas(CD95) and caspase 8 activation, suggesting that these compounds activate both the mitochondrial and death receptor pathways working together to induce apoptosis. These findings provide insight into the mechanism of apoptotic action of capsaicin and resveratrol based on p53 status and indicate manipulation of NO may offer exciting opportunities to improve the effectiveness of colon cancer treatment.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Arginine; Capsaicin; Caspase 3; Caspase 9; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; HCT116 Cells; Humans; Isoenzymes; Nitric Oxide; Nitric Oxide Synthase; Nucleosomes; Resveratrol; Stilbenes; Tumor Suppressor Protein p53

Conjugated linoleic acid (CLA), which is naturally present in a variety of foods such as milk fat and the meat of ruminant animals, has been demonstrated to exert chemoprotective effects in several tissues in experimental animals. CLA is a collective term, which denotes one or more positional and geometric isomers of octadecadienoic acid, with cis-9,trans-11 (c9t11) and trans-10,cis-12 CLA (t10c12) being the principal isomers in commercial preparations. We observed previously that physiological levels of CLA inhibited HT-29 cell growth, and the growth inhibitory effects of CLA were attributed to the effect of t10c12, but not c9t11. In the present study, we assessed the mechanisms by which physiological levels of CLA and t10c12 induce apoptosis in HT-29 cells. HT-29 cells were cultured for 3 days in serum-free medium in the presence of various concentrations of CLA (0-20 micromol/L) or t10c12 (0-4 micromol/L). Addition of CLA or t10c12 to culture medium resulted in a dose-dependent increase in the numbers of apoptotic cells. The results of western blot analysis of total cell lysates showed that CLA and t10c12 increased the levels of cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase but did not alter the levels of Bcl-2 family member proteins. However, these fatty acids were shown to increase the translocation of Bad and Bax to the mitochondria, increase mitochondrial membrane permeability, and induce the release of cytochrome c and Smac/Diablo from the mitochondria. In addition, CLA and t10c12 diminished Akt content and Akt phosphorylation. These findings indicate that physiological levels of t10c12 induce apoptosis in HT-29 colon cancer cells, which is mediated via mitochondrion-mediated events associated with a decline in Akt activity, an increase in the translocation of the pro-apototic Bad and Bax to the mitochondria, and the subsequent disruption of normal mitochondrial membrane potential.

Topics: Apoptosis; Biological Transport; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; HT29 Cells; Humans; Intracellular Signaling Peptides and Proteins; Isomerism; Linoleic Acids, Conjugated; Membrane Potentials; Mitochondrial Membranes; Mitochondrial Proteins; Poly(ADP-ribose) Polymerases

Evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cycloxygenase (COX) and production of the proinflammatory prostaglandin, PGE2, and thus prevent carcinogenesis in the colon. Indeed, one of the specific COX-2 inhibitors, celecoxib, had been accepted by the US FDA for the treatment of familial adenomatous polyposis. However, the molecular mechanism of such inhibition is not clear, although apoptosis appears to be the dominant antiproliferative end effect. The present study delineates the intracellular ionic milieu in the colonocytes that could generate strong apoptotic signals where DMH-induced carcinogenesis was studied in the initiation stage in rats and its regression with the COX inhibitors. While DMH treatment produced a significant elevation in the Na+/H+ exchanger activity and resultant proton efflux, this was reversed by the NSAIDs, particularly so with celecoxib and etoricoxib compared to aspirin. Similarly, the intracellular pH was changed, with more alkalosis noted in DMH, which was reversed by NSAIDs. Also, an intracellular Ca2+ build up was noted by Fura 2 AM, which was also supported by a reduced Ca2+ ATPase and an enhanced inward movement of Ca2+. Further, mitochondrial dysfunction-related cyt C release, increased DNA ladder formation, activation of caspase-3, and cleavage product of poly (ADP-ribose) polymerase (PARP) were not seen in DMH but well noted in NSAIDs. Our results indicate that NSAIDs can induce apoptosis through a change in the colonic Na+/H+ exchange, intracellular pH, and an unfavorable Ca2+ homeostasis.

Topics: 1,2-Dimethylhydrazine; Animals; Apoptosis; Calcium; Caspases; Celecoxib; Colon; Colonic Neoplasms; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cytochromes c; Enzyme Activation; Epithelial Cells; Homeostasis; Hydrogen-Ion Concentration; Ion Transport; Male; Poly(ADP-ribose) Polymerases; Pyrazoles; Rats; Rats, Sprague-Dawley; Sodium; Sodium-Hydrogen Exchangers; Sulfonamides

MDM2 oncoprotein binds directly to the p53 tumor suppressor and inhibits its function in cancers retaining wild-type p53. Blocking this interaction using small molecules is a promising approach to reactivate p53 function and is being pursued as a new anticancer strategy. The spiro-oxindole MI-43, a small-molecule inhibitor of the MDM2-p53 interaction, was designed and examined for its cellular mechanism of action and therapeutic potential in colon cancer. MI-43 binds to MDM2 protein with a K(i) value of 18 nmol/L and is 300 times more potent than a native p53 peptide. MI-43 blocks the intracellular MDM2-p53 interaction and induces p53 accumulation in both normal and cancer cells, with wild-type p53 without causing p53 phosphorylation. Induction of p53 leads to modulation of the expression of p53 target genes, including up-regulation of p21 and MDM2 in normal primary human cells and in colon cancer cells with wild-type p53. Using HCT-116 isogenic colon cancer cell lines differing only in p53 status or RNA interference to knockdown expression of p53 in the RKO colon cancer cell line, we show that the cell growth inhibition and cell death induction by MI-43 is p53 dependent. Furthermore, induction of cell cycle arrest by MI-43 is dependent on p53 and p21. In normal cells, MI-43 induces cell cycle arrest but not apoptosis. This study suggests that p53 activation by a potent and specific spiro-oxindole MDM2 antagonist may represent a promising therapeutic strategy for the treatment of colon cancer and should be further evaluated in vivo and in the clinic.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Drug Design; Enzyme Activation; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Phosphoserine; Protein Binding; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Tumor Suppressor Protein p53

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 microg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.

Topics: Antibodies, Blocking; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Humans; Mitochondrial Membranes; Models, Biological; Neoplasm Metastasis; Oxidative Stress; Permeability; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Terpenes; TNF-Related Apoptosis-Inducing Ligand

To investigate the reversing effect of Bcl-XL small interfering RNA (siRNA) on the acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon cancer.. Human colon cancer cells DLD1-TRAIL/R, with acquired resistance to TRAIL, were firstly transfected with Bcl-XL siRNA for 24 h followed by the treatment of TRAIL protein. The survival rate of DLD1-TRAIL/R cells was assessed by FACS analysis and cell number counting, respectively, and activation of its apoptotic signaling was evaluated by Western blot.. Bcl-XL siRNA effectively downregulated the expression of Bcl-XL protein and reversed the acquired resistance to TRAIL in DLD1-TRAIL/R cells. After combination treatment of Bcl-XL siRNA and TRAIL protein, the apoptotic rate of DLD1-TRAIL/R cells was more than 50% and survival rate was less than 40%, whereas there was no effect on the survival of DLD1-TRAIL/R cells after treatment with control treatment or TRAIL protein treatment alone (P < 0.05). Western blot analysis demonstrated that caspase-8, caspase-9, Bid, caspase-3, and poly (ADP-ribose) polymerase (PARP) were obviously activated after combination treatment with Bcl-XL siRNA and TRAIL protein, and the release of cytochrome C was also significantly increased.. Bcl-XL siRNA can effectively reverse the acquired resistance to TRAIL in human colon cancer cells, suggesting that it might be a new strategy for overcoming the resistance in cancer therapy.

