Apoptosis In Colorectal Carcinoma Cells Research Articles

3,3′,4,5,5′-pentahydroxy-trans-stilbene, a resveratrol derivative, induces apoptosis in colorectal carcinoma cells via oxidative stress

Resveratrol exhibits anti-tumor properties against different types of cancer. In this study, several polyhydroxylated resveratrol derivatives were prepared with the aim of discovering new leading compounds with clinical potential for human colon cancer chemotherapy. Among these compounds, 3,3',4,5,5'-pentahydroxy-trans-stilbene (PHS) displayed the most potent cytotoxicity and triggered apoptosis in HT-29 cells as evidenced by increased poly(ADP-ribose) polymerase (PARP) cleavage, elevated levels of cytoplasmic nucleosomes and DNA fragmentation. Further mechanistic analysis revealed that PHS-induced apoptosis was caspase-dependent and mediated by its pro-oxidative action through up-regulation of reactive oxidative species generation and depletion of intracellular glutathione.

Docosahexaenoic acid induces apoptosis in colorectal carcinoma cells by modulating the PI3 kinase and p38 MAPK pathways

Numerous studies have shown that long-chain polyunsaturated fatty acids can kill cancer cells in vitro as well as in vivo, while normal cells remain unaffected. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. The aim of this study was to investigate the potential chemopreventative/antiproliferative potential of docosahexaenoic acid (DHA) in an adenocarcinoma cell line (CaCo 2 cells) and to evaluate the signalling pathways modulated by it. DHA (5-50 μM) significantly inhibited cell viability in a dose-dependent manner in CaCo 2 cells, while the viability of normal colon cells (NCM460 cells) was not compromised. DHA also induced apoptosis in CaCo 2 cells, as indicated by increases in caspase-3 activation and poly-ADP-ribose polymerase cleavage. Signalling proteins, which include extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Akt and p53 were analysed by Western blotting using phosphospecific and total antibodies. The protein inhibitors wortmannin (phosphoinositide 3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB 203580 (p38 inhibitor) as well as silencing RNA [small interfering RNA (siRNA)] of the p38 MAPK protein, were used to investigate cross-talk between signalling pathways. DHA supplementation significantly suppressed Akt phosphorylation, which also correlated with decreased cell viability and increased apoptosis in CaCo 2 cells. Furthermore, siRNA experiments suggested a possible role for p38 MAPK in the phosphorylation of p53 at Ser 15, a site which is associated with DNA damage. DHA might thus exert its beneficial effects by means of increased apoptosis and suppression of the important survival-related kinase, Akt.

The 15‐lipoxygenase‐1 expression may enhance the sensitivity to non‐steroidal anti‐inflammatory drug‐induced apoptosis in colorectal cancers from patients who are treated with the compounds

Non-steroidal anti-inflammatory drugs (NSAIDs) can prevent colorectal cancer (CRC), but their effect is limited. Recent studies have shown the involvement of 15-lipoxygenase-1 (15-LOX-1) in NSAID-induced apoptosis in colorectal carcinoma cells. We evaluate whether 15-LOX-1 expression influences the sensitivity of NSAID-induced apoptosis in CRCs. In 22 CRC surgical samples from NSAID users who had been constant for more than 5 years and 28 CRC surgical samples from NSAID non-users, the expressions of 15-LOX-1, cyclooxygenase-2 (COX-2), beta-catenin, and p53 were analyzed using immunohistochemistry. TUNEL assay was also performed for samples. The effects of the transient transfection of 15-LOX-1 cDNA on indomethacin-induced apoptosis were certified in HCT-116 cells. The effects of adding 13-S-hydroxyoctadecadinoic acid (13-S-HODE) on indomethacin-induced apoptosis were also examined in HCT-116 cells. The levels of apoptosis were determined by the analysis of the floating-cells ratio and DNA gel electrophoresis. The expression of 15-LOX-1 on CRCs from NSAID users was significantly decreased compared with those from NSAID non-users; however, the expressions of other molecules were not significantly different between two groups. The levels of TUNEL scoring in samples from NSAID users were similar to those from NSAID non-users. Indomethacin (100 microM) induced less apoptosis in mocked cells, whereas the same concentrations of indomethacin enhanced the level of apoptosis in 15-LOX-1-transfected cells. 13-S-HODE also increased the level of indomethacin-induced apoptosis in cells. Results suggest that 15-LOX-1 expression may be one of the mechanisms which enhance the sensitivity to NSAID-induced apoptosis in CRCs from patients who are treated with the compounds.

