Mechanism of antiproliferative activity of tolfenamic acid

Non-steroidal anti-inflammatory drugs have been shown to exhibit anti-tumor activities primarily through the inhibition of cyclooxygenases (COX-2). Among them, tolfenamic acid (TA) has also demonstrated potent anti-cancer effects across multiple cancer cell lines but acts through COX independent pathways. The proposed mechanism of action of TA involves its ability to target specificity protein transcription factors (Sp1, Sp3, and Sp4) which in turn downregulate potential oncogenic proteins such as survivin, VEGF, and c-MET. Recent work from our laboratory demonstrated, in medulloblastoma cells, the ability of TA to induce DNA double-strand breaks (DSBs). In this study we investigated the DNA damaging effects of TA across multiple cancer cell lines (including neuroblastoma, ovarian, breast, pancreatic, and prostate) and examined the activation of the DNA damage response pathways. Cell lines representing various cancers were treated with increasing concentration of TA for 48h and DNA damage assessed by monitoring phosphorylation of histone variant H2AX (gammaH2AX) by Western blot analysis. Activation of the DSB response following TA treatment, in a medulloblastoma cell line was investigated by examining the activation of ATM / ATR and Chk1 / Chk2 by Western blotting. Apoptosis was detected by cleavage of PARP-1 protein. Consistent with our findings in medulloblastoma, we observed a dose dependent increase in gammaH2AX, following TA treatment for 48h, in all cancer cell lines tested. Levels of cleaved PARP also increased with TA treatment in all cell lines. Furthermore, in medulloblastoma cells, gammaH2AX was detected as early as 24h post-treatment. We also demonstrated a dose dependent increase in phosphorylation of ATM and Chk2 proteins. Taken together, these results suggest that TA treatment induces DNA DSBs in cancer cells resulting in the activation of ATM and Chk2, a major DNA damage response pathway, ultimately leading to cell death via activation of apoptosis. The results highlight TA's multi-targeted effects and the potential to develop effective combination strategies with other small molecules or drugs to achieve synergistic therapeutic outcomes.