Capsaicin exerts therapeutic effects by targeting tNOX-SIRT1 axis and augmenting ROS-dependent autophagy in melanoma cancer cells.

Although considered a sporadic type of skin cancer, malignant melanoma has regularly increased internationally and is a major cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effect of capsaicin on melanoma growth. The cellular thermal shift assay (CETSA), isothermal dose-response fingerprint curves (ITDRFCETSA), and CETSA-pulse proteolysis were utilized to confirm the direct binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice. Our data show that capsaicin directly engaged with cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin was accompanied by the attenuation of SIRT1, a NAD+-dependent deacetylase. The suppression of tNOX and SIRT1 then enhanced ULK1 acetylation and induced ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Taken together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo, and that inhibition of the tNOX-SIRT1 axis contributes to inducting ROS-dependent autophagy in melanoma cells.

Inhibitory Effects of Digoxin and Digitoxin on Cell Growth in Human Ovarian Cancer Cell Line SKOV-3.

BACKGROUND: Cardiac glycosides (CGs) possess a chemical structure similar to steroids, and are inhibitors of the sodium potassium pump. An anti-tumor effect of CGs in breast and prostate cancers has been reported, but the effect of CGs on ovarian cancer is still unclear. AIMS: In this study, the effects of CGs on proliferation, cytotoxicity and cell cycle of ovarian cancer cell line (SKOV-3) have been investigated. PROCEDURE: The cell proliferation and cytotoxicity were detected by MTT assay and LDH activity assay, respectively. CGs, at concentrations higher than IC50, decreased cell proliferation and showed increased cytotoxicity toward SKOV-3 cells. The colony-formation ability was reduced after treatment with digoxin and digitoxin for 10 days. Furthermore, we explored the effect of digoxin and digitoxin on the distribution of cell cycle by flow cytometry. RESULTS: Results revealed that both digoxin and digitoxin led to cell cycle arrest in G0/G1 phase with 24 or 48 hours, but the arrest of G0/G1 phase was not observed at 72 hours. We evaluated the percentage of hypodiploid cell population as an index of the cellular fragments through flow cytometry. The data indicated that cellular fragments were significantly increased by treating with digitoxin at the concentrations of IC50 and 10-6 M for 72 hours. CONCLUSION: Taken together, these data suggest that CGs decreased cell proliferation and increased cytotoxicity through cell cycle arrest at the G0/G1 phase. CGs have anti-tumor effect in SKOV-3 cells and might be a potential therapeutic drug for ovarian cancer. Since this study is a preliminary investigation of CGs on SKOV-3 cells, more experiments might be performed in the future. Furthermore, more ovarian cancer cell lines might also be employed in the future studies to confirm the effect of CGs in ovarian cancer.

Regulation of extracellular and intracellular prolactin on cell proliferation and survival rate through GHR/JAK2/STAT3 pathway in NSCLC.

Styrene increases serum prolactin (PRL) concentration. Hyperprolactinemia is associated with poor prognosis in lung cancer patients, but the mechanism of PRL action is unclear. The aims of this study were to (i) investigate the mechanism of PRL-action receptor in NSCLC cells (ii) measure whether PRL was secreted by NSCLC cells and its stimulatory mechanism in vitro and in vivo. We found that cell proliferation was increased after treatment of a pharmacological dose of PRL in A549 cells, which through up regulation of growth hormone receptor (GHR) and downstream of JAK2/STAT3/VEGF pathway. All NSCLC cells in the present study secreted PRL and expressed GHR, but not PRLR. Inhibition of GHR protein level led to decrease the PRL-induced cell proliferation. PRL was detected in NSCLC cells culture medium. Knockdown of intracellular PRL downregulated JAK2/STAT3 protein activities and GHR and VEGF protein levels. Furthermore, knockdown of intracellular PRL reduced the cell proliferation and the ability of colony-forming. In lung cancer tissues, PRL, GHR and VEGF levels were higher in the tumor tissues than in normal tissues and the protein expressions of these three proteins are positively correlated, respectively. High expression levels of both PRL and GHR cause a poor survival rate in lung cancer patients. Taken together, our results suggested that extracellular and intracellular PRL were involved in cell proliferation through GHR. Combination of in vitro and in vivo results, GHR and PRL are important targets for suppressing NSCLC cell proliferation, which might improve the survival rate in NSCLC patients.

