Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

Cancer Cell enters reversible quiescence through Intracellular Acidification to resist Paclitaxel Cytotoxicity.

Cancer cells can enter quiescent or dormant state to resist anticancer agents while maintaining the potential of reactivation. However, the molecular mechanism underlying quiescence entry and reactivation remains largely unknown. In this paper, cancer cells eventually entered a reversible quiescent state to resist long-term paclitaxel (PTX) stress. The quiescent cells were characterized with Na+/H+ exchanger 1 (NHE1) downregulation and showed acidic intracellular pH (pHi). Accordingly, decreasing pHi by NHE1 inhibitor could induce cell enter quiescence. Further, acidic pHi could activate the ubiquitin-proteasome system and inhibiting proteasome activity by MG132 prevented cells entering quiescence. In addition, we show that after partial release, the key G1-S transcription factor E2F1 protein level was not recovered, while MCM7 protein returned to normal level in the reactivated cells. More importantly, MCM7 knockdown inhibited G1/S genes transcription and inhibited the reactivated proliferation. Taken together, this study demonstrates a regulatory function of intracellular acidification and subsequent protein ubiquitination on quiescence entry, and reveals a supportive effect of MCM7 on the quiescence-reactivated proliferation.

Norcantharidin enhances antitumor immunity of GM-CSF prostate cancer cells vaccine by inducing apoptosis of regulatory T cells.

Norcantharidin (NCTD) is a promising antitumor drug with low toxicity. It was reported to be able to regulate immunity, but the mechanism is not yet clear. Here we explored whether NCTD could enhance the antitumor immunity induced by prostate cancer cell vaccine. The results of the in vitro study showed that NCTD induced apoptosis and inhibited proliferation of regulatory T cells (Tregs). Mechanistic research showed that NCTD inhibited Akt activation and activated FOXO1 transcription, resulting in a pro-apoptotic effect. The results of the in vivo study showed that more tumor-infiltrating Tregs existed within peripheral blood and tumor tissue after treatment with the vaccine. Adding NCTD to vaccine treatment could decrease the number of tumor-infiltrating Tregs and increase the number of CD4+ and CD8+ T cells. Combination therapy with NCTD and vaccine was more effective in inhibiting tumor growth than the vaccine alone. In general, this is the first report that NCTD could induce apoptosis of Tregs and enhance the vaccine-induced immunity.

Depletion of regulatory T cells by anti-ICOS antibody enhances anti-tumor immunity of tumor cell vaccine in prostate cancer.

ICOS+Treg cells exert important immunosuppressive effects in tumor immunity. We adopt a combination approach of ICOS+Treg cells depletion with tumor cell vaccine to evaluate anti-tumor immunity in mouse prostate cancer model. Streptavidin (SA)-mGM-CSF surface-modified RM-1 cells were prepared as the vaccine and the mouse subcutaneous prostate tumor model was used to evaluate the immunity. Tumor growth, flow cytometry, immunohistochemistry, immunofluorescence and enzyme linked immunosorbent assay (ELISA) were performed to evaluate the therapeutic effects. Our results demonstrated that SA-mGM-CSF vaccine was prepared successfully and tumor growth was inhibited. The tumor size in the combination group was much smaller than that in the vaccine with IgG mAb group. The portions of dendritic cells, CD8+ and CD4+T cells in the mice blood and tumor tissues were increased after treatment with vaccine. There were more immune-suppressing Tregs infiltrated into tumor after treatment with tumor cell vaccine, and ICOS blocking could deplete the infiltrated Tregs, and T lymphocytes increased more dramatically in the combination therapy group. The concentrations of interferon-γ were increased in all vaccine group, the concentrations of Interleukin-10 and Interleukin-4 were much lower in the combination group. Our study demonstrated that ICOS blocking could deplete the tumor-infiltrated ICOS+Treg cells. Combining GM-CSF surface-modified RM-1 cell vaccine with Anti-ICOS antibody could induce better antitumor immunity than a vaccine alone.

Pre-RC Protein MCM7 depletion promotes mitotic exit by Inhibiting CDK1 activity.

MCM7, a subunit of mini-chromosome maintenance proteins (MCM) complex, plays an important role in initiating DNA replication during the G1 phase and extending DNA strands during the S phase. Here, we demonstrated that MCM7 is not only sustained but maintains association with chromatin during M phase. Remarkably, MCM7 siRNA can accelerate mitotic exit. MCM7 depletion leads to CDK1 inactivation and promotes subsequent cohesin/RAD21 cleavage, which eventually leads to sister chromatin segregation. Moreover, MCM7 is co-localized with tubulin in the mitotic cells and MCM7 depletion results in aberrant mitosis. Our results indicate that MCM7 may exert certain functions on spindle formation to prevent cytokinesis during early mitosis by regulating CDK1 activity.

Cell Division Cycle 6 Promotes Mitotic Slippage and Contributes to Drug Resistance in Paclitaxel-Treated Cancer Cells.

Paclitaxel (PTX) is an antimitotic drug that possesses potent anticancer activity, but its therapeutic potential in the clinic has been hindered by drug resistance. Here, we report a mechanism by which cancer cells can exit from the PTX-induced mitotic arrest, i.e. mitotic slippage, and avoid subsequent death resulting in drug resistance. In cells experiencing mitotic slippage, Cdc6 protein level was significantly upregulated, Cdk1 activity was inhibited, and Cohesin/Rad21 was cleaved as a result. Cdc6 depletion by RNAi or Norcantharidin inhibited PTX-induced Cdc6 up-regulation, maintained Cdk1 activity, and repressed Cohesin/Rad21 cleavage. In all, this resulted in reduced mitotic slippage and reversal of PTX resistance. Moreover, in synchronized cells, the role of Cdc6 in mitotic exit under PTX pressure was also confirmed. This study indicates that Cdc6 may promote mitotic slippage by inactivation of Cdk1. Targeting of Cdc6 may serve as a promising strategy for enhancing the anticancer activity of PTX.

Cdc6 contributes to cisplatin-resistance by activation of ATR-Chk1 pathway in bladder cancer cells.

High activation of DNA damage response is implicated in cisplatin (CDDP) resistance which presents as a serious obstacle for bladder cancer treatment. Cdc6 plays an important role in the malignant progression of tumor. Here, we reported that Cdc6 expression is up-regulated in bladder cancer tissues and is positively correlated to high tumor grade. Cdc6 depletion can attenuate the malignant properties of bladder cancer cells, including DNA replication, migration and invasion. Furthermore, higher levels of chromatin-binding Cdc6 and ATR were detected in CDDP-resistant bladder cancer cells than in the parent bladder cancer cells. Intriguingly, down-regulation of Cdc6 can enhance sensitivity to CDDP both in bladder cancer cells and CDDP-resistant bladder cancer cells. Cdc6 depletion abrogates S phase arrest caused by CDDP, leading to aberrant mitosis by inactivating ATR-Chk1-Cdc25C pathway. Our results indicate that Cdc6 may be a promising target for overcoming CDDP resistance in bladder cancer.