| Chapter One Differential proteomic profile of Baicalin on human keratincyte Haca T cells irradiated with ultraviolet BBackground The skin is the biggest organ of human body and has very important barrier function against the harmful effects of UV radiation.Overexposure to solar ultraviolet(UV)radiation,especially UVB(290~320 nm)component,induces a multitude(multiple or many)of acute and delayed responses in human skin,including the generation of free radicals and relevant reactive oxygen species(ROS),secretion of inflammatory cytokines,immunosuppression of topical and general immune systems,DNA damage and mutation,at last contributes to photoaging and photocarcinogenesis development.Especially non-melanoma skin cancer has become the most common malignancy in humans.For the past few decades,there has been great interest in chemoprevention of photodamage by using naturally botanical agents to prevent the initial UV-introduced damage.One agent that has been receiving increasing attention and could protect skin against UVB irradiation is Baicalin,isolated from baical skullcap root.Our previous studies in vivo and in vitro demonstrated that Baicalin can reduced the increased apoptosis rate,CPDs formation as well as IL-6 secretion level induced by UVB irradiation on human skin cells(keratinocytes and fibroblasts)and mice epidermis.However,the systemic and concrete mechanism among Baicalin,skin cells and UVB irradiation remains inconclusive.To date,proteomic techniques have been used as a powerful tool for the qualitative and quantitative measurement of diversity of total proteins related to specific cellular responses.2D-PAGE and proteomic analysis are still the main choice to separate and visualize the proteins of cells or tissues in most of studies.Hundreds of proteins could be separated on a 2D-PAGE and then identified by mass Spectrometer.Therefore,the proteomic scheme was implemented to search globally for the differentially expressed proteins.The aim of the present study is to identify,by a proteomic strategy,the new molecular targets of protein expressions in human skin keratinocyte Ha Ca T cells in response to UVB and Baicalin exposure.This study may be helpful to illustrate the complex responses of total cellular protein to UVB irradiation and/or Baicalin treatment.Methods We used Ha Ca T cells,a transformed non tumoral continuous epidermis cell line,as a convenient experimental cell model to observe the molecular mechanisms of UVB and Baicalin on skin damage and cell responses.Ha Ca T cells were divided into six groups: control group,Baicalin group,UVB 24 h group,UVB 48 h group,UVB 72 h group,Baicalin+UVB group.Cells were washed and irradiated in phosphate-buffered saline(PBS)with 30 m J/cm2 UVB.Following UVB irradiation,cells were collected and solubilized in lysis buffer.The proteins were harvested and seperated in two-dimensional gel electrophoresis(2-DE).The differential protein expressions between the two groups of cell were analyzed using image analysis software.The differentially expressed protein spots were selected,digested in-gel with trypsin,and measured by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MADI-TOF-MS),the data obtained from peptide mass fingerprinting(PMF)were used for protein database search.Results We successfully obtained the 2-DE images with a high reproducibility.The resulting maps were quantitatively and statistically analyzed.(1)There were 76 differential protein spots were identified between the control group,and UVB 24 h group.Among these spots,19 protein spots were expressed only in the cells before UVB irradiation and 24 protein spots were expressed only in the Ha Ca T cells after UVB irradiation.The expression levels of 22 protein spots in the Ha Ca T cells were increased by UVB irradiation,when the expression levels of 11 protein spots were decreased by UVB irradiation.All the 76 protein spots were analyze by MALDI-TOF-MS.There were 65 protein spots were identified.And we also compared the UVB 48 h group and the UVB 72 h group with the control group respectively.There were 4 protein spots in UVB 48 h group and 10 protein spots in UVB 72 h group were identified.These proteins were involved in cytoskeleton,stress response,metabolism,cells proliferation,apoptosis and tumorigenesis,including prohibitin,Serpin B5 precursor,Heat shock protein beta-1,and etc.