MI-319

GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis. Our data demonstrate a complex partnership between GSK-3β and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.

Background/Aims: Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. Methods: Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. Results: Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. Conclusion: Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs.