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MP35-08 TYROSINE-KINASE INHIBITORS TREATMENT INDUCES IL-6 SECRETION ON RENAL CARCINOMA CELLS
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
then tumor infiltrating lymphocyte was analyzed by immunohistochemistry. RESULTS: All three treatment groups ((1), (2) and (3)) showed significant decrease in tumor size compared with vehicle group. In particular, combination of PD-1 Ab and CTLA-4 Ab exhibited the most significant anti-tumor effect. The percentage of PCNA+ tumor cells were very similar amongst all therapies, but the percentage of Ki-67+ tumor cells were significantly decreased with combination of PD-1 Ab and CTLA-4 Ab. Combination of PD-1 Ab and CTLA-4 Ab demonstrated the most potent effect to trigger the infiltration of both CD4+ lymphocyte and CD8+ lymphocytes. These in vivo results indicate that combination of PD-1 Ab and CTLA-4 Ab may have a significant immunostimulatory effect to exert the anti-tumor effect to renal cell carcinoma. CONCLUSIONS: Combination therapy of anti-CTLA-4 antibody and anti-PD-1 antibody can be a potential therapeutic option for advanced kidney cancer patients.
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Leptin, HGF, HB-EGF and EGF secretions from RCC cells cultured with TKIs were measured by VersaMAP Development System. Western blot analyses were applied for detection of phosphorylated STAT3, ERK Akt, mTOR and NFkB proteins. Alterations of cell proliferation and IL-6 signaling pathway by humanized IL-6R antibody Tocilizumab was analyzed by BrdU assay, MTT assay and Western blot analysis. RESULTS: Among the measured cytokines, all the RCC cell lines secreted IL-6 when they were cultured with TKIs. IL-10, IL-23, HB-EGF and leptin were decreased by TKI treatment. Among the RCC cell lines, 786-O and Dibo showed high level of IL-6 production 1 hour after TKIs treatment. VEGF secretion was also observed when the 786O cells were treated by TKIs. In 786-O cells, Western blot analysis revealed that STAT3 and ERK were phosphorylated by TKI treatment even at a low concentration of 0.5 mM. Furthermore, Akt and mTOR were also activated by TKIs treatment. HIF2a expression and phosphorylation of NFkB which would lead to VEGF and IL-6 expression were also observed at the same concentration of TKIs. Tocilizumab treatment in combination with TKIs reduced activation of IL-6 signaling pathway and also suppressed cell proliferation. VEGF secretion following 1 hour TKIs treatment were 3-fold enhanced compared with non treated cells, and the VEGF secretion from RCC cells decreased to 40% by blocking of IL-6 receptor by tocilizumab. CONCLUSIONS: Our results indicate that TKI treatment induces IL-6 secretion in concentration under therapeutical level on RCC cells. Autocrine secretion of IL-6 activates STAT3 and Akt-mTOR pathway and it leads to VEGF secretion. IL-6 could be a possible target for overcoming the development of resistance to TKIs. Source of Funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No.22591773, No.25462492)
MP35-09 BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT TO CAUSE UBIQUITINATED PROTEIN ACCUMULATION IN RENAL CANCER CELLS Takako Asano*, Akinori Sato, Makoto Isono, Keiichi Ito, Tomohiko Asano, Tokorozawa, Japan
Source of Funding: none
