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Helianthus Annuus L. (HAL) is composed of flavonoids and polysaccharides. Flavonoids have demonstrated beneficial effects on atherosclerosis (AS). The objective of this study was to investigate the anti-atherosclerosis effect and the related mechanism of HAL. In this study, the AS model induced by high-fat diet (HFD) mice that lacked apolipoprotein E (Apoe[Formula: see text] received feed containing 5% HAL for 24 weeks. After administration, the analysis of plaque on aorta was conducted, and the possible mechanisms were further explored. With HAL treatment, the size of atherosclerotic lesions in HFD-induced AS model mice was reduced. HAL ameliorated dyslipidemia and decreased the combined ratio. HAL up-regulated concentrations of superoxide dismutase (SOD), nitric oxide (NO) and glutathione peroxidase (GSH-Px) and down-regulated concentrations of malondialdehyde (MDA) in the aorta. In addition, 16S rRNA analysis showed that HAL also reduced diversity of the intestinal microbiota, decreased the Firmicutes-to-Bacteroidetes ratio, and increased the relative abundance of probiotics such as Akkermansia muciniphila and Lactobacillus. In the end, HAL decreased the permeability of intestine by increasing the levels of occludin and tight junction protein 1 (ZO-1) in the colon, consequently decreasing concentration of interleukin (IL)-6, IL-1[Formula: see text] and tumor necrosis factor-alpha (TNF-[Formula: see text] in serum and mRNA expressions in the aorta. Data showed that HAL alleviates AS by restraining oxidative stress, regulating intestinal microbiota, decreasing intestinal permeability and inhibiting inflammation. Our findings provided novel insights into the role and mechanism of anti-atherogenic potential of HAL.

Acquired perforating dermatoses (APDs) are a group of diverse skin disorders in patients with systemic disease, most commonly chronic renal failure and diabetes mellitus. APD induced by medication has seldom been reported. Anti-PD-1 monoclonal antibody has recently been used as a broad-spectrum, effective, durable, and relatively safe antitumor therapy for various malignancies. Thus far, known side effects involving skin have included rash, pruritus, and vitiligo. Here, we present a rare case of a unilateral linear eruption with histopathologic features of APD in a 36-year-old man during treatment with Terepril monoclonal antibody. To the best of our knowledge, APD induced by the PD-1 inhibitor has not been described in the medical literature.

Rationale Coronary artery ligation to induce myocardial infarction (MI) and ischemia injury in mice is typically performed in normal mice, but This is not consistent with disease progression. There should be atherosclerosis (AS) first, followed by MI. Objective We tried a novel model to induce MI that was established on atherosclerosis in mice. This approach was much more consistent with disease progression. Methods In this study, Mice lacking apolipoprotein E (ApoE−/-) were randomly divided into four groups. The mice of the control and MI groups were fed normal diet for 24-weeks, while the mice of AS and AS + MI groups were fed high-fat diet (HFD). After 23 weeks, the mice of MI and AS + MI groups were ligated with coronary arteries. A week later, after echocardiography, analysis of plaque and myocardium were conducted on aortic and heart, then the serum, aorta and heart tissues were further detected. Results Our results showed that AS model mice exhibited significant body weight gain, dyslipidemia and atherosclerotic lesions formation which were in accordance with the pathological changes of AS. Co-treatment with AS and MI led to higher operative mortality and heart pathological were in accordance with the pathological changes of MI. In addition, Echocardiography and NT pro-BNP revealed co-treatment with AS and MI led to deterioration of cardiac function. AS also aggravated myocardial inflammatory cell infiltration and fibrosis post-MI. Conclusions Together, it is feasible to establish myocardial infarction model based on atherosclerosis model.

Vitiligo is a skin depigmentation disorder with an increasing prevalence. Among recognized mechanisms is the oxidative stress that affects melanocytes which are responsible for skin pigmentation. Studies have shown that high concentration of hydrogen peroxide, or H2O2, induces apoptotic activities. Few studies have been done with lower doses of H2O2. Using human skin melanocytes, we investigated the effect of moderate concentration of H2O2 on melanocyte dendrites. Confocal data show that H2O2 at 250 µM induces loss of dendrites, as indicated by cytoskeletal proteins. α-melanocyte stimulating hormone or α-MSH pretreatment protects against H2O2-induced loss of dendrites, while α-MSH alone enhances dendrites. PI3K/AKT inhibitor LY294002 and mTORC1 inhibitor Rapamycin inhibit α-MSH-induced dendrites. In this study, we also investigated the effect of TNFα on cultured human skin melanocytes, since TNFα plays important roles in vitiligo. Confocal data demonstrate that TNFα induces NFκB activation. Western blot analysis shows that TNFα induces IκB phosphorylation and degradation. Interestingly, α-MSH does not have any effect of TNFα-induced IκB degradation and NF-κB activation. α-MSH, however, activates mTORC1 pathway. TNFα induces p38 but not AMPKα activation. Collectively, our data suggest that modulation of mTOR and NF-κB pathways may be a novel approach for better clinical management of vitiligo.