Topics: Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Humans; Poly(ADP-ribose) Polymerases; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Transfection

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In the previous study [Yoo and Lee, 2007], we have reported that hyperthermia could enhance the cytotoxicity of TRAIL-induced apoptosis. We observed in human colorectal cancer cell line CX-1 that TRAIL-induced apoptotic death and also that mild hyperthermia promoted TRAIL-induced apoptotic death through caspase activation and cytochrome-c release. Although its effects in vivo are not clear, hyperthermia has been used as an adjunctive therapy for cancer. Hyperthermia is often accompanied by chemotherapy to enhance its effect. In this study, CX-1 colorectal adenocarcinoma cells were treated with TRAIL concurrently with hyperthermia and oxaliplatin or melphalan. To evaluate the cell death effects on tumor cells via hyperthermia and TRAIL and chemotherapeutic agents, FACS analysis, DNA fragmentation, and immunoblottings for PARP-1 and several caspases and antiapoptotic proteins were performed. Activities of casapse-8, caspase-9, and caspase-3 were also measured in hyperthermic condition. Interestingly, when analyzed with Western blot, we detected little change in the intracellular levels of proteins related to apoptosis. Clonogenic assay shows, however, that chemotherapeutic agents will trigger cancer cell death, either apoptotic or non-apoptotic, more efficiently. We demonstrate here that CX-1 cells exposed to 42 degrees C and chemotherapeutic agents were sensitized and died by apoptotic and non-apoptotic cell death even in low concentration (10 ng/ml) of TRAIL.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Humans; Melphalan; Mitochondria; Organoplatinum Compounds; Oxaliplatin; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Temperature; TNF-Related Apoptosis-Inducing Ligand

Acetylcholinesterase (AChE) is emerging as an important component in leading to apoptosis. Our previous study demonstrated that silencing of the AChE gene blocked the interaction between cytochrome c and apoptotic protease-activating factor-1 (Apaf-1) in etoposide-induced apoptosis of HT-29 cells. We undertook this study to further dissect the molecular role of AChE in apoptosome formation. The present study elicited that small interfering RNA (siRNA) to cytochrome c gene blocked the interaction of AChE with Apaf-1, whereas siRNA to Apaf-1 gene did not block the interaction of AChE with cytochrome c, indicating that the interaction of AChE with cytochrome c is required for the interaction between cytochrome c and protease-activating factor-1. We further observed that AChE is localized to caveolae via interacting with caveolin-1 during apoptosis and that the disruption of caveolae prevented apoptosome formation. These data indicate that the interactions of AChE with caveolin-1 and subsequently with cytochrome c appear to be indispensable for apoptosome formation.

Topics: Acetylcholinesterase; Apoptosis; Base Sequence; Caveolin 1; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Primers; Humans; Molecular Sequence Data; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Sulindac; Transfection

Considerable researches have been done about integrin alphanubeta6 and carcinomas, but little information has been shown about the relationship between integrin alphanubeta6 and apoptosis. In this study, we investigated the apoptosis and its related signal pathways to integrin alphavbeta6 in colon cancer cells. After we blocked the function of integrin alphavbeta6 in HT29 cells used the monoclonal antibody, the apoptotic cells increased markedly. Meanwhile, cytochrome C released from mitochondria into cytosol, Bcl-2 decreased while Bax increased significantly, and Fas and Fas-ligand had no change. The activities of caspase-3 and caspase-9 increased, while caspase-8 remained no change. Moreover, the expression of phosphorylated extracellular signal-related kinase (P-ERK) decreased. We confirmed that integrin alphavbeta6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involved the mitochondrial pathway.

Topics: Antibodies; Antigens, Neoplasm; Apoptosis; bcl-2-Associated X Protein; Caspases; Colonic Neoplasms; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; HT29 Cells; Humans; Integrins; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Insulin-induced gene 2 (Insig2) was recently identified as a putative positive prognostic biomarker for colon cancer prognosis. Insig2 has been previously reported to be an endoplasmic reticulum (ER) membrane protein, and a negative regulator of cholesterol synthesis. Here we report that Insig2 was validated as a gene with univariate negative prognostic capacity to discriminate human colon cancer survivorship. To investigate the functional roles it plays in tumor development and malignancy, Insig2 was over-expressed in colon cancer cells resulting in increased cellular proliferation, invasion, anchorage independent growth and inhibition of apoptosis. Over-expression of Insig2 appeared to suppress chemotherapeutic drug treatment-induced Bcl2 associated X protein (Bax) expression and activation. Insig2 was also found to localize to the mitochondria/heavy membrane fraction and associate with conformationally changed Bax. Moreover, Insig2 altered the expression of several additional apoptosis genes located in mitochondria, further supporting its new functional role in regulating mitochondrial mediated apoptosis. Our findings show that Insig2 is a novel colon cancer biomarker, and suggest, for the first time, a reasonable connection between Insig2 and Bax-mediated apoptosis through the mitochondrial pathway.

Topics: Apoptosis; bcl-2-Associated X Protein; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; DNA, Complementary; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Membrane Proteins; Microscopy, Confocal; Mitochondria; Polymerase Chain Reaction; Protein Array Analysis; RNA Interference

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.

Topics: Antimetabolites, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; fas Receptor; Fluorouracil; Gene Expression; HCT116 Cells; Humans; Mitochondria; RNA, Small Interfering; Tumor Suppressor Protein p53

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a pleiotropic enzyme that is overexpressed in apoptosis and in several human chronic pathologies. Here, we report that the protein accumulates in mitochondria during apoptosis, and induces the pro-apoptotic mitochondrial membrane permeabilization, a decisive event of the intrinsic pathway of apoptosis. GAPDH was localized by immunogold labeling and identified by matrix-assisted laser desorption/ionization-time of flight and nano liquid chromatography mass spectroscopy/mass spectroscopy in the mitochondrion of various tissues and origins. In isolated mitochondria, GAPDH can be imported and interact with the voltage-dependent anion channel (VDAC1), but not the adenine nucleotide translocase (ANT). The protein mediates a cyclosporin A-inhibitable permeability transition, characterized by a loss of the inner transmembrane potential, matrix swelling, permeabilization of the inner mitochondrial membrane and the release of two pro-apoptotic proteins, cytochrome c and apoptosis-inducing factor (AIF). This novel function of GAPDH might have implications for the understanding of mitochondrial biology, oncogenesis and apoptosis.

