Apoptosis In Human Colorectal Cancer Research Articles

Engulfment and Cell Motility 1 (ELMO1) Regulates Tumor Cell Behavior and Predicts Prognosis in Colorectal Cancer.

Engulfment and cell motility 1 (ELMO1) plays a crucial role in the process of migration, chemotaxis, and metastasis of tumor cells. ELMO1 has been implicated in the pathogenesis of various cancers. However, the distinct function of ELMO1 in colorectal cancer (CRC) is unclear. We determined whether ELMO1 affects the oncogenic behavior of CRC cells and investigated its prognostic value in CRC patients. We investigated the impact of ELMO1 on tumor cell behavior using small interference RNA (siRNA) in CRC cell lines, including SW480 and DLD1. The expression of ELMO1 was investigated by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) in cancer tissues and sera obtained from CRC patients. ELMO1 knockdown suppressed tumor cell proliferation in SW480 and DLD1 cells. ELMO1 knockdown-induced apoptosis through up-regulation of caspase-3, -7, and PARP activities and down-regulation of the anti-apoptotic Mcl-1 protein. ELMO1 knockdown-induced cell-cycle arrest by decreasing cyclin D1, cyclin-dependent kinase 2, 4 and 6, and the 25C cell division cycle (CDC25C). ELMO1 knockdown suppressed tumor cell invasion and migration. The expression of E-cadherin was increased, while that of Vimentin and Claudin 1 decreased following ELMO1 knockdown. The phosphorylation levels of PDK1, Akt, and GSK-3β and were down-regulated after ELMO1 knockdown. The expression of ELMO1 was found up-regulated in cancer tissues and sera taken from CRC patients. ELMO1 expression was significantly associated with tumor stage, lymph node metastasis, distant metastases, and poor survival. ELMO1 mediates tumor progression by increasing tumor cell motility and inhibiting apoptosis in human CRC.

Co-inhibition of Aurora A and Haspin kinases enhances survivin blockage and p53 induction for mitotic catastrophe and apoptosis in human colorectal cancer

Colorectal cancer (CRC) is a leading cause and mortality worldwide. Aurora A and haspin kinases act pivotal roles in mitotic progression. However, the blockage of Aurora A and Haspin for CRC therapy is still unclear. Here we show that the Haspin and p-H3T3 protein levels were highly expressed in CRC tumor tissues of clinical patients. Overexpression of Haspin increased the protein levels of p-H3T3 and survivin in human CRC cells; conversely, the protein levels of p-H3T3 and survivin were decreased by the Haspin gene knockdown. Moreover, the gene knockdown of Aurora A induced abnormal chromosome segregation, mitotic catastrophe, and cell growth inhibition. Combined targeted by co-treatment of CHR6494, a Haspin inhibitor, and MLN8237, an Aurora A inhibitor, enhanced apoptosis and CRC tumor inhibition. MLN8237 and CHR6494 induced abnormal chromosome segregation and mitotic catastrophe. Meanwhile, MLN8237 and CHR6494 inhibited survivin protein levels but conversely induced p53 protein expression. Ectopic survivin expression by transfection with a survivin-expressed vector resisted the cell death in the MLN8237- and CHR6494-treated cells. In contrast, the existence of functional p53 increased the apoptotic levels by treatment with MLN8237 and CHR6494. Co-treatment of CHR6494 and MLN8237 enhanced the blockage of human CRC xenograft tumors in nude mice. Taken together, co-inhibition of Aurora A and Haspin enhances survivin inhibition, p53 pathway induction, mitotic catastrophe, apoptosis and tumor inhibition that may provide a potential strategy for CRC therapy.

Brg1 is required to maintain colorectal cancer stem cells.