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 m...

This study investigated the role of LOX in promoting invasion and metastasis of epithelial ovarian cancer in a hypoxic environment and its specific signal transduction pathway. Immunohistochemical detection of HIF-1α and LOX protein expression was performed on formalin-fixed paraffin sections of normal ovary, benign ovarian tumors, borderline and malignant epithelial ovarian tumor paraffin sample, using Mann-Whitney U test for independent comparisons and Wilcoxon signed-ranks test for paired comparisons. HIF-1α and LOX were knocked down in epithelial ovarian cancer cells (EOC), and HIF-1α/LOX regulation mechanism and LOX catalytic activity under hypoxia/reoxygenation microenvironment were explored. Cell migration and invasion ability in LOX inhibited HO8910 cells were investigated under hypoxia/reoxygenation conditions, using matrigel cell invasion and migration assays. We found that HIF-1α and LOX are highly expressed in epithelial ovarian cancer tissues, and the expression of both proteins is significantly correlated with the tumor grade, tumor diameter and lymph node metastasis. HIF-1α expression is positively correlated with the expression of LOX. Specifically, the expression of LOX and HIF-1α markedly increases under hypoxic conditions and decreases after reoxygenation. siRNA knockdown of LOX or β-aminoproprionitrile (βAPN), an inhibitor of LOX activity, that attenuates LOX activity, downregulates HIF-1α protein expression and inhibits HO8910 migratory and invasive abilities. LOX catalytic activity is significantly reduced under hypoxic conditions. Moreover, EOC cells display a marked increase in LOX-dependent FAK/AKT activation and cell migration following hypoxia/reoxygenation. Collectively, our study demonstrates that the hypoxia-HIF-1α, LOX-FAK/AKT pathway regulates the migration and invasion of epithelial ovarian cancer cells under hypoxia/reoxygenation conditions, thus, promoting metastasis of ovarian cancer.

One of the major characteristics of human skin photoaging induced by ultraviolet (UV) radiation is the dehydration of the skin. Water movement across plasma membrane occurs via diffusion through lipid bilayer and via aquaporins (AQPs). We find that UV induces aquaporin-3 (AQP3) down-regulation in human skin keratinocytes. MEK/ERK inhibitors PD98059 and U0126 inhibit UV-induced down-regulation of AQP3. Antioxidant N-acetyl-L-cysteine or NAC blocks UV-induced MEK/ERK activation and down-regulation of AQP3. All-trans retinoic acid or atRA, while alone inducing AQP3 expression, attenuates UV-induced down-regulation of AQP3 and water permeability. Using special inhibitors, we find that activation of EGFR and inhibition on ERK activation are involved in atRA's protective effects against UV-induced AQP3 down-regulation. Using specific AQP3's water transport inhibitors and siRNA knockdown, we observe that AQP3 is involved in cell migration and in vitro wound healing. UV-induced AQP3 down-regulation results in reduced water permeability, decreased cell migration, and delayed wound healing, which are attenuated by atRA pretreatment. We conclude that atRA protects against UV-induced down-regulation AQP3 and decrease in water permeability, reduction in cell migration and delayed in vitro wound healing via trans-activation of EGFR and inhibition on ROS-mediated MEK/ERK pathway. This novel finding provides evidence to support possible involvement of AQP3 in UV induced skin dehydration.

Ovarian cancer is one of the most aggressive types of cancer in women. While the molecular mechanisms of ovarian cancer have been extensively studied, treatments for ovarian cancer are still limite...

UV radiation and oxidative stress have been shown to be related to skin aging and skin cancer. Our previous studies have demonstrated that UV radiation induces generation of reactive oxygen species...

Cancer’s use of alternate cellular pathways is necessary in order to proliferate and expand so aggressively but may make it possible to target using certain cancer therapies. We studied the effects...

Ovarian cancer accounts for three percent of all cancers that affect women world-wide and it is regarded at the deadliest form of gynecological cancer. Alteration in cellular metabolism is a manifestation of cancer that typically results in a shift from oxidative phosphorylation to aerobic glycolysis. This change is defined as the Warburg Effect. In this study, we used cell culture techniques to investigate the metabolic behavior of an ovarian cancer cell line, CaOV3. We determined metabolic activity through XF Cell Mito Stress and XF Glycolysis stress tests using a Seahorse Bioscience XFe96 Analyzer. After titrations were completed for optimal cell number and concentration of Oligomycin, we pretreated CaOV3 cells with PI3K/AKT and mTOR inhibitors and repeated stress tests. We found that under the influence of Rapamycin (mTOR inhibitor) and LY294002 (PI3K/AKT inhibitor), the cells exhibited an increase in oxygen consumption by OCR (pMoles/min) and ECAR (mpH/min) analysis, suggesting an increase in capacit...