Topics: Amino Acid Sequence; Animals; Apoptosis; Caspase 3; Cell Membrane Permeability; Cells, Cultured; Colonic Neoplasms; Cyclosporine; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Glyceraldehyde-3-Phosphate Dehydrogenases; HeLa Cells; Humans; Immunosuppressive Agents; Kidney; Male; Membrane Potentials; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Membranes; Molecular Sequence Data; Protein Interaction Mapping; Rats; Rats, Wistar; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions; Voltage-Dependent Anion Channel 1

We reported previously that 7beta-hydroxysitosterol and 7beta-hydroxycholesterol induced apoptosis in Caco-2 cells. Apoptosis caused by 7beta-hydroxysitosterol but not by 7beta-hydroxycholesterol was related to a caspase-dependent process. In the present report, we compared the effects of both compounds on mitochondria integrity and on various modulators of apoptosis. When Caco-2 cells were exposed to both hydroxysterols, no changes in Bcl-2 and Bax expressions were detected indicating a Bcl-2/Bax-independent cell death pathway, whereas loss of mitochondrial membrane potential and cytochrome c release were observed. Endonuclease G expression and enhanced production of reactive oxygen species were detected in 7beta-hydroxycholesterol treated cells, but not with 7beta-hydroxysitosterol. Loss of mitochondrial membrane potential and cell death produced by both hydroxysterols were prevented by vitamin C. Lysosomal membrane integrity was altered with both hydroxysterols, but 7beta-hydroxysitosterol was significantly more active on than 7beta-hydroxycholesterol. Both hydroxysterols induced apoptosis by mitochondrial membrane permeabilization. However, 7beta-hydroxycholesterol exhibited a specific enhancement of oxidative stress and of endonuclease G expression despite its closely related chemical structure with 7beta-hydroxysitosterol. The two hydroxysterols exhibit different lipophilic properties which may explain their different biological effects.

Topics: Apoptosis; Ascorbic Acid; bcl-2-Associated X Protein; Caco-2 Cells; Colonic Neoplasms; Cytochromes c; Endodeoxyribonucleases; Humans; Hydroxycholesterols; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sitosterols

Highly polar xanthophylls of 9'-cis-neoxanthin (neoxanthin) and fucoxanthin, which have the characteristic structure of an epoxy group and an allenic bond, were previously found to induce apoptosis in human prostate cancer cells. In the present study, we found apoptosis induction by neoxanthin in HCT116 human colon cancer cells and examined the induction mechanism. The cells exposed to 20 microM neoxanthin clearly showed chromatin condensation, DNA fragmentation, and an increase in hypodiploid cells. Neoxanthin treatment increased the activities of caspase-3, -8 and -9, and the protein levels of their active subunits, except in the case of caspase-8. The treatment also caused the loss of mitochondrial transmembrane potential at an early stage and subsequently the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol. The exposure of neoxanthin directly to mitochondria isolated from the cells enhanced the release of cytochrome c and AIF in a dose-dependent manner. Approximately 50% of the neoxanthin taken up into the HCT116 cells accumulated in the mitochondrial fraction. These results suggest that the accumulation of neoxanthin in mitochondria causes the loss of mitochondrial transmembrane potential and thereafter releases cytochrome c and AIF, leading to the execution of apoptosis.

Topics: Apoptosis; Apoptosis Inducing Factor; Caspases; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; HCT116 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Subcellular Fractions; Time Factors; Xanthophylls

Bioflavanoids are the major pigments in plants with multitude of biological activities including inhibition of proliferation or induction of apoptosis in tumor cells. Even though the safety records of most flavanoids are exceptional, its therapeutic use is still in its infancy. We have isolated pinocembrin (5,7-dihydroxyflavanone) from Alpinia galanga that showed cytotoxicity against a variety of cancer cells including normal lung fibroblasts with relative nontoxicity to human umbilical cord endothelial cells. The compound induced loss of mitochondrial membrane potential with subsequent release of cytochrome c and processing of caspase-9 and -3 in colon cancer cell line HCT 116. Processing of caspase-8 was minimal. The initial trigger for mitochondrial apoptosis appears to be by the translocation of cytosolic Bax protein to mitochondria. Overexpression of proapoptotic Bax protein sensitized the colon cancer cells to pinocembrin-induced apoptosis and Bax knockout cells were resistant to pinocembrin-induced apoptosis. Antiapoptotic protein Bcl-X(L) only partially prevented apoptosis induced by this compound. The Bax-dependent cell death involving classical cytochrome c release and processing of caspase-9 and -3 suggests that pinocembrin is a classical mitochondrial apoptosis inducer. But the failure of Bcl-X(L) overexpression to completely prevent apoptosis induced by this compound suggests that pinocembrin is capable of triggering mitochondrial-independent cell death that needs to be clarified. The existence of cell death upon Bcl-X(L) overexpression is a promising feature of this compound that can be exploited against drug resistant forms of cancer cells either alone or in combination with other drugs.

Topics: Alpinia; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Breast Neoplasms; Caspase Inhibitors; Caspases; Chromatin; Colonic Neoplasms; Cytochromes c; Endothelial Cells; Fibroblasts; Flavanones; Fluorescent Antibody Technique; HCT116 Cells; Humans; Lung; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Transfection; Umbilical Cord

Tissue injury induces the acute phase response, aimed at minimizing damage and starting the healing process. Polymorphonuclear leukocytes (PMNs) respond to the presence of specific chemoattractants and begin to appear in large numbers. The aim of this study was to investigate the influence of reactive oxygen species (ROS) produced by PMNs on the interaction between colon carcinoma cells and mesothelial cells. An experimental human in vitro model was designed using Caco-2 colon carcinoma cells and primary cultures of mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with stimulated PMNs and unstimulated PMNs. Mesothelial cells were also incubated with xanthine/xanthine oxidase (X/XO) complex producing ROS after which adhesion of Caco-2 cells was investigated and the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) by means of enzyme immunoassay. In the control situation the average adhesion of Caco-2 cells to the mesothelial monolayers was 23%. Mesothelial monolayers incubated with unstimulated PMNs showed a 25% increase of tumor cell adhesion (P < 0.05). The adhesion of tumor to the monolayers incubated with the N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs increased with 40% (P < 0.01). Incubation of the mesothelium with X/XO resulted in an enhancement of adhesion of Caco-2 cells of 70% and an up-regulation of expression of ICAM-1, VCAM-1, and CD44. This study reveals an increase of tumor cell adhesion to the mesothelium induced by incubating the mesothelial monolayers with PMNs. PMNs are producing a number of products, like proteolytic enzymes, cytokines, and ROS. These factors up-regulate the expression of adhesion molecules and in that way stimulate the adhesion of tumor to the mesothelium.

Topics: Acute-Phase Reaction; Caco-2 Cells; Cell Adhesion; Colonic Neoplasms; Cytochromes c; Epithelium; Humans; Immunoenzyme Techniques; In Vitro Techniques; Neoplasm Recurrence, Local; Neutrophils; Reactive Oxygen Species; Reproducibility of Results; Xanthine; Xanthine Oxidase

Approximately 25% of patients with colorectal cancer will develop metastatic disease exclusively or largely confined to the liver, and the vast majority of these cases are not amenable to surgical resection. These unresectable cases of liver metastatic disease can be treated with isolated hepatic perfusion (IHP), which involves a method of complete vascular isolation of the liver to allow treatment of liver tumors with toxic systemic doses of chemotherapeutic agents. To improve the efficacy of IHP, hyperthermia and biological agents have been applied along with the chemotherapeutic agents. In this study, we investigated whether hyperthermia in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) enhances mortality in human colorectal carcinoma CX-1 cells. Cells were treated with various concentrations of TRAIL (0-200 ng/ml) at various temperatures (40-46 degrees C) for 1 h and further incubated at 37 degrees C in the presence of TRAIL. We observed that hyperthermia at 42-43 degrees C effectively promoted TRAIL-induced apoptosis, as indicated by cell death, poly (ADP-ribose) polymerase (PARP) cleavage, and activation of caspase-8, -9, and -3. In contrast, hyperthermia at 45-46 degrees C suppressed TRAIL-induced apoptosis. We also observed that mild hyperthermia, but not acute hyperthermia, promoted cytochrome c release during treatment with TRAIL. Our data suggest that promotion of cytochrome c release during mild hyperthermia is responsible for the enhancement of TRAIL cytotoxicity.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Collagen Type XI; Colonic Neoplasms; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Humans; Immunoblotting; In Situ Nick-End Labeling; Mitochondria; Receptors, TNF-Related Apoptosis-Inducing Ligand; Temperature; TNF-Related Apoptosis-Inducing Ligand

