naphthoquinones and Carcinoma--Renal-Cell

To investigate the effect of dioxonaphthoimidazolium analog YM155 on cell cycle progression of the clear-cell variant of renal cell carcinoma (ccRCC).. Cell cycle analysis was performed using bromodeoxyuridine (BrdU) and PI, apoptosis initiation was monitored using Annexin V and proteins expression was determined using western immunoblotting.. Here, we showed that YM155 activated stress-related molecules (histone H2AX, checkpoint kinases Chk1 and Chk2, p53) that mediate DNA damage checkpoint responses. The coordinated activation of these effector molecules disrupts progression of the cell cycle at the S phase as deduced from BrdU pulsing experiments and the ensuing changes in the levels of proteins (cyclins, CDKs, CDK inhibitors, phosphatases) that control cell cycle progression. Notably, we found increases in cyclin E and Cdc2 which regulate transition of cells from G1 to S, even as losses were observed for other CDKs and their cyclin partners. Furthermore, by inducing a loss in total pRb possibly by promoting its degradation, YM155 promoted the E2F transcription of genes that regulate entry into the S phase. After 24 h, cell cycle arrest to repair YM155-inflicted DNA damage was overtaken by p53-mediated apoptosis. YM155 induced increases in pro-apoptotic proteins (Bax and Bad), diminished anti-apoptotic proteins (Mcl-1, Bcl-xl, XIAP, survivin) and initiated cleavage of apoptotic marker proteins caspase 3 and PARP.. Taken together, the added insight provided on the cell cycle perturbative effects of YM155 may assist clinicians in framing rational choices for combining YM155 with other anti-cancer drugs or treatment modalities in ccRCC.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Humans; Imidazoles; Kidney Neoplasms; Naphthoquinones; Tumor Cells, Cultured

Constitutive activation of the NF-κB signaling cascade is associated with tumourigenesis and poor prognosis in many human cancers including RCC. YM155, a small molecule inhibitor of survivin, was previously shown to potently inhibit the viability of immortalized and patient derived renal cell carcinoma (RCC) cell lines. Here we investigated the role of NF-κB signaling to the anti-cancer properties of YM155 in RCC786.0 cells. YM155 diminished nuclear levels of p65 and phosphorylated p65 and attenuated the transcriptional competencies of the p65/p50 heterodimers. Accordingly, we found that YM155 diminished the transcription of NF-κB target gene survivin. Events that led to the interception of the nuclear translocation of p65/p50 were the activation of the deubiquinating enzyme CYLD by YM155, which led to the inhibition of IKKβ, stabilization of IκBα and retention of NF-κB heterodimers in the cytosol. Importantly, the suppressive effects of YM155 were time-dependent and observed at the 24 h time point, and not earlier. TNF-α, a stimulator of NF-κB signaling did not affect its inhibitory properties. The ability of YM155 to intercept a major transcriptional pathway like NF-κB, would have important ramifications on the pharmacodynamics effects elicited by this unusual molecule.

Topics: Carcinoma, Renal Cell; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Kidney Neoplasms; Naphthoquinones; NF-kappa B; Phosphorylation; Protein Transport; Signal Transduction; Survivin; Tumor Cells, Cultured

The dioxonapthoimidazolium YM155 is a survivin suppressant which has been investigated as an anticancer agent in clinical trials. Here, we investigated its growth inhibitory properties on a panel of immortalized and patient derived renal cell carcinoma (RCC) cell lines which were either deficient in the tumour suppressor von Hippel-Lindau (VHL) protein or possessed a functional copy. Neither the VHL status nor the survivin expression levels of these cell lines influenced their susceptibility to growth inhibition by YM155. Of the various RCC lines, the papillary subtype was more resistant to YM155, suggesting that the therapeutic efficacy of YM155 may be restricted to clear cell subtypes. YM155 was equally potent in cells (RCC786.0) in which survivin expression had been stably silenced or overexpressed, implicating a limited reliance on survivin in the mode of action of YM155. A follow-up in-vitro high throughput RNA microarray identified possible targets of YM155 apart from survivin. Selected genes (ID1, FOXO1, CYLD) that were differentially expressed in YM155-sensitive RCC cells and relevant to RCC pathology were validated with real-time PCR and western immunoblotting analyses. Thus, there is corroboratory evidence that the growth inhibitory activity of YM155 in RCC cell lines is not exclusively mediated by its suppression of survivin. In view of the growing importance of combination therapy in oncology, we showed that a combination of YM155 and sorafenib at ½ x IC50 concentrations was synergistic on RCC786.0 cells. However, when tested intraperitoneally on a murine xenograft model derived from a nephrectomised patient with clear cell RCC, a combination of suboptimal doses of both drugs failed to arrest tumour progression. The absence of synergy in vivo highlighted the need to further optimize the dosing schedules of YM155 and sorafenib, as well as their routes of administration. It also implied that the expression of other oncogenic proteins which YM155 may target is either low or absent in this clear cell RCC.

