Expression Of Survivin, PTEN And Livin Proteins In Basal Cell Carcinoma And Its Significance And The Effects Of As₂O₃ On It

Objectives:Epidermal tumors are ones of the most common malignant or benign tumors in the world. The incidence of basal cell carcinoma (BCC) and seborrheic keratosis (SK) is high in China.They are a great threaten to human health. Therefore, it is extremely important to investigate pathogenesis and search new target and effective drugs for BCC and SK therapy to decrease mortality and prolong life span of patients with epidermal tumors.Survivin is a novel member of the apoptosis protein family member. Survivin has correlation with tumor development and progress in many human tumors. Its main biological functions include inhibiting cell apoptosis and enhancing cell proliferation. Survivin selectively expresses in most common hunman noeplasms. More and more researchers study it as an ideal target for cancer treatment. Down-regulation of survivin expression can induce cancer cell apoptosis and enhances radiosensitivity in many human cancers. It can be served as marker of prognosis.PTEN is a dual-specificity lipid phosphase originally identified as a tumor suuressor gene. It frequently mutates and is absent in a number of human cancers, which leads to the weaking or absence of tumor suppressing functions. PTEN plays a critical role in the development and progression of many cancers. PTEN regulates complex signal transduction pathways (such as P13K/AKT pathway, FAK/MAPK cacade, PTEN/P53/MDM2 network), induces G1 cell-cycle arrest and apoptosis, suppresses the vscular formation and inhibits invasion and metastasis of tumors. With further study on the important function of PTEN, an earlier diagnosis, appropriate clinical surveillance, gene-targeted immunotherapy, prognostic evaluation, new drug development and population-oriented first-line prevention of cancers might be possible. Livin, as a new member of IAPs, plays an important role in anti-cancers. Livin regulates complex signal transduction pathways (such as TAK1/JNK1, et al) in antiapoptotic functions. Many studies have discovered that livin protein and livin mRNA are expressed in many malignant human tumors, but not or down-regulatedly expressed in normal tissues and benign tumors. With further sdudy on the important functions of livin, it can be a gene target in immunotherapy for cancers.Clinical trials have demonstrated that arsenic trioxide is effective for acute promyelocytic leukemia; it also has the action of induction of apoptosis and inhibition of growth of some solid cancer cells. It can induce differentiation of human nasopharyngeal carcinoma in BALB/C nude mice xenograft model. But the mechanisms are not clearly understood. Based on these facts, studies have been carried out to study its roles in the treatment of solid cancers. Many studies have discovered that arsenic trioxide can be used in many therapies for solid neoplasm.Therefore, in the present study we examined the expression of survivin, PTEN proteins in the process of carcinogenesis of epidermal tumors and determined the correlation between the levels of these proteins and various clinical and pathological features. Then, we also examined the expression of livin in the process of carcinogenesis of BCC and SK and determined the correlation between the levels of these proteins and various clinical and pathological features;We also analysed the correlation between linvin and survivin, PTEN in BCC and SK. Next, we studied the effects of arsenic trioxide on proliferation, cell cycle distribution, and expression of survivin, PTEN and livin in human epidermal cancer A-431cells.It may deepen our knowledge about epidermal tumors such as basal cell carcinoma and Sk,et al.It might provide theoretical and experimental evidence for utilization of arsenic trioxide in epidermal carcinoma therapy.Methods:1 Expression of survivin, PTEN in epidermal tumors and normal skin tissues and the correlation between the levels of these proteins and clinical-pathological features.Basal cell carcinoma tissues, seborrheic keratosis tissues and normal skin tissues were obtained from resected surgical specimens of epidermal tumors in our dermatology department. There were thirty cases of basal cell carcinoma including 18males and 12 females, who were aged from 45 to 83 years old (averaged 67.40 years old).There were 30 cases of SK including 16 males and 14 females, who were aged from 38 to 70 years old (averaged 54.80 years old).There were 20 cases of normal skin tissues which were from the patients who received surgical treatments in our dermatology department.All the specimens were verified by pathologic diagnosis. Survivin, PTEN protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were examined by immunohistochemistry (IHC). Survivin, PTEN protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were also examined by flow cytometry (FCM). Relationship between their expressions and clinical pathological features was analyzed.2 Expression of livin protein in epidermal tumors and normal skin tissues and the correlation between the levels of this protein and clinical-pathological features and the correlation with survivin and PTEN based on part one study.In this study, basal cell carcinoma tissues, seborrheic keratosis tissues and normal skin tissues were obtained from resected surgical specimens of epidermal tumors in our dermatology department. All the specimens were verified by pathologic diagnosis. They were the same as in part one study. Livin protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were examined by immunohistochemistry (IHC). Livin protein expression in normal skin tissues and the lesions of basal cell carcinoma and seborrheic keratosis were also examined by flow cytometry (FCM). Relationship between its expressions and clinical pathological features was analyzed. Statistical treatment with SPSS was applied to deal with all the experimental data.We also analysed the correlation with survivin and PTEN based on part one study. 