Transformation from disciplined melanocytes to untamed melanoma cells remains an enigma. Our previous studies have demonstrated that melanoma cells are more resistant to oxidative stress and melanoma cells exhibit constitutive mTOR activity. Surprisingly, further studies have failed to present the expression and activity of EGFR using conventional anti-EGFR antibodies. We hypothesized that constitutive mTOR activity in melanoma cells may be due to mutated EGFR variants and membrane channel activities. Using patch clamping technique, we have shown that melanoma cells (WM 266-4) but not human skin melanocytes exhibit Na+ current which is blocked by TTX. Interestingly, mTOR inhibitor, rapamycin, blocks Na+ current. Western blot and confocal microscopy data further revealed that Na+/Ca2+ exchanger blocker KB-R7943 (KBR), and L-type Ca2+ channel blocker Nifedipine (NIF) inhibits mTOR activity in melanoma cells in a dose dependent manner. To further characterize Na+ channels, we used commercially available channel antibodies. Western blot analysis data showed that melanoma cells but not human melanocytes express Na+ v1.5 and Na+ v1.6 and NCX3. Functional studies also indicated that KBR and NIF inhibit melanoma cell proliferation and migration. Taken all together, our data suggest that aberrant Na+ current contributes to constitutive mTOR activity in melanoma cells and channel blockers may be potential for the treatment of melanoma. Citation Format: Benjamin Gallant, Nicole Lizza, Ryan Garrity, Audrey Madigan, Zachary Jost, Alfredo Gonzalez, Jeanine Justiniano, An Xie, Yinsheng Wan. Aberrant sodium current contributes to constitutive mTOR activity in malignant melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 234A.

Cell-mediated autoimmunity has been suggested to be involved in the melanocyte apoptosis that occurs in vitiligo. We investigated the cytotoxicity to autologous melanocytes of CD8+ T cells from the perilesional margins and peripheral blood samples of vitiligo patients. CD8+ T cells isolated from skin biopsied from the edges of depigmented skin patches of vitiligo patients or from peripheral blood samples of the same donors were proliferated in culture medium. The primary cultures of CD8+ T cells and autologous melanocytes were mixed at ratios of 1:1, 1:2 or 1:5 and incubated for 3 days. The apoptosis of the melanocytes was analyzed by flow cytometry. Secreted cytokines in selected samples were measured by cytokine arrays. The results show that the CD8+ T cells were successfully isolated from the vitiligo perilesional margins. This cell population showed a significantly higher percentage of CD69 expression (56.13±3.55 versus 29.93±2.35%, p

When rhegmatogenous retinal detachment occurs, tumor necrosis factor-alpha (TNF-α) among other cytokines leaks into the subretinal space, induces resident retinal pigment epithelial (RPE) cells to migrate, which is the initial step of proliferative vitreoretinopathy (PVR). In the current study, we aim to understand how this is regulated by focusing the cellular mechanisms involved. Here we identified an Akt/Tuberous sclerosis protein 2 (TSC2)/mTOR complex1 (mTORC1) signaling pathway after TNF-α treatment to mediate RPE cell migration. Suppression of mTORC1 activation, either by its inhibitor rapamycin, or by activation of its suppressor AMP activated protein kinase (AMPK), inhibited TNF-α-mediated RPE cell migration, while RNA interference (RNAi)-mediated knocking-down of SIN1 or Rictor, two key components of mTOR complex 2 (mTORC2), had no significant effect on TNF-α-induced RPE cell migration. Our data provide initial evidence that TNF-α-mediated in vitro RPE cell migration mainly requires Akt/mTORC1, but not mTORC2 signaling. The results of this study may lead to indentify novel signaling targets against PVR.

