GRP75-Mediated Mitochondria-Endoplasmic Reticulum Contacts Modulate Mitochondrial Ca2+ Signalling and Apoptotic Sensitivity to Sorafenib in HepG2 Cells
Year: 2026
Type: preprint
Abstract: Background Systemic treatment for hepatocellular carcinoma (HCC) remains limited by incomplete therapeutic responses and dysregulated apoptosis. Mitochondria-endoplasmic reticulum contacts (MERCs) coordinate Ca2 + signalling, mitochondrial function and cell-death control. GRP75, encoded by HSPA9 and also known as mortalin, is a molecular chaperone in the IP3R-GRP75-VDAC1 complex. However, whether GRP75-mediated MERC remodelling directly links mitochondrial Ca2 + responses to sorafenib sensitivity in HCC cells remains unclear. Methods Transmission electron microscopy (TEM) was used to compare MERC morphology between HepG2 cells and non-malignant THLE-2 liver epithelial cells. HSPA9 mRNA expression and its association with survival were analysed in the TCGA-LIHC cohort. GRP75-overexpressing and GRP75-knockdown HepG2 cells were assessed for MERC morphology, mitochondrial Ca2 + transients measured with Rhod-2 AM, JC-1-labelled mitochondrial membrane potential, Annexin V-FITC/PI apoptosis, apoptotic marker expression and Caspase-3/7 activity. A HepG2 xenograft model, together with HSPA9/GRP75 qPCR and immunohistochemistry, was used to examine the relationship between GRP75 knockdown, endpoint tumour burden, and sorafenib-associated apoptotic markers in vivo. Results Compared with THLE-2 cells, HepG2 cells showed a greater fraction of the mitochondrial perimeter occupied by MERCs and a shorter mean contact distance. In TCGA-LIHC, HSPA9 mRNA was upregulated in HCC tumour tissues and was associated with worse overall survival. GRP75 overexpression increased MERC contact length and reduced mean contact distance, whereas GRP75 knockdown had the opposite effect. Rhod-2 AM assays showed enhanced mitochondrial Ca2 + transients in GRP75-overexpressing cells and weaker transients in GRP75-knockdown cells. Upon sorafenib challenge, GRP75 knockdown increased apoptosis, aggravated mitochondrial membrane-potential loss, increased Bax and cleaved caspase-3 expression, reduced Bcl-2 expression and elevated Caspase-3/7 activity. Conversely, GRP75 overexpression conferred a relative protective effect against sorafenib-induced mitochondrial injury. In the HepG2 xenograft model, GRP75 knockdown was associated with lower final tumour burden and reduced Ki-67 immunoreactivity. Combined treatment with sorafenib further increased cleaved caspase-3 immunoreactivity. Conclusions GRP75 expression was associated with MERC remodelling in HepG2 cells and parallel alterations in mitochondrial Ca2 + transients, mitochondrial membrane-potential maintenance and apoptotic sensitivity to sorafenib. Together, the in vitro and in vivo data suggest that the GRP75-MERC axis primes the mitochondrial apoptotic threshold following drug exposure. Additional cell models, GRP75 rescue experiments and dynamic Ca2+-flux analyses are needed to establish the causal mechanism.
Source: Research Square
Institution: Guangxi University of Chinese Medicine
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7225610766