(2)Baicalin could alter the proteome of Ha Ca T cells.18 differential protein spots were were analyze by MALDI-TOF-MS,and 14 protein spots were identified as Heat shock protein beta-1,Actin,Dihydropyrimidinase-related protein 2,and etc.(3)The Baicalin treatment showed that there was a general trend that Baicalin inhibited the protein expression mode induced by UVB irradiation.The 20 down-regulated proteins were all up-regulated by Baicalin to some degree,while the 26 up-regulated proteins were all down-regulatedConclusions By using a proteomic approach,we have established a global proteinic mapping and identified selective proteins which may be molecular targets of Baicalin and UVB in the present study.In conclusion,our results shed light on the mechanisms ofBaicalin for protecting the skin against the adverse effects of UVB irradiation,though further study is needed to elucidate the exact roles of these proteins.Chapter two Upregulation of TCTP expression in human skin squamous cell carcinoma increases tumor cell viability through anti-apoptotic action of the proteinBackgroud and Aim Human cutaneous squamous cell carcinoma(SCC)is the sixth most common cancer in the world.It arises from the keratinocytes of the epidermis or its appendages.Skin SCC may arise de novo or develop from precursor lesions,such as actinic keratosis(AK)or Bowen’s disease.Most of primary skin SCCs are good prognosis and are usually curable in comparison with other cancer types,but the potential for invasion and metastases significantly contributes to the mortality.Based on the degree of tumor differentiation,SCC can be graded into three histological categories: well-differentiated,moderately-differentiated and poorly-differentiated SCCs.Poorly differentiated SCCs usually exhibit a higher risk of recurrence and metastasis.However,biological behaviors can be difficult to be predicted by the status of differentiation of tumor cells.Therefore,a novel molecular biomarker that shows associations between the aggressive,invasive and metastatic behaviors of cutaneous SCC needs to be identified.Translationally controlled tumor protein(TCTP),a 172 amino acid anti-apoptotic polypeptide,was originally identified in the Ehrlich ascites tumor cell line.It is a cell growth-associated protein that is ubiquitously present in a wide variety of organisms and plays a pivotal role in the development of different organisms.It is present both extra-and intracellular which has been implicated in many cellular functions that are related to cell growth and apoptosis.The TCTP gene is significantly downregulated inrevertant tumor cells and it was therefore suggested that inhibiting the expression of TCTP could revert cancer cells back into normal phenotypes.Certain stimuli,including dioxin,heavy metals,growth factors,and vitamin D,can regulate the expression of TCTP.TCTP also interacts with many cellular proteins,including tubulin,translation elongation factors(e EF1A)and its guanine nucleotide exchange factor(e EF1B-b),Mcl-1,TSAP6,Na,K-ATPase,and Bcl-XL.The anti-apoptotic activity of TCTP has been reported recently,which may be related to its interaction with Mcl-1 and/or Bcl-XL.TCTP is highly expressed in several cancer types,but the expression of TCTP has not been investigated in human cutaneous SCC.Its roles in skin carcinogenesis also need to be elucidated.In this study,we investigated the expression of TCTP in human cutaneous squamous cell carcinoma and its function in skin carcinogenesis by si RNA gene silencing approach.Methods We used immunohistochemical to observe the expression of TCTP in SCC.Western-Blot was used to detect TCTP in SCC cell lines A431 and SCL-1.RNA interference technology was used to knock down the expression of TCTP in A431,RT-PCR and Weatern-Blot were used to confirm the efficiency of transfection.Flow cytometric detection and MTT method were used to detect cell apoptosis and proliferation.Results(1)The result shows that TCTP was overexpressed in SCC specimen,and the expression level and the grading of cancer tissue has a positive relationship.(2)Western blot results confirmed that the expression of TCTP in A431 and SCL-1 were higher than human keratinocytes cell line(Hacat).(3)RT-PCR and Western-Blot results showed that the si RNA can knock downTPT1 and TCTP expression in A431 cells effectively,Conclusion we found that the downregulation of TCTP expression was associated with decreased cell proliferation and increased apoptosis of A431 cells. |
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