Study On The Mechanism Of Pinellia Extract On Proliferation Inhibition And Apoptosis Of Drug-resistant Chronic Myeloid Leukemia Cells K562

Research backgroundIn the past decade,the incidence of leukemia in China has increased at a rate of 2%.Leukemia is a malignant tumor that originates from hematopoietic precursor cells.After a complete remission of chemotherapy,hematopoietic stem cell transplantation is the main means to treat leukemia.However,the side effects of chemotherapy and the generation of multidrug resistance have brought many difficulties to the complete remission and consolidation of leukemia.Traditional Chinese medicine is a unique theory and method and it has obvious advantages of individualized and dialectical treatment.Practice has proved that traditional Chinese medicine play an important role in the treatment of leukemia.In recent years,with the increasing clinical and experimental research of traditional Chinese medicine and its active ingredients,it has found that the effective ingredients in the traditional Chinese medicine can change the biological activity of leukemia cells and induce the maturation and apoptosis of leukemia cells.Pinellia is a traditional Chinese herbal medicine that contains a variety of effective active ingredients.Pinellia extract and alkaloids have related research on the inhibition of leukemia cell proliferation,but its mechanism of action is not yet clear,and it is worth further research.This paper attempts to change the biological characteristics of leukemia cells through some active ingredients of traditional Chinese medicine,thereby increasing the sensitivity of chemotherapy drugs and obtaining early and better complete remission.It is necessary to create conditions for stem cell transplantation and other technical means that may cure leukemia.PurposeTo explore the effective components of pinellia extract to inhibit the proliferation of drug-resistant leukemia cell line K562-ADR(Adriamycin-resistant chronic myeloid leukemia cell line K562)and induce apoptosis;To explore the molecular level mechanism of its effect;To explore the possible application value of Pinellia extract in the treatment of drug-resistant leukemia.Research methodThe chloroform low-temperature ultrasonic method was used to extract the active components of Pinellia.The K562-ADR cell lines treated with adriamycin were respectively interfered with 7concentrations of 0.1 mg/ml,0.3 mg/ml,0.5 mg/ml,0.7 mg/ml,1 mg/ml,2 mg/ml,and 3 mg/m L.Then the IC50 was used to calculate the half inhibitory concentration and the concentration range was optimized.To observe the morphological changes of K562-ADR induced by Pinellia extract in chronic myeloid leukemia drug resistance;CCK-8 method was used to detect the inhibitory effect of Pinellia extract on the proliferation of K562-ADR cells.Real-time fluorescence quantitative PCR was used to detect the expression of BAX,BCL-2,MDM2,BCR-ABL and PML genes in K562-ADR cells.Research results1.Adriamycin resistance test results show that the multidrug resistance ratio of K562-ADR cells is 59.39 times that of K562.2.CCK-8 test results show that Pinellia ternate extract has strong inhibitory effect on proliferation of K562 and K562-ADR leukemia cells.3.The IC50 of Pinellia ternata extract on K562 group was 1.83mg/ml calculated by Graphpad Prism respectively,and the IC50 of K562-ADR group was 2.36mg/ml.4.RT-PCR and Western blot were used to detect PML expression in K562-ADR cells.The results showed that PML expression in K562-ADR cells was higher than that in K562 cells.5.RT-PCR showed:(1)BCL-2 had the maximum value when 5 mg/m L intervened at 48 h and 72h(P<0.01)BAX expression level is down-regulated,suggesting that BAX/BCL-2pathway may not participate in K562-ADR cell apoptosis at this concentration.(2)1,3,5 mg/ml Pinellia ternate extract affected the transcription and expression levels of MDM2,BCR-ABL and PML in K562 and K562-ADR cells 48 hours after intervention.The results showed that 1,3 mg/ml Pinellia ternate extract may activate MDM2,BCR-ABL and PML expression in K562-ADR cells.However,when 5mg/ml was used,the expression levels of BCR-ABL and PML were down-regulated.(3)after 0.5 and 0.7mg/ml Pinellia ternate extract intervened for 48 and 72 h,it affected the transcription BAX and BCL-2 expression level of K562-ADR cells,and the BCL-2expression level was down-regulated(p < 0.05 or p < 0.01).Conclusions1.Pinellia extract can inhibit the proliferation of adriamycin-resistant chronic myeloid leukemia cells,promote cell apoptosis through BAX and BCL-2 pathways at a lower concentration of 0.5 and 0.7 mg / ml,and has the most stable growth inhibition curve and apoptosis protein expression for 48 to 72 hours.2.The expression of BAX and BCL-2 m RNA was affected by the concentration and duration of the Pinellia extract in adriamycin-resistant chronic myeloid leukemia cell with a higher concentration of 1,3,5 mg/ml.3.The expression levels of MDM2,BCR-ABL and PML are affected by the concentration and duration of Pinellia ternate extract in the higher concentration range of 1,3 and 5mg/ml,and m RNA expression levels are different at different concentrations.4.The resistance of chronic myeloid leukemia cells may be mediated by PML,and intervention of 5mg/ml Pinellia ternate extract for 48 hours can reduce the expression level of PML.