Proliferation Of Colon Carcinoma Cells Research Articles

Down-regulation of EZH2 genes targeting RUNX3 affects proliferation, invasion, and metastasis of human colon cancer cells by Wnt/β-catenin signaling pathway.

In order to detect the effect of EZH2 genes on proliferation, migration, invasion, and apoptosis of colon carcinoma cell strains HCT116 and HT29 by the Wnt/β-catenin signaling pathway, qRT-PCR was applied to measure relative expressions of EZH2, RUNX3, CEA, CA199, MMP-9, VEGF, β-catenin, and CyclinD1 in each group; Western-blot was employed with the intention of exploring relative expressions of these proteins in vivo and in vitro; monoclonal proliferation experiments and CCK-8 assay was adopted so as to check cell proliferation; the effect on cell migration was investigated via Transwell assay and cell scratch wound assay; flow cytometry was applied with a view to determining the effect on cell apoptosis. Transfected HCT116 cells are injected subcutaneously into nude mice. In colon cell strains HCT-116 and HT29, contrasted to the si-NC group, the RUNX3 expression was prominently up-regulated in the si-EZH2 group. Besides, expressions of CEA, CA199, MMP-9, and VEGF were significantly reduced; down-regulation of EZH2 genes remarkably inhibited cell proliferation, invasion and migration when facilitating apoptosis; down-regulation of EZH2 genes also significantly reduced expressions of essential proteins β-catenin and CyclinD1 on the Wnt pathway. The subcutaneous tumor body of nude mice was reduced. EZH2-OE is the opposite trend to si-EZH2; The EZH2 gene may target regulatory RUNX3 regulation via that Wnt/β-catenin signaling pathway, hence affecting colon carcinoma cell proliferation, invasion, migration, and apoptosis. Therefore, EZH2 may become a promising target for the clinical therapy of colon carcinoma.

The Pro-Oncogenic Protein IF1 Promotes Proliferation of Anoxic Cancer Cells during Re-Oxygenation.

Cancer cells overexpress IF1, the endogenous protein that inhibits the hydrolytic activity of ATP synthase when mitochondrial membrane potential (ΔμH+) falls, as in ischemia. Other roles have been ascribed to IF1, but the associated molecular mechanisms are still under debate. We investigated the ability of IF1 to promote survival and proliferation in osteosarcoma and colon carcinoma cells exposed to conditions mimicking ischemia and reperfusion, as occurs in vivo, particularly in solid tumors. IF1-silenced and parental cells were exposed to the FCCP uncoupler to collapse ΔμH+ and the bioenergetics of cell models were validated. All the uncoupled cells preserved mitochondrial mass, but the implemented mechanisms differed in IF1-expressing and IF1-silenced cells. Indeed, the membrane potential collapse and the energy charge preservation allowed an increase in both mitophagy and mitochondrial biogenesis in IF1-expressing cells only. Interestingly, the presence of IF1 also conferred a proliferative advantage to cells highly dependent on oxidative phosphorylation when the uncoupler was washed out, mimicking cell re-oxygenation. Overall, our results indicate that IF1, by allowing energy preservation and promoting mitochondrial renewal, can favor proliferation of anoxic cells and tumor growth. Therefore, hindering the action of IF1 may be promising for the therapy of tumors that rely on oxidative phosphorylation for energy production.

Highly expressed miR-144-3p promotes the proliferation, migration and invasion of colon carcinoma cells by activating the Wnt/β-catenin signaling pathway through targeting SFRP1.

