Induced Apoptosis In Colorectal Cancer Cells Research Articles

The role of MARCH9 in colorectal cancer progression.

Colorectal cancer (CRC) is the third most common cancer with a high global incidence and mortality. Mutated genes or dysregulated pathways responsible for CRC progression have been identified and employed as biomarkers for diagnosis and prognosis. In this study, a ubiquitination regulator, MARCH9, was shown to accelerate CRC progression both in vitro and in vivo. CRC samples from The Cancer Genome Atlas (TCGA) showed significantly upregulated MARCH9 expression by individual cancer stage, histological subtype, and nodal metastasis status. Knockdown of MARCH9 inhibited, while MARCH9 overexpression promoted, CRC cell proliferation and migration. Knockdown of MARCH9 also induced CRC cell apoptosis and caused cell cycle arrest. Further investigation showed that MARCH9 promoted CRC progression by downregulating the expression of a deubiquitinase cylindromatosis (CYLD) gene and activating p65, a member of the nuclear factor-κB (NF-κB) protein family. Finally, in vivo xenograft studies confirmed that MARCH9 knockdown suppressed tumor growth in nude mice. Thus, this study demonstrated that MARCH9 may be a novel and effective therapeutic target for CRC therapy.

Circ_0000395 Promoted CRC Progression via Elevating MYH9 Expression by Sequestering miR-432-5p.

Colorectal cancer (CRC) has been listed as the fourth deadly cancer. Circular RNA hsa_circRNA_001046, also termed as hsa_circ_0000395 (circ_0000395), has been shown to be upregulated in CRC. Nevertheless, the function of circ_0000395 in CRC progression is unclear. 42 CRC patients were enrolled in the study. Detection of circ_0000395 expression in tissues and cells was executed using real-time quantitative polymerase chain reaction (RT-qPCR). Evaluation of circ_0000395 function was performed using loss-of-function experiments in vitro and in vivo. The regulatory mechanism of circ_0000395 was predicted by bioinformatics analysis and validated by dual-luciferase reporter and RIP assays. Exosomes were isolated by ultracentrifugation and validated by western blotting, TEM, and NTA. Circ_0000395 was strongly expressed in CRC samples and cell lines. Also, circ_0000395 repressed CRC growth in mouse models in vivo and induced CRC cell apoptosis, restrained CRC cell proliferation, migration, invasion, and EMT in vitro. Mechanistically, circ_0000395 sequestered miR-432-5p to regulate MYH9 expression. Furthermore, miR-432-5p knockdown reversed circ_0000395 silencing-mediated effects on CRC cell malignant phenotypes. MYH9 overexpression counteracted the inhibiting effects of miR-432-5p upregulation on CRC cell malignant phenotypes. Additionally, CRC cells derived from exosomal circ_0000395 promoted cancer cell malignant phenotypes. Our findings demonstrated that circ_0000395 sequestered miR-432-5p to elevate MYH9 expression, resulting in facilitating CRC progression, manifesting a potential therapeutic target for CRC.

Auriculasin enhances ROS generation to regulate colorectal cancer cell apoptosis, ferroptosis, oxeiptosis, invasion and colony formation

Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, and Chinese herbal medicine plays an important role in tumor treatment. The in-depth study of auriculasin isolated from Flemingia philippinensis showed that auriculasin promoted reactive oxygen species (ROS) generation in a concentration-dependent manner; when ROS scavenger NAC was added, the effects of auriculasin in promoting ROS generation and inhibiting cell viability were blocked. Auriculasin induced CRC cell apoptosis, led to mitochondrial shrinkage, and increased the intracellular accumulation of Fe2+ and MDA. When auriculasin and NAC were added simultaneously, the levels of apoptosis, Fe2+ and MDA returned to the control group levels, indicating that auriculasin activated apoptosis and ferroptosis by inducing ROS generation. In addition, auriculasin promoted the expression of Keap1 and AIFM1, but significantly reduced the phosphorylation level of AIFM1, while NAC significantly blocked the regulation of Keap1 and AIFM1 by auriculasin, which indicates that auriculasin can also induce oxeiptosis through ROS. When Z-VAD-FMK, Ferrostatin-1, Keap1 siRNA, PGAM5 siRNA and AIFM1 siRNA were added respectively, the inhibitory effect of auriculasin on cell viability was significantly weakened, indicating that auriculasin inhibits cell viability by inducing apoptosis, ferroptosis and oxeiptosis. Auriculasin also inhibited the invasion and clone forming ability of CRC cells, while NAC blocked the above effects of auriculasin. Therefore, auriculasin can promote CRC cell apoptosis, ferroptosis and oxeiptosis by inducing ROS generation, thereby inhibiting cell viability, invasion and clone formation, indicating that auriculasin has a significant antitumor effect.

