| Objective:Malignant tumors are a major threat to human health along with a high morbidity and mortality all around the world.Recent studies have shown that cancer stem cells(CSCs)have strong specific biological behaviors of self-renewal,anti-apoptosis,tumor formation,invasion and metastasis.They could be considered as the 'seeds' of tumor.With the deepening of the study of biological behaviors of CSCs,more and more evidence shows that the biological behaviors of CSCs are not controlled by a gene and a pathway,but are controlled by a complex network regulation system.With decades of exploration and practice in prevention and treatment of cancer by traditional Chinese medicine(TCM),a large number of clinical studies have shown that TCM has a significant advantage in preventing tumor recurrence and metastasis in many kinds of tumors.It is considered to be spontaneous that good curative effect is rooted in good theoretical guidance.In terms of the theoretical research,Professor Lin Hongsheng put forward the theory of 'Gu Ben Qing Yuan' based on the identification of pathological factors such as 'deficiency','toxin','stasis' in cancer development according to traditional Chinese medicine theory and on the summary of 'Fu Zheng Pei Ben' theory.To further explain this theory,on one hand,'Gu Ben' represents the method of strengthening right qi to improve the ability of disease prevention and resistance.On the other hand,'Qing Yuan' means to eliminate pathological factors and control the tumor form the origin.Modern medical research has shown that cancer stem cells are the most fundamental'seed' that causes tumor progression and even progress,so traditional Chinese medicine is likely to achieve the goal of controlling tumors from the source by removing cancer stem cells.According to the understanding of the pathological factors of 'stasis'in cancer initiation and development,the treatment principle of'invigorating blood and dissolving stasis' is a basic treatment principle in cancer treatment based on the theory of 'Gu Ben Qing Yuan' according to traditional Chinese medicine theory.So it may be reasonable to speculate that Chinese herb based on the treatment principle of 'invigorating blood and dissolving stasis' undertake the intervention of cancer stem cells to achieve the role of prevention and treatment of cancer.In this study,we investigated the effect of cryptotanshinone(CT)which is extracted from Salvia miltiorrhiza based on the treatment principle of 'invigorating blood and dissolving stasis' on prostate cancer stem cells derived from DU 145 cells,and explored the mechanism of cryptotanshinone on regulation of prostate cancer stem cells.The high-throughput method of microarray reveals the anti-tumor characteristics of multi-target and complex intervention of traditional Chinese medicine monomer,in order to enrich the theoretical connotation of 'Gu Ben Qing Yuan'theory,so as to try to apply modern medical mechanism to restore traditional Chinese medicine theory,and provide a new scientific basis for further study on the biological behaviors and molecular mechanism of CSCs.Methods:In order to explore and clarify the effect and mechanism of cryptotanshinone on the prostate cancer,DU 145 cell line and prostate CSCs derived from DU 145 were used to investigate the anti-tumor effect of cryptotanshinone and analyze underlying mechanism.1.Cell culture,purification and identification of prostate cancer stem cells derived from DU 145 cell line.Human prostate cancer cell line DU 145 was selected and the cancer stem cells were obtained by serum-free suspension culture and further be purified and enriched.The phenotype of cancer stem cells was identified by FACS.And we use xenograft animal model to further evaluate its tumorigenic ability.Specifically,DU145 cells and DU145-CSC(prostate cancer stem cells)were inoculated into NOD/SCID mice with bilateral axillary,and the rate/time of tumor formation were observed.2.The discussion of regulation mechanism of cancer stem cells with strong self-renewal ability and anti-apoptotic ability.The regulation mechanism of biological behaviors of prostate cancer stem cells was studied by protein chip analysis method by