Yunfeng He

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.

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.

Functional significance of the hepaCAM gene in bladder cancer.

BackgroundThe hepaCAM gene encodes a new immunoglobulin-like cell adhesion molecule, and its expression is suppressed in a variety of human cancers. Additionally, hepaCAM possesses properties often observed in tumor suppressor genes. However, the expression and biological function of hepaCAM has not been investigated in bladder cancer. Therefore we sought to examine hepaCAM expression and the relationship between its structure and function in human transitional cell carcinoma of bladder (TCCB).Materials and methodsHepaCAM expression was evaluated in 28 normal and 34 TCCB bladder specimens and 2 TCCB cell lines using semi-quantitative RT-PCR. The wild-type hepaCAM and the extracellular domain-truncated mutant gene were transfected into the TCCB cell line T24, and the biological properties of both the wild-type gene and the domain-truncated mutant were then assessed.ResultsHepaCAM expression was down-regulated in 82% (28/34) of TCCB specimens and undetectable in the 2 TCCB cell lines tested. The localization of hepaCAM appeared to be dependent on cell density in T24 cells. In widely spread cells, hepaCAM accumulated on the perinuclear membrane and the cell surface protrusions, whereas in confluent cells, hepaCAM was predominantly localized at the sites of cell-cell contacts on the cell membrane. Functionally, hepaCAM expressed not only increased cell spreading, delayed cell detachment, enhanced wound healing and increased cell invasion; it also inhibited cell growth (P < 0.01). When the extracellular domain was deleted, the localization of hepaCAM was significantly altered, and it lost both its adhesive function and its influence on cell growth.ConclusionsHepaCAM is involved in cell adhesion and growth control, and its expression is frequently silenced in TCCB. The extracellular domain of hepaCAM is essential to its physiological and biological functions.

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

Impaired Delta Np63 Expression is Associated with Poor Tumor Development in Transitional Cell Carcinoma of the Bladder

The oncogenic isoform of the p63 protein, delta Np63 (ΔNp63), plays an important role in the pathogenesis of many epithelial carcinomas, and emerging evidences suggest that ΔNp63 is a promising drug target. However, the functions of ΔNp63 in transitional cell carcinoma of bladder (TCCB) are poorly defined. In this study, a ΔNp63 shRNA expression vector was transfected into TCCB cell line 5637 and cell cycling, cell proliferation and protein expression were assessed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-dimethyl tetrazolium bromide (MTT) assay, and immunohistochemistry, respectively. The ΔNp63 shRNA expression vector was also injected into 5637 cell xenograft tumors in nude mice, and tumor size was measured, tumor tissue morphology was assessed by immunohistopathology and transmission electron microscopy. In the in vitro study, ΔNp63 shRNA transfection caused successful ΔNp63 gene silencing and resulted in significant arrest of cell cycling and cellular proliferation (p<0.05) as well as cyclin D1 expression. In the nude mouse xenograft model, ΔNp63 shRNA greatly inhibited tumor growth, induced tumor cell apoptosis (p<0.05) and resulted in cyclin D1 downregulation. Our data suggest that ΔNp63 may play an oncogenic role in TCCB progression through promoting cell survival and proliferation. Intratumoral administration of ΔNp63-specific shRNA suppressed tumor ΔNp63 expression and cellular proliferation while promoted tumor cellular apoptosis, and therefore inhibited tumor growth and improved survival of xenograft-bearing mice, which was not accompanied by significant signs of systemic toxicity.

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.

Interleukin 6 induces cell proliferation of clear cell renal cell carcinoma by suppressing hepaCAM via the STAT3-dependent up-regulation of DNMT1 or DNMT3b.

