Solamargine Alleviated UVB-Induced Inflammation and Melanogenesis in Human Keratinocytes and Melanocytes via the p38 MAPK Signaling Pathway, a Promising Agent for Post-inflammatory Hyperpigmentation.

Post-inflammatory hyperpigmentation (PIH) is a common acquired pigmentary disorder occurring after skin inflammation or injury. Ultraviolet B irradiation could exaggerate PIH clinically due to its effect on promoting cutaneous inflammation and melanogenesis in keratinocytes and melanocytes, respectively. Solamargine (SM), a steroidal alkaloid glycoside extracted from Solanum undatum, significantly inhibits Ultraviolet B (UVB)-induced pro-inflammatory cytokines IL-1α, IL-1ß, IL-8, and IFN-γ, as well as paracrine melanogenic factors ET-1, α-MSH, and bFGF in human keratinocytes. Additionally, SM significantly attenuated UVB-induced melanin synthesis in human epidermal melanocytes through down-regulation of tyrosinase activity and expression of MITF, TRP-1, TRP-2, and tyrosinase. SM exerted an anti-inflammatory effect in UVB-irradiated keratinocytes through the p38 MAPK/Nrf2/HO-1 signaling pathway. With its anti-inflammatory and whitening effect, SM may improve PIH through paracrine regulations of keratinocytes and direct action on melanocytes, making it a promising agent for PIH.

Effects of Solanum undatum extract (SR-T100) on photocarcinogenesis and photoaging of actinic keratosis.

SR-T100 gel, containing solamargine extracted from Solanum undatum (synonym: Solanum incanum), had good therapeutic effects on actinic keratosis (AK) in human and ultraviolet B-induced papilloma in mice. This study aimed to investigate the immunohistochemical changes in the human skin after SR-T100 treatment. An immunohistochemical study was performed and the changes in photocarcinogenesis and photoaging markers after 16-week SR-T100 gel treatment were documented. SR-T100 gel treatment for 16 weeks resulted in complete remission in nine AK lesions and partial remission in four AK lesions. SR-T100 gel abolished the expression of mutant p53 and SOX2 and restored the expression of NOTCH1. Additionally, SR-T100 gel improved wrinkling in human skin, while restoring the expression of lamin B1 and increasing synthesis of new elastic fibers. SR-T100 gel had therapeutic effects on photocarcinogenesis and photoaging of photodamaged skin with AK.

Real-world efficacy of biological agents in moderate-to-severe plaque psoriasis: An analysis of 75 patients in Taiwan.

BACKGROUND: Real-world clinical data on psoriasis patients receiving different biological agents is needed, especially in Asian populations. OBJECTIVES: Our aim is to compare and analyze the efficacy and safety profile of four biological agents (etanercept, adalimumab, ustekinumab and secukinumab) in a real-world setting in Taiwan. METHODS: We retrospectively analyzed the clinical data of all patients with moderate-to-severe plaque psoriasis (Psoriasis Area and Severity Index (PASI) ≥ 10) who received etanercept, adalimumab, ustekinumab or secukinumab between January 2011 and December 2018 in a tertiary hospital in Taiwan. RESULTS: A total of 119 treatment episodes in 75 patients were included in this study. Ustekinumab was used in 49 treatment episodes, followed by secukinumab in 46 treatment episodes, adalimumab in 14 treatment episodes and etanercept in 10 treatment episodes. The proportion of the biologic-naïve was highest in etanercept (100%) and lowest in secukinumab (23.9%). The PASI-75, -90 and -100 were the highest in secukinumab (91.3%, 82.6%, 41.3%, respectively), followed by ustekinumab (79.6%, 44.9%, 16.3%), adalimumab (64.3%, 28.6%, 7.1%) and etanercept (50.0%, 30.0%, 0%). The rate of adverse events that required treatment was highest for secukinumab (15.2%), followed by adalimumab (14.3%), ustekinumab (8.2%), and etanercept (0%), including 4 cases of infections, 2 cases of cardiovascular diseases and 4 cases of cancers. CONCLUSIONS: This real world data showed differential efficacy and safety of the four biological agents.

By protecting against cutaneous inflammation, epidermal pigmentation provided an additional advantage for ancestral humans.