Tumor cells capable of self-renewal and continuous production of progeny cells are called tumor stem cells (TSCs) and are considered to be potential therapeutic targets. However, the mechanisms underlying the survival and function of TSCs are not fully understood. We previously reported that chromatin remodeling regulator Brg1 is essential for intestinal stem cells in mice and Dclk1 is an intestinal TSC marker. In this study, we investigated the role of Brg1 in Dclk1+ intestinal tumor cells for the maintenance of intestinal tumors in mice. Specific ablation of Brg1 in Dclk1+ intestinal tumor cells reduced intestinal tumors in ApcMin mice, and continuous ablation of Brg1 maintained the reduction of intestinal tumors. Lineage tracing in the context of Brg1 ablation in Dclk1+ intestinal tumor cells revealed that Brg1-null Dclk1+ intestinal tumor cells did not give rise to their descendent tumor cells, indicating that Brg1 is essential for the self-renewal of Dclk1+ intestinal tumor cells. Five days after Brg1 ablation, we observed increased apoptosis in Dclk1+ tumor cells. Furthermore, Brg1 was crucial for the stemness of intestinal tumor cells in a spheroid culture system. BRG1 knockdown also impaired cell proliferation and increased apoptosis in human colorectal cancer (CRC) cells. Microarray analysis revealed that apoptosis-related genes were upregulated and stem cell-related genes were downregulated in human CRC cells by BRG1 suppression. Consistently, high BRG1 expression correlated with poor disease-specific survival in human CRC patients. These data indicate that Brg1 plays a crucial role in intestinal TSCs in mice by inhibiting apoptosis and is critical for cell survival and stem cell features in human CRC cells. Thus, BRG1 represents a new therapeutic target for human CRC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A Disintegrin and Metalloprotease 12 Promotes Tumor Progression by Inhibiting Apoptosis in Human Colorectal Cancer.

Simple SummaryA disintegrin and metalloprotease 12 (ADAM12) has been associated with tumor development and progression. The aim of the current study was to evaluate the impact of ADAM12 on cancer progression, prognosis, and therapeutic targets in colorectal cancer (CRC). Our results show that ADAM12 overexpression enhanced proliferation, inhibited apoptosis, and acted as a positive regulator of cell cycle progression in CRC cells. Phosphorylation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was decreased and that of Akt was increased by ADAM12 overexpression. These results were reversed upon ADAM12 knockdown. ADAM12 overexpression was significantly associated with the cancer stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival in CRC patients. In a mouse xenograft model, tumor area, volume, and weight were significantly greater for the ADAM12 overexpression group and significantly lower for the ADAM12 knockdown group. In conclusion, ADAM12 may serve as a promising biomarker and/or therapeutic target in CRC.A disintegrin and metalloprotease 12 (ADAM12) has been implicated in cell growth, tumor formation, and metastasis. Therefore, we evaluated the role of ADAM12 in colorectal cancer (CRC) progression and prognosis, and elucidated whether targeted downregulation of ADAM12 could lead to therapeutic sensitization. The effect of ADAM12 on tumor cell behavior was assessed in CRC cell lines, CRC tissues, and a mouse xenograft model. ADAM12 overexpression enhanced proliferation, inhibited apoptosis, and acted as positive regulator of cell cycle progression in CRC cells. Phosphorylation of PTEN was decreased and that of Akt was increased by ADAM12 overexpression. These results were reversed upon ADAM12 knockdown. ADAM12 overexpression was significantly associated with the cancer stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival in CRC patients. In a mouse xenograft model, tumor area, volume, and weight were significantly greater for the ADAM12-pcDNA6-myc-transfected group than for the empty-pcDNA6-myc-transfected group, and significantly lower for the ADAM12-pGFP-C-shLenti-transfected group than for the scrambled pGFP-C-shLenti-transfected group. In conclusion, ADAM12 overexpression is essential for the growth and progression of CRC. Furthermore, ADAM12 knockdown reveals potent anti-tumor activity in a mouse xenograft model. Thus, ADAM12 may serve as a promising biomarker and/or therapeutic target in CRC.

Inhibition of Autophagy Amplifies Baicalein-Induced Apoptosis in Human Colorectal Cancer

Baicalein is a Chinese herbal compound extracted from Scutellaria baicalensis that has anti-tumor properties. The aim of this study was to elucidate the mechanisms of action of baicalein against human colorectal cancer cell lines and to assess whether the anti-proliferative effects of baicalein may be amplified with autophagy inhibition. Human colon cancer cell lines (HT-29, HCT-116, SW480, and SW620) were treated with baicalein alone and in combination with the autophagy inhibitor chloroquine (CQ). Baicalein reduced cell viability in all four colon cancer lines in a dose-dependent fashion. Combination treatment of baicalein and the autophagy inhibitor CQ significantly decreased cell viability compared with baicalein alone in HT-29 and HCT-116 cell lines. Western blot analysis of the HCT-116 cell line treated with both baicalein and CQ demonstrated increased expression of LC3-II, a component of autophagy. The combination of baicalein with CQ culminated in activation of caspase-3-mediated apoptosis. These findings demonstrate that inhibition of autophagy enhanced apoptotic cell death induced by baicalein treatment in colon cancer cell lines. Future work will assess other targetable apoptotic pathways activated by baicalein and autophagy inhibition.