Background: To investigate the influence of miR-144-3p on the proliferation, migration and invasion of colon carcinoma by targeting secreted frizzled-related protein 1 (SFRP1) as well as of the Wnt/β-catenin signaling pathway. Methods: Based on the TCGA database, the association between the expression of miR-144-3p and the clinical information and prognosis of patients with colon carcinoma was examined, and SFRP1 was selected as the target gene for further studies based on bioinformatics prediction tools. CCK8 assay, wound healing assay and transwell invasion assay were employed to examine the impact of miR-144-3p on colon carcinoma cells. The regulation of SFRP1 by miR-144-3p was investigated using a dual-luciferase reporter system, and a rescue experiment was conducted to further elucidate whether miR-144-3p promotes the migration of colon carcinoma cells through targeting SFRP1 or not. The Wnt/β-catenin signaling pathway-mediated effect of miR-144-3p in colon carcinoma was finally validated through the targeting of SFRP1. Results: The bioinformatics analysis showed that the miR-144 expression levels were substantially greater in colon carcinoma tissue than in para-carcinoma tissue and were closely with clinical stage and prognosis. The findings obtained from the trial indicated that miR-144-3p substantially expressed in colon carcinoma tissue sample and the colon carcinoma cells, and the overexpressed miR-144-3p boosted the colon carcinoma cells' proliferation, migration and invasion. The results of dual-luciferase reporter gene assay revealed that miR-144-3p targeted SFRP1, and rescue experiment was carried out and its results indicated that miR-144-3p increased colon carcinoma cells' migration through targeting SFRP1. In addition, the molecular axis of miR-144-3p/SFRP1 may over-activate the Wnt/β-catenin signaling pathway. Conclusions: The present study has identified a novel malignant biological behavior, namely the ability of miR-144-3p to enhance the proliferation, migration and invasion of colon carcinoma cells by targeting SFRP1 and activating the Wnt/β-catenin signaling pathway. Consequently, miR-144-3p emerges as a promising diagnostic and therapeutic target for colon carcinoma.

Preclinical Efficacy and Toxicity Analysis of the Pan-Histone Deacetylase Inhibitor Gossypol for the Therapy of Colorectal Cancer or Hepatocellular Carcinoma.

Gossypol, a sesquiterpenoid found in cotton seeds, exerts anticancer effects on several tumor entities due to inhibition of DNA synthesis and other mechanisms. In clinical oncology, histone deacetylase inhibitors (HDACi) are applied as anticancer compounds. In this study, we examined whether gossypol harbors HDAC inhibiting activity. In vitro analyses showed that gossypol inhibited class I, II, and IV HDAC, displaying the capability to laterally interact with the respective catalytic center and is, therefore, classified as a pan-HDAC inhibitor. Next, we studied the effects of gossypol on human-derived hepatoma (HepG2) and colon carcinoma (HCT-116) cell lines and found that gossypol induced hyperacetylation of histone protein H3 and/or tubulin within 6 h. Furthermore, incubation with different concentrations of gossypol (5–50 µM) over a time period of 96 h led to a prominent reduction in cellular viability and proliferation of hepatoma (HepG2, Hep3B) and colon carcinoma (HCT-116, HT-29) cells. In-depth analysis of underlying mechanisms showed that gossypol induced apoptosis via caspase activation. For pre-clinical evaluation, toxicity analyses showed toxic effects of gossypol in vitro toward non-malignant primary hepatocytes (PHH), the colon-derived fibroblast cell line CCD-18Co, and the intestinal epithelial cell line CCD 841 CoN at concentrations of ≥5 µM, and embryotoxicity in chicken embryos at ≥2.5 µM. In conclusion, the pronounced inhibitory capacity of gossypol on cancer cells was characterized, and pan-HDACi activity was detected in silico, in vitro, by inhibiting individual HDAC isoenzymes, and on protein level by determining histone acetylation. However, for clinical application, further chemical optimization is required to decrease cellular toxicity.

Design, synthesis, and evaluation of novel combretastatin A‐4 based chalcone derivatives as anticancer agents

AbstractA series of combretastatin A‐4 based chalcones (14a‐l) were designed, synthesized and these compounds examined for inhibitory effects on the proliferation of human lung (A549), breast (MCF‐7), melanoma (A375), and colon (HT‐29) carcinoma cells. Compounds 14b, 14c, 14e, 14h, and 14i (tri/dimethoxy, methyl, and mono/dinitro derivatives) have exhibited the most potent antiproliferative activity with IC50 < 2 μM and the hexa methoxy derivative 14b, the most promising one, which displayed the potent inhibitory activities in MCF‐7 (IC50: 10 nM), A375 (IC50: 12 nM), and A549 (IC50: 65 nM) cell lines, and is 18 times more potent than the CA‐4. Compound 14b represents a new scaffold and the results provide insights into further development of anticancer agents.

LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis.

Low density lipoprotein receptor related protein-1 (LRP-1) is a large ubiquitous endocytic receptor mediating the clearance of various molecules from the extracellular matrix. Several studies have shown that LRP-1 plays crucial roles during tumorigenesis functioning as a main signal pathway regulator, especially by interacting with other cell-surface receptors. Discoïdin Domain Receptors (DDRs), type I collagen receptors with tyrosine kinase activity, have previously been associated with tumor invasion and aggressiveness in diverse tumor environments. Here, we addressed whether it could exist functional interplays between LRP-1 and DDR1 to control colon carcinoma cell behavior in three-dimensional (3D) collagen matrices. We found that LRP-1 established tight molecular connections with DDR1 at the plasma membrane in colon cancer cells. In this tumor context, we provide evidence that LRP-1 regulates by endocytosis the cell surface levels of DDR1 expression. The LRP-1 mediated endocytosis of DDR1 increased cell proliferation by promoting cell cycle progression into S phase and decreasing apoptosis. In this study, we identified a new molecular way that controls the cell-surface expression of DDR1 and consequently the colon carcinoma cell proliferation and apoptosis and highlighted an additional mechanism by which LRP-1 carries out its sensor activity of the tumor microenvironment.

ARHGAP10 Inhibits the Proliferation and Metastasis of CRC Cells via Blocking the Activity of RhoA/AKT Signaling Pathway.

IntroductionARHGAP10 belongs to the ARHGAP family, which is downregulated in certain human tumors. However, the detailed function of ARHGAP10 remains unclear in human colon carcinoma (CRC). In the current study, we aimed to explore the role of ARHGAP10 in the growth and metastasis of CRC cells.MethodsARHGAP10 was induced silencing and overexpression using RNA interference (RNAi) and lentiviral-vector in CRC cells. Quantitative real-time PCR (qRT-PCR) and Western blot were used to quantify the mRNA and protein contents of ARHGAP10. Cell proliferation was determined by using Cell counting kit-8 (CCK-8). Transwell assay was utilized to examine the role of ARHGAP10 in the migration and invasion of CRC cells.ResultsOur results indicated that ARHGAP10 was downregulated in human CRC tissues and low expression of ARHGAP10 was associated with poor prognosis of patients with CRC. Moreover, ARHGAP10 overexpression significantly inhibited the proliferation and metastasis of CRC cells. Moreover, a PI3K/AKT inhibitor LY294002 was utilized to examine the connection between ARHGAP10 and AKT. Our findings demonstrated that the AKT inhibitor LY294002 could rescue the function of ARHGAP10 in CRC cells.DiscussionIt was the first time to elucidate that AKT involved in the ARHGAP10 signaling pathway and ARHGAP10 negatively mediated the phosphorylation of AKT (p-AKT) and RhoA activity in CRC cells. Interestingly, the Rho/MRTF/SRF inhibitor CCG-1423 significantly inhibited the phosphorylation of AKT in ARHGAP10 siRNA transfected CRC cells. Much importantly, overexpression of ARHGAP10 deeply suppressed the metastasis of CRC cells in the lung in vivo. Taken together, our findings not only enhanced the understanding of the anti-cancer effect of ARHGAP10 in CRC cells but also indicated its underlying pathway in CRC.