Ultraviolet (UV) irradiation induces skin photoaging associated with up-regulated matrix metalloproteinase (MMP) expression. Inhibition of MMP expression is suggested to alleviate photoaging induced by UV irradiation. Astragaloside IV (As-IV), one of the main active ingredients of Astragalus membranaceus (Fisch) Bge, has been reported to have various biological activities. However, its anti-photoaging effect has not been examined to date. In the present study, we observed the effect of As-IV on matrix metalloproteinase-1 (MMP-1) expression in UV-irradiated human dermal fibroblasts (HDFs). We found that treatment with As-IV significantly decreased UV-induced MMP-1 expression at the messenger RNA and protein levels. In addition, western blotting analysis revealed that As-IV concentration-dependently suppressed UV-induced phosphorylation of extracellular-regulated protein kinase, Jun-N-terminal kinase and p38 mitogen-activated protein kinase (MAPK). Furthermore, treatment with As-IV markedly inhibited UV-induced nuclear factor kappaB (NF-κB) activity. These results suggest that As-IV down-regulates UV-induced MMP-1 expression, perhaps through suppression of MAPK and NF-κB activation in HDFs. As-IV is thus a potential agent for the management of skin photoaging.

Human vitiligo is an acquired depigmenting skin disorder characterized by milk-white skin macules resulting from a chronic and progressive loss of melanocytes. It has been suggested that autoimmune mechanisms are involved in this disorder. We undertook this project to obtain an MCHR1-C linear peptide containing four main MCHR1 B-cell epitopes in an attempt to detect the IgG antibody against MCHR1 in vitiligo. The target gene encoding the MCHR1-C peptide was cloned into a pGEX-4T-2 expression vector, expressed in Escherichia coli BL21, and purified using a GST column. The molecular weight was analyzed by SDS-PAGE and western blotting. The IgG antibody response to MCHR1 was detected by ELISA with MCHR1-C as a coated antigen, and the absorption experiment was used to assess the binding ability of the Ab detected by MCHR1-C with a membrane protein in human epidermal melanocytes. The purified peptide MCHR1-C with a molecular weight of 33 kDa was obtained, and could bind to the MCHR1 antibody in human sera. Of the vitiligo patient sera examined, 24 of 145 (16.55%) tested positive for the MCHR1 antibody, and the frequency in the vitiligo patient group was significantly greater compared to the normal control. However, no significant difference between gender, disease duration, clinical subtype or family history between the two groups was observed. In addition, the Ab detected by MCHR1-C in sera was specifically absorbed by the membrane protein obtained from human epidermal melanocytes. Collectively, our data suggest that the MCHR1-C peptide can be used to detect the MCHR1 antibody in vitiligo patients as a coated antigen instead of MCHR1 protein. We conclude that the level of MCHR1 antibody in active vitiligo patients is significantly higher than that in healthy individuals, while gender, disease duration, clinical subtype and family history have no association with the level of MCHR1 antibody in vitiligo patients.

Solar ultraviolet (UV) irradiation is one of the most significant extrinsic factors contributing to skin photoaging. One major characteristic of photoaging induced by UV is water loss of the skin. Water movement across the plasma membrane can occur via two pathways: by diffusion through the lipid bilayer and by membrane-inserted water channels (aquaporins). In this study we demonstrate that UV induces aquaporin-3 (AQP3) downregulation in cultured keratinocytes (HaCaT cells). PD98059 and U0126, MEK/ERK inhibitors, inhibit UV-induced AQP3 loss. Trans-Zeatin (tZ), which alone induces AQP3 expression, attenuates UV-induced loss of AQP3. We found that tZ inhibits UV-induced MEK/ERK activation; the latter serves as the key signal pathway mediating UV-induced AQP3 loss. Using specific AQP3 siRNA knockdown, we found AQP3 is involved in wound healing in human skin keratinocytes. Loss-of-AQP3-mediated delayed wound healing in UV-radiated skin keratinocytes is attenuated by tZ pretreatment. tZ pretreatment also attenuates UV-induced decreased water permeability in HaCaT cells. We concluded that UV radiation downregulates AQP3 in HaCaT cells. MEK/ERK activation is involved in this process. tZ treatment attenuates UV-induced AQP3 loss, in vitro wound healing delay and water permeability decrease. This work provides a new explanation for the anti-photoaging potential of tZ.

Aquaporins (AQPs) are a family of 13 small ( approximately 30 kDa/monomer), hydrophobic, integral membrane proteins. AQPs are expressed in various epithelial and endothelial cells involved in fluid transport. Here, we demonstrated for the first time that AQP1 is expressed in cultured human retinal pigment epithelial (RPE) cells (ARPE-19 cell line). Ultraviolet radiation (UVB) and H2O2, two major factors causing RPE cell damage, induced AQP1 downregulation which was mediated by MEK/ERK activation. UV and H2O2 as well as AQP1-specific siRNA knockdown impaired water permeability of ARPE-19 cells. Notably, pretreatment with all-trans retinoic acid attenuated UV- and H2O2-induced AQP1 downregulation and water permeability impairment. Considering that water permeability is involved in multiple functions of RPE cells such as cellular junction formation, fluid or protein exchange and barrier formation, our data elucidated a novel mechanism through which UV radiation and oxidative stress induce eye cell damage. Our results further support the notion that all-trans retinoic acid might be useful for protection against UV or oxidative stress-induced eye cell damage.

