Three-dimensional (3D) cell culture models are used in cancer research because they mimic physiological responses in vivo compared with two-dimensional (2D) culture systems. Recently, cross-resistance of butyrate-resistant (BR) cells and chemoresistance in colorectal cancer (CRC) cells have been reported; however, effective treatments for BR cells have not been identified. In this study, we investigated the cytotoxicity of metformin (MET), an anti-diabetic drug, on BR CRC cells in a 3D spheroid culture model. The results demonstrate that MET decreases spheroid size, migration, and spheroid viability, while it increases spheroid death. The molecular mechanism revealed that AMP-activated protein kinase (AMPK) and Akt serine/threonine kinase 1(Akt) were significantly upregulated, whereas the acetyl-CoA-carboxylase (ACC) and mammalian target of rapamycin (mTOR) were downregulated, which led to caspase activation and apoptosis. Our findings show the potential cytotoxicity of MET on CRC-BR cells. The combination of MET and conventional chemotherapeutic drugs should be addressed in further studies to reduce the side effects of standard chemotherapy for CRC.

Colorectal cancer (CRC) is the third most common cancer worldwide. The gut microbiota plays a critical role in homeostasis and carcinogenesis. Butyrate, a short‐chain fatty acid produced by the gut microbiota, plays a role in intestinal homeostasis and acts as an anticancer agent by inhibiting growth and inducing apoptosis. However, microbiota studies have revealed an abnormally high abundance of butyrate‐producing bacteria in patients with CRC and indicated that it leads to chemoresistance. We characterized butyrate resistance in HCT‐116 and PMF‐K014 CRC cells after treatment with a maximum butyrate concentration of 3.2 mM. The 50% inhibitory concentration of butyrate was increased in butyrate‐resistant (BR) cells compared with that in parental (PT) cells. The mechanism of butyrate resistance was initially investigated by determining the expression of butyrate influx‐ and drug efflux‐related genes. We found the increased expression of influx‐ and efflux‐related genes in BR cells compared with that in PT cells. Proteomic data showed both identical and different proteins in PT and BR cells. Further analysis revealed the crossresistance of HCT‐116 cells to metformin and oxaliplatin and that of PMF‐K014 cells to 5‐fluorouracil. Our findings suggest that the acquisition of butyrate resistance induces the development of chemoresistance in CRC cells, which may play an important role in CRC development, treatment, and metastasis.

Cervical cancer (CC) remains a major global health challenge due to chemotherapy resistance and recurrence. Mesenchymal stem cell-derived exosomes (MSC-exosomes) have dual roles, as they can act as therapeutic agents and contribute to chemoresistance. However, their role in response to chemotherapy in CC remains unclear. Therefore, our study investigated the effects of MSC-exosome pretreatment on chemotherapy sensitivity using three-dimensional spheroid models generated from HeLa and SiHa CC cell lines. Proteomic profiling of MSC-exosomes identified key proteins, including ANXA1, ANXA2, EEF2, LGALS1, and PKM2, associated with tumor regeneration and chemotherapy response. MSC-exosomes exhibited context-dependent effects in both chemoresistance and chemosensitization by modulating drug efflux, metabolic reprogramming, stress adaptation, apoptosis, DNA damage response, and integrin-mediated signaling. MSC-exosome pretreatment altered spheroid responses to paclitaxel in combination with cisplatin or carboplatin. MSC-exosomes significantly enhanced chemotherapy-induced cytotoxicity in HeLa spheroids, as evidenced by reduced cell viability, increased caspase activity, and upregulation of the pro-apoptotic marker Bax. In contrast, SiHa spheroids represented selective responses: MSC-exosome pretreatment did not enhance sensitivity to paclitaxel–cisplatin but improved responsiveness to paclitaxel–carboplatin, particularly within the spheroid core. Overall, MSC-exosome pretreatment exerts cell type and drug-specific effects in CC spheroids, supporting their potential to modulate chemotherapy response.

Background: Because of the limited efficacy of existing therapeutic strategies and the emergence of treatment resistance, cervical cancer remains a major global health burden. Recently, marine-derived natural compounds have attracted attention as potential sources of novel anticancer agents. We aimed to investigate the cytotoxic effects of crude Halimeda macroloba extract on cervical cancer spheroid cells and to explore its underlying molecular mechanisms. Methods: Three-dimensional spheroid cultures of SiHa cervical cancer cells were treated with varying concentrations of H. macroloba extract, followed by assessment of migration, cell viability, caspase activity, and protein expression profiles using liquid chromatography–tandem mass spectrometry, with selected findings validated by Western blotting. Results: Treatment with H. macroloba extract significantly reduced spheroid migration and decreased cell viability in a dose-dependent manner, accompanied by increased caspase activity. Proteomic analysis demonstrated altered expression of proteins associated with mitochondrial function, apoptosis-related pathways, and oxidative stress-related processes. Mechanistic studies further revealed downregulation of phosphorylated mTOR (p-mTOR) and total mTOR, together with increased expression of pro-apoptotic markers. Conclusion: The H. macroloba crude extract exerts anticancer effects in cervical cancer spheroids, primarily through apoptosis-associated mechanisms and modulation of the mTOR signaling pathway, supporting its potential for further investigation as a marine-derived bioactive compound.