Hsin-Su Yu

Molecular Mechanisms of UV-Induced Apoptosis and Its Effects on Skin Residential Cells: The Implication in UV-Based Phototherapy

The human skin is an integral system that acts as a physical and immunological barrier to outside pathogens, toxicants, and harmful irradiations. Environmental ultraviolet rays (UV) from the sun might potentially play a more active role in regulating several important biological responses in the context of global warming. UV rays first encounter the uppermost epidermal keratinocytes causing apoptosis. The molecular mechanisms of UV-induced apoptosis of keratinocytes include direct DNA damage (intrinsic), clustering of death receptors on the cell surface (extrinsic), and generation of ROS. When apoptotic keratinocytes are processed by adjacent immature Langerhans cells (LCs), the inappropriately activated Langerhans cells could result in immunosuppression. Furthermore, UV can deplete LCs in the epidermis and impair their migratory capacity, leading to their accumulation in the dermis. Intriguingly, receptor activator of NF-κB (RANK) activation of LCs by UV can induce the pro-survival and anti-apoptotic signals due to the upregulation of Bcl-xL, leading to the generation of regulatory T cells. Meanwhile, a physiological dosage of UV can also enhance melanocyte survival and melanogenesis. Analogous to its effect in keratinocytes, a therapeutic dosage of UV can induce cell cycle arrest, activate antioxidant and DNA repair enzymes, and induce apoptosis through translocation of the Bcl-2 family proteins in melanocytes to ensure genomic integrity and survival of melanocytes. Furthermore, UV can elicit the synthesis of vitamin D, an important molecule in calcium homeostasis of various types of skin cells contributing to DNA repair and immunomodulation. Taken together, the above-mentioned effects of UV on apoptosis and its related biological effects such as proliferation inhibition, melanin synthesis, and immunomodulations on skin residential cells have provided an integrated biochemical and molecular biological basis for phototherapy that has been widely used in the treatment of many dermatological diseases.

Narrow-band ultraviolet-B stimulates proliferation and migration of cultured melanocytes.

Abstract:  Narrow‐band ultraviolet‐B (UVB) radiation is an effective treatment for vitiligo vulgaris. However, the mechanisms of narrow‐band UVB in inducing repigmentation of vitiligo lesions are not thoroughly clarified. The purpose of our study was to investigate the effects of narrow‐band UVB irradiation on melanocyte proliferation and migration in vitro. Our results showed that the cell counts as well as [3H]thymidine uptake of melanocytes were significantly enhanced by narrow‐band UVB‐irradiated keratinocyte supernatants. In these supernatants, a significant increase in basic fibroblast growth factor (bFGF) and in endothelin‐1 (ET‐1) release was observed. bFGF is a natural mitogen for melanocytes, whereas ET‐1 can stimulate DNA synthesis in melanocytes. This stimulatory effect of melanocyte proliferation by supernatants derived from narrow‐band UVB‐irradiated keratinocytes was significantly reduced by a selective endothelin‐B (ET‐B) receptor antagonist (BQ788), suggesting an essential role of ET‐1 on melanocyte proliferation. Our results of time‐lapse microphotography revealed a stimulatory effect of narrow‐band UVB irradiation on melanocyte migration. Focal adhesion kinase (FAK) plays a pivotal role in cell migration. Phosphorylated FAK (p125FAK) expression on melanocyte was enhanced by narrow‐band UVB irradiation. In this study, narrow‐band UVB irradiation stimulated a significant increase in matrix metalloproteinase‐2 (MMP‐2) activity in melanocyte supernatants. Narrow‐band UVB‐irradiation‐induced migration of melanocytes was significantly annihilated by the addition of p125FAK inhibitor (herbimycin‐A) or MMP‐2 inhibitor (GM6001). These results suggest that p125FAK and MMP‐2 activity play important roles in narrow‐band UVB‐induced migration of melanocytes. Our results provide a theoretical basis for the effectiveness of narrow‐band UVB irradiation in treating vitiligo.

