Xiaohou Wu

shRNA targeting PLCε inhibits bladder cancer cell growth in vitro and in vivo.

OBJECTIVES To investigate the role of phospholipase Cε (PLCε) by silencing PLCε with short hairpin RNA (shRNA) in human bladder cancer cells BIU-87 in vitro and in vivo. METHODS A PLCε shRNA expression vector was transfected into BIU-87 cells, and the expression of PLCε protein was detected by Western blotting. Cell proliferation was determined using the MTT assay, and the cell cycle was detected using flow cytometry. A tumor xenograft experiment was established to evaluate the tumor growth under the condition of PLCε knockdown, and the expression of PLCε, proliferating cell nuclear antigen, and cyclin D1 were detected by Western blotting or immunohistrochemistry. RESULTS PLCε shRNA reduced the protein level of PLCε, leading to marked proliferation inhibition and significant cell cycle arrest. Furthermore, PLCε shRNA reduced the tumor xenograft growth implanted with BIU-87 cells. The protein expression of PLCε, proliferating cell nuclear antigen, and cyclin D1 were downregulated in the bladder tumor xenograft. CONCLUSIONS The knockdown of PLCε by shRNA could inhibit bladder tumor growth and might be an alternative approach for human bladder cancer therapy.

Exosomal Hsp70 mediates immunosuppressive activity of the myeloid-derived suppressor cells via phosphorylation of Stat3

Myeloid-derived suppressor cells (MDSCs), one of the main cell populations, are responsible for regulating the immune response, which accumulates in tumor-bearing mice and humans contributing to cancer development. Exosomes produced by tumor cells have been involved in tumor-associated immune suppression. However, the role of exosomes is unclear in the activation of MDSCs. Here, we have purified tumor-derived exosomes from the supernatants of Renca cell cultures. Transmission electron microscopy was used to confirm their morphology, and Western blot analysis showed that Hsp70 was rich in these isolated exosomes compared with the whole-cell lysates of Renca cells. Then, we demonstrated that there was a more powerful activity of exosomal Hsp70-mediated induction of proinflammation cytokines, tumor growth factors of MDSCs and tumor progression than exosomes pre-incubated with anti-Hsp70 antibody. Furthermore, we show that an interactive exosomal HSP70 and MDSCs determine the suppressive activity of the MDSCs via phosphorylation of Stat3 (p-Stat3). Finally, we show that exosomal Hsp70 triggers p-Stat3 in MDSCs in a TLR2-MyD88-dependent manner. Meanwhile, we also find that there is a more significant increase in the percentage of CD11b+Gr-1+ cells in the mice, which are treated with exosomal Hsp70 than that exosomes pre-incubated with anti-Hsp70 antibody. Hence, we believe that the signaling pathway activation by exosomal Hsp70 within MDSCs may be a significant target in future treatment of renal cell carcinoma.

HepaCAM induces G1 phase arrest and promotes c‐Myc degradation in human renal cell carcinoma

Hepatocyte cell adhesion molecule (hepaCAM) encodes a generally inactive phosphorylated glycoprotein which mediates cancer cell proliferation, migration, and differentiation. We have reported that hepaCAM is down‐regulated in renal cell carcinoma (RCC) and takes responsibility of cell growth inhibition. However, the precise mechanisms of hepaCAM inhibits cell growth is still unknown. In this study, we demonstrated that re‐expression of hepaCAM can cause an accumulation in G0/G1 phase in 786‐0 cells. This reaction was accompanied by a substantial reduction of c‐Myc expression through using an ectopic hepaCAM expression system. Furthermore, we found a comparable decrease in proliferation and G0/G1 accumulation of 786‐0 and RC‐2 cells after treatment with a small molecule c‐Myc inhibitor, 10058‐F4. This indicated that the down regulation of c‐Myc was an essential process in controlling growth inhibitory actions of hepaCAM. Nevertheless, re‐expression of hepaCAM results in apparent reduction of c‐Myc protein with no corresponding reduction of c‐Myc mRNA. This suggests that this reaction might take place at a post‐transcriptional level rather than transcriptional one. Consistent with these findings, hepaCAM decreased c‐Myc stability by increasing the proportion of c‐Myc phosphorylation on T58 which can be abrogated by a proteasomal inhibitor (MG132). Thus, our research implies that the decrease in c‐Myc protein expression, resulting from ectopic expression of hepaCAM, may contribute to the inhibition of proliferation in these cells. J. Cell. Biochem. 112: 2910–2919, 2011.

