The Mechanisms Of Apoptosis Of Skin Squamous Cell Carcinoma Induced By IFN-γ And ATRA

The mechanisms of apoptosis of skin squamous cell carcinoma induced by IFN-γand ATRAIntroduction and objectiveSkin squamous cell carcinoma (SCC) is a malignant neoplasm derived from epidermal keratinocyte and squamous cells of skin appendant organ .It is one of the most common skin carcinoma, and the incidence rate of SCC is 20% in malignant non-melanoma. It is a common disease that seriously harms health of people in our country, so we should pay more attention to it.At present, surgical operation is still a preferred method of treatment of SCC of skin,and surgical operation and assisted preoperative and postoperative radiotherapy and chemotherapy have become a standard mode for the treatment of skin SCC. But skin SCC at the progressive stage is still a difficult problem that needs to be solved in the treatment, so the search for promising gene therapy technology has an important significance.The key to gene therapy is the selection of the target gene. With the completion of the Human Genome Project, many genes have been discovered and cloned, giving great promise to the gene therapy of tumors. Survivin is a recently discovered apoptosis inhibiting protein, belonging to the family of apoptosis protein inhibiting factors, with unique structure, only expressed in the embryonic tissue and most tumorous tissues, such as colon cancer, lung cancer, breast cancer, stomach cancer and liver cancer, and is related to the progress of disease and bad prognosis. The specific distribution of surviving makes it become a gene therapy target.The aim of this research was to approach the expression of survivin and bcl-2 in human skin squamous cell carcinoma, and the effect of ATRA andγ-IFN on the growth,apoptosis,differentiation and the expression of suvivin.Methods1. Expression of survivin in the skin SCC tissue by the immunohistochemical method and its clinical significance.2. Detection of cell growth inhibition with the MTT analysis method.3. To detect the morphous of the apoptosis cell by transmission electron microscope(TEM) and iodinate tritroche stain.4. To detect the apoptosis rate by FCM.5. To detect the expression of survivin before and afer the treatment of ATRA andγ-IFN by immunohistochemical method and Western blot.6. To detect the expression of survivin mRNA before and afer the treatment of ATRA andγ-IFN by RT-PCR method.Results1. Survivin and bcl-2 expression in the skin SCC tissueIn SCC, the positive rates of bcl-2 and survivin were 70% and 60%,respectively(P＞0.05). Survivin expression was not related to clinical staging of patients, but related to the lymph node metastasis.2. To detect growth inhibition rate of SCL-1 cells by MTT:ATRA andγ-IFN could significantly inhibit the proliferation of SCL-1 cells and showed dose dependence.(1)In group ATRA The difference of inhibition rate among the six groups was significant (p=0.000＜0.05). There has no difference between the control and the group 0.5μmol/1, 0.1μmol/1 ATRA (p= 0.345, 0.077＞0.05).The difference of inhibition rate between the control and the grouplμmol/1, 5μmol/1ATRA and 10μmol/1 ATRA was significant (p=0.000＜0.05). (2)In groupγ-IFN The difference of inhibition rate among the four groups were significant (p=0.000＜0.01). There has difference between the control and the experiment groups (p＜0.01).The difference of inhibition rate between the group 1000 U/mlγ- IFN and the group 100 U/ml or 500U/mlγ- IFN, was significant (p=0.000,0.001＜0.01).(3)In group combination of ATRA andγ-IFN:①Different concentration of ATRA+1000 U/mlγ- IFNThe difference of inhibition rate among the six groups were significant (p=0.000＜0.01) There has no difference between 1000 U/mlγ- IFN+0.1μmol/1 ATRA and the group 1000 U/mlγ- IFN+0.5μmol/1 ATRA(p=0.391＞0.05).The difference of inhibition rate between the group 1000 U/mlγ- IFN+ 1μmol/1 ATRA and group 1000 U/mlγ- IFN+0.1μmol/1 ATRA,1000 U/mlγ- IFN+0.5μmol/1 ATRA was significant (p=0.000＜0.01), but no difference between the group 1000 U/mlγIFN+1μmol/1 ATRA and group 1000 U/mlγ- IFN+5μmol/1 ATRA,1000 U/mlγIFN+10μmol/1 ATRA (p=0.294, 0.536＞0.05).②Different concentration ofγ- IFN+1μmol/1 ATRAThe difference of inhibition rate among the five groups were significant (p=0.000＜0.01). There has difference between control group and experiment groups (p=0.000＜0.01).3. To survey the morphous of the cell apoptosis by transmissionelectron microscope(TEM):The shape of the cells in control were as follow lower tracing: the intranuclear chromoplasma were well-distributed,the nuclear membrane were clear, mitochondria ribosome,solvent can be seen.The shape of experiment were as follow lower tracing:The nuclus were pycnosis,cell membrane were damaged,the nucleolemma were shrinkage.Some of the cells were as followed:chromatic agglutination in the intranuclear,the chromatin were side,looking like crescent,the intermembrancee were not identical.Some of the cells were as followed:nuclus chromatins were side,nuclear type had several notches,nuclear membranes were clear.4. The cells that are stained by 1% iodinate tritroche can be observed by fluorescence microscope:Control group:the conformation of nucleous is satiation and present a yellow red color of fluorescence. 1000 U/mlγ- IFN+1μmol/1 ATRA group: the nucleous of most cells are presenting karyopyknosis,becoming punctuate or massiveness and concentrating at one side of the cell to form crescent shape or granular shape.The nucleous/plasma is decreading.5. To observe the differentiation of SCL- 1 cell:Cells in the control group were well grew adhesively, most in shuttle shape, moderately sized, clear nucleolus, nuclear division phase found.ATRA-stimulated cells displayed a somewhat bipolar appearance.γ- IFN- stimulated cells showed the number of bipolar cells increased.The cells treated by ATRA andγ-IFN were present in the supematant.The density of adherent cells was diminished when compared with the control.6. To detect the cell apoptosis rate by FCM:The difference of the early apoptosis rate among the five groups were significant (p=0.000＜0.01).7. To detect the expression of survivin protein by cell immunochemistryThe expression of survivin protein was positive in SCL-1 cells untreated.It was weak stained after treatment of 1000 U/mlγ- IFN.We can't detect the te expression of survivin protein after treatment of 1000 U/mlγ- IFN and 1μmol/1 ATRA.8. Survivin mRNA analysis by RT-PCR: The results showed that in the ATRA andγ-IFN treatment group, survivin mRNA cells were significantly lower than that in control.It shows the dependence of the time-dose.9. To detect the expression of survivin protein by western blot:γ- IFN could significantly inhibit the expression of survivin(131.60±1.52 in the control group)in dose-dependent and time-dependent manner.γ-IFN combined with ATRA could enhance the inhibition of survivin's expression.Conclusions1. High survivin expression in the skin SCC tissue is related to the lymph node metastasis, suggesting that survivin plays an important role in the incidence.Survivin and bcl-2 may induce cell apoptosis through different pathways.2. Survivin might play an important role in the apoptosis of squamous cell carcinoma cell induced byγ- IFN and ATRA.3. ATRA andγ-IFN induce cell apoptosis of SCL-1 dependent of time-dose.The role can be enhanced by the combination of the drugs.4. One of the mechanisms of apoptosis of skin squamous cell carcinoma induced by IFN-γand ATRA may be through decreasing the expression of survivin.