Concentration- and stage-specific effects of nitrite on colon cancer cell lines

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM–100 μM and inhibits stage 3 HCT15 proliferation at 100 nM–1 μM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 μM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 μM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 μM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.     

MicroRNA-375 inhibits colorectal cancer growth by targeting PIK3CA

Colorectal cancer (CRC) is the second most common cause of death from cancer. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by triggering RNA degradation or interfering with translation. Aberrant miRNA expression is involved in human disease including cancer. Herein, we showed that miR-375 was frequently down-regulated in human colorectal cancer cell lines and tissues when compared to normal human colon tissues. PIK3CA was identified as a potential miR-375 target by bioinformatics. Overexpression of miR-375 in SW480 and HCT15 cells reduced PIK3CA protein expression. Subsequently, using reporter constructs, we showed that the PIK3CA untranslated region (3′-UTR) carries the directly binding site of miR-375. Additionally, miR-375 suppressed CRC cell proliferation and colony formation and led to cell cycle arrest. Furthermore, miR-375 overexpression resulted in inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. SiRNA-mediated silencing of PIK3CA blocked the inhibitory effect of miR-375 on CRC cell growth. Lastly, we found overexpressed miR-375 effectively repressed tumor growth in xenograft animal experiments. Taken together, we propose that overexpression of miR-375 may provide a selective growth inhibition for CRC cells by targeting PI3K/Akt signaling pathway.     

The research on the mechanism of Tsoong inhibiting for colon cancer

The roots of Codonopis bulleynana Forest ex diels (cbFed), locally known as Tsoong, have been used as a tonic food. Tsoong has wide range of pharmacological effects, including anticancer effects. In the present study, the anticancer activity of Tsoong and its potential molecular mechanisms were investigated. Using high throughput sequencing the apoptotic pathway was ranked as one of the most important pathways and the differential expressions of apoptosis-related genes such as Casp3, Casp6 and Apaf1 were identified. The following experiments were qRT-PCR which were used to verify the genes. In vitro, cell counting kit-8 (CCK-8) assays and flow cytometry in HCT116 and SW480 colon cancer cell were used to assess the anti‐proliferation and apoptosis-promoting activities of Tsoong. In vivo, the antitumor effect of Tsoong was assessed in colon cancer-bearing nude mice as a xenograft model. H&E staining was performed with oxaliplatin set as a positive control. The results showed that Tsoong up-regulated apoptosis-related genes, inhibited tumor cell proliferation, promoted tumor cellapoptosis in a dose-dependent manner and restrained the growth of colon neoplasm. The effects of a high dose of Tsoong on colon cancer cells were similar to those of oxaliplatin. Our results may ultimately help in the development of diagnostic and therapeutic strategies to control this devastating disease. Therefore, Tsoong may be a promising Chinese herbal compound for development for use in cancer therapy.     

Overexpression of the Promigratory and Prometastatic PTK7 Receptor Is Associated with an Adverse Clinical Outcome in Colorectal Cancer

Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。     

Effect of RSK4 on biological characteristics of colorectal cancer

Background

This study aimed to investigate the expression of P90 Ribosomal Protein S6 kinase 4 (RSK4) in colorectal cancer cells and its biological function.

Methods

We selected early SW480 and HCT116 colorectal cancer cell lines, using Lipofectamine? 2000 transfection reagent carrying RSK4 gene transfected into cells to establish the colorectal cancer cell lines with high expression of RSK4. RT-PCR and western blot (WB) analysis confirmed RSK4 expression in SW480 and HCT116 cancer cell lines. We used methylthiazoltetrazolium (MTT) assay and flow cytometry to detect the proliferation of colorectal cancer cells. After transfection of RSK4, the effect of RSK4 on the RNA levels associated with epithelial–mesenchymal transition (EMT) of colorectal cancer cells was analyzed by real-time fluorescence quantitative PCR and the expression of EMT-related protein was detected by WB analysis.

