Extraction And Isolation Of Piperlongumine And Its Anti-breast Cancer Mechanism

Breast cancer has become the highest incidence of malignant tumors in women worldwide,causing heavy medical and social burdens.Current treatment options include surgery,radiation therapy,endocrine therapy,targeted therapy and chemotherapy.At present,chemotherapy is still the main method of breast cancer treatment.However,its severe side effects and problem of causing drug resistance have not been improved,resulting in a poor prognosis.Therefore,it is urgent to find new medicines which could combine with these chemotherapy drugs to improve treatment effect.Piperlongumine(PL)has been reported showing selective anticancer activity,while its distribution in Chinese Piper longum roots and its mechanism of killing breast cancer when treated alone or in combination remains to be fully investigated.Here,we studied following contents,including the method of extraction and isolation of PL,the anticancer activity and mechanism of PL alone as well as combined with piperine(PP)and doxorubicin(DOX).Firstly,we investigated the natural alkaloids of Piper longum roots growing in China.4.5 kg of Piper longum roots were purchased from Yunnan and identified by morphology.470 g of extract was obtained after pulverization,extraction and concentration.The extract was dissolved and coarsely separated by silica gel column chromatography.Gradient elution was performed with petroleum ether,ethyl acetate and methanol as eluents to obtain a three-phase crude extract,including 24 g of a petroleum ether phase,203 g of an ethyl acetate phase and 232 g of a methanol phase.Each phase was then eluted with petroleum and ether-ethyl acetate in different proportions,and separated phase by phase to obtain fifteen components.Four of them were identified as alkaloids by the color reaction of bismuth potassium iodide.These alkaloids were identified by nuclear magnetic resonance(NMR)analysis as PL(12.8 mg),PP(8.59 g),piperolactam A(70.4 mg)and piperolactam D(20.4 mg).The above results showed that domestic Piper longum root was rich in alkaloids,including anticancer components PL and PP.Secondly,we investigated the anticancer activity and mechanism of PL against breast cancer in vitro and in vivo.Cell proliferation experiments,colony formation experiments,apoptosis experiments and cell cycle arrest experiments were performed,data showed that PL effectively inhibited proliferation of MDA-MB-231 and MCF-7 cells,thereby leading to apoptosis.However,there was only a weak effect was observed in MCF-10A normal cells,indicating an anticancer selectivity of PL.Western Blot experiments showed that PL effectively inhibited activation of signal transducer and activator of transcription 3(STAT3)in MDA-MB-231 and MCF-7 cells,regulated expression of several downstream signaling molecules and finally induced apoptosis,which was depended on reactive oxygen species(ROS).Tumor xenograft model experiments showed that 4 mg/kg and 12 mg/kg of PL effectively reduced tumor volume as well as tumor weight.PL downregulated p-STAT3 expression in tumor tissues,however,no significance was observed between the two doses.Hematoxylin-eosin(HE)staining experiments showed that PL had no significant organ toxicity in nude mice at both doses.The above results demonstrated that PL effectively inhibited breast cancer cell proliferation,with good selectivity and in vivo safety.Thirdly,we investigated the anticancer activity and mechanism of PL and PP against breast cancer in vitro and in vivo.Cell proliferation experiments showed that PL had certain anticancer selectivity and stronger activity than that of PP,and these results were confirmed in cell apoptosis experiments.Combination index(CI)analysis experiments showed that CI values of PL and PP at lower doses(PP 50?M+PL 5 ?M,PP 100 ?M+PL 5 ?M)in MDA-MB-231 and MCF-7 cells were smaller than 1,and bigger than 1 in MCF-10A cells,indicating a synergistic inhibitory effect between the two agents.Also,these results were confirmed in cell apoptosis experiments.Western Blot experiments showed that PL inhibited p-STAT3 expression in MDA-MB-231 cells,while PP showed the opposite effect.However,the expression of p-STAT3 was significantly decreased when treating with PL and PP in combination,indicating PL and PP produced a synergistic inhibitory effect on STAT3 activation.Moreover,this synergistic effect was not observed in normal breast cells.Tumor xenograft model experiments showed that the combination of 4 mg/kg PL and 10 mg/kg PP effectively reduced tumor volume as well as tumor weight.In addition,both agents synergistically inhibited p-STAT3 expression in tumor tissues.HE staining experiments showed that neither single agent nor combination of PL and PP had significant organ toxicity in nude mice.The above results demonstrated that PL and PP synergistically inhibited breast cancer cell proliferation,with good selectivity and in vivo safety.Fourthly,we investigated the anticancer activity and mechanism of PL and DOX against triple-negative breast cancer(TNBC)in vitro and in vivo.Cell proliferation experiments showed that DOX had stronger activity than that of PL,and these results were confirmed in cell apoptosis experiments.CI analysis experiments showed that CI values of PL and DOX at lower doses(PL 5 ?M+DOX 0.5 ?M)in MDA-MB-231 and MDA-MB-453 cells were 0.57 and 0.61,respectively,indicating a synergistic inhibitory effect between the two agents.Also,these results were confirmed in cell apoptosis experiments.Western Blot experiments showed that PL inhibited p-STAT3 expression in both cells,while DOX showed the opposite effect.However,the expression of p-STAT3 was decreased when treating with two agents together compared to DOX alone,indicating DOX-mediated upregulation of p-STAT3 could be reversed by PL.Tumor xenograft model experiments showed that the combination of 4 mg/kg PL and 0.8 mg/kg DOX effectively reduced tumor volume as well as tumor weight.In addition,the effect of DOX-mediated upregulation of p-STAT3 was reversed by PL in tumor tissues.HE staining experiments showed that neither single agent nor combination of PL and DOX had significant organ toxicity in nude mice.The above results demonstrated that PL and DOX synergistically inhibited triple-negative breast cancer cell proliferation,with good in vivo safety.Taken together,we isolated PL from the root of Piper longum L.grown in China;revealed the anti-breast cancer mechanism of PL:inhibiting STAT3 activation through oxidative stress,thereby mediating three signaling pathways to induce cell apoptosis;demonstrated the anti-breast cancer activity and mechanism of PL synergized with PP or DOX.Our study demonstrated that inactivation of STAT3 by PL may help reduce side effects of DOX and improve drug resistance.This thesis provided potential strategy for future clinical therapy of breast cancer.