Meclizine (MEC), a histamine H1 antagonist, is used for the treatment of motion sickness and vertigo. In this study, we demonstrate that MEC dose-dependently induced apoptosis in human colon cancer cell lines (COLO 205 and HT 29 cells). Results of a DNA ladder assay revealed that DNA ladders appeared with MEC treatment in COLO 205 cells at dosage of >50 microM. In addition, the total cell number decreased dose-dependently after treatment with MEC in COLO 205 and HT 29 cells. Using flow cytometry, the percentage of COLO 205 cells arrested at G0/G1 phase increased dose-dependently. Analysis of changes in cell-cycle arrest-associated proteins with Western blotting showed that p53 and p21 were upregulated after treatment with MEC. The kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were suppressed in MEC-treated cells. As for apoptosis, MEC may induce upregulation of p53 and downregulation of Bcl-2, thus causing the release of cytochrome C from mitochondria and the translocation of apoptosis-inducing factor (AIF) to the nucleus. This resulted in the activation of caspase 3, 8, and 9. Our results provide the molecular basis of MEC-induced apoptosis and cell-cycle arrest in human colon cancer cells.

Topics: Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cyclin-Dependent Kinases; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Histamine H1 Antagonists; HT29 Cells; Humans; Meclizine

Targeting the ubiquitin-proteasome degradation pathway has become a promising approach for cancer therapy. Previous studies have shown that proteasome inhibition leads to apoptosis in various cancer cells. The mechanism by which apoptosis occurs are not fully understood and can be cell type and/or inhibitor specific. In this study, we investigated the mechanism of mitochondrial activation by proteasome inhibitors in colon cancer cells. We found that Bax activation and mitochondria translocation were required for apoptosis induced by multiple proteasome inhibitors. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. In contrast, treatment with MG132 or bortezomib induced a significant accumulation of p53 and PUMA. Genetic deletion of either p53 or PUMA led to a marked suppression of apoptosis induced by these inhibitors, accompanied with reduced Bax activation and cytochrome c release. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. These findings thus strongly indicate the critical involvement of p53-, PUMA-, and Bax-mediated mitochondrial activation in proteasome inhibitor-induced apoptosis in colon cancer cells.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Boronic Acids; Bortezomib; Caspase Inhibitors; Caspases; Cell Nucleus; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Cytochromes c; Humans; Leupeptins; Mice; Mitochondria; Proteasome Inhibitors; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Reactive Oxygen Species; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Berberine is the major constituent of Coptidis Rhizoma with multiple pharmacological activities, including anti-inflammation, promotion of apoptosis and anticancer potential effect. Mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) may contribute to the causal relationship between tumorigenesis and pro-apoptotic function. Berberine is studied for the mechanism of its action in apoptotic pathway in human colonic carcinoma cell. Treatment of SW620 cells with 50 microM berberine resulted in activation of the caspase 3 and caspase 8, cleavage of poly ADP-ribose polymerase (PARP) and the release of cytochrome c; whereas, the expression of BID and anti-apoptosis factor c-IAP1, Bcl-2, and Bcl-(XL) were decreased markedly. Berberine-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK and p38 MAPK, as well as generation of the ROS. Furthermore, the induction of apoptosis was alleviated by inhibitors specific for JNK and p38. In addition, there was an increase in the cellular levels of phospho-c-Jun, FasL and t-BID in the berberine-induced apoptosis via the activation of JNK and p38 signaling modules. NAC administration, a scavenger of ROS, reversed berberine-induced apoptosis effects via inhibition of JNK, p38 and c-jun activation, and FasL and t-BID expression. These results leads us to speculate that berberine may play an apoptotic cascade in SW620 cells by activation of the JNK/p38 pathway and induction of ROS production, providing a new mechanism for berberine-induced cell death in human colon cancer cells.

Topics: Apoptosis; bcl-X Protein; Berberine; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 8; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Fas Ligand Protein; Humans; Inhibitor of Apoptosis Proteins; JNK Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

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

Tetracycline analogues (TCNAs) possess cytotoxic activities as well as matrix metalloproteinase (MMP) inhibitory properties. Previously, we demonstrated that doxycycline (DOXY) could induce apoptosis in human HT29 colon cancer cells. In present study, the molecular apoptotic mechanisms induced by two kinds of TCNAs, designated as DOXY and COL-3 (chemically modified tetracycline-3; 6-demethyl, 6-deoxy, 4-dedimethylamino tetracycline), were evaluated in cultured HT29 cells. Both TCNAs inhibited the proliferation of 6 different colorectal cancer cell lines in a dose-dependent manner. Especially, COL-3 had a stronger effect on cancer cells than DOXY. Apoptotic changes were actually observed by 10 mug/ml COL-3 and 20 mug/ml DOXY in a time-dependent manner. COL-3 produced the increase in cytosolic cytochrome c and the loss of mitochondrial membrane potential after 3 hr treatment, and thereafter activated caspases. In case of DOXY, these changes were observed after 24 hr. Bax translocation was not a prerequisite for cytochrome c releasing in COL-3 treatment. Pretreated pancaspase inhibitor (Z-VAD-FMK) reduced COL-3 and DOXY mediated apoptosis up to 81.3 and 35.3%, as compared with nontreated cells, respectively. These data indicated that TCNAs could induce mitochondria-mediated apoptosis through both caspase-dependent and -independent pathway. In fact, endonuclease G and apoptosis-inducing factor were released into cytosol after the treatment of TCNAs, which indicated that caspase-independent apoptotic pathway is also one of the key mechanisms for the treatment of TCNAs. Taken together, we believe that TCNAs could have strong potentials for clinical application in treating colorectal cancers and improve cancer chemotherapy.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Doxycycline; Humans; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction; Protease Inhibitors; Protein Transport; Tetracyclines

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

Photodynamic therapy (PDT) typically involves systemic or topical administration of a tumor-localizing photosensitizer or prodrug and its subsequent activation by visible light. This results primarily in singlet oxygen-induced photodamage to the tumor. 5-Aminolevulinic acid (ALA) and its derivatives have recently been widely used for PDT due to their selective induction in tumor of endogenous protoporphyrin IX (PpIX), a potent photosensitizer. Although ALA-PDT has achieved successful results in the treatment of several clinical oncological and nononcological diseases, the mechanisms of this modality are still not fully elucidated. In the present study, the human colon carcinoma cell line 320DM was treated in vitro with PDT using hexaminolevulinate (HAL), a hexylester of ALA known to be 50 to 100 times more efficient at producing PpIX formation than ALA itself. PpIX production increased with increasing HAL concentrations in the cells and phototoxicity of the cells was enhanced with increasing light (450 nm) doses. HAL-PDT induced apoptotic cell death, as measured by nuclear staining of Hoechst 33342 for fluorescence microscopy, DNA electrophoresis and TdT staining for flow cytometry. PDT with 5 muM of HAL and a light dose of 640 mJ/cm2 produced a 75% apoptotic cell population 40 hr after the treatment. Furthermore, the loss of mitochondrial membrane potential coincident with the release of cytochrome c from the mitochondria into the cytosol led to a rapid activation of caspase-9 and caspase-3 (an executioner), indicating that the selective damage to the mitochondria by HAL-PDT can induce a cytochrome-c-mediated apoptotic response in the 320DM cells.