comparing the differences in protein phosphorylation of the DU 145 and the DU145-CSC.3.The effect of proliferation inhibition of compounds derived from traditional Chinese medicine towards prostate cancer cells was explored.3.1 Drug screening of anti-tumor compounds based on invigorating blood and detoxifying toxin principle.DU 145 cells were treated with different concentrations of 5 compounds,including cryptotanshinone(CT),curcumol,permine,curcumin and gambogic acid(GA)for 24h,48h and 72h.CCK8 assay was used to analyze the cell viability.3.2 Growth inhibition of CT in prostate cancer stem cells.Prostate cancer stem cells obtained and further purified by serum-free suspension culture were treated with different concentrations of CT for 6h,12h,24h,48h,72h and 96h.CCK8 assay was used to analyze the cell viability.3.3 Growth inhibition of GA in prostate cancer stem cells.Prostate cancer stem cells obtained and further purified by serum-free suspension culture were treated with different concentrations of GA for 6h,12h,24h,48h,72h and 96h.CCK8 assay was used to analyze the cell viability.4.Pro-apoptotic effect of CT in prostate cancer stem cells.Flow cytometry was used to analysis the proportion of apoptotic cells of CSCs treated with CT 3.46?M,6.91?M,13.82?M for 48h with AV/PI staining.5.Cell cycle intervention effect of CT in prostate cancer stem cells.Flow cytometry was used to analysis the cell cycle distribution of CSCs treated with CT 3.46?M,6.91?M and 13.82?M for 48h with PI staining.6.Mechanism study of CT intervention on the specific biological behaviors of prostate cancer stem cells.Protein chip analysis method was used to compare the differences in protein phosphorylation of DU145-CSC and CT-CSC in order to explore functional mechanism of CT on prostate cancer stem cells.7.Effect and mechanism of CT on prostate cancer stem cells in vivo.NOD/SCID mice were inoculated with human prostate cancer stem cells,and the prostate cancer xenograft model was established.Tumor-bearing nude mice were injected with CT(6.25mg/kg)intraperitoneally every other day for 4 weeks.The tumor volumes and the body weights of mice were measured weekly,and tumor weights measured at the end of the experiment.The tumor inhibition rate was observed.Tissue lysates were prepared from tumor tissues isolated from treated-mice.Western Blot analysis was performed to measure the expression of related proteins to explore its mechanism of action in vivo.So CT was used to observe the tumor inhibiton effect in vivo and to explore its mechanism.Results:1.Tumor Spheres obtained by serum-free suspension culture have the surface markers of prostate cancer stem cells and strong tumorigenic ability in vivo.1.1 DU145 cells were cultured in serum-free culture medium for 7 days to obtain stem cell spheres(Tumor Spheres).Tumor Spheres were purified when they were passaged more than three times.The CD44+ CD24-/low phenotype of DU 145 cells and Tumor Spheres were evaluated by flow cytometry.The results of CD44+ CD24'/low surface markers in DU 145 group was(35.45 ± 5.05)%and the third generation of Tumor Spheres was(74.16 ± 6.25)%(P<0.01).1.2 The tumorigenic ability of DU 145 and Tumor Spheres was evaluated with xenograft model.DU 145 cells and DU145-CSC(prostate cancer stem cells)were inoculated into NOD/SCID mice with bilateral axillary,and the rate/time of tumor formation were observed.The tumor formation rate of Tumor Spheres was 100%on the 30th day,while that of DU145 cells was 20%after 2×103cells injection.It was confirmed that Tumor Spheres had tumor stem cell characteristics.2.Using protein chip technique to compare the differential expression of protein phosphorylation of DU 145 and DU145-CSC,we explored the specific biological behaviors regulation mechanism of prostate cancer stem cells.It was found that when the cut off value is set to 1.2(the ratio>1.2 and the ratio<0.833)compared with DU 145,DU145-CSC expressed 125 differential phosphorylated proteins with upregulated 98 phosphorylated proteins and downregulated 27 phosphorylated proteins.Searching their corresponding swissprot numbers through the online database KEGG respectively,it was found that JAK/STAT,PI3K/AKT,RAS/MAPK/ERK