Low Expression of CyclinH and Cyclin-Dependent Kinase 7 Can Decrease the Proliferation of Human Esophageal Squamous Cell Carcinoma

Background

Increased expression of cyclinH (CCNH) and cyclin-dependent kinase 7 (CDK7) has a relationship with poor prognosis in most human cancers.

Aim

Investigate the expression of CCNH and CDK7 in human esophageal squamous cell carcinoma (ESCC) and the effect of chemotherapy on their expression.

Methods

Western blotting and immunohistochemistry were used to measure the expression of CCNH and CDK7 proteins in ESCC and adjacent normal tissue in 98 patients. We use Cell Counting Kit-8 and cell flow to analyze the effects of cisplatin and interference of CCNH and CDK7 in cell cycle process.

Results

Immunohistochemical analysis showed that CCNH and CDK7 expression were significantly associated with unfavorable clinicopathologic variables. CCNH and CDK7 protein levels were elevated in ESCC tissues in comparison with adjacent normal tissues. Survival analysis revealed that CCNH and CDK7 overexpression were significantly associated with overall survival (P < 0.001). Cisplatin or interference of CCNH or CDK7 led cells to grow slowly. Overexpression of CCNH and CDK7 in TE1 cells can lead to resistance to cisplatin.

Conclusions

We can conclude that CCNH and CDK7 may play an important role in the tumorigenesis and development of ESCC. CCNH and CDK7 expression affected the chemotherapy of tumor.     

Higher expression of SIRT1 induced resistance of esophageal squamous cell carcinoma cells to cisplatin

Background

High expression of Sirtuin type 1 (SIRT1) exists in some cancer cells. However, it is still unclear whether SIRT1 affects the sensitivity of esophageal cancer cells to cisplatin. This study was designed to explore the relationship between SIRT1 expression and resistance of esophageal squamous cell carcinoma (ESCC) cells to cisplatin and reveal the underlying mechanism.

Methods

The tissue samples of 68 ESCC patients were collected from Nanjing Drum Tower Hospital, China. All the patients had undergone cisplatin based combination chemotherapy. The expression of SIRT1and Noxa in tissue samples were analyzed by quantitative real-time reverse PCR (qRT-PCR) and Western blot. Human ESCC cell line (ECa9706 cells) was cultured and a cisplatin-resistant subline (ECa9706-CisR cells) was established by continuous exposure to cisplatin at different concentrations. The expression of SIRT1 and Noxa in both cell lines was analyzed by qRT-PCR and Western blot. siRNA technology was utilized to down-regulate the SIRT1 expression in ECa9706-CisR cells. The influence of SIRT1 silence on sensitivity of ECa9706-CisR cells to cisplatin was confirmed using CCK-8 assay and flow cytometry. Furthermore, the level change of Noxa after SIRT1 silence in ECa9706-CisR cells was determined by qRT-PCR and Western blot.

Result

SIRT1 and Noxa expression in chemo-resistant patients was significantly increased and decreased respectively, compared with chemo-sensitive patients. SIRT1 expression in ECa9706-CisR cells was significantly increased with a lower Noxa level, compared with normal ECa9706 cells. Cisplatin 5 µM could cause proliferation inhibition, G2/M phase arrest and apoptosis in ECa9706-CisR cells and these effects could be enhanced dramatically by SIRT1 silencing. Moreover, Noxa expression was increased after treated with SIRT1 siRNA.

Conclusions

Over-expression of SIRT1 may cause resistance of ESCC cells to cisplatin through the mechanism involved with Noxa expression.     

miR-449a Regulates Proliferation and Chemosensitivity to Cisplatin by Targeting Cyclin D1 and BCL2 in SGC7901 Cells

Background

Recently, several miRNAs have been determined as tumor suppressors in various cancers, such as microRNA-449a. However, the exact molecular mechanisms underlying miR-449a regulated cell proliferation and chemosensitivity in gastric cancer cells have not been well documented.

Aim

The present study was designed to test whether miR-449a mediates cell proliferation and chemosensitivity in gastric cancer cells via regulating cyclin D1 and BCL2.

Methods

In vitro, the ability of cell proliferation and cell viability were measured by MTT assay; cell cycle and cell apoptosis was detected by FCM. qRT-PCR was used to measure the expression of miR-449a. Western blot and real-time PCR assays were used to detect the expression of cyclin D1 and BCL2 in gastric cancer cell line SGC7901.

