Resveratrol Enhanced Arhi Expression And Inhibited STAT3 Signaling In Human Cutaneous Squamous Cell Carcinomas

Backgrounds and Objectives:Non-melanoma skin cancers(NMSC) is the most common type of skin cancer, representing about 1/3 of all malignancies diagnosed worldwide each year. NMSC has a large number of patients and a significant impact on public health. The most common NMSC are basal cell carcinoma(BCC) and squamous cell carcinoma(SCC).In China cutaneous squamous cell carcinoma(cSCC) accountes the proportion of all malignant skin cancer higher than basal cell carcinoma. Ultraviolet radiation and chemical carcinogen could initiate and promote a well-characterized progression of histologic changes in keratinocytes that may ultimately result in cSCC. In the majority of cases, cSCC can be treated successfully with loco-regional methods including surgical and/or radiation therapy, however high-risk subtypes may recur and lead to lymphovascular invasion as well as nodal and distant metastasis. Topical self-administered treatments may be a highly desirable alternative, both in aged and unhealthy patients(who may be poor surgical candidates) and in relatively young subjects with lesions located on cosmetically sensitive areas and who wish to avoid disfiguring scars. Exploring effective preparation has practical significance of tumor treatment.Resveratrol is a natural polyphenolic phytoalexin extracted from various herbs and plants, including grapes, peanuts, and certain other fruits. Resveratrol has been extensively studied and shown to exhibit significant anti-oxidative and cardioprotective effects as well as anticancer properties. Recent reports have provided evidence that resveratrol possesses anti-cancer properties against some cancer types by regulating a number of cell signaling pathways such as p53,STAT3,ERK1/2 etc. Resveratrol is likely to become an ideal drug in the clinical treatment of skin cancer. However, the specific role of the signaling pathway of resveratrol and other tumors have proven regulatory mechanisms in skin squamous cell carcinoma rarely reported. Therefore, the research of cutaneous squamous cell carcinoma is very important.Aplasia ras homolog member I(ARHI) is a maternally imprinted tumor suppressor gene. ARHI inhibition the growth, proliferation and invasion of cancer cells in a variety of cancers and ARHI promote the apoptosis of cancer cells. ARHI regulating multiple signaling pathways, which involved in signal transduction and activator of transcription 3(STAT3) signaling pathway, preventing the phosphorylation of STAT3 translocating to the nucleus to induce gene expression, inhibits the proliferation and migration of cells. Studies have found that over-expression of p-STAT3 may play an important role in the development of skin cancer. The expression of ARHI has not been reported in cSCC. In summary,we will explore the expression of ARHI in cSCC and the contact of resveratrol, ARHI and STAT3 signaling pathway in cSCC,for studying the pathogenesis of cutaneous squamous cell carcinoma and for providing a new theoretical basis of the strategies for further treatment of cutaneous squamous cell carcinoma. Materials and Methods:84 cases of skin cancer and 74 cases of adjacent normal tissues were supplied by the Dalian Dermatology Hospital. Cell lines Colo16, SCC-1, SCC-12, SCC-13 were kindly provided by Professor Liu Zhili, Department of dermatology Hospital of Dalian. Immortalized human keratinocyte cell line HaCa T was provided by Dr. Martin Hofman the German Institute of Genetics. The different expression of ARHI in normal skin and skin cancer tissue were analyzed by paraffin-embedded tissue array and immunohistochemical analysis(IHC).The chemo-sensitivities of Each cell line to resveratrol were analyzed by MTT assay and flow cytometry(FCM). mRNA expression levels of ARHI, STAT3 signaling pathway genes in each cell line was detected in astrocyte by RT-PCR and the expression of ARHI, STAT3 signaling pathway protein in each cell line with/without resveratrol treatment were detected by Immunocytochemistry analysis(ICC) and Western blot. Positioning expression of ARHI, STAT3 protein in each cell line with/without resveratrol treatment were analyzed by Immunofluorescence analysis(IFC). Results:1.The expression of ARHI in squamous cell carcinoma was significantly lower than in normal skin tissue, statistical analysis P <0.05.2.The chemo-sensitivities of each cell line to resveratrol.2.1Resveratrol could inhibit cell proliferation in each cell lines analyzed by MTT assay.2.2Colo16, SCC-1 and SCC-13 cell lines occurred G1 phase arrest with resveratrol treatment detected by FCM.3.RT-PCR analysis.The mRNA expression of ARHI in each cell line with resveratrol treatment were all increased, and the mRNA expression of STAT3 did not change significantly with/without resveratrol treatment. We detected the mRNA expression of some downstream target genes of STAT3 signaling pathway, the results show: resveratrol could downregulate the mRNA expression of survivin in Colo16, SCC-1,SCC12 and SCC-13 cell lines; downregulate the mRNA expression of VEGF in HaCaT, SCC12 and SCC-13 cell lines; downregulate the m RNA expression of c-Myc only in Colo16;the mRNA expression of cyclinD1 did not change significantly with resveratrol treatment.4.Western blot analysis and immunocytochemistry analysis.The protein expression of ARHI in each cell line with resveratrol treatment were all increased. The protein expression of STAT3 did not change significantly with/without resveratrol treatment, but the protein expression of p-STAT3 were decreased in HaCaT, Colo16 and SCC-1.The protein expression of survivin were decreased in SCC-12 and SCC-13 cell lines. The protein expression of VEGF were decreased in all cell lines. The protein expression of c-Myc were decreased only in HaCaT cell line. The protein expression of cyclinD1 were decreased in HaCaT, Colo16 and SCC-13 cell lines.5.Immunofluorescence analysis.ARHI and STAT3 concentrated around the nucleus with resveratrol treatment, thus resveratrol could inhibit STAT3 into the nucleus. Conclusions:1. ARHI is low expression in cutaneous squamous cell carcinoma.2. Resveratrol could effectively inhibit the proliferation ofColo16, SCC-1, SCC-12, SCC-13 cell lines and cell lines occurred G1 phase arrest.3. Resveratrol could promote the expression of ARHI in each cell line and inhibit the phosphorylation of STAT3. The expression of ARHI maybe prevent the p-STAT3 transferring into the nucleus.4. Resveratrol partially inhibited the expression of STAT3 signal transduction pathways downstream target genes in different cell lines and there may be the possibility of other signaling pathways compensatory complementary.