Apoptosis Of CRC Cells Research Articles

AMPK-autophagy inhibition sensitizes icaritin-induced anti-colorectal cancer cell activity.

The current research studied the potential effect of autophagy on icaritin-induced anti-colorectal cancer (CRC) cell activity. Treatment of icaritin in both primary and established (HT-29) CRC cells induced feedback activation of autophagy, evidenced by p62 degradation, Beclin-1 and autophagy-related gene-5 (ATG-5) upregulation, as well as light chain 3B (LC3B)-GFP puncta formation. Pharmacological inhibiting of autophagy dramatically potentiated icaritin-induced CRC cell death and apoptosis. Meanwhile, shRNA-mediated knockdown of Beclin-1 or ATG-5 also sensitized icaritin-induced CRC cell death and apoptosis. Icaritin activated AMP-activated protein kinase (AMPK) signaling in CRC cells, functioning as the upstream signaling for autophagy activation. shRNA/siRNA-mediated knockdown of AMPKα1inhibited icaritin-induced autophagy activation, but exacerbated CRC cell death. On the other hand, the AMPK activator compound 13 (C13) or the autophagy activator MHY1485 attenuated icaritin-induced cytotoxicity. In nude mice, icaritin (oral administration)-induced HT-29 tumor growth inhibition was potentiated when combined with AMPKα1 shRNA knockdown in tumors. We conclude that feedback activation of AMPK-autophagy pathway could be a primary resistance factor of icaritin.

Preclinical study of cinobufagin as a promising anti-colorectal cancer agent.

Here, we assessed the anti-colorectal cancer (CRC) cell activity of cinobufagin (CBG). We found that CBG exerted potent cytotoxic and anti-proliferative activity against CRC lines (HCT-116 and HT-29) and primary human CRC cells. Meanwhile, it activated apoptosis, and disrupted cell-cycle progression in the cells. At the signaling level, CBG treatment in CRC cells provoked endoplasmic reticulum stress (ER stress), the latter was evidenced by caspase-12 activation, CHOP expression, as well as PERK and IRE1 phosphorylations. Contrarily, the ER stress inhibitor salubrinal, the caspase-12 inhibitor and CHOP shRNA remarkably attenuated CBG-induced CRC cell death and apoptosis. Further, CBG in-activated mammalian target or rapamycin complex 1 (mTORC1), which appeared responsible for proliferation inhibition in CRC cells. Introduction of a constitutively-active S6K1 (“ca-S6K1”) restored proliferation of CBG-treated CRC cells. Finally, CBG intraperitoneal injection suppressed HCT-116 xenograft tumor growth in the nude mice. CHOP upregulation and mTORC1 in-activation were also noticed in CBG-treated HCT-116 tumors. The results of this preclinical study suggest that CBG could be tested as promising anti-CRC agent.

MTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells.

MiR-450b-5p induced by oncogenic KRAS is required for colorectal cancer progression.

The development and progression of CRC are regarded as a complicated network and progressive event including genetic and/or epigenetic alterations. Recent researches revealed that MicroRNAs are biomarkers and regulators of CRC progression. Analyses of published microarray datasets revealed that miR-450b-5p was highly up-regulated in CRC tissues. In addition, high expression of miR-450b-5p was significantly associated with KRAS mutation. However, the role of miR-450b-5p in the progression of CRC remains unknown. Here, we sought to validate the expression of miR-450b-5p in CRC tissues and investigate the role and underlying mechanism of miR-450b-5p in the progression of CRC. The results revealed that miR-450b-5p was up-regulated in CRC tissues, high expression level of miR-450b-5p was positively associated with poor differentiation, advanced TNM classification and poor prognosis. Moreover, miR-450b-5p was especially high in KRAS-mutated cell lines and could be up-regulated by KRAS/AP-1 signaling. Functional validation revealed that overexpression of miR-450b-5p promoted cell proliferation and tumor growth while inhibited apoptosis of CRC cells. Furthermore, we demonstrated that miR-450b-5p directly bound the 3′-UTRs of SFRP2 and SIAH1, and activated Wnt/β-Catenin signaling. In conclusion, miR-450b-5p induced by oncogenic KRAS is required for colorectal cancer progression. Collectively, our work helped to understand the precise role of miR-450b-5p in the progression of CRC, and might promote the development of new therapeutic strategies against CRC.