Results

miR-449a expression was downregulated in gastric cancer cell line SGC7901 and human gastric cancer tissues, compared to the gastric epithelial cell line GES-1 and matched non-tumor associated tissues. Upregulation of miR-449a reduced the proliferation of SGC7901 cells. Ectopic expression of miR-449a decreased the percentage of S phase cells, increased the percentage of G1/G0 phase cells and increased the apoptosis induced by cisplatin. Moreover, miR-449a inhibited SGC7901 cells proliferation and enhanced cisplatin chemosensitivity by downregulating expression of BCL2 and cyclin D1, respectively, via directly targeting the 3′-untranslated regions of BCL2 and cyclin D1 mRNA.

Conclusions

This is the first report to provide evidence that miR-449a could modulate cell cycle and apoptosis through regulating cyclin D1 and BCL2 expression in SGC7901 cells.     

MYO5B Is Epigenetically Silenced and Associated with MET Signaling in Human Gastric Cancer

Background

Our previous study has shown that MYO5B is downregulated in gastric cancer. However, the mechanism by which the expression of MYO5B was inhibited remains unknown.

Methods

Inspection of the human MYO5B locus uncovered a large and dense CpG island within the 5′ region of this gene. Methylation-specific PCR (MSP) and bisulfite sequencing (BSP) were used for determination of MYO5B promoter methylation in gastric cancer cell lines and gastric cancer samples. Involvement of histone H3 methylation in those cell lines were examined by ChIP assay.

Results

The densely methylated MYO5B promoter region was confirmed by MSP and BSP. Enhanced gene expression was detected when the cells were treated with the DNA-demethylating agent 5-aza-2′-deoxycytidine (DAC) and trichostatin A (TSA), a histone deacetylase inhibitor. Knockdown of MYO5B expression in gastric cancer cells expressing endogenous MYO5B inhibits HGF-stimulated MET degradation, concomitant with sustained c-MET levels and signaling.

Conclusion

The results of our study showed for the first time that MYO5B is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification. Inactivation of MYO5B expression in gastric cancer cells expressing endogenous MYO5B inhibits HGF-stimulated MET degradation, concomitant with sustained c-MET levels and signaling.     

Uncovering a key to the process of metastasis in human cancers: a review of critical regulators of anoikis

Purpose

Anoikis (‘homelessness’ in Greek) is a form of apoptosis following the detachment of cells from the appropriate extracellular matrix (Chiarugi and Giannoni in Biochem Pharmacol 76:1352–1364, 2008). Resistance to anoikis is a critical mediator of metastasis in cancer by enabling cancer cells to survive during invasion and transport in the blood and lymph. Numerous regulators and mechanisms of anoikis in human cancer have been proposed to date. Consequently, the identification of key regulators of anoikis that can be targeted to at least partially restore anoikis sensitivity in cancer cells is important in the development of therapies to treat metastatic cancer.

Methods

A literature search focusing on the regulators of anoikis in human cancer was performed on the Medline, Embase and Scopus databases.

Results

Mcl-1, Cav-1, Bcl-x_L, cFLIP, 14-3-3ζ and Bit1 appear to regulate anoikis in human cancer by participating in the intrinsic apoptotic pathway, extrinsic apoptotic pathway or caspase-independent pathways. Mcl-1, Cav-1, Bcl-x_L, cFLIP and 14-3-3ζ are suppressors of anoikis, and their upregulation confers anoikis resistance to cancer cells. Bit1 is a promoter of anoikis and is downregulated to confer anoikis resistance in metastatic cancer.

Conclusion

Anoikis is a complex process involving the crosstalk between different signalling pathways. The dysregulated expression of key regulators of anoikis that participate in these signalling pathways promotes anoikis resistance in human cancer. These regulators of anoikis might therefore be the targets for developing therapies to overcome anoikis resistance in metastatic cancer.     

SULF1 Inhibits Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells by Decreasing Heparin-Binding Growth Factor Signaling

Background

Heparin-binding growth factor signaling is involved in the pathogenesis and development of human cancers. It can be regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPG). SULF1 is a heparin-degrading endosulfatase which can modulate the sulfation of HSPGs.

Aim

The purpose of this study was to elucidate the role of SULF1 in modulating proliferation and invasion of esophageal squamous cell carcinoma (ESCC) by decreasing heparin-binding growth factor signaling.

Methods

We restored SULF1 expression in the ESCC cell line KYSE150, and examined the effects of SULF1 expression on the proliferation and invasion of KYSE150 cells. In addition, we investigated the expression of SULF1 in human ESCC tissues and analyzed the correlation of SULF1 expression with clinicopathologic characteristics of ESCC.

