Bacille Calmette-Guerin can induce cellular apoptosis of urothelial cancer directly through toll-like receptor 7 activation.

Immunotherapy using bacille Calmette-Guerin (BCG) instillation is the mainstay treatment modality for superficial urothelial cancer (UC) through toll-like receptor (TLR) activation of cognitive immune response. We investigated the roles of TLR7 in the activation of apoptosis in UC cells after BCG treatment. The in vitro cytotoxicity effect of BCG on UC cells was measured by a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium assay. Expressions of TLR7 mRNA and protein in native UC cells prior to and after BCG treatment were analyzed using real-time quantitative polymerase chain reaction and western blot methods. Phagocytotic processes after BCG treatment in UC cells were observed microscopically using a specific immunostain, subsequent cellular apoptosis-related signals induced by TLR7 were analyzed by western blot. Low-grade UC cells, TSGH8301, showed significant cellular death (4.23-fold higher than the high-grade UC cells T24 and J82) when treated with BCG and the BCG cytotoxicity was displayed in a dose-time-dependent manner. TSGH8301 cells had the highest content of TLR7 mRNA, 7.2- and 4.5-fold higher than that of T24 and J82 cells, respectively. TLR7 protein expression was also significantly increased in TSGH8301 cells. Phagocytosis-related markers, including beclin 1, ATG2, and LC3, were increased when TSGH8301 cells were treated by BCG. Interleukin-1 receptor-associated kinases 2 and 4 were also increased markedly in TSGH8301 cells. On the contrary, cellular apoptosis of TSGH8301 cells decreased by 34% when TLR7 activation was suppressed by the TLR antagonist IRS661 after BCG treatment. Our findings suggest that well differentiated TCC cells have higher expression of TLR7 and BCG can drive cellular death of TCC cells directly via TLR7 activation and related apoptotic pathway.

NGAL can alternately mediate sunitinib resistance in renal cell carcinoma.

PURPOSE: Serum NGAL is highly expressed in patients with advanced renal cancer treated with sunitinib. We investigated the role of NGAL in sunitinib resistance in renal cell carcinoma to identify potential tactics to overcome it. MATERIALS AND METHODS: NGAL expression was correlated with sunitinib sensitivity. Vascular endothelial growth factor related upstream Ras, Erk1/2 and STAT1 phosphorylation activity in Caki-1 and NGAL transfected Caki-1 cells after sunitinib treatment was analyzed using Western blot. NGAL and vascular endothelial growth factor-A interaction with sunitinib therapeutic efficacy was monitored in renal cell carcinoma tumor xenografted mice by tumor growth inhibition, serum NGAL and vascular endothelial growth factor-a levels, and microscopic examination of tumor microvascular density. RESULTS: Sunitinib cytotoxicity in various renal cell carcinoma cell lines was reversibly related to NGAL expression. Sunitinib showed the lowest 50% inhibitory concentration (5.53 µM) in Caki-1 cells, which had the lowest NGAL expression of these renal cell carcinoma cell lines. After sunitinib treatment adding NGAL inhibited Ras and Erk1/2 phosphorylation but activated STAT1α phosphorylation in Caki-1 cells and Caki-1 cells transfected with NGAL. In a xenograft mouse model sunitinib significantly inhibited tumor growth in Caki-1 mice. NGAL transfected Caki-1 mice had higher serum NGAL and lower vascular endothelial growth factor-A than Caki-1 mice. Microvascular density was decreased in Caki-1 mice with sunitinib treatment. CONCLUSIONS: NGAL in tumor cells may show crosstalk with vascular endothelial growth factor-a and alternative activation in stimulating tumor growth during sunitinib treatment. It may become a therapeutic target to reverse sunitinib resistance in renal cell carcinoma.

Epigenetic regulation of vascular endothelial growth factor a dynamic expression in transitional cell carcinoma.

Vascular endothelial growth factor A (VEGF-A) is a key mediator in the neovascularization of cancers. We have found that VEGF-A was expressed at significantly higher levels in high-grade transitional cell carcinoma (TCC) cells than low-grade TCC cells in our previous study. In the present study, promoter methylation pattern was assessed and quantified by bisulfite genomic sequencing (BGS) and specific VEGF-A CpG sites in low-grade, but not in high-grade, TCC cells were observed. Reporter assays indicated that hypermethylation of nine CpG sites can inhibit the transcriptional activity of the VEGF-A gene. Subsequent chromatin immunoprecipitation (ChIP) assay revealed down-regulation of transcription activity of VEGF-A with increasing binding of methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in low-grade TCC cells during hypermethylation. Furthermore, treatment of low-grade TCC cells with DNA methyltransferase inhibitor and histone deacetylase inhibitor can restore the expression of VEGF-A and promote the invasive ability of low-grade TCC cells. Hypermethylation with lower expression levels of VEGF-A in low-grade TCC tumors than high-grade TCC tumors was also confirmed in clinical specimens by reverse transcriptase-PCR and pyrosequencing analyses. Our findings are the first results indicating that VEGF-A expression is suppressed in low-grade TCC tumors by promoter hypermethylation. This offers a new perspective on the role of VEGF-A in TCC tumor behavior.

Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer.

