Effect of CLIC1 gene silencing on proliferation, migration, invasion and apoptosis of human gallbladder cancer cells.

This study aimed to explore the effects of CLIC1 gene silencing on proliferation, migration, invasion and apoptosis of human gallbladder cancer (GBC). GBC and normal gallbladder tissues were extracted for the detection of mRNA and protein expressions of CLIC1. GBC-SD and NOZ cells in the logarithmic growth phase were selected to conduct the experiment. Three different siRNA recombined expression vectors were established using CLIC1 as a target at different sites. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were, respectively, used to detect the CLIC1 mRNA and protein expressions. MTT assay was performed to detect the cell proliferation. Flow cytometry was applied to measure the cell apoptosis and cell cycle distribution. The variations of cell migration and invasion were evaluated using Transwell assay. GBC tissues showed higher CLIC1 mRNA and protein expressions than normal gallbladder tissues. The CLIC1 mRNA and protein expressions in the CLIC1 siRNA group were significantly lower than those in the NC and blank groups. Compared with the NC and blank groups, the CLIC1 siRNA group showed a significant decrease in cell proliferation but an obvious increase in apoptosis rate in GBC cells. Besides, in the CLIC1 siRNA group, cell percentage in G0/G1 and G2/M phase was gradually increased but decreased in S phases. The migration and invasion abilities in GBC cells were significantly lower than those in the NC and blank groups. Our study demonstrates that CLIC1 gene silencing could promote apoptosis and inhibit proliferation migration and invasion of GBC cells.

Erythrocyte membrane-coated gold nanocages for targeted photothermal and chemical cancer therapy.

Recently, red blood cell (RBC) membrane-coated nanoparticles have attracted much attention because of their excellent immune escapability; meanwhile, gold nanocages (AuNs) have been extensively used for cancer therapy due to their photothermal effect and drug delivery capability. The combination of the RBC membrane coating and AuNs may provide an effective approach for targeted cancer therapy. However, few reports have shown the utilization of combining these two technologies. Here, we design erythrocyte membrane-coated gold nanocages for targeted photothermal and chemical cancer therapy. First, anti-EpCam antibodies were used to modify the RBC membranes to target 4T1 cancer cells. Second, the antitumor drug paclitaxel (PTX) was encapsulated into AuNs. Then, the AuNs were coated with the modified RBC membranes. These new nanoparticles were termed EpCam-RPAuNs. We characterized the capability of the EpCam-RPAuNs for selective tumor targeting via exposure to near-infrared irradiation. The experimental results demonstrate that EpCam-RPAuNs can effectively generate hyperthermia and precisely deliver the antitumor drug PTX to targeted cells. We also validated the biocompatibility of the EpCam-RAuNs in vitro. By combining the molecularly modified targeting RBC membrane and AuNs, our approach provides a new way to design biomimetic nanoparticles to enhance the surface functionality of nanoparticles. We believe that EpCam-RPAuNs can be potentially applied for cancer diagnoses and therapies.

Upregulation of DARS2 by HBV promotes hepatocarcinogenesis through the miR-30e-5p/MAPK/NFAT5 pathway.

BACKGROUND: Infection with the hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma (HCC). The osmoregulatory transcription factor nuclear factor of activated T-cells 5 (NFAT5) has been shown to play an important role in the development of many types of human cancers. The role of NFAT5 in HBV-associated HCC has never previously been investigated. METHODS: We compared expression profiles of NFAT5, DARS2 and miR-30e-5p in HCC samples, adjacent nontumor tissues and different hepatoma cell lines by quantitative real-time polymerase chain reaction and /or Western blot. Clinical data of HCC patients for up to 80 months were analyzed. The regulatory mechanisms upstream and convergent downstream pathways of NFAT5 in HBV-associated HCC were investigated by ChIP-seq, MSP, luciferase report assay and bioinformation anaylsis. RESULTS: We first found that higher levels of NFAT5 expression predict a good prognosis, suggesting that NFAT5 is a potential tumor-suppressing gene, and verified that NFAT5 promotes hepatoma cell apoptosis and inhibits cell growth in vitro. Second, our results showed that HBV could suppress NFAT5 expression by inducing hypermethylation of the AP1-binding site in the NFAT5 promoter in hepatoma cells. In addition, HBV also inhibited NFAT5 through miR-30e-5p targeted MAP4K4, and miR-30e-5p in turn inhibited HBV replication. Finally, we demonstrated that NFAT5 suppressed DARS2 by directly binding to its promoter. DARS2 was identified as an HCC oncogene that promotes HCC cell cycle progression and inhibits HCC cell apoptosis. CONCLUSION: HBV suppresses NFAT5 through the miR-30e-5p/mitogen-activated protein kinase (MAPK) signaling pathway upstream of NFAT5 and inhibits the NFAT5 to enhance HCC tumorigenesis via the downstream target genes of DARS2.

