The Anti-cancer Effect Of TouNongSan And Its Possible Mechanism

AIM:To investigate the anti-cancer effects of Tou Nong San extracts (TNSEs) and its possible mechanism.METHODS:LC-MS Assay for TNS extract. The cytotoxic effect of TNSE on Raji cells and LOVO cells was measured by3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle and apoptosis were determined by flow cytometry(FCM).The molecular mechanisms of TNSE-mediated apoptosis were further investigated by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the mRNA expression of nuclear factor-KB (NF-κB), Bcl-xL and Bcl-2-associated death promoter (Bad), caspase-9and caspase-3,PI3K, Akt, mTOR, p70s6k1, and by Western blotting to detect the proteins expression of NF-κB, Bad,PI3K,p-Akt, p-mTOR, p-p70s6k1, caspase-9, caspase-3.The safty of TNSEs was detected by acute toxicity testing.The inhibition ratio was gained on the base of the weight and volume of transplanted tumor, morphosis of tumor was detected by hematoxylin and eosin (HE)stain, the proteins expression of PI3K,p-Akt,p-mTOR,p-p70s6k1, caspase-9, caspase-3,VEGF,CD31were detect by immuno-histochemical(IHC) method and the content of cytokine of Thl/Th2was tested by Enzyme-Linked Immunosorbent Assay(ELISA).RESULTS:The contents of7major components in TNSEs were Calycosin-7-O-b-D-glucoside, Formononetin-7-O-b-D-glucoside senkyunolide I, Calycosin,formononetin, Astragaloside IV, Z-Ligustilide.TNSEs inhibited Raji cells and LOVO cells proliferation in dose-and time-dependent manners. The Half maximal inhibitory concentration(IC50) of Raji cells was817.142μg/mL at72h,and that of LOVO cells was347.603μg/mL at48h. The apoptosis and Glaresst of these two cell lines were found after treated with TNSEs. After48h treatment with various concentrations of TNSEs (125,250and500μg/mL), the apoptosis rates of Raji cells were12.23±1.98%(P<0.05),20.97±3.96%(P<0.01) and30.4±4.87%(P<0.01), respectively, compared with that of the control (6.02±1.01%).and the G1arrest was also found that was The proportion of G1cells was44.80%±2.36%(P<0.05),55.48%±2.71%(P<0.01),66.76%±2.25%(P<0.01) respectively, compared with that of the control (39.66%±2.01%).After48h treatment with various concentrations of TNSE (62.5μg/mL,125μg/mL,and250μg/mL),the apoptosis rates of LOVO cell were12.8%±1.58%(P<0.05),19.9%±2.16%,(P<0.01)and29.57%±2.76%(P<0.01), respectively, compared with that of the control(7.3%±1.06%).The proportion of G1cells was45.45%±2.54%(P<0.05),56.13%±2.54%(P<0.01),66.05%±2.54%(P<0.01) respectively, compared with that of the control (40.02%±0.69%).RT-PCR demonstrated that NF-κB mRNA expression was significantly downregulated in Raji cells treated with250μg/mL TNSEs for48h (P<0.05), while Bad, caspase-9and caspase-3mRNA levels were upregulated (P<0.05). Moreover, TNSEs treatment resulted in down-regulation of NF-κB protein expression and strikingly up-regulated proteins expression of Bad, cleaved caspase-9, cleaved caspase-3in a dose-dependent manner, as determined by western blot.Furthermore,the similar results were found in LOVO cells treated with125μg/mL TNSEs for48h. The mRNA expression of PI3K, AKT, mTOR, P70s6k1was significantly downregulated (P<0.05), while caspase-9and caspase-3mRNA levels were upregulated (P<0.05,), which was accompanied by significantly down-regulation of the proteins of PI3K,p-AKT,p-mTOR, p-P70s6k1and strikingly up-regulation of the proteins of cleaved caspase-9, cleaved caspase-3in a dose-dependent fashion, as determined by western blot.There is no any toxicity at the maximum administration40000mg/kg for the mice, so40000mg/kg is safe dose and used for the next experiments. The inhibition rate of the implanted tumor in these three groups(high dosage group,middle dosage group,low dosage group)was67.11%,48.68%,36.84%respectively.The proteins expression of PI3K, p-Akt, p-mTOR,p-P70S6K1, VEGF,CD31were decreased and that of caspase-9,caspase-3were increased in implanted tumor of nude mouse treated with different concentration TNSEs as detected with IHC. Further-moer,TNSEs could regulate the nude mouses’immunity through upragulating Th1cytockines including IL-2, IL-12and down-regulating Th2cytockines, including IL-6IL-10, rectifying the drift of Thl/Th2,which was also in a dose dependant manner.Conclusion:1TNSEs could reduce proliferation, cause apoptosis and cell-cycle arrest in Raji cells and LOVO cells, which might be associated with the regulation of PI3K/AKT signaling pathway from two levels of gene transduction and protein expression.2TNSEs inhibited the growth of implanted tumor of nude mouse which was increasingly associated with regulating multiple molecules in the P3K/AKT pathway and with rectifying the drift of Thl/Th2.3TNS is a hopeful classic Chinese Medical Formula for tumor therapy.