Exploration of the correlations between interferon-γ in patient serum and HEPACAM in bladder transitional cell carcinoma, and the interferon-γ mechanism inhibiting BIU-87 proliferation.

PURPOSE Interferon-γ inhibits cancer cell proliferation and induces re-expression of different tumor suppressor genes. As a candidate, HEPACAM is almost lost in bladder transitional cell carcinoma. To our knowledge whether interferon-γ inhibits BIU-87 proliferation and re-expresses HEPACAM mRNA is still unknown. Thus, we probed the mechanism and examined the correlations between interferon-γ in patient serum and HEPACAM in bladder transitional cell carcinoma. MATERIALS AND METHODS Using enzyme-linked immunosorbent assay we measured serum interferon-γ in 27 men and 6 women, and 15 volunteers. Disease was Ta-T1 in 12 patients, T2-T4 in 21, low grade in 25, high grade in 8, primary in 13 and recurrent in 20. A total of 33 cancer and 26 adjacent tissues were examined by immunohistochemistry to detect HEPACAM protein and ensure the position. Under interferon-γ stimulation we detected BIU-87 proliferation by MTT assay. Cell cycles were examined by flow cytometry. HEPACAM mRNA expression was determined by reverse transcription-polymerase chain reaction. Western blot was used to detect p21(WAF1). RESULTS Interferon-γ was remarkably low in patients with bladder transitional cell carcinoma vs volunteers (p <0.01). HEPACAM protein was highly expressed in adjacent tissue, mainly at the cytomembrane, but it was almost absent in bladder transitional cell carcinoma (p <0.01). The interferon-γ decrease in the serum of patients with bladder transitional cell carcinoma and the low HEPACAM expression in tumors correlated linearly (r = 0.899, p <0.01). In vitro interferon-γ inhibited BIU-87 proliferation (p <0.01) and slightly re-expressed HEPACAM mRNA (p <0.05). The cell cycle was arrested at G(0)/G(1) and p21(WAF1) was concurrently increased in response to interferon-γ (p <0.01). CONCLUSIONS Results suggest an important connection between HEPACAM and interferon-γ, which may inhibit BIU-87 proliferation through HEPACAM re-expression and p21(WAF1) up-regulation to arrest cells at the G(0)/G(1) phase.

In vitro and in vivo studies of pirarubicin-loaded SWNT for the treatment of bladder cancer

Intravesical chemotherapy is an important part of the treatment for superficial bladder cancer. However, the response to it is limited and its side effects are extensive. Functional single-walled carbon nanotubes (SWNT) have shown promise for tumor-targeted accumulation and low toxicity. In the present study, we performed in vivo and in vitro investigations to determine whether SWNT-based drug delivery could induce high tumor depression in rat bladder cancer and could decrease the side effects of pirarubicin (tetrahydropyranyl-adriamycin, THP). We modified SWNT with phospholipid-branched polyethylene glycol and constructed an SWNT-THP conjugate via a cleavable ester bond. The cytotoxicity of SWNT-THP against the human bladder cancer cell line BIU-87 was evaluated in vitro. Rat bladder cancer in situ models constructed by N-methyl-N-nitrosourea intravesical installation (1 g/L, 2 mg/rat once every 2 weeks for 8 weeks) were used for in vivo evaluation of the cytotoxicity of SWNT and SWNT-THP. Specific side effects in the THP group including urinary frequency (N = 12), macroscopic hematuria (N = 1), and vomiting (N = 7) were identified; however, no side effects were observed with SWNT-THP treatment. Flow cytometry was used to assess the cytotoxicity in vitro and in vivo. Results showed that SWNT alone did not yield significant tumor depression compared to saline (1.74 ± 0.56 and 1.23 ± 0.42%) in vitro. SWNT-THP exhibited higher tumor depression than THP-saline in vitro (74.35 ± 2.56 and 51.24 ± 1.45%) and in vivo (52.46 ± 2.41 and 96.85 ± 0.85%). The present findings indicate that SWNT delivery of THP for the treatment of bladder cancer leads to minimal side effects without loss of therapeutic efficacy. Therefore, this nanotechnology may play a crucial role in the improvement of intravesical treatment of bladder cancer. Key words: Single-walled carbon nanotubes; Bladder cancer; Drug vehicle; THP; Intravesical chemotherapy

