ABSTRACT The ability of tumor cells to migrate and invade adjacent tissue is a key property underlying the metastatic process. To ensure greater deformability and to facilitate movement, migratory cells undergo multiple changes in biophysical parameters, including those of stiffness and membrane viscosity. However, reports on correlations between cell motility and stiffness, or between cell motility and membrane microviscosity are rather limited and conflicting. Here, using atomic force microscopy (AFM) and fluorescence lifetime imaging (FLIM), we have investigated alterations in the mechanical properties of cancer cells and in the microviscosity of their plasma membranes that are associated with the migration process. It was found that upon activation of migration either through a “wound healing” test or by inducing epithelial-mesenchymal transition, human colorectal cancer cells undergo profound biomechanical remodeling characterized by simultaneous decreases in cell stiffness and in plasma membrane microviscosity. Our findings, therefore, support the results of previous studies that have shown cell softening and membrane fluidization to be critical adaptive responses enabling cell movement and that these can be regarded as potential biomarkers of tumor cell motility, offering scope for identifying new therapeutic targets.

ABSTRACT Mutations in ATP8B1 cause a spectrum of cholestatic liver disease, ranging from Progressive-Familial-Intrahepatic-Cholestasis type-1 (PFIC1) to Benign-Recurrent-Intrahepatic-Cholestasis type-1 (BRIC1). Manifestations of PFIC1 include severe pruritus, jaundice, and liver damage. Extrahepatic features sometimes observed in PFIC1 include sensorineural hearing loss, diarrhea, pancreatitis, and short stature. ATP8B1 was shown to translocate phospholipids across the plasma membrane; however, expression of ATP8B1 in many tissues and the range of pathological manifestations in ATP8B1 deficiency suggest diverse physiological functions of ATP8B1, and pleiotropic mechanisms regulating its activity. Recent studies suggest that phosphoinositides, including PIP2 and PIP3, can function as regulators, substrates, and binding partners of ATP8B1. New research shows that ATP8B1 modulates host immune system by regulating cleavage of pyroptotic-executioner Gasdermin D (GSDMD), and inflammation-resolution pathways such as phagocytosis/efferocytosis. Further mechanistic insights can accelerate development of new therapies for restoring membrane integrity, reducing inflammasome activity, and correcting metabolic imbalances caused by ATP8B1 dysfunction.

ABSTRACT Previous studies demonstrated that integrase-strand-transfer-inhibitors (INSTIs) promote adipocyte differentiation, while nucleoside-reverse-transcriptase-inhibitors (NRTIs) tenofovir-alafenamide-fumarate (TAF) and tenofovir-disoproxil-fumarate (TDF), inhibit adipogenesis. NRTIs were shown to counteract the pro-adipogenic effects of INSTIs[6]. However, the effects of non-nucleoside-reverse-transcriptase-inhibitors (NNRTIs) and of the novel long-acting INSTI cabotegravir (CAB), on adipogenesis, alone or in combination with NRTIs or other INSTIs, remain unclear. This study aims to elucidate the impact of NNRTIs and recent INSTIs on adipogenesis. 3T3-L1 cells were used as an adipogenesis in vitro model. The NNRTIs doravirine (DOR) and rilpivirine (RPV) were tested alone and in combination with DTG, CAB, TDF, TAF. Adipogenesis was assessed by Oil-Red-O-staining and by measuring expression-levels of peroxisome-proliferator-activated-receptor-gamma (PPARγ) and CCAAT/enhancer-binding-protein-alpha (C/EBPα). Moreover, Fibroblast-marker ER-TR7 was assessed by immunohistochemistry. CAB, DOR, and RPV promoted adipogenesis, with CAB and DOR showing greater effects. In combination, NNRTIs enhanced the adipogenic effects of CAB and DTG. Conversely, TAF and TDF, when paired with RPV or DOR, inhibited adipogenesis. NNRTIs and CAB increased ER-TR7 expression, suggesting fibroblastic differentiation. Finally, NNRTIs and INSTIs promote adipogenesis and induce fibroblastic features in 3T3-L1 cells. Contrarily, TAF and TDF exhibited an antagonistic effect on adipogenesis when combined with certain antiretrovirals, supporting our previous research.

ABSTRACT Background: This study aimed to investigate the expression pattern of astrocyte-derived STC1 in TLE and elucidate the molecular mechanisms by which STC1 regulates neuroinflammation and seizures through the NF-κB signaling pathway. Methods: A TLE model was established by intrahippocampal injection of kainic acid (KA) in mice. STC1 expression levels and cellular localization in the hippocampus of TLE mice were examined. Adeno-associated virus-mediated gene overexpression and shRNA knockdown approaches were employed to investigate the effects of STC1 on neuroinflammatory responses, neuronal survival, and seizure activity. qRT-PCR and immunofluorescence methods were further utilized to evaluate inflammatory cytokine levels and NF-κB signaling pathway activity. Results: STC1 expression was upregulated in hippocampal tissues of TLE mice, with double immunofluorescence showing STC1 predominantly localized in GFAP-positive reactive astrocytes. STC1 overexpression significantly exacerbated KA-induced neuroinflammation, along with enhanced microglial activation. STC1 knockdown attenuated neuroinflammatory responses. Nissl staining and NeuN immunohistochemistry confirmed that STC1 aggravated KA-induced neuronal loss. STC1 overexpression promoted p65 phosphorylation and nuclear translocation, activating the NF-κB signaling pathway. Conclusion: This study reveals the molecular mechanism by which astrocyte-derived STC1 promotes TLE-associated neuroinflammation and neuronal injury through activation of the NF-κB signaling pathway, elucidating the crucial role of the astrocyte-STC1-NF-κB axis in epileptogenesis.