Abstract 1222: Fluorinated N, N-diarylureas treatment as effective strategy to target colorectal cancer stem cells

Abstract Colorectal (CRC) cancer is the second leading cause of cancer deaths in the US; treatment of metastatic CRCs is limited due to drug resistance and eventual relapse. Cancer stem cells and PI3K mutation (i.e., pik3ca mutation) have been implicated in the relapse and lack of treatment response for many cancers including CRCs; therefore, it is critical to develop a therapeutic strategy that eliminates both the fast-growing cancer cells and the more resistant cancer stem cells. The purpose of the present study was to evaluate the anticancer activity of recently-described, novel AMPK activators, fluorinated N, N-diarylureas (FNDs), against CRC metastatic cell lines, pik3ca mutant cell lines, and CRC stem cells. METHODS. i) First, to assess AMPK activation, metastatic CRC cell lines (HT29 and KM20 cells), HCT116 pik3ca wild-type (WT) and mutant (MUT) cell lines, and CRC stem cell lines (from Celprogen) were treated with varying concentrations of 8 FNDs (4a, 4b, 4h, 4j, 4k, 4z, 4aa, 4bb). AMPK activation and activation of the upstream regulator, LKB1, was assessed by western blot. ii) Next, we determined the effect of the FNDs and, for comparison, metformin (an AMPK activator) on induction of cell cycle suppression and induction of apoptosis as assessed by cyclin D1 expression and PARP cleavage, respectively; β-actin antibody was used as a loading control. RESULTS. i) Treatment with the FNDs resulted in AMPK activation in HT29 and KM20 cells and CRC stem cells at concentrations as low as 10 μM with a peak activation at 12 h. We observed LKB1-independent AMPK kinase activation. Decreased LKB1 phosphorylation after FND treatment was dose dependent (i.e., decreased by ∼50% at 10 μM and undetectable at 50 μM). ii) All 8 FNDs (at a 25 μM dosage) completely suppressed cyclin D1 expression in HT29 and KM20 cells; a similar cyclin D1 suppression was noted with metformin at a 500x higher dosage (i.e., 10mM). Five of the 8 FNDs (4b, 4j, 4z, 4aa, 4bb) increased PARP cleavage in HT29 and KM20 cells. We used these 5 FNDs (at a dosage of 50 μM each) to treat the CRC stem cell lines; cyclin D1 suppression was noted for all 5 FNDs but strong induction of apoptosis was only identified using the 4b FND. Finally, we treated HCT116 pik3ca WT and MUT cell lines with 4b FND and found that cyclin D1 expression was completely suppressed at 40 μM and induction of apoptosis was noted at a dosage of 20 μM. CONCLUSIONS. We demonstrate cell cycle suppression and apoptosis in CRC cells and stem cells using the recently-developed FND compounds with the 4b compound as the most effective. These compounds appear to have considerable promise as a targeted cancer stem cell-specific agents and offer a potentially novel strategy for the treatment of CRC metastases. Citation Format: Piotr Rychahou, Dasha Kenlan, Vitaliy M. Sviripa, David S. Watt, B. Mark Evers. Fluorinated N, N-diarylureas treatment as effective strategy to target colorectal cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1222.

Nitidine chloride inhibits proliferation and induces apoptosis in colorectal cancer cells by suppressing the ERK signaling pathway.

