Jintao Zhuang

The expression and role of protein kinase C (PKC) epsilon in clear cell renal cell carcinoma

Protein kinase C epsilon (PKCε), an oncogene overexpressed in several human cancers, is involved in cell proliferation, migration, invasion, and survival. However, its roles in clear cell renal cell carcinoma (RCC) are unclear. This study aimed to investigate the functions of PKCε in RCC, especially in clear cell RCC, to determine the possibility of using it as a therapeutic target. By immunohistochemistry, we found that the expression of PKCε was up-regulated in RCCs and was associated with tumor Fuhrman grade and T stage in clear cell RCCs. Clone formation, wound healing, and Borden assays showed that down-regulating PKCε by RNA interference resulted in inhibition of the growth, migration, and invasion of clear cell RCC cell line 769P and, more importantly, sensitized cells to chemotherapeutic drugs as indicated by enhanced activity of caspase-3 in PKCε siRNA-transfected cells. These results indicate that the overexpression of PKCε is associated with an aggressive phenotype of clear cell RCC and may be a potential therapeutic target for this disease.

Association between RASSF1A Promoter Methylation and Prostate Cancer: A Systematic Review and Meta-Analysis

Prostate cancer (PCa) remains as one of the most common cause of cancer related death among men in the US. The widely used prostate specific antigen (PSA) screening is limited by low specificity. The diagnostic value of other biomarkers such as RAS association domain family protein 1 A (RASSF1A) promoter methylation in prostate cancer and the relationship between RASSF1A methylation and pathological features or tumor stage remains to be established. Therefore, a meta-analysis of published studies was performed to understand the association between RASSF1A methylation and prostate cancer. In total, 16 studies involving 1431 cases and 565 controls were pooled with a random effect model in this investigation. The odds ratio (OR) of RASSF1A methylation in PCa case, compared to controls, was 14.73 with 95% CI = 7.58–28.61. Stratified analyses consistently showed a similar risk across different sample types and, methylation detection methods. In addition, RASSF1A methylation was associated with high Gleason score OR=2.35, 95% CI: 1.56–3.53. Furthermore, the pooled specificity for all included studies was 0.87 (95% CI: 0.72–0.94), and the pooled sensitivity was 0.76 (95% CI: 0.55–0.89). The specificity in each subgroup stratified by sample type remained above 0.84 and the sensitivity also remained above 0.60. These results suggested that RASSF1A promoter methylation would be a potential biomarker in PCa diagnosis and therapy.

Vasculogenic Mimicry in Prostate Cancer: The Roles of EphA2 and PI3K.

BACKGROUND. Aggressive tumor cells can form perfusable networks that mimic normal vasculature and enhance tumor growth and metastasis. A number of molecular players have been implicated in such vasculogenic mimicry, among them the receptor tyrosine kinase EphA2, which is aberrantly expressed in aggressive tumors. Here we study the role and regulation of EphA2 in vasculogenic mimicry in prostate cancer where this phenomenon is still poorly understood. METHODS. Vasculogenic mimicry was characterized by tubules whose cellular lining was negative for the endothelial cell marker CD34 but positive for periodic acid-Schiff staining, and/or contained red blood cells. Vasculogenic mimicry was assessed in 92 clinical samples of prostate cancer and analyzed in more detail in three prostate cancer cell lines kept in three-dimensional culture. Tissue samples and cell lines were also assessed for total and phosphorylated levels of EphA2 and its potential regulator, Phosphoinositide 3-Kinase (PI3K). In addition, the role of EphA2 in vasculogenic mimicry and in cell migration and invasion were investigated by manipulating the levels of EphA2 through specific siRNAs. Furthermore, the role of PI3K in vasculogenic mimicry and in regulating EphA2 was tested by application of an inhibitor, LY294002. RESULTS. Immunohistochemistry of prostate cancers showed a significant correlation between vasculogenic mimicry and high expression levels of EphA2, high Gleason scores, advanced TNM stage, and the presence of lymph node and distant metastases. Likewise, two prostate cancer cell lines (PC3 and DU-145) formed vasculogenic networks on Matrigel and expressed high EphA2 levels, while one line (LNCaP) showed no vasculogenic networks and lower EphA2 levels. Specific silencing of EphA2 in PC3 and DU-145 cells decreased vasculogenic mimicry as well as cell migration and invasion. Furthermore, high expression levels of PI3K and EphA2 phosphorylation at Ser897 significantly correlated with the presence of vasculogenic mimicry and in vitro inhibition of PI3K by LY294002 disrupted vasculogenic mimicry, potentially through a reduction of EphA2 phosphorylation at Ser897. CONCLUSIONS. The expression levels of PI3K and EphA2 are positively correlated with vasculogenic mimicry both in vivo and in vitro. Moreover, phosphorylation levels of EphA2 regulated by PI3K are also significantly associated with vasculogenic mimicry in vivo. Based on its functional implication in vasculogenic mimicry in vitro, EphA2 signaling may be a potential therapeutic target in advanced prostate cancer.