It has been shown that the VIT1 gene is down-regulated in vitiligo melanocytes, but the expression of the VIT1 gene in melanocytes of Chinese patients with vitiligo and the potential function of VIT1 in pathogenesis of vitiligo are not as well known. In this study, we first compared VIT1 gene expression in melanocytes cultured from non-lesional skin of generalized vitiligo patients, normal melanocytes and melanomas in China. The cloned VIT1 ORF cDNA fragments were sequenced and then compared. VIT1 siRNAs were transfected into mouse B10BR melanocytes. The results from semi-quantitative RT-PCR demonstrated that the expression levels of VIT1 in non-lesional melanocytes from vitiligo patients are significantly lower than those in normal melanocytes. In contrast, the expression levels of VIT1 in melanoma are higher. The results also demonstrated that VIT1 is not a novel gene including a fragment of 81 bp flanking by consensus GT and AG sequences on the 5' and 3' ends, which is regarded as an intron. VIT1 without the extra intron is a known gene called FBXO11. Our findings revealed abnormal morphology by light and electron microscopes in FBXO11 siRNA transfected melancytes, which display large epithelioid perikaryon and stubby dendrites with occasional multidendricity as opposed to slender perikaryon and bipolar dendrites of controls. The electron microscopic observation indicated swelling mitochondria and endoplasmic reticulum (ER), and increased lysosomes and bubbles. Further, transfection of FBXO11 siRNA significantly promoted cell apoptosis. Collectively, this study provides comprehensive morphological proof of the relationship between dilation of ER and decreased levels of the FBXO11 gene in vitiligo melanocytes.

Ultraviolet (UV) irradiation induces the expression of matrix metalloproteinases (MMPs), disturbing the metabolism of extracellular matrix (ECM), and causes the characteristic changes of photoaging in skin. Inhibition of induction of MMPs is suggested to alleviate photoaging induced by UV irradiation. Zeatin, purified from Zea mays, is a member of the cytokinin group of plant growth factors, the activity of which is attributed to its more stable trans form. In this study, we investigated the effect of trans-Zeatin on UVB-induced matrix metalloproteinase-1 (MMP-1) expression in cultured human skin fibroblasts (HSFs) and studied the mechanisms of its actions. We found that pretreatment with trans-Zeatin significantly inhibits UVB-induced MMP-1 expression and c-Jun activation in a dose-dependent manner. We also observed that trans-Zeatin inhibits UVB-induced phosphorylation of ERK, JNK and p38 MAP kinases (MAPKs) dose-dependently. As expected, PD98059, an ERK inhibitor, SP600125, a JNK inhibitor and SB203580, a p38 MAPK inhibitor effectively inhibit UVB-induced phosphorylation of ERK, JNK and p38 MAPKs, respectively. Moreover, the inhibitory mechanism of trans-Zeatin was further demonstrated in MMP-1 secretion using MAPK-specific inhibitors. PD98059, SP600125 and SB203580 suppressed UVB-induced MMP-1 secretion, which is consistent with the above results. Collectively, our results suggest that trans-Zeatin inhibits UVB-induced MMP-1 expression, which may be mediated by inhibition of ERK, JNK and p38 MAPKs signaling pathways in HSFs. Trans-Zeatin is a potential agent for the management of skin photoaging.

Hypoxia inducible factor 1alpha (HIF-1alpha) regulates the transcription of a number of genes under hypoxia and other extracellular or intracellular stimulations. It also promotes angiogenesis, tumor metastasis and invasion. To investigate the effect of hypoxia and reoxygenation on cell proliferation, invasion and adhesion, which are all related to ovarian cancer, we applied chemically-induced hypoxia in the cultured human ovarian carcinoma cell line, HO-8910PM. Semi-quantitative RT-PCR results show that CoCl2 induces the expression of HIF-1alpha in a time- and dose-dependent manner. MTT assay results show that CoCl2-induced hypoxia inhibits cell proliferation which is recovered by reoxygenation. The Boyden and cell adhesion test results indicate that CoCl2-induced hypoxia inhibits cell invasion and adhesion which are markedly enhanced by reoxygenation in the human ovarian carcinoma cell line, HO-8910PM. Collectively, our data provide insight into understanding molecular mechanisms of the invasiveness of ovarian cancer under the conditions of hypoxia and reoxygenation.

