1,25-Dihydroxyvitamin D3 (1,25D3) exhibits potent antitumor activity in the murine
squamous cell carcinoma (SCC) SCCVII/SF, and the combination of 1,25D3 with
cisplatin (1,25D3/
cisplatin) demonstrates even greater activity. Because these agents possess different mechanisms of cytotoxicity, studies were initiated to define the mechanism by which the combination displays enhanced activity. Median dose-effect analysis demonstrates that 1,25D3 and
cisplatin act synergistically to inhibit SCC growth. When SCC cells were treated with 1,25D3 (10 nM) and/or
cisplatin (0.5 microg/ml), greater
caspase-3 activation was observed for the combination than for either agent alone. This suggests that the enhanced cytotoxicity is, at least in part, due to greater induction of apoptosis. No alterations in cellular
platinum concentration or
platinum-
DNA adducts were observed for 1,25D3/
cisplatin cotreatment compared with
cisplatin treatment alone. Effects of the combination on
cisplatin and 1,25D3 signaling pathways in adherent (nonapoptotic) and floating (apoptotic) cells were explored.
Cisplatin induced p53 and its downstream targets, p21(Cip1) (p21) and Bax, in both cell populations. In contrast, 1,25D3 reduced p53, p21, and Bax to nearly undetectable levels in adherent cells. In the floating cells, 1,25D3 reduced levels of p53 and p21, but Bax expression was maintained at control levels. Expression of these
proteins in cells treated with 1,25D3/
cisplatin was similar to treatment with 1,25D3 alone. The two agents also had divergent effects on survival and stress signaling pathways. Phospho-
extracellular signal-regulated kinase 1/2 and phospho-Jun levels increased
after treatment with
cisplatin but decreased
after treatment with 1,25D3 and 1,25D3/
cisplatin. Moreover,
cisplatin decreased levels of
mitogen-activated protein kinase kinase kinase (MEKK-1), whereas 1,25D3 up-regulated MEKK-1, and 1,25D3/
cisplatin further up-regulated MEKK-1. We propose that the increased cytotoxicity for 1,25D3/
cisplatin results from
cisplatin enhancement of 1,25D3-induced apoptotic signaling through MEKK-1.