Effects of CLIC4 on Fucoxanthinol-Induced Apoptosis in Human Colorectal Cancer Cells

Fucoxanthin is a marine xanthophyll found in edible brown algae, and a metabolite, fucoxanthinol (FxOH), possesses a potent apoptosis inducing effect in many cancer cells. Chloride intracellular channel 4 (CLIC4) is a member of the CLIC family that plays an important role in cancer development and apoptosis. However, the role of CLIC4 in FxOH-induced apoptosis is not well understood. In this study, we investigated whether CLIC4 affects the apoptotic properties of FxOH in human colorectal cancer (CRC) cells under FxOH treatment. Treating human CRC DLD-1 cells with 5.0 μmol/L FxOH significantly induced apoptosis. FxOH downregulated CLIC4, integrin β1, NHERF2 and pSmad2 (Ser465/467) by 0.6-, 0.7-, 0.7-, and 0.5-fold, respectively, compared with control cells without alteration of Rab35 expression. No colocalizing change was observed in CLIC4-related proteins in either control or FxOH-treated cells. CLIC4 knockdown suppressed cell growth and apoptosis. Interestingly, apoptosis induction by FxOH almost disappeared with CLIC4 knockdown. Our findings suggested that CLIC4 could be involved in FxOH-induced apoptosis in human CRC.

Novel Oral Derivative UD-017, a Highly Selective CDK7 Inhibitor, Exhibits Anticancer Activity by Inducing Cell- Cycle Arrest and Apoptosis in Human Colorectal Cancer

Novel Oral Derivative UD-017, a Highly Selective CDK7 Inhibitor, Exhibits Anticancer Activity by Inducing Cell- Cycle Arrest and Apoptosis in Human Colorectal Cancer

Deoxyelephantopin induces ROS-mediated autophagy and apoptosis in human colorectal cancer in vitro and in vivo

Objective: To systematically map the stepwise events leading to deoxyelephantopin-induced cell death of HCT116 human colorectal cancer cells and evaluate the effectiveness of deoxyelephantopin in vivo.Methods: HCT116 cells were treated with deoxyelephantopin at various concentrations and time points. Autophagy was confirmed by the detection of autophagosomes and autophagosomal proteins by electron microscopy and Western blotting assays, respectively, and then validated by siRNA knockdown. In addition, apoptosis was confirmed by the detection of apoptosis-related proteins. The intracellular reactive oxygen species (ROS) level was measured using flow cytometry. The growth inhibitory effect of deoxyelephantopin was further evaluated in vivo using a mouse xenograft model.Results: Deoxyelephantopin firstly elevated ROS production, which then triggered autophagic flux with the accumulation of autophagosomal proteins including LC3A/B, ATG5, and ATG7, followed by the induction of apoptosis via the intrinsic and extrinsic pathways. Pre-treatment with N-acetyl-L-cysteine, a ROS inhibitor, reversed both apoptosis and autophagy. The knockdown of LC3 prevented apoptosis induction which confirmed that deoxyelephantopin induced autophagy-dependent apoptosis in HCT116 cells. Accumulation of ROS also activated apoptosis via the mitogen-activated protein kinases signaling pathway. Furthermore, deoxyelephantopin also inhibited the PI3K/AKT/mTOR pathway, which then released the inhibition of autophagy. In vivo study further showed that deoxyelephantopin significantly suppressed the growth of HCT116 subcutaneous xenograft in nude mice.Conclusions: Our findings revealed that deoxyelephantopin elevates oxidative stress and induces ROS-dependent autophagy followed by apoptosis in HCT116 cells via the concerted modulation of multiple signaling pathways. These findings further support the development of deoxyelephantopin as a therapeutic agent for colorectal cancer.