Topics: Aminolevulinic Acid; Apoptosis; Carcinoma; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; DNA Fragmentation; Humans; Membrane Potentials; Mitochondria; Photochemotherapy; Photosensitizing Agents; Protoporphyrins

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 2; Caspase 3; Caspase 8; Caspase 9; Caspases; Colonic Neoplasms; Cytochromes c; Cytosol; Endodeoxyribonucleases; Fibroblasts; Green Fluorescent Proteins; HCT116 Cells; Humans; Mice; Mitochondria; Poly(ADP-ribose) Polymerases; Protein Conformation; Resveratrol; Stilbenes

Ceramides are sphingolipid second messengers that are involved in the mediation of cell death. There is accumulating evidence that mitochondria play a central role in ceramide-derived toxicity. We designed a novel cationic long-chain ceramide [omega-pyridinium bromide D-erythro-C16-ceramide (LCL-30)] targeting negatively charged mitochondria. Our results show that LCL-30 is highly cytotoxic to SW403 cells (and other cancer cell lines) and preferentially accumulates in mitochondria, resulting in a decrease of the mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 and caspase-9. Ultrastructural analyses support the concept of mitochondrial selectivity. Interestingly, levels of endogenous mitochondrial C16-ceramide decreased by more than half, whereas levels of sphingosine-1-phosphate increased dramatically and selectively in mitochondria after administration of LCL-30, suggesting the presence of a mitochondrial sphingosine kinase. Of note, intracellular long-chain ceramide levels and sphingosine-1-phosphate remained unaffected in the cytosolic and extramitochondrial (nuclei/cellular membranes) cellular fractions. Furthermore, a synergistic effect of cotreatment of LCL-30 and doxorubicin was observed, which was not related to alterations in endogenous ceramide levels. Cationic long-chain pyridinium ceramides might be promising new drugs for cancer therapy through their mitochondrial preference.

Topics: Antibiotics, Antineoplastic; Caspase 3; Caspase 9; Caspases; Cations; Cell Death; Ceramides; Colonic Neoplasms; Cytochromes c; Doxorubicin; Drug Combinations; Drug Synergism; Enzyme Activation; Humans; Lysophospholipids; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Sphingosine; Tumor Cells, Cultured

Several studies have shown that violacein, a purple pigment extracted from Chromobacterium violaceum, is capable to induce apoptosis in a variety of cancer cells, including those leukemia cell lines. Herein, we examined the effects of violacein on reactive oxygen species (ROS) production during the apoptotic colon cancer cell death. We demonstrate that violacein mediates ROS production followed by activation of Caspase-3, release of cytochrome c, and calcium release to citosol in Caco-2 cells. Moreover, presence of ROS scavengers such as N-acetyl-cysteine (NAC) diminishes ROS cytotoxicity induced by violacein in Caco-2 cells, indicating that violacein mediates cellular critical mechanisms in the triggering of apoptotic tumor cell death. These data also imply that violacein-induced ROS are collectively key mediators of mitochondrial membrane collapse, leading to cytochrome c release, and culminating in tumor apoptosis. Unlike in Caco-2 cells, violacein was incapable of increasing ROS levels in HT29 cells, suggesting the existence of violacein cell-type specific mechanisms. Those findings bring light to the violacein cytotoxic mechanism studies, indicating that oxidative stress play a role in the violacein-induced cytotoxicity.

Topics: Apoptosis; Caco-2 Cells; Calcium; Caspase 3; Cell Death; Cell Survival; Colonic Neoplasms; Cytochromes c; Enzyme Activation; HT29 Cells; Humans; Indicators and Reagents; Indoles; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

Amooranin (AMR), a natural triterpenoid drug isolated and characterized from Amoora rohituka stem bark, is cytotoxic to SW620 human colon carcinoma cell line with an IC(50) value of 2.9 microg/ml. This novel compound caused depolarization of mitochondrial membrane and decrease of membrane potential, indicating initial signal of apoptosis induction. The percentage of cells with decreased mitochondrial potential ranged from 7.4% at 1 microg/ml to 60.5% at 100 microg/ml AMR. Flow cytometric analysis of apoptosis using Annexin-V-FITC staining showed that the percentage of apoptotic cells ranged from 7.5% at 1 microg/ml to 59.2% at 100 microg/ml AMR. AMR-induced apoptosis was accompanied by redistribution of cytochrome c from mitochondria to cytosol as well as down regulation of Bcl-2 and Bcl-X(L) proteins in a dose-dependent manner. SW620 human colon carcinoma xenograft mice treated with AMR showed significant reduction in tumor growth rates compared to saline- and doxorubicin-treated groups. The reduction in tumor growth rate was better in xenografts treated with 2 mg/kg AMR than 5 and 10 mg/kg treated mice. The analysis of global gene expression changes induced by AMR in xenograft tumors by microarray hybridization revealed that several genes involved in energy pathways, transport, apoptosis, immune response, nucleic acid metabolism, protein metabolism, cell growth and/or maintenance, signal transduction and cell communication, were affected by this natural cancer drug. These results suggest that the anticancer properties of AMR in SW620 human colon carcinoma cell line are mediated through its effects on functional genomics, targeting the apoptotic process.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Triterpenes

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

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Caspase Inhibitors; Caspases; Colonic Neoplasms; Complement Membrane Attack Complex; Complement System Proteins; Curcumin; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Flavoproteins; Glycoproteins; Humans; Membrane Proteins; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

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

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

Topics: Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; HT29 Cells; Humans; Kinetics; Membrane Potentials; Mesoporphyrins; Mitochondria; Necrosis; Photochemotherapy; Photosensitizing Agents; Spheroids, Cellular

We previously found that a change in the balance between mitochondrial pro- and anti-apoptotic proteins caused by ectopic expression of the Bax gene led to increased induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To investigate whether a similar effect can be elicited by down-regulating Bcl-X(L), an anti-apoptotic protein, we tested the effects of a small interfering RNA (siRNA) specific for Bcl-X(L) in TRAIL-resistant cells. The down-regulation of Bcl-X(L) by siRNA inhibited cell proliferation and sensitized TRAIL-induced apoptosis in human cancer cells with both acquired and intrinsic TRAIL resistance. Combining the Bcl-X(L) siRNA with TRAIL protein treatment resulted in an increase in the percentage of apoptotic cells and increased cleavage of caspase-8, caspase-9, caspase-3 and PARP. Furthermore, the release of cytochrome c but not Smac from mitochondria was induced by Bcl-X(L) siRNA alone, and this release was dramatically amplified by combining the Bcl-X(L) siRNA and TRAIL protein treatment. Together, our data suggest that simultaneous triggering of the death receptor and mitochondrial apoptotic pathways leads to enhanced induction of apoptosis, which makes it potentially useful for the treatment of resistant cancers.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Collagen Type XI; Colonic Neoplasms; Cytochromes c; Down-Regulation; Drug Synergism; Enzyme Activation; Female; Humans; Ligands; Membrane Glycoproteins; Mitochondria; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear.. We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests.. Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006).. Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Apoptotic Protease-Activating Factor 1; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cardiolipins; Caspases; Coenzyme A Ligases; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Female; Flavoproteins; Gene Transfer Techniques; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Mitochondria; Neoplasms, Experimental; Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Stomach Neoplasms; Transfection; Transplantation, Heterologous; Triazenes; Tumor Suppressor Protein p53