Results

Our study shows that re-expression of SULF1 in ESCC cell line results in the downregulation of hepatocyte growth factor-mediated activation of MAPK pathways with a resultant decrease in cell invasiveness. Cell proliferation was also inhibited in SULF1-transfected KYSE150 cells. Immunohistochemical assays reveal that SULF1 is expressed in nearly half of the human ESCC tissues but not in normal esophageal epithelial cells. SULF1 expression in human ESCC tissues is negatively correlated with tumor size and tumor invasion.

Conclusion

This study identified that SULF1 inhibits proliferation and invasion of ESCC by decreasing heparin-binding growth factor signaling and suggested that SULF1 plays an inhibiting role in the pathogenesis of ESCC.     

GPER functions as a tumor suppressor in triple-negative breast cancer cells

Background

The orphan, membrane-bound estrogen receptor (GPER) is expressed at high levels in a large fraction of breast cancer patients and its expression is favorable for patients’ survival.

Methods

We investigated the role of GPER as a potential tumor suppressor in triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 using cell cycle analysis and apoptosis assay. The constitutive activity of GPER was investigated.

Results

GPER-specific activation with G-1 agonist inhibited breast cancer cell growth in concentration-dependent manner via induction of the cell cycle arrest in G2/M phase, enhanced phosphorylation of histone H3 and caspase-3-mediated apoptosis. Analysis of the methylation status of the GPER promoter in the triple-negative breast cancer cells and in tissues derived from breast cancer patients revealed that GPER amount is regulated by epigenetic mechanisms and GPER expression is inactivated by promoter methylation. Furthermore, GPER expression was induced by stress factors, such as radiation, and GPER amount inversely correlated with the p53 expression level.

Conclusions

Overall, our results establish the protective role in breast cancer tumorigenesis, and the cell surface expression of GPER makes it an excellent potential therapeutic target for triple-negative breast cancer.     

Aberrant methylation of the MSH3 promoter and distal enhancer in esophageal cancer patients exposed to first-hand tobacco smoke

Purpose

Polymorphisms in MSH3 gene confer risk of esophageal cancer when in combination with tobacco smoke exposure. The purpose of this study was to investigate the methylation status of MSH3 gene in esophageal cancer patients in order to further elucidate possible role of MSH3 in esophageal tumorigenesis.

Methods

We applied nested methylation-specific polymerase chain reaction to investigate the methylation status of the MSH3 promoter in tumors and matching adjacent normal-looking tissues of 84 esophageal cancer patients from a high-risk South African population. The Cancer Genome Atlas data were used to examine DNA methylation profiles at 17 CpG sites located in the MSH3 locus.

Results

Overall, promoter methylation was detected in 91.9 % of tumors, which was significantly higher compared to 76.0 % in adjacent normal-looking esophageal tissues (P = 0.008). When samples were grouped according to different demographics (including age, gender and ethnicity) and smoking status of patients, methylation frequencies were found to be significantly higher in tumor tissues of Black subjects (P = 0.024), patients of 55–65 years of age (P = 0.032), males (P = 0.037) and tobacco smokers (P = 0.015). Furthermore, methylation of the MSH3 promoter was significantly more frequent in tumor samples from smokers compared to tumor samples from non-smokers [odds ratio (OR) = 31.9, P = 0.031]. The TCGA data confirmed significantly higher DNA methylation level at the MSH3 promoter region in tumors (P = 0.0024). In addition, we found evidence of an aberrantly methylated putative MSH3-associated distal enhancer element.

Conclusion

Our results suggest that methylation of MSH3 together with exposure to tobacco smoke is involved in esophageal carcinogenesis. Due to the active role of the MSH3 protein in modulating chemosensitivity of cells, methylation of MSH3 should further be examined in association with the outcome of esophageal cancer treatment using anticancer drugs.     

Expression of MUC5AC, an early marker of pancreatobiliary cancer, is regulated by DNA methylation in the distal promoter region in cancer cells

Background and purpose

High de novo expression of MUC5AC (a gastric-type secreted mucin) is observed in many types of pancreatobiliary neoplasms, including precursor lesions. In this study, we show that the DNA methylation pattern is intimately correlated with MUC5AC expression in ten cancer cell lines (breast, lung, pancreas, and colon).

Methods

The CpG methylation status of the MUC5AC promoter from ?3855 to +321 was mapped using MassARRAY analysis, which utilizes base-specific cleavage of nucleic acids. ChIP assays and micro-RNA (miRNA) microarray expression profiling were also carried out in both MUC5AC-positive cells and in those with no or low MUC5AC expression.

