Functionial And Mechanical Research Of Resveratrol As Anti-cancer Chemicals On Skin Squamous Cell Carcinoma In Vitro And In Vivo

Background and Objectives:Squamous cell carcinoma(SCC)is a common skin malignant tumor with high cure rate after definite diagnosis.Nevertheless,slow development of patient symptom and out of self-conscience would easily result in hysteretic diagnosis and increased risk.At the same time,SCC could also cause large wound surface and defective outlook.Operation is the first choice of skin cancer treatment and always followed with chemotherapeutic agent and radiotherapy in metastatic states.Long-term use of chemotherapeutics will result in various complications,and several tumor cells will develop drug resistance to specific chemotherapeutics.Therefore the researchers devoted to discovering natural molecules with anticancer potential from daily food in the past decades.Resveratrol,a common polyphenols,is widely discovered in vegetable,nuts and fruits.As reported,resveratrol possess various pharmacological functions,such as anti-aging,anti-oxidation,anti-viral,anti-epileptic,anti-neurodegenetive disorders and anti-tumor.At the same time,resveratrol is also revealed as effective chemoprophylaxis for multiple cancers.Although resveratrol is regarded as active broad-spectrum anti-cancer chemical,little is known about the effect of resveratrol on skin tumor,especially on animal models.Therefore,our study explored the time-effect and concentration-effect of resveratrol on two typical SCC cell lines in vitro and the potential cellular as well as molecular mechanism.Nextly,we constructed chemical-induced rat skin tumor,and the in vivo pharmacological activity and mechanism of resveratrol on SCC were investigated.The research explored the relationship between resveratrol pharmacological function with administration time span and concentration to provide experimental data for the choice of resveratrol dosage in SCC prevention and therapy.At the same time,undelying anti-tumor mechanism of resveratrol was revealed to offer new strategy in SCC treatment.Research methods:1、anti-tumor research of resveratrol in vitroⅠ: The inhibitory effect of resveratrol on cell viability was tested in SCC cell lines-SCC1 and Colo-16 via CCK-8 assay;Ⅱ: The apoptosis induction function of resveratrol was detected in SCC1 by Annexin-V/PI double staining followed with flow cytometry;Ⅲ: The mRNA and protein level of pivotal genes in cell cycle modulation and cell apoptosis were indicated via western blotting,semi-quantitative PCR and quantitative PCR in SCC1 to explore the signal pathway of resveratrol in SCC.2、anti-tumor research of resveratrol in vivoⅠ: Rat skin SCC model was constructed via classical two-stage DMBA/TPA induction method,and the rat was divided into 4 groups: control group(Control),model group(DMBA),low-dosage resveratrol treatment group(DMBA+RES-1);high-dosage resveratrol treatment group(DMBA+RES-2).Then,the rat weight,tumor incidence rate,tumor inhibition rate,tumor volume and tumor weight were detected to indicate the pharmacological anti-tumor activity of resveratrol in vivo;Ⅱ: The impact of resveratrol on cell cycle and cell apoptosis were tested via cell cycle distribution assay,Ki-67 immunohistochemical staining,Annexin-V/PI double staining followed flow cytometry,TUNEL apoptotic staining;Ⅲ: The impact of resveratrol on key genes in cell cycle regulation and apoptosis initiation was tested by western blotting to verify the anti-tumor mechanism of resveratrol in vivo.Results:1、 anti-tumor research of resveratrol in vitroⅠ: Obvious cytotoxic effect of resveratrol on SCC1 was showed in our results,and the highest inhibitory percentage was up to 80%(72h,31.25μM).Resveratrol inhibited SCC1 cell viability in time-dependent and concentration-dependent manner.Nevertheless,high concentration of resveratrol could only inhibit Colo-16 viability to 85%.Ⅱ: Resveratrol could stimulated cell apoptosis in dosage-dependent manner in SCC1 cell line;Ⅲ: Resveratrol could up-regulate the mRNA level and protein level of p53,p21,Bax and caspase-3 in apoptosis pathway and down-regulate transcription and translation level of cdc2 as well as cyclin B in G2/M cell cycle checkpoint.2、anti-tumor research of resveratrol in vivoⅠ: All rats in model group were discovered with solid tumor and abnormal Keratinized cells,which indicated that the two-stage DMBA/TPA model was effective in our experiments;Ⅱ: Compared with model rats,average body weight decreasing was blocked in low-dosage and high-dosage treated groups.At the same time,tumor incidence rate,tumor inhibition rate,tumor volume and tumor weight were decreased in resveratrol administration groups,and the tumor inhibition efficiency was more obvious in high-dosage resveratrol treated rats.Ⅲ: Compared with DMBA group,1 or 2 mg/kg b.w of resveratrol treated rats were significantly arrest 45.6% and 52.3% of cells at G2/M,and the proliferation indices indicated by Ki-67 positive percentage was decreased in both groups.Furthermore,resveratrol elevated early-apoptotic and late-apoptotic cell proportions under DMBA/TPA induction.Thus,resveratrol could act as effective proliferation inhibitory and apoptosis inductive agent for SCC tumor cells.IV: Apoptotic proteins(p53 、 p21 、 Bax and caspase-3)were up-regulated in resveratrol administration rats in tow dosages,and anti-apoptotic protein survivin was inhibited by resveratrol when compared with that in model rats.Cdc2 and cyclin B in G2/M cell cycle checkpoint were also down-regulated in vivo.CONCLUSIONS:1、anti-tumor research of resveratrol in vitroⅠ: Resveratrol showed differential cytotoxic effect on SCC cell lines.High inhibitory efficiency of resveratrol on SCC1 was found in both time-dependent and concentration-dependent manners,while effect of resveratrol on Colo-16 viability was obscurely.Ⅱ: Resveratrol could dosage-dependent stimulated SCC1 cell into apoptotic progress and the resveratrol induced apoptosis might contribute to its cytotoxic activity.Ⅲ: Resveratrol might stimulate cell apoptosis via p53-p21-Bax-caspase-9 axis.On the other hand,resveratrol inhibited expression of cyclin B and cdc2 without affecting cell cycle distribution,which indicated that more pathways were regulated by resveratrol to modulate cell cycle in corporate fashion.2、 anti-tumor research of resveratrol in vivoⅠ: Resveratrol could prevent tumor growth in both low and high concentrations with more inhibitory efficiency in tumor incidence rate,tumor inhibition rate,tumor volume and tumor weight was demonstrated in high-dosage group;Ⅱ: Prohibition of tumor cell proliferation,arrest of cell at G2/M phase and stimulation of tumor cell apoptosis might contribute to anti-tumor function of resveratrol rhythmically;Ⅲ: Resveratrol might induce apoptosis by p53-p21-Bax-caspase-9 axis and modulate cell cycle via p53-cyclin B/cdc2 axis in vitro as well as in vivo.In summary,our findings suggest that resveratrol might be used as an anti-tumor candidate.However,further study about dosage broad,underlying mechanism and combination pattern with chemotherapeutics would accelerate application of resveratrol in SCC chemoprevention and therapy.