Proliferation Of Human Colorectal Cancer Cells Research Articles

Gab3 is required for human colorectal cancer cell proliferation

Here, we focused on the potential function of Gab3, an uncommon Gab family protein, in human colorectal cancer (CRC) cells. We found that Gab3 was only expressed in human colon cancer tissues as well as in established (HCT-116 and HT-29 lines) and primary human CRC cells. It was however absent in normal human colon cancer tissues and in FHC colon epithelial cells. Knockdown of Gab3 by targeted-shRNAs inhibited proliferation of the CRC cells. Reversely, exogenous over-expression of Gab3 promoted CRC cell proliferation. At the signaling level, Gab3 co-precipitated with p85 and SHP2 in CRC cells, which was required for subsequent Akt and Erk activation. Gab3 shRNA knockdown inhibited Akt and Erk activation, yet Gab3 over-expression augmented it. In vivo, HCT-116 xenograft tumor growth in severe combined immune deficient (SCID) mice was suppressed following expressing Gab3 shRNAs. Meanwhile, Akt and Erk activation in Gab3 shRNA-expressing tumors was also largely inhibited. Together, our results suggest that Gab3 expression in CRC cells is important for Akt-Erk activation and cell proliferation.

Abstract 4606: Novel mechanism for suppression of human colorectal cancer cell proliferation through induction of Wnt9A and suppression of β-catenin by resveratrol

Abstract Dysregulation of the Wnt pathway in histologically distinct cancers has been studied extensively. The canonical Wnt pathway (CWP) utilizing β-catenin is one of the major drivers for proliferation. In contrast, the capacity of the non-canonical Wnt pathway (NCWP) to regulate proliferation has not been studied extensively in neoplastic tissues. Our earlier studies demonstrated that RV-mediated suppression of colorectal cancer (CRC) cell proliferation correlated with upregulated expression of several NCWP components. These results are consistent with the hypothesis that these upregulated NCWP components suppress CWP activity leading to proliferation inhibition. Of the NCWP components measured, the ligand Wnt9A was most prominently upregulated. Since the CWP utilizes β-catenin for its activity, the purpose of the present study was to determine whether induction of Wnt9A and suppression of CRC proliferation by RV can be correlated directly to modulation of β-catenin message and protein levels. This study was conducted on human CRC cells derived from surgical specimens (n = 5). Cell proliferation and apoptosis were determined by MTS and Caspase 3 assays, respectively. β-catenin protein levels were determined by ELISA and gene expression using quantitative RT-PCR Wnt pathway gene expression arrays. RV increased Wnt9A expression in CRC cells in a concentration-dependent manner. Wnt9A expression was increased by a mean of 8, 36 and 260 fold (p<0.004, ANOVA) relative to media controls with RV concentrations of 5, 10 and 20 μg/ml, respectively. The increase in Wnt9A expression was associated with decreased total β-catenin protein by 21, 51 and 71% (p<0.001, ANOVA), and active β-catenin protein by 17, 22 and 50% (p<0.001, ANOVA). These effects were associated with significant inhibition of proliferation by 35, 54 and 66% relative to media controls with RV concentrations of 5, 10 and 20 μg/ml, respectively (p<0.006, ANOVA) and a significant increase in apoptosis (p<0.01, ANOVA). Significantly, mRNA levels for β-catenin were not affected by any concentration of RV. Finally, the suppression of CRC cell proliferation by RV was reversed by Wnt9A antibody and IWP-2, a Wnt ligand secretion inhibitor (p<0.02, ANOVA). The induction of Wnt9A with subsequent suppression of β-catenin protein levels by resveratrol represents a novel mechanism for the suppression of canonical Wnt pathway-mediated CRC cell proliferation. These results suggest that interventions that stimulate the induction of non-canonical Wnt pathway components, and specifically Wnt9A, may be therapeutically beneficial for controlling proliferation of colorectal cancers. Citation Format: Irshad Ali, Bani Medegan, Donald Braun. Novel mechanism for suppression of human colorectal cancer cell proliferation through induction of Wnt9A and suppression of β-catenin by resveratrol. [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 4606.

Wnt9A Induction Linked to Suppression of Human Colorectal Cancer Cell Proliferation.

Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP’s role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation (p < 0.001) and increased apoptosis (p < 0.01) coincided with 23-fold increase (p < 0.025) in the expression of the NCWP ligand, Wnt9A. LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation.

PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.

Suppression of human colorectal cancer cell proliferation by noncanonical Wnt 9A ligand induction.

e14621 Background: Malignant tissues are often characterized by dysfunctional Wnt signaling with the predominant feature involving the regulation of β-catenin. β-catenin is utilized by the canonica...

Recombinant lentivirus with enhanced expression of caudal-related homeobox protein 2 inhibits human colorectal cancer cell proliferation in vitro.

