Effects Of Green Tea Extract And Epigallocatechin-3-gallate On Ceil Proliferation And Signaling Pathway In Oral Squamous Cell Carcinoma

Objective:Oral squamous cell carcinoma（OSCC）is the sixth most common cancer reportedglobally with an annual incidence of over300000cases and an increase of62%has beenreported in the developing countries.5-year survial rate is less than50%. OSCC is seriouslythreatening the human life and health. Now the standard treatment of OSCC is comprehensivetreatment including surgery primarily, chemotherapy and radiation therapy. However thechemotherapy drug injury tumor cells not only, but also has an effect on the normal cells.Most chemotherapy drugs have a maximum safe dose, if the dose exceeds the limited extent,it will produce side effects and some drug can induce multidrug resistance of tumor. It’s verynecessary to find a kind of natural drug, that is high-effective and low toxity to tumor. Greentea is very popular beverage in everyday life. In recent years the research found that green teaextract (GTE) has shown greater promise and efficacy against multiple cancers, including skincancer, lung cancer, gastric cancer, breast cancer, colon cancer and head and neck cancer, andit shows effects on. However, it is seldom found that GTE act on OSCC. In the present studywe explored the effects of GTE and (-)-epigallocatechin-3-gallate (EGCG) on proliferationand signaling networks of squamous carcinoma cells, and observed the inhibition of GTE andEGCG on OSCC xenografted tumor.Methods:The three experiments were performed. In the first part, the three cells (CAL-27, SCC-25,KB) were cultured in vitro and the cells were treated with different doses of GTE and EGCG.Its growth inhibition was detected by MTT. The most sensitive cell was screened by MTT. Thesensitive cell was treated with different doses of GTE and EGCG, and cell cycle was analyzedby flow cytometry. The action mechanisms of GTE and EGCG were investigated. In the second part, Protein pathway array was used to assess the effects of GTE andEGCG on the protein expressions in CAL-27, SCC-25and KB cells. The differential proteinwas analyzed by protein pathway array after treatment with GTE and EGCG in the three cells.The machanism and the affected signaling pathway of GTE and EGCG were clearly studiedin oral squamous cell carcinoma cells.In the third part, the sensitive cell for GTE and EGCG was subcutaneously transplantedto establish tumor-bearing nude mice model.When transplantable tumor grow into4-6mm.The mice were randomly divided into GTE, EGCG and control groups. After treatment for4weeks by GTE (50mg/kg, intragastric administration)，EGCG (25mg/kg, intragastricadministration), Control group (equivalent saline), the tumor volume and weight weremeasured and tumor growth inhibiting rate was calculated to assess the effects of GTE andEGCG on the growth of transplanted tumors.Results:In the first part, GTE and EGCG showed significantly proliferation inhibition in threeoral squamous carcinoma cells in a dose-dependent manner. Different sensitivities to GTE andEGCG were observed in different cells, CAL-27was the most sensitive cell. Differentsensitivities to GTE and EGCG in the three oral squamous carcinoma cells were observed witha50%growth inhibition concentrations (IC50), which were70,125,145μg/ml for GTE (35,62.5,72.5μg/ml for GTE at EGCG equivalent concentration) and59,102.5,102.5μg/ml forEGCG.. The GTE at an EGCG equivalent concentration exhibited a stronger inhibition on allthree cells than EGCG alone. Flow cytometric analysis showed that different concentrations ofGTE (0、25、50and100μg/ml) and EGCG (0,12.5,25and50μg/ml) caused a significantarrest at S phase and G2/M phase with concurrent decrease of G0/G1phase in GTE andEGCG-treated CAL-27cell as compared with the untreated cell after72-hour incubation.In the second part, to better understand the molecular mechanisms of GTE and EGCG’seffect on cell growth, we surveyed signaling network changes used pathway array technologyafter treated with100μg/ml GTE and50μg/ml EGCG.. After48-hr treatment with either GTEor EGCG, we extracted the total proteins and performed a global screening of signalingnetwork using pathway array technology. Protein pathway array revealed that a total46 proteins were expressed in oral squamous carcinoma cells. Of those46proteins andphosphorylations,23were differentially expressed in these cells. The pattern also indicated thatSCC-25and KB cells were more closely related to each other than with CAL-27, which mayexplain their difference in response to GTE and EGCG. After SCC-25, KB and CAL-27cellswere treated with either GTE or EGCG for48hour, Pathway Array assessment indicated atotal of21proteins and phosphorylations altered significantly based on ANOVA analysis(P<0.05). Of21proteins and phosphorylations, only CDK6was consistently down-regulatedby GTE and EGCG in all3cells. Furthermore, down-regulation of cell proliferation relatedproteins by GTE and EGCG was a dominant finding, although there was a remarkabledifference among different cells. For example, the phosphorylation or expression of p-PDK1,p-ERK, p-CDC2, CDK6, CDK4, CDK2and Cyclin E in CAL-27cells were down-regulated byGTE and EGCG. There was a decreased phosphorylation or expression of p-PDK1, p-CDC2,CDK6, Cyclin E and cPKCα in SCC-25and CDK6and CDC2in KB cells.The dose-dependentinhibition of GTE on p-PDK1, CDK4and CDK6in CAL-27cells were also confirmed byWestern Blot. In addition to down-regulation, an increased expression of someproliferation-related proteins was also observed after treatment with GTE and EGCG. Forexample, the expression of Notch4increased in CAL-27, CDK2and CDC2in SCC-25andfinally p-ERK, p-CDC2, CDK2, cPKCα and ERK1/2increased in KB cells. After all3cellswere treated with GTE or EGCG, the expression of some known tumor suppressors weredown-regulated, such as PTEN, p53and RB. A heterogeneity exists at signaling network levelin squamous carcinoma cells. GTE and EGCG target multiple pathways or global network incancer cells and they significantly affected EGFR signal network, resulted in collectiveinhibition of cancer cell growth. GTE had additional benefit on cancer inhibition than EGCG.In the third part, the animal experiment showed that GTE and EGCG effectivelyinhibited the growth of xenografted human mouth epidermal carcinoma in mice (P<0.01). TheGTE at an EGCG equivalent concentration exhibited a stronger inhibition on the growth ofxenografted tumor than EGCG. Conclusions:Both GTE and EGCG inhibit the proliferation of human squamous carcinoma cellCAL-27, SCC-25and KB. GTE and EGCG cause arrest at S phase and G2/M phase. Aheterogeneity exists at signaling network level in squamous carcinoma cells. Differentsensitivities to GTE and EGCG in different cells are found, CAL-27is the most sensitive cell.After the three cells were treated with either GTE or EGCG, a total of21proteins andphosphorylations significantly alter. Of these21proteins and phosphorylations,7are related tosignaling transductions,3are known tumor suppressors,1apoptosis-inhibiting protein, and7are cell cycle/proliferation related proteins. Furthermore,5proteins are affected by GTE andEGCG at phosphorylation level. These data suggest that both agents can alter the expression oractivation of many signal transduction pathways. However, the major pathway affected by GTEand EGCG may be EGFR pathway which transmitted their inhibitory effects to cell cyclerelated proteins and results in S and G2/M arrest in the squamous carcinoma cells. Animalexperiment indicates that GTE and EGCG effectively inhibit the growth of xenograftedhuman mouth epidermal carcinoma in mice. The GTE at an EGCG equivalent concentrationexhibits a stronger action against tumor than EGCG.