miR-150-504-519d inhibits the growth of human colorectal cancer cell line SW48 and downregulates c-FLIP receptor

microRNAs (miRNAs) are noncoding RNAs that regulates the expression of target messenger RNAs (mRNAs). c-FLIP is an inhibitor of cell apoptosis through inhibition of caspase 8. miR-150, miR-504, and miR-519d were related to cancer cell proliferation, invasion, and migration in colorectal cancer (CRC). However, the role of miR-150-504-519d in CRC has not been studied and the relationship between miR-150-504-519d and c-FLIP remains unclear. In this study, we found that c-FLIP was upregulated in CRC tissues, without detectable expression in normal CRC tissues. Using SW48 cell line, we further showed that miR-150-504-519d inhibited migration, invasion, and promoted apoptosis of SW48 cells. Moreover, in SW48 cell line transfected with miR-150-504-519d, the protein expression of c-FLIP was significantly lower compared with cells transfected with scramble. Our results demonstrated upregulation of c-FLIP in CRC, which was downregulated in SW48 cells after the transfection of miR-150-504-519d, suggesting that manipulation of miR-150-504-519d expression might be a novel approach for the treatment of colorectal cancer.     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

STAT3-siRNA对人结直肠癌细胞的干涉作用

目的:研究STAT3-siRNA对STAT3基因表达阳性的结直肠癌细胞凋亡的影响。方法:应用脂质体转染试剂将STAT3-siRNA表达盒(STAT3-siRNA expression cassettes,STAT3-SECs)体外转染至人结直肠癌SW480细胞及人成纤维细胞中,同时分别设立人成纤维对照组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组。于48h后收集细胞,先经荧光染色方法观察细胞表象变化,再通过流式细胞仪检测人结直肠癌SW480细胞凋亡情况,后分别提取细胞总RNA,用RT-PCR测定STAT3基因在mRNA水平的表达。结果:SW480STAT3-SECs组的细胞可见凋亡小体,出现明显的凋亡现象,而人成纤维对照组、人成纤维STAT3-SECs组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组未出现明显的凋亡现象。SW480STAT3-SECs组细胞的凋亡比率较SW480对照组、SW480错配链-SECs组和SW480空转染试剂组有明显的增高。RT-PCR所得数据经统计学处理得出:SW480STAT3-SECs组细胞的STAT3基因表达在mRNA水平上显著低于SW480对照组(P0.01);而人成纤维对照组与人成纤维STAT3-SECs组,SW480细胞对照组与SW480错配链-SECs组、SW480空转染试剂组之间无明显差异(P0.05)。结论:应用RNAi技术沉默STAT3基因可以降低人结直肠癌SW480细胞中STAT3的表达,诱导细胞的凋亡。     

Effects of silk sericin on the proliferation and apoptosis of colon cancer cells

Sericin is a silk protein woven from silkworm cocoons (Bombyx mori). In animal model, sericin has been reported to have anti-tumoral action against colon cancer. The mechanisms underlying the activity of sericin against cancer cells are not fully understood. The present study investigated the effects of sericin on human colorectal cancer SW480 cells compared to normal colonic mucosal FHC cells. Since the size of the sericin protein may be important for its activity, two ranges of molecular weight were tested. Sericin was found to decrease SW480 and FHC cell viability. The small sericin had higher anti-proliferative effects than that of the large sericin in both cell types. Increased apoptosis of SW480 cells is associated with increased caspase-3 activity and decreased Bcl-2 expression. The anti-proliferative effect of sericin was accompanied by cell cycle arrest at the S phase. Thus, sericin reduced SW480 cell viability by inducing cell apoptosis via caspase-3 activation and down-regulation of Bcl-2 expression. The present study provides scientific data that support the protective effect of silk sericin against cancer cells of the colon and suggests that this protein may have significant health benefits and could potentially be developed as a dietary supplement for colon cancer prevention.     

