Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines.
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ABSTRACT:
SUBMITTER: Leroy K
PROVIDER: S-EPMC8623148 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAlthough metabolism is profoundly altered in human liver cancer, the extent to which experimental models, e.g. cell lines, mimic those alterations is unresolved. Here, we aimed to determine the resemblance of hepatocellular carcinoma (HCC) cell lines to human liver tumours, specifically in the expression of deregulated metabolic targets in clinical tissue samples.MethodsWe compared the overall gene expression profile of poorly-differentiated (HLE, HLF, SNU-449) to well-differentiated (HUH7, HEPG2, HEP3B) HCC cell lines in three publicly available microarray datasets. Three thousand and eighty-five differentially expressed genes in ?2 datasets (P?<?0.05) were used for pathway enrichment and gene ontology (GO) analyses. Further, we compared the topmost gene expression, pathways, and GO from poorly differentiated cell lines to the pattern from four human HCC datasets (623 tumour tissues). In well- versus poorly differentiated cell lines, and in representative models HLE and HUH7 cells, we specifically assessed the expression pattern of 634 consistently deregulated metabolic genes in human HCC. These data were complemented by quantitative PCR, proteomics, metabolomics and assessment of response to thirteen metabolism-targeting compounds in HLE versus HUH7 cells.ResultsWe found that poorly-differentiated HCC cells display upregulated MAPK/RAS/NFkB signaling, focal adhesion, and downregulated complement/coagulation cascade, PPAR-signaling, among pathway alterations seen in clinical tumour datasets. In HLE cells, 148 downregulated metabolic genes in liver tumours also showed low gene/protein expression - notably in fatty acid ?-oxidation (e.g. ACAA1/2, ACADSB, HADH), urea cycle (e.g. CPS1, ARG1, ASL), molecule transport (e.g. SLC2A2, SLC7A1, SLC25A15/20), and amino acid metabolism (e.g. PHGDH, PSAT1, GOT1, GLUD1). In contrast, HUH7 cells showed a higher expression of 98 metabolic targets upregulated in tumours (e.g. HK2, PKM, PSPH, GLUL, ASNS, and fatty acid synthesis enzymes ACLY, FASN). Metabolomics revealed that the genomic portrait of HLE cells co-exist with profound reliance on glutamine to fuel tricarboxylic acid cycle, whereas HUH7 cells use both glucose and glutamine. Targeting glutamine pathway selectively suppressed the proliferation of HLE cells.ConclusionsWe report a yet unappreciated distinct expression pattern of clinically-relevant metabolic genes in HCC cell lines, which could enable the identification and therapeutic targeting of metabolic vulnerabilities at various liver cancer stages.
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Project description:BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with beta2-adrenergic antagonist ICI 118,551 (100 microM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the beta-adrenergic agonist isoproterenol (10 microM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 microM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and beta-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results.
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Project description:Impaired expression of connexins, the gap junction subunits that facilitate direct cell-cell communication, have been implicated in prostate cancer growth. To elucidate the crucial role of connexins in prostate cancer progression, we performed a systematic quantitative RT-PCR screening of connexin expression in four representative prostate cancer cell lines across the spectrum of malignancy. Transcripts of several connexin subunits were detected in all four cell lines, and connexin 43 (Cx43) showed marked elevation at both RNA and protein levels in cells with increased metastatic potential. Analysis of gap-junction-mediated intercellular communication revealed homocellular coupling in PC-3 cells, which had the highest C x 43 expression, with minimal coupling in LNCaP cells where C x 43 expression was very low. Treatment with the gap junction inhibitor carbenoxolone or connexin mimetic peptide ACT-1 did not impair cell growth, suggesting that growth is independent of functional gap junctions. PC-3 cells with C x 43 expression reduced by shRNA showed decreased migration in monolayer wound healing assay, as well as decreased transwell invasion capacities when compared to control cells expressing non-targeting shRNA. These results, together with the correlation between C x 43 expression levels and the metastatic capacity of the cell lines, suggest a role of C x 43 in prostate cancer invasion and metastasis.
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Project description:Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),1 Lichtenstein et al. (2020),2 and Knebel et al. (2019).3.
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Project description:Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBP? was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.
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Project description:IntroductionGlioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43.Materials and methodsF98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ER?, ER? and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.ResultsE2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ER? was found to be high in C6, but low in F98 cells. ER? was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ER? in C6 cultures, while it decreased ER? expression in F98 glioma cells.DiscussionThese findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.
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