Chaperone-mediated autophagy regulates apoptosis and the proliferation of colon carcinoma cells

Chaperone-mediated autophagy (CMA) is one of the three types of autophagy. In recent years, CMA has been shown to be associated with the pathogenesis of several types of cancer. However, whether CMA is involved in the pathogenesis of colorectal cancer (CRC) remains unclear. In this study, we investigated CMA activity in tissue specimens from CRC patients and mouse models of colitis-associated CRC (induced by administration of AOM plus DSS). In addition, we down-regulated CMA in CT26 colon carcinoma cells stably transfected with a vector expressing a siRNA targeting LAMP-2A, the limiting component in the CMA pathway, to explore the role of CMA in these cells. Apoptosis was detected using TUNEL assay, and the apoptosis-related proteins were detected using western blotting. Cell proliferation was assessed using MTT assay, Ki-67 labelling and western blotting for PCNA. We found that LAMP-2A expression was significantly increased in CRC patients and mouse models and varied according to the stage of the disease. Inhibition of CMA in CT26 cells facilitated apoptosis, as evidenced by increased TUNEL immunolabeling, increased expression of Bax and Bnip3, and decreased expression of Bcl-2. Cell proliferation assays showed that inhibition of CMA impeded the proliferation of CT26 cells. These data support the hypothesis that CMA is up-regulated in CRC, and inhibition of CMA may be a new therapeutic strategy for CRC patients.

Reduction of inflammation and colon injury by a Pennyroyal phenolic extract in experimental inflammatory bowel disease in mice

PurposeLittle is known about the pharmacological effects of the phenolic compounds of Pennyroyal (Mentha pulegium). This Mediterranean aromatic plant, used as a gastronomic spice and as food preservative by the food industry has been studied mainly due to its essential oil antibacterial properties, composed primarily by monoterpenes. With this work, we aimed to evaluate the effects of a phenolic extract of pennyroyal in the impairment of inflammatory processes in Inflammatory Bowel Diseases (IBD) and in the potential inhibition of progression to colorectal cancer (CRC). MethodsTo that purpose, we evaluated the effect of pennyroyal extract administration in a model of TNBS-induced colitis in mice and further determined its effect on human colon carcinoma cell proliferation and invasion. ResultsThe phenolic extract of pennyroyal exhibited antioxidant properties in in vitro assays and administration of the extract in a rat model of carrageenan-induced paw oedema led to significant anti-inflammatory effects. Further results evidenced a beneficial effect of the phenolic extract in the attenuation of experimental colitis and a potential antiproliferative effect on cultured colon cancer cells, effects not previously described, to our knowledge. A reduction in several markers of colon inflammation was observed following administration of the extract to colitis-induced mice, including functional and histological indicators. A successful inhibition of cancer cell invasion and proliferation was also observed in in vitro studies with HT-29 cells. Furthermore, the extract also led to a reduced expression of iNOS/COX-2 in the colon of colitis-induced mice, both being crucial mediators of intestinal inflammation. ConclusionsTaking into consideration the central role of inflammation in the pathophysiology of CRC and the recognised connection between inflammatory events and cancer, these results enlighten the relevance of the phenolic constituents of pennyroyal as important pharmacological sources in the investigation of new treatment options for patients with inflammatory bowel diseases.

Increased glucocorticoid receptor activity and proliferation in metastatic colon cancer

Metastasis is regarded as the fatal hallmark for colon cancer, but molecular mechanisms responsible for it have remained poorly defined. Glucocorticoid receptor (GR) within the tumor microenvironment mediates the effects of stress hormones which are used in clinics for their inflammation-modulatory and immunosuppressive properties. Further, epigenetic activation of GR promotes tumor heterogeneity and metastasis. Here, we sought to investigate the correlation between GR activation and proliferation and invasion in metastatic colon cancer microenvironment. We used proliferation/invasion assays, western blot, RT-qPCR, immunofluorescence staining and quantitative methylation to study glucocorticoid-GR signaling, including the involvement of CDK1, in human colon adenocarcinoma cell lines HT29 and T84 (a representative metastatic cell line). Nuclear expression levels of GR were significantly upregulated in metastatic T84 cells, and glucocorticoid derivative, dexamethasone (DEX) treatment caused increased proliferation and invasion in T84 cell, compared to HT29 cell. DEX treatment induced CDK1 expression which was accompanied by reduced CDK1 methylation, indicating epigenetic regulation. Depletion of GR suppressed proliferation of metastatic colon carcinoma cells and depletion of CDK1 had similar suppressing effects on proliferation as well as invasion of metastatic cells. Our study suggests that glucocorticoid-GR-CDK1 signaling induces proliferation and invasion of colon cancer cells and therapies involving the use of glucocorticoids need to exercise caution and re-evaluation.