Distribution of convergent afferents innervating bladder and prostate at dorsal root Ganglia in rats.

OBJECTIVES To investigate the distribution of dichotomizing afferents supplying both the prostate and urinary bladder, and to discern the effects of noxious stimulation of the prostate on urinary bladder function in rats. METHODS Dual retrograde fluorescence labeling was used to investigate the neurogenic aspect of urinary bladder function. The dual distribution of dorsal root ganglia (DRG) cells was determined by propidium iodide (PI) and propidium bisbenzimide (Bb) staining into the prostate and bladder. To examine mechanical sensitivity of the bladder, conscious filling cystometry was performed before and after completion of Freund adjuvant injection into the prostate. RESULTS Double-labeled positive cells were found in the lumbosacral DRG, predominantly in L1-L2 and L6-S1, with distribution varyinig from 7.5% to 14%. Most of the double-labeled cells were classified as small and medium in size. Prostatic irritation had no effect on the number of labeled cells. With the use of cystometry, prostatic irritation was found to shorten mean micturition interval (P <.05), decrease mean volume threshold inducing micturition, and increase baseline pressure and threshold pressure (P <.05), but to lower peak micturition pressure compared with that in controls (P <.01). CONCLUSIONS These findings suggest that bladder-prostate convergent DRG neurons may play a role in bladder-prostate cross-sensitization after prostatitis. This study also provided neuronal anatomical evidence for voiding dysfunction associated with chronic prostatitis/chronic pelvic pain syndrome.

Expression of hepaCAM and its effect on proliferation of tumor cells in renal cell carcinoma.

OBJECTIVES To evaluate hepaCAM (hepatocyte cell adhesion molecule) gene expression in patients with renal cell carcinoma (RCC) and to explore its effect on proliferation of 786-0 cells. hepaCAM is a tumor suppressor gene, which has been identified as a member of immunoglobulin superfamily cell adhesion molecule. METHODS Two-step reverse transcription-polymerase chain reaction was used to determine hepaCAM expression in 30 paired (RCC and the adjacent non-RCC) renal specimens. Transfection studies were carried out by expressing green fluorescent protein and green fluorescent protein-fused hepaCAM in 786-0 cells. RESULTS Significant downregulation of hepaCAM was detected in 25 of 30 RCC patients tested. When transfected into 786-0 cells, the number of colony formation was reduced by 5-fold according to colony formation assay. MTT (3-diphenyltetrazolium bromide) showed the inhibition rates on the fourth, fifth, and sixth days of culturing were 26.5%, 38.1%, and 35.7%, respectively. CONCLUSION Our data show that hepaCAM is frequently downregulated in RCC, and that exogenous hepaCAM exhibits antiproliferative effect on 786-0 cells, suggesting that silencing of hepaCAM may be associated with carcinogenesis of RCC.

