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Background: The increased risk of post-menopausal women developing abnormalities of heart function emphasises the requirement to understand the effect of declining oestrogen levels on cardiac electrophysiology and structure, and investigate possible therapeutic targets, namely the G protein-coupled oestrogen receptor 1 (GPER).Methods: Female guinea pigs underwent sham or ovariectomy (OVx) surgeries. Cardiomyocytes were isolated 150-days post-operatively. Membrane structure was assessed using di-8-ANEPPs staining and scanning ion conductance microscopy. Imunnohistochemistry (IHC) determined the localisation of oestrogen receptors. The effect of GPER activation on excitation-contraction coupling mechanisms were assessed using electrophysiological and fluorescence techniques. Downstream signalling proteins were investigated by western blot.Results: IHC staining confirmed the presence of nuclear oestrogen receptors and GPER, the latter prominently localised to the peri-nuclear region and having a clear striated pattern elsewhere in the cells. Following OVx, GPER expression increased and its activation reduced Ca2+ transient amplitude (by 40%) and sarcomere shortening (by 32%). In these cells, GPER activation reduced abnormal spontaneous Ca2+ activity, shortened action potential duration and limited drug-induced early after-depolarisation formation.Conclusion: In an animal species with comparable steroidogenesis and cardiac physiology to humans, we show the expression and localisation of all three oestrogen receptors in cardiac myocytes. We found that following oestrogen withdrawal, GPER expression increased and its activation limited arrhythmogenic behaviours in this low oestrogen state, indicating a potential cardioprotective role of this receptor in post-menopausal women.

Abstract CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization‐dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.

Abstract Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145FP). These DU145FP cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.

Our earlier study revealed that long-term ethidium bromide application causes mitochondrial DNA depletion in human prostate cancer DU145 cell line (DU145 MtDP ), and this DU145 MtDP subline appears to have expanded CD44 Bright cell population than its parental wild type DU145 cells (DU145 WT ). Increasing evidence suggests that CD44 Bright cells are highly cancer stem cell like, but it is not clear about their dynamic transition between CD44 Dim and CD44 Bright phenotypes in prostate cancer cells, and how it is affected by mitochondrial DNA depletion. To address these questions, four cell subpopulations were isolated from both DU145 WT and DU145 MtDP cell lines based on their CD44 expression level and mitochondrial membrane potential. The cell motility and colony formation capability of the fluorescence activated cell sorting–sorted cell subpopulations were further examined. It was discovered in the DU145 WT cells that CD44 Dim cells could transit into both CD44 Dim and CD44 Bright phenotypes and that CD44 Bright cells were prone to sustain their CD44 Bright phenotype as renewal. However, such transition principle was altered in the DU145 MtDP cells, in which CD44 Bright cells showed similar capability to sustain a CD44 Bright phenotype, while the transition of CD44 Dim cells to CD44 Bright were suppressed. It is concluded that mitochondrial DNA depletion in the human prostate cancer DU145 cells influences their renewal and CD44 subphenotype transition. Such alterations may be the driving force for the enrichment of CD44 Bright DU145 cells after the mitochondrial DNA depletion, although the molecular mechanisms remain unclear.