Nitidine chloride (NC) is a natural bioactive phytochemical alkaloid that has displayed anticancer activity in various types of cancer. However, no evidence has been reported for the direct effect of NC on CRC cell proliferation and apoptosis, and the underling mechanisms to be fully elucidated. The present study aimed to investigate the influence of NC on the apoptosis and proliferation of CRC cells. The viability and proliferation of CRC cells was measured by MTT assay and a [3H] thymidine uptake assay. Apoptosis was measured using a flow cytometric apoptosis assay and TUNEL staining. The expression levels of apoptotic-regulated proteins in addition to extracellular signal-regulated kinase (ERK) were measured by western blot analysis following stimulation with NC. The results indicated that NC inhibited the proliferation of HCT116 cells in a dose- and time-dependent manner. Additionally, apoptotic induction by NC treatment was confirmed. Furthermore, NC was demonstrated to significantly upregulate the expression of Bax, p53, cleaved caspase-3 and -9 and downregulate the expression of Bcl-2. Treatment with NC reduced the phosphorylation of ERK and by using an ERK inhibitor, U0126, the roles of NC in apoptotic induction and the inhibition of proliferation were further demonstrated. These results demonstrated that NC inhibited the proliferation and induced the apoptosis of CRC cells via the ERK signaling pathway.

Up-regulation of miR-592 correlates with tumor progression and poor prognosis in patients with colorectal cancer

miR-592, as a potential biomarker, has been linked to several cancers. However, the expression level and prognostic value of miR-592 in CRC have not been elucidated. In this study, we detected the miR-592 expression in CRC serum, tumor tissues, adjacent non-tumor tissues (NATs) and four colorectal cancer cell lines by RT-PCR. Our data proved that miR-592 expression was up-regulated in clinical CRC serum and tissues (P<0.05). Serum or tissue miR-592 in CRC metastatic patients also maintained a high level, compared to that in non-metastatic CRC patients (P<0.05). After radical surgery, postoperative serum miR-592 level in CRC patients significantly decreased (P<0.05). Our clinicopathological analysis revealed that high miR-592 was significant associated with the tumor size (P=0.008), TNM stage (P=0.026), distant metastasis (P=0.004) and preoperative CEA level (P=0.022), which led to a shorter overall survival rate in CRC patients (P=0.032). Furthermore, we designed and transfected miR-592 mimics or inhibitors into the corresponding CRC lines, and our experiments in vitro demonstrated that miR-592 could promote cell proliferation, wound healing and invasion ability of CRC cells (P<0.05), while miR-592 did not influence the CRC cell apoptosis (P>0.05). All these results suggested that miR-592 functioned as a novel and potential carcinogen-initiated and metastasis-related biomarker in CRC, and down-regulation of miR-592 might be considered as a potentially significant molecular treatment strategy for CRC patients.

Abstract B06: Functional characterization of K-RAS A146T in colorectal cancer

Abstract Mutational activation of the Kras oncogene occurs in 40% of colon cancers, which affects over 150,000 people and approximately 45,000 people will die of the disease in the United States per year. Although advances in early detection and clinical management have led to improved survival, the future of colon cancer therapy lies in personalized molecular medicine, where physicians tailor an individual's treatment to the mutation(s) that have occurred in their cancer. While activating mutations at codons 12, 13, and 61 of K-Ras were originally identified in cancers, a broader assessment of RAS mutations has led to the identification of weaker, non-canonical activating mutations at codon 146 (typically an alanine to threonine substitution), which occurs in approximately 4% of colorectal cancers. Kras mutations at codons 12 or 13 are associated with poor prognosis and show resistance to both conventional and targeted therapies, while mutations at codon 146 is associated with a more favorable clinical outcome in patients with colorectal cancer. Identification of a K-RAS mutant that would prove to be a potential target for therapeutic intervention in colorectal cancer would clearly be valuable. In order to fully understand the implications of these observations, and to utilize the information for therapeutic benefit, we need to study the different mutant forms of K-Ras in an experimentally tractable system. Using both genetically engineered mouse models and human colorectal cancer cell lines we will assess the molecular and phenotypic similarities and differences between distinct forms of mutant K-Ras. Consistent with previous reports of the biochemical activities of the different mutants, we found that K-RasA146T exhibited enhanced GTP-binding relative to wild type, but significantly lower GTP-binding relative to K-RasG12D. Interestingly, A146T animals exhibited an intermediate tissue-level hyperplasia phenotype, consistent with the intermediate biochemical phenotype, but exhibit a hyper-proliferation phenotype that is equal to G12D. Unlike our observation from genetically engineered mice, we found that cell lines carrying codon 146 mutations had levels of K-Ras-GTP that were equal to cell lines carrying codon 12/13 mutations. At a functional level, we found that codon 146 mutations enhanced sodium butyrate-induced apoptosis in CRC cells, just like codon 12 mutations, and that loss of K-RasA146T suppressed proliferation at a level similar to loss of K-RasG12D. In the context of Apc-mutant colonic tumors, activation of K-RasA146T led to an increase in tumor number and a decrease in animal life span compared to control animals, similar to K-RasG12D animals. In the future we will employ computational modeling and network-level signaling pathway measurements to characterize how signaling pathways are affected by the mutation of K-RAS A146T compared to both wild type and other K-Ras mutants. We expect that these studies will provide a rational for broader K-RAS testing in the clinic beyond the common alleles at codon 12 and 13 and allow for the development of novel therapies to treat K-RAS mutant colorectal cancer patients. Citation Format: Jessica J. Gierut, Kevin M. Haigis. Functional characterization of K-RAS A146T in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B06. doi: 10.1158/1557-3125.RASONC14-B06

