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.

Figure 1

Cytotoxic Potential of RAJI in 3 Different Cell Lines, 2 TNBC cell lines - MDA-MB-231 & MDA-MB-468 and a normal epithelial breast cell line MCF 10A.

Figure 2

Live/Dead Staining of TNBC Cell Lines with Acridine Orange and Ethidium Bromide

Figure 3

Effect of RAJI in Cell Migration in the TNBC Cell Lines where the Invasion was Measured by Wound Healing Assay

Figure 4

RAJI Induced Cell Cycle Arrest in TNBC Cell Lines where Cells Stained with PI in Different Concentrations of RAJI were Analysed via Flow Cytometry

Figure 5

Flow Cytometric Analysis of Apoptosis by Staining Annexin-V-FITC in RAJI treated TNBC Cell Lines MDA-MB-231 & MDA-MB-468.

Figure 6

Flow Cytometric Analysis of ROS Levels in RAJI Treated TNBC Cell Lines MDA-MB-231 and MDA-MB-468.

Figure 7

RAJI Treated TNBC Cell Lines MDA-MB-231 and MDA-MB-468 Imaged under Fluorescent Microscope Post Hoechst and JC-1 Staining.

Figure 8

Estimation of Caspase 3/7 and 9 Activities in the RAJI treated TNBC Cell Line – MDA-MB-468.

Figure 9

Relative mRNA Expressions ofGenes (A) AKT1, (B) PTEN, (C) mTOR, (D) Cyclin D1, (E) BCl-2 and (F) Bax – Untreated, Doxorubicin and RAJI treated MDA-MB-231 cells.

Figure 10

(A) Macroscopic visualization of tumour size reduction post treatment. (B) Significant reduction in the tumour weight in all the treated groups in comparison to the untreated or control group. The statistical difference is demoted as * (p<0.05). (C) Significant reduction in the tumour volume in all the treated groups in comparison to the untreated or control group when observed on a weekly basis. The statistical difference is demoted as * (p<0.05).