| Malignant tumor is one of the major chronic diseases that endanger our health.In traditional Chinese medicine(TCM),it is regarded as 'zhengJia','Jiju','Yan','Liu',' Yege '.Professor Lin Hongsheng has been working for nearly 40 years on the research and clinic concerning the integration of traditional and western medicine for malignant tumor treatment.Professor Lin believes that tumor is the partial reflection of systemic disease namely asthenia in whole and excess in partial.'Asthenia','Poison' and 'Stasis' were the main factors of induced tumor.Based on the TCM workers' study on TCM tumor's etiology and pathogenesis and principle-method-recipe-medicines throughout the country,Professor Lin inherited and summed up previous theory 'Fuzheng Peiben'and further put forward new theory 'Guben Qingyuan' that was more suitable for solving 'asthenia','poison','stasis' of tumors'pathogenic characteristics.'Guben' meant to protect the body's vital qi to strengthen the body,to improve the ability of disease prevention and cure,and to correct the pathological state of deficiency of vital qi.'Qingyuan' meant to eliminate the pathogenic factors of tumor development and to control the tumor from the source.Professor Lin has proved that'Fuzheng Huoxue Jiedu' method under the guidance of the theory 'Guben Qingyuan' could prolong the survival period and improve the quality of life,of non-small cell lung cancer patients,by the way of evidence-based medicine.It was confirmed that 'Fuzheng Huoxue Jiedu' under the guidance of 'Guben Qingyuan' theory had clinical effectiveness.In order to further clarify the biological connotation of 'Guben Qingyuan' theory,and verify the mechanism of Chinese medicine of 'Tuzheng Huoxue Jiedu' on tumors,Professor Lin's team regarded the tumor microenvironment and tumor stem cells as the breakthrough points to further study that 'Fuzheng Huoxue Jiedu' Chinese medicine intervened the biological behaviors of cancer stem cells.In previous researches,the regulation of Chinese medicine compound,namely medicine with 'Fuzheng Huoxue Jiedu',on the biological behaviors of tumor stem cells has been confirmed from'Guben'and'Qingyuan'.In order to further study the roles of Chinese medicine based on different treatment methods played in 'Guben'and'Qingyuan' theories of TCM tumor,and their intervention mechanisms on tumor stem cells'biological behaviors.The research group performed researches studied on each single treatment method,and so far,it had been confirmed that Chinese medicine of 'Jiedu' and'Huoxue' could regulate the tumor stem cells' biological behaviors under the guidance of'Qingyuan' theory.However,the research group had not done a systematic study on the biological behavior of tumor stem cells intervened by TCM of 'Fuzheng' theory.Given the lack of such research,this trial was designed as using GRh2(Ginsenoside Rh2,GRh2),which was extracted from Chinese medicine of Ginsheng,taking DU 145 stem cell from prostate cancer as study subjects,exploring the regulation of Chinese medicine with effect of'Fuzheng' on the biological behaviors of tumor stem cells under the guidance of 'Qingyuan'theory.Hoping to further clear the effect of intervention on the occurrence and development of tumor with'Fuzheng Huoxue Jiedu' theory.'Guben Qingyuan'theory was enriched,at the same time,laboratory basis for how Chinese Medicine works for tumor stem cells by multi-channels and multi-targets was provided.Objective1.Figure out the mechanism that GRh2 regulates the biological behaviors of tumor stem cells by downregulating the Notch1 signaling pathway,and thus inhibits the growth and breeding of tumors.2.Explore the mechanisms that GRh2 combined with mTOR targeted drugs can improve the anti-tumor effect.Methods1.In vivo,Tumor Spheres of tumor stem cells were isolated,cultured and purified by the method of sphere formation with prostate cancer cell line of DU145.The stem cell phenotypes were identificated by FACS.Animal