MP35-08 TYROSINE-KINASE INHIBITORS TREATMENT INDUCES IL-6 SECRETION ON RENAL CARCINOMA CELLS Kei Ishibashi*, Yoshiyuki Kojima, Fukushima, Japan; Tobias Haber, €roff, Walburgis Brenner, Mainz, Germany Joachim W. Thu INTRODUCTION AND OBJECTIVES: Tyrosine-kinase inhibitors (TKI) treatment targeted at the vascular endothelial growth factor (VEGF) pathways represents the standard of care in advanced renal cell carcinoma (RCC). Although the TKI treatment has promising effect against RCC, the development of resistance to TKIs should be solved. We have been reported that insufficient concentration of TKI treatment would lead to RCC cell proliferations. We investigated the role of IL-6 in cellular tumor biology of RCC cells treated by TKIs. METHODS: Human renal cell carcinoma cell lines 786-O, Caki-1, Caki-2, CCF-RC1, A498 and Dibo were used for this study. Sorafenib, sunitinib and pazopanib at concentrations of 0.5, 1.0, 5.0, 10.0 mM were used as TKIs. Cytokine IL-6, IL-10, IL-1 beta, IL-1 ra, IL-17, IL-19, IL-23, IL-18BPa and Cripto-1, and growth factors VEGF,
INTRODUCTION AND OBJECTIVES: The histone deacetylase (HDAC) inhibitor belinostat suppresses heat shock protein 90 by inhibiting HDAC6, and this increases the amount of unfolded proteins in the cell. If the proteasome functions normally, however, these unfolded proteins are ubiquitinated and rapidly degraded. We thought that combining the proteasome inhibitor bortezomib with belinostat would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. METHODS: Renal cancer cells (769-P, 786-O, ACHN) were treated with belinostat (0.5-5 mM) and/or bortezomib (5-10 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. Combination indexes (CI) were calculated using the Chou-Talalay method. Flow cytometry was used for annexin-V assay and cell cycle analysis. The pancaspase inhibitor Z-VAD-FMK was used to evaluate whether the combination-induced apoptosis was caspase dependent. Induction of the unfolded protein response and the expression of ubiquitinated proteins and cleaved poly(ADP-ribose) polymerase were evaluated by western blotting. RESULTS: Bortezomib in combination with belinostat inhibited the growth of renal cancer cells synergistically (CI <1) and colony formation significantly (P <0.05). The combination perturbed the cell cycle, leading to the accumulation of the cells in the sub-G1 fraction (up to 63.8%). The combination induced drastic apoptosis and, because ZVAD-FMK changed the number of annexin-V-positive cells (decreasing them in 769-P cells and increasing them in 786-O and ACHN cells), this apoptosis was thought to be caspase dependent. Mechanistically, the
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
then tumor infiltrating lymphocyte was analyzed by immunohistochemistry. RESULTS: All three treatment groups ((1), (2) and (3)) showed significant decrease in tumor size compared with vehicle group. In particular, combination of PD-1 Ab and CTLA-4 Ab exhibited the most significant anti-tumor effect. The percentage of PCNA+ tumor cells were very similar amongst all therapies, but the percentage of Ki-67+ tumor cells were significantly decreased with combination of PD-1 Ab and CTLA-4 Ab. Combination of PD-1 Ab and CTLA-4 Ab demonstrated the most potent effect to trigger the infiltration of both CD4+ lymphocyte and CD8+ lymphocytes. These in vivo results indicate that combination of PD-1 Ab and CTLA-4 Ab may have a significant immunostimulatory effect to exert the anti-tumor effect to renal cell carcinoma. CONCLUSIONS: Combination therapy of anti-CTLA-4 antibody and anti-PD-1 antibody can be a potential therapeutic option for advanced kidney cancer patients.