Long non-coding RNA PTENP1 interacts with miR-193a-3p to suppress cell migration and invasion through the PTEN pathway in hepatocellular carcinoma.

Long non-coding RNA PTENP1, the pseudogene of PTEN tumor suppressor, was previously reported to be a tumour suppressor in some cancer types. However, the precise effects mediated by PTENP1 transcripts within intricate regulatory networks involving molecular interactions with PTEN and tumorigenicity in hepatocellular carcinoma (HCC) remains elusive. Here, we identify the critical biological functions of PTENP1 and discuss whether PTENP1 could directly interact with miR-193a-3p to affect the progression of HCC both in vitro and in vivo. We demonstrated that PTENP1 level in the HCC tissues was significantly lower compared with those in the adjacent normal tissues. And PTENP1 was able to repress cell invasion, metastasis, and proliferation capacity in HCC cell lines. The overexpression of PTENP1 inhibited HCC growth both in vitro and in vivo. There were a binding sequence and direct interaction between PTENP1 and miR-193a-3p. PTENP1 as an endogenous sponge interacted with miR-193a-3p, leading to regulate the downstream PTEN/Akt pathway. These results suggested that PTENP1 with its suppression effect might serve as novel biomarkers and potent therapeutic strategies in HCC.

Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer.

Cyclin-dependent kinase (CDK) family members have been considered as attractive therapeutic targets for cancer. In this study, we aim to investigate the anticancer effects of a selective CDK7 inhibitor, BS-181, in gastric cancer (GC) cell line. Human GC cells (BGC823) were cultured with or without BS-181 at different concentrations for 24-72 hours. BS-181 significantly reduced the activity of CDK7 with downregulation of cyclin D1 and XIAP in GC cells. Treatment with BS-181 induced cell cycle arrest and apoptosis. The expression of Bax and caspase-3 was significantly increased, while Bcl-2 expression was decreased in cells treated with BS-181. In addition, the inhibition of CDK7 with BS-181 resulted in reduced rates of proliferation, migration, and invasion of gastric cells. Those results demonstrated the anticancer activities of selective CDK7 inhibitor BS-181 in BGC823 cells, suggesting that CDK7 may serve as a novel therapeutic target or the treatment of GC.

The long noncoding RNA, EGFR-AS1, a target of GHR, increases the expression of EGFR in hepatocellular carcinoma.

LncRNA has provided an important new perspective regarding gene regulation. Both the expression and activation of EGFR have been proven to be under the tight control of the GHR pathway. EGFR-AS1 has been found to inhibit the expression of EGFR. GHR-siRNA and EGFR-AS1-siRNA were transfected into HCC cell lines, and a series of WB, q-PCR, and IF experiments was conducted to evaluate whether EGFR-AS1 participated in the regulation of GHR and EGFR. We found that impeded expression of GHR decreased the expression of EGFR and EGFR-AS1 in vivo and in vitro. Then, it was verified that EGFR and EGFR-AS1 were relatively upregulated in HCC tissue, and they were significantly related to some clinical characteristics and patient prognosis. Furthermore, EGFR-AS1 was determined to promote HCC development by improving the ability of invasion and proliferation of HCC cells in vitro, and it was also found to affect the cell cycle. Our study identified that EGFR-AS1 may promote HCC genesis and development. EGFR-AS1 may act as a prognostic factor in HCC. More importantly, we observed that the inhibition of EGFR-AS1 in HCC cells significantly impeded cell proliferation and invasion in vivo, which might provide a potential possibility for targeted therapy of HCC.