Pigmentation evolved in ancestral humans to protect against toxic, ultraviolet B irradiation, but the question remains: "what is being protected?" Because humans with dark pigmentation display a suite of superior epidermal functions in comparison with their more lightly pigmented counterparts, we hypothesized and provided evidence that dark pigmentation evolved in Africa to support cutaneous function. Because our prior clinical studies also showed that a restoration of a competent barrier dampens cutaneous inflammation, we hypothesized that resistance to inflammation could have provided pigmented hominins with yet another, important evolutionary benefit. We addressed this issue here in two closely related strains of hairless mice, endowed with either moderate (Skh2/J) or absent (Skh1) pigmentation. In these models, we showed that (a) pigmented mice display a markedly reduced propensity to develop inflammation after challenges with either a topical irritant or allergen in comparison with their nonpigmented counterparts; (b) visible and histologic evidence of inflammation was paralleled by reduced levels of pro-inflammatory cytokines (i.e., IL-1α and INFα); (c) because depigmentation of Skh2/J mouse skin enhanced both visible inflammation and pro-inflammatory cytokine levels after comparable pro-inflammatory challenges, the reduced propensity to develop inflammation was directly linked to the presence of pigmentation; and (d) furthermore, in accordance with our prior work showing that pigment production endows benefits by reducing the surface pH of skin, acidification of albino (Skh1) mouse skin also protected against inflammation, and equalized cytokine levels to those found in pigmented skin. In summary, pigmentation yields a reduced propensity to develop inflammation, consistent with our hypothesis that dark pigmentation evolved in ancestral humans to provide a suite of barrier-linked benefits that now include resistance to inflammation.

Efficacy and safety of topical SR-T100 gel in treating actinic keratosis in Taiwan: A Phase III randomized double-blind vehicle-controlled parallel trial.

BACKGROUND: Currently available topical treatments for actinic keratosis (AK) are associated with substantial side-effects. OBJECTIVES: To evaluate the efficacy and safety of topical SR-T100 gel in treating AK. METHODS: A multicenter, randomized, double-blinded phase III trial was conducted. Patients with at least two clinically visible AK were enrolled and a punch biopsy was performed on one of the AK to confirm the diagnosis. This study consisted of up to 16-week treatment and 8-week post-treatment periods. Medication was applied daily with occlusive dressing. RESULTS: 123 subjects were recruited and 113 were randomized. 76 subjects were in the SR-T100 and 37 in the vehicle arms. In SR-T100 and vehicle groups, 32.39% and 17.14% of subjects achieved complete clearance, respectively. For 75% partial clearance of lesions, 71.83% and 37.1% of subjects achieved this goal in SR-T100 and vehicle group, respectively. When comparing SR-T100 to vehicle, the odds ratio of complete clearance was 2.14 (p = 0.111), and odds ratio of partial clearance was 4.36 (p < 0.001). Severe local reactions were reported by only one subject using SR-T100. CONCLUSION: The imitation of the study was that not all the treated AK lesions were confirmed by histopathology. The diagnostic uncertainty may contribute to the high partial clearance rate in the vehicle group since the clinical-diagnosed AK showed higher clearance rate compared to histopathology-confirmed AK. The use of occlusive dressing was another possible explanation for high placebo effects. The results suggested that topical SR-T100 gel may be an effective and safe treatment for field therapy of AK.

Solanum incanum extract (SR-T100) induces melanoma cell apoptosis and inhibits established lung metastasis.

Melanoma, a cancer derived from melanocytes, is one of the most chemoresistant cancers and tends to metastasize. Once it metastasizes, the prognosis is poor. Even with the recent advancement of targeted therapy and immunotherapy, the prognosis remains discouraging. SR-T100, a Solanum incanum extract, shows anticancer effects against several cancers; however, its therapeutic efficacy against melanoma and established metastasis remains unknown. In this study, we showed that SR-T100 induces apoptosis, DNA damage, and G0/G1 cell cycle arrest in murine B16 melanoma cells in vitro. In vivo, intralesional injection of SR-T100 decreased the tumor size of the regional melanoma in the foot pad. Moreover, intraperitoneal injection of SR-T100 inhibited the growth and the number of established melanoma metastases in the lungs. Our study highlights SR-T100 as a potential novel treatment for established tumors from regional and metastatic melanoma.

UV irradiation/cold shock-mediated apoptosis is switched to bubbling cell death at low temperatures.

When COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10-30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause "bubbling death". Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

Human sebum extract induces barrier disruption and cytokine expression in murine epidermis.

