Molecular Pathways Affected by Sulfonylpurine Derivatives in 2D and 3D HeLa Cell Models

This study investigates two sulfonylpurine derivatives, Pur-6-NH₂-SS and Pur-6-Mor-SS, which contain amino and morpholino substituents, for their anticancer potential in 2D and 3D models of human cervical adenocarcinoma (HeLa) cells. Cell cycle distribution, apoptosis, mitochondrial membrane potential, and ROS accumulation were evaluated by flow cytometry. Both Pur-6-NH₂-SS and Pur-6-Mor-SS reduced the proportion of cells in the G0/G1 phase (to 39.65 ± 5.59% and 28.25 ± 1.20%, respectively) when compared with untreated cells. Pur-6-NH₂-SS additionally increased the proportion of cells in the S phase (7.41 ± 0.32%), whereas Pur-6-Mor-SS increased the number of cells in subG0 (21.05 ± 6.15%). Additionally, Pur-6-NH₂-SS triggered early apoptosis in 79.6 ± 8.5% of cells, accompanied by mitochondrial membrane depolarisation in 64.3 ± 9.0%. In comparison, Pur-6-Mor-SS elicited an even stronger apoptotic response, inducing early apoptosis in 87.4 ± 15.6% of cells and mitochondrial membrane potential disruption in 86.8 ± 9.0%, relative to untreated cells. RT-PCR analysis assessed the expression of key regulators, including miR-21, miR-210, and genes involved in survival and stress-response pathways (<i>Akt</i>, <i>CAIX</i>, <i>caspase-3</i>, and <i>cytochrome C</i>). In the 2D model, both derivatives increased <i>CAIX</i>, <i>Akt</i>, and <i>Cyp C</i> expression compared with untreated cells. In contrast, <i>p53</i> expression remained unchanged in Pur-6-NH₂-SS-treated cells and was slightly decreased in Pur-6-Mor-SS-treated cells. Casp3 expression was slightly elevated following Pur-6-NH₂-SS treatment and remained nearly unchanged in Pur-6-Mor-SS-treated cells. In the 3D model, Pur-6-NH₂-SS exerted a stronger inhibitory effect on <i>CAIX</i>, <i>Akt</i>, <i>p53</i>, <i>Cyp C</i>, and <i>Casp3</i> expression than Pur-6-Mor-SS, which showed weaker inhibition overall. Both derivatives had a comparable impact on miR-21 and miR-210 expression in 2D and 3D HeLa models. These findings provide mechanistic insight into amino- and morpholino-substituted sulfonylpurine derivatives and highlight how 2D and 3D tumour models influence drug response, offering a basis for further development of purine-based anticancer agents.