Caudal-related homeobox protein 2 (CDX2), a tumor suppressor in the adult colon, is overexpressed under a non-cancer specific cytomegalovirus promoter in certain tumor cells; furthermore, non-specific expression of CDX2 may result in aberrant side effects in normal cells. The human telomerase reverse transcriptase (hTERT) promoter is active in the majority of cancer cells but not in normal cells. Hypoxia is a key feature of solid tumors, and targeted genes may be significantly upregulated by five copies of hypoxia-response elements (HREs) under hypoxic conditions. However, the effect of CDX2 overexpression, as controlled by five copies of HREs and the hTERT promoter, on human colorectal cancer (CRC) cell proliferation in vitro remains to be fully elucidated. In the current study, a recombinant lentivirus containing the CDX2 gene under the control of five HREs and the hTERT promoter was generated. An immunofluorescence assay was used to detect CDX2 expression by the 5HhC lentivirus, whereas an MTT assay was used to detect the effects of CoCl2 on the viability of LoVo cells. Western blot analysis was conducted in order to determine the relative ratios of recombinant CDX2 protein to the internal control β-actin, following 5HhC/LoVo cell culture under normoxic and hypoxic conditions (100, 200, 300, 400 or 500 µmol/l CoCl2) for 24 h, then for 12, 24 or 36 h with the optimal concentration (300 µmol/l) of CoCl2. Reverse transcription polymerase chain reaction analysis was used to determine the transcription of recombinant CDX2 mRNA following culture of 5HhC/LoVo cells under normoxic or hypoxic conditions. Finally, a cloning assay was used to detect the proliferative ability of 5HhC/LoVo and 5Hh cells. High CDX2 expression was observed in hTERT-positive LoVo cells under hypoxic conditions, an effect which was mimicked by treatment with CoCl2 to inhibit LoVo cell proliferation in vitro. High expression of CDX2 therefore provides a promising strategy for the development of novel targeted treatments and gene therapy for CRC.

Glargine Promotes Human Colorectal Cancer Cell Proliferation via Upregulation of miR-95.

Several studies have shown a correlation between glargine use and cancer risk. However, the role of glargine in carcinogenesis, especially in colorectal cancer (CRC), is still inconclusive. The aim of this study was to investigate the influence of glargine on proliferation of CRC cells and its possible mechanism. Effect of glargine on the cell proliferation was tested in HCT-116 and SW480 cells by MTT assay, and apoptosis was measured by flow cytometry. The expression of microRNA-95 (miR-95) and sorting nexin 1 (SNX1) protein was also determined by real-time PCR and Western blotting, respectively. The results showed that high dose glargine (from 150 to 300 nM) promoted proliferation and inhibit2ed apoptosis of CRC cells compared with untreated cells. Moreover, glargine could upregulate miR-95 and downregulate SNX1 protein expression in CRC cells. These data show that glargine may indeed trigger cellular proliferation in CRC, probably by regulating miR-95.

MicroRNA-100 regulates SW620 colorectal cancer cell proliferation and invasion by targeting RAP1B

MicroRNAs (miRNAs) have been demonstrated to play important roles in tumorigenesis of human cancer. Fewer studies have explored the roles of miR-100 on human colorectal cancer cell proliferation and invasion. In this study, we utilized real-time PCR to verify whether miR-100 was downregulated in human colorectal cancer tissues compared with matched adjacent normal tissues. Functional studies demonstrated that ectopic expression of miR-100 inhabits cell growth and invasion and induce apoptosis, whereas knockdown of miR-100 yielded the reverse phenotype. Mechanistic studies reveal that miR-100 repressed the activity of a reporter gene fused to the 3'-untranslated region (3'-UTR) of RAP1B, whereas miR-100 silencing upregulated the expression of the reporter gene. Furthermore, we also detected that RAP1B mRNA was inversely expressed with miR-100 in colorectal cancer tissues. These data indicate that the miR-100 plays a tumor suppressor role by regulating colorectal cancer cell growth and invasion phenotype, and could serve as a potential maker for colorectal cancer therapy.

Abstract 1940: Reg4 induces the expression of D-type cyclins and associated kinases to induce mitotic division of human colorectal cancer cells