Chronic intermittent hypoxia stimulates testosterone production in rat Leydig cells.

AIMS: The hypoxia-stimulated response of the endocrine system depends on the kind and duration of hypoxia. Hypoxia has been reported to stimulate testosterone (T) production in rats, but the mechanisms remain to be investigated. MATERIALS AND METHODS: Male rats were divided into two groups. The rats exposed to chronic intermittent hypoxia (CIH) at 8 h/day were housed in a hypoxic chamber (12% O2) for 14 days. Normoxic rats were used as control animals. T was measured after challenging the rat Leydig cells (LCs) with different stimulators, including hCG (0.01 IU/ml), forskolin (10-5 M), 8-bromo-cAMP (10-4 M), A23187 (10-5 M), cyclopiazonic acid (10-4 M), and androstenedione (10-8 M). Meanwhile, the LCs were incubated with trilostane (10-5 M) and/or 25-OH-hydroxycholesterol (10-5 M); thereafter the media were collected for pregnenolone assay. KEY FINDINGS: In the CIH group, plasma T levels were increased, but the serum luteinizing hormone (LH) was decreased. Furthermore, at several time intervals after hCG injection, plasma T levels were higher in the CIH group. The evoked-release of T and pregnenolone were significantly increased in the CIH group. Compared with the normoxic group, the CIH group had higher mRNA and protein expression levels of the LH receptor and CYP11A1 but not StAR. The plasma and testicular microvasculature VEGF levels were increased in the CIH group. The testicular vessel distribution was more obvious in CIH rats. SIGNIFICANCE: CIH-induced T secretion might be partially mediated by mechanisms involving the induction of LH receptor expression, testicular angiogenesis, CYP11A1 activity, 17ß-HSD activity, and calcium-related pathway.

17α-Ethynylestradiol and 4-nonylphenol stimulate lung adenocarcinoma cell production in xenoestrogenic way.

Lung cancer has been one of the major cancers leading to mortalities worldwide. In addition to smoking, estrogen is considered to play an important role in the lung cancer development because women have a higher proportion of adenocarcinoma than men. In the environment, there are many metabolites and waste products that mimic human estrogen structurally and functionally. 17α-Ethynylestradiol (EE2) which is used as an oral contraceptive is released into wastewater after being utilized. Moreover, 4-nonylphenol (NP) which is found in the petrochemical products and air pollutants reveals estrogenic activity. In the present study, 17ß-estradiol (E2), EE2, and NP are administered to stimulate male lung adenocarcinoma cells (A549) and female lung adenocarcinoma cells (H1435). The results demonstrate that EE2 and NP stimulate A549 and H1435 cells proliferation in a dose- and time-dependent manner. Both estrogen receptors α and ß are simultaneously activated. In response to estrogens, up-regulation of the epidermal growth factor receptor and extracellular signal-regulated kinase expression occurs. In conclusion, this is the first study to report that EE2 and NP exert a biotoxic effect to stimulate the proliferation of both male and female lung cancer cell in a dose- and time- dependent manner. The environmental hormones posing new challenges for lung cancer deserve further investigation.

Stimulatory Effect of Intermittent Hypoxia on the Production of Corticosterone by Zona Fasciculata-Reticularis Cells in Rats.

Hypoxia or intermittent hypoxia (IH) have known to alter both synthesis and secretion of hormones. However, the effect of IH on the production of adrenal cortical steroid hormones is still unclear. The aim of present study was to explore the mechanism involved in the effect of IH on the production of corticosterone by rat ZFR cells. Male rats were exposed at 12% O2 and 88% N2 (8 hours per day) for 1, 2, or 4 days. The ZFR cells were incubated at 37 °C for 1 hour with or without ACTH, 8-Br-cAMP, calcium ion channel blockers, or steroidogenic precursors. The concentration of plasma corticosterone was increased time-dependently by administration of IH hypoxia. The basal levels of corticosterone production in cells were higher in the IH groups than in normoxic group. IH resulted in a time-dependent increase of corticosterone production in response to ACTH, 8-Br-cAMP, progesterone and deoxycorticosterone. The production of pregnenolone in response to 25-OH-C and that of progesterone in response to pregnenolone in ZFR cells were enhanced by 4-day IH. These results suggest that IH in rats increases the secretion of corticosterone via a mechanism at least in part associated with the activation of cAMP pathway and steroidogenic enzymes.