Topics: Animals; Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Survival; Deubiquitinating Enzyme CYLD; Dose-Response Relationship, Drug; Drug Combinations; Forkhead Box Protein O1; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Inhibitor of Differentiation Protein 1; Kidney Neoplasms; Male; Mice; Mice, SCID; Naphthoquinones; Niacinamide; Phenylurea Compounds; Primary Cell Culture; Signal Transduction; Sorafenib; Survivin; Tumor Suppressor Proteins; Von Hippel-Lindau Tumor Suppressor Protein; Xenograft Model Antitumor Assays

The purpose of the present study was to clarify whether treatment with YM155, a novel small-molecule inhibitor of survivin, reverses statin resistance in statin-resistant renal cell cancer (RCC).. We induced simvastatin resistance in a renal clear cell carcinoma cell line (Caki-1-staR). In vitro and in vivo models were used to test the efficacy of YM155 and simvastatin.. Survivin gene expression was significantly stronger in Caki-1-staR cells than in its parent cells (Caki-1). In Caki-1-staR cells, YM155 significantly reduced expression of survivin gene and cell proliferation in a dose-dependent manner. Treatment with YM155 significantly reversed simvastatin resistance in Caki-1-staR cells. YM155 significantly inhibited the growth of Caki-1-staR tumors in a nude mouse tumor xenograft model. Furthermore, YM155 significantly enhanced the antitumor effects of simvastatin on Caki-1-staR tumors.. Our results indicate that inhibition of survivin by YM155 overcomes statin resistance in RCC cells.

Topics: Animals; Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Imidazoles; Inhibitor of Apoptosis Proteins; Kidney Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; RNA Interference; Simvastatin; Survivin; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

YM155, a small-molecule survivin inhibitor, has been reported for its anti-cancer activity in various cancer cells. In this study, we investigated the effect of YM155 to enhance TRAIL-mediated apoptosis in human renal carcinoma cells. We found that YM155 alone had no effect on apoptosis, however, combined treatment with YM155 and TRAIL markedly induced apoptosis in human renal carcinoma cells (Caki, ACHN, and A498), breast cancer cells (MDA-MB231), and glioma cells (U251MG), but not normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. YM155 induced down-regulation of Mcl-1 expression at the post-translational levels, and the overexpression of Mcl-1 markedly inhibited YM155 plus TRAIL-induced apoptosis. Furthermore, YM155 induced down-regulation of c-FLIP mRNA expression through inhibition of NF-κB transcriptional activity. Ectopic expression of c-FLIP markedly blocked YM155-induced TRAIL sensitization. Taken together, our results suggested that YM155 sensitizes TRAIL-mediated apoptosis via down-regulation of Mcl-1 and c-FLIP expression in renal carcinoma Caki cells.

Topics: Apoptosis; Carcinoma, Renal Cell; CASP8 and FADD-Like Apoptosis Regulating Protein; Cathepsins; Cell Line, Tumor; Down-Regulation; Flow Cytometry; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Kidney Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; NF-kappa B; Recombinant Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Survivin; TNF-Related Apoptosis-Inducing Ligand