Thymol Isolated from Thymus vulgaris L. Inhibits Colorectal Cancer Cell Growth and Metastasis by Suppressing the Wnt/β-Catenin Pathway.

PurposeColorectal cancer (CRC) is one of the most commonly occurring cancers and is associated with high morbidity and mortality. Nevertheless, there is currently no safe and effective treatment for this condition. Thymol is a phenolic compound that is recognized as safe for use in food as well as medical and cosmetic fields. Increasing evidence has indicated that thymol exerts prominent antitumor effects in a variety of cancers, including CRC. However, how thymol elicits these effects on CRC and the associated underlying mechanisms remains unclear.MethodsHCT116 and Lovo cells were treated with different concentrations of thymol. Cell Counting Kit-8 (CCK-8) and transwell migration and invasion assays were used to evaluate cell proliferation, migration, and invasion, respectively. Cell apoptosis and cell cycle distribution were measured by flow cytometry. RT-qPCR, Western blot, and immunohistochemistry were used to detect the expression of related genes and their protein products.ResultsIn this study, we tested the antitumor activity of thymol extracted from a Chinese medicinal herb, Thymus vulgaris L. We show that thymol treatment in vitro inhibited cell proliferation and induced apoptosis and cell cycle arrest in CRC. Furthermore, in vivo treatment with 75 and 150 mg/kg thymol led to a significant decrease in tumor volume. Thymol administration induced CRC cell apoptosis through activation of the BAX/Bcl-2 signaling pathway. In addition, thymol suppressed CRC cell epithelial–mesenchymal transition (EMT), invasion, and metastasis via inhibiting the activation of the Wnt/β-catenin pathway, both in vitro and in vivo.ConclusionThymol may prevent CRC progression through inhibition of the Wnt/β-catenin signaling pathway, highlighting its potential as a novel therapeutic option for the treatment of CRC.

ROS/JNK/c-Jun axis is involved in oridonin-induced caspase-dependent apoptosis in human colorectal cancer cells

Colorectal cancer (CRC) is one of the most common malignant neoplasms with high mortality worldwide. Oridonin, a diterpenoid isolated from the Chinese medicinal herb Rabdosia rubescens, has been proved to have anticancer effect on various types of cancer cells. However, the detailed mechanisms of oridonin in CRC cells remain unclear and if oridonin can overcome 5-FU resistance have not been investigated yet. In this study, we investigated the anticancer effect of oridonin in both 5-FU sensitive and resistant CRC cells and illuminated the underlying mechanisms. We showed that oridonin induced proliferation inhibition and caspase-dependent apoptosis in both 5-FU sensitive and resistant CRC cells. Oridonin induced reactive oxygen species (ROS) accumulation in both 5-FU sensitive and resistant CRC cells, which resulted in cell apoptosis as oridonin-induced apoptosis was almost abolished when cells were co-treated with the ROS scavenger N-acetyl-L-cysteine (NAC). Moreover, we found that oridonin induced CRC cell apoptosis via the c-Jun N-terminal kinase (JNK)/c-Jun pathway as oridonin activated JNK/c-Jun pathway and the JNK inhibitor SP600125 restored oridonin-induced apoptosis in CRC cells. Interestingly, when CRC cells were co-treated with NAC, the activation of JNK/c-Jun pathway induced by oridonin was nearly reversed, indicating that oridonin induced JNK/c-Jun pathway activation through the accumulation of ROS. Taken together, these data reveal that oridonin induces apoptosis through the ROS/JNK/c-Jun axis in both 5-FU sensitive and resistant CRC cells, suggesting that oridonin could be a potential agent for CRC treatment.