Association of DSC3 mRNA down-regulation in prostate cancer with promoter hypermethylation and poor prognosis.

Background Desmocollin 3 (DSC3), a member of the cadherin gene superfamily, is associated with pathogenesis of some cancers, but its role in prostate cancer (PCa) remains largely unknown. Methods DSC3 gene expression level in available PCa microarray dataset was examined using the Oncomine database. DSC3 transcript expression in prostate cell line panel and an independent tissue cohort (n = 52) was estimated by quantitative PCR (Q-PCR). Epigenetic status of DSC3 gene promoter in PCa was investigated by uploading three dataset (ENCODE Infinium 450K array data and two methylation sequencing) in UCSC genome browser. While pyrosequencing analysis measured promoter DNA methylation, Q-PCR estimates were obtained for DSC3 transcript re-expression after 5-Aza-deoxycytidine (5-Aza) treatment. Clinical relevance of DSC3 expression was studied by Kaplan-Meier survival analysis. Finally, functional studies monitoring cell proliferation, migration and invasion were performed in prostate cell lines after siRNA mediated DSC3 knockdown or following 5-Aza induced re-expression. EMT markers Vimentin and E-cadherin expression was measured by Western Blot. Results Microarray data analyses revealed a significant decrease in DSC3 transcript expression in PCa, compared to benign samples. Q-PCR analysis of an independent cohort revealed DSC3 transcript down-regulation, both in PCa cell lines and tumor tissues but not in their benign counterpart. Examination of available NGS and Infinium data identified a role for epigenetic regulation DSC3 mRNA reduction in PCa. Pyrosequencing confirmed the increased DSC3 promoter methylation in cancer cell lines and restoration of transcript expression upon 5-Aza treatment further corroborated this epigenetic silencing mechanism. Importantly Kaplan-Meier analysis of an outcome cohort showed an association between loss of DSC3 expression and significantly increased risk of biochemical recurrence. Functional studies indicate a role for epithelial–mesenchymal transition in DSC3 regulated cell migration/invasion. Conclusion Taken together, our data suggests that DNA methylation contributes to down-regulation of DSC3 in prostate cancer, and loss of DSC3 predicts poor clinical outcome.

The expression and role of tyrosine kinase ETK/BMX in renal cell carcinoma.

BackgroundExpression of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors, but the underlying molecular mechanisms and its clinical significance in renal cell carcinoma (RCC) remain to be elucidated.MethodsETK expression in 90 human RCC and 30 human normal renal tissue samples was examined by immunohistochemistry and compared with several clinicopathologic parameters. To further demonstrate the biological function of ETK in RCC, Western blot was used to test the expression level of ETK protein in RCC cell lines. Subsequent to the downregulation of ETK by small interfering RNA, the effects of ETK on RCC cell growth, apoptosis, migration and invasion were assessed by methyl thiazol tetrazolium assay, flow cytometry and transwell assay. And the varying expression of VEGF, STAT3 and phosphorylated STAT3 (p-STAT3) in RCC were evaluated by Western blot.ResultsImmunohistochemistry analysis showed that ETK expression was highly increased in RCC and was positively correlated with clinical stage, grade and metastasis. Simultaneously, the overall survival time in patients with higher ETK expression was obviously shorter than that in patients with lower ETK expression. ETK was also detected in RCC cell lines. Moreover, the down-regulating ETK significantly inhibited RCC cell growth, migration, invasion and promoted apoptosis. The expression of VEGF and p-STAT3 were also decreased.ConclusionsOur study suggests that the overexpression of ETK is associated with the malignancy and disease progression of RCC. Since ETK is also involved in RCC cell biological function and VEGF-ETK-STAT3 loop, ETK may be used as a potential therapeutic target for RCC.