Interleukin 6 (IL-6), a tumor promoting cytokine, has been largely implicated in the development of renal cell carcinoma (RCC). Hepatocyte cell adhesion molecule (hepaCAM) is a novel tumor suppressor, which is lost or down-regulated in many cancer types including RCC. In the present study, we intensively investigated the connection between IL-6 and hepaCAM in RCC. Our analysis of RCC tissues, adjacent tissues and paired serum samples from RCC patients revealed that IL-6 was elevated in patient serum and RCC tissue, whereas hepaCAM was completely lost or significantly down-regulated. Furthermore, we observed an association between IL-6 increase and hepaCAM decrease in RCC tissue samples. In the section of cytological researches, we found in RCC cell lines that IL-6 was a direct upstream regulator of hepaCAM, and that hepaCAM down-regulation was involved in IL-6-driven cell proliferation. We also demonstrated that IL-6-mediated promoter hypermethylation largely accounted for the hepaCAM loss in RCC, and it was STAT3-dependent. Additionally, our data showed that DNMT1 up-regulation induced by IL-6/STAT3 signaling was indispensable for IL-6-mediated hepaCAM loss in RCC cell lines ACHN and 769-P, while DNMT3b up-regulation was crucial for hepaCAM loss in A498. Our findings provide a novel signal pathway regulating cell proliferation, potentially representing a therapeutic target for RCC.

Effects of silencing transforming growth factor-β1 by RNA interference plasmid on rat renal allograft fibrosis using Smads pathway.

OBJECTIVES To evaluate the effects of transforming growth factor (TGF)-β1 RNA interference plasmid on rat renal allograft fibrosis and to explore its mechanisms. METHODS A Sprague-Dawley to Wistar rat transplant kidney-sclerosis accelerated model was constructed and transfected with short hairpin RNA-TGF-β1 based on the hydromechanics. Kidney and blood samples were collected at the first, second, and third months after transplantation. Reverse transcriptase-polymerase chain reaction and Western blotting were used to detect the expression of TGF-β1, phosphorylated Smad3/7, E-cadherin, and type I collagen. The fibrosis extent was assessed using Masson staining. The immunohistochemical staining of E-cadherin and α-smooth muscle actin were used to label the tubular epithelial cells and fibroblast, respectively. RESULTS The blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P <.05 and P <.01, respectively). The expression of TGF-β1 was significantly inhibited by the plasmid and its target gene type I collagen (P <.05 or P <.01), in which the signal proteins of phosphorylated Smad3 was downregulated and phosphorylated Smad7 was upregulated. Also, the fibrosis of the renal allograft was improved and milder fibrosis was present in the plasmid group. In addition, short hairpin RNA-TGF-β1 plasmid maintained the expression of E-cadherin on tubular epithelial cells, resulting in inhibition of cell transdifferentiation from epithelial cells to fibroblast. CONCLUSIONS Our results suggest that short hairpin RNA-TGF-β1 plasmid could prevent the fibrosis of renal allografts. The mechanism might be associated with its effects of downregulating phosphorylated Smad3 and upregulating phosphorylated Smad7, leading to the suppression of epithelial-myofibroblast transdifferentiation and extracellular matrix synthesis.

Upregulation of cell adhesion through delta Np63 silencing in human 5637 bladder cancer cells

Some researchs have demonstrated that the loss of delta Np63 is associated with aggressive phenotypes and poor prognosis. However, other research indicates that delta Np63 is considered to have oncogenic properties. Delta Np63 overexpression is often observed in association with the oncogenic growth of squamous cell carcinomas and bladder cancer. In this study, we investigated the oncogenic role of delta Np63 in regulating cell adhesion in transitional cell carcinoma of the bladder (TCCB). The cells were stably transfected with the delta Np63 short hairpin RNA (shRNA) plasmid. Immunocytochemistry was performed to determine the knockdown efficiency. Tumour cells were studied for their ability to adhere to vascular endothelial cells. Confocal microscopy was used to analyse the changes in cytoskeletal F-actin. F-actin expression was measured by flow cytometry. Cell invasion ability was assessed using transwell chambers. The delta Np63-silenced tumour cells were shown to adhere more tightly than controls to vascular endothelial cells (P<0.05). The content of F-actin in the delta Np63-silenced cells was enhanced (P<0.05). The Matrigel invasion assays showed that human 5637 bladder cancer cells had a lower degree of motility when transfected with pdelta Np63-shRNA (P<0.05). In conclusion, silencing of the delta Np63 expression can enhance the adhesiveness of 5637 cells by inducing F-actin cytoskeleton production, and it will possibly inhibit the TCCB invasion and metastasis.