Evaluating the Cytotoxic Potential of 3-(2-(3,4 dimethoxyphenyl)-2-oxoethylidene) indolin-2-one) (RAJI) on Triple Negative Breast Cancer Cells

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive and treatment-resistant subtype of breast cancer (BC) that is a leading global malignancy. A novel drug candidate, 3-(2-(3,4-dimethoxyphenyl)-2-oxoethylidene)indolin-2-one (RAJI), was synthesized using piperidine, isatin, and 3,4-dimethoxy acetophenones. Although these components have established roles in various drug syntheses and malaria treatment, their anti-cancer potential remains underexplored. Hence, the RAJI was designed to bridge this gap.

Methods: The cytotoxic effects of RAJI on TNBC cell lines (MDA-MB-231 and MDA-MB-468) were evaluated using MTT assay, cell migration assay, apoptosis analysis (Annexin V), mitochondrial membrane potential tests, qRT-PCR, and tumor-induced mouse model evaluation.

Results: RAJI exhibited cytotoxicity against TNBC cells, with IC50 values of 20 and 25 µg/mL for MDA-MB-231 and MDA-MB-468 cells, respectively. It reduced cell migration and induced apoptosis, as evident from the cell populations in the early and late apoptotic stages. Mitochondrial membrane potential assays revealed mitochondrial depolarization and cellular stress. Gene expression analysis via RT-PCR revealed that RAJI significantly downregulated Akt, PTEN, mTOR (AKT/PI3K signaling), Cyclin D1, indicating the induction of apoptosis in MDA-MB-231 cells via modulation of apoptotic genes such as Bax and Bcl-2. In the in In-vivo analysis, RAJI significantly reduced tumor volume in BALB/c athymic nude mice implanted with MDA-MB-231 cells over four weeks, with no notable toxicity.

Conclusion: RAJI demonstrated potent anticancer activity, induced apoptosis, and reduced TNBC tumor progression by altering the Akt/PI3K pathway, making it a promising therapeutic candidate for breast cancer treatment.

Keywords: Apoptosis; Cell migration; Cytotoxicity; breast cancer; triple negative breast cancer.