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Cytosol; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Ligands; Membrane Glycoproteins; Mitochondria; Ovarian Neoplasms; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

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

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

Proteasome inhibitors can resensitize cells that are resistant to tumor necrosis factor-related apoptotic-inducing ligand (TRAIL)-mediated apoptosis. However, the underlying mechanisms of this effect are unclear. To characterize the mechanisms of interaction between proteasome inhibitors and TRAIL protein, we evaluated the effects of combined treatment with the proteasome inhibitors bortezomib and MG132 and TRAIL protein on two TRAIL-resistant human colon cancer cell lines, DLD1-TRAIL/R and LOVO-TRAIL/R. Both bortezomib and MG132 in combination with TRAIL enhanced apoptotosis induction in these cells, as evidenced by enhanced cleavage of caspases 8, 9, and 3, Bid, poly(ADP-ribose) polymerase and by the release of cytochrome C and Smac. Subsequent studies showed that combined treatment with bortezomib or MG132 resulted in an increase of death receptor (DR) 5 and Bik at protein levels but had no effects on protein levels of DR4, Bax, Bak, Bcl-2, Bcl-XL or Flice-inhibitory protein (FLIP). Moreover, c-Jun N-terminal kinase (JNK) is activated by these proteasome inhibitors. Blocking JNK activation with the JNK inhibitor SP600125 attenuated DR5 increase, but enhancement of apoptosis induction and increase of Bik protein were not affected. However, bortezomib-mediated TRAIL sensitization was partially blocked by using siRNA to knockdown Bik. Thus, our data suggests that accumulation of Bik may be critical for proteasome inhibitor-mediated resensitization of TRAIL.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Boronic Acids; Bortezomib; Caspases; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Cytochromes c; Drug Resistance, Neoplasm; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Membrane Glycoproteins; Membrane Proteins; Mitochondrial Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Apoptosis is a crucial mechanism to eliminate harmful cells in which growth factors and cytokines are key regulators. In HT29-D4 cells, a model of human colon carcinoma, IFNgamma presensitization is essential to induce an apoptotic response to TNFalpha whereas it only slightly enhances TRAIL-induced apoptosis. To compare the transcriptional profiles induced by TNFalpha and TRAIL and their regulation by IFNgamma, we optimized a cDNA array analysis on targeted signaling pathways and confirmed the gene expression modulations by comparative RT-PCR. Although the two TNFSF ligands induced a same strong up-expression of pro-apoptotic Bax gene, the expression of anti-apoptotic Bcl-xL gene was more strongly up-regulated in TNFalpha- than in TRAIL-stimulated cells. Thus, TRAIL but not TNFalpha induced apoptotic mitochondrial cascade as highlighted by cytochrome c release into cytosol. IFNgamma presensitization of TRAIL-stimulated cells did not induce any change in cytochrome c release, suggesting that the increase of IFNgamma/TRAIL-induced apoptosis is independent of this pathway. In contrast, IFNgamma pretreatment prevented Bcl-xL gene up-expression in TNFalpha-stimulated cells and allowed cytochrome c release. Thus, we hypothesize that the Bcl-xL/Bax ratio can block the apoptotic response in TNFalpha-stimulated cells but allows cell death initiation when it is altered by a crosstalk between IFNgamma presensitization and TNFalpha induced signalings.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Humans; Interferon-gamma; Membrane Glycoproteins; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release.

Topics: Apoptosis; Caspase 9; Caspases; Colonic Neoplasms; Cytochromes c; Deoxycholic Acid; HCT116 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Mitochondria; Ursodeoxycholic Acid

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Topics: Acetylcysteine; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Respiration; Colonic Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Membrane Glycoproteins; Mitochondria; Mitochondrial Proteins; Oxidative Phosphorylation; Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Uncoupling Agents; X-Linked Inhibitor of Apoptosis Protein

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

Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) that induces apoptosis in cultured colon cancer cells and in intestinal epithelia in association with its chemopreventive efficacy. Resistance to sulindac is well documented in patients with familial adenomatous polyposis; however, the molecular mechanisms underlying such resistance remain unknown. We determined the effect of ectopic Bcl-2 expression upon sulindac-induced apoptotic signaling in SW480 human colon cancer cells. Sulindac sulfide activated both the caspase-8-dependent and mitochondrial apoptotic pathways. Ectopic Bcl-2 attenuated cytochrome c release and apoptosis induction compared with SW480/neo cells. Coadministration of sulindac sulfide and the small-molecule Bcl-2 inhibitor HA14-1 increased apoptosis induction and enhanced caspase-8 and caspase-9 cleavage, Bax redistribution, and cytochrome c and second mitochondria-derived activator of caspase release. Given that sulindac sulfide activated caspase-8 and increased membrane death receptor (DR4 and DR5) protein levels, we evaluated its combination with the endogenous death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Coadministration of sulindac sulfide and TRAIL cooperatively enhanced apoptotic signaling as effectively as did HA14-1. Together, these data indicate that HA14-1 or TRAIL can enhance sulindac sulfide-induced apoptosis and represent novel strategies for circumventing Bcl-2-mediated apoptosis resistance in human colon cancer cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Benzopyrans; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Humans; Membrane Glycoproteins; Mitochondria; Nitriles; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Sulindac; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Necrosis Factor-alpha

We have shown earlier that heat shock renders human colon cancer cells resistant to curcumin-induced apoptosis, but the contribution of individual heat shock proteins (hsps) to this resistance has not been tested. High expression of hsp27 and hsp70 in breast, endometrial and gastric cancers has been associated with metastasis, poor prognosis and resistance to chemo- or radiotherapy. In this study, SW480 cells were transfected with hsp70 cDNA in either the sense or antisense orientation and stable clones were selected and tested for their sensitivity to curcumin. The cells were protected from curcumin-induced cell death by hsp70 while cells harboring antisense hsp70 (Ashsp70) were highly sensitive to curcumin. Curcumin-induced nuclear condensation was less in hsp70 but more in Ashsp70 cells when compared with control vector-transfected cells. Loss of mitochondrial transmembrane potential induced by curcumin was further accelerated by antisense hsp70 expression and hsp70 restored it partly. Ashsp70 cells released more cytochrome c, AIF and Smac from mitochondria upon curcumin treatment than control cells. hsp70 partly prevented the release of AIF but not the other proteins. Activation of caspases 3 and 9 induced by curcumin was also inhibited by hsp70, whereas more activation could be seen in Ashsp70 cells, although caspase 8 activation was unaffected by changes in hsp70 expression. Curcumin-induced cleavage of PARP and DFF45 was inhibited by hsp70 but enhanced in Ashsp70 cells. The present study demonstrates the potential of hsp70 in protecting SW480 cells from curcumin-induced apoptosis and highlights that silencing the expression of hsp70 is an effective approach to augment curcumin-based therapy in cancers that are resistant due to hsp70 expression.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Caspases; Chromatin; Colonic Neoplasms; Complement Membrane Attack Complex; Complement System Proteins; Curcumin; Cytochromes c; DNA, Antisense; Drug Resistance, Neoplasm; Enzyme Activation; Flavoproteins; Gene Expression; Gene Silencing; Glycoproteins; HSP70 Heat-Shock Proteins; Humans; Membrane Potentials; Membrane Proteins; Mitochondria; Poly(ADP-ribose) Polymerases; Transfection; Tumor Cells, Cultured

