MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53.,A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma.,However, there are no approved drugs, which could translate these new findings into clinical applications.,We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation.,We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing.,A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells.,They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing.,Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells.,We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX.,We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage.,On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX.,Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX.,Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.
Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF.,Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions.,Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels.,Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis.,Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression.,Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP.,To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector.,Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage.,Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.