Peri-operative use of sorafenib in liver transplantation: a time-to-event meta-analysis.

AIM: To evaluate whether the application of sorafenib during the peri-operative period of liver transplantation improves prognosis in liver cancer patients. METHODS: We searched PubMed, EMBASE and MEDLINE for eligible articles. A total of 4 studies were found that fulfilled the previously agreed-upon standards. We then performed a systematic review and meta-analysis on the enrolled trials that met the inclusion criteria. RESULTS: Out of the 104 studies identified in the database, 82 were not clinical experiments, and 18 did not fit the inclusion standards. Among the remaining 4 articles, only 1 was related to the preoperative use of sorafenib, whereas the other 3 were related to its postoperative use. As the heterogeneity among the 4 studies was high, with an I(2) of 86%, a randomized effect model was applied to pool the data. The application of sorafenib before liver transplantation had a hazard ratio (HR) of 3.29 with a 95% confidence interval (CI) of 0.33-32.56. The use of sorafenib after liver transplantation had an HR of 1.44 (95%CI: 0.27-7.71). The overall pooled HR was 1.68 (95%CI: 0.41-6.91). CONCLUSION: The results showed that the use of sorafenib during the peri-operative period of liver transplantation did not improve patient survival significantly. In fact, sorafenib could even lead to a worse prognosis, as its use may increase the hazard of poor survival.

Triptolide induces apoptosis of gastric cancer cells via inhibiting the overexpression of MDM2.

Triptolide has been reported to exhibit antitumor effects in several cancers. This study investigates the mechanism by which triptolide induces apoptosis of gastric cancer cells. Gastric biopsies were collected for histological evaluation and detection of murine double minute 2 (MDM2) expression. Gastric cancer cells were cultured and treated with different concentrations of triptolide at indicated time points. The expression of MDM2, p53 protein, and target proteins including p21, PUMA, and X-linked inhibitor of apoptosis protein (XIAP) was detected. Apoptosis of cells treated with or without triptolide was evaluated. Our results showed that MDM2 protein was overexpressed in gastric cancer (p < 0.01, resp.). Triptolide induced significant apoptosis of gastric cancer cells in a dose- and time-dependent manner (p < 0.05). In addition, treatment with triptolide strongly inhibited the overexpression of MDM2 in gastric cancer cells, and this MDM2 inhibition led to increased levels of p53 protein and inhibition of XIAP (p < 0.05). However, triptolide failed to increase the expression of p53 target protein p21 and PUMA (p > 0.05). In conclusion, triptolide may induce apoptosis of gastric cancer cells via the inhibition of MDM2 overexpression in a p53-independent manner.

Molecular mechanisms of gender disparity in hepatitis B virus-associated hepatocellular carcinoma.

Chronic hepatitis B virus (HBV) infection is one of the most common causes of hepatocellular carcinoma (HCC), a malignant tumor with high mortality worldwide. One remarkable clinical feature of HBV-related HCC is that its incidence is higher in males and postmenopausal females compared to other females. Increasing evidence indicates that HBV-associated HCC may involve gender disparity and that it may be a type of hormone-responsive malignant tumor. Sex hormones, such as androgen and estrogen, have been shown to play very different roles in the progression of an HBV infection and in the development of HBV-related HCC. Through binding to their specific cellular receptors and affecting the corresponding signaling pathways, sex hormones can regulate the transactivation of HBx, cause the chronic release of inflammatory cytokines in the hepatocellular microenvironment, and participate in epigenetic and genetic alternations in hepatocytes. All of these functions may be related to the initiation and progression of HBV-associated HCC. A thorough investigation of the molecular mechanisms underlying the gender-related disparity in HBV-related HCC should provide a new perspective for better understanding its pathogenesis and exploring more effective methods for the prevention and treatment of this disease.

Suppression of angiogenesis and tumor growth in vitro and in vivo using an anti-angiopoietin-2 single-chain antibody.

