| Prostate cancer is the most common tumor in old man,it is also one of the three reasons causing death of men in European and America. For advanced metastasis prostate cancer,it is sensitive for androgen deprivation therapy at first,but afer the median of 13-22 months,most people would turn into castration resistant prostate cancer(CRPC),When the first and second line of thrapy dugs fail,people who are in the stage of CRPC are deficient of effective method,Chinese medicine has the character of overall regulation,different treatment according to different syndrome and treating disease through multiply targets,approaches,levels.Because of these,Chinese medicine With unique efficacy has been used extensively in the treatment of cancer.Qianliexiaozheng Decotion(QD) is an effective Chinese medicine prescriptio n treating CRPC,The Decotion has been used for 10 years in the urinary surge ry of Guang anmen Hospital,China Academy of Chinese Medical Science.In the guide of "mass accumulation because of the lack of healthy atmosphere and t hen squat of evil""chronic illness injury Qi"and other traditional Chinese medici ne theory, Liu YouFang who is a famous expert in combination of Chinese an d Western medicine in urology in our country make use of the combining of macroscopic disease differentiation and microscopic syndrome differentiation thr ough continuous clinical practice,and summarize the heart of advanced prostate cancer pathogenesis as"lack of righteousness, inherence is full by dampness and poison" He use the method of tonifying Qi,detoxification,removing dampness t herapies to formulate Qianliexiaozheng Decotion(QD). The decotion is compose d by Coix seed, rhizoma polygonati,astragalus membranaceus,Oldenlandia diffusa, Curcuma Zedoary, Rhizoma Bolbostemmae,grifola.In the prescription, The sover eign drug is Coix seed to tonify spleen and excrete dampness, The associate dr ugs are Oldenlandia diffusa to detoxify and soften hard mass,The compatible d rugs are astragalus membranaceus and rhizoma polygonati to Tonify Qi and sple en and nourish yin.The decotion is also include Curcuma Zedoary,Rhizoma Bo lbostemmae,grifola to detoxicate,remove stasis,dispers blood stasis and relieve p ain.with above all, The achivement of decotion is tonifying Qi and spleen, pro ducing Blood, detoxicating,removing stasis and excreting dampness.All of these reflect that the basic rule of traditional Chinese medicine treating advanced ca ncer in the treatment is strengthening the body resistance to eliminate pathogenic f actorsPre-clinical studies have shown the QD can improve the quality of life,increase the score of physiological status,emotion situation and additional attention in Functional assessment of cancer therapy-prostate(FACT-P), and decrease Psychological anxiety levels related with disease in memorial anxiety scale for prostate cancer(MAX-PC).It also can improve the symptoms of difficulty of urination, urinary urgency, dysuria and weak,decrease the double time of prostate-specific antigen (PSA) levels.Pre-basic studies have shown QD can inhibit the proliferation of nude mice transplantation tumors of PC-3 cells, Drug serum of QD can inhibit the proliferation of PC-3 cells,reduce the invasiveness of PC-3,regulate the key protein in PI3/AKT signal pathway.Now,We want to fully understand the effect of QD on prostate cancer cell lines, dicuss the rationality and effectiveness of the theory of tonifying Qi and spleen, producing Blood, detoxicating,removing stasis and excreting dampness treating CRPC and explore the mechanism of Chinese compound formula treating tumor.So We choose DU-145 as object of study in this experiment study,We research the proliferation of prostate cancer DU-145 cells dealing with QD by MTT assay; and find out the IC50 of QD acting to DU-145 cells; We measure the ability of DU-145 cells migration and invasion with method of cell wound scratch assay, plate clone formation assay,Transwell assay,and resarch the Cell cycle and apoptosis through flow cytometry,and study the key protein in the pathway of TGF-β and Wnt/p-catenin signals in DU-145 dealing with QD by RT-PCR,Western Blot Immunofluorescence technologies.Purpose:1.QD could inhibit proliferation of prostate cancer DU-145 cells in vitro.2.QD could affect the invasion,migration,proliferation and apoptosis of DU-145 cells in vitro.3.QD could inhibit the proliferation and induce apoptosis of DU-145 in vitro,The mechanism may act through the the interaction of TGF-(3 and Wnt/β-catenin signals probably.Method:Measure the proliferation of prostate cancer DU-145 cells dealing with QD by MTT assay; and find out the IC50 of QD acting to DU-145 cells;Study the migration and invasion DU-145 cells dealing with QD cell by wound scratch assay, plate clone formation assay,Transwell assay.Resarch the Cell cycle and apoptosis of DU-145 cells under different concertration of QD through flow cytometry.We divided into five groups:High dose group,Medium dose group,Low dose group, Blockers group,control group, study key protein and gene Smad3,a-SMA,Smad2,E-cadherin in the pathway of TGF-β signals and key protein and gene Wnt,β-catenin,Axin,SFRP-1 in the pathway of Wnt/β-catenin signals in DU-145 dealing with QD by RT-PCR,Western Blot technologies.Result:1.Max concertation of QD in MTT assay was 1920μg/ml,then measured the PH value and osmotic pressure three times, and took the average, the range of PH value is 6.912±0.074, the range of osmotic pressure value is 285.673±5.057.Both them were within the normal range.MTT assay showed that QD could inhibit proliferation of prostate cancer DU-145 cells. IC50 