Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract

Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4222.