BACKGROUND: Previous studies have shown that human sebum may play a role in barrier function but with much debate. OBJECTIVE: To elucidate the effects of human sebum on skin barrier function. METHODS: We used hairless mouse skin to study the functional and morphological alternation of epidermis after the application of human sebum. RESULTS: The results showed a significant increase in transepidermal water loss and erythema value, and a decrease in skin hydration, accompanied by epidermal hyperplasia with parakeratosis following sebum application. Nile red staining together with electron microscopic examination confirmed the underlying mechanisms for sebum-induced barrier disruption are related directly to the interaction of sebum with the intracellular lipid lamellae of the SC, thereby leading to the increase in the fluidity of SC intracellular lipids as demonstrated by ATR-FTIR measurement. An inflammatory reaction characterized by an enhanced cytokine cascade, including up-regulation of TNF-α, IL-1α and IL-6, was also observed. On the other hand, there were insignificant expression of thymic stromal lymphopoietin and unchanged serum levels of IgE, suggesting non-immunogenic stimulation by sebum treatment. CONCLUSION: It may be concluded that inflammation induced by excess amount of sebum is more likely an irritant contact dermatitis rather than an allergic one. Moreover, these findings implicated possible relationships between sebum, irritant contact dermatitis, and seborrheic dermatitis.

Leptin of dermal adipose tissue is differentially expressed during the hair cycle and contributes to adipocyte-mediated growth inhibition of anagen-phase vibrissa hair.

Adipose tissue encircles the lower portion of anagen hair follicles and may regulate hair cycle progression. As leptin is a major adipokine, its level of expression from the dermal white adipose tissue during hair cycle progression was studied. The result shows that leptin level is differentially expressed during hair cycle, the lowest in early anagen phase, upregulated in late anagen phase and the highest in the telogen phase. On the other hand, leptin receptor is detected in keratin 15-positive hair bulge epithelium of both anagen- and telogen-phase hair follicles of mice pelage and vibrissa hair, and hair from human scalp. Leptin contributes to adipocyte-mediated growth inhibition of anagen-phase vibrissa hair as demonstrated in organ culture and coculture system. Our data suggest that leptin of dermal white adipose tissue might regulate hair growth and, therefore, hair cycle progression via leptin receptor on the hair follicle epithelium.

N-acetylcysteine attenuates hexavalent chromium-induced hypersensitivity through inhibition of cell death, ROS-related signaling and cytokine expression.

Chromium hypersensitivity (chromium-induced allergic contact dermatitis) is an important issue in occupational skin disease. Hexavalent chromium (Cr (VI)) can activate the Akt, Nuclear factor κB (NF-κB), and Mitogen-activated protein kinase (MAPK) pathways and induce cell death, via the effects of reactive oxygen species (ROS). Recently, cell death stimuli have been proposed to regulate the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). However, the exact effects of ROS on the signaling molecules and cytotoxicity involved in Cr(VI)-induced hypersensitivity have not yet been fully demonstrated. N-acetylcysteine (NAC) could increase glutathione levels in the skin and act as an antioxidant. In this study, we investigated the effects of NAC on attenuating the Cr(VI)-triggered ROS signaling in both normal keratinocyte cells (HaCaT cells) and a guinea pig (GP) model. The results showed the induction of apoptosis, autophagy and ROS were observed after different concentrations of Cr(VI) treatment. HaCaT cells pretreated with NAC exhibited a decrease in apoptosis and autophagy, which could affect cell viability. In addition, Cr (VI) activated the Akt, NF-κB and MAPK pathways thereby increasing IL-1α and TNF-α production. However, all of these stimulation phenomena could be inhibited by NAC in both of in vitro and in vivo studies. These novel findings indicate that NAC may prevent the development of chromium hypersensitivity by inhibiting of ROS-induced cell death and cytokine expression.

Higher body mass index is associated with greater severity of alopecia in men with male-pattern androgenetic alopecia in Taiwan: a cross-sectional study.

BACKGROUND: Obesity is a risk factor for multiple health problems, but its association with androgenetic alopecia (AGA) remains controversial. OBJECTIVE: We sought to determine the association between body mass index (BMI) and alopecia severity in men with AGA and early-onset AGA. METHODS: A cross-sectional study was conducted. The medical charts and photographs of men with a clinical diagnosis of AGA were reviewed. RESULTS: In all, 189 men were enrolled with a mean age of 30.8 years. In male-pattern AGA (n = 142), men with severe alopecia (grade V-VII) had higher BMI than those with mild to moderate alopecia (grade I-IV) (25.1 vs 22.8 kg/m(2), P = .01). After multivariate adjustments, the risk for severe alopecia was higher in the overweight or obese (BMI ≥24 kg/m(2)) subjects with male-pattern AGA (odds ratio 3.52, P < .01). In early-onset male-pattern AGA (n = 46), the risk for having severe alopecia was also higher in the overweight or obese subjects (odds ratio 4.97, P = .03). LIMITATIONS: Parameters used to evaluate obesity were limited because of the retrospective nature of the study. CONCLUSIONS: Higher BMI was significantly associated with greater severity of hair loss in men with male-pattern AGA, especially in those with early-onset AGA.