Interactive Effect of Corticosterone and Lactate on Regulation of Testosterone Production in Rat Leydig Cells.

The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5-20 mM), cortiosterone (10-9 -10-4 M) and lactate plus corticosterone. The production of testosterone were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR), and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by Western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. J. Cell. Physiol. 232: 2135-2144, 2017. © 2016 Wiley Periodicals, Inc.

Attenuation of exercise effect on inflammatory responses via novel role of TLR4/PI3K/Akt signaling in rat splenocytes.

Moderate exercise diminishes proinflammation cytokine production in various types of immune cells, but the intracellular signaling pathways involved are not completely understood. Phosphoinositide 3-kinase (PI3K)/Akt, a crucial downstream protein of toll-like receptor 4 (TLR4), may modulate inflammation. The present study aimed to investigate the effects of exercises on lipopolysaccharide (LPS)-stimulated inflammatory response in splenocytes and to explore potential mechanisms of the PI3K/Akt pathway. Male rats were divided into sedentary and exercise groups. Animals in the exercise group underwent endurance training 30 min/day, 7 days/wk, at the speed of 20 m/min on a treadmill for 1 wk. Here, we showed that exercise 1) attenuated TLR4, 2) increased PI3K/phospho-Akt (p-Akt), and 3) diminished phospho-nuclear factor-κB (p-NF-κB) expression. In addition, administration of splenocytes isolated from trained rats with LPS in vitro showed 1) reduced tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and nitric oxide secretion and 2) decreased splenocyte proliferation. The plasma corticosterone (CCS) level in the exercise group was higher than that in the sedentary group. We confirmed that CCS down-regulated TNF-α and IL-6 secretion in response to LPS in rat splenocytes. Dexamethasone also significantly attenuated LPS-evoked release of TNF-α and IL-6 in a dose-dependent manner. These findings suggested that exercise dampened the secretion of inflammation mediators probably through partial inhibition of TLR4 and p-NF-κB and activation of PI3K/p-Akt expression in the spleen.

Induction of renal senescence marker protein-30 (SMP30) expression by testosterone and its contribution to urinary calcium absorption in male rats.

The aim of this study was to investigate the involvement of androgen, mainly testosterone, in the expression of renal senescence marker protein-30 (SMP30) in male rats. We found that the renal SMP30 expression was up-regulated by endogenous testosterone stimulation during puberty. Interestingly, androgen-deficient orchidectomized (ORX) rats exhibited lower SMP30 mRNA and protein expression in the kidney, and that was restored by testosterone propionate (TP) replacement. Abrogation of androgen receptor (AR) activity by co-treatment with flutamide abolished testosterone-induced SMP30 expression in the kidney as well as in the NRK52E cells. However, SMP30 expression was unaltered in the liver of ORX rats. We also showed a positive correlation between renal SMP30 expression and plasma testosterone level during the aging process. TP-induced SMP30 expression in ovariectomized (OVX) rats was observed and was an evidence to explain the gender difference of SMP30 levels. Immunofluorescence assay showed that renal SMP30 was specifically expressed in the proximal tubular segments of the kidney. The urinary Ca(2+) level was increased in both ORX and male aging rats. Taken together, our results indicate a novel role of testosterone in regulating SMP30 expression specifically in the kidney to contribute to urinary calcium absorption.

Effects of acrolein on the production of corticosterone in male rats.