e373
Leptin, HGF, HB-EGF and EGF secretions from RCC cells cultured with TKIs were measured by VersaMAP Development System. Western blot analyses were applied for detection of phosphorylated STAT3, ERK Akt, mTOR and NFkB proteins. Alterations of cell proliferation and IL-6 signaling pathway by humanized IL-6R antibody Tocilizumab was analyzed by BrdU assay, MTT assay and Western blot analysis. RESULTS: Among the measured cytokines, all the RCC cell lines secreted IL-6 when they were cultured with TKIs. IL-10, IL-23, HB-EGF and leptin were decreased by TKI treatment. Among the RCC cell lines, 786-O and Dibo showed high level of IL-6 production 1 hour after TKIs treatment. VEGF secretion was also observed when the 786O cells were treated by TKIs. In 786-O cells, Western blot analysis revealed that STAT3 and ERK were phosphorylated by TKI treatment even at a low concentration of 0.5 mM. Furthermore, Akt and mTOR were also activated by TKIs treatment. HIF2a expression and phosphorylation of NFkB which would lead to VEGF and IL-6 expression were also observed at the same concentration of TKIs. Tocilizumab treatment in combination with TKIs reduced activation of IL-6 signaling pathway and also suppressed cell proliferation. VEGF secretion following 1 hour TKIs treatment were 3-fold enhanced compared with non treated cells, and the VEGF secretion from RCC cells decreased to 40% by blocking of IL-6 receptor by tocilizumab. CONCLUSIONS: Our results indicate that TKI treatment induces IL-6 secretion in concentration under therapeutical level on RCC cells. Autocrine secretion of IL-6 activates STAT3 and Akt-mTOR pathway and it leads to VEGF secretion. IL-6 could be a possible target for overcoming the development of resistance to TKIs. Source of Funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No.22591773, No.25462492)
MP35-09 BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT TO CAUSE UBIQUITINATED PROTEIN ACCUMULATION IN RENAL CANCER CELLS Takako Asano*, Akinori Sato, Makoto Isono, Keiichi Ito, Tomohiko Asano, Tokorozawa, Japan
Source of Funding: none
MP35-08 TYROSINE-KINASE INHIBITORS TREATMENT INDUCES IL-6 SECRETION ON RENAL CARCINOMA CELLS Kei Ishibashi*, Yoshiyuki Kojima, Fukushima, Japan; Tobias Haber, €roff, Walburgis Brenner, Mainz, Germany Joachim W. Thu INTRODUCTION AND OBJECTIVES: Tyrosine-kinase inhibitors (TKI) treatment targeted at the vascular endothelial growth factor (VEGF) pathways represents the standard of care in advanced renal cell carcinoma (RCC). Although the TKI treatment has promising effect against RCC, the development of resistance to TKIs should be solved. We have been reported that insufficient concentration of TKI treatment would lead to RCC cell proliferations. We investigated the role of IL-6 in cellular tumor biology of RCC cells treated by TKIs. METHODS: Human renal cell carcinoma cell lines 786-O, Caki-1, Caki-2, CCF-RC1, A498 and Dibo were used for this study. Sorafenib, sunitinib and pazopanib at concentrations of 0.5, 1.0, 5.0, 10.0 mM were used as TKIs. Cytokine IL-6, IL-10, IL-1 beta, IL-1 ra, IL-17, IL-19, IL-23, IL-18BPa and Cripto-1, and growth factors VEGF,
INTRODUCTION AND OBJECTIVES: The histone deacetylase (HDAC) inhibitor belinostat suppresses heat shock protein 90 by inhibiting HDAC6, and this increases the amount of unfolded proteins in the cell. If the proteasome functions normally, however, these unfolded proteins are ubiquitinated and rapidly degraded. We thought that combining the proteasome inhibitor bortezomib with belinostat would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. METHODS: Renal cancer cells (769-P, 786-O, ACHN) were treated with belinostat (0.5-5 mM) and/or bortezomib (5-10 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. Combination indexes (CI) were calculated using the Chou-Talalay method. Flow cytometry was used for annexin-V assay and cell cycle analysis. The pancaspase inhibitor Z-VAD-FMK was used to evaluate whether the combination-induced apoptosis was caspase dependent. Induction of the unfolded protein response and the expression of ubiquitinated proteins and cleaved poly(ADP-ribose) polymerase were evaluated by western blotting. RESULTS: Bortezomib in combination with belinostat inhibited the growth of renal cancer cells synergistically (CI <1) and colony formation significantly (P <0.05). The combination perturbed the cell cycle, leading to the accumulation of the cells in the sub-G1 fraction (up to 63.8%). The combination induced drastic apoptosis and, because ZVAD-FMK changed the number of annexin-V-positive cells (decreasing them in 769-P cells and increasing them in 786-O and ACHN cells), this apoptosis was thought to be caspase dependent. Mechanistically, the