Melagenine, extracted from human placenta, has been shown to be effective in treating patients with vitiligo, yet the mechanisms of melagenine in inducing the repigmentation of vitiligo patients have not been fully investigated. Recent studies have suggested that melagenine stimulates melanocyte proliferation and melanogenesis. In this study, we utilized the NCCmelb4M5 melanoblast cell line to investigate the effects of melagenine on proliferation and differentiation of immature melanocytes or melanoblasts. NCCmelb4M5 cells were treated with different concentrations of melagenine (50-400 microg/ml), and MTT assay was performed to evaluate the effects of melagenine on proliferation of melanoblasts. RT-PCR and Western blotting were used to determine the expression of c-KIT and tyrosinase (TYR). Our results show that melagenine stimulates proliferation of NCCmelb4M5 cells in a dose-dependent manner with an optimal concentration of 100 microg/ml. Multipolar and highly branched dendritic network, as well as cluster-like growing cell assembly were visible in melagenine-treated NCCmelb4M5 cells. Melagenine induced expression of c-KIT, TYR and MITF. Our results provide insights into the molecular mechanism of the beneficial effect of melagenine in the treatment of vitiligo.

Vitiligo is an acquired and progressive disorder manifested by the selective destruction of melanocytes in the skin. An extremely high level of hydrogen peroxide (H2O2) in plasma as well as in lesional skin has been reported in vitiligo patients. High H2O2 level has been suggested to be responsible for the disappearance of melanocytes in vitiligo. JNK and p38 MAPK are strongly induced by oxidative stress and related to neuron loss in neurodegenerative disorders. Minocycline, an antibiotic possessing antioxidant activity, is capable of attenuating oxidative stress-induced neurotoxicity. To investigate whether minocycline rescues melanocytes from H2O2-induced apoptosis, cultured mouse melanocytes (B10BR) were treated with H2O2 in the presence or absence of minocycline. Our data showed that H2O2 decreases cell viability in a concentration-dependent manner which is attenuated by minocycline. Also, H2O2 treatment activates JNK and p38 MAPK, and executive caspase 3 in B10BR cells. Minocycline significantly inhibits H2O2-induced activation of JNK, p38 MAPK and caspase 3. Collectively, we concluded that minocycline protects melanocytes against H2O2-induced apoptosis in vitro. Its protective effect is associated with the inhibition of JNK and p38 MAPK. Our findings suggest that minocycline, a clinically well-tolerated, safe antibiotic, may be used to prevent melanocyte loss in the early stage of vitiligo.

Chronic exposure to solar UV irradiation leads to photoaging, immunosuppression, and ultimately carcinogenesis. Cellular senescence is thought to play an important role in tumor suppression and apoptosis resistance. However, the relationships among stress-induced premature senescence (SIPS), tumorigenesis and apoptosis induced by UVB remain unknown. We developed a model of UVB-induced premature senescence in human skin fibroblasts (HSFs). After five repeated subcytotoxic UVB exposures at a dose of 10 mJ/cm2, the following biomarkers of senescence were markedly present: senescence-associated beta-galactosidase (SA beta-gal) activity, growth arrest, and the overexpression of senescence-associated genes. Firstly, there was an increase in the proportion of cells positive for SA beta-gal activity. Secondly, there was a loss of replicative potential as assessed by MTT assay. FACS analysis showed that UVB-stressed HSFs were blocked mostly in the G1 phase of the cell cycle, and replicative senescence, and protein expression of p53, p21(WAF-1) and p16(INK-4a) increased significantly. Thirdly, the mRNA levels of three senescence-associated genes, fibronectin, osteonectin and SM22, also increased. A real time PCR array to investigate the mRNA expression of p53-related genes involved in growth arrest, apoptosis and tumorigenesis indicated that p53, p21, p19, Hdm2, and Bax were up-regulated, and bcl, HIF-1alpha and VEGF were down-regulated. Collectively, our data suggest that UVB-induced SIPS plays an important role in p53-related apoptosis resistance and tumor suppression activity.

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.

Ovarian cancer remains the leading cause of fatality among all gynecologic cancers, although promising therapies are in the making. It has been speculated that metastasis is critical for ovarian cancer, and yet the molecular mechanisms of metastasis in ovarian cancer are poorly understood. Growth factors have been proven to play important roles in cell migration associated with metastasis, and inhibition of growth factor receptors and their distinct cell signaling pathways has been intensively studied, and yet the uncovered interaction or crosstalk among various growth factor receptors complicates this otherwise promising approach. We investigated the crosstalk between EGFR and TrkB, both of which have been known to be important in cell survival and migration in response to EGF and BDNF. Our results showed that both EGF and BDNF induced cell migration and cell proliferation in cultured human ovarian cancer cells (Caov3 cell line). EGF and BDNF transactivated TrkB and EGFR respectively, and activated downstream cell survival components such as Akt. EGFR and TrkB kinase inhibitors inhibited EGF- and BDNF-induced TrkB and EGFR activation and Akt phosphorylation, and cell proliferation and migration. Using EGFR knockout cells, we further demonstrated that EGFR is required for EGF-induced cell migration. Collectively, our data indicate that EGFR and TrkB crosstalk each other in response to EGF and BDNF, leading to cell survival pathway activation in ovarian cancer cells. Our data suggest that a combination of inhibitors of both receptors with cell survival pathway inhibitors would provide a better outcome in the clinical treatment of ovarian cancer.