Matrix metalloproteinase-9 is required for vasculogenic mimicry by clear cell renal carcinoma cells☆

BACKGROUND Vasculogenic mimicry (VM), a new pattern of tumor microcirculation system, has been proved to be important for tumor growth and progression and may be one of the causes of antiangiogenesis resistance. Matrix metalloproteinase-9 (MMP9) was shown to correlate with VM formation in some other cancers. However, the relationship between VM formation and MMP9 in renal cell carcinoma (RCC) has not been determined. METHODS The VM formation and MMP9 expressions were analyzed by CD34/periodic acid-Schiff dual staining and immunohistochemistry in 119 RCC specimens. We used a well-established 3-dimention culture model to compare VM formation in 786-O, 769-P, and HK-2 cell lines in vitro. MMP9 expressions on either messenger RNA or protein levels were compared among the cell lines by quantitative polymerase chain reaction or Western blot. To determine further the relationship between MMP9 and VM in RCC, 786-O and 769-P were treated with specific MMP9 inhibitor or small interfering RNA. VM formation, cell migration, and invasion were subsequently assessed by 3-dimention culture, wound-healing, and transwell assays. RESULTS Immunohistochemistry demonstrated both VM formation and MMP9 overexpression were positively associated with clinical staging, pathological grade, and metastasis (P<0.01). VM formation was closely correlated with MMP9 overexpression in RCC (r = 0.602, P<0.01). Lower MMP9 expression level was observed in normal kidney cell line HK-2, which was unable to form VM on Matrigel, whereas higher expression of MMP9 was found in VM-forming cancer cell lines 786-O and 769-P. Inhibition of MMP9 not only disrupted VM formation in 786-O and 769-P but also reduced cell migration and invasion. CONCLUSIONS These results indicate an intimate relationship between MMP9 overexpression and VM formation in RCC. Treatments targeting VM formation by inhibiting the activity of MMP9 could be beneficial in RCC therapy.

Screening and identification of a renal carcinoma specific peptide from a phage display peptide library

BackgroundSpecific peptide ligands to cell surface receptors have been extensively used in tumor research and clinical applications. Phage display technology is a powerful tool for the isolation of cell-specific peptide ligands. To screen and identify novel markers for renal cell carcinoma, we evaluated a peptide that had been identified by phage display technology.MethodsA renal carcinoma cell line A498 and a normal renal cell line HK-2 were used to carry out subtractive screening in vitro with a phage display peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the A498 cells, and the output/input ratio of phages increased about 100 fold. A group of peptides capable of binding specifically to the renal carcinoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied.ResultsThrough a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specifically bind to the tumor cell surfaces of A498 and incision specimens, but not to normal renal tissue samples.ConclusionA peptide ZT-2, which binds specifically to the renal carcinoma cell line A498 was selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of renal carcinoma or targeted drug delivery in chemotherapy.

Sunitinib monotherapy instead of mitotane combination therapy for the treatment of refractory adrenocortical carcinoma.