Results

In the distal region from ?3718 to ?3670 of the promoter, MUC5AC-negative cancer cells (e.g., MDA-MB-453) were highly methylated, whereas MUC5AC-positive cells (e.g., MCF-7) had low methylation levels. The modification status of histone H3 lysine 9 (H3-K9) was also closely related to MUC5AC expression. Expression levels of miRNAs in the cancer cells were not correlated with MUC5AC expression.

Conclusion

Our results indicate that MUC5AC is regulated by CpG methylation and histone H3-K9 modification of the MUC5AC promoter distal region, but not by miRNAs. An understanding of the epigenetic regulation of MUC5AC may be of importance for the diagnosis of carcinogenic risk in the pancreatobiliary system.     

DNA Methylation of NDRG2 in Gastric Cancer and Its Clinical Significance

Background

Gastric cancer is one of the most common digestive malignancies worldwide. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation-related gene that is considered to be a metastasis suppressor gene. In this study, we examined the expression and DNA methylation of NDRG2 in gastric cancer cell lines and tissues, as well as its clinical significance.

Methods

Six gastric cancer cell lines and 42 paired normal and gastric cancer tissue samples were used to assess NDRG2 mRNA expression using RT-PCR. NDRG2 DNA methylation status was evaluated by methylation-specific PCR (MSP) in gastric cancer cell lines and tissues. The suppression of NDRG2 in BGC823 cells by siRNA transfection was utilized to detect the role of NDRG2 in gastric cancer progression.

Results

NDRG2 mRNA was down-regulated in gastric cancer cell lines and tissues, and its expression was just related to lymph node metastasis (p = 0.032). MSP showed methylation of NDRG2 in 54.0 % (47/87) of primary gastric cancer specimens and in 20.0 % (16/80) of corresponding nonmalignant gastric tissues. NDRG2 methylation was related to depth of tumor invasion, Borrmann classification and TNM stage (p < 0.05). Upon treatment with 5-aza-2′-deoxycytidine and trichostatin A, NDRG2 expression was upregulated in HGC27 cells, and demethylation of the highly metastatic cell line, MKN45, inhibited cell invasion. Furthermore, the suppression of NDRG2 by siRNA transfection enhanced BGC823 cells invasion.

Conclusions

Our results suggest that the aberrant methylation of NDRG2 may be mainly responsible for its downregulation in gastric cancer, and may play an important role in the metastasis of gastric cancer.     

Effect of shRNA targeting survivin on ovarian cancer

Purpose

This study investigated the effect of shRNA targeting survivin on cultured ovarian cancer cells and on a murine ovarian cancer xenograft.

Methods

An RNAi plasmid for survivin was transfected into SKOV3 cells, and the effect of shRNA targeting survivin on the expression of survivin was determined. Transmission electron microscopy (TEM), flow cytometry, and TUNEL staining were used to assess apoptosis. The MTT assay was used to measure cell growth and changes in cisplatin sensitivity. SKOV3 cells were injected into nude mice, and the effect of shRNA targeting the survivin gene on tumor growth was assessed.

Results

SKOV3 cells transfected with an RNAi plasmid against survivin had increased apoptosis and slower growth. At the molecular level, these cells also had lower expression of survivin. Nude mice inoculated with SKOV3 cells developed cancers, and treatment with shRNA targeting survivin markedly inhibited the growth of these cancers with no obvious side effects.

Conclusions

Our studies of SKOV3 cells and ovarian cancer xenografts in nude mice indicate that shRNA targeting survivin has potential for the treatment of ovarian cancer.     

Egr-1 Promotes Cell Proliferation and Invasion by Increasing β-Catenin Expression in Gastric Cancer

Background

Abnormal expression of early growth response gene 1 (Egr-1) and β-catenin may play a crucial role in the development and progression of human cancer. However, little is known about the expression and underlying molecular mechanisms in which Egr-1 and β-catenin are involved in the development and progression of gastric cancer.

Aims

The purpose of this study was to elucidate the potential relationship between Egr-1 and β-catenin expression in gastric cancer, which contributes to finding new molecular carcinogenesis as a potential therapeutic target for gastric cancer.

Methods

In a sample of 102 cases of human gastric cancer, the expression of Egr-1 and β-catenin was detected using immunohistochemistry. Egr-1 gene was transfected into gastric cancer SGC7901 cells and its role in proliferation and cell invasion was detected by MTT assay, flow cytometry, wound-healing and transwell invasion assay. Western blot analysis was used to study the expression of β-catenin and cyclin D1 proteins.

