Mechanisms Of Skin Diseases Associated With Arsenic And Antagonistic Effects Of Retinoic Acid

Recently,there were many studies about diseases associated with arsenic and the application of arsenic carried out deeply and extensively by domestic or foreign researchers.Correspondingly,foundation research and clinical application of retinoic acid(RA)also urge certain subject forward.In the field of hematology,RA is effective to treat some leukemia,especially acute promyelocytic leukemia (APL). Arsenic Trioxide (As2O3) is also an effective agent for RA-resistant APL,which means that a strong synergy exists between As2O3 and RA.Arsenic compounds ever used to treat psoriasis,up to now they are still the important composition of some Chinese traditional medicines.It is the fact that accumulation of arsenic in skin may cause arsenic keratosis,even the development of skin tumor.RA is the primary agent to treat psoriasis and approve to be effective to treat skin diseases associated with arsenic. Both of RA and arsenic, some relationships between them are certain, can regulate the proliferation and differentiation of keratinocyte in skin through different signal transduction path.Long time exposure arsenic produces tumors of the skin,bladder,lung,liver and prostate due to drinking water,burning coal,drugs and professional factor.Arsenic accumulated in skin cause hyperpigmention, hyperkeratosis, BCC and SCC.Arsenic carcinogenesis is a long and complex process and lack of good animal model.Keratinocyte cultured in vitro and stimulated by arsenic is a relative ideal model.Recently,Microarray analyses indicated most of the aberrant genes are associated with oxidative stress,cell proliferation and cell differentiation.Among the gene expression correlated with cell growth regulation,ERK path of EGFR is important. In keratinocyte,Cooper'study indicated that arsenic can stimulate p38 and ERK activation in a time and concentration-dependent manner,though the mechanism is different.Activation of ERK depend on EGFR,partly on Src kinase.On the contrary,activation of p38 don't depend on EGF and Src.MAPK signal transduction stimulated by arsenic result into the production MMP-9.Our past studies revealed that arsenic of certain concentration range might stimulate KC proliferation presenting with increasing DNA synthesis,which maybe correlated with activation of ERK and increasing E2F1 expression.In this study,we investigated the alteration of cell cycle regulator after treatment of arsenic in KC.The results showed that As2O3 didn't affect the expression of CDK2,but the expression of CDK4, cyclinE was increased significantly.In order to recognize the effects of arsenic for keratinocyte, we investigated the expression profile of keratinocytes gene in response to low concentration and long time arsenic treated by cDNA microarrays.A total of 176 genes expressed differently,of which 120 genes were down-regulated and 56 genes were up-regulated.These genes primarily encoded signal transduction protein,DNA binding and transcription factor,cell cycle protein and some tumor related genes.More and more evidences indicated that patients suffered from skin diseases associated with arsenic had abnomal immunologic function.Certain concentration arsenic led to downregulation of JAK3 and inhibited lymphocytes proliferation so as to affect the immune function,which may be one of the mechanisms of arsenic remedy arsenic associated diseases.Soto-Pena GA examine the immunotoxic effects of arsenic in humans, Therefore, they collected peripheral blood mononuclear cells (PBMCs) of children exposed to arsenic through their drinking water. Proportions of lymphocyte subpopulations, PBMC mitogenic proliferative response, and urinary arsenic levels were evaluated.The results showed that increased urine arsenic levels were associated with a reduced proliferative response to phytohemaglutinin (PHA) stimulation, CD4 subpopulation proportion, CD4/CD8 ratio, and IL-2 secretion levels. Real-time PCR analysis showed that pretreatment of certain concentration arsenic led to downregulation of JAK3 and inhibited lymphocytes proliferation so as to affect the immune function,which may be one of the mechanisms of arsenic associated diseases.RA,which was called"the third milestone"in the history of dermatological therapeutics, was used broadly to treat various of abnormal keratinization skin diseases,photoaging skin diseases and many kind of skin tumors.Many reports demonstrated that RA approved to be effective drugs to treat arsenic associated diseases, the detail mechanisms are not clear.We stimulated the proliferation of KC by arsenic,then investigated the effects of various concentration of arotinoid trometamol and acitretin on the cell cycle and proliferation in KC.This study closely link the clinic,and make a review about the relationship of arsenic,arsenic