Acrolein, an α, ß-unsaturated aldehyde, exists in a wide range of sources. Acrolein can be not only generated from all types of smoke but also produced endogenously from the metabolism by lipid peroxidation. The cellular influence of acrolein is due to its electrophilic character via binding to and depleting cellular nucleophiles. Although the toxicity of acrolein has been extensively studied, there is relatively little information about its impact on hormone release. This study aimed at the effect of acrolein on hypothalamic-pituitary-adrenal (H-P-A) axis. In an in vivo study, male rats were administrated with acrolein for 1 or 3days. The plasma corticosterone in response to a single injection of adrenocorticotropic hormone (ACTH) increased slowly in acrolein-pretreated rats than in control rats. Further investigating the steroidogenic pathway, the protein expressions of steroidogenic acute regulatory protein (StAR) and the upper receptor-melanocortin 2 receptor (MC2R) were attenuated in acrolein-treated groups. Another experiment using trilostane showed less activity of P450scc in zona fasciculata-reticularis (ZFR) cells in acrolein-treated groups. In addition to the suppressed ability of corticosterone production in ZFR cells, acrolein even had extended influence at higher concentrations. The lower ACTH was observed in the plasma from acrolein-pretreated rats. In an in vitro study, ZFR cells were incubated with acrolein and the results showed that corticosterone concentrations in media were decreased in a dose-dependent manner. Acrolein also desensitized the response of the ZFR cells to ACTH. These results suggested that acrolein decreased the releasing ability of corticosterone via an inhibition on the response of ZFR cells to ACTH and the reduction of protein expressions of StAR and MC2R as well as the activity of P450scc in rat ZFR cells. The present evidences showed that the H-P-A axis was affected by the administration of acrolein.

Effects of acrolein on aldosterone release from zona glomerulosa cells in male rats.

A positive correlation between smoking and hypertension has been well established. Acrolein is a major toxic volatile compound found in cigarette smoke. Human exposure to low levels of acrolein is unavoidable due to its production in daily activities, such as smoke from industrial, hot oil cooking vapors, and exhaust fumes from vehicles. The toxicity and the action mechanism of acrolein to induce apoptosis have been extensively studied, but the effects of acrolein on hypertension are still unknown. The present study aimed to examine the effects of acrolein on aldosterone release both in vivo and in vitro. Male rats were divided into three groups, and intraperitoneally injected with normal saline, or acrolein (2mg/kg) for 1 (group A-1) or 3 (group A-3) days, respectively. After sacrificing, rat blood samples were obtained to measure plasma aldosterone and angiotensin II (Ang II) levels. Zona glomerulosa (ZG) cells were prepared from rat adrenal cortex, and were incubated with or without stimulants. We found that the serum aldosterone was increased by 1.2-fold (p<0.05) in A-3 group as compared to control group. Basal aldosterone release from ZG cells in A-3 group was also increased significantly. Moreover, acrolein enhanced the stimulatory effects of Ang II and 8-bromo-cyclic AMP on aldosterone secretion from ZG cells prepared in both A-1 and A-3 groups. Furthermore, the enzyme activity of P450scc, the rate-limiting step of aldosterone synthesis, was elevated after acrolein injection. Plasma level of Ang II was increased in both A-1 and A-3 groups. These results suggested that acrolein exposure increased aldosterone production, at least in part, through elevating the level of plasma Ang II and stimulating steroidogenesis pathways.

Regulation of Intermittent Hypoxia on Brain Dopamine in Amphetaminized Rats.

We investigated intermittent hypoxia (IH) on dopamine (DA) release in rat brain treated with or without amphetamine (AMPH). Rats were divided into four groups including normoxia, IH, AMPH, and AMPH + IH treatments. The cerebrospinal fluid (CSF) was collected and the DA levels were detected by high performance liquid chromatography (HPLC). The plasma prolactin (PRL) concentration was measured by radioimmunoassay (RIA). We found that IH reduced basal DA concentration in media prefrontal cortex (mPFC), but increased that in striatum, where DA level was also increased in rats treated with AMPH or AMPH + IH. Angiotensin II (Ang II) increased the DA release in mPFC and striatum and this effect was enhanced in AMPH + IH group. The stimulatory effect of IH on plasma PRL was attenuated in presence of AMPH. Tyrosine hydroxylase (TH) expression was decreased by IH, but increased by AMPH + IH in mPFC. IH or AMPH treatment decreased the expression of vesicular monoamine transporter-2 (VMAT-2) in rat brain. These data suggested that IH altered the DA release and changed the protein expression levels in different parts of rat brain treated with AMPH. IH may play a role in regulating DA metabolism in AMPH addiction.

Cytotoxic effect of s-petasin and iso-s-petasin on the proliferation of human prostate cancer cells.

