Nitric Oxide Donors Sensitize Resistant Cancer Cells to Apoptosis Induced by Chemotherapy: Molecular Mechanisms of Sensitization

Abstract The role of nitric oxide (NO) in cancer has been the subject of many contradictory reports. Both NO-mediated protumorigenic effects and antitumorigenic effects have been reported. It has been proposed that the dual contradictory effects by NO may be due to both the levels and the tumor type. High levels of NO are shown to be antitumorigenic in several cancer cell lines examined. We and others have reported that high levels of NO donors sensitized chemotherapy-resistant cell lines to drug-induced cell death and apoptosis. Analyses were performed to delineate the underlying potential mechanisms of NO-mediated chemosensitization to various chemotherapeutic drugs. Treatment of resistant cell lines with NO donors (e.g., DETANONOate) resulted in the inhibition of the constitutively hyperactivated proliferative/antiapoptotic NF-κB signaling pathway. The NF-κB inhibition was the result of S-nitrosylation of p50 and p65 polypeptide chains of NF-κB. The inhibition of NF-κB by NO, thus, resulted in also the inhibition downstream of several antiapoptotic gene products. Further analyses identified in cancer cells a dysregulated NF-κB/Snail/YY1/RKIP/PTEN loop that was shown to regulate cell survival, cell growth, EMT/metastasis, and resistance to both chemo- and immunotherapeutic drugs. Treatment with the NO donors resulted in the inhibition of NF-κB, Snail, and Yin Yang 1 (YY1), an induction of the repressed Raf-1-kinase inhibitor protein (RKIP) and PTEN expressions. Hence, the treatment of drug-resistant cancer cells with NO resulted in the disruption of this loop on one hand and sensitization to drug-induced apoptosis on the other hand. We, then, examined the individual role of each of the gene products of the loop in the regulation of chemoresistance following NO treatment and their role in the reversal of resistance. The role of YY1 in chemosensitization was shown to result in both the inhibition of YY1 transcription (due to NF-κB inhibition) and the S-nitrosylation of YY1 that prevented its DNA-binding activity. Treatment of drug-resistant tumor cells with siRNA YY1 sensitized the cells to drug-induced apoptosis, mimicking the treatment with NO donors. The inhibition of Snail by NO was the result of both the inhibition of NF-κB and the inhibition of YY1 that also regulate Snail transcription. The role of Snail in the regulation of resistance was demonstrated by treatment of the cancer cells with siRNA Snail and mimicking NO-mediated chemosensitization. The induction of RKIP expression by NO treatment resulted from the inhibition of its repressor Snail. The direct role of RKIP in chemosensitization was shown following overexpression of RKIP in drug-resistant cells and resulting in chemosensitization similar to NO. Likewise, the induction of PTEN by NO was the result, in part, of the inhibition of its repressor YY1 by NO. The induction of PTEN inhibited the activity of the PI3K/AKT survival/antiapoptotic pathway and sensitized the cells to drug-induced apoptosis. Overall, treatment of drug-resistant cells with NO interfered with the dysregulated survival antiapoptotic NF-κB/Snail/YY1/RKIP/PTEN loop, and each modified gene product expression in the loop was involved in the regulation of chemoresistance. The sensitization of drug-resistant tumor cells by NO in vitro was corroborated in vivo in mice bearing human tumor xenografts treated with a combination of an NO donor and a chemotherapeutic drug. This treatment resulted in the inhibition of tumor growth and tumor tissues derived from the treated mice examined ex vivo showed the modification of the dysregulated loop that was observed in vitro with the cell lines. It is of interest, therefore, that the above findings in vivo and in vitro with tumor cell lines need to be validated in tumor specimens from patients treated with NO donors and chemotherapy. In addition, studies are warranted to examine the clinical significance of a number of NO donors in vivo in patients that have tumor-specific chemosensitizing activities and minimal toxic side effects. It is also suggested to design NO-drug conjugates that are specifically targeted to tumor cells via antibody/ligand incorporated into the NO-drug complex.