associated diseases,RA and keratinocyte.we cultured KC,PBMC in vitro and explore the mechanisms of skin diseases associated with arsenic and antagonistic effects of retinoic acid using western-blot,Real-time PCR and microarray technique.Part I Effects of Arsenite on CDK2, CDK4 and CyclinE Expression in Human Epidermal Keratinocytes.Objective: To investigate the effects of arsenite on CDK2, CDK4 and cyclinE expression in human epidermal keratinocytes.Methods: Human epidermal keratinocytes (cell line HaCaT) were cultured in vitro. After treatment with certain concentration.arsenite, the expression of CDK2, CDK4, cyclinE and the effect of arsenite on its expression in human epidermal keratinocytes was determined by immunoblot analysis.Results: HaCaT cell of control group showed the expression of CDK2, CDK4, cyclinE steadily. In addition, the expression of CDK4, cyclinE was increased significantly when cells were treated by 1nmol/L arsenic trioxide for 24h, though the expression of CDK2 was not so.Conclusions: Certain concentration arsenite may stimulate proliferation of HaCaT cell, which may be associated with increasing expression of CDK4, cyclinE and leading to higher percentage cells in S phase. All of these provide some evidence for the mechanisms of arsenite's carcinogenicity. Part II Arsenic Inhibits Proliferation of Lymphocytes via down- regulating JAK3 Expression.Objective To study the effects of arsenic on cell proliferation and expression of JAK3 in lymphocytes.Methods Human peripheral blood lymphocytes were separated and cultured in vitro., 3H-TdR method was used to detect proliferation of lymphocytes treated with various concentration of arsenic. The expression of JAK3 in lymphocytes was assessed by Realtime-PCR method to explore the mechanism of lymphocytes proliferation.Results In the groups treated with arsenic trioxide (0.01, 0.1, and 1 microM),the amount of incorporated 3H-TdR decreased.There was no significant difference between 0.01μM arsenic group and the control group(P>0.05).Significant differences were noted between 0.1, 1 microM arsenic groups and the control group(P>0.05)Real-time PCR analysis showed that levels of JAK3 mRNA were downregulated in lymphocytes whose proliferation were inhibited .Conclusion Certain concentration arsenic led to downregulation of JAK3 and inhibited lymphocytes proliferation so as to affect the immune function, which may be one of the mechanisms of arsenic treating arsenic associated diseasesPart III Expression profile of Keratinocyte Gene in Response to low Concentration and Long time Arsenic Treatment by cDNA microarrays.Objective To investigate the expression profile of keratinocytes gene in response to low concentration and long time arsenic treatment by cDNA microarrays.Methods Total RNA of keratinocytes cultured in vivo before and after arsenic treatment were extracted and subjected to reverse transcription into cDNA.The cDNA probes were labeled with Cy3 and Cy5 fluorescence and hybridized to the cDNA chips.Differential expression genes were analyzed by screening.Results A total of 176 genes expressed differently,of which 120 genes were down-regulated and 56 genes were up-regulated.These genes primarily encoded signal transduction protein,DNA binding and transcription factor,cell cycle protein and some tumor related genes. Conclusions The expressions of many kinds of genes in keratinocytes will be altered after low concentration and long time arsenic treatment,which may provide some evidences to further studies on carcinogenesis of arsenic.Part IV Antagonistic Effects of Retinoic Acid on Keratinocyte Proliferation Stimulated by Trivalent ArsenicalsObjective To investigate the antagonistic effects of retinoic acid on keratinocyte proliferation stimulated by trivalent arsenicals and its mechanism. Methods Human epidermal keratinocytes (cell line HaCaT) were cultured in vitro. After treatment with trivalent arsenicals for 3 days, keratinocyte proliferation and the antagonistic effects of different retinoid acid were determined by 3H-TdR method.Cell cycle distribution and apoptosis were assessed by flow cytometry. Results Certain concentration trivalent arsenicals stimulated keratinocyte proliferation.After incubation with various concentration of arotinoid trometamol or acitretin for 24h, keratinocyte proliferation stimulated by arsenic were antagonisted by retinoic acid ranging from 0.01μM to 1μM and the role of arotinoid trometamol was stronger than acitretin. DNA flow cytometric analysis indicated that the proportion of S phase decreased and that of G0-G1 phase increased.Conclusion Low concentration trivalent arsenicals stimulated keratinocyte proliferation,which were antagonisted by retinoic acid through decreasing the cell proportion of S phrase.It may be one of the mechanisms that retinoid acid treated skin diseases associated with arsenic.