of QD was 830μg/ml approximately.2.The migration distance of DU-145 cells after 24h culturing in the wound scratch assay:high dose group>medium dose group>low dose group>control dose group.The migration distance in low dose group had statistical differences compared with control dose group(P<0.05).The migration distance in high dose group had statistical differences compared with control dose group(P<0.01).The migration distance in medium dose group had statistical differences compared with control dose group(P<0.01).3.The ratio of plate clone formation of DU-145 cells after 24h culturing in the plate clone formation assay:high dose group<medium dose group<low dose group <control dose group. The ratio of plate clone formation in low dose group had no statistical differences compared with control dose group(P>0.05). The ratio of plate clone formation in high dose group had notably statistical differences compared with control dose group(P<0.01). The ratio of plate clone formation in medium dose group had statistical differences compared with control dose group(P<0.05).4. The number of DU-145 cells transferring into membrane after 24h culturing in the Transwell assay:high dose group<medium dose group<low dose group<control dose group.The number of cells transferring into membrane in low dose group had no statistical differences compared with control dose group(P> 0.05).The number of cells transferring into membrane in high dose group had notably statistical differences compared with control dose group(P<0.01).The number of cells transferring into membrane in medium dose group had statistical differences compared with control dose group(P<0.05).5.The early stage of apoptosis,late stage of apoptosis in low dose group both had no statistical differences compared with control dose group(P>0.05). The early stage of apoptosis,late stage of apoptosis in medium dose group both had statistical differences compared with control dose group(P<0.05). The early stage of apoptosis plus late stage of apoptosis in medium dose group had notably statistical differences compared with control dose group(P<0.01). The early stage of apoptosis,late stage of apoptosis in high dose group both had notably statistical differences compared with control dose group(P<0.01). The early stage of apoptosis plus late stage of apoptosis in high dose group had notably statistical differences compared with control dose group(P<0.01).6.The result of Cell cycle showed that the ratio of cell in S period in control dose group and low dose group was (31.69±1.29)%,(31.51±2.61)% respectively, the difference between them was not statistically significant(P>0.05). the ratio of cell in S period in control dose group and medium dose group was (31.69±1.29)%,(37. 96±2.24)% respectively, the difference between them was statistically significant(P< 0.05). the ratio of cell in S period in control dose group and high dose group was (31.69±1.29)%,(43.77±2.48)% respectively,the difference between them was notably statistically significant(P<0.01).7. RT-PCR result:(1)There was no statistical difference between the low-dose group and the controlled group in the RQ of Smad3 mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), andthe difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01). (2)There was no statistical difference between the low-dose group and the controlled group in the RQ of a-SMA mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01). (3)There was no statistical difference between the low-dose group and the controlled group in the RQ of Smad2 mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was notably statistically significant (P<0.01), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01).(4)There was no statistical difference between the low-dose group and the controlled group in the RQ of E-cadherin mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was notably statistically significant (P<0.01), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), andthe difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01).(5)There was no statistical difference between the low-dose group and the controlled group in the RQ of Wnt mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the RQ was statistically significant (P<0.05).(6)There was no statistical difference between the low-dose group and the controlled group in the RQ of β-catenin mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01).(7)There was no statistical difference between the low-dose group and the controlled group in the RQ of Axin mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01).(8)There was no statistical difference between the low-dose group and the controlled group in the RQ of SFRP-1 mRNA (P>0.05); the difference between the mid-dose group and the controlled group in the RQ was notably statistically significant (P<0.01), the difference between the blocker group and the controlled group in the RQ was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the RQ was notably statistically significant (P<0.01).8. Western blot result:(1)There was no statistical difference between the low-dose group and the controlled group in the Smad3 protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the Smad3 protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the Smad3 protein expression was statistically significant (P<0.05), andthe difference between the high-dose group and the controlled group in the Smad3 protein expression was notably statistically significant (P<0.01). (2)There was no statistical difference between the low-dose group and the controlled group in the a-SMA protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the a-SMA protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the α-SMA protein expression was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the a-SMA protein expression was notably statistically significant (P<0.01). (3)There was no statistical difference between the low-dose group and the controlled group in the Smad2 protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the Smad2 protein expression was notably statistically significant (P<0.01), the difference between the blocker group and the controlled group in the Smad2 protein expression was notably statistically significant (P<0.01), andthe difference between the high-dose group and the controlled group in the Smad2 protein expression was notably statistically significant (P<0.01). (4)There was no statistical difference between the low-dose group and the controlled group in the E-cadherin protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the E-cadherin protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the E-cadherin protein expression was notably statistically significant (P<0.01), andthe difference between the high-dose group and the controlled group in the E-cadherin protein expression was notably statistically significant (P<0.01). (5)There was no statistical difference between the low-dose group and the controlled group in the Wnt protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the Wnt protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the Wnt protein expression was statistically significant (P<0.05), and the difference between the high-dose group and the controlled group in the Wnt protein expression was notably statistically significant (P<0.01). (6)There was no statistical difference between the low-dose group and the controlled group in the β-catenin protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the β-catenin protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the β-catenin protein expression was notably statistically significant (P<0.01), and the difference between the high-dose group and the controlled group in the β-catenin protein expression was notably statistically significant (P<0.01). (7)There was no statistical difference between the low-dose group and the controlled group in the Axin protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the Axin protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the Axin protein expression was statistically significant (P<0.05),and the difference between the high-dose group and the controlled group in the Axin protein expression was statistically significant (P<0.05). (8)There was no statistical difference between the low-dose group and the controlled group in the SFRP-1 protein expression (P>0.05); the difference between the mid-dose group and the controlled group in the SFRP-1 protein expression was statistically significant (P<0.05), the difference between the blocker group and the controlled group in the SFRP-1 protein expression was statistically significant (P<0.05), and the difference between the high-dose group and the controlled group in the SFRP-1 protein expression was statistically significant (P<0.05).Innovations:We research the biological behavior of prostate cancer DU-145 cells dealing with Chinese compound formula QD with Molecular biological technology, We verify the rationality the theory of tonifying Qi and spleen, producing Blood, detoxicating,removing stasis and excreting dampness treating CRPC. QD can inhibit the proliferation and induce apoptosis of DU-145,It realize above function may via the mechanism of regulating the signal pathway of Wnt/β-catenin and TGF-β/Smad.It reveals that Chinese medicine treating diease have character of multi targets, multi-paths, multi-links, multilevel.It helps to illuminate scientific connotation of famous TCM doctor’s experience through cells-gene-protein level.Conclusion:l.The MTT assay showed that QD could inhibit proliferation of prostate cancer DU-145 cells, IC50 was 830μg/ml approximately.2.The wound scratch assay showed that QD could inhibit migration of prostate cancer DU-145 cells,The plate clone formation assay showed that QD could inhibit the ability of clone formation of prostate cancer DU-145 cells, The Transwell assay showed that QD could inhibit the ability of invasion of prostate cancer DU-145 cells,the all above inhibition augmented which was followed by the concetration increasing.3.QD could induce the apoptosis of early stage and late stage of DU-145 cells, the induction augmented which was followed by the concetration increasing. QD could decrese the percentage of G0/G1 period of Cell cycle in tumor cells and increase the percentage of S period,unchange the percentage of G2/M period.These predicted thay QD stop the Cell cycle of DU-145 cells turning S period into G2 period4.QD could inhibit proliferation and induce the apoptosis of prostate cancer DU-145 cells,increase the expression of E-cadherin, Smad2,decrease the expression of α-SMA,Smad3.It is similar to retardant of TGF-β pathway.It indicated that QD could inhibit proliferation and induce the apoptosis of prostate cancer DU-145 cells through regulatingTGF-β pathway.5.QD could increase the expression of Axin,SFRP-1,decrease the expression of Wnt,β-catenin.It is similar to retardant of Wnt/β- catenin pathway.lt indicated that QD could regulate TGF-β pathway.6.QD could inhibit proliferation and induce the apoptosis of prostate cancer DU-145 cells,The mechanism may act through the the interaction of TGF-β and Wnt/β-catenin signals probably. |
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