Abstract 235: Combination of the probiotic Lactobacillus rhamnosus GG with grape antioxidant resveratrol for the management of colorectal cancer

Abstract Colorectal cancer (CRC), also known as colon/bowel cancer, is the third most commonly diagnosed cancer in the world. In the United States, 142,820 new cases of CRC and 50,830 CRC-related deaths are predicted for the year 2013. CRC has been widely associated with lifestyle and aging. Treatment for CRC varies greatly based on disease severity and is curable with surgery, only if detected very early. It is being increasingly appreciated that nutritional and lifestyle modification might prevent or delay certain cancers, including CRC. Imbalance in gut microbiome, due to lifestyle and dietary habits, can lead to a variety of intestinal disorders including excessive immune response and inflammation that might ultimately lead to CRC. The probiotics, which are live microorganisms, have been shown to promote healthy microbiome balance in gut to support healthy colon. For an optimal functioning, the probiotics need to remain viable. This might be achieved via prebiotics and prebiotics-like substances that confer beneficial effects by promoting the microbiota. Therefore, novel means to balance the gut microbiota by prebiotic-probiotic combination could be useful in the management of inflammation related CRC. The grape antioxidant resveratrol (RSV) has been shown to support the health of beneficial gut bacteria, thus functioning essentially as a prebiotic. The goal of this study was to determine if a combination of the resveratrol and the probiotic Lactobacillus rhamnosus GG (LGG) imparts anti-proliferative response in human CRC cell lines, DLD 1 and RKO. For our experiments, DLD1 and RKO cells (∼50% confluent) were treated with RSV and cell free supernatants of LGG. RSV was used at a single concentration (10 µM) whereas LGG cell-free supernatants were used at two concentrations: LGG1 (2.125 x 105 CFU/mL) and LGG2 (4.35 x 105 CFU/mL). We found that a combination of LGG and RSV resulted in a significant decrease in the growth and viability of DLD 1 and RKO cells, as assessed by MTT- and Trypan Blue exclusion- assays. Further, LGG-RSV combination resulted in a marked decrease in the clonogenic survival of CRC cells, as assessed by colony formation abilities of of DLD 1 and RKO cells. Furthermore, LGG-RSV combination resulted in an induction of apoptosis in CRC cells, as determined by cleavage of protein poly (ADP-ribose) polymerase (PARP). Overall, our study provides a proof of principle for the potential use of RSV-LGG combination in CRC management. However, further in vivo studies in appropriate animal model(s) are needed to determine the efficacy of RSV-LGG combination in prevention of CRC. Citation Format: Chandra K. Singh, Jasmine George, Megan Duster, Nasia Safdar, Nihal Ahmad. Combination of the probiotic Lactobacillus rhamnosus GG with grape antioxidant resveratrol for the management of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 235. doi:10.1158/1538-7445.AM2014-235