Antitumor effect of proanthocyanidin induced apoptosis in human colorectal cancer (HT-29) cells and its molecular docking studies

Proanthocyanidin (PAC) is a promising compound that has displayed its potent antineoplastic properties with a specific intrinsic pathway. This precise us to explore the phyto-preventive effect of PAC against colon cancer (HT-29). The results showed that PAC inhibited the cell growth and GI50 value was found to be 6.25 μM for 24 h exposure, when correlated to the normal cell line does not have toxicity was noticed. The linguistic differences, similarly membrane blebbing, cell shrinkage fragmented nuclear bodies and mitochondrial membrane were observed in AO/EtBr and DAPI staining. The features of regular mechanical apoptotic characterization was analyzed by DNA fragmentation. The cell cycle arrest at G2/M phases was detected using FACS analysis. The early and late apoptotic cells were observed by using Annexin V/PI staining. The ligand–protein interaction and docking studies were performed using Schrodinger’s software. The QPLD analysis of docking studies revealed that PAC exhibited better binding affinity of − 5.23, − 5.17 and − 4.43, − 4.47 kcal/mol against BCL-XL, CDK2 and were compared with 5-FU respectively, which significantly reveals the anticancerous activity of Proanthocyanidin compound. Thus, the PAC compound provides future application of therapeutic option in the treatment of colon cancers.

Raddeanin A inhibits growth and induces apoptosis in human colorectal cancer through downregulating the Wnt/β-catenin and NF-κB signaling pathway

AimsColorectal cancer (CRC) remains one of the most lethal human malignancies with high incidence and lack of effective therapy. Raddeanin A (RA), an active triterpenoid saponins, has been demonstrated the ability to inhibit the growth of tumor. But the therapeutic effects and mechanisms of RA in CRC remain elusive. Here, we investigated the efficacy and mechanism of RA in CRC both in vitro and in vivo. Main methodsCell viability was investigated to evaluate cytotoxic activity by MTT method. Apoptosis induced by RA was studied using Annexin V-FITC/PI binding and JC-1 staining by flow cytometry analysis. The xenograft mouse model of CRC was used to investigate anti-tumor effects in vivo. The key proteins involved in mitochondrial apoptotic, Wnt/β-catenin and NF-κB pathway were detected by Western blotting, Immunofluorescence, and Immunohistochemistry. Key findingsRA induced apoptosis and inhibited cell proliferation of SW480 and LOVO cells in a concentration-dependent manner. Moreover, RA efficiently inhibited tumor growth in xenograft mouse model. RA could down regulate the Wnt/β-catenin signaling to display anti-tumor effects via suppression of p-LRP6, induction of AKT inactivation, removal of GSK-3β inhibition and attenuation of β-catenin. Meanwhile, RA also suppressed the NF-κB pathway by decreasing the phosphorylation of IKBα to induce subsequently mitochondrial apoptotic pathway. SignificanceIn summary, RA suppressed the growth and triggered the apoptosis of CRC through discontinuing Wnt/β-catenin signaling and inhibiting the NF-κB pathway. These findings suggested that RA may hold a promise as a novel therapeutic agent for CRC therapy.

Downregulation of Lgr6 inhibits proliferation and invasion and increases apoptosis in human colorectal cancer.

The aim of the present study was to analyze the role of leucine‑rich repeat‑containing G‑protein coupled receptor6(Lgr6) in the proliferation and invasion of colorectal cancer (CRC) cells, and to investigate its possible mechanisms. The expression of Lgr6 in CRC tissues was observed by real time‑quantitative polymerase chain reaction and western blotting. Then cell viability, apoptosis and cell invasion was measured by MTT, flow cytometry or Matrigel‑Transwell system, respectively in CRC cells after transfected with Lgr6 siRNA or Lgr6 vector. Furthermore, the expression of apoptosis‑associated protein and PI3K/AKT signaling (phosphorylated‑PI3K, phosphorylated‑AKT, t‑PI3K, t‑AKT) were measured by real‑time PCR/or western blot analysis. The results demonstrated that the level of Lgr6 was higher in CRC tissues than that in adjacent tissues, and Lgr6 overexpression increased CRC proliferation, and invasion of CRC cells invitro. Notably, suppressing the expression of Lgr6 in CRC cells increased the expression of B‑cell lymphoma-2(Bcl‑2)‑associated X protein and caspase‑3, but decreased the expression of Bcl‑2 at the mRNA and protein levels. Lgr6 also had the ability to regulate the phosphoinositide 3‑kinase/AKT signaling pathway. It was concluded that Lgr6 has a tumor‑promoting role in the development of CRC, and may serve as a potential diagnostic and prognostic biomarker for the disease.

