Cytotoxic and Pro‐Apoptotic Effects of the Compound 2‐Carboxy‐2‐Heptano‐Indolin‐3‐One in HeLa Cell Spheroids

ABSTRACT Indolinones are compounds with recognized antineoplastic properties and can act as inhibitors of multiple kinases. However, the antitumor effects of 2‐carboxy‐2‐heptano‐indolin‐3‐one (IDC) have not yet been investigated in cervical cancer models. This study investigated the cytotoxic and pro‐apoptotic effects of IDC in HeLa cells, with emphasis on a three‐dimensional spheroid model, and evaluated its effects on spheroid growth, cell cycle distribution, apoptosis, and the expression of genes associated with cell death and DNA damage responses. Cell viability was assessed in monolayer cultures using the resazurin assay, while spheroid growth and morphology were evaluated by microscopy and volumetric analysis. Apoptosis and cell cycle distribution were investigated by flow cytometry, and gene expression was assessed by RT‐qPCR. IDC reduced cell viability by up to 80% in monolayer cultures and significantly inhibited spheroid growth, with loss of structural integrity and absence of recovery after treatment withdrawal. Flow cytometry revealed 81.7–83.6% apoptotic cells following IDC treatment, compared with 35.8% in the vehicle control. No significant changes in G1, S, or G2/M cell cycle distribution were observed. RT‐qPCR showed increased mRNA levels of CASP8 , CASP9 , CASP3 , BBC3 , and GADD45A and decreased levels of TP53 , CDKN1A , and MYC . These transcriptional changes suggest the involvement of apoptotic signaling and DNA damage response mechanisms. Overall, IDC exhibited cytotoxic and pro‐apoptotic effects in HeLa cells and impaired three‐dimensional spheroid growth and integrity, supporting further investigation of its antineoplastic potential in cervical cancer.