Apoptosis induced by photodynamic therapy (PDT) is considered to be an important factor defining the treatment outcome. Nevertheless, the relevance of apoptotic events in overall cell death should be established for every given photosensitizer. The present study addresses the contribution of Foscan-(meta-tetra(hydroxyphenyl)chlorine; mTHPC) photosensitized apoptosis in overall cell death in a model of cultured HT29 adenocarcinoma cells. Early events of cell death were assessed by the evaluation of mitochondrial response to mTHPC-mediated PDT, cytochrome c release and membrane depolarization. Apoptosis was measured through the activity of caspase-3 and the binding of the fluorescent conjugate Ca2+-dependent protein Annexin-V on membrane externalized phosphatidylserine at 2, 4, and 24 h post-PDT. Immediately after mTHPC-PDT, from 28 to 57% cells exhibited cytochrome c release concomitantly with mitochondrial membrane depolarization for light doses inducing more than 90% overall cell death. The maximum of caspase-3 activation (12-fold more than control) was reached 24 h after irradiation at fluence inducing 90% cell death (LD(90)). The corresponding measurement of apoptotic cells (12% of Annexin-V bound cells) confirmed the mild and delayed apoptotic response of HT29 cells to mTHPC-PDT.

Topics: Apoptosis; Caspase 3; Caspases; Colonic Neoplasms; Cytochromes c; Endopeptidases; HT29 Cells; Humans; Membrane Potentials; Mesoporphyrins; Mitochondria; Photochemotherapy; Photosensitizing Agents

Diosgenin-3-O-alpha-L-rhamnopyranosyl-(1 --> 4)-beta-D-glucopyranoside (DRG) is a well-known pentacyclic triterpene glycoside newly isolated from the rhizomes of Dioscorea futschauensis R. Kunth (Dioscoreaceae) by our group. In the present work, the inhibitory effect of DRG on the cell proliferation of human cancer cell lines was examined to reveal for the first time that DRG shows stronger anticancer activity than that of the positive control cisplatin. DRG inhibited the proliferation of human cancer cells, A431, A2780, A549, K562, and HCT-15, with IC50 (micromol L(-1)) values of 9.33 +/- 0.22, 18.7 +/- 0.16, 9.98 +/- 0.38, 6.44 +/- 0.10, and 5.86 +/- 0.14 respectively. It was then found, by morphological observation, "DNA ladder" detection and flow cytometric analysis, that DRG exerts its anticancer effect through inducing apoptosis on HCT-15 cells. Furthermore, it has been demonstrated that DRG triggers a mitochondria-controlled apoptotic pathway to induce apoptosis on HCT-15 cells, which involves the reduction of the mitochondrial potential (deltapsim), the release of cytochrome c from mitochondria into the cytosol, and the down-regulation of the ratio of Bcl-2/Bax expression level. The present results reasonably suggest that regulating the balance of Bcl-2/Bax expression level plays a key role in the DRG-induced apoptosis. Such findings provide novel knowledge to elucidate the biological properties of DRG, even though DRG was discovered early in the late 1960s. These results suggest that DRG may be a good candidate as a chemotherapeutic agent to treat human colon carcinoma.

Topics: Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Dioscorea; Diosgenin; Down-Regulation; Flow Cytometry; Humans; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Saponins

We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo "necrotic" cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.

Topics: Animals; Apoptosis; Cisplatin; Colonic Neoplasms; Cytochromes c; DNA, Antisense; Down-Regulation; Doxorubicin; Etoposide; HCT116 Cells; HT29 Cells; Humans; Macrophages; Mice; Rats; Staurosporine; Transfection

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

Curcumin, the yellow pigment derived from Curcuma longa, is known to induce apoptosis of several cancer cells. However, many cancer cells protect themselves by over-expressing antiapoptotic proteins such as Bcl-XL or Ku70. To study their role in curcumin-induced apoptosis, human colon cancer cells (SW480) were made to over-express or under-express Bcl-XL (by stable transfection) and Ku70 (by transient transfection) using plasmid constructs that express their genes in sense or antisense orientation, respectively. Stable cells that express Bax [Bax-GFP (green fluorescent protein)], a proapoptotic member of the Bcl-2 family, were also established. Curcumin-induced cell death and nuclear condensation was more in AsBcl-XL and AsKu70 cells that under-express Bcl-XL and Ku70, respectively, compared with the vector-transfected cells. Bcl-XL and Ku70 protected the cells by inhibiting the release of cytochrome c, Smac (second mitochondria derived activator of caspase) and apoptosis inducing factor (AIF), and the activation of caspases 9, 8 and 3 triggered by curcumin. AsBcl-XL and AsKu70 cells were more sensitive to curcumin through enhanced activation of caspases 9 and 3 and release of cytochrome c, Smac and AIF. Curcumin-induced activation of caspase 8 was blocked by Ku70 but not by Bcl-XL. However, caspase 8 activation by curcumin was accelerated in both AsBcl-XL and AsKu70 cells suggesting a possible feedback activation of caspase 8 by caspase 3. Bax-GFP cells were highly sensitized when Ku70 was down-regulated supporting the reported role of Ku70 in the retention of Bax within the cytosol. The study reveals the potential of antisense inhibition of antiapoptotic proteins as an effective strategy to tackle chemoresistant cancers with curcumin.

Topics: Antigens, Nuclear; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Caspase Inhibitors; Caspases; Colonic Neoplasms; Complement Membrane Attack Complex; Complement System Proteins; Curcumin; Cytochromes c; DNA Repair; DNA-Binding Proteins; Down-Regulation; Enzyme Activation; Flavoproteins; Gene Expression Regulation, Neoplastic; Glycoproteins; Green Fluorescent Proteins; Humans; Ku Autoantigen; Luminescent Proteins; Membrane Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

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

Nonsteroidal antiinflammatory drugs (NSAIDs) form a paradigm for the chemoprevention of cancer, preventing colonic tumor progression in both experimental animals and humans. However, the mechanisms underlying the antineoplastic effects of NSAIDs are currently unclear. We found that the mitochondrial second mitochondrial-derived activator of caspase (SMAC)/direct inhibitor of apoptosis protein-binding protein with low pI (Diablo) protein translocates into the cytosol during NSAID-induced apoptosis in colon cancer cells. When SMAC/Diablo is disrupted by homologous recombination and RNA interference in these cells, the NSAID-induced apoptosis is abrogated. Biochemical markers of apoptosis, such as caspase activation, cytosolic release of cytochrome c and apoptosis-inducing factor, and mitochondrial membrane potential change, are accordingly decreased. These results establish that SMAC/Diablo is essential for the apoptosis induced by NSAIDs in colon cancer cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Base Sequence; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; DNA Primers; Flavoproteins; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitochondrial Proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that induces apoptosis in multiple tumor cell types while sparing most normal cells. We determined the effect of ectopic Bcl-2 expression on TRAIL-induced apoptosis and whether the small molecule Bcl-2 inhibitor, HA14-1, could increase TRAIL sensitivity.. SW480 human colon cancer cells were stably transfected with the PC3-Bcl-2 plasmid or vector alone. Cells were incubated with recombinant human TRAIL +/- HA14-1 or caspase-9 inhibitor (Z-LEHD-FMK). Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate labeling and DNA fragmentation factor 45 (DFF45) cleavage. Clonigenic survival was also studied. Caspase activation was determined by immunoblotting or colorimetric assay. The cytosolic expression of Bid, Bax, and XIAP and release of cytochrome c and Smac/DIABLO were determined by immunoblotting.. Bcl-2 overexpression partially protected SW480 cells from a dose-dependent induction of apoptosis by TRAIL, as did a caspase-9 inhibitor, and increased their clonogenic survival. Bcl-2 overexpression attenuated TRAIL-induced cleavage of caspase-8, indicating its activation upstream and downstream of mitochondria, as well as cleavage of Bid and caspase-3. Bcl-2 inhibited TRAIL-induced Bax translocation, cytosolic release of cytochrome c and Smac/DIABLO, and the downstream cleavage of XIAP and DFF45. Coadministration of HA14-1 and TRAIL increased apoptosis in SW480/Bcl-2 cells by restoring Bax redistribution and cytochrome c release.. Bcl-2 confers apoptosis resistance to TRAIL by inhibiting a mitochondrial amplification step and by inactivating downstream XIAP in SW480 cells. HA14-1 reversed Bcl-2-mediated TRAIL resistance, suggesting a novel strategy for increasing TRAIL sensitivity in Bcl-2-overexpressing colon cancers.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzopyrans; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 9; Caspase Inhibitors; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Mitochondrial Proteins; Nitriles; Oligopeptides; Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Stem Cell Assay; X-Linked Inhibitor of Apoptosis Protein