The molecular and cellular mechanism of the development of pancreatic cancer is under constant and intensive study, and yet the cure is still out of reach. While surgical treatment is optional, conventional chemotherapy or chemo-radiotherapy remains the best choice. Among others, paclitaxel is proven to be a popular and, to a certain extent, effective chemotherapy agent. We proposed that the combination of paclitaxel and membrane permeable ceramide would enhance the fatality of cancer cells, and reported that the combination increased cell death of both head and neck and leukemic cancer cells. In this study, we treated pancreatic cancer cells (L3.6 cells) with paclitaxel and ceramide at the concentrations of clinical relevance, and treatment was then followed up with an investigation of the molecular mechanism of the synergism of paclitaxel and ceramide. The results of Western blot analysis indicated that the combo synergistically induced ERK and JNK phosphorylation, but not p38 and Akt phosphorylation. We also found that the combination (combo) induced EGFR phosphorylation in a synergistic manner. Furthermore, we observed that paclitaxel, ceramide, or combo-induced EGFR phosphorylation was inhibited by EGFR inhibitor, PD153035, while paclitaxel, ceramide, or combo-induced JNK and ERK phosphorylation was blocked by EGFR inhibitor, PD153035 and ERK inhibitor, U126. Taken together, our results demonstrated that the combination of paclitaxel and ceramide synergistically induced pancreatic cancer cell death through differential activation of EGFR-mediated MAP kinases. EGFR and ERK inhibitors may further enhance the paclitaxel and ceramide effect.

Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.

Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. UV radiation-induced proinflammatory cytokines mediated by NF-kappaB reportedly play important roles in photoaging and cancer. NF-kappaB and cytokines have been thus perceived as molecular targets for pharmacological intervention. With an increasing amount of knowledge of the actions of green tea extracts at cellular and molecular levels, the beneficial effect of drinking green tea has become well recognized if not completely accepted. The components in green tea have even been added to skin-care products unregulated, while the molecular mechanisms of the actions of those components on human skin are being unraveled. Using cultured human keratinocytes, we investigated the effects of (-)-epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent in green tea, on UV-induced activation of transcription factor NF-kappaB and proinflammatory pathway by measuring nuclear translocation of NF-kappaB and IL-6 secretion in vitro. Immunohistochemical and Western blot analysis and ELISA indicated that both nuclear p65 and secreted IL-6 were significantly (p

Emerging studies have suggested that transient activation of the cell survival pathway may be the strategy for cancer cells to fight against chemotherapy and eventually mysteriously evade paclitaxel-induced cell death. Modulation of the EGFR-mediated survival pathway in addition to the utilization of paclitaxel renders a promise of better clinical management. The objective of this study was to understand the molecular mechanism of transient induction of EGFR-mediated cell survival by paclitaxel. We utilized ovarian cancer cell line, Caov3, cells to investigate the effect of paclitaxel on EGFR-mediated MAP kinase and AKT activation, and the expression of survivin. We found that paclitaxel transiently induced EGFR phosphorylation and ERK and AKT activation but not JNK and p38. Paclitaxel-induced ERK and AKT activity was inhibited by the EGFR inhibitor, PD153035; ERK inhibitor, U0126; and PI3 kinase inhibitor, LY294002, respectively. We observed that paclitaxel transiently induced expression of survivin in the early hours of treatment. Paclitaxel-induced survivin expression was inhibited by the EGFR inhibitor, PD153035. Inhibitors of EGFR, ERK and PI3 kinase all enhanced paclitaxel-induced cell death. We conclude that paclitaxel transiently transactivates EGFR, leading to activation of cell survival factors, such as ERK and AKT, and expression of survivin, which are all inclusively accountable for ovarian cancer cell resistance to paclitaxel treatment. A combination of these inhibitors with paclitaxel may be a better option for ovarian cancer treatment.