A study of the impact of inhibitors of DNA binding-1 on proliferation and migration in human colon carcinoma cells.

This study aims to explore the effect of an inhibitor of DNA binding-1 (Id-1) on the proliferation and migration of human colon carcinoma cell line SW480 and HT-29. SW480 and HT-29 cells transfected with Id-1-interference sequence were assigned to the experimental groups (inhibition groups 1 and 2), and SW480 and HT-29 cells with blank interference sequence (blank groups) and blank load transfection (blank load groups) were assigned as the control groups. The expression of Id-1 in six groups was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation in vitro was assessed by MTT assay. RT-PCR and Western blot results demonstrated that the mRNA and protein expressions of Id-1 in the inhibition group 1 were lower than those in the blank group 1 and blank load group 1. RT-PCR and Western blot results revealed that the mRNA and protein expressions of Id-1 were lower in the inhibition group 2 than in the blank group 2 and blank load group 2. The results of the growth curve revealed that proliferation ability was significantly weaker from the third day in the inhibition groups 1 and 2 than in the blank group and blank load group. Transwell chamber experiment and Matrigel invasion assay revealed that the number of Transwell cells significantly decreased in the inhibition groups 1 and 2 than in the blank groups and blank load groups (P < 0.01). Id-1 significantly promotes the proliferation and migration of human colon carcinoma cell lines SW480 and HT-29.

Yin Yang 1 promotes the Warburg effect and tumorigenesis via glucose transporter GLUT3.

Cancer cells typically shift their metabolism to aerobic glycolysis to fulfill the demand of energy and macromolecules to support their proliferation. Glucose transporter (GLUT) family‐mediated glucose transport is the pacesetter of aerobic glycolysis and, thus, is critical for tumor cell metabolism. Yin Yang 1 (YY1) is an oncogene crucial for tumorigenesis; however, its role in tumor cell glucose metabolism remains unclear. Here, we revealed that YY1 activates GLUT3 transcription by directly binding to its promoter and, concomitantly, enhances tumor cell aerobic glycolysis. This regulatory effect of YY1 on glucose entry into the cells is critical for YY1‐induced tumor cell proliferation and tumorigenesis. Intriguingly, YY1 regulation of GLUT3 expression, and, subsequently, of tumor cell aerobic glycolysis and tumorigenesis, occurs p53‐independently. Our results also showed that clinical drug oxaliplatin suppresses colon carcinoma cell proliferation by inhibiting the YY1/GLUT3 axis. Together, these results link YY1's tumorigenic potential with the critical first step of aerobic glycolysis. Thus, our novel findings not only provide new insights into the complex role of YY1 in tumorigenesis but also indicate the potential of YY1 as a target for cancer therapy irrespective of the p53 status.

PO-374 LINC00152 long non-coding RNA promotes the proliferation of SW480 colon carcinoma cells through regulation of cell cycle and WNT signalling pathway