曲美替尼与GSK2126458联合用药逆转结直肠癌SW480细胞的耐药

近年来, 肿瘤靶向药物因其特异性强与对正常细胞损伤小等特点,已成为癌症治疗的热点药物。但由肿瘤异质性导致的靶向药物的耐受现象,成为癌症治疗需要解决的难题之一。为解决单一药物的耐受现象,可以通过药物组合来达到理想的治疗效果。本课题以结直肠癌为研究对象,评估8种结直肠癌细胞对30种靶向药物的敏感性,并筛选可逆转耐药的药物组合,探究药物组合的作用。通过MTT实验测定细胞存活率,计算IC50值进行敏感性分析,敏感标准为IC50值≤100 nmol/L。对敏感的单药进行组合筛选,选取细胞存活率最小的组合。采用流式细胞术和Western印迹检测联合用药对细胞凋亡及MAPK、PI3K通路相关蛋白质表达水平的影响。MTT结果显示,结直肠癌SW480细胞耐受30种肿瘤靶向药物,经联合用药筛选,SW480细胞对曲美替尼与GSK2126458组合最为敏感,与对照组和单药组相比,该组合可使SW480细胞凋亡明显增加。免疫印迹结果显示,ERK、Akt和mTOR磷酸化水平降低,Cleaved PARP表达增加。上述结果表明,8种结直肠癌细胞存在不同程度耐受靶向抑制剂的现象,曲美替尼与GSK2126458联合应用可逆转结直肠癌SW480细胞的耐药现象。     

曲美替尼与GSK2126458联合用药逆转结直肠癌SW480细胞的耐药

近年来, 肿瘤靶向药物因其特异性强与对正常细胞损伤小等特点,已成为癌症治疗的热点药物。但由肿瘤异质性导致的靶向药物的耐受现象,成为癌症治疗需要解决的难题之一。为解决单一药物的耐受现象,可以通过药物组合来达到理想的治疗效果。本课题以结直肠癌为研究对象,评估8种结直肠癌细胞对30种靶向药物的敏感性,并筛选可逆转耐药的药物组合,探究药物组合的作用。通过MTT实验测定细胞存活率,计算IC50值进行敏感性分析,敏感标准为IC50值≤100 nmol/L。对敏感的单药进行组合筛选,选取细胞存活率最小的组合。采用流式细胞术和Western印迹检测联合用药对细胞凋亡及MAPK、PI3K通路相关蛋白质表达水平的影响。MTT结果显示,结直肠癌SW480细胞耐受30种肿瘤靶向药物,经联合用药筛选,SW480细胞对曲美替尼与GSK2126458组合最为敏感,与对照组和单药组相比,该组合可使SW480细胞凋亡明显增加。免疫印迹结果显示,ERK、Akt和mTOR磷酸化水平降低,Cleaved PARP表达增加。上述结果表明,8种结直肠癌细胞存在不同程度耐受靶向抑制剂的现象,曲美替尼与GSK2126458联合应用可逆转结直肠癌SW480细胞的耐药现象。     

20(S)-protopanaxadiol induces apoptosis in human umbilical vein endothelial cells by activating the PERK-eIF2alpha-ATF4 signaling pathway