Results

After transfection of RSK4 overexpression, the MTT assay detected that RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro; flow cytometry detected that S-phase cells decreased significantly, and G0/1 cells increased significantly (P?<?0.05). The invasion ability of SW480 and HCT116 cells transfected with RSK4 was markedly lower than that in the control group, and the difference was statistically significant (P?<?0.05). Fluorescent quantitative PCR and WB analysis showed that the expression of EMT-associated molecular E-cadherin was remarkably increased and the expression of Snail was significantly decreased (P?<?0.01).

Conclusion

RSK4 gene in colorectal cancer cell lines with low expression of RSK4 after transfection can inhibit the growth and invasion of tumor cells. RSK4 gene may inhibit EMT and inhibit metastasis of colorectal cancer cells, may be a potential tumor suppressor gene and inhibit tumor distant metastasis, and may provide the biological basis for new therapeutic targets.

     

Inhibition of colon cancer cell proliferation by the dietary compound conjugated linoleic acid is mediated by the CDK inhibitor p21CIP1/WAF1

Our previous studies indicated that dietary conjugated linoleic acid (CLA) inhibits colon tumor cell proliferation in vitro and in vivo. To identify mechanisms by which CLA regulates growth arrest, the HT-29 human colon carcinoma cell line was treated with various physiological concentrations of CLA and analyzed by flow cytometry. We detected a dose-dependent increase in the percentage of cells arrested in G1 after CLA treatment that was accompanied by induction of the cyclin dependent kinase (CDK) inhibitor p21CIP1/WAF. CLA addition also led to increased p21 expression in HCT116 and SW480 cells, indicating that p21 induction is a general consequence of CLA treatment in colon cancer cells. Since both HT-29 and SW480 cells have mutant p53, our data indicate that p53 is not essential for induction of p21. In addition to an increase in p21 levels, HT-29 cell growth arrest was also accompanied by moderate decreases in Cyclin A, D1, E, and proliferating cell nuclear antigen (PCNA) levels. Following CLA treatment, p21 associated with and inhibited CDK4 and CDK2, and this correlated with reduced phosphorylation of retinoblastoma proteins. Increased association of p21 with PCNA was also detected. Dietary CLA inhibits cell cycle progression by inducing p21, which negatively regulates the growth promoting activities of CDK/cyclins and PCNA. These studies indicate that physiological concentrations of CLA inhibit growth of colon cancer cells with either wild-type or mutant p53, and may have therapeutic benefits in vivo.     

Genistein affects histone modifications on Dickkopf-related protein 1 (DKK1) gene in SW480 human colon cancer cell line

Genistein (GEN) is a plant-derived isoflavone and can block uncontrolled cell growth in colon cancer by inhibiting the WNT signaling pathway. This study aimed to test the hypothesis that the enhanced gene expression of the WNT signaling pathway antagonist, DKK1 by genistein treatment is associated with epigenetic modifications of the gene in colon cancer cells. Genistein treatment induced a concentration-dependent G2 phase arrest in the human colon cancer cell line SW480 and reduced cell proliferation. Results from several other human colon cancer cell lines confirmed the growth inhibitory effects of genistein. Overexpression of DKK1 confirmed its involvement in growth inhibition. Knockdown of DKK1 expression by siRNA slightly induced cell growth. DKK1 gene expression was increased by genistein in SW480 and HCT15 cells. DNA methylation at the DKK1 promoter was not affected by genistein treatment in all the cell lines tested. On the other hand, genistein induced histone H3 acetylation of the DKK1 promoter region in SW480 and HCT15 cells. This indicates that increased histone acetylation is associated with the genistein-induced DKK1 expression. The association between histone acetylation and DKK1 gene expression is confirmed by the histone deacetylase inhibitor trichostatin A (TSA) treatment. In conclusion, genistein treatment decreases cell growth and proliferation in colon cancer cell lines. The effect is associated with the increased DKK1 expression through the induction of histone acetylation at the DKK1 promoter region.     

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.     