Downregulation of myosin VI reduced cell growth and increased apoptosis in human colorectal cancer.

Downregulation of myosin VI reduced cell growth and increased apoptosis in human colorectal cancer.

Glycolysis is essential for chemoresistance induced by transient receptor potential channel C5 in colorectal cancer

BackgroundElevated intracellular Ca2+ ([Ca2+]i) level could lead to [Ca2+]i overload and promote apoptosis via different pathways. In our previously study, up-regulated expression of transient receptor potential canonical channel (TRPC5) was proven to increase [Ca2+]i level, and resulted in chemoresistance whereas not apoptosis in human colorectal cancer (CRC) cells. The ATP-dependent homeostatic maintenance of resting [Ca2+]i should be important in this process. Increased glycolysis was found to be an important adenosine triphosphate (ATP) source in cancer. This study aimed to explore the potential mechanism of aerobic glycolysis in transient receptor potential channel TRPC5 induced chemoresistance.MethodsIn this study, we examined glucose transporter 1 (GLUT1) expression, glucose consumption and celluar ATP production to determine glycolytic activity. Real-time PCR and western blot were analyzed to determine TRPC5 expression at the mRNA and protein levels in human CRC cells (HCT-8, LoVo), and fluorouracil (5-Fu) resistant CRC cells (HCT-8/5-Fu, LoVo/5-Fu). 3-bromopyruvate (3-BP) and 2-Deoxy-D-glucose (2DG) were used to inhibit glycolysis. Glycolytic activity, intracellular Ca2+ ([Ca2+]i) and the half maximal inhibitory concentration of 5-Fu (5-Fu IC50) were measured. Western blot was analyzed to determine cleaved Caspase-3 protein level. Flow cytometry was performed to detect the apoptosis rates. Immunohistochemistry staining was performed to determine TRPC5 and GLUT1 expression level in human CRC tissues.ResultsOverproduced of TRPC5 and increased glycolysis were found in HCT-8/5-Fu and LoVo/5-Fu than in HCT-8 and LoVo cells. Compared to HCT-8 cells, the HCT-8/5-Fu cells showed higher [Ca2+]i levels which decreased after treated with TRPC5-specific shRNA. Furthemore, inhibition of glycolysis resulted in decreased ATP production, elevation of [Ca2+]i level and cleaved caspase-3, increased apoptotic cells rate, and a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu cells, while showed no effect in HCT-8 cells. BAPTA-AM, a [Ca2+]i chelator, could reduce the elevation of cleaved caspase-3 and increased apoptotic cells rate due to glycolysis inhibition. Advanced CRC patients with high expression of TRPC5/GLUT1 displayed poorer chemotherapy outcome, and notably, the significant association between high TRPC5 expression and chemoresistance is GLUT1 expression level dependent.ConclusionsWe demonstrated the essential role of glycolysis in TRPC5 induced chemoresistance in human CRC cells via maintaining [Ca2+]i homeostasis.

Cytotoxic activity of the twigs of Cinnamomum cassia through the suppression of cell proliferation and the induction of apoptosis in human colorectal cancer cells