20(S)-protopanaxadiol (PPD)-type ginsenosides are generally believed to be the most pharmacologically active components of Panax ginseng. These compounds induce apoptotic cell death in various cancer cells, which suggests that they have anti-cancer activity. Anti-angiogenesis is a promising therapeutic approach for controlling angiogenesis-related diseases such as malignant tumors, age-related macular degeneration, and atherosclerosis. Studies showed that 20(S)-PPD at low concentrations induces endothelial cell growth, but in our present study, we found 20(S)-PPD at high concentrations inhibited cell growth and mediated apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism by which high concentrations of 20(S)-PPD mediate endothelial cell apoptosis remains elusive. The present current study investigated how 20(S)-PPD induces apoptosis in HUVECs for the first time. We found that caspase-9 and its downstream caspase, caspase-3, were cleaved into their active forms after 20(S)-PPD treatment. Treatment with 20(S)-PPD decreased the level of Bcl-2 expression but did not change the level of Bax expression. 20(S)-PPD induced endoplasmic reticulum stress in HUVECs and stimulated UPR signaling, initiated by protein kinase R-like endoplasmic reticulum kinase (PERK) activation. Total protein expression and ATF4 nuclear import were increased, and CEBP-homologous protein (CHOP) expression increased after treatment with 20(S)-PPD. Furthermore, siRNA-mediated knockdown of PERK or ATF4 inhibited the induction of CHOP expression and 20(s)-PPD-induced apoptosis. Collectively, our findings show that 20(S)-PPD inhibits HUVEC growth by inducing apoptosis and that ATF4 expression activated by the PERK-eIF2α signaling pathway is essential for this process. These findings suggest that high concentrations of 20(S)-PPD could be used to treat angiogenesis-related diseases.     

Mitochondrial DNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells

We investigated the potential role of mitochondrial DNA (mtDNA) in colorectal carcinogenesis by constructing a eukaryotic expression vector of the mitochondrial D-loop gene from colorectal cancer cell SW480 and transfected NIH3T3 cells. The NIH3T3/SW480 cells exhibited a significantly increased growth rate and colony formation rate, and also had a decreased apoptotic rate. Polyploidy and aberrant chromosomes were detected in the NIH3T3/SW480 cells by chromosome karyotype analysis. Our results suggested that mtDNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells. Further study of the biological functions of NIH3T3/SW480 cells might be helpful in understanding the role of mtDNA in colorectal carcinogenesis.     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.     

PEG-liposomal oxaliplatin induces apoptosis in human colorectal cancer cells via Fas/FasL and caspase-8

Since cellular uptake of PEG [poly(ethylene glycol)]-liposomal L-OHP (oxaliplatin) induces bioactive changes in CRC (colorectal cancer), we have investigated its apoptotic effect and anticancer mechanism. Human CRC SW480 cells were treated with PEG-liposomal L-OHP and a caspase-8 inhibitor [Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-dl-Asp-fluoromethylketone)]. Apoptosis was measured by FCM (flow cytometry) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. Expression of Fas/FasL and cytochrome c was detected using FCM and an immunofluorescence assay. Expression of caspase-8, Bid, caspase-9, caspase-7 and activated caspase-3 (P17) was examined by Western blot analyses. The results indicated that PEG-liposomal L-OHP (28 μg/ml L-OHP) induced marked apoptosis in SW480 cells compared with 28 μg/ml free L-OHP. The expression levels of Fas, FasL, cytochrome c, caspase-9, caspase-7 and activated caspase-3 proteins were up-regulated, with a corresponding increase in apoptosis; however, expression of caspase-8 and Bid were down-regulated as apoptosis increased. When cells were treated with Z-IETD-FMK, apoptosis was inhibited, but there was little impact on the expression of Fas, FasL, cytochrome c, Bid, caspase-9, caspase-7 and activated caspase-3. These findings indicate that PEG-liposomal L-OHP enhances the anticancer potency of the chemotherapeutic agent; moreover, Fas/FasL and caspase-8 signalling pathways play a key role in mediating PEG-liposomal L-OHP-induced apoptosis.     