Nicotinamide phosphoribosyl transferase regulates cell growth via the Sirt1/P53 signaling pathway and is a prognosis marker in colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancy, and the metabolic properties of CRC cells include enhanced aerobic glycolysis (the Warburg effect). Nicotinamide phosphoribosyl transferase (NAMPT) is one of the crucial enzymes that regulate the activity of nicotinamide adenine dinucleodinucleotide dependent enzymes. Targeting NAMPT is a potential method of CRC therapy. Nevertheless, the underlying clinical implications and regulatory mechanisms of NAMPT in CRC remain unclear. In this study, we showed that NAMPT protein expression was increased in subjects with rectal localization compared with those with colon localization, and NAMPT was a poor prognostic marker for the overall survival rate in patients with CRC. In addition, the NAMPT inhibitor FK866 or lentivirus-mediated silencing induced CRC cell growth inhibition. Mechanistically, NAMPT regulated Sirt1 and P53 expression and induced G0/G1 cell cycle arrest, along with the upregulation of downstream p21 and downregulation of cyclin D1, cyclin E1, and cyclin E2 expression. FK866 administration or knockdown of NAMPT induced CRC cell apoptosis via upregulation of caspase-3. In conclusion, NAMPT regulated Sirt1/P53 signaling during CRC cell growth and warrants further investigation for clinical administration in CRC.

Sanguinarine triggers intrinsic apoptosis to suppress colorectal cancer growth through disassociation between STRAP and MELK

BackgroundPrevious studies showed sanguinarine induced apoptosis in CRC cells but did not define the underlying mechanisms. The purpose of this work was to determine the in vivo and in vitro effects of sanguinarine on CRC tumors and to elucidate the mechanism in regulating the intrinsic apoptosis.MethodsCell viability of CRC cell lines treated with sanguinarine was measured by MTT assay. Apoptotic cells stained with Annexin V and 7-AAD were detected by flow cytometry. Mitochondrial membrane potential and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. The in vitro kinase activity of MELK was analyzed by using HTRF® KinEASE™-STK kit. The expression of proteins were determined using Western blotting and immunohistochemistry. Co-immunoprecipitation and immunofluorecence were used to study the interaction between STRAP and MELK. The anti-neoplastic effect of sanguinarine was observed in vivo in an orthotopic CRC model.ResultsSanguinarine decreased the tumor size in a dose-dependent manner in orthotopical colorectal carcinomas through intrinsic apoptosis pathway in BALB/c-nu mice. It significantly increased cleavage of caspase 3 and PARP in implanted colorectal tissues. Sanguinarine increased mitochondrial ROS and triggered mitochondrial outer membrane permeabilization in multiple colorectal cancer (CRC) cell lines. NAC pretreatment lowered ROS level and downregulated apoptosis induced by sanguinarine. The intrinsic apoptosis induced by sanguinarine was Bax-dependent. The elevated expression and association between serine-threonine kinase receptor-associated protein (STRAP) and maternal embryonic leucine zipper kinase (MELK) were observed in Bax positive cells but not in Bax negative cells. Sanguinarine dephosphorylated STRAP and MELK and disrupted the association between them in HCT116 and SW480 cells. The expression and association between STRAP and MELK were also attenuated by sanguinarine in the tumor tissues. Importantly, we found that STRAP and MELK were overexpressed and highly phosphorylated in colorectal adenocarcinomas and their expression were significantly correlated with tumor stages. Furthermore, the expression of MELK, but not STRAP, was associated with lymph node metastasis.ConclusionsSanguinarine dephosphorelates STRAP and MELK and disassociates the interaction between them to trigger intrinsic apoptosis. Overexpression of STRAP and MELK may be markers of CRC and their disassociation may be a determinant of therapeutic efficacy.