DOI: 10.1111/iju.12903 ACC is a rare malignancy with a poor prognosis. Only complete surgical resection can offer the potential for cure; however, even after successful excision, local recurrences and distant metastases frequently occur. Thus, adjuvant therapy options need to be considered for advanced ACC, but the effectiveness remains controversial. As is well known, mitotane is the only recognized adrenal cytotoxic agent currently available, and might prolong progressionfree survival. However, the objective response rate was just 23%, and long-term disease control was achieved in <15% of patients. Therefore, several studies have investigated targeted therapies, such as imatinib, erlotinib, sorafenib and sunitinib, in patients with ACC, but the responses have been disappointing. Here, we report a case of refractory ACC that showed a good response to sunitinib treatment. A 33-year-old woman presented to First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, with a palpable mass in her right upper quadrant of the abdomen. CT scan showed a 13.8 9 10.4-cm mass in the right adrenal gland and liver involvement; a tumor thrombus in the inferior vena cava and lung metastases were also found (Fig. S1a,b). Endocrine examination was almost normal. She was diagnosed with advanced ACC and received surgical resection in March 2011. The pathological evaluation confirmed an ACC with liver invasion, and immunohistochemical staining for VEGF was positive in the tumor cells. The Weiss score was nine (Fig. S1c-f). Then she received adjuvant chemotherapy. Mitotane plus etoposide, doxorubicin and cisplatin chemotherapies were initiated, but failed after two cycles of chemotherapy. After the failure of mitotane-based cytotoxic chemotherapy, targeted therapy with sunitinib was chosen after careful discussion with the patient’s family. She received sunitinib plus mitotane treatment from August 2011. However, after 8 months of sunitinib treatment, the patient developed a severe cough, and a CT scan showed progressive disease in April 2012. Her entire lungs were bestrewn with increasing pulmonary nodules (Fig. 1a,b). Because of the disease progression and adverse events, mitotane was withdrawn in May 2012 and sunitinib monotherapy was continued. In May 2013, a CT scan showed a significant remission (Fig. 1c,d) and the patient’s condition improved. In May 2014, a further CT scan showed that there were fewer metastatic pulmonary nodules and their sizes were smaller (Fig. 1e,f). The patient’s condition stabilized and she survived postoperatively for 4 years. Sunitinib is an oral multitargeted tyrosine kinase inhibitor that targets VEGFR1, VEGFR2, c-KIT and Fms-like tyrosine kinase 3, and thus combines direct antitumor effects with antiangiogenic activity. The results of a phase II study in patients progressing after mitotane and one to three cytotoxic chemotherapies showed that sunitinib had modest activity in advanced refractory ACC. Furthermore, the results were highly suggestive of a negative impact of concomitant mitotane therapy on the outcome. Fortunately, we have gained great encouragement from our successful experience with that refractory ACC patient. After the failure of mitotane-based chemotherapy, we continued her treatment with a combination of sunitinib and mitotane, but the tumor still progressed. This result was in good agreement with published data. Mitotane was then withdrawn and sunitinib monotherapy was continued. A year later, unexpected results were observed. A CT scan showed that the patient’s metastatic pulmonary nodules had nearly disappeared and her condition stabilized. The clinical benefit of sunitinib has persisted until the present time. However, the present results again suggest that a drug interaction between sunitinib and mitotane might have a major influence on their effects. The pharmacokinetic properties of the two drugs could be the reason for this interaction. It is well established that sunitinib is metabolized in the liver and intestine by CYP3A4 monooxygenase to its active metabolite SU12662, which is then inactivated in a second step by CYP3A4 monooxygenase. However, mitotane is an extraordinarily strong inducer of CYP3A4, which will markedly increase the inactivation of sunitinib, and therefore reduce

Varicocele anatomy during subinguinal microsurgical varicocelectomy in Chinese men.

Knowledge of subinguinal microsurgical varicocelectomy is of fundamental importance to ensure that varicocele is resolved and testicular function is preserved. Our study aimed to describe the number of veins, arteries and lymphatics in the subinguinal spermatic cord and to clarify their differences between two sides, between patients with different complaints and between varicoceles with different clinical grades. A total of 102 consecutive patients underwent 162 primary subinguinal microsurgical varicocelectomies, during which the number of vessels with different diameters was recorded. A mean number of 12.9 internal spermatic veins, 0.9 external spermatic veins, 1.8 internal spermatic arteries and 2.9 lymphatics were identified per cord. 88.2% of the internal spermatic arteries were surrounded by a dense complex of adherent veins. The external spermatic vein or veins were found in 49.4% of the cases. The mean number of medium (1–3 mm in diameter) internal spermatic veins on the left was larger than that on the right (P < 0.001). The mean number of medium internal spermatic veins in grade III varicocele was larger than that in grade I or grade II (P < 0.015). There was no significant anatomical difference between the men presenting for infertility, chronic testicular pain and both the two complaints.

Vasculogenic mimicry plays an important role in adrenocortical carcinoma

To determine the prognostic role of vasculogenic mimicry in adrenocortical carcinoma, and to explore its relationship with vascular endothelial growth factor receptor 2 expression.