Fuqing Zeng

Downregulation of XIAP expression induces apoptosis and enhances chemotherapeutic sensitivity in human gastric cancer cells.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) gene family in terms of its ability to inhibit caspases and suppress apoptosis. Recent evidence has suggested that XIAP is a key determinant in chemoresistance of cancer cells. To explore a novel approach for ameliorating chemotherapy of gastric cancer, the antisense expression vector for the XIAP gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). This transfer resulted in significant downregulation of XIAP expression, decreased in vitro cell viabilities, and induced apoptosis. In transferred cells, inactive caspase-3 precursors were cleaved into the active subunits (p20 and p17) during apoptosis induced by downregulation of XIAP. The inhibitory effects of cisplatin and mitomycin C on the growth of XIAP downregulated cancer cells were significantly enhanced. In addition, this process occurred only in wild-type p53 (MKN-45), but not in mutant-type p53 (MKN-28) gastric cancer cells. The data presented suggest that downregulation of XIAP via antisense RNA can lead to apoptosis of gastric cancer cells in vitro, correlating with cellular p53 status and activation of caspase-3. This finding could lead to a potential strategy for improving the efficiency of therapies for gastric cancer.

Long Non-Coding RNA MEG3 Inhibits Cell Proliferation and Induces Apoptosis in Prostate Cancer

Background/Aims: Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as cell growth, apoptosis and migration. Although downregulation of lncRNA maternally expressed gene 3 (MEG3) has been identified in several cancers, little is known about its role in prostate cancer progression. The aim of this study was to detect MEG3 expression in clinical prostate cancer tissues, investigate its biological functions in the development of prostate cancer and the underlying mechanism. Methods: MEG3 expression levels were detected by qRT-PCR in both tumor tissues and adjacent non-tumor tissues from 21 prostate cancer patients. The effects of MEG3 on PC3 and DU145 cells were assessed by MTT assay, colony formation assay, western blot and flow cytometry. Transfected PC3 cells were transplanted into nude mice, and the tumor growth curves were determined. Results: MEG3 decreased significantly in prostate cancer tissues relative to adjacent normal tissues. MEG3 inhibited intrinsic cell survival pathway in vitro and in vivo by reducing the protein expression of Bcl-2, enhancing Bax and activating caspase 3. We further demonstrated that MEG3 inhibited the expression of cell cycle regulatory protein Cyclin D1 and induced cell cycle arrest in G0/G1 phase. Conclusions: Our study presents an important role of MEG3 in the molecular etiology of prostate cancer and implicates the potential application of MEG3 in prostate cancer therapy.

CircHIPK3 sponges miR-558 to suppress heparanase expression in bladder cancer cells.

Increasing evidences suggest that circular RNAs (circRNAs) exert crucial functions in regulating gene expression. In this study, we perform RNA‐seq and identify 6,154 distinct circRNAs from human bladder cancer and normal bladder tissues. We find that hundreds of circRNAs are significantly dysregulated in human bladder cancer tissues. We further show that circHIPK3, also named bladder cancer‐related circular RNA‐2 (BCRC‐2), is significantly down‐regulated in bladder cancer tissues and cell lines, and negatively correlates with bladder cancer grade, invasion as well as lymph node metastasis, respectively. Over‐expression of circHIPK3 effectively inhibits migration, invasion, and angiogenesis of bladder cancer cells in vitro and suppresses bladder cancer growth and metastasis in vivo. Mechanistic studies reveal that circHIPK3 contains two critical binding sites for the microRNA miR‐558 and can abundantly sponge miR‐558 to suppress the expression of heparanase (HPSE). Taken together, our findings provide evidence that circRNAs act as “microRNA sponges”, and suggest a new therapeutic target for the treatment of bladder cancer.

Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells

The ubiquitin specific peptidase 22 (USP22) is a positive regulator of the growth of tumors. However, little is known about the impact of USP22 knockdown on the growth of human bladder cells. In the present study, we designed a series of asymmetric interfering RNAs (aiRNAs) and compared the efficacy of aiRNA and conventional symmetric interfering RNA (siRNA) in the silencing of USP22 expression and the growth of human bladder EJ cells in vitro and in vivo. In comparison with transfection with the USP22-specific siRNA, transfection with 15/21 aiRNA was more potent in down-regulating the USP22 expression and inhibiting EJ cell proliferation in vitro. Furthermore, transfection with 15/21 aiRNA induced higher frequency of EJ cells arrested at the G0/G1 phases, but did not trigger EJ cell apoptosis. Moreover, transfection with either the siRNA or 15/21 aiRNA up-regulated the expression of p53 and p21, but down-regulated the expression of cyclin E and Mdm2 in EJ cells. The up-regulated p53 expression induced by the specific siRNA or aiRNA was abrogated by induction of Mdm2 over-expression. In addition, treatment with the specific siRNA or aiRNA inhibited the growth of implanted human bladder tumors in mice and the aiRNA had more potent anti-tumor activity in vivo. Therefore, our data suggest that knockdown of USP22 expression by the aiRNA may down-regulate the expression of Mdm2 and cyclin E, resulting in the up-regulated expression of p53 and p21 and leading to cell cycling arrest and inhibition of human bladder EJ cell proliferation. Our findings indicate that the USP22-specific aiRNA may be a novel approach for the intervention of human bladder tumors.

Small RNAs Targeting Transcription Start Site Induce Heparanase Silencing through Interference with Transcription Initiation in Human Cancer Cells

Heparanase (HPA), an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in human cells. In this study, transfection of siRNA against −9/+10 bp (siH3), but not −174/−155 bp (siH1) or −134/−115 bp (siH2) region relative to transcription start site (TSS) locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3) or active chromatin marker acetylated histone H3 (AcH3). The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB), but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (−9/+10 bp) into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells.

Gambogic acid inhibits TNF-α-induced invasion of human prostate cancer PC3 cells in vitro through PI3K/Akt and NF-κB signaling pathways

Aim:To investigate the mechanisms underlying the inhibitory effect of gambogic acid (GA) on TNF-α-induced metastasis of human prostate cancer PC3 cells in vitro.Methods:TNF-α-mediated migration and invasion of PC3 cells was examined using migration and invasion assays, respectively. NF-κB transcriptional activity and nuclear translocation were analyzed with luciferase reporter gene assays, immunofluorescence assays and Western blots. The ability of p65 to bind the promoter of Snail, an important mesenchymal molecular marker, was detected using a chromatin immunoprecipitation (ChIP) assay. After treatment with Snail-specific siRNA, the expression of invasiveness-associated genes was measured using quantitative real-time PCR and Western blot.Results:GA significantly inhibited the viability of PC3 cells at 1–5 μmol/L, but did not produce cytotoxic effect at the concentrations below 0.5 μmol/L. GA (0.125–0.5 μmol/L) dose-dependently inhibited the migration and invasion of PC3 cells induced by TNF-α (10 ng/mL). Moreover, the TNF-α-mediated activation of phosphatidylinositol-3-OH kinase/protein kinase B (PI3K/Akt) and NF-κB pathways was suppressed by GA (0.5 μmol/L). Furthermore, this anti-invasion effect of GA was associated with regulation of Snail. Snail expression was significantly down-regulated by treatment with GA (0.5 μmol/L) in the TNF-α-stimulated PC3 cells.Conclusion:GA inhibits TNF-α-induced invasion of PC3 cells via inactivation of the PI3K/Akt and NF-κB signaling pathways, which may offer a novel approach for the treatment of human prostate cancer.

Tumor cell-specific blockade of CXCR4/SDF-1 interactions in prostate cancer cells by hTERT promoter induced CXCR4 knockdown: A possible metastasis preventing and minimizing approach.

tromal cell-derived factor-1 (SDF-1)/CXCR4 pathway has been shown to play an important role in prostate cancer (PCa) metastasis and siRNA expression using cell-specific promoters has been demonstrated to be a potential tool for targeted gene therapy. Here, we illustrate that human telomerase reverse transcriptase (hTERT) promoter-induced CXCR4 knockdown inhibits PCa bone metastasis. We first investigated CXCR4 expressions and interactions of CXCR4/SDF-1 in PCa cells, developed a retrovirus system that could stably express CXCR4 small hairpin RNA driven by hTERT promoter and then determined the inhibitory effects of cell-specific blockade of CXCR4/SDF-1 pathway on PCa metastasis. It was shown that both PCa tissues and cell lines expressed CXCR4 and the expression in PCa tissue had a positive correlation to clinical stages while not to Gleason scores or serum PSA level. PCa metastases most presenting human tissues expressed high levels of SDF-1. Exogenous SDF-1 enhanced in vitro adhesion, migration and invasion of PCa cells and these bioeffects were repressed by hTERT promoter-induced CXCR4- shRNA expression. This CXCR4 knockdown was also found to significantly inhibit bone metastasis in vivo. We conclude that CXCR4/SDF-1 pathway plays an important role in PCa bone metastasis. hTERT promoter-induced tumor cell-specific CXCR4 gene silencing may prevent in vitro invasiveness and in vivo bone metastasis of PCa. These findings may enable new avenues of prevention and treatment for PCa metastasis.