Abstract Background: Cyclins and cyclin-dependent kinases (CDK) are important component of the cell cycle machinery. D-type cyclins are positive regulators of cell cycle and a known target of Wnt/β-Catenin signaling. Cyclin D1 and cyclin D3 play a prominent role in differentiation and proliferation and is highly upregulated in many cancers. Recent studies also showed frequent upregulation of regenerating (Reg) gene 4 expression in many human gastrointestinal (GI) malignancies including colorectal cancer (CRC). We further observed a Reg4-mediated increase in β-Catenin expression and its subsequent nuclear translocation. Therefore, we tested the hypothesis that Reg4 induces the expression of D-type cyclins and associated kinases to induce the mitotic division of human CRC cells. Methods: Real time RT-PCR and western blot analyses were performed to determine the effects of Reg4 treatments on the expression of D-type cyclins and associated kinases. Mitotic and MTT assays were performed to determine its association with regulation of mitotic division and proliferation of human CRC cells. Results: Following real-time RT-PCR and western blot analyses we observed a time-dependent increase in cyclin D1 and D3 expression following the addition of recombinant human Reg4 (rhR4) to the cultures of HCT116 and HT29 human CRC cells. Furthermore, addition of rhR4 to the cell cultures also led to an increased expression of CDK4 and CDK6, the known kinase partners of cyclin D. We previously observed the involvement of Akt kinase, GSK-3β, and β-Catenin activities in mediating Reg4-signaling. Therefore, in the present study we utilized a specific Akt inhibitor as an antagonist and SB216763, an inhibitor of GSK-3β as an agonist of Reg4-signaling and examined their effects on expression of D-type cyclins and mitotic index of human CRC cells. Treatments of specific Akt inhibitor (10 μM) to the human CRC cells significantly inhibited the expression of cyclin D1 and D3, and subsequently inhibited their mitotic index. Furthermore, an addition of rhR4 (200 nM) to the cultures of human CRC cells pretreated with specific Akt inhibitor (10 μM) did not increase the expression of these cyclins, hence the mitotic index of human CRC cells. In addition, the treatments of SB216763 (10 μM) positively modulated the Reg4-signaling and led to significant increases in the expression of D-type cyclins, hence mitotic index of human CRC cells. Conclusions: These results suggest that Reg4 is a potent regulator of mitotic division of human CRC cells and involves Akt kinase and GSK-3β as important mediators to induce the expression of D-type cyclins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2011-1940

Impact of fragile histidine triad gene transfection on the proliferation and apoptosis of human colorectal cancer cell

To investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression. The eukaryotic expression plasmid containing FHIT, pRc/CMV2-FHIT was prepared and purified, and then identified by restrictive enzyme digestion. pRc/CMV2-FHIT was transfected into SW480 cells, and positive cell clones (SW480-FHIT, study group) were selected and amplified. Empty plasmid-transfected SW480 cells(SW480-pRc/CMV2, negative control) and normal SW480 cells (bland control) were used as control. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of SW480 cells. Cell-cycle kinetics and apoptosis were analyzed by flow cytometry (FCM). The changes of pro-caspase-8, caspase-8 mRNA and caspase-8 relative activity were analyzed by Western blot, semi-quantitative RT-PCR and colorimetric assay with pan labeled substrate, respectively. At 96 hours after transfection, cell inhibition rates of the study group and the negative control group were 71.7% and 16.9%. G0/G1 ratio was (63.2±3.5)% and (50.6±2.1)%, optical density of caspase-8 mRNA band 107 and 41, and relative activity of caspase-8 0.43 and 0.25, respectively. All the differences above were statistically significant (P<0.05). When FHIT inhibitor was added, the relative activity of caspase-8 decreased to 0.22, comparable to that in the control group. FHIT gene transfection can significantly inhibit the proliferation and induce G0/G1 arrest in human colon cancer cell line SW480. The mechanism is related to the up-regulation of caspase-8 expression.

Mitogenic CA2+ entry through ICRAC channels reciprocally regulates the tumor suppressor guanylyl cyclase C (GCC)

Guanylyl cyclase C (GCC) through cyclic GMP (cGMP) inhibits colorectal cancer cell proliferation, serving as a tumor suppressor in intestine. Conversely, Ca2+ entry through calcium release-activated calcium (Icrac) channels promotes colorectal cancer growth. The present study explored the functional relationship between mitogenic signaling by Ca2+ entry through Icrac channels and antiproliferative signaling by GCC in T84 human colon carcinoma cells. The E. coli heat-stable enterotoxin ST, a specific GCC ligand, induced intracellular accumulation of cGMP, Ca2+ entry through CNG channels and inhibition of human colorectal cancer cell proliferation. In contrast, influx of extracellular Ca2+ through Icrac channels, induced by thapsigargin, UTP or carbachol, inhibited GCC-dependent cGMP accumulation. Chelating intracellular Ca2+, removing extracellular Ca2+, or specifically blocking Icrac channels with the antagonist 2-APB reversed this inhibition. Moreover, 2-APB blocked Ca2+-dependent cell proliferation and amplified the anti-proliferative effects of ST. These data suggest that an imbalance of pro- and anti-mitogenic signals mediated by Ca2+ may contribute to mechanisms underlying colorectal carcinogenesis. In addition, they suggest that the combination of GCC agonists and Icrac channel inhibitors represents a novel paradigm for cGMP-directed therapy for colorectal tumors. Clinical Pharmacology & Therapeutics (2004) 75, P48–P48; doi: 10.1016/j.clpt.2003.11.180