were the core signal pathways.3.In vitro observation of the inhibitory rate of CT,permine,curcumin,curcumol and GA on DU145 and/or DU145-CSC.3.1 It was showed that CT could significantly inhibit proliferation of DU 145 cells.It was dose-time dependence.The IC50 value was 6.91?M at 48h.When treated with 6.25?M-50?M CT after 72h,the inhibition rate achieved 95%.The proliferation of DU 145 cells was significantly inhibited by GA,and the IC50 value was 0.65?M at 48h,and the proliferation inhibition rate of DU145 cells was close to 100%when treated with 3.13?M-25?M CT after 48h and 72h.Pennine and curcumol had no strong inhibitory effect on DU 145 cell proliferation.The inhibitory rates of different concentrations and intervention time were not more than 50%.Curcumin could inhibit the proliferation of DU145 cells,and the IC50 was 19.99?M at 48h,and the inhibition of cell proliferation was more obvious with the prolongation of time.3.2 To investigate the effect of CT and GA on the proliferation of DU145-CSC cells,we found that the IC50 value of CT was 4.093?M at 48h.The inhibition rate of DU145-CSC was nearly 100%with 6.25p,M-25?M CT intervention at 72h-96h.The IC50 value of GA on DU145-CSC cells was 0.388?M at 48h,and the inhibition rate was close to 100%with 1.56?M-12.5?M CT at 48h.Compared with DU145 cells,CT and GA had a stronger inhibitory effect on DU145-CSC.4.To investigate the effect of CT on the apoptosis of DU145-CSC cells,the results showed that when treated with 3.46?M,6.91?M and 13.82?M of CT for 48 hours,CT promoted the apoptosis of DU145-CSC cells in a dose-dependent manner.After the intervention of each dose of CT,the apoptotic rate in CT group was significantly different with the control group(control group:7.27±3.34%,3.46?M group:34.82±9.83%,6.91 pM group:42.72±11.58%,13.82?M group:48.34±10.73%,p<0.01).5.To investigate the effect of CT on the cell cycle of DU145-CSC cells,the results showed that when treated with 3.46?M,6.91?M and 13.82?M CT for 48 hours,(66.43±0.20)%,(66.63±0.63)%and(71.95±1.64)%of the cells were blocked in G0/G1 phase,respectively.The proportion of G0/G1 phase cells was(56.56±0.49)%in the control group(p<0.01).6.The protein microarray technique was used to compare the differential expression of CT-CSC and CSC protein phosphorylation to explore the regulation mechanism of cryptotansetine on the specific biological behaviors of prostate cancer stem cells.The results showed that when the cut off value is set to 1.2(the ratio>1.2/the ratio<0.833),FAK(Phospho-Tyr861,Phospho-Tyr397),PYK2(Phospho-Tyr402),AKT(Phospho-Ser473),CREB(Phospho-Ser133),BCL-2(Phospho-Ser70),p70 S6 Kinase(Phospho-Ser424),14-3-3 zeta(Phospho-Ser58)were down-regulated in CT-CSC group.According to the online library KEGG,the related down-regulated phosphorylated proteins were based on PI3K/AKT pathway as the core signal pathway.7.Effect and mechanism of CT on prostate cancer stem cells in vivo.NOD/SCID mice were inoculated with human prostate cancer stem cells,and the prostate cancer xenograft model was established.Tumor-bearing nude mice were injected with CT(6.25mg/kg)or vehicle intraperitoneally every other day for 4 weeks.The tumor inhibiton rate of CT in vivo was 35.4%compared to the control group.The expression of FAK,p-FAK,PYK2,p-PYK2,p-AKT and p-BCL-2 protein were significantly lower in CT group than those of the control group by Western Blot(P<0.05).Conclusion:1.Prostate cancer stem cells(DU145-CSC)could be isolated and enriched from DU 145-prostate cancer cell line and expressed CD44 + CD24-/low phenotype,and had the ability of self-renewal and tumorigenictity.2.The specific biological behaviors of DU145-CSC cells were regulated by interoperable network regulation system with JAK/STAT,PI3K/AKT,RAS/MAPK/ERK as the core pathways.3.Cryptotanshinone can inhibit the proliferation,induce apoptosis and cell cycle arrest in G0/G1 phase of DU145-CSC in vitro and inhibit tumor formation of DU145-CSC in vivo.4.Intervention mechanism of Cryptotanshinone on human prostate cancer stem cell biology behaviors was related to the regulation of the network system with PI3K/AKT as the core pathway. |
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