In-Vitro Study on the Mechanism of Antiproliferative and Apoptotic Effect of Teekshna Apamarga Kshara on Oral Squamous Cell Carcinoma (KB) Cells

Abstract

Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy worldwide, and India contributes nearly one-third of the global burden of oral cancer. Apamarga (Achyranthes aspera), a pan-tropical weed described in classical Ayurveda, is processed into Kshara, an alkaline preparation credited with Ksharana (excision, incision and scraping) properties capable of destroying morbid tissue while sparing normal cells. Objectives: To evaluate the anti-proliferative and apoptotic effect of Teekshna Apamarga Kshara on oral squamous cell carcinoma (KB) cells and to explore its underlying molecular mechanism. Materials and Methods: Apamarga Kshara was prepared as per classical reference and standardized physico-chemically. Its effect on KB cells was evaluated using the MTT assay for cell viability, Acridine Orange/Ethidium Bromide (AO/EtBr) dual fluorescent staining for apoptosis, flow cytometry for cell-cycle distribution, and quantitative real-time PCR (qRT-PCR) for expression of ERK1 and PCNA genes. Results: Apamarga Kshara produced a concentration-dependent decline in KB cell viability, from 91.44% at 6.25µg/mL to 58.90% at 100µg/mL, with an LC50 of 118.373µg/ml. AO/EtBr staining showed increased early and late apoptotic populations relative to control. Flow cytometry revealed an elevated S-phase fraction in treated cells (16.7%) compared to control (8.1%), indicating S-phase arrest. qRT-PCR showed downregulation of ERK1 (~47%) and PCNA (~15%) gene expression relative to control, confirmed by agarose gel electrophoresis. Conclusion: Teekshna Apamarga Kshara exerts a significant, concentration-dependent antiproliferative and apoptotic effect on KB oral carcinoma cells, plausibly mediated through suppression of the ERK1 pro-survival pathway and PCNA-dependent proliferation, supporting its potential as a botanical anticancer agent that warrants further mechanistic and in-vivo investigation.