Mast cell degranulation and elastolysis in the early stage of striae distensae

The lesions of nine patients with early striae distensae (SD) during puberty were examined by light and electron microscopy. Specific changes were seen in very early stage SD, and in clinically uninvolved skin 0.5 to 3 cm remote from the edge of the long axis of the SD lesions. Sequential changes of elastolysis accompanied by mast cell degranulation appeared first, followed by an influx of activated macrophages that enveloped fragmented elastic fibers. The relationships among elastic fibers, mast cells, and macrophages seen in the present work suggest their critical roles in the process of SD formation, especially in the early stage. Our results also indicate that the elastic fiber is the primary target of the pathological process, and the abnormalities extend as far as 3 cm beyond the lesion into normal skin.

Traffic-Related Air Pollution, Climate, and Prevalence of Eczema in Taiwanese School Children

The prevalence of childhood eczema is increasing in many countries. Epidemiological studies, however, say little of its association to outdoor air pollution and climate factors. We conducted a nationwide survey of middle-school students in Taiwan from 1995 to 1996. The 12-month prevalence of eczema was compared with air monitoring station data of temperature, relative humidity, and criteria air pollutants. A total of 317,926 children attended schools located within 2 km of 55 stations. Prevalence rates of recurrent eczema were 2.4 and 2.3% in boys and girls, respectively, with prevalence rates of flexural eczema 1.7% in both sexes. After adjustment for possible confounders, flexural eczema was found to be associated with traffic-related air pollutants, including nitrogen oxides and carbon monoxide. Recurrent eczema was associated with traffic-related air pollution only in girls. There were no associations for the highest monthly means of temperature, whereas the annual means and the lowest monthly means of temperature were negatively related to flexural eczema, but only in girls. The lowest monthly mean relative humidity was positively related to eczema. The results suggest that air pollution and climatic factors, which showed stronger associations in girls than boys, may affect the prevalence of childhood eczema.

Aberrant Cell Proliferation by Enhanced Mitochondrial Biogenesis via mtTFA in Arsenical Skin Cancers

Arsenic-induced Bowens disease (As-BD), a cutaneous carcinoma in situ, is thought to arise from gene mutation and uncontrolled proliferation. However, how mitochondria regulate the arsenic-induced cell proliferation remains unclear. The aim of this study was to clarify whether arsenic interfered with mitochondrial biogenesis and function, leading to aberrant cell proliferation in As-BD. Skin biopsy samples from patients with As-BD and controls were stained for cytochrome c oxidase (Complex IV), measured for mitochondrial DNA (mtDNA) copy number and the expression levels of mitochondrial biogenesis-related genes, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (mtTFA). The results showed that expression of cytochrome c oxidase, mtTFA, NRF-1, and PGC-1α was increased in As-BD compared with in healthy subjects. Treatment of primary keratinocytes with arsenic at concentrations lower than 1.0 μmol/L induced cell proliferation, along with enhanced mitochondrial biogenesis. Furthermore, we observed that the mitochondrial oxygen consumption rate and intracellular ATP level were increased in arsenic-treated keratinocytes. Blocking of mitochondrial function by oligomycin A (Complex V inhibitor) or knockdown of mtTFA by RNA interference abrogated arsenic-induced cell proliferation without affecting cyclin D1 expression. We concluded that mtTFA up-regulation, augmented mitochondrial biogenesis, and enhanced mitochondrial functions may contribute to arsenic-induced cell proliferation. Targeting mitochondrial biogenesis may help treat arsenical cancers at the stage of cell proliferation.

Synthesis, and biological evaluation of 2-(4-aminophenyl)benzothiazole derivatives as photosensitizing agents.

Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric structure and absorb light in the UVA (315-400 nm). These results encouraged us to design and synthesize a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosensitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated cells displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochondrial membrane potential (Deltapsi(mt)) and ATP via enhanced ROS generation and promoted phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation, and ultimately lead to BCC cell apoptosis.

Peripheral Vascular Diseases Resulting from Chronic Arsenical Poisoning

Drinking water contaminated by arsenic remains a major public health problem. Long‐term arsenic exposure has been found to be associated with peripheral vascular diseases in a variety of studies. Reports of vascular effects of arsenic in drinking water, which span almost 100 years, have been published in Taiwan, Chile, Mexico, and China. This paper reviewed the association of peripheral vascular diseases resulting from arsenic exposure to drinking water from the clinical and pathological points of view. An endemic peripheral vascular disorder called “blackfoot disease” has been noticed in a limited area in Taiwan. This disease results in gangrene in the extremities. It has been associated with the ingestion of high concentrations of arsenic‐tainted artesian well water. Epidemiological studies confirmed a dose‐response relationship between long‐term arsenic exposure and the occurrence of blackfoot disease. Whereas arsenic has induced various clinical manifestations of vascular effects in Chile, Mexico and China, they do not compare in magnitude or severity to the blackfoot disease found in Taiwan. The pathogenesis of vascular effects induced by arsenic is still controversial. The possible mechanisms include endothelial cell destruction, arsenic‐associated atherogenesis, carotene and zinc deficiency, and/or some immunological mechanism. Microcirculatory assessments revealed that deficits of capillary blood flow and permeability exist in clinically normal skin of patients with chronic arsenical poisoning. The vascular effects of chronic arsenic poisoning may involve cardiovascular and cerebrovascular systems as well. In view of the increasing public health problems caused by arsenic exposure, vascular effects should be included in the future study of health effects of arsenic.

Expression of nitric oxide synthases in keratinocytes after UVB irradiation

The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.

Prediction of heat-induced collagen shrinkage by use of second harmonic generation microscopy

Collagen shrinkage associated with denaturation from thermal treatment has a number of important clinical applications. However, individualized treatment is hindered by the lack of reliable noninvasive methods to monitor the process of collagen denaturation. We investigate the serial changes of collagen denaturation from thermal treatment of rat tail tendons at 58 degrees C by use of second harmonic generation (SHG) microscopy. We find that rat tail tendon shrinks progressively from 0 to 9 min of thermal treatment, and remains unchanged in length upon further thermal treatment. The SHG intensity also decreases from 0 to 9 min of thermal treatment and becomes barely detectable from further thermal treatment. Collagen shrinkage and the SHG intensity are well correlated in a linear model. In addition, SHG imaging reveals a tiger-tail-like pattern of collagen denaturation. The bands of denatured collagen progressively widen from increased thermal treatment and completely replace the adjacent bands of normal collagen after 9 min of thermal treatment. Our results show that collagen denaturation in rat tail tendon from thermal treatment is inhomogeneous, and that SHG intensity can be used to predict the degree of thermally induced collagen shrinkage. With additional development, this approach has the potential to be used in biomedical applications.

Synthesis and biological evaluation of thiobenzanilides as anticancer agents

A series of novel thiobenzanilides is described. These compounds have been previously found to show strong biological activity such as antimycotic and antifungal actions. This is the first demonstration on the mechanism of the anticancer effect of thiobenzanilide agents (4a-c) on human melanoma A375 cells. The cytotoxic studies of compounds 4a-c on human melanoma A375 cells indicate thiobenzanilides induced higher cytotoxicity than nitrobenzanilides (3a-c). In addition, DNA flow cytometric analysis shows that 4a-c displays a significant G2/M phase arrest, which progresses to early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide (PI). Because cellular apoptosis is often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in 4a-c-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with 4a-c resulted in the loss of mitochondrial membrane potential (DeltaPsi(mt)), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and activation of caspase-3. Thus, we suggest that 4a-c agents are potent inducers of cell apoptosis in A375 cells.