Vascular basement membrane-derived multifunctional peptide, a novel inhibitor of angiogenesis and tumor growth

Vascular basement membrane-derived multifunctional peptide(VBMDMP)gene(fusion geneof the human immunoglobulin G3 upper hinge region and two tumstatin-derived fragments)obtained bychemical synthesis was cloned into vector pUC19,and introduced into the expression vector pGEX-4T-1 toconstruct a prokaryotic expression vector pGEX-4T-1-VBMDMP.Recombinant VBMDMP produced inEscherichia coli has been shown to have significant activity of antitumor growth and antimetastasis inLewis lung carcinoma transplanted into mouse C57B1/6.In the present study,we have studied the ability ofrVBMDMP to inhibit endothelial cell tube formation and proliferation,to induce apoptosis in vitro,and tosuppress tumor growth in vivo.The experimental results showed that rVBMDMP potently inhibited prolif-eration of human endothelial(HUVEC-12)cells and human colon cancer(SW480)cells in vitro,with noinhibition of proliferation in Chinese hamster ovary(CHO-K1)cells.rVBMDMP also significantly inhibitedhuman endothelial cell tube formation and suppressed tumor growth of SW480 cells in a mouse xenograftmodel.These results suggest that rVBMDMP is a powerful therapeutic agent for suppressing angiogenesisand tumor growth.     

pTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)

Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and in cell motility and invasion. CTTN gene is amplified and its protein is overexpressed in several types of cancer. The phosphorylated form of cortactin (pTyr⁴²¹) is required for cancer cell motility and invasion. In this study, we demonstrate that a majority of the tested primary colorectal tumor specimens show greatly enhanced expression of pTyr⁴²¹-CTTN, but no change at the mRNA level as compared to healthy subjects, thus suggesting post-translational activation rather than gene amplification in these tumors. Curcumin (diferulolylmethane), a natural compound with promising chemopreventive and chemosensitizing effects, reduced the indirect association of cortactin with the plasma membrane protein fraction in colon adenocarcinoma cells as measured by surface biotinylation, mass spectrometry, and Western blotting. Curcumin significantly decreased the pTyr⁴²¹-CTTN in HCT116 cells and SW480 cells, but was ineffective in HT-29 cells. Curcumin physically interacted with PTPN1 tyrosine phosphatases to increase its activity and lead to dephosphorylation of pTyr⁴²¹-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr⁴²¹-CTTN. Transduction with adenovirally-encoded CTTN increased migration of HCT116, SW480, and HT-29. Curcumin decreased migration of HCT116 and SW480 cells which highly express PTPN1, but not of HT-29 cells with significantly reduced endogenous expression of PTPN1. Curcumin significantly reduced the physical interaction of CTTN and pTyr⁴²¹-CTTN with p120 catenin (CTNND1). Collectively, these data suggest that curcumin is an activator of PTPN1 and can reduce cell motility in colon cancer via dephosphorylation of pTyr⁴²¹-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr⁴²¹-CTTN expression.     

RNF168 facilitates proliferation and invasion of esophageal carcinoma,possibly via stabilizing STAT1

Oesophageal cancer ranks as one of the most common malignancy in China and worldwide. Although genome‐wide association studies and molecular biology studies aim to elucidate the driver molecules in oesophageal cancer progression, the detailed mechanisms remain to be identified. Interestingly, RNF168 (RING finger protein 168) shows a high frequency of gene amplification in oesophageal cancer from TCGA database. Here, we report an important function for RNF168 protein in supporting oesophageal cancer growth and invasion by stabilizing STAT1 protein. RNF168 gene is amplified in oesophageal cancer samples, which tends to correlate with poor prognosis. Depletion RNF168 causes decreased cell proliferation and invasion in oesophageal cancer cells. Through unbiased RNA sequencing in RNF168 depleted oesophageal cancer cell, we identifies JAK‐STAT pathway is dramatically decreased. Depletion RNF168 reduced JAK‐STAT target genes, such as IRF1, IRF9 and IFITM1. Immuno‐precipitation reveals that RNF168 associates with STAT1 in the nucleus, stabilizing STAT1 protein and inhibiting its poly‐ubiquitination and degradation. Our study provides a novel mechanism that RNF168 promoting JAK‐STAT signalling in supporting oesophageal cancer progression. It could be a promising strategy to target RNF168 for oesophageal cancer treatment.     