Sorafenib treatment of clear-cell renal cell carcinoma (CCRCC) and colorectal carcinoma (CRC) cells: Differential effects on gene expression and cell death pathways

15612 Background: The von Hippel Lindau gene (VHL) is often mutated in CCRCC and leads to loss of VHL protein (pVHL) expression. Sorafenib is a TKI with clinical activity in metastatic CCRCC. Studies to define mechanisms governing anti-tumor activity of this agent in CCRCC or CRC cell lines that express wild-type pVHL were conducted. Methods: We evaluated CAKI-1 (CCRCC) and HCT- 116/p53 +/+ (CRC) cell lines as model systems expressing wild-type pVHL. Cells were treated at 37°C in an atmosphere of normoxia (21% O2) or hypoxia (1% O2), 5% CO2 and the remainder N2 in the absence (control) or presence of sorafenib (2.5–20 μM) for 24–96 hours. Expression of target angiogenesis, apoptotic and anti-apoptotic genes was determined by real-time RT- PCR. Fluorescence microscopy following staining with Hoechst 33342 plus propidium iodide was used to analyze cell death by apoptosis and/or necrosis. Caspase-3 activity was measured using the target substrate DEVD-AFC. Results: In CAKI-1 and HCT-116 cells, exposure to 1% O2 relative to 21% O2, led to increased expression (2 to 6-fold) of angiogenesis (VEGF) and anti-apoptosis (TNFAIP3 & MCF2) genes. However, in an atmosphere of 1% O2 relative to 21% O2, a decreased (>2-fold) and increased (>3-fold) expression of the apoptotic (TNFRSF25) gene was observed in CAKI-1 cells and HCT-116 cells. Sorafenib treatment (7.5 μM) of CAKI-1 cells in 1% O2 led to a >3–4-fold decrease in expression of the VEGF and TNFAIP3 and a 3-fold increase TNFRSF25 genes. Following treatment with 10 μM sorafenib for 48h, cell death was >80% by necrosis in CAKI-1 cells and >95% by apoptosis in HCT-116 cells. Apoptotic cell death in the HCT-116 was also confirmed by increased caspase-3 activity in cell extracts following sorafenib treatment. Apoptotic cell death or necrotic cell death induced by sorafenib was unaffected by normoxia or hypoxia. Conclusions: In contrast to CCRCC cells, hypoxia led to upregulation of the apoptotic gene TNFRSF25 in the CRC cells. Anti- proliferative effects of sorafenib were primarily by necrosis in CCRCC cells and by apoptosis in CRC cells. No significant financial relationships to disclose.

Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive growth and overcomes p38 MAPK-mediated apoptosis

Hepatocyte growth factor (HGF) induces invasive growth, a biological program that confers tumor cells the capability to invade and metastasize by integrating cell proliferation, motility, morphogenesis, and survival. We here demonstrate that HGFR activation promotes survival of colorectal carcinoma (CRC) cells exposed to conditions that mimic those met during tumor progression, i.e. nutrient deprivation or substrate detachment, and following chemotherapeutic treatment. In all these conditions, a sustained activation of p38 MAPK delivers a main death signal that is overcome by cell treatment with HGF. HGF-driven survival requires the engagement of the PI3K/Akt/mTOR/p70S6K and ERK MAPK transduction pathways. Abrogation of p38 MAPK activity prevents CRC cell apoptosis also when these transduction pathways are inhibited, and treatment with HGF further increases survival. Engagement of these signaling cascades is also needed for HGF to induce CRC cell scattering, morphogenesis, motility and invasion. Activation of p38 MAPK signaling is therefore a main apoptotic switch for CRC cells in the stressful conditions encountered during tumor progression. Conversely, HGF orchestrates several biochemical pathways, which allow cell survival in these same conditions and promote the biological responses required for tumor invasive growth. Both p38 MAPK and HGF/HGFR signaling constitute potential molecular targets for inhibiting colorectal carcinogenesis.

