Guanhong Luo

MiR-150 promotes gastric cancer proliferation by negatively regulating the pro-apoptotic gene EGR2

Accumulating evidence suggests small non-coding RNAs (microRNAs) play important roles in human cancer progression. In the present study, we found miR-150 was overexpressed in gastric cancer cell lines and tissues. Ectopic expression of miR-150 promoted tumorigenesis and proliferation of gastric cancer cells. Luciferase reporter assay demonstrated that EGR2 was a direct target of miR-150. Collectively, our study demonstrated that overexpression of miR-150 in gastric cancer could promote proliferation and growth of cancer cells at least partially through directly targeting the tumor-suppressor EGR2, suggesting a potential strategy for the development of miRNA-based treatment of gastric cancer.

Inhibition of PI3K/Akt partially leads to the inhibition of PrP(C)-induced drug resistance in gastric cancer cells.

Cellular prion protein (PrPC), a glycosyl‐phosphatidylinositol‐anchored membrane protein with unclear physiological function, was previous found to be upregulated in adriamycin (ADR)‐resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Overexpression of PrPC in gastric cancer has certain effects on drug accumulation through upregulation of P‐glycoprotein (P‐gp), which is suggested to play an important role in determining the sensitivity of tumor cells to chemotherapy and is linked to activation of the phosphatidylinositol‐3‐kinase/Akt (PI3K/Akt) pathway. In the present study, we further investigate the role of the PI3K/Akt pathway in PrPC‐induced multidrug‐resistance (MDR) in gastric cancer. Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrPC and phosphorylated Akt (p‐Akt) expression in gastric cancer. Using established stable PrPC transfectant cell lines, we demonstrated that the level of p‐Akt was increased in PrPC‐transfected cells. Inhibition of PrPC expression by RNA interference resulted in decreased p‐Akt expression. Inhibition of the PI3K/Akt pathway by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P‐gp induced by PrPC. Taken together, our results suggest that PrPC‐induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrPC‐induced drug resistance and P‐gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrPC regulates gastric cancer cell survival.

miR‐17‐5p promotes proliferation by targeting SOCS6 in gastric cancer cells

This study aimed to test the exact functions and potential mechanisms of miR‐17‐5p in gastric cancer. Using real‐time PCR, miR‐17‐5p was found to be expressed more highly in gastric cancer compared with‐normal tissues. Gain‐ and loss‐of‐function assays demonstrated that miR‐17‐5p increased the proliferation and growth of gastric cancer cells in vitro and in vivo. Through reporter gene and western blot assays, SOCS6 was shown to be a direct target of miR‐17‐5p, and proliferative assays confirmed that SOCS6 exerted opposing function to that of miR‐17‐5p in gastric cancer. In short, miR‐17‐5p might function as a pro‐proliferative factor by repressing SOCS6 in gastric cancer.

RhoE enhances multidrug resistance of gastric cancer cells by suppressing Bax.

We have previously reported that RhoE is overexpressed in the SGC7901/VCR cell line. However, the potential role of RhoE in the development of multidrug resistance of gastric cancer is unknown. In the present study, RhoE enhanced the resistance of SGC7901 cells to several kinds of antitumor drugs. RhoE overexpression did not alter the intracellular adriamycin accumulation of SGC7901 cells nor the expression of P-gp and MRP-1, but protected SGC7901 cells from vincristine-induced apoptosis. RhoE was found to downregulate the expression of Bax at a posttranscriptional level. Western blot revealed no effects of RhoE on the activities of the Caspase family of proteins. In brief, our study demonstrated that RhoE may promote the multidrug resistance phenotype of gastric cancer cells by decreasing the expression of Bax at posttranscriptional level, thus inhibiting vincristine-induced apoptosis.

Proteasome inhibitor MG132 reverses multidrug resistance of gastric cancer through enhancing apoptosis and inhibiting P-gp.

ABSTRACT Multidrug resistance (MDR) is a major impediment to the effective chemotherapy of many human malignancies. Although much effort has been devoted to develop new drugs for overcoming MDR, until now, still no useful method of reversing MDR, suitable for clinical use, has emerged from this large quantity of work. Some researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells. In the present study, we found that, in vincristine-resistant human gastric cancer cell line SGC7901/VCR, proteasome inhibitor MG132 was an effective inducer of apoptosis, and also had the capacity of downregulating the expression of anti-apoptotic Bcl-2 and MDR1 (P-gp), by which MG132 resensitized tumor cells to the apoptosis induced by anticancer drugs. Data presented by drug sensitivity assay further demonstrated that MG132 could reverse the resistant phenotype of gastric cancer cells effectively through both enhancing drug-induced apoptosis and inhibiting P-gp. The further study of the effectiveness and safety of proteasome inhibitor in vivo may be helpful for developing a new possible strategy to treat gastric cancer MDR.

The miR27b-CCNG1-P53-miR-508-5p axis regulates multidrug resistance of gastric cancer.

Multidrug resistance (MDR) correlates with treatment failure and poor prognosis among gastric cancer (GC) patients. In a previous study using high-throughput functional screening, we identified 11 microRNAs (miRNAs) that regulate MDR in GC and found that miR-508-5p reversed MDR by targeting ABCB1 and ZNRD1. However, the mechanism by which miR-508-5p was decreased in chemo-resistant GC cells was unclear. In this study, we found that ectopic miR-27b is sufficient to sensitize tumors to chemotherapy in vitro and in vivo. Moreover, miR-27b directly targets the 3′ untranslated regions (3′-UTRs) of CCNG1, a well-known negative regulator of P53 stability. Interestingly, miR-27b up-regulation leads to increased miR-508-5p expression, and this phenomenon is mediated by CCNG1 and P53. Further investigation indicated that miR-508-5p is directly regulated by P53. Thus, the miR-27b/CCNG1/P53/miR-508-5p axis plays important roles in GC-associated MDR. In addition, miR-27b and miR-508-5p expression was detected in GC tissues with different chemo-sensitivities, and we found that tissues in which miR-27b and miR-508-5p are up-regulated are more sensitive to chemotherapy. Together, these data suggest that the combination of miR-27b and miR-508-5p represents a potential marker of MDR. Restoring the miR-27b and miR-508-5p levels might contribute to MDR reversion in future clinical practice.