Exon 2 Methylation Inhibits hepaCAM Expression in Transitional Cell Carcinoma of the Bladder

Aim: We aimed to investigate the mechanisms of hepaCAM inactivation in transitional cell carcinoma of the bladder through the analysis of hepaCAM exon 2 methylation. Methods: The methylation of hepaCAM exon 2 and the expression of hepaCAM were determined by methylation-specific restriction PCR assay and RT-PCR in bladder cancer cells (T24, BIU-87) as well as in 55 paired bladder cancer specimens. The methylated bladder cancer cells were treated with 5-Aza- 2′-deoxycytidine (5-Aza-CdR), a demethylating agent. MTT was used to detect the proliferation of T24 and BIU-87 cells. Results: The proliferation of T24 and BIU-87 cells was suppressed by treatment with different concentrations of 5-Aza-CdR; the expression of hepaCAM was absent in T24 and BIU-87 cells, and we found that exon 2 of hepaCAM was methylated in the 2 cells. hepaCAM mRNA was re-expressed and the methylation status of hepaCAM exon 2 was reversed after treatment with 5-Aza-CdR. The expression of hepaCAM mRNA in bladder cancer tissues was significantly lower than that in adjacent tissues. The methylation rate of hepaCAM exon 2 was significantly higher in bladder cancer tissues than in adjacent tissues. The methylation of hepaCAM exon 2 was related to hepaCAM expression in bladder cancer tissues. Conclusions: Downregulation of hepaCAM expression plays an important role in the tumorigenesis and development of bladder cancer. DNA methylation may be important for downregulation of hepaCAM expression in bladder cancer.

Synergistic immunotherapeutic effects of Lycium barbarum polysaccharide and interferon-α2b on the murine Renca renal cell carcinoma cell line in vitro and in vivo.

Novel therapeutic strategies to improve clinical efficacy in patients with renal cell carcinoma (RCC) are required. The possibility of combination therapy with Lycium barbarum polysaccharides (LBP) and recombinant interferon (IFN)-α2b remains to be elucidated in RCC. The present study investigated the putative synergistic immunotherapeutic roles of LBP and IFN-α2b against RCC in vitro and in vivo. The mouse RCC cell line, Renca, was used for in vitro experiments. Treatment of the cells with a combination of LBP and IFN-α2b markedly inhibited cell proliferation, retarded cell cycle growth and promoted apoptosis in the Renca cells. Western blot analysis revealed that LBP and IFN-α2b synergistically downregulated the expression levels of cyclin D1, c-Myc and Bcl-2, and upregulated the expression of the antiapoptotic protein, Bax. Myeloid-derived suppressor cells (MDSCs) were markedly upregulated during tumour progression and promoted tumour growth by inhibiting the T-cell-mediated immune response. In vivo, a marked reduction in the MDSC ratio and tumour volume was observed in a group receiving combined treatment with LBP and IFN-α2b in a xenograft tumour model. In conclusion, the present study suggested that the combination of LBP and IFN-α2b is likely to be more effective in treating murine RCC compared with the less pronounced immunotherapeutic effects of administering LBP or IFN-α2b alone.

Hypoxia promotes 786-O cells invasiveness and resistance to sorafenib via HIF-2α/COX-2.

Accumulating evidences indicated that hypoxia-induced factors and COX-2 play a important role in tumorigenesis in various human cancer. Yet, the relationship between HIFs and COX-2 in human renal cancer remains unclear. The present study was to examine the role of HIFs and COX-2 in the invasiveness and the resistance to target agent in renal cancer cell line (786-O). In 786-O cells, hypoxia induced the increase in the protein expression of HIF1 and HIF2. We also demonstrate that hypoxia up-regulated the protein expression of COX-2 and Snail, but down-regulation of E-cadherin expression in 786-O cells promoted the invasiveness of 786-O cells and enhanced the resistance of 786-O cells to sorafenib. siRNA target to HIF1α, HIF2α and NS398, a selective inhibitor of COX-2, were used in this study. Only siRNA-HIF2α significantly suppressed the protein expression of HIF2 and COX-2, then decreased the invasive ability and resistance of 786-O cells to sorafenib under hypoxia. NS398 attenuated the increase in invasive cells number and the IC50 value of sorafenib induced by hypoxia. In conclusion, our results demonstrated that hypoxia promoted the invasiveness and resistance of 786-O cells to sorafenib via HIF2 and COX-2 and induced the activation of Snail/E-cadherin, suggesting that a signalling mechanism involving HIF2/COX2 modulates invasiveness and resistance to sorafenib in 786-O cells under hypoxia.