| Background:Leukemia is often considered a clonal stem cell disease,In this disease,self-renewing,LSCs（Leukemia stem cells,LSC）are considered to be an important factor in initiating tumor formation.Currently,chemotherapy using cytotoxic drugs to destroy cancer cells is the most effective treatment.However,multidrug resistance（Multidrug resistance,MDR）is an important cause of the difficulty in curing tumors and is a major problem that needs to be addressed in cancer treatment.Among them,the presence of leukemia stem cells is the main cause of multidrug resistance and recurrence of leukemia.Therefore,the search for a new treatment for leukemia stem cells（LSC）is the key to curing leukemia.The promyelocytic leukemia protein（Promyelcocytic leukemia protein,PML）gene was first described in the early 1990s as a target for chromosomal translocation t（15;17）in acute promyelocytic leukemia,which produces carcinogenic PML-retinant Acid receptor alpha（RAR alpha）fusion protein（PML-RAR alpha）.PML has been reported to be overexpressed in MDR chronic myeloid leukemia（CML）.Therefore,PML may be in MDR leukemia.However,the mechanism of PML resistance to leukemia cells remains unclear.In order to solve the problem of multi-drug resistance,recent studies have found that new aconitine is the main biological active ingredient in aconite,with strong analgesic,anti-rheumatic and anti-tumor pharmacological effects.Previous studies have shown that new aconitine has a significant inhibitory effect on the growth of leukemia cell K562.Therefore,this study mainly used the drug-resistant chronic myeloid leukemia cell line K562-ADR as a research object to investigate whether PML gene is involved in the development of CML resistance.Based on this,we further explored whether the drug resistance of K562-ADR in CML cell line can be reduced by the action of aconitine.Objective:To investigate the expression of PML in B562-ABL-positive chronic myeloid leukemia cells K562 and K562-ADR,and to explore the effect of aconitine,a Chinese aconite component,on K562-ADR cell resistance.Methods:First of all,The two chronic myeloid leukemia cells K562 and K562-ADR were cultured for a period of time to stabilize,and the resistance of K562-ADR cell line to ADR（doxorubicin）was detected by CCK8 method.Secondly,the expression of PML between K562 and K562-ADR cells was detected by RQ-PCR and Western Blot.Thirdly,The growth inhibition rate of K562 and K562-ADR cells treated with different concentrations of aconitine at 24h,48h and 72h was detected by CCK8 method.The growth inhibition rate of K562 and K562-ADR cells treated with different concentrations of new aconitine at 24h,48h and 72h was detected by CCK8method.Three new aconitine concentrations of 50μg/mL,75μg/ml and 100μg/ml were selected for subsequent experiments.Fourth,the experiment was divided into five groups:A:K562-ADR;B:K562-ADR+ADR;C:K562-ADR+50μg/ml new aconitine;D:K562-ADR+75μg/ml new aconitine;E:K562-ADR+100μg/ml new aconitine;Doxorubicin is 3μg/ml,5×106cells were added to per hole.After treatment with cells for 48h and 72h by adding drugs,the expression of PML was detected by RQ-PCR and Western Blot.Fifth,the experiment was divided into five other groups:A:K562-ADR;B:K562-ADR+ADR C:K562-ADR+50μg/ml new aconitine+ADR;D:K562-ADR+75μg/ml new aconitine+ADR;E:K562-ADR+100μg/ml new aconitine+ADR;Doxorubicin is 3μg/ml,5×106cells were added to per hole.After treatment with cells for 48h by adding drugs,the expression level of PML was detected by RQ-PCR and Western Blot.Sixth,the experiment was divided into seven groups:A:K562 B:K562+ADR C:K562-ADR D:K562-ADR+ADR E:K562-ADR+50μg/ml new aconitine+ADR F:K562-ADR+75μg/ml new aconitine+ADR G:K562-ADR+100μg/ml new aconitine+ADR;Doxorubicin is 3μg/ml,1500 cells were added to per hole.After treatment with cells for 24h、48h and 72h by adding drugs,Cell inhibition rate was detected by CCK8 method.Finally,the experiment was divided into four groups:A:K562-ADR+ADR;B:K562-ADR+50μg/ml new aconitine+ADR;C:K562-ADR+75μg/ml new aconitine+ADR;D:K562-ADR+100μg/ml new aconitine+ADR;Doxorubicin is 3μg/ml,5×106cells were added to per hole.After treatment with cells for 48h by adding drugs,Apoptosis was detected by flow cytometry.The results are as follows:The drug resistance of K562-ADR was verified by CCK8.It was found that doxorubicin was concentration-dependent on K562.With the increase of doxorubicin concentration,the inhibitory effect was enhanced,and the IC50 was 2.521μg/mL.However,doxorubicin had no concentration-dependent effect on K562-ADR,and the inhibitory effect did not change significantly with increasing doxorubicin concentration,and there was no the value of IC50;The expression of PML in K562-ADR cells was detected by RQ-PCR and Western blot.The results showed that the expression of PML in K562-ADR cells was higher than that in K562 cells;The inhibition of proliferation of K562 and K562-ADR cells,treated by different concentrations of new aconitine,was detected by CCK8 method.It was found that new aconitine had the most obvious inhibitory effect on the proliferation of K562 and K562-ADR cells,used the concentration range of50-100μg/ml;The effects of on the expression of PML in K562-ADR cells,treated by different concentrations of new aconitine,were detected by RQ-PCR and Western blot.It was found that new aconitine significantly inhibited the expression of PML in K562-ADR cells.However,there was no significant difference in the inhibitory effect of different concentrations of new aconitine on PML expression in K562-ADR cells;The inhibition of proliferation of K562-ADR cells,treated by different concentrations of new aconitine combined with 3μg/ml doxorubicin,was detected by CCK8 method.It was found that new aconitine combined with 3μg/ml doxorubicin significantly inhibited the proliferation of K562-ADR cells,and as the concentration of new aconitine increases,the inhibitory effect is more pronounced;The effects of different concentrations of new aconitine combined with3μg/ml doxorubicin on the expression of PML in K562-ADR cells were detected by RQ-PCR and Westen Blot.As a result,it was found that compared with the experimental group co-cultured with K562-ADR and 3μg/ml of doxorubicin,100μg/ml neoconophylline combined with 3μg/ml doxorubicin can reduce the high expression of PML;The effect of different concentrations of new aconitine combined with 3μg/ml doxorubicin on apoptosis of K562-ADR cells was detected by flow cytometry.It was found that 100μg/ml new aconitine combined with 3μg/ml doxorubicin could promote apoptosis of K562-ADR cells.Conclusion:PML is highly expressed in BCR-ABL-postive adriamycin-resistant chronic myeloid leukemia cell K562-ADR.The Chinese aconite component new aconitine has a significant inhibitory effect on the proliferation of K562 and K562-ADR in the concentration range of 50-100μg/ml and can reduce the high expression of PML.100μg/ml new aconitine combined with 3μg/ml doxorubicin can reduce the high expression of PML and promote the apoptosis of K562-ADR cells.Therefore,the Chinese aconite component new aconitine can reduce the drug resistance of K562-ADR by decreasing the high expression of PML,and then promote the apoptosis of K562-ADR cells. |