Human skin, like all other organs, undergoes chronological aging. In addition, unlike other organs, skin is in direct contact with the environment and therefore undergoes aging as a consequence of environmental damage. The primary environmental factor that causes human skin aging is UV irradiation from the sun. This sun-induced skin aging (photoaging), like chronological aging, is a cumulative process. However, unlike chronological aging, which depends on the passage of time per se, photoaging depends primarily on the degree of sun exposure and skin pigment. Individuals who have outdoor lifestyles, live in sunny climates, and are lightly pigmented will experience the greatest degree of photoaging. During the last decade, substantial progress has been made in understanding cellular and molecular mechanisms that bring about chronological aging and photoaging. This emerging information reveals that chronological aging and photoaging share fundamental molecular pathways. These new insights regarding convergence of the molecular basis of chronological aging and photoaging provide exciting new opportunities for the development of new anti-aging therapies. This article reviews our current understanding and presents new data about the molecular pathways that mediate skin damage by UV irradiation and by the passage of time.

Introduction: Pancreatic adenocarcinoma a highly lethal malignancy (5 yr survival ∼ 5%) is poorly responsive to all chemotherapy, in part related to presence of chemo resistant pathways: prosurvival (AKT/PI3K/mTOR and mutant KRAS. We have previously demonstrated reversal of these resistant pathways by C6 Ceramide and now are pursuing optimum intracellular delivery using selected liposomal preparations ofC6Ceramide. Methods: C6 Ceramide housed in 4 different liposomal vesicle formulations were tested against pancreatic cell lines L3.6, PANC1 and MIA, using combination therapy with chemotherapy agents Gemcitabine, Paclitaxel and Cetuximab with determination of the MTT anti tumor response. Liposomal preparations included : (#1)18:1 PC(1,2-dioleoyl-sn-glycero-3-phosphocholine; (#3),18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine;(#5) DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl); and (#7) 18:0 PEG2 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000. (#1)18:1 PC- has low transition temp and is fluid at bodyT*(# 3). 18:0 PC- has high transition Temperature(T*) is rigid at body T*;#5 DPGG- avoids RE System (is fluid at bodyT*, induces long circulations)#7. 18:0 PEG 2 PE - industry standard (also avoids RE system) Biologic effects of the 4 lipid formulations with/without C6 Ceramide were initially assessed invitro. Results: Dose response MTT assay demonstrated no distinctive response of C6 combined liposomes vesicles # 1 & 3, but confirmed high activity in preparations #5 & 7. Conclusion: Liposomal C6 preparations show high activity in potentiating chemo toxicity by Gemcitabine, Paclitaxel and Cetuximab against Pancreatic Cancer. Confirmatory in vivo studies are in process. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B104. Citation Format: Harold J. Wanebo, Cong Cao, Shaun Lu, David Shrayer, Yinsheng Wan, Wayne Bowen, Wayne Bowen. Liposomal C6 Ceramide appears to potentiate chemotoxicity of paclitaxel, gemcitabine and cetuximab against aggressive pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B104.

Melanoma is considered as one of the most aggressive and less treatable cancers. While various models of mechanisms have been proposed, the transformation from tranquil melanocytes to invasive melanoma cells remains an enigma. Alteration of cellular redox status has been suggested to be associated with cellular transformation. We compared primary human melanocytes and melanoma cells and their responses to oxidative stress. Both cells were treated with H2O2 at doses between 5 and 250μM. Microscopic data showed that melanoma cells are more resistant to H2O2 treatment. Western blot and confocal microscopic data showed that in melanoma cells, H2O2 does not induce CRT translocation which is considered as a mechanism through which melanocytes surrender to oxidative stress and induces immunogenic cell death. Furthermore, Western blot analysis showed that melanoma cells express EGFR, responsive to EGF treatment and have constitutive activation of AKT and mTOR, which are all related to better survival. In contrast, in the primary human melanocytes, EGFR expression is almost undetectable. And confocal microscopic data confirm this observation. Treatments of cells with MEK/ERK inhibitor U0126 and PI3K/AKT inhibitor LY294002 attenuates S6 phosphorylation. We also observed that in melanoma cells, but not in melanocytes, low dose (5 to 250μM) of H2O2 induces melanin production that is recognized to protect from further oxidative stress. Collectively, our data suggest that active MEK/ERK and AKT/mTOR pathways and up-regulation of melanin production contribute to the resistance of melanoma cells to oxidative stress and attribute to the aggressiveness of melanoma. Our data provides insights into the understanding of the molecular mechanisms of the transformation from melanocytes to melanoma and support the notion from the most recently published proteomic studies that EGFR remains one of the most important targets for better clinical management of melanoma. Citation Format: David Calianese, Garrett Cammarata, Gabriella Brum, Kevin Szulak, Charles Best, Katelyn Higgins, Yinsheng Wan. Human melanoma cells resist oxidative stress due to overactive MEK/ERK and AKT/mTOR pathways and overproduction of melanin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2013-5247 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.