TanshinoneI induces human colorectal cancer cell apoptosis: The potential roles of AuroraA-p53 and survivin-mediated signaling pathways.

Colorectal cancer (CRC) is one of the most common malignancies worldwide and a leading cause of cancer death. Despite decades of intensive investigations, effective interventional options are still limited and patient prognosis remains poor. TanshinoneI, an active compound from traditional Chinese herbal medicine Salvia miltiorrhiza Bunge, has been shown to inhibit cell growth of leukemia, lung, and breast cancers. However, whether and how TanshinoneI exerts similar effects on CRC needs to be elucidated. TanshinoneI induced CRC cell apoptosis was characterized and the roles of AuroraA-p53 and survivin-mediated pathways were analyzed in different CRC cell lines. TanshinoneI markedly inhibited CRC cell growth and induced apoptosis in CRC cells with functional p53 protein. Interestingly, TanshinoneI did not exert as much inhibitory effect on normal colon epithelial cells or CRC cells with mutant p53, indicating relative selectivity toward colorectal cancer cells with full presence of p53. In tse cells with wild-type p53, data showed that TanshinoneI mediated AuroraA inhibition results in p53 upregulation, which is required for cell apoptosis. In CRC cells with mutant p53 protein (not able to localize to the nucleus), however, AuroraA knockdown failed to induce CRC cell apoptosis. Instead, data showed that protein level of survivin decreased following TanshinoneI treatment. These observations were further substantiated by the pivotal role of survivin in TanshinoneI mediated apoptosis in CRC cells with p53 mutant. TanshinoneI, a novel natural compound, exerts significant inhibitory effect on CRC cell growth via a mechanism involving either AuroraA-p53 axis or survivin-involving mechanism depending on different intrinsic characteristics of tumor cells.

ApoG2 inhibits the antiapoptotic protein, Mcl‑1, and induces mitochondria‑dependent apoptosis in human colorectal cancer cells.

Colorectal cancer (CRC) is a worldwide malignancy of high incidence and mortality. At present, there is a lack of effective drugs against CRC. The B‑cell leukemia/lymphoma 2 (Bcl‑2) protein family members are considered to be closely associated with tumorigenesis and the chemoresistance of CRC. As a novel gossypol derivative targeting antiapoptotic proteins of the Bcl‑2 family, apogossypolone (ApoG2) exhibits antitumor properties in various cancer types, although its effects against CRC remain to be fully elucidated. In the present study, the cytotoxicity of ApoG2 in vitro on CRC cells was investigated, with the aim of elucidating the underlying mechanism. Using an MTT assay, ApoG2 was revealed to inhibit the growth of the HT29, SW480 and HCT116 CRC cell lines in a dose‑ and a time‑dependent manner. Hoechst staining revealed that ApoG2 induced CRC cell apoptosis, marked by morphological changes, including cell shrinkage and nuclear fragmentation. Flow cytometric analysis also detected a higher apoptotic ratio following treatment with ApoG2. The ratio was dependent upon the concentration of ApoG2, which the cells were exposed to, and the duration of the exposure. Western blot analysis and immunoprecipitation experiments revealed that ApoG2 treatment led to the downregulation of the protein expression of Mcl‑1, and the interruption of the binding of Mcl‑1 to the protein Bax. Furthermore, treatment with ApoG2 led to the release of cytochrome c into the cytoplasm and the activation of caspases 3 and 7. The present study revealed that ApoG2 inhibited the proliferation of the CRC cell lines through mitochondrial signaling pathway‑dependent apoptosis, which may be associated with the disruption of the function of the Mcl‑1 protein by ApoG2.

Silencing of JMJD2B induces cell apoptosis via mitochondria-mediated and death receptor-mediated pathway activation in colorectal cancer.