Heterocyclic complexes of ruthenium(III) induce apoptosis in colorectal carcinoma cells

The ruthenium complex salt indazolium trans-[tetrachlorobisindazole-ruthenate(III)] (KP1019) and the analogous sodium salt KP1339 are effective tumor-inhibiting drugs in experimental therapy of autochthonous colorectal carcinomas in rats. This paper examines the cell biological mechanisms underlying their antineoplastic effects. Colorectal tumor cell lines were used to analyze uptake of the ruthenium(III) complexes into the cells and the mechanism as well as the efficacy of their cytotoxic effects. KP1019 and KP1339 are efficiently taken up into the cells: 100 microM ruthenium(III) complex in the growth medium led to the uptake of 120-160 ng ruthenium per 10(6) cells within 30 min. Uptake of KP418 was tenfold lower correlating with its lower cytotoxic efficiency. KP1019 and KP1339 induced apoptosis in SW480 and HT29 cells predominantly by the intrinsic mitochondrial pathway as indicated by loss of mitochondrial membrane potential. Correspondingly sensitivity of the cells paralleled expression of bcl(2) while it was only slightly affected by mutations in Ki-ras. Our data demonstrate that trans-[tetrachlorobisindazole-ruthenate(III)] complex salts are promising candidate drugs in the second-line treatment of colorectal cancers resistant to other cytostatic drugs and has been introduced into phase I clinical trials.

Flavone inhibition of tumor growth via apoptosis in vitro and in vivo

Colorectal carcinoma is a human malignant tumor, which is very resistant to currently available methods of treatment. Therefore, developing an effective agent with anti-colorectal carcinoma activity is important. In the present study, 8 structurally related flavones including flavone, 3-OH flavone, 5-OH flavone, 7-OH flavone, quercetin, kaempferol, quercetin, and morin were used to study their effects on colorectal carcinoma cells (HT29, COLO205, COLO320-HSR). Results of MTT assay indicated that flavone shows the most potent cytoxic effect among them on these three cell types. The cytotoxicity induced by flavone is mediated by inducing the occurrence of apoptosis characterized by the appearance of DNA ladders, apoptotic bodies and hypodiploid cells. Activation of caspase 3 protein procession and enzyme activity with inducing cleavage of caspase 3 substrates PARP was identified in flavone-treated cells, and an inhibitory peptide Ac-DEVD-FMK for caspase 3, but not Ac-YVAD-FMK for caspase 1, attenuates the cytotoxic effect of flavone in COLO205 and HT29 cells. Elevation of p21 but no p53 protein was observed in flavone-treated cells. Increasing intracellular peroxide level was detected in flavone-treated cells by DCHF-DA assay, and antioxidants such as tiron, catalase, SOD, PDTC, but not DPI, suppress flavone-induced cytotoxic effect. In vivo anti-tumor study indicates that flavone exhibits ability to inhibit tumor formation elicited by s.c. injection of COLO205 cells in nude mice, and apoptotic cells and an increase in p21, but not p53, protein were observed in tumor tissues derived from flavone-treated group. Additionally, flavone induced apoptosis in primary colon carcinoma cells COLO205-X with appearance of DNA ladders, caspase 3 protein procession, PARP protein cleavage, and an increase in p21 (not p53) protein. These data provide evidence to suggest that flavone is an effective agent to induce apoptosis in colorectal carcinoma cells in vitro and in vivo; activation of caspase 3, ROS production, and increasing p21 protein are involved.

Linear relationship between Wnt activity levels and apoptosis in colorectal carcinoma cells exposed to butyrate.