BackgroundBecause twigs of Cinnamomum cassia (TC) have been reported to exert anti-cancer activity, the mechanistic study for TC’s anti-cancer activity is required. Thus, we elucidated the potential molecular mechanism of TC’s anti-proliferative effect and the induction of apoptosis in human colorectal cancer cells.MethodsHow water extracts form TC (TC-HW) was used in this study. Anti-cell proliferative effect of TC-HW was evaluated by MTT assay. The change of protein or mRNA level by TC-HW was evaluated by Western blot and RT-RCR, respectively. The promoter construct for ATF3, NF-κB, TOP-FLASH or FOP-FLASH was used for the investigation of the transcriptional activity for ATF3, NF-κB or Wnt. siRNA for ATF3 or p65 was used for the knockdown of ATF3 and p65.ResultsTC-HW reduced the cell viability in human colorectal cancer cells. TC-HW decreased cyclin D1 protein level through cyclin D1 degradation via GSK3β-dependent threonine-286 (T286) phosphorylation of cyclin D1, indicating that cyclin D1 degradation may contribute to TC-HW-mediated decrease of cyclin D1 protein level. TC-HW downregulated the expression of cyclin D1 mRNA level and inhibited Wnt activation through the downregulation of β-catenin and TCF4 expression, indicating that inhibition of cyclin D1 transcription may also result in TC-HW-mediated decrease of cyclin D1 protein level. In addition, TC-HW was observed to induce apoptosis through ROS-dependent DNA damage. TC-HW-induced ROS increased NF-κB and ATF3 activation, and inhibition of NF-κB and ATF3 activation attenuated TC-HW-mediated apoptosis.ConclusionsOur results suggest that TC-HW may suppress cell proliferation through the downregulation of cyclin D1 via proteasomal degradation and transcriptional inhibition, and may induce apoptosis through ROS-dependent NF-κB and ATF3 activation. These effects of TC-HW may contribute to the reduction of cell viability in human colorectal cancer cells. From these findings, TC-HW has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Downregulation of myosin VI reduced cell growth and increased apoptosis in human colorectal cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with the mortality increasing steadily over the last decade. Myosin VI (MYO6) expression is found to be elevated in some types of human carcinoma cell types, suggesting that it may be a sensitive biomarker for the diagnosis and follow-up. In this study, we first used the Oncomine database to explore the expression of MYO6 in CRC tissues, and then constructed a plasmid of RNA interference targeting MYO6 gene. After transfection of lentivirus targeting MYO6 into SW1116 cells, cell viability and proliferation were measured with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Cell cycle distribution was assayed by flow cytometry and apoptosis was evaluated by Annexin V. MYO6 expression was detected by quantitative real-time polymerase chain reaction and western blot analysis. It was found that MYO6 mRNA was upregulated in CRC tissues using data mining of public Oncomine microarray datasets. Depletion of MYO6 significantly inhibited cell proliferation and colony formation. In addition, knockdown of MYO6 slightly arrested cell cycle in G0/G1 phase, but remarkably increased the proportion of the sub-G1 phase of cell with the increase of apoptotic cells. These results suggest that MYO6 may promote cell growth and may be used as a potential target for anticancer therapy of CRC.

Water extract of brewers' rice induces apoptosis in human colorectal cancer cells via activation of caspase-3 and caspase-8 and downregulates the Wnt/β-catenin downstream signaling pathway in brewers' rice-treated rats with azoxymethane-induced colon carcinogenesis.

BackgroundBrewers’ rice, is locally known as temukut, is a mixture of broken rice, rice bran, and rice germ. The current study is an extension of our previous work, which demonstrated that water extract of brewers’ rice (WBR) induced apoptosis in human colorectal cancer (HT-29) cells. We also identified that brewers’ rice was effective in reducing the tumor incidence and multiplicity in azoxymethane (AOM)-injected colon cancer rats. Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells. To further determine whether the in vitro mechanisms of action observed in the HT-29 cells inhibit the downstream signaling target of the Wnt/β-catenin pathway, we evaluated the mechanistic action of brewers’ rice in regulating the expressions and key protein markers during colon carcinogenesis in male Sprague–Dawley rats.MethodsThe mRNA levels of several upstream-related genes in the Wnt signaling pathway in HT-29 cells treated with WBR were determined by quantitative real-time PCR analyses. Caspase-3 and −8 were evaluated using a colorimetric assay. Male Sprague–Dawley rats were administered two intraperitoneal injections of AOM in saline (15 mg/kg body weight) over a two-week period and received with 10, 20, and 40 % (w/w) brewers’ rice. The expressions and protein levels of cyclin D1 and c-myc were evaluated by immunohistochemical staining and western blotting, respectively.ResultsThe overall analyses revealed that the treatment of HT-29 cells with WBR inhibited Wnt signaling activity through upregulation of the casein kinase 1 (CK1) and adenomatous polyposis coli (APC) mRNA levels. We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and −8 activities compared with the control (P < 0.05). In vivo analyses indicated that brewers’ rice diminished the β-catenin, cyclin D1, and c-myc protein levels.ConclusionsWe provide evidence that brewers’ rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway in vivo. We suggest that brewers’ rice may be a useful dietary agent for colorectal cancer.

Inhibition of protein phosphatase 5 (PP5) suppresses survival and growth of colorectal cancer cells.