Matrine promotes apoptosis in SW480 colorectal cancer cells via elevating MIEF1-related mitochondrial division in a manner dependent on LATS2-Hippo pathway

Matrine, an alkaloid compound isolated from Sophora flavescens Ait, has been shown to exert cancer-killing actions in a variety of tumors; however, its anticancer mechanism in colorectal cancer (CRC) is not clear. The goal of our study was to characterize the anticancer effects and molecular mechanisms of matrine in SW480 CRC cells in vitro. Matrine treatment reduced mitochondrial metabolic function and ATP levels, repressed mitochondrial membrane potential, evoked mitochondrial reactive oxygen species accumulation, and promoted cyt-c-related mitochondrial apoptosis activation. In addition, we found that matrine treatment activated mitochondrial fission through upregulating mitochondrial elongation factor 1 (MIEF1); silencing of MIEF1 prevented matrine-mediated mitochondrial damage and reversed the decrease in SW480 cell viability. Moreover, matrine treatment affected MIEF1 expression via the large tumor suppressor-2 (LATS2)-Hippo axis, and LATS2 deficiency suppressed the anticancer actions exerted by matrine on SW480 cancer cells. In summary, we show for the first time that matrine inhibits SW480 cell survival by activating MIEF1-related mitochondrial division via the LATS2-Hippo pathway. These findings explain the anticancer mechanisms of matrine in CRC and also identify the LATS2-MIEF1 signaling pathway as an effective target for the treatment of CRC.     

Protective effects of the ethanolic extract of Melia toosendan fruit against colon cancer

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.     

Triggering of death receptor apoptotic signaling by human papillomavirus 16 E2 protein in cervical cancer cell lines is mediated by interaction with c-FLIP

Human papillomavirus (HPV) E2 gene disruption is one of the key features of HPV-induced cervical malignant transformation. Though it is thought to prevent progression of carcinogenesis, the pro-apoptotic function of E2 protein remains poorly understood. This study shows that expression of HPV16 E2 induces apoptosis both in HPV-positive and -negative cervical cancer cell lines and leads to hyperactivation of caspase-8 and caspase-3. Activation of these signaling factors is responsible for the observed sensitivity to apoptosis upon treatment with anti-Fas antibody or TNF-α. In addition, immunoprecipitation experiments clearly show an interaction between HPV16 E2 and c-FLIP, a key regulator of apoptotic cell death mediated by death receptor signaling. Moreover, c-FLIP and a caspase-8 inhibitor protect cells from HPV16 E2-mediated apoptosis. Overexpression of c-FLIP rescues cervical cancer cells from apoptosis induced by HPV16 E2 protein expression. The data suggest that HPV16 E2 abrogates the apoptosis-inhibitory function of c-FLIP and renders the cell hypersensitive to the Fas/FasL apoptotic signal even below threshold concentration. This suggests a novel mechanism for deregulation of cervical epithelial cell growth upon HPV-induced transformation, which is of great significance in developing therapeutic strategies for intervention of cervical carcinogenesis.     

Down-regulation of AMP-activated protein kinase sensitizes DU145 carcinoma to Fas-induced apoptosis via c-FLIP degradation

The death receptor Fas (APO-1/CD95) induces apoptosis in many tissues upon cross-linking by its ligand (FasL), but a number of cancer cells exhibit resistance to such apoptosis. Indeed, resistance to apoptosis seems to be one of the hallmarks of cancer, and therefore, it is clinically important to understand the underlying mechanisms by which cancer cells acquire such resistance. In the present study, we demonstrate that Fas signaling in DU145 human prostate cancer cells leads to rapid activation of AMP-activated protein kinase (AMPK), which plays a major role in adaptive responses to ATP-depleting conditions; prostate cancer is resistant to Fas-mediated apoptosis despite high levels of Fas surface expression and no mutation in the Fas gene. We further demonstrate that inhibition of AMPK sensitizes DU145 cells to Fas-induced apoptosis via enhancement of ubiquitination and consequent proteasome degradation of the apoptosis inhibitory protein c-FLIP. These findings thus reveal a novel anticancer property of AMPK inhibition and support the synergistic application of AMPK inhibition in cancer therapy to overcome Fas resistance.     

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP_L, and the short c-FLIP isoform, c-FLIP_R, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.     

PQ1, a quinoline derivative, induces apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9

Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.