BACKGROUND: Petasin (Petasides hybridus) is a perennial shrub that is found in Europe as well as parts of Asia and North America and is being used to treat hypertension, tumors and asthma. In a previous study, we reported that petasin possesses biological effects including inhibition of testosterone production and the release of corticosterone from rat zona fasciculata-reticularis cells, and anti-proliferative effect on human T24 bladder carcinoma cells. MATERIALS AND METHODS: In the present study, we assessed the effects of S-petasin and iso-S-petasin on the growth and proliferation of two hormone-independent DU145 and PC3 and one hormone-dependent LNCaP prostate cancer cell line at concentrations of 10(-7)-10(-5) mol/l. The cell proliferation index, cell number index, expression of caspases and apoptosis-associated proteins and cell morphology were measured. RESULTS: S-Petasin and iso-S-petasin reduced the viable cell number and increased the numbers of apoptotic cells in the tested cell lines in a dose-dependent manner. Western blot analysis revealed that S-petasin and iso-S-petasin reduced the protein levels of procaspase 3, 8, and 9 and cleaved poly(ADP-ribose) polymerase (PARP) in all tested prostate cancer cell lines, and reduced that of procaspase 7 in LNCaP and PC3 cells. At the same time, S-petasin and iso-S-petasin increased mitochondrial membrane permeability and cytochrome c release from mitochondria to the cytosol via reducing the ratio of BCL2/BAX in DU145 and PC3 cells, and up-regulating the levels of p53 in DU145 cells but down-regulating it in PC3 cells. CONCLUSION: These results indicate that S-petasin and iso-S-petasin induce apoptosis via the activation of mitochondria-related pathways in prostate cancer cells, suggesting S-petasin and iso-S-petasin could be potential anticancer agents.

Association of caveolin-1 genotypes with renal cell carcinoma risk in Taiwan.

The alteration of caveolin-1 (Cav-1) during carcinogenesis is of great interest and its over-expression in the tumor cell cytoplasm can predict a poor prognosis of renal cell carcinoma (RCC). However, whether the over-expression in RCC is associated with inherited polymorphism is not clear. In this hospital-based case-control study, the association of Cav-1 genotypes with RCC risk in a central Taiwanese population was investigated. Ninety-two patients with RCC and five hundred and eighty of age/gender-matched healthy controls were recruited and genotyped for six polymorphic sites at Cav-1, C521A (rs1997623), G14713A (rs3807987), G21985A (rs12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992). The results showed that there were statistically different distributions of the genotypic (P = 0.0170 and 0.0011) and allelic (P = 0.0033 and 0.0352) frequencies for the Cav-1 G14713A and T29107A polymorphisms among RCC patients and control subjects, respectively. As for the haplotype analysis, subjects carrying "GG/AT or GG/AA" at Cav-1 G14713A/T29107A showed a 2.06-fold increased odds ratio of RCC compared to those with GG/TT, while those of any other combinations were of unaltered odds ratios. In conclusion, this is the first report providing evidence showing that Cav-1 genotype is associated with RCC. The results showed that the G allele of the Cav-1 G14713A and the A allele of the Cav-1 T29107A are risky genetic factors for RCC susceptibility and the combinative GG/AT or GG/AA haplotype at Cav-1 G14713A/T29107A can serve as one of the RCC predictors for Taiwanese.

Phosphorylation of serine-504 of tNOX (ENOX2) modulates cell proliferation and migration in cancer cells.

Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. Consistent with this function, tNOX knockdown by RNA interference leads to a significant reduction in cell proliferation and migration in HeLa cells, whereas tNOX overexpression confers an aggressive phenotype. Here, for the first time, we report that tNOX is phosphorylated by protein kinase Cδ (PKCδ) both in vitro and in vivo. Replacement of serine-504 with alanine significantly reduces phosphorylation by PKCδ. Co-immunoprecipitation experiments reveal an interaction between tNOX and PKCδ. Moreover, whereas overexpression of wild-type tNOX in NIH3T3 cells increases cell proliferation and migration, overexpression of the S504A tNOX mutant leads to diminished cell proliferation and migration, reflecting reduced stability of the unphosphorylatable tNOX mutant protein. Collectively, these results suggest that phosphorylation of serine-504 by PKCδ modulates the biological function of tNOX.