Resting membrane potential (RMP) and intracellular Ca2+ concentration [(Ca2+)i] are involved in tumorigenesis and metastasis. The present study investigated whether functional cardiac Na+ channels are expressed in human melanoma cells (WM 266-4) and its nonmalignant human melanocytes (HMC), as well as whether they participate in RMP maintenance and Ca2+ homeostasis. Confocal microscopy and western blot analysis were used to detect Na+ channels. The patch-clamp technique was employed to record Na+ currents and action potentials. Cytoplasmic Ca2+ was measured by loading Fluo-4. Cardiac (Nav1.5) Na+ channels were expressed in HMCs and WM 266-4 cells. Tetrodotoxin (TTX) dose-dependently blocked Na+ currents in WM 266-4 while HMCs had no Na+ currents. Ultraviolet light induced similar action potentials in HMCs and WM 266-4 cells, which were abolished by transient receptor potential A1 channel-specific blocker, HC-030031. Compared with HMCs, RMP was substantially depolarized in WM 266-4. TTX hyperpolarized RMP in WM 266-4 cells at a concentration of 30 µM, which facilitated Ca2+ influx. Compared with HMCs, (Ca2+)i was significantly higher in WM 266-4 cells and was elevated by 30 µM TTX. Collectively, Cardiac Na+ channels depolarize RMP and inhibit Ca2+ uptake in melanoma cells possibly contributing to tumorigenesis and metastasis. Na+ channel agonists may be developed to treat melanoma such as WM 266-4.

Ovarian cancer remains the most prevalent type in gynecological cancer, with drug resistance still being a challenge. Our previous studies have demonstrated that ovarian cancer cells are resilient due to their active EGFR and AKT/mTOR pathways. Also, abundant data suggests that similar to other cancer types, ovarian cancer cells grow and proliferate remarkably well under hypoxia situation. We hypothesize that the application of oxygen to ovarian cancer cells may render unfavorable conditions, thus leading to the enhanced cell death, when combined with chemotherapy drugs. Recent studies have shown that polydimethylsiloxane (PDMS)‐encapsulated calcium peroxide, or PDMS‐CaO2, potently deliver oxygen and eliminate hypoxia‐induced cell dysfunction and cell death in normal pancreatic cells and other types of normal cells. In this study, we followed the published protocol, produced PDMS‐CaO2 disks and tested in vitro the effects of released oxygen on cultured ovarian cancer cells (CaOV3 cells). The results showed that our manufactured disks sustainably release O2 in a time dependent manner. MTT assay results indicated that PDMS‐CaO2 disks induce cell death in an oxygen dependent manner, compared to PDMS disks only. Combination of inhibitors of EGFR, MEK/ERK and AKT/mTOR with PDMS‐CaO2 disks increases cell death. Interestingly, PDMS disks significantly enhance the effects of cisplatin, oxaliplatin and doxorubicin, but not that of paclitaxel or taxol. PDMS‐CaO2 disks inhibit exosome inhibitor GW4869 induced cellular proliferation. Also, those disks significantly augment ionophore monensin A (for Na+/H+ antiporter), and calcium ionophore A23187‐induced cell death. Confocal microscope data showed that PDMS‐CaO2 disks alter mitochondria activities. Collectively, our data suggests that PDMS‐CaO2 disks releasing O2 and inducing cell death may affect cell membrane ion channels and mitochondria activities. The combination of cancer drugs or ionophores or EGFR/MEK/AKT/mTOR inhibitors with PDMS‐O2 disks may provide novel approaches for ovarian cancer clinical management.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Our previous studies have demonstrated that ultraviolet radiation‐induced skin photo‐aging is associated with collagen degradation, dehydration, and inflammation. While the cellular and molecular mechanisms through which UV induces skin aging are still being unraveled, active compounds, including those natural products, are being sought to attenuate UV‐induced skin cell damage and inflammation. Recent studies have shown that maple extracts having active ingredients may have beneficial effects for the development of cosmetics products. In this study, five samples including black cumin extracts (BCE), thymoquinone (TQ, a major component of black cumin), Maple syrup extracts (MSX), Maple leave extracts (MAP), gennalin A (GA), were investigated whether those abstracts or compounds have effects on cytokine‐induced activation of NFkB pathway, which is critical for UV‐induced skin inflammation and skin aging. First, we used MTT assay to determine the proper concentration in cultured skin keratinocytes. We found that 5 mg/ml is the concentration that does not affect cellular proliferation. Second, we treated both keratinocytes and fibroblasts with TNFa and IL1b (10 ng/ml) and measured the degradation of IkB and NFkB subunit, p65 translocation from cytoplasm to nucleus, using confocal microscope and Western blot analysis. The results showed that both TNFa and IL1b induce IkB degradation and p65 translocation in both types of skin cells, while fibroblasts are more responsive to TNFa and IL1b. Under UV radiation, keratinocytes are activated and release cytokines that further activate skin fibroblasts. Third, we tested the effects of all five extracts or compounds on TNFa and IL1b p65 translocation and IkB degradation by confocal and Western blot. The results showed that p65 translocation was inhibited after administering 10 mg/ml of the BCE, and inhibition improved as the dosage of BCE increased. IkB level was elevated at the following dosages of BCE 25 and 50 mg/ml. In addition, p65 translocation appeared to be inhibited exclusively at 50 mg/ml in fibroblast cells. IkB level was observed to be consistent as the dosage of BCE increased, suggesting that BCE may block IkB degradation in fibroblast cells, with a result of inhibition of p65 translocation. Other samples are still being further investigated. Taken all attained data together, we conclude that some of those tested extracts or compounds may be added to the cosmetics products to attenuate or prevent from UV‐induced skin cell damage and inflammation.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract

Melanoma can be one of the most aggressive and lethal forms of skin cancer. Despite the recent improvements in targeted therapies, many patients with advanced disease fail to achieve lasting tumor regression. Therefore, it is important to develop novel druggable targets that can be exploited to improve clinical outcome. Here we studied the role of c-CBL, an E3 ubiquitin ligase, in human melanoma. Employing Western blot analysis and quantitative real-time PCR in a panel of human melanoma cell lines (A375, G361, Hs-294T, SK-Mel-2, SK-Mel-28 and 451Lu), we found that c-CBL is strongly expressed in human melanoma cells at the protein and mRNA levels. Further, we determined c-CBL levels in clinical samples of melanomas and benign melanocytic nevi, using quantitative Nuance multispectral imaging. Compared to benign nevi, melanomas showed a trend toward higher c-CBL immunoreactivity. Small interfering RNA (siRNA)-mediated knockdown of c-CBL resulted in decreased proliferation, clonogenic survival and trans-well migration and invasion of melanoma cells. Furthermore, it also resulted in decreased cellular invasion in a 3D spheroid assay system. C-CBL and FAK are regulated by SRC, and FAK binds to SRC and GRB2. The c-CBL E3 ligase domain regulates receptor tyrosine kinase internalization through ubiquitination and its ring finger domain stabilizes the FAK-SRC-actin cytoskeleton thereby promoting cellular motility. C-CBL knockdown was associated with decreased protein and/or mRNA levels of SRC, FAK and GRB2. Taken together, we have provided evidence that c-CBL plays a role in proliferation, migration and invasion as well as modulation of the FAK-GRB2-SRC nexus. Additional mechanistic experiments and in-vivo studies are needed to further dissect the role of c-CBL in melanoma and determine the potential therapeutic benefit of its inhibition.