Results

Upregulated Egr-1 and β-catenin protein expression were strongly correlated with cancer progression and depth of invasion in gastric cancer. β-catenin, present mainly in cytoplasmic and nucleus of gastric cancer cells, was also positively correlated with Egr-1 expression in gastric cancer. Furthermore, the overexpression of Egr-1 upregulated β-catenin expression level, promoted cell proliferation, increased cell population in S-phase and enhanced gastric cancer cell migration and invasion in vitro.

Conclusions

Egr-1 might contribute to gastric cancer proliferation and invasion through activation of the β-catenin signaling pathway.     

Epigenetic silencing of WIF-1 in hepatocellular carcinomas

Purpose

To examine the expression profile and promoter methylation status of WIF-1 in hepatocellular carcinoma (HCC) and identify the possible relationship between the WIF-1 expression pattern and promoter methylation status.

Methods

Quantitative real-time PCR was performed to detect mRNA level of WIF-1 in 4 HCC cell lines, 15 paired HCC clinical samples and 3 normal liver tissues. Methylation-specific PCR and bisulfite DNA sequencing were used in methylation analysis. In vitro assays for HCC cells, colony formation and cell proliferation assay were carried out to observe the effect of WIF-1 on cell growth; TOP-flash luciferase analysis was employed to determine its role in the Wnt pathway.

Results

Quantitative real-time PCR analysis showed the extensive low expression of WIF-1 mRNA in HCC, and this down-regulation was generally dependent on the degree of methylation at its promoter region. In vitro assays indicated WIF-1 can inhibit cell growth by blocking Wnt signaling in HCC cells.

Conclusions

WIF-1 silencing as a result of its promoter hypermethylation may be a frequent event in HCC.     

Role of IGFBP4 and IGF-I expression in esophageal squamous cell carcinoma

Background

The purpose of this research is to determine the correlation between IGFBP-4/IGF-I expression and the clinical status of esophageal squamous cell carcinoma (ESCC).

Materials and methods

Immunohistochemical staining for IGFBP-4 and IGF-I was performed in 61 clinical samples. The correlation between IGFBP-4/IGF-I staining and clinicopathological features was examined statistically. In addition, we compared the mRNA expression levels of IGFBP-4 and IGF-I between ESCC cancer tissues and the normal esophageal epithelium using a quantitative real-time RT-PCR.

Results

According to the results of immunohistochemical staining, IGFBP-4 expression in the region of normal epithelial cells showed an increase of 52.5 % compared to expression in the tumor cells. There was no statistically significant correlation between IGFBP-4/IGF-I and clinicopathological features. The correlation between IGF-I and IGFBP-4 was not significant either. RT-PCR assay showed IGF-I expression in ESCC tissue has a tendency to be higher than that of normal esophageal epithelium. On the other hand, IGFBP-4 expression levels in cancer tissue were lower in comparison with normal epithelium. These results were not significant.

Conclusions

There were the differences of IGFBP-4 expression between the region of ESCC and the region of normal epithelium cells. One suggested possibility is that IGF-independent action of IGFBP-4 might have taken part in these results. We need further exploratory experiments to elucidate the details of the actual function of IGFBP-4 expression in ESCC.     

Effect of miR-451 on the Biological Behavior of the Esophageal Carcinoma Cell Line EC9706

Background

MicroRNAs play important roles in coordinating a variety of cellular processes. Abnormal expression of miRNAs has been linked to several cancers. However, the functional role of miR-451 in esophageal squamous cell carcinoma remains unclear.

Aims

The present study explored the effects of miR-451 on the biological behavior of the esophageal carcinoma cell line EC9706.

Methods

Synthetic miR-451 mimics were transfected into EC9706 cells using Lipofectamine? 2000. The expression of miR-451 was analyzed by RT–PCR and the expressions of Bcl-2, AKT and phosphorylated AKT were analyzed by Western blotting. The MTT assay, soft agar colony formation assay, transwell assay and FACS were used to assess the effect of miR-451 on EC9706 cell proliferation, invasion, metastasis and apoptosis. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice.

Results

In comparison to the controls, a significant increase in the expression of miR-451 was associated with significantly decreased expressions of Bcl-2, AKT and p-AKT, and a significant increase in the apoptosis rate. The number of cell clones was significantly decreased by miR-451 expression, which also caused the inhibition of cell proliferation. The average number of cells penetrating the matrigel was significantly lower than the controls. Injection of miR-451 inhibited tumor growth in a xenograft model.

Conclusions

Upregulated expression of miR-451 induced apoptosis and suppressed cell proliferation, invasion and metastasis in the esophageal carcinoma cell line EC9706. In addition, injection of miR-451 inhibited tumor growth in a xenograft model of esophageal cancer.