Hepatocellular carcinomas (HCCs) are tumors with a highly developed vascular architecture. HCC cells require access to blood vessels for growth and metastasis; therefore, the inhibition of angiogenesis represents a potential therapeutic target for HCC that may reduce the mortality and morbidity from HCC. Various attempts to develop an anti-angiogenic therapy have been made in past decades; however, modest results have been achieved in clinical trials and the challenge of HCC treatment remains. Single-chain antibodies (scFv) are characterized by low molecular weight, low immunogenicity, high penetration and a short half-life, and are easy to produce on a large scale by genetic engineering. Accordingly, an scFv against a specific angiogenic regulator, such as angiopoietin (Ang), may be a promising anti-angiogenic therapy for HCC. Our previous study indicated that an imbalanced expression of angiopoietin-2 (Ang-2) vs. angiopoietin-1 (Ang-1) in HCCs contributes to initiation of neovascularization and promotes the angiogenesis and progression of HCCs. Therefore, we suggest that specific Ang-2-targeting interventions may be valuable in the treatment of HCC via remodeling the neovascular network and changing the tumor microenvironment. In this study, a prokaryotic expression vector of Ang-2 was constructed and purified human Ang-2 protein was isolated. An scFv against human Ang-2 (scFv-Ang2) was identified and purified via phage display technology, and the effects of scFv-Ang2 in vitro and in vivo on HCC in nude mice were evaluated. The results show that scFv-Ang2 inhibits vascular endothelial growth factor (VEGF) and Ang-2 induces the proliferation, migration and tubule formation of human umbilical vein endothelial cells (HUVECs) in vitro. In the in vivo assay, statistical indices, including tumor weight and volume, metastases to lungs, CD31 expression and the microvessel density (MVD) count in the scFv-Ang2-treated group of mice were significantly lower than those in the control group (P<0.05). In conclusion, the successfully generated scFv-Ang2 showed significant inhibitory effects on the angiogenesis and tumor growth of human HCC in vitro and in vivo.

5-Aza-2<-deoxycytidine induces hepatoma cell apoptosis via enhancing methionine adenosyltransferase 1A expression and inducing S-adenosylmethionine production.

In hepatocellular cancer (HCC), lack of response to chemotherapy and radiation treatment can be caused by a loss of epigenetic modifications of cancer cells. Methionine adenosyltransferase 1A is inactivated in HCC and may be stimulated by an epigenetic change involving promoter hypermethylation. Therefore, drugs releasing epigenetic repression have been proposed to reverse this process. We studied the effect of the demethylating reagent 5-aza-2<-deoxycitidine (5-Aza-CdR) on MAT1A gene expression, DNA methylation and S-adenosylmethionine (SAMe) production in the HCC cell line Huh7. We found that MAT1A mRNA and protein expression were activated in Huh7 cells with the treatment of 5-Aza-CdR; the status of promoter hypermethylation was reversed. At the same time, MAT2A mRNA and protein expression was significantly reduced in Huh7 cells treated with 5-Aza-CdR, while SAMe production was significantly induced. However, 5-Aza-CdR showed no effects on MAT2A methylation. Furthermore, 5-Aza-CdR inhibited the growth of Huh7 cells and induced apoptosis and through down-regulation of Bcl-2, up-regulation of Bax and caspase-3. Our observations suggest that 5-Aza- CdR exerts its anti-tumor effects in Huh7 cells through an epigenetic change involving increased expression of the methionine adenosyltransferase 1A gene and induction of S-adenosylmethionine production.

[The efficiency and safety of laparoscopic surgery for colorectal carcinoma: a systematic review].