Functional characterization of the tumor-suppressor MARCKS in colorectal cancer and its association with survival

The myristoylated alanine-rich C-kinase substrate (MARCKS) acts as a tumor suppressor in a variety of human neoplasms. In colorectal cancers (CRCs), MARCKS has been shown to be a preferential target of mutational inactivation in tumors following the microsatellite instability (MSI-H) pathway but little is known about its impact on intestinal carcinogenesis. To investigate the relevance of MARCKS inactivation in more detail, we analyzed 926 MSI-typed CRCsfor MARCKS expression by immunohistochemistry and studied the functional consequences of MARCKS depletion in colorectal cancer cell lines. We found that loss of MARCKS expression was not restricted to MSI-H cancers but also occurred in microsatellite stable (MSS) tumors, where it was associated with an adverse outcome regarding overall survival, cancer-specific and disease-free survival (P=0.002, P=0.0018, P=0.0001, respectively; univariate analysis). In MARCKS-positive MSScolon cancer cell lines (SW480 andSW707) small interfering RNA (siRNA)-mediated knockdown of MARCKS conferred resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. This was accompanied by the downregulation of the TRAIL receptors DR4 and DR5 at the cell surface and activation of AKT signaling. Inhibition of AKT signaling and transient overexpression of wild-type MARCKS, but not of MARCKS lacking the effector domain (ED), abolished the anti-apoptotic effect. In conclusion, our data show that inactivation of MARCKS is common in CRCsand is associated with adverse outcome in MSS cancers. The finding that MARCKS acts as a mediator of apoptosis in MSS CRCcells adds a novel tumor-suppressing function to the so far established roles of MARCKS in cell motility and proliferation and can explain the prognostic effect of MARCKS depletion in MSS CRC.

Ambra1 Is an Essential Regulator of Autophagy and Apoptosis in SW620 Cells: Pro-Survival Role of Ambra1

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.

MicroRNA-16 represses colorectal cancer cell growth in vitro by regulating the p53/survivin signaling pathway

Dysregulated expression of microRNAs (miRNA) is a hallmark of cancer. miR-16 has been reported to be downregulated and to act as a tumor suppressor in different cancer types. In the present study, we sought to investigate the possible roles and mechanisms of miR-16 and its relationship with p53 and survivin in CRC cells. We showed that miR-16 was downregulated in 67% of CRC tissues and was correlated with the degree of histological differentiation. Experiments invitro showed that overexpression of miR-16 inhibited the proliferation and induced apoptosis of CRC cells through the intrinsic apoptosis pathway. We further showed that miR-16 repressed survivin expression at both the mRNA and protein levels and the survivin gene was a direct target of miR-16. In addition, miR-16 reduced p53 expression and p53 increased miR-16 levels, with downregulation of miR-16 targets survivin, cyclin D1 and CDK6. Our findings suggest that miR-16 represses colorectal cancer cell growth invitro by regulating the p53/survivin signaling pathway. Our findings provide further evidence for the involvement of dysregulated miRNAs in CRC, and miR-16 could serve as a molecular target for CRC therapy.

A Truncated Form of IKKα Is Responsible for Specific Nuclear IKK Activity in Colorectal Cancer

Nuclear IKKα regulates gene transcription by phosphorylating specific substrates and has been linked to cancer progression and metastasis. However, the mechanistic connection between tumorigenesis and IKKα activity remains poorly understood. We have now analyzed 288 human colorectal cancer samples and found a significant association between the presence of nuclear IKK and malignancy. Importantly, the nucleus of tumor cells contains an active IKKα isoform with a predicted molecular weight of 45kDa (p45-IKKα) that includes the kinase domain but lacks several regulatory regions. Active nuclear p45-IKKα forms a complex with nonactive IKKα andNEMO that mediates phosphorylation of SMRT and histone H3. Proteolytic cleavage of FL-IKKα into p45-IKKα is required for preventing the apoptosis of CRC cells invitro and sustaining tumor growth invivo. Our findings identify a potentially druggable target for treating patients with advance refractory CRC.