Early intervention with high-dose steroid pulse therapy prolongs disease-free interval of severe alopecia areata: a retrospective study.

BACKGROUND: Spontaneous recovery of severe alopecia areata is rare and the condition is difficult to treat. OBJECTIVE: The aim of this study is to investigate and compare the effects and safety of steroid pulse therapy between oral and intravenous administrations between 1999 and 2010 at the Department of Dermatology, National Cheng Kung University Hospital. METHODS: Data were retrospectively retrieved. A satisfactory response was defined as more than 75% hair regrowth in the balding area. RESULTS: A total of 85 patients with more than 50% hair loss were identified and treated, with an overall satisfactory response rate of 51.8%. The mean follow-up time was 37.6 months, with a relapse rate of 22.7%. Patients with alopecia areata (hereafter, AA) of recent onset within one year showed higher response rates (p<0.001) and lower relapse rates compared to patients with AA persisting for more than 1 year. Further, even in patients with alopecia totalis, alopecia universalis or ophiasis type, early treatment resulted in a satisfactory response rate of 47% among the treated patients. In general, oral therapy was as effective and well-tolerated as intravenous therapy. CONCLUSION: The response rate is determined by disease severity and time of intervention, not by the administration form of steroid pulse therapy. Oral steroid pulse therapy can be considered as the first-line treatment for patients with severe AA of recent onset within one year.

Low concentration of arsenic-induced aberrant mitosis in keratinocytes through E2F1 transcriptionally regulated Aurora-A.

Chronic exposure to low-concentration arsenic promotes cell proliferation and carcinogenesis both in vitro and in vivo. Centrosome amplification, the major cause of chromosome instability, occurs frequently in cancers. Aurora-A is a mitotic kinase and causes centrosome amplification and chromosome instability when overexpressed. Our previous study revealed that low-concentration arsenic induces Aurora-A overexpression in immortalized bladder cells. In this study, we hypothesized that low-concentration arsenic induces aberrant mitosis in keratinocytes due to Aurora-A overexpression. The specimen of Bowen's disease (BD) and squamous cell carcinoma obtained from arseniasis-endemic areas in Taiwan showed Aurora-A overexpression. The mRNA/protein levels and kinase activity of Aurora-A were increased in immortalized keratinocyte HaCaT cells after arsenic treatment at low concentration (< 1µM). Aberrant spindles, multiple centrosomes, and multinucleated cells were detected under fluorescent microscopy in HaCaT cells after arsenic treatment. These findings were associated with increased expression of Aurora-A. We further revealed that Aurora-A was regulated by arsenic-induced transcriptional factor E2F1 as demonstrated by chromosome immunoprecipitation, promoter activity, and small interfering RNA assays. Finally, in arsenic-treated HaCaT cells and in BD, a significant increase of dysfunctional p53 was found, and this event correlated with the increase in expression of Aurora-A. Altogether, our data suggest that low concentration of arsenic induces activation of E2F1-Aurora-A axis and results in aberrant mitosis of keratinocytes. Overexpression of Aurora-A and dysfunctional p53 may act synergistically to trigger skin tumor formation. Our findings suggest that Aurora-A may be a potential target for the prevention and treatment of arsenic-related cancers.

Solanum incanum extract (SR-T100) induces human cutaneous squamous cell carcinoma apoptosis through modulating tumor necrosis factor receptor signaling pathway.