tumor experiment was performed that DU 145 and Tumor Spheres of different orders of magnitude were inoculated into NOD/SCID mice under bilateral alar to observe the tumor formation rate,in order to confirm the characteristics of cancer stem cells on Tumor Spheres.NOD/SCID mice were inoculated into Tumor Spheres to establish the tumor bearing mice model by xenotransplantation experiment.Tumor bearing mice model was treated by different doses of GRh2 with tail vein injection,and then the body weight and tumor volume were monitored.The tumor weight was weighted after 4 weeks when the mice sacrificed.The optimal dose was determined to inhibite tumor.The proteins of Notch1 HES1,Sox2,HIF-la,jaggedl were tested by Western blot,IHC,Real-time PCR to determine the regulatory effects of different drug doses on the related proteins and genes.And with GRh2 of the optimal drug dose,the enhanced effect of GRh2 combined with cyclophosphamide or mTOR inhibitor rapamycin on tumor suppression was further explored.2.In vitro,Tumor Spheres that were isolated,cultured and purified by DU 145 were tested on the differentiation,proliferation,reactive oxygen species,cell cycle,and specific proteins and genes.The tumor stem cell characteristics were further proved.CCK8 method was used to detect the proliferation of DU 145 or tumor stem cells at different time points and different concentrations of GRh2.The effects of GRh2 on the tumor cell cycle,apoptosis induction,the regulation of reactive oxygen species and the surface markers were detected by the kits.In order to further explore the mechanism of GRh2 regulating the biological behavior of tumor stem cells,and to verify the results of animal experiments,Notch 1,HES1,Sox2,HIF-la,jaggedl of the cancer stem cells that were intervened by 50uM or 75uM GRh2 for 24h or 48h were tested by Western blot and Real-time PCR.CCK8 method was used to observe the effect of GRh2 combined with mTOR inhibitor or PI3K inhibitor on the proliferation of cancer stem cells.In vitro,the mechanism of inhibition of GRh2 combined with mTOR inhibitor on cancer stem cells was further explored.And the results of animal experiments were further verified.Results1.Suspension culture was used to isolate the DU 145 cells in logarithmic growth phase to form Tumor Spheres in serum-free medium.Tumor Spheres were cultured for three generation could achieve the purpose of purification.Flow cytometry was used to identify the phenotype of prostate cancer stem cells,CD44(+)CD24(-/low)(p<0.05)2.The tumor formation rate of Tumor Spheres side were higher than that of DU145 cells(p<0.05)?3.There was no significant difference in the weight of tumor bearing mice under different doses of GRh2(p>0.05).The tumor volume and weight of tumor showed that the tumor weight inhibition of tail vein of 0.5mg/kg mice was the highest.4.Western blot and IHC results showed that the concentrations of tail vein of 0.5mg/kg mice could downregulate the proteins of JAG1,HES1,Sox2,HIF-l?,NICD,NTM,p-mTOR.5.Real-time PCR results showed that GRh2 with the concentrations of tail vein of 0.5mg/kg mice could downregulate the mRNA of Notch 1,HES1 jaggedl,but upregulate the mRNA of Sox2 and HIF-1?.6.GRh2 combined with CTX could enhanced the rate of tumor inhibition.7.GRh2 combined with mTOR targeted drugs could enhanced the rate of tumor inhibition.And Western blot results showed that combination of both could enhance the inhibition effect of p-mTOR,NTM,NICD.IHC results showed that combination of both could enhance the inhibition effect of HES1 and HIF-1?.8.Cancer stem cells had stronger self-renewal ability and differentiation potential,and the cell cycle test showed that most of the tumor stem cells were in the G0/G1 phase of the cell cycle.The level of reactive oxygen species was found to have a lower level of reactive oxygen species.9.Cancer stem cells highly expressed HES1,p-mTOR protein,and expressed Notch1,HES1 HIF-1,Sox2,Jagged1 mRNA.10.CCK8 method