Elevated expression of the antiapoptotic factor survivin has been implicated in cancer cell survival and disease progression. However, its specific contribution to renal cell carcinoma (RCC) pathogenesis is not well defined. We investigated the roles of survivin in RCC tumor progression, resistance to mTOR inhibitors, and evaluated the therapeutic activity of the survivin suppressant YM155 in RCC models. Here, we report that survivin expression levels were significantly higher in RCC cell lines compared with normal renal cells. Stable targeted knockdown of survivin completely abrogated the ability of 786-O RCC tumors to grow in mice, thus demonstrating its importance as a regulator of RCC tumorigenesis. We next explored multiple strategies to therapeutically inhibit survivin function in RCC. Treatment with the mTOR inhibitor temsirolimus partially diminished survivin levels and this effect was augmented by the addition of YM155. Further analyses revealed that, in accordance with their combined anti-survivin effects, YM155 significantly improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in xenograft models in vivo. Similar to pharmacologic inhibition of survivin, shRNA-mediated silencing of survivin expression not only inhibited RCC tumor growth, but also significantly sensitized RCC cells to temsirolimus therapy. Subsequent experiments demonstrated that the effectiveness of this dual survivin/mTOR inhibition strategy was mediated by a potent decrease in survivin levels and corresponding induction of apoptosis. Our findings establish survivin inhibition as a novel approach to improve RCC therapy that warrants further investigation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Renal Cell; Cell Proliferation; Cell Survival; Disease Progression; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Imidazoles; Immunoblotting; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Kidney Neoplasms; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sirolimus; Survivin; Xenograft Model Antitumor Assays

YM155, a small molecule inhibitor of the antiapoptotic protein survivin, has been developed as a potential anti-cancer drug. We investigated a combination therapy of YM155 and interleukin-2 (IL-2) in a mouse model of renal cell carcinoma (RCC). YM155 caused cell cycle arrest and apoptosis in renal cancer (RENCA) cells. Next, luciferase-expressing RENCA cells were implanted in the left kidney and the lung of BALB/c mice to develop RCC metastatic model. In this orthotopic renal and metastatic lung tumors models, YM155 and IL-2 additively decreased tumor weight, lung metastasis, and luciferin-stained tumor images. Also, the combination significantly suppressed regulatory T cells and myeloid-derived suppressor cells compared with single agent treatment. We suggest that a combination of YM155 and IL-2 can be tested as a potential therapeutic modality in patients with RCC.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Renal Cell; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cell Separation; Disease Models, Animal; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Interleukin-2; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Naphthoquinones; Neoplasm Metastasis; Neoplasm Transplantation; Survivin; T-Lymphocytes, Regulatory

Mammalian target of rapamycin inhibitor has exhibited promising anticancer activity for the treatment of renal cell carcinoma (RCC). However, many patients acquire resistance to therapeutic agents leading to treatment failure. The objective of this study was to determine whether treatment with YM155, a novel small molecule inhibitor of survivin, could reverse rapamycin resistance in a rapamycin-resistant RCC.. We induced a rapamycin-resistant clear cell carcinoma cell line (Caki-1-RapR). We showed that survivin gene expression was significantly up-regulated in Caki-1-RapR compared with that in its parent cells (Caki-1). Therefore, we hypothesized that targeting of survivin in Caki-1-RapR could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of rapamycin. We used both in vitro and in vivo models to test the efficacy of YM155 either as a single agent or in combination with rapamycin.. In Caki-1-RapR cells, YM155 significantly decreased survivin gene and protein expression levels and cell proliferation in a dose-dependent manner in vitro. In addition, YM155 treatment significantly reversed rapamycin resistance in cancer cells. In a nude mouse tumor xenograft model, YM155 significantly inhibited the growth of Caki-1-RapR tumor. In addition, YM155 significantly enhanced the antitumor effects of rapamycin in Caki-1-RapR tumor.. Our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of molecular target therapy in RCC.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Kidney Neoplasms; Mice; Mice, Nude; Naphthoquinones; Real-Time Polymerase Chain Reaction; Sirolimus; Survivin; Up-Regulation; Xenograft Model Antitumor Assays

Serial dilutions of standardised water, ethanol, and dichloromethane extracts of the stembark and fruits of Kigelia pinnata were tested for their growth inhibitory effects against four melanoma cell lines and a renal cell carcinoma line (Caki-2) using two different (MTT and SRB) assays. Lapachol, a possible constituent of these extracts, together with known therapeutic antineoplastic agents, was also tested in the same way. The IC50 of each extract was measured after extracts were diluted to 100 micrograms/ml in 1% ethanol or water. Significant inhibitory activity was shown by the dichloromethane extract of the stembark and lapachol (continuous exposure). Moreover, activity was dose-dependent, the extract being less active after 1 h exposure. Chemosensitivity of the melanoma cell lines to the stembark was greater than that seen for the renal adenocarcinoma line. In marked contrast, sensitivity to lapachol was similar amongst the five cell lines. Lapachol was not detected in the stembark extract.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Renal Cell; Cell Line; Humans; Kidney Neoplasms; Melanoma; Naphthoquinones; Plants, Medicinal; Tissue Extracts; Tumor Cells, Cultured; Vincristine