Overexpression of hypoxia-inducible factor-1 alpha is noted during the invasive and metastatic process of transitional cell carcinoma. It will upregulate vascular endothelial growth factor (VEGF) and drive proliferation, invasiveness, metastasis, and antiapoptotic ability of cancer cells. We proposed that tyrosine kinase receptor inhibitor, sunitinib malate-(Sutent; Pfizer Inc., Taiwan), combined with chemotherapeutic drug may present synergistic cytotoxic enhancement to transitional cell carcinoma cells with subsequent inhibition of their cellular behaviors, including proliferation, invasiveness, and metastatic activity. The contents of VEGF-A in mouse bladder tumor cells (MBT-2) and culture medium were detected by quantification-polymerase chain reaction and Western blot individually. The inhibitory concentrations of various chemotherapeutic drugs, sunitinib, and their combination treatment in MBT-2 were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Microchamber transmembrane migration assay was applied in evaluation of the inhibitory effects of different dosages of sunitinib and combination treatment on tumor cells. The cell cycle and apoptosis were analyzed after combination therapy by flow cytometry. Variation in apoptotic pathway was elucidated by Western blot using specific antibodies with cleaved PARP and caspase-3. Metastatic animal model mimicked by tail vein injection of MBT-2 cells was used to evaluate the treatment efficiency in tumor weight and survival rate. The mRNA and protein level of VEGF-A in MBT-2 cells increased by 70% at 48 hours interval under hypoxia stress condition. In MTT assay, MBT-2 cells had shown the highest sensitivity to epirubicin. Sunitinib combined with epirubicin had shown a synergistic cytotoxic effect to MBT-2 cells. Sunitinib and its combination with epirubicin showed significant inhibition on MBT-2 cells migration in microchambers. G2/M phase arrest and increased subG1 in cell cycle was seen in the epirubicin and sunitinib combination treatment group. The activation of apoptosis pathway was confirmed by increased cleaved caspase-3 and cleaved PARP in MBT-2 cells. In tail vein tumor inoculation C3H mice model, epirubicin alone and sunitinib combination therapy decreased tumor growth in lungs with marginal effect. Sunitinib and epirubicin combination had shown a synergistic cytotoxic effect and inhibited cell migration ability in MBT-2 cells. The combination can induce cell cycle arrest at G2/M phase and increase subG1 cells. Metastatic animal study also showed that sunitinib combined with epirubicin has a marginal effect on inhibition of tumor growth of lungs. The tyrosine kinase receptor inhibitor-targeted combined chemotherapy regimen may provide as a new treatment modality for advanced bladder cancer in the future.

Sunitinib can enhance BCG mediated cytotoxicity to transitional cell carcinoma through apoptosis pathway.

Bacillus Calmette-Guerin (BCG)-refractory generated a high risk to patients with bladder cancer during treatment. Tyrosine kinase receptor (TKR) and TKR-mediated signal transduction pathways play an important role in tumor initiation, maintenance, angiogenesis, and vascular proliferation. Theoretically, it is helpful in adjuvant treatment for transitional cell carcinoma (TCC). Hence, we proposed that sunitinib, a endothelial growth factor receptor (VEGFR) inhibitor, may have a synergistic effect with BCG in enhancing its cytotoxicity to bladder cancer. The level of VEGF in various TCC cell lines was quantified by real time PCR. High grade TCC-T24 cell line with high level of VEGF expression was selected as representative tumor cells for further study. The single drug and combined inhibitory effects of BCG and sunitinib in T24 cells were determined by MTT method. The drug mediated cell apoptosis in T24 cells was characterized by flow cytometry with PI and annexin V stain. Bcl-2 apoptotic pathway induction by BCG and sunitinib treatment was evaluated by Western blotting method. Inhibitory ability of sunitinib in BCG induced cell migration was verified by cell migration assay. The results shown that expression level of VEGF mRNA in high grade T24 cells was higher than low grade J82, TSGH 8301, and TCC 9202 cell lines. Both BCG and sunitinib treatment presented cytotoxic effect to T24 cells in a dose-dependent manner. Combination of BCG and sunitinib revealed superior cytotoxicity effect than single agent when cells were pretreated with low dosage BCG before sunitinib treatment. By Annexin V analysis it was observed that cell death associated with increased early and late apoptosis process individually. Furthermore, the bcl-2 expression was significant reduced in T24 cells in metachronous BCG and sunitinib combination treatment than single agent. Tumor cell migration activity was also markedly inhibited with BCG and sunitinib combination treatment. In conclusion, these results suggested that during BCG and sunitinib combination treatment both reagents interacted with each other and caused TCC cells apoptosis in addition to direct cytotoxicity. This combination therapeutic model may have the potential for future clinical application to bladder cancer treatment.

Taxol synergizes with antioxidants in inhibiting hormal refractory prostate cancer cell growth.

Taxanes are chemotherapeutic agents commonly used to treat various carcinomas. Dietary antioxidants, such as vitamin E, green tea extracts, and isoflavones have been used against prostate cancer, and exhibit anticancer effects both in vitro and in vivo. We evaluated the combined effect of taxol (paclitaxel) with pyrrolidine dithiocarbamate, vitamin E, epigallocatechin gallate, and genistein in killing hormone-refractory prostate cancer cells. Those agents were tested on the hormone-refractory prostate cancer cell line PC-3, and the viability of the cells was determined using MTT {3 (4, 5-dimethylthiazo-2-yl)-2, 5-diphenyl tetrazolium} assay after drug treatment. PC-3 cells were sensitive to these drugs with 50% inhibitory concentrations of 0.1, 23, 220, 1122, and 260 microM, for taxol, pyrrolidine dithiocarbamate, epigallocatechin gallate, genistein, and vitamin E, respectively. Genistein, pyrrolidine dithiocarbamate, and epigallocatechin gallate showed synergistic cytotoxicity to PC-3 cells when combined with 0.01 microM taxol. Only high concentration of vitamin E showed a synergistic effect with this dose of taxol. Further study revealed that 3 combinations could induce sub-G1 phase of cell cycle, induce apoptosis, and increase caspase activity and decrease Bcl-2 expression simultaneously. In conclusion, in addition to vitamin E, incorporation of these antioxidants with taxan-based cytotoxic therapies offers encouraging strategies for combating hormone-refractory prostate cancers.