IntroductionLong non-coding RNAs (lncRNAs) contribute to different cancers including colorectal cancer (CRC). Previous studies have shown altered LINC00152 expression in CRC, but the detailed mechanism of its effects during CRC development and progression is not well studied. We aimed to study the effects of LINC00152 silencing on the cell cycle regulation and whole transcriptome in colon carcinoma cells. We also analysed the DNA methylation alterations caused by LINC00152 knockdown.Material and methodsLINC00152 were silenced in SW480 colon carcinoma cells using Stealth siRNAs. Flow cytometric cell cycle analysis was performed using propidium-iodide DNA staining. Cyclin D1 protein expression was detected using flow cytometry after labelling with anti-cyclin D1 antibody. The effect of LINC00152 silencing to genome-wide gene expression was studied on Human Transcriptome Array 2.0 microarrays. DNA methylation alterations after LINC00152 knockdown were evaluated using Reduced Representation Bisulfite Sequencing (RRBS) method using NextSeq500 device.Results and discussionsSilencing of LINC00152 significantly suppressed cell growth compared to negative control cells (p<0.05). LINC00152 knockdown caused approximately two-fold increase in apoptosis (48 hour: si-NEG: 4%, si-LINC00152: 8%; 72 hour: si-NEG: 11%, si-LINC00152: 23%) (p<0.05). In parallel with the growth inhibitory effect, silencing of LINC00152 could reduce cyclin D1 expression already after 48 hours, however, significant decrease was detected after 72 hours in the LINC00152 siRNA-treated cells (si-NEG MFI=0.70 and si-LINC00152 MFI=0.56) without attenuation of phospho-S6 protein. Whole transcriptome microarray analysis of LINC00152 silenced cells revealed significant under-expression of genes with oncogenic and/or metastasis promoting function (e.g. STC1, YES1, HES1, KLK6, PORCN) and up-regulation of tumour suppressor genes (e.g. DKK1, PERP) (FDR p<0.05, absolute value of logFC >1). Using RRBS, DNA methylation alterations after LINC00152 silencing could be detected genome-widely including hypomethylation in SFRP2 and ALDH1A3 gene promoters.ConclusionOur results suggest that LINC00152 lncRNA can contribute to CRC pathogenesis by facilitating cell proliferation through up-regulation of several oncogenes/metastatic genes in WNT, Notch and TP53 pathways and of cyclin D1 cell cycle progression gene, furthermore, by affecting the promoter methylation status of certain CRC associated genes.

Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G₀/G₁ or G₂/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

Formononetin inhibits colon carcinoma cell growth and invasion by microRNA‑149‑mediated EphB3 downregulation and inhibition of PI3K/AKT and STAT3 signaling pathways.

Formononetin (Form), a phytoestrogen extracted from the roots of Astragalus membranaceus, is one of the fundamental herbs used in traditional Chinese medicine because of its protective effects against certain malignant tumors. However, its role in colon carcinoma cells and the underlying molecular mechanisms have not been completely elucidated. The present study aimed to demonstrate that Form significantly inhibited the proliferation and invasion of the colon carcinoma cell lines SW1116 and HCT116. Mechanistic studies have suggested that Form suppresses colon carcinoma cell growth by downregulating cell cycle-associated protein (cyclin D1) expression and arresting the cell cycle at the G0-G1 checkpoint. Further studies revealed that treatment with Form inhibits matrix metalloproteinase (MMP)2 and MMP9 expression. Aditionally, the results demonstrated that Form significantly increased microRNA (miR)-149 expression. Following miR-149 overexpression in SW1116 and HCT116 cells using an miR-149 mimic, cell viability and Ephrin type-B receptor 3 (EphB3) levels decreased. Furthermore, the inhibitory effects of Form were associated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. These results indicated the suppressive effect of Form on colon carcinoma cell proliferation and invasion, possibly via miR-149-induced EphB3 downregulation and the inhibition of the PI3K/AKT and STAT3 signaling pathways. Overall, Form may be used as a novel candidate for the clinical treatment of colorectal cancer in the future.

Asiatic acid inhibits proliferation, migration and induces apoptosis by regulating Pdcd4 via the PI3K/Akt/mTOR/p70S6K signaling pathway in human colon carcinoma cells.