OBJECTIVE: To evaluate and compare the efficiency and safety of laparoscopic surgery (LS) and open surgery (OS) in the treatment of colorectal carcinoma. METHODS: Randomized controlled trials on laparoscopic surgery and open surgery for colorectal carcinoma from January 2000 to October 2010 were searched in the databases of EMbase, PubMed, Cochrane Library, Sciencedirect, Springer, VIP, CNKI, CBMdisc. The methodological quality was assessed according to the standard of Cochrane systematic review. For homogeneous studies, RevMan5.0 software was used for meta-analysis. RESULTS: A total of 13 RCTs involving 4603 patients were included in this study, and among those 6 were multi-center randomized controlled trials. The meta-analysis showed that: the operation time of the LS group was longer than that of the OS group (WMD = 38.91, 95%CI: 33.89 - 43.93, P < 0.001), the blood loss (WMD = -138.14, 95%CI: -195.79 - -80.50, P < 0.001) and the length of hospital stay (WMD = 2.91, 95%CI: -4.65 - -1.17, P = 0.001) of the LS group was less than those in OS group. There was no significant differences between the two groups in the number of dissected lymph nodes (WMD = -0.62, 95%CI: -1.47 - 0.23, P = 0.150). There was no significant differences between the two groups in terms of the postoperative complications (30 days) (RR = 0.78, 95%CI: 0.59 - 1.01, P = 0.06). There was no significant differences between the two groups in 3-year overall survival (RR = 1.00, 95%CI: 0.96 - 1.04, P = 0.970). There was no significant differences between the two groups in 5-year overall survival (RR = 1.03, 95%CI: 0.99 - 1.08, P = 0.140). There was no significant differences between the two groups in 5-year overall recurrence (RR = 0.89, 95%CI: 0.74 - 1.07, P = 0.200). CONCLUSIONS: Laparoscopic surgery for colorectal carcinoma is a safe and effective therapy as open surgery in the short term or long term outcomes. It could be an acceptable alternative to open surgery for colorectal carcinoma.

Inhibition of hepatocelluar carcinoma MAT2A and MAT2beta gene expressions by single and dual small interfering RNA.

RNA interference (RNAi) has been successfully applied in suppression of hepatic cancer genes. In hepatocelluar carcinoma cell, one methionine adenosyltransferase (MAT) isozyme, MATII was found to have two catalytic subunits which were encoded by MAT2A and MAT2beta respectively. During tumorigeness of hepatocelluar carcinoma, expressions of the two genes were discovered to be increased combining with a switch of MAT (form MATI to MATII), To figure out the role played by MATII in hepatic cancer, In this study, for the first time we established a dual small interfering RNA (siRNA) expression system, which could simultaneously express two different siRNA molecules specifically targeting two genes. To test the effectiveness of this system, we applied this approach to express simultaneously two different siRNA duplexes that specifically target MAT2A and MAT2beta genes of hepatocelluar carcinoma respectively in HepG2 cell. Results indicated that dual siRNA could simultaneously inhibit the expression of MAT2A and MAT2beta gene by 89.5% and 97.8% respectively, In addition, dual siRNA molecules were able to significantly suppress growth of hepatocelluar carcinoma cell in vitro as well as induce apoptosis which was involved in arrest cell cycle at the G1/S checkpoint and the expressions of p21, p27 and Bax.

Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma.

AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2beta gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-x(L) and bcl-x(S) were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved in down-regulating bcl-x(L) and up- regulating bcl-x(S). CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

[RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells].

OBJECTIVES: To investigate the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells. METHODS: The four siRNA against MAT 2A gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilence-2.1-U6. We inserted the target sequence of MAT 2A gene into the upstream of the reporter gene in order to construct the recombinant plasmid vector plucA-MAT 2A. The recombinant plasmid and siRNA-producing plasmid were co-transfected into 293 T cells using this construct via lipofectamine methods. The inhibition effect was detected by measuring luciferase activity in the cell lysate to screen the effective siRNA, and then, the effective siRNA was transfected into Bel-7402 cells. The effect of siRNA treatment on the MAT 2A mRNA level and the MAT activity of hepatoma cells were measured. In order to study the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells, the tumor cell killing rate was analyzed by MTT method and the rate of apoptosis of hepatoma cells was evaluated by flow cytometry. RESULTS: The two siRNA among the four siRNA displayed inhibitory effect on the lucifermase expression with the inhibitory rates of 81% and 89% respectively. The expression of MAT 2A mRNA in Bel-7402 cells was specifically inhibited and the MAT activity in Bel-7402 cells was decreased. Furthermore, silencing of the MAT 2A gene by RNAi significantly inhibited hepatoma cell growth and led to induction of apoptosis. CONCLUSION: RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells; MAT 2A is an ideal target of gene-specific therapy for liver cancer.