Retracted: MicroRNA-204-3p represses colon cancer cells proliferation,migration, and invasion by targeting HMGA2

Colon cancer is a detrimental neoplasm of the digestive tract. MicroRNAs (miRNAs) as central regulators have been discovered in colon cancer. Nonetheless, the impact of miR-204-3p on colon cancer remains indistinct. The research attempted to uncover the impacts of miR-204-3p on colon cancer cells growth, migration, and invasion. miR-204-3p expression level in colon cancer tissues and diverse colon cancer cell lines were testified by the quantitative real-time polymerase chain reaction. Exploration of the impacts of miR-204-3p on cell growth, migration, invasion, and their associated factors through assessment of CCK-8, flow cytometry, Transwell, and western blot, respectively. High mobility group AT-hook 2 (HMGA2) expression was then detected in Caco-2 cells after miR-204-3p mimic and inhibitor transfection, additionally dual-luciferase activity was implemented to further uncover the correlation between HMGA2 and miR-204-3p. The impact of HMGA2 on Caco-2 cell growth, migration, and invasion was finally assessed. We found that repression of miR-204-3p was discovered in colon cancer tissues and HCT116, SW480, Caco-2, HT29 and SW620 cell lines. MiR-204-3p overexpression mitigated Coca-2 cell viability, facilitated apoptosis, simultaneously adjusted CyclinD1 and cleaved caspase-3 expression. Cell migration, invasion, and the associated factors were all suppressed by miR-204-3p overexpression. Reduction of HMGA2 was presented in Caco-2 cells with miR-204-3p mimic transfection, and HMGA2 was predicated to be a target gene of miR-204-3p. Besides, HMGA2 silence showed the inhibitory effect on Caco-2 cells growth, migration, and invasion. In conclusion, miR-204-3p repressed colon cancer cell growth, migration, and invasion through targeting HMGA2.     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

Zinc stabilizes adenomatous polyposis coli (APC) protein levels and induces cell cycle arrest in colon cancer cells

In the present study, we investigated the mechanisms by which zinc causes growth arrest in colon cancer cells. The results suggest that zinc treatment stabilizes the levels of the wild-type adenomatous polyposis coli (APC) protein at the post-translational level since the APC mRNA levels and the promoter activity of the APC gene were decreased in HCT-116 cells (which express the wild-type APC gene) after treatment with ZnCl2. Increased levels of wild-type but not truncated APC proteins were required for the ZnCl2-mediated G2/M phase arrest in different colon cancer cell lines. We further tested whether serum-stimulation, which induces cell cycle arrest in the S phase, can relieve ZnCl2-induced G2/M phase arrest of HCT-116 cells. Results showed that in the HCT-116 cells pretreated with ZnCl2, the serum-stimulation neither changed the distribution of G2/M phase arrested cells nor the increased levels of APC protein. The G2/M phase arrest correlated with retarded growth of HCT-116 cells. To further establish that wild-type APC protein plays a role in ZnCl2-induced G2/M arrest, we treated SW480 colon cancer cells that express truncated APC protein. We found that ZnCl2 treatment did not induce G2/M phase arrest in SW480 cells; however, the cell growth was retarded due to the loss of E-cadherin and alpha-tubulin levels. These results suggest that ZnCl2 inhibits the proliferation of colon cancer cells (which carry the wild-type APC gene) through stabilization of the APC protein and cell cycle arrest in the G2/M phase. On the other hand, ZnCl2 inhibits the proliferation of colon cancer cells (which carry the mutant APC gene) by disrupting cellular attachment and microtubule stability.