Previous studies have demonstrated that asiatic acid (AA), the major component of Centella asiatica, is able to meditate cytotoxic and anticancer effects on various types of carcinoma cells. In order to investigate the molecular mechanism that underlies the antitumor effect of AA, the present study investigated the effects of AA on proliferation, migration and apoptosis of SW480 and HCT116 colon cancer cells. Viability and changes in cell morphology in the cells were assessed by MTT assay and transmission electron microscopy, respectively. Colony formation analysis was used to observe proliferation of the single cell, and migratory ability of the cells was assessed by performing Transwell migration assay. Hoechst 33342 nuclear staining and flow cytometry were used to assess apoptosis in colon carcinoma cells. The expression of proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K signaling pathway and epithelial-mesenchymal transition (EMT) marker were analyzed by western blotting. The present study revealed that proliferation and migration of colon carcinoma cells were inhibited by AA in a dose-dependent and time-dependent manner. Numerous apoptotic bodies were observed, and G2/M and S phase progression were delayed in colon cancer cells treated with AA, but not in the control group. A number of phosphorylated proteins, including PI3K, Akt (Ser473), mTOR, ribosomal protein S6 kinase (p70S6K) downregulated, while the expression of Pdcd4 was upregulated following treatment with AA. Additionally, AA affects expression of EMT markers in a dose-dependent manner. On the basis of these results, it was concluded that AA inhibited proliferation, migration and induced apoptosis of colon cancer cells by regulating Pdcd4 via the PI3K/Akt/mTOR/p70S6K signaling pathway. These observations suggest that AA may be a potential therapeutic agent for the treatment of colon carcinoma.

CXCL12 gene silencing down-regulates metastatic potential via blockage of MAPK/PI3K/AP-1 signaling pathway in colon cancer

BackgroundTo investigate the effect of CXCL12 gene silencing on proliferation,invasion, angiogenesis and the relationship of MAPK/PI3K/AP-1 signaling pathway in colon cancer cells.MethodsRT-PCR and Western-blot were used to detect the expression of CXCL12 mRNA and protein in four colon cancer cell lines. Human colon cancer cells were transfected with CXCL12 siRNA carrying by Lipofectamine 2000. The expression of CXCL12 protein was confirmed by immunoblotting. WST-1, invasion and angiogenesis assay were used to examine the effect on proliferation, invasion and angiogenesis in colon cancer cells after CXCL12 siRNA silence, respectively. The phosphorylation of MAPK/PI3K/AP-1 protein levels was detected by Western blotting in CXCL12 siRNA suppression DLD-1 cell.ResultsCXCL12 mRNA and proteins were only expressed in DLD-1 colon cancer cell lines. CXCL12 siRNA were transfected into DLD-1 cells, the expression CXCL12 proteins was significantly inhibited (P < 0.01), and the proliferation, invasion and angiogenesis of DLD-1 cells were inhibited significantly (P < 0.01). CXCL12 gene silencing resulted in blockage of MAPK, PI3K and AP-1 phosphorylation by CXCL12-induced in DLD-1 colon cancer cell.ConclusionThe silencing CXCL12 gene significantly inhibits the proliferation, invasion and angiogenesis ability of some types colon carcinoma cells through down-regulation of MAPK/PI3K/AP-1 signaling pathway.

Abstract B148: Activity of the TAM kinase-targeting compound, SLC-391, is mediated by the engagement of the immune system in CT-26 syngeneic mouse model

Abstract Overexpression of TAM family receptor tyrosine kinases such as Axl has been reported in numerous types of human cancer, and found to correlate with tumor progression and prognosis, metastasis, and drug resistance. Aside from the role in cell growth and survival, TAM kinases have long been recognized for their immunosuppressive activity as well. Hence, downregulation of their activity is expected to unleash antitumor immunity in the tumor microenvironment. As one of the several TAM-targeting small-molecule inhibitors identified, SLC-391 displays a relatively strong activity against Axl, as evidenced by the reductions of phosphotransferase activity in radiometric biochemical assay and the level of Axl Y779 phosphorylation in the cell-based assay. Contradictorily, proliferation of CT-26 murine colon carcinoma cells seemed to be unaffected by the compound in the thymidine incorporation assay with an IC50 of ~10 μM. Interestingly, SLC-391 inhibited CT-26 tumor growth by 37% in a 15-day efficacy study in which the compound was administered at 50 mg/kg p.o.. In comparison, a PD-1 antibody delayed tumor growth by 27%. Tumor-infiltrating lymphocyte phenotyping revealed increases in the number of NK cells and the ratio of M1/M2-polarized macrophages in SLC-391 treatment group, followed by the rise of CD8+ T/Treg ratio and reduction in immunosuppressive myeloid cells. This is indicative of sequential engagement and stimulation of proinflammatory innate immune response and adaptive immune response. In addition, a synergistic antitumor effect was observed when the anti-PD-1 insensitive CT-26 syngeneic model was treated with a combination of SLC-391 and an anti-PD-1 antibody and the overall survival rate of the combination group was prolonged dramatically in comparison with the vehicle control group. To summarize, the antitumor activity of SLC-391 is at least in part mediated by reversing the immunosuppressive tumor microenvironment in CT-26 colon carcinoma model. Citation Format: Shenshen Lai, Rick Li, Paromita Raha, Yuxiang Hu, Jun Yan, Hong Zhang, Anthony Marotta, Zaihui Zhang. Activity of the TAM kinase-targeting compound, SLC-391, is mediated by the engagement of the immune system in CT-26 syngeneic mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B148.