ABSTRACT Hepatocellular carcinoma (HCC) is characterized by high metastatic potential and poor prognosis. Ring finger protein 220 (RNF220) has been implicated in tumorigenesis across various cancers; however, its role and associated regulatory mechanisms in HCC remain unclear. In this study, analysis of The Cancer Genome Atlas (TCGA) database revealed that RNF220 expression was significantly elevated in liver hepatocellular carcinoma (LIHC) tissues and was associated with poor prognosis. Further experiments confirmed the upregulation of RNF220 mRNA and protein in HCC tissues. Functional assays demonstrated that RNF220 overexpression promoted cell proliferation, migration and stemness, whereas RNF220 knockdown suppressed these processes in HCC cells. Mechanistically, RNF220 enhanced ubiquitin-specific protease 22 (USP22) expression, leading to activation of the protein kinase B (Akt) pathway. Furthermore, the knockdown of RNF220 inhibited HCC progression, an effect that could be reversed by SC79 (an Akt activator), an Akt activator. In vivo experiments further confirmed that RNF220 aggravated tumor growth and metastasis. In summary, these findings indicate that RNF220 promotes HCC progression by regulating USP22 and activating the Akt pathway, suggesting that RNF220 may serve as a potential biomarker and therapeutic target for HCC.

ABSTRACT NRF2, a crucial antioxidant transcription factor in ovarian cancer (OC), is closely associated with CEBPB activation. However, the regulatory mechanism of NRF2 by CEBPB in OC remains poorly understood. In this study, we systematically evaluated the malignant behavior of SKOV3 and A2780 cells through comprehensive approaches, including CCK-8 kits, clone formation assays, and flow cytometry analysis. Cellular antioxidant capacity was quantitatively assessed using the DCFH-DA and total-antioxidant capacity (T-AOC) assays. Molecular mechanisms were investigated through multiple experimental approaches: the interaction between NRF2 and the DUSP1 promoter was examined using dual fluorescence reporter assays, while the activation status of CEBPB, NRF2, DUSP1, antioxidant proteins, and MAPK pathway components was analyzed via immunofluorescence and western blotting. Our findings demonstrate that CEBPB overexpression significantly enhanced cellular proliferation, clone formation, cell cycle progression, and antioxidant capacity, while simultaneously reducing apoptosis rates and reactive oxygen species (ROS) levels. Conversely, CEBPB knockdown or NRF2 inhibition produced opposing effects. These results establish that CEBPB-mediated NRF2 activation promotes OC cell proliferation and antioxidant defense mechanisms. Mechanistically, we identified that NRF2 directly binds to the DUSP1 promoter, as confirmed by dual-luciferase reporter assays. NRF2 activation led to upregulation of DUSP1 and phosphorylated ERK1/2 levels, while downregulating JNK and p38 phosphorylation. These findings were further validated in vivo, confirming that CEBPB activates NRF2 to regulate the MAPK pathway through DUSP1, thereby promoting OC cell proliferation and antioxidant capacity. In conclusion, our study reveals a novel regulatory axis in which CEBPB activates NRF2 to regulate the MAPK pathway via DUSP1, driving malignant progression and enhancing antioxidant activity in OC.

ABSTRACT Ultraviolet radiation is a major factor in causing skin aging. Compared to younger individuals, older adults exhibit a significant imbalance in the M1/M2 macrophage ratio, with an elevated proportion of M1 macrophages, but little is known about the role of macrophages in skin aging. Here, we report the critical role of M2 macrophages and PKM2 in preventing fibroblast photoaging. UVB-treated photoaged fibroblasts showed a reduction in PKM2. Compared to M1 macrophages, treatment with M2 macrophage significantly alleviated this photoaging and enhanced PKM2 synthesis in fibroblasts. Mechanistically, this is due to the secretion of CCL1 by M2 macrophages, which acts on the CCR8 receptor on the cell surface, promoting PKM2 production in photoaged fibroblasts. This further activates the TGF-β1/Smad2 pathway, thereby reducing cellular aging. This provides a potential strategy for the treatment of skin photoaging.

ABSTRACT α-Fodrin, a known scaffolding protein for cytoskeleton stabilization, performs various functions including cell adhesion, cell motility, DNA repair and apoptosis. Based on our previous results revealing its role in mitosis in glioblastoma, we have examined its effect in pancreatic cancer, which is often linked to mitotic aberrations including aneuploidy and chromosome instability. Here, we show that the expression of α-Fodrin increases in pancreatic adenocarcinoma tissues compared to its normal counterpart, suggesting its tumor promoting role. shRNA-mediated knock-down of α-Fodrin significantly reduces the xenograft growth in immunocompromised mice underscoring the importance of α-Fodrin in tumor progression. CENP-E (centromere-associated protein E) is a motor protein essential for chromosomal alignment and segregation during mitosis. We have found that α-Fodrin interacts with CENP-E to recruit it to the kinetochore and depletion of α-Fodrin has a crucial role in controlling aneuploidy. As these mitotic defects can lead to apoptosis, we have further evaluated the activation of possible upstream pathways. Paclitaxel, a chemotherapeutic agent that stabilizes microtubules, disrupts mitosis and induces apoptosis. We found that Paclitaxel triggered stronger activation of JNK, ERK, and P38 MAPKs, altered BCL2/BAX ratios, cytochrome C release causing increased apoptosis in α-Fodrin knockdown cells compared to cells with wild-type α-Fodrin. This enhanced sensitivity to paclitaxel is consistent with improved survival in pancreatic cancer patients with low α-Fodrin (SPTAN1) and low CENP-E expression compared to poor prognosis with high expressions of both the genes. Taken together, this study provides the molecular mechanism by which α-Fodrin – CENP-E axis regulates pancreatic cancer progression and drug response.