showed IC50 of 24h,48h,72h with GRh2 intervening DU145 were 62.833uM,54.746uM,51.796uM.IC50 of 24h,48h with GRh2 intervening the single cell of CSCs were 71.834u,70.579uM.IC50 of 24h,48h with GRh2 intervening tumor spheres were 83.969uM,73.562uM.11.The results of cell cycle indicated that 50uM GRh2 blocked the DU 145 cell cycle in S phase,and 75uM GRh2 blocked DU145 cell cycle in G1 phase.50uM and 75uM could promote the development of tumor stem cells from quiescent phase G1 to S phase and G2 phase,100uM blocked CSCs in the G2/M phase.12.75uM of GRh2 could induce the apoptosis of cancer stem cells.13.GRh2 could increase the level of reactive oxygen species in DU 145 cells and tumor stem cells.14.GRh2 could downregulate the level of tumor stem cell surface marker CD44(+)CD24(-/low),and the higher the dose,the more obvious down-regulation.15.Western blot results showed that 50uM and 75uM of GRh2 intervening CSCs for 24h and 48h could downregulate the proteins of NTM,HES1,Sox2.16.Real-time PCR results showed that 50uM and 75uM of GRh2 intervening CSCs for 24h could downregulate the mRNA of Notch1,HES1,HIF-1?,Sox2,JAG1.50uM of GRh2 intervening CSCs for 48h could upregulate the mRNA of Sox2 and jaggedl,but there was no significant effect on Notchl,HES1,HIF-1?.75uM of GRh2 intervening CSCs for 48h could upregulate the mRNA of Notch1,HES1,HIF-1?,Sox2,JAG1.17.CCK8 methods showed that,in 24h and 48h group,the inhibition rate of LY was higher than RAPA(p<0.01),the inhibition rate of RAPA combined with GRh2 was higher than RAPA(p<0.01),the inhibition rate of LY combined with GRh2 was higher than LY(p<0.01).Compared with 24h group,the inhibition rates of RAPA and 24h of LY were lower than 48h(p<0.01),but there was no significant difference between 24h and 48h in the two groups of RAPA+GRh2 and LY+GRh2(p>0.05).18.HES1 protein tested by Western blot showed that GRh2 group,combined group and RAPA group all can downregulate the protein of HES1.All had statistical difference(p<0.01).And the combined group's inhibition effect was the most obvious.19.p-mTOR protein tested by Western blot showed that GRh2 group,combined group and RAPA group all could downregulate the protein of p-mTOR.All had statistical difference(p<0.01).And the combined group's inhibition effect was the most obvious.Conclusion1.Animal tumorigenesis experiments and biological function tests of tumor stem cell had both confirmed that Tumor Spheres had the biological characteristics of self-renewal,differentiation and proliferation.2.In vivo,GRh2 with tail vein injection for twice a week,0.5mg/kg mice weight of the drug doses induced the highest rate of self-renewal inhibition to cancer stem cells.3,In vivo,it was proved that GRh2 can regulate the biological behavior of tumor stem cells by downregulating the Notch 1/HES1 signaling pathway,and thus inhibit the growth of tumor.4.In vivo,it was proved that GRh2 combined with chemotherapeutic drugs would improve the anti-tumor effect and increase the efficacy of chemotherapy.5.In vivo,it was proved that GRh2 combined with mTOR inhibitors could further reduce the inhibitory effect by further downregulating the Notchl signaling pathway and mTOR signaling pathway.6.In vitro,GRh2 could inhibit the proliferation of tumor stem cells;GRh2 could promote tumor stem cells' cell cycle from the stationary phase G1 phase to S or G2 phase.GRh2 could induce tumor stem cell apoptosis.GRh2 could increase the level of intracellular reactive oxygen species.GRh2 could inhibit the expression of tumor stem cell surface markers.7.In vitro,it was proved that GRh2 could regulate the biological behavior of cancer stem cells by downregulating Notch1/HES1 signaling pathway.Animal experimental conclusions were verified.8.In vitro,it was proved that GRh2 combined with mTOR inhibitors could further downregulate HES1 and p-mTOR proteins,and improve the inhibition of tumor stem cells. |
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