We have reported that butyrate, a fatty acid produced by dietary fiber that induces cell cycle arrest, differentiation and/or apoptosis in colorectal carcinoma (CRC) cells in vitro, modulates Wnt activity in 2 CRC cell lines (Bordonaro et al., Int. J. Cancer, 2002; 97:42-51). Our study determines how changes in the levels of Wnt activity induced by butyrate relate to the effects of butyrate on apoptosis, cell cycle arrest and differentiation of CRC cells. In 10 human CRC cell lines a direct relationship was shown between apoptosis and butyrate-induced increase in Wnt activity, as well as between suppressed clonal growth and increased Wnt activity. No correlation existed between butyrate-induced increase in Wnt activity and differentiation. The direct relationship between apoptosis and Wnt activity was supported by analyses of DLD-1 and HCT-116 cells expressing a dominant negative form of Tcf4, and therefore, with repressed Wnt activity, as well as by measuring the ratio of apoptotic to live cells in flow cytometry-sorted cell fractions with high and low Wnt activity. Novel flow cytometric methodology was utilized to show that butyrate differentially increases the number of cells with Wnt activity in different CRC cell lines. Thus, CRC cell lines in which butyrate upregulated Wnt activity to relatively high levels were most susceptible to the apoptotic effects of butyrate, whereas cell lines in which butyrate modestly modulated Wnt activity were less affected.

Induction of apoptosis in colorectal carcinoma cells treated with 4-hydroxy-2-nonenal and structurally related aldehydic products of lipid peroxidation.

The oxidation of polyunsaturated fatty acids during oxidative stress gives rise to a series of toxic alpha,beta-unsaturated aldehydes, including the electrophile 4-hydroxy-2-nonenal (4-HNE) and the related aldehydes, 4-hydroperoxy-2-nonenal (4-HPNE) and 4-oxo-2-nonenal (4-ONE). We synthesized these compounds, as well as the resolved enantiomers of 4-HNE, and compared their toxicities and apoptotic responses in the human colorectal cancer cell line RKO. All of these molecules execute similar death responses at comparable doses over almost identical time frames in RKO cells. The apoptotic response induced by 4-HPNE, 4-ONE, and 4-HNE enantiomers involves activation of caspases, proteolysis of downstream caspase targets, and nucleosomal DNA fragmentation. The results presented herein suggest that these molecules commonly activate certain signaling pathways that control cell death irrespective of their reactive properties.

Overadditive anti-proliferative and pro-apoptotic effects of a combination therapy on colorectal carcinoma cells

The prognosis of advanced colorectal carcinoma (CC) is poor. Established chemotherapy shows only limited efficacy but significant side effects. We investigated how far a combination of tamoxifen (TAM), 9-cis-retinoic acid (CRA) and the fluoroquinolone ciprofloxacin (CIP) synergize to inhibit proliferation and promote apoptosis of CC cells in vitro. The CC cell lines LOVO, CC-531 and SW-403 were incubated with TAM, CRA and CIP (10(minus;4)-10(minus;6) M) as single agents and in combination. Cell proliferation was assessed by bromodeoxyuridin incorporation. Apoptosis was quantified immunohistochemically and by FACS analysis after staining with propidium iodide. Changes in the expression of caspase 3, bax, bcl-2 and p21cip/waf were assessed by quantitative Western blotting. CRA and TAM monotherapy was moderately effective. Their combination enhanced apoptosis from 60% to more than 80% in all cell types. Apoptosis was paralleled by inhibition of proliferation and further potentiated by addition of CIP. The combination effectively up-regulated caspase 3 and bax and down-regulated bcl-2 and p21cip/waf. Combinations of biomodulaters act synergistically to inhibit proliferation and promote apoptosis in CC cells. Due to their known safety profile, this justifies clinical trials for colorectal cancer using combinations of these biological response modifiers.

Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor

Background Spontaneous apoptosis has been shown to predict tumor response to radiochemotherapy in rectal cancer in vivo. It remains to be elucidated, however, which genetic profile determines whether a tumor is more or less prone to apoptosis. Recently, a novel member of the inhibitor of apoptosis protein family, designated survivin, was identified. We investigated the impact of surviving expression on tumor cell apoptosis in three colorectal cell lines of different intrinsic radiosensitivities. Methods and materials Survivin protein expression was measured by Western blot analysis, and survivin mRNA expression by quantitative TaqMan reverse transcription polymerase chain reaction, both in untreated cell and after irradiation with 2 and 8 Gy. The expression profile was then correlated to spontaneous and radiation-induced apoptosis (Tunel-Assay, DAPI-staining) in three colorectal cell lines of low (SW 480), intermediate (HCT-15), and high radiosensitivity (SW 48), as determined by the colony-forming assay. Results In vitro analysis revealed higher spontaneous and higher radiation-induced apoptosis rates in the radiosensitive line (SW 48), as compared with the more resistant line (SW 480). In Western blot analysis and in TaqMan analysis, SW 480 was characterized by a higher spontaneous expression and a pronounced induction of survivin 48 h after irradiation, whereas survivin expression was low when untreated and not increased after irradiation in the most radiosensitive line SW 48. HCT-15 was intermediate, both with respect to the level of survivin mRNA and protein expression. Conclusion The inverse correlation of survivin-expression with spontaneous and radiation-induced apoptosis suggests that survivin is an important inhibitor of apoptosis in colorectal cancer cell lines. Analysis of survivin mRNA or protein expression may therefore provide predictive information on radio- and chemoresistance of individual colorectal tumors.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

Role of Bax in resveratrol-induced apoptosis of colorectal carcinoma cells.

BackgroundThe natural plant polyphenol resveratrol present in some foods including grapes, wine, and peanuts, has been implicated in the inhibition, delay, and reversion of cellular events associated with heart diseases and tumorigenesis. Recent work has suggested that the cancer chemoprotective effect of the compound is primarily linked to its ability to induce cell division cycle arrest and apoptosis, the latter possibly through the activation of pro-apoptotic proteins such as Bax.MethodsThe expression, subcellular localization, and importance of Bax for resveratrol-provoked apoptosis were assessed in human HCT116 colon carcinoma cells and derivatives with both bax alleles inactivated.ResultsLow to moderate concentrations of resveratrol induced co-localization of cellular Bax protein with mitochondria, collapse of the mitochondrial membrane potential, activation of caspases 3 and 9, and finally, apoptosis. In the absence of Bax, membrane potential collapse was delayed, and apoptosis was reduced but not absent. Resveratrol inhibited the formation of colonies by both HCT116 and HCT116 bax -/- cells.ConclusionResveratrol at physiological doses can induce a Bax-mediated and a Bax-independent mitochondrial apoptosis. Both can limit the ability of the cells to form colonies.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria.

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.

The possible involvement of 15-lipoxygenase/leukocyte type 12-lipoxygenase in colorectal carcinogenesis.

The initial genetic alteration in colorectal carcinoma is mutation of the APC gene. As a result, the expected rate of apoptosis in colorectal cells is attenuated leading to the expansion of the progeny of the APC mutant cells and hence tumor formation1. The up-regulation of cyclooxygenase-2 (Cox-2) occurs at this early stage of colorectal carcinoma and high expression is observed in multiple intestinal neoplasm (Min) mice, the model of familiar adenomatous polyposis2,3, and in 85% of human colorectal carcinomas4. The over-expression of Cox-2 inhibits apoptosis in rat intestinal cells5 and non-steroidal anti-inflammatory drugs (NSAIDs), such as sulindac sulfide, indomethacin and aspirin promote apoptosis in colorectal carcinoma cells. NSAIDs serve in chemoprevention of colorectal cancer in experimental animal models indicating Cox as a target for preventing colorectal tumors in man6,7. However, the functional role of the mutated APC, the overexpression Cox-2 and the inhibition of apoptosis is not clearly understood.