Methyl jasmonate sensitizes human bladder cancer cells to gambogic acid-induced apoptosis through down-regulation of EZH2 expression by miR-101.

Gambogic acid (GA) and methyl jasmonate (MJ) are increasingly being recognized as novel natural anticancer compounds. Here, we investigated the antitumour effects of GA in combination with MJ on human bladder cancer cells.

Apoptosis-inducing effects of curcumin derivatives in human bladder cancer cells

Our aim was to prepare curcumin derivatives and study their apoptosis-inducing effects on bladder cancer cells in order to establish a basis for targeted chemotherapy of cancer. n-Maleoyl-L-valine-curcumin (NVC) and n-maleoyl-glycine-curcumin (NGC) were chemically synthesized. Intracellular esterase activity of the human bladder cancer EJ cell line and renal tubular epithelial (HKC) cells was examined by 6-carboxyfluorescein diacetate fluorometry. After incubation with NVC or NGC for 6–24 h, cell viability was detected by MTT colorimetry. Cell apoptosis and apoptotic rates were measured by acridine orange/ethidium bromide staining, TUNEL labeling and flow cytometry. Intracellular caspase-3 activities were determined by spectrophotometry. The esterase activity within EJ cells was 10.2-fold higher than that of HKC cells, which was abolished by bis-p-nitrophenylphosphate, an esterase inhibitor, resulting in decreases in NVC- and NGC-mediated cell viability arrest. For EJ cells, the IC50 values of NVC (20.1 μmol/l) and NGC (18.7 μmol/l) were close to curcumin (16.5 μmol/l). Meanwhile, their IC50 values on HKC cells were, respectively, 4.06- and 3.23-fold higher than curcumin. Moreover, NVC and NGC induced apoptosis of EJ cells by 10.13–23.36 and 12.42–28.56%, respectively. Administration of these two derivatives resulted in decreased apoptosis of HKC cells compared with curcumin. The caspase-3 activities of EJ cells, but not of HKC cells, were 5.21- and 5.63-fold enhanced by NVC and NGC, respectively. Thus, novel esterase-sensitive curcumin derivatives were synthesized, which induced extensive apoptosis of bladder cancer EJ cells, but not normal cells.

BRD4 Regulates EZH2 Transcription through Upregulation of C-MYC and Represents a Novel Therapeutic Target in Bladder Cancer

People who develop bladder cancer frequently succumb to the intractable disease. Current treatment strategies are limited presumably due to the underlying molecular complexity and insufficient comprehension. Therefore, exploration of new therapeutic targets in bladder cancer remains necessary. Here, we identify that bromodomain-4 protein (BRD4), an important epigenome reader of bromodomain and extraterminal domain (BET) family member, is a key upstream regulator of enhancer of zeste homologue 2 (EZH2), and represents a novel therapeutic target in bladder cancer. We found that BRD4 was significantly overexpressed in bladder cancer cells and tissues. Inhibition of BRD4 decreased bladder cancer cell proliferation concomitantly with the accumulation of cell apoptosis in vitro and suppressed tumor growth in vivo. We further found that suppression of BRD4 decreased the mRNA and protein levels of EZH2, which was reversed by ectopic expression of C-MYC. In particular, individual silencing of BRD4 using shRNA or the BET inhibitor JQ1 strikingly diminished the recruitment of C-MYC to EZH2 promoter in bladder cancer. Briefly, our research reveals that BRD4 positively regulates EZH2 transcription through upregulation of C-MYC, and is a novel promising target for pharmacologic treatment in transcriptional program intervention against this intractable disease. Mol Cancer Ther; 15(5); 1029–42. ©2016 AACR.