Protein phosphatase 5 (PP5) is a unique member of the protein phosphatases family that functions in multiple signaling pathways involved in DNA damage, cell cycle control, cell growth, and apoptosis. Recent evidence indicated that PP5 may play a role in cancer progression. In this study, we aimed to examine the biological effect of PP5 on cell growth and apoptosis in human colorectal cancer (CRC). We first knocked down PP5 expression in RKO cells via a short hairpin RNA containing lentivirus system. Then, methylthiazoletetrazolium assay, colony formation assay, and flow cytometry analysis were performed. The proliferation and colony formation ability of RKO cells were remarkably suppressed in PP5-silenced groups, as compared with control groups. Moreover, downregulation of PP5 resulted in a significant G0/G1 phase cell cycle arrest and an induction of apoptosis. In all, these results demonstrated the importance of PP5 in CRC cell growth, and it might be used as a potential therapeutic target for the treatment of CRC.

Increase in apoptosis by combination of metformin with silibinin in human colorectal cancer cells.

To investigate the effect of metformin on silibinin-induced apoptosis in human colorectal cancer (COLO 205) cells. MTT assays were performed to quantify cell viability. Western blot assays were applied to identify the expression of signaling proteins. The combined treatment of COLO 205 cells with metformin and silibinin decreased cell survival at a dose insufficient to influence the non-malignant cells [Human colonic epithelial cells (HCoEpiC)]. Silibinin and metformin increased phosphatase and tensin homolog and 5'-adenosine monophosphate-activated protein kinase expression in COLO 205 cells and inhibited the phosphorylation of mammol/Lalian target of rapamycin. This combined treatment resulted in an increase in the expression of activated caspase 3 and apoptosis inducing factor, indicating apoptosis. The combined treatment of human colorectal cancer cells with silibinin and metformin may induce apoptosis at a dose that does not affect HCoEpiC. This finding reveals a potential therapeutic strategy for the treatment of colorectal cancer.

MicroRNA-378 inhibits cell growth and enhances L-OHP-induced apoptosis in human colorectal cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that participate in a variety of biological processes, and dysregulation of miRNAs is widely associated with cancer development and progression. MiR-378 is frequently downregulated in colorectal cancer (CRC) and colorectal cell lines; however, it has high serum levels. Bioinformatics analysis further deduced that CDC40 is a potential target of miR-378, and luciferase reporter assays confirmed the direct regulation of CDC40 by miR-378. CDC40 plays a key role in cell cycle progression through G1/S and G2/M and pre-mRNA splicing. Subsequently, we determined that miR-378 inhibits cell growth and the G1/S transition in CRC cells and that these effects were CDC40-dependent. Finally, miR-378 increased cell apoptosis induced by the chemotherapeutic drug L-OHP. Our data highlight the potential application of miR-378 as a tumor suppressor for CRC therapy and overcoming chemoresistance, and it may also be a potential tumor marker for CRC prognosis.

3,3′-Diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4 in human colorectal cancer cells

3,3′-Diindolylmethane (DIM) is a major in vivo condensation product of indole-3-carbinol, which is present in cruciferous vegetables. Although these compounds have been widely implicated in antitumorigenic and proapoptotic properties in animal as well as in vitro models of cancer, the underlying cellular mechanisms regulated by DIM are only partially understood. Activating transcription factor 3 (ATF3) is a member of the ATF/c-AMP response element-binding (CREB) subfamily of the basic-region leucine zipper family and has been known to induce apoptosis in human colorectal cancer (CRC) cells. The present study was performed to elucidate the molecular mechanism of ATF3 induction by DIM in human CRC cells. The DIM treatment induced apoptosis and induced ATF3 gene expression at protein and messenger RNA levels. DIM increased ATF3 promoter activity, and the region of −84 to +34 within ATF3 promoter was responsible for promoter activation by DIM. This region contained an ATF binding site. Deletion and point mutation of the ATF binding site (−23 to −16) abolished ATF3 promoter activation by DIM, and overexpression of ATF4 enhanced ATF3 transactivation. Chromatin immunoprecipitation assay confirmed the binding of ATF4 in the ATF3 promoter. Inhibition of ATF4 expression by small interference RNA results in repression of DIM-induced ATF3 expression. The current study demonstrates that DIM stimulates ATF3 expression through ATF4-mediated pathway and subsequently induces apoptosis in human CRC cells.