A novel soil bacterial heat shock extract enhances fingolimod chemosensitivity in colorectal cancer through modulation of PI3K/AKT/mTOR signaling and caspase-dependent apoptosis

Colorectal cancer (CRC) remains a major therapeutic challenge due to drug resistance, activation of survival pathways, and limited selectivity of conventional anticancer agents. Bacterial-derived bioactive molecules have recently emerged as promising biological modulators capable of enhancing anticancer responses. This study aimed to evaluate the chemosensitizing potential of a heat shock-derived extract from Heyndrickxia coagulans in combination with Fingolimod against HT-29 colorectal cancer cells. A soil-derived bacterial isolate was recovered from agricultural samples collected in East Azerbaijan Province, Iran, and identified by 16 S rRNA gene sequencing. The validated sequence was deposited in the NCBI GenBank database as Heyndrickxia coagulans strain Ab.369 TZ (Accession No. PZ576482). A heat shock-derived intracellular extract was prepared following thermal stress induction and sonication of bacterial cells. The antiproliferative effects of the extract, Fingolimod, and their combination were evaluated using the MTT assay. Molecular mechanisms were investigated by quantitative real-time PCR analysis of apoptosis, survival, inflammatory, and stress-related genes. Apoptotic cell death was confirmed by Annexin V-FITC/PI flow cytometry. The bacterial extract selectively inhibited HT-29 cell proliferation while exhibiting minimal toxicity toward HUVEC normal cells. Combination treatment markedly enhanced Fingolimod activity, reducing its IC₅₀ from 25.7 µg/mL to 138 ng/mL, corresponding to an approximately 186-fold reduction in the effective concentration. Molecular analysis demonstrated that the combination treatment promoted apoptotic signaling, enhanced tumor suppressor responses, and suppressed major survival and inflammatory pathways associated with colorectal cancer progression. Flow cytometry confirmed apoptosis induction, with 12.83% early apoptotic and 46.65% late apoptotic cells following combination treatment. Heat shock-derived Heyndrickxia coagulans extract enhances Fingolimod-mediated anticancer activity through coordinated regulation of apoptosis, survival signaling, and tumor-associated pathways. This bacterial-derived biological adjuvant represents a promising strategy for improving colorectal cancer therapy. Not applicable. Illustrating the proposed anticancer mechanism of the heat shock-derived Heyndrickxia coagulans extract in combination with Fingolimod against HT-29 colorectal cancer cells. Heyndrickxia coagulans strain Ab.369 TZ was isolated from soil, identified by 16S rRNA sequencing, and deposited in the NCBI GenBank database. Following heat shock treatment, the bacterial extract was evaluated alone or in combination with Fingolimod. The extract selectively inhibited HT-29 cell proliferation while exhibiting minimal cytotoxicity toward normal HUVEC cells. Combination treatment markedly enhanced Fingolimod chemosensitivity, reducing its IC₅₀ from 25.7 μg/mL to 138 ng/mL (approximately 186-fold reduction). Mechanistically, the combination activated both intrinsic (BAX, APAF1, CASP9, CASP3) and extrinsic (FAS, CASP8) apoptotic pathways, upregulated PTEN, P53, P21, MAPK (p38α), and IκB, and suppressed AKT, mTOR, BCL-2, BIRC5 (Survivin), and COX-2. Flow cytometry supported apoptosis as the major observed mode of cell death, with a predominance of Annexin V-positive populations and minimal primary necrosis. Overall, the heat shock-derived Heyndrickxia coagulans extract acts as a promising biological chemosensitizer that enhances the anticancer efficacy of Fingolimod through coordinated modulation of apoptosis and survival signaling pathways.