P27 Disrupts Mitochondrial Function and Induces Apoptosis in OSCC

Objectives: Oral squamous cell carcinoma (OSCC) is a deadly disease with 45,000 new cases reported annually and no efficacious therapies for advanced stage disease Our previous studies demonstrated Transient Receptor Potential Vanilloid Subtype 1 (TRPV1) agonist, polygodial, has significant anti-tumor effects against OSCC mouse xenografts. Furthermore, we developed two novel analogs, P3 and P27. P27 proved to be more potent than the parent compound with no observable adverse effects in vivo. We hypothesize that P27 induces free radicals and subsequent apoptosis by disrupting mitochondrial function in OSCC. Objective: To delineate the anti-cancer mechanism-of-action of P27.
Methods: Mitochondrial membrane depolarization studies were performed to determine effects on mitochondrial function. Staining with Mito-tracker red was used to analyze mitochondrial subcellular localization following treatment with P27. Apoptosis was evaluated by western blot analysis of cleaved PARP (cPARP). MTS assays were performed to determine anti-proliferative effects of P27 across a panel of cancer cell lines including OSCC. Pre-treatment with the TRPV1 antagonist capsazepine (CPZ) was performed to assess if TRPV1 activation is a potential mechanism-of-action of P27. In addition, pre-treatment with the anti-oxidant N-acetyl-cysteine (NAC) was performed to assess if free radicals play a role in P27 anti-proliferative effects.
Results: P27 significantly reduced cell proliferation in all cancer cell lines tested with a GC₅₀ between 2 and 75 μM depending on the cell line. Pre-treatment with CPZ had no effect on P27 actions however NAC dramatically reversed the effects of P27 in Cal27 OSCC cells. Mitochondrial staining using MitoTracker red revealed transient perinuclear localization following P27 treatments. In addition, we found a significant dose-dependent depolarization of the mitochondrial membrane potential (p<0.01) and induction of apoptosis in response to P27.
Conclusions: P27 has significant anti-proliferative effects against OSCC that is not mediated by TRPV1 activation. P27 induces mitochondrial membrane potential depolarization in OSCC and subsequent apoptosis.