3 Effects of arsenic trioxide on cell proliferation, apoptosis, cell cycle distribution and gene expression in human epidermis cancer cell line A-431Human epidermal carcinoma cells (cell line A431) were conventionally cultured. After treatment with arsenic trioxide in different concentrations, inhibition of cellular growth was determined by measuring MTT dye absorption of living cells in 24,48,72h. We calculated the OD values.Then, apoptosis, cell cycle distribution and survivin expression were assessed by flow cytometry and western Blot methods.Results:1 Expression of survivin, PTEN in epidermal tumors and normal skin tissues and the correlation between the levels of these proteins and clinical-pathological features(1) The protein of survivin was positively expressed in 18 cases of the 30 SK specimens while none was expressed in the 20 normal controls (P<0.05). The protein of PTEN was positively expressed in 17 cases of the 30 SK specimens and in 13 cases of the 20 normal controls (P>0.05).(2) The protein of survivin was highly expressed in BCC lesions than in SK lesions and in normal controls (P<0.05), while the protein of PTEN was lowly expressed in BCC than in SK lesions and normal controls (P<0.05).(3) There had been a significant negative correlation between the expressions of survivin and PTEN (r= -0.546, P<0.01) in BCC and SK.(4) The expressions of survivin and PTEN were related with the patients'age, areas of the lesions and the disease duration (P<0.05), but not related with the patients'genders, sites of lesions and numbers of lesions (P>0.05) in patients with BCC.2 Expression of livin protein in epidermal tumors and normal skin tissues and the correlation between the levels of this protein and clinical-pathological features and the correlation with survivin and PTEN.(1) The protein of Livin was highly positively expressed in basal cell carcinoma than in seborrheic keratosis. There was significant difference (P<0.05) and it was also highlier expressed in BCC tissues than in normal controls (P<0.05).(2) The protein of Livin was highlier expressed in SK lesions than in normal controls, there was significant difference (P<0.05).(3) From normal skin to seborrheic keratosis and to basal cell carcinoma, the expressions of Livin were getting gradually highlier.(4) The expressions of livin were related with the patients'age, areas of the lesions and the disease duration (P<0.05), but not related with the patients'gender, sites of lesions and numbers of lesions(P>0.05) in patients with basal cell carcinoma..(5) There had been a significant positive correlation between the expressions of Livin and Survivin (r= 0.557, P<0.05) in basal cell carcinoma and seborrheic keratosis. There had been a significant positive correlation between the expressions of Livin and PTEN (r=- 0.453, P<0.05) in basal cell carcinoma and seborrheic keratosis.3 Effects of arsenic trioxide on cell proliferation, apoptosis, cell cycle distribution and gene expression in human epidermis cancer cell A-431MTT assay showed that with concentrations from 10umol/L to 40umol/L, arsenic trioxide can significantly inhibited the proliferation of A431 cells in a dose- and time-dependent manner (P<0.05). After A-431 cells were dealed with different concentration of arsenic trioxide in 24-72hours, the OD values of the treatment groups decreased to some extent compared with the control group. There were statistically significant differences between them. Survivin expression was significantly downed regulated by arsenic trioxide in A-431 cell line (P<0.05). Arsenic trioxide can inhibit cell proliferation and induce apoptosis in A-431 cells. Arsenic trioxide can also down-regulate the expression of Survivin in A-431 cells. The anti-proliferation effect of arsenic trioxide on human epidermoid carcinoma A-431 maybe is related to down-regulated expression of Survivin gene (P<0.05). Arsenic trioxide can also affect the cell cycle distribution in A-431 cells in our FCM assays. Every group of Arsenic trioxide blocked A-431 cell cycle at S-phase (P<0.05).Cellular morphology was observed under light microscope: cells shrinkage after treatment, the breakdown was irregular in shapes. Pseudo-foot lenders appeared in some cells at both ends. A-431 cells showed morphological changes.Conclusions:(1)Our study found that in the process of carcinogenesis of the basal cell carcinoma, the expression of survivin may play a role in the pathgenesis of BCC and SK while the expression of PTEN may take a part in the protection of BCC and SK. There propably is an interaction between the two genes in BCC and SK. Detecting the two protein levels can provide certain reference value in evaluating the malignant probability and in judging the prognosis of seborrheic keratosis.(2) It was first found that the abnormally high expressions of Livin protein may play a role in the pathogenesis of seborrheic keratosis and basal cell carcinoma through inhibiting cell apoptosis. From normal skin tissues to seberric kerotosis and to basal cell carcinoma, livin expressions are increasing gradually. Our study suggests that Livin can be a gene target in immunotherapy for epidermal tumors such as BCC and SK. There had been a significant positive correlation between the expressions of Livin and Survivin in BCC and SK.There may be a co-operation between the two genes in basal cell carcinoma and seborrheic keratosis. There had been a significant negtive correlation between the expressions of Livin and PTEN in BCC and SK.(3) In our study, it was first found that arsenic trioxide can inhibit cell proliferation and induce apoptosis in A-431 cells. Arsenic trioxide can also down-regulate the expression of survivin in A-431 cells. The anti-proliferation effect of arsenic trioxide on human epidermoid carcinoma A-431 maybe is related to down-regulated expression of survivin gene. Arsenic trioxide can also affect the cell cycle distribution in A-431 cells.