Resveratrol, a polyphenol found in grape skin and various other food products, may function as a cancer chemopreventive agent for colon and other malignant tumors and possesses a chemotherapeutic potential through its ability to trigger apoptosis in tumor cells. The present study analyses the molecular mechanisms of resveratrol-induced apoptosis in colon cancer cells, with special attention to the role of the death receptor Fas in this pathway. We show that, in the 10-100 microm range of concentrations, resveratrol activates various caspases and triggers apoptosis in SW480 human colon cancer cells. Caspase activation is associated with accumulation of the pro-apoptotic proteins Bax and Bak that undergo conformational changes and relocalization to the mitochondria. Resveratrol does not modulate the expression of Fas and Fas-ligand (FasL) at the surface of cancer cells, and inhibition of the Fas/FasL interaction does not influence the apoptotic response to the molecule. Resveratrol induces the clustering of Fas and its redistribution in cholesterol and sphingolipid-rich fractions of SW480 cells, together with FADD and procaspase-8. This redistribution is associated with the formation of a death-inducing signaling complex (DISC). Transient transfection of either a dominant-negative mutant of FADD, E8, or MC159 viral proteins that interfere with the DISC function, decreases the apoptotic response of SW480 cells to resveratrol and partially prevents resveratrol-induced Bax and Bak conformational changes. Altogether, these results indicate that the ability of resveratrol to induce the redistribution of Fas receptor in membrane rafts may contribute to the molecule's ability to trigger apoptosis in colon cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Flow Cytometry; Genes, Dominant; Genetic Vectors; Humans; Immunoblotting; Ligands; Lipid Metabolism; Membrane Glycoproteins; Membrane Microdomains; Microscopy, Fluorescence; Mitochondria; Precipitin Tests; Protein Conformation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Signal Transduction; Stilbenes; Time Factors; Transfection

Transformation of colonic epithelial cells is characterized by decreased mitochondrial activity, increased mitochondrial membrane potential (Deltapsi(m)), and disruptions in the equilibrium between cell proliferation and death by apoptosis. We have previously shown that an intact Deltapsi(m) is essential for growth arrest and apoptosis induced by butyrate, a physiological regulator of maturation in these cells, suggesting a role for the Deltapsi(m) in the initiation and integration of proliferation and apoptotic pathways. To extend this work, we have generated isogenic cell lines, from SW620 human colonic carcinoma cells, which exhibit significant differences in intrinsic Deltapsi(m). These differences in Deltapsi(m) are not linked to alterations in viability, Bcl-2 levels, or the differentiation status of the cells. However, compared with parental cells and those with increased Deltapsi(m), cells with decreased intrinsic Deltapsi(m) exhibit significantly higher levels of steady-state mitochondrial mRNA and butyrate-induced p21(WAF1/Cip1) and G(0)-G(1) arrest. Moreover, despite butyrate-mediated translocation of proapoptotic Bax and Bak to the mitochondria, fewer cells with elevated intrinsic Deltapsi(m) exhibit concomitant cytochrome c release, and cells with elevated Deltapsi(m) undergo significantly lower levels of Deltapsi(m) dissipation and apoptosis than parental cells, or cells with decreased Deltapsi(m). Homeostasis of the colonic mucosa depends on balancing cell proliferation with apoptosis, and mitochondrial abnormalities are associated with disruptions in this balance. Thus, by affecting steady-state mitochondrial activity and the extent to which cells enter growth arrest and apoptotic cascades, these data establish a role for the intrinsic Deltapsi(m) in contributing to the probability of colonic tumorigenesis and progression.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Butyrates; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Humans; Intracellular Membranes; Membrane Potentials; Membrane Proteins; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

Flavopiridol is a cyclin-dependent kinase inhibitor currently under development by the National Cancer Institute both as a single agent and in combination with chemotherapy. There have been numerous reports that flavopiridol potently enhances the induction of apoptosis by chemotherapy. However, the effect of flavopiridol on radiotherapy (RT)-induced apoptosis has been largely untested. RT has become the cornerstone of adjuvant treatment of colorectal and gastric cancer. In view of this, we elected to evaluate the effect of flavopiridol on potentiating RT-induced apoptosis in the human colon cancer cell line HCT-116 and the gastric cancer cell line MKN-74.. The efficacy of combination of gamma-irradiation and flavopiridol was tested in vitro in MKN-74 and HCT-116 cells and correlated to changes in p21 expression. HCT-116 cells were also established as tumors in nude mice and treated with gamma-irradiation and flavopiridol either as single agents or in sequential combinations such that flavopiridol was either given 7 h before, concomitantly, or 3 and 7 h after gamma-irradiation.. Flavopiridol significantly enhanced the induction of apoptosis by gamma-irradiation in both cell lines as measured by quantitative fluorescent microscopy, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release. To achieve the best effect, it was important to expose the tumor cells to gamma-irradiation before the flavopiridol. This sequence dependence was confirmed in vivo. When gamma-irradiation was administered 7 h before flavopiridol, 42% of the tumor-bearing animals were rendered disease free, compared with no animals treated with either gamma-irradiation or flavopiridol alone. Examination of the p21 status of HCT-116 and MKN-74 cells, after treatment with sequential gamma-irradiation and flavopiridol, indicated a loss of p21 protein expression. Loss of p21 was mainly due to cleavage by caspases. HCT-116 cells that lack p21 (p21(-/-)) also exhibited sensitization to gamma-irradiation and showed an even greater enhancement of gamma-irradiation-induced apoptosis by flavopiridol when compared with the parental HCT-116 cells.. These studies indicate that gamma-irradiation followed by flavopiridol enhances apoptosis and yields significantly increased tumor regressions and cures that are not achievable with radiation alone. These results indicate that flavopiridol can potently enhance the effect of gamma-radiation both in vitro and in vivo and may provide a new means to treat patients with locally advanced gastrointestinal cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Division; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Gamma Rays; Male; Mice; Mice, Nude; Microscopy, Fluorescence; Piperidines; Poly(ADP-ribose) Polymerases; Stomach Neoplasms; Transplantation, Heterologous; Tumor Cells, Cultured

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Adhesion; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Deoxyadenosines; Enzyme Precursors; Flow Cytometry; Humans; Microscopy, Fluorescence; Pentostatin; Thioinosine

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