Hypoxia induced overexpression of PrP(C) in gastric cancer cell lines.

Cellular prion protein (PrPC), a copper-binding glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that is expressed predominantly in neurons can be induced in ischemia/hypoxic brain tissues. It was also found to be overexpressed and conferred multidrug resistance, promoting cancer metastasis and inhibiting apoptosis in gastric cancer in our lab. In solid tumors, hypoxia can promote malignant progression and confer resistance to chemotherapy by altering gene expression. In present study, we investigated the molecular mechanisms and signaling pathway involved in the induction of the PrPC gene by hypoxia in cancer cell lines. PrPC was detected to be upregulated in several cancer cell lines at both mRNA and protein level, and then found to be induced by hypoxia in a time-dependent manner. After hypoxia treatment, gastric cancer MKN28 cells transfected with luciferase reporter constructs of the human PrPC promoter, which contained HSE, expressed higher luciferase activities (4.3-fold) than those cells transfected with the constructs containing no HSE. In addition, the upregulation of PrPC was reduced by MERK/ERK inhibitor (PD98059). siRNA knockdown of PrPC could make the cells more sensitive to hypoxia induced drug sensitivity. In conclusion, from these findings, we can propose that some transcriptional factors phosphorylated by ERK1/2, could in turn interact with HSE in the promoter of PrPC resulting in upregulation of PrPC in gastric cancer cell line MKN28 during hypoxia. Downregulation of PrPC makes gastric cancer cells more sensitive to hypoxia induced drug sensitivity. However, other mechanisms might also be responsible for hypoxia induced overexpression of PrPC in gastric cancer.

Inhibition of the p53–MDM2 interaction by adenovirus delivery of ribosomal protein L23 stabilizes p53 and induces cell cycle arrest and apoptosis in gastric cancer

Inhibiting MDM2 activity in tumors that express wild‐type (wt) p53 but have high levels of MDM2 protein has been considered an attractive anticancer strategy for many years. Previous studies revealed that human ribosomal protein L23 (RPL23) inhibited MDM2‐mediated p53 degradation and thus induced p53 levels as well as its activity, suggesting that it might be a candidate for use as a gene therapy for cancer. In the present study, we evaluated whether targeting this pathway could be of therapeutic value against human gastric carcinoma.

Effect of a novel inhibitory mAb antibody against β-subunit of F1F0 ATPase on HCC

Hepatocellular carcinoma (HCC) represents a worldwide health problem. F1F0 ATPase, one of the most unique supermolecule enzymes in the inner mitochondrial membrane, was recently found located also on the plasma membrane of some tumor and epithelial cells. Ecto-F1F0 ATPase might play the major role in maintaining the normal average intracellular pH in those cells relative to tumor acidic extracellular microenviroment. Inhibiting the extracellular F1F0 ATPase on tumor exhibits both antiangiogenic and antitumorigenic activities. We found previously a strain of murine mAb, mAb6F2C4, which binds with β-catalytic subunit of F1F0 ATPase. Immunofluorescence and flow cytometry assay showed that mAb6F2C4 could bind with plasma membrane of diverse hepatoma cells and HUVEC. Moreover, it could markedly block extracellular ATP generation of SMMC-7721 cells under extracellular acidic condition. In vitro, mAb6F2C4 retarded not only the proliferation and colony forming ability of SMMC-7721 cells, but also the proliferation and tube formation ability of HUVEC. mAb6F2C4 was located on plasma membrane of some hepatoma cells and attenuated dramaticly tumor growth in tumor xenograft models in nude mice. Therefore, we concluded that mAb6F2C4 binding with ecto-β-subunit of F1F0 ATPase, could inhibit extracellular ATP synthesis and exhibit both antiangiogenic and antitumorigenic activities, which could be further developed for HCC therapy.

Adenoviral-mediated RNA interference targeting URG11 inhibits growth of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common malignancy in Asia, with a 5‐year survival rate of less than 5% due to high recurrence after surgery and resistance to chemotherapy. A variety of therapeutic interventions to treat HCC, particularly gene therapy, have recently been investigated in tumor model systems to provide a more complete understanding of hepatocarcinogenesis and effectively design therapeutic strategies to treat this disease. In our study, we constructed an adenoviral vector expressing small interfering RNA (siRNA) targeting a newly discovered gene named upregulated gene 11 (URG11). We introduced this vector into HCC cells to investigate the role of URG11 in HCC carcinogenesis. We observed that upon URG11 knockdown, HCC cell proliferation was inhibited through downregulation of several G1‐S phase related molecules including cyclin D1 and apoptosis was induced as a result of Bcl‐2 downregulation. Besides decreased expression of cyclin D1, CDK4, pRb and Bcl‐2, URG11 also suppressed several other proteins including CAPN9, which was identified by cDNA microarray and 2D gel electrophoresis. Moreover, Ad‐URG11‐siRNA significantly suppressed HCC tumor growth in nude mice. In conclusion, Ad‐URG11‐siRNA can significantly suppress HCC tumor growth in vitro and in vivo by silencing the URG11 gene, and the use of this vector for gene therapy may represent a novel strategy to treat human HCC.