BACKGROUND: The Solanum species herbs have been used to treat cancer for centuries; however, the underlying mechanisms and effectiveness in vivo remain unclear. OBJECTIVES: SR-T100, extracted from the Solanum incanum, contains solamargine alkaloid as the main active ingredient. Here, we investigated the apoptosis-inducing effects of SR-T100 for targeting squamous cell carcinoma (SCC) in vitro and in vivo. METHODS: We elucidated the mechanism by which SR-T100 induces apoptosis of human SCCs (A431, SCC4, SCC9, and SCC25) cells. The efficacy and safety issues were addressed regarding topical treatment of SR-T100 on UVB-induced cutaneous SCC of hairless mice and actinic keratoses (AKs) of human. RESULTS: SR-T100 induces apoptosis in human SCCs cell lines by up-regulating the expressions of tumor necrosis factor receptors (TNFRs) and Fas, and downstream adaptors FADD/TRADD of the TNF-α and Fas ligand signaling cascades. SR-T100 also triggered the mitochondrial apoptotic pathway, as up-regulated cytochrome c and Bax, down-regulated Bcl-X(L). Animal experiments showed that all papillomas (35/35) and 27 of 30 UVB-induced microinvasive SCCs in hairless mice disappeared within 10 weeks after once-daily application of topical SR-T100. Furthermore, 13 patients, who suffered with 14 AKs, were treated with once-daily topical SR-T100 gel and 10 AKs cured after 16 weeks, showing negligible discomforts. CONCLUSION: Our studies indicate that SR-T100 induces apoptosis of SCC cells via death receptors and the mitochondrial death pathway. The high efficacy of SR-T100 in our preclinical trial suggests that SR-T100 is a highly promising herb for AKs and related disorders.

Downregulation of thrombomodulin, a novel target of Snail, induces tumorigenesis through epithelial-mesenchymal transition.

The expression of thrombomodulin (TM), a calcium-dependent adhesion molecule, is frequently downregulated in various cancer types. However, the mechanism responsible for the low expression level of TM in tumorigenesis is unknown. Here, an inverse expression of TM and Snail was detected in different cancer cell lines. We further confirmed this inverse relation using the epithelial-mesenchymal transition cell model in HaCaT and A431 cells. We demonstrated that Snail suppressed TM expression by binding to E-box (CACCTG) in TM promoter. Moreover, TM knockdown by short hairpin RNA disrupted E-cadherin-mediated cell junctions and contributed to tumorigenesis. In the calcium switch assay, E-cadherin lost the ability to associate with ß-catenin and accumulated in cytoplasm in TM knockdown cells. Meanwhile, wound healing and invasive assays showed that TM knockdown promoted cell motility. A subcutaneous injection of TM knockdown transfectants into immunocompromised mice induced squamous cell carcinoma-like tumors. Besides, forced expression of murine TM in TM knockdown cells made the cells reassume epithelium-like morphology and increased calcium-dependent association of E-cadherin and ß-catenin. In conclusion, TM, a novel downstream target of Snail in epithelial-mesenchymal transition, is required for maintaining epithelial morphology and functions as a tumor suppressor.

Hexavalent chromium induced ROS formation, Akt, NF-kappaB, and MAPK activation, and TNF-alpha and IL-1alpha production in keratinocytes.

In certain cell types, it has been found that, hexavalent chromium could increase ROS formation, activate cell signaling and stimulate the release of cytokines. But, in keratinocytes, these effects have not yet fully been demonstrated. Our aim is to observe the above effects of hexavalent chromium on keratinocytes. By utilizing HaCaT cells and the skin of albino guinea pigs, we showed that hexavalent chromium could increase ROS formation, activate the Akt, NF-kB, and MAPK pathways as well as increase the production of cytokines, including TNF-alpha and IL-1alpha. The release of these cytokines from keratinocytes is considered to be a key participant in the pathogenesis of contact hypersensitivity. Among cement workers, chromium hypersensitivity is an important occupational skin disease issue. Therefore, the observations of our study help us better understand the role of hexavalent chromium on the development of chromium hypersensitivity, which might provide clues for clinicians in the development of chemopreventative agents for the prevention of chromium hypersensitivity among cement workers.

Testosterone synthesized in cultured human SZ95 sebocytes derives mainly from dehydroepiandrosterone.

Human sebaceous gland possesses all the steroidogenic enzymes required for androgen synthesis. It remains unclear whether the testosterone produced in situ mainly derives from circulating dehydroepiandrosterone (DHEA) or from de novo synthesis utilizing serum cholesterol. Using testosterone radioimmunoassay, we found that testosterone was barely detectable in the supernatant of cultured human SZ95 sebocytes when cholesterol was added alone, indicating a low basal expression of steroidogenic acute regulatory protein (StAR) in SZ95 cells. Human chorionic gonadotropin and fibroblast growth factor-9 were as potent as forskolin in activating StAR to enhance testosterone production, while interleukin-1 beta, dexamethasone, insulin and insulin-like growth factor-1 showed no stimulatory effect. A two-fold increase of testosterone production was observed in supplementation of DHEA as compared to pregnenolone, progesterone or 17 alpha-hydroxyprogesterone. Based on our findings, testosterone synthesized in cultured sebocytes derived mainly from DHEA and inhibition of 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase may be a new target of androgen suppression for acne treatment.