To investigate the molecular mechanism of colorectal cancer (CRC) cell apoptosis induced by the Jumonji domain containing 2B (JMJD2B) silencing. Both HCT116 and LoVo CRC cell lines were used for analyses. Cell apoptosis after JMJD2B silencing was determined by flow cytometry. JC-1 fluorescence probe was applied to measure the mitochondrial outer membrane permeabilization by flow cytometry and fluorescence microscopy. Immunofluorescence was used to detect cytochrome C translocation from mitochondria to cytosol after JMJD2B silencing. The efficacy of JMJD2B silencing on the protein levels of Bcl-2 family, caspase proteins, CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were detected by Western blot. JMJD2B silencing induced CRC cell apoptosis via a decrease of the anti-apoptotic gene Bcl-2 family expression, leading to the translocation of Bak and Bax proteins and the promotion of mitochondrial membrane disruption, resulting in the release of cytochrome C from mitochondria and subsequent caspase-9 and caspase-3 cleavage. It also increased the amount of cleaved caspase-8 involved in the death receptor-related apoptotic pathway. Bcl-2 homology 3 interacting-domain death agonist (Bid), a specific caspase-8 substrate involved in the Fas signaling pathway, subsequently induced cleavage via caspase-8 activation. However, levels of CHOP and GRP78 remained unchanged after JMJD2B silencing. JMJD2B silencing induced CRC cell apoptosis via both mitochondria-related and death receptor-related pathways. The cleavage of Bid activated by caspase-8 might serve as a crosstalk mediator between these two pathways in CRC.

The p38 MAPK-regulated PKD1/CREB/Bcl-2 pathway contributes to selenite-induced colorectal cancer cell apoptosis in vitro and in vivo

Supranutritional selenite has anti-cancer therapeutic effects in vivo; however, the detailed mechanisms underlying these effects are not clearly understood. Further studies would broaden our understanding of the anti-cancer effects of this compound and provide a theoretical basis for its clinical application. In this study, we primarily found that selenite exposure inhibited phosphorylation of cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB), leading to suppression of Bcl-2 in HCT116 and SW480 colorectal cancer (CRC) cells. Moreover, the selenite-induced inhibitory effect on PKD1 activation was involved in suppression of the CREB signalling pathway. Additionally, we discovered that selenite treatment can upregulate p38 MAPK phosphorylation, which results in inhibition of the PKD1/CREB/Bcl-2 survival pathway and triggers apoptosis. Finally, we established a colorectal cancer xenograft model and found that selenite treatment markedly inhibits tumour growth through the MAPK/PKD1/CREB/Bcl-2 pathway in vivo. Our results demonstrated that a supranutritional dose of selenite induced CRC cell apoptosis through inhibition of the PKD1/CREB/Bcl-2 axis both in vitro and in vivo.

MiR-7 inhibits colorectal cancer cell proliferation and induces apoptosis by targeting XRCC2

BackgroundAnalysis using publicly available algorithms predicts that X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2), a key component in the homologous recombination repair pathway, is a potential target of micro-ribonucleic acid-7 (miR-7). Some studies have shown that both miR-7 and XRCC2 are associated with cancer development. For this purpose, we searched for the possible relationship between miR-7 and XRCC2 in the development of colorectal cancer (CRC).MethodsmiR-7 expression was assessed in CRC specimens and cell lines using real-time polymerase chain reaction (PCR). Luciferase reporter assay was used to confirm the target associations. The effect of miR-7 on cell proliferation and apoptosis was confirmed in vitro by the methylthiazol tetrazolium (MTT) assay, colony formation assay, and flow cytometry. Gene and protein expression were examined using real time PCR and western blotting, respectively.ResultsmiR-7 was downregulated in CRC specimens and cell lines, and targeted the 3′ untranslated region of XRCC2. miR-7 overexpression reduced cyclin D1 expression and increased p21, caspase-3, and BAX expression, which subsequently inhibited CRC cell proliferation and induced CRC cell apoptosis. However, XRCC2 can repress the inhibitory effects of miR-7 on proliferation.ConclusionOur findings suggest that miR-7 plays a protective role by inhibiting proliferation and increasing apoptosis of CRC cells. It may identify new targets for anti-cancer treatment.