Mono-ADP-ribosylation of histone 3 at arginine-117 promotes proliferation through its interaction with P300.

Relatively little attention has been paid to ADP-ribosylated modifications of histones, especially to mono-ADP-ribosylation. As an increasing number of mono-ADP-ribosyltransferases have been identified in recent studies, the functions of mono-ADP-ribosylated proteins have aroused research interest. In particular, histones are substrates of some mono-ADP-ribosyltransferases and mono-ADP-ribosylated histone have been detected in physiological or pathological processes. In this research, arginine-117 (Arg-117; R-117) of hsitone3(H3) is identified as the a site of mono-ADP-ribosylation in colon carcinoma(the first such site to be identified); this posttranslational modification may promote the proliferation of colon carcinoma cells in vitro and in vivo. Using a point-mutant lentivirus transfection and using an activator of P300 allowed us to observe the mono-ADP-ribosylation at H3R117 and enhancement of the activity of P300 to up-regulate the level of acetylated β-catenin, which could increase the expression of c-myc and cyclin D1.

Danshen improves survival of patients with colon cancer and dihydroisotanshinone I inhibit the proliferation of colon cancer cells via apoptosis and skp2 signaling pathway

Ethnopharmacological relevanceDanshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. However, it's definite clinical effect and mechanism on colon carcinoma is unclear. Aim of the studyTo test the hypothesis that the protective effect of danshen on colon cancer and discover the bioactive compounds through in vitro study. Materials and methodsWe conducted a nationwide cohort study by using population-based data from the Taiwan National Health Insurance Research Database (NHIRD). The study cohort comprised patients diagnosed with malignant neoplasm of colon (ICD-9-CM codes:153) in catastrophic illness database between January 1, 2000, and December 31, 2010. We used the Kaplan–Meier method to estimate lung cancer cumulative incidences. Next, human colon cancer cells (HCT 116 cells and HT29 cells) were used to investigate the effect of dihydroisotanshinone I (DT) on the proliferation and apoptosis of human colon cancer cells and the underlying mechanism through XTT assay and flow cytometry. The in vivo effect of DT treatment was investigated through a xenograft nude mouse model. ResultsIn our study, the in vivo protective effect of danshen in the different stage of colon cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro, we found that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the proliferation of colon carcinoma cells, HCT 116 cells and HT-29 cells. Moreover, DT induced apoptosis of colorectal cancer cells. DT also repressed the protein expression of Skp2 (S-Phase Kinase Associated Protein 2) and the mRNA levels of its related gene, Snail1 (Zinc finger protein SNAI1) and RhoA (Ras homolog gene family, member A). In addition, DT also blocked the colon cancer cells recruitment ability of macrophage by decreasing CCL2 secretion in macrophages. DT treatment also significantly inhibited the final tumor volume in a xenograft nude mouse model. ConclusionDanshen has protective effects in colon cancer patients, which could be attributed to DT through blocking the proliferation of colon cancer cells through apoptosis.