Chemotherapy Sensitizing Effect Of Tan ?A Combined With ATO On Acute Promyelocyte Leukemia And Its Mechanism In Vivo And In Vitro

Background: Acute promyelocyte leukemia(APL),a distinct subtype of acute myelogenous leukemia,is characterized by differentiation development stagnation and malignant clone proliferation of hematopoietic progenitors at the promyelocyte stage.All-trans retinoic acid(ATRA)and arsenic trioxide(ATO)are effective drugs for APL,the clinical complete remission(CR)rate has reached up to 90%~95%.However,it has been reported there have some drug resistance and relapse cases of APL and the resistant mechanism is related to the defect of apoptosis,moreover,these patients could not achieve remission again using ATRA and ATO.So far,refractory APL is still a sever challenge faced by many scholars and experts.Therefore,it is imminent to actively explore new antineoplastic mechanism and invent safe and effective chemotherapy drugs for APL.Apoptosis is a programmed process of cell death which is triggered by activating the stored death programe in cells,and its disorder is an important mechanism of tumorigenesis.In recent 10 years,it has been found that,besides apoptosis,autophagy is also involved in the pathogenesis of tumor.Autophagy is a catabolic process in cell depending on lysosome degradation pathway.It eliminates potential cytotoxic substance by degradation of aberrantly folded protein? damaged organelles and so on,which make cells quickly adapted to the change of internal and external environment avoiding dysfunction or death,so in some extent,autophagy is a defensive mechanism helping cell survive.Autophagy has double-edged sword effects on cell survival,moderate autophagy could maintain cell energe renewal and structure reconstruction,which is helpful for cell to survive,but excessive autophagy could induce cell autophagic death,so called “type-II programed cell death”.Activating autophagy or inducing cell autophagic death would be a valuable new method to increase the antitumor efficacy.Except apoptosis,inducing cell autophagic death is another important antitumor mechanism of most chemotherapeutic drugs.Tanshinone IIA(Tan IIA),as a diterpene quinone compounds extract from Tanshinone,was mainly used for clinical treatment of cardiovascular disease and cerebral ischemic stroke.Recently it has been found that Tan IIA has antiproliferative activity for a wide range of tumor cells,but its mechanism is still not clear.Previous studies mostly focused on apoptosis,reports on the role of autophagic death in antitumor mechanism of Tan IIA are very rare.Objective: From the perspective of apoptosis and autophagy,to explore the influence of Tan II A? Tan IIA combined with ATO on the growth of APL cell line NB4 and its possible molecular mechanisms and regulatory mechanism;Establishing nude mice model of human APL,in order to explore the influence of Tan IIA combined with ATO on the growth of NB4 xenograft in vivo and its molecular mechanism,in order to provide experimental and theoretical basis for clinical use of Tan IIA as an adjuvant therapy strategy for leukemia treatment.Method:Part of expement in vitro(1)the APL cell line NB4 was cultured in vitro,then divided into 6 groups: 4?8?16mg/ml Tan IIA?0.5mg/ml ATO and 8mg/ml Tan IIA+0.5mg/ml ATO?normal saline(NS)control group;(2)Assay the cell proliferation ability of each group with the method of CCK-8 at time point of 12h? 24h?48h?72h after cells being treated;NB4 cells was preincubated with the apoptosis inhibitor z-VAD-fmk or autophagy inhibitor3-MA for 1h,then being treated with Tan IIA,the cell proliferation ability was measured using CCK-8;(3)Flow Cytometry(FCM)assayed the cell apoptosis and autophagy rate respectively;(4)The apoptosis-related protein caspase-3 and autophagy-related protein Beclin-1?LC3-II?p62 were measured by using Western blotting method;(5)The PI3K-I/Akt/m TOR signaling pathway phosphorylated proteins were also measured using Western blotting method. Part of expement in vivo(1)The human APL NB4 cells were subcutaneously inoculated in BALB/C-nu/nu mice to establish the NB4 xenograft model.(2)The mices were randomly divided into four groups : Tan IIA group,ATO group,Tan IIA combined with ATO group and NS control group,and accepted designed drugs by intraperitoneal injection;(3)Measure the maximum diameter(a)and the minor axis(b)of xenograft with vernier caliper every other day,then calculate the average tumor volume of each group and draw the growth curve of xenograft;(4)The 15 th day after the administration,the mices were killed by cervical dislocation,then xenografts were stripped and weighed,the inhibition rate(%)was compared;(5)Oberve pathologic changes of bone marrow?heart?liver?kidney and lymph nodes of mices under optical microscope after wright stain and HE stain;(6)The apoptosis-related protein caspase-3 and autophagy-related protein Beclin-1in xenografts were measured using Western blotting method.Result:Part of expement in vitro(1)Tan IIA could antiproliferate NB4 cells in a time and dose dependent manner,the NB4 cell proliferation inhibition rate of Tan IIA combined with ATO group is greater than the Tan IIA or ATO single group,which is of statistic significance(P< 0.01);(2)Under the intervention of apoptosis inhibitor z-VAD-fmk,the proliferation inhibition rate of Tan IIA monotherapy group was decreased,but still higher than the control group',the difference was of statistic significance(P < 0.05);(3)The proliferation inhibition rate of NB4 cells in Tan IIA monotherapy group increased more than 10% after treated with autophagy inhibitor 3-MA,the difference was statistically significant(P <0.05).(4)Tan IIA could induce both apoptosis and autophagic cell death of NB4 cells in a time and dose dependent manner;the apoptosis and autophagic cell death rate induced by Tan IIA combined with ATO was higher than Tan IIA or ATO monotherapy group(P < 0.01);(5)Western blotting results showed that the special apoptotic protein caspase-3 and the special autophagic protein Beclin-1?LC3-II in NB4 cells of Tan IIA group were more than control group,and Tan IIA combined with ATO group ‘s were higher than the Tan IIA or ATO monotherapy group,the difference was statistically significant(P<0.05);the expression level of autophagy-related protein P62 in each treated group was less than the control group,especially the combination group was least,the difference was statistically significant(P<0.05). Part of expement in vivo(1)Human APL cell line NB4(4?106)were subcutaneously inoculated in BALB/C-nu/nu mice,and tumor formation rate is 100%,indicating that a human APL xenograft animal model was successfully established;(2)Tumor growth rate of Tan IIA group was slightly slower than the control group,but the difference was not statistically significant(P > 0.05),tumor growth rate of combination group were not only slower than the control group,but also slower than Tan IIA group's and ATO group's,the difference was statistically significant(P <0.05).(3)The NB4 xenografts weight of Tan II group was lighter than the control group,but the difference was not statistically significant(P > 0.05);The xenografts weight of the combination group was not only less than the control group but also less than the combination group,the diference was of significance(P <0.01);The tumor inhibition rate of the combination group was 24.38%,dramaticly higher than the ATO group(15.74%)and the Tan IIA group(6.79%);(4)It was not found that there was pathologic change in heart,liver,kidney and lymph node of nude mices in the combination group;(5)Western blotting results showed that,the relative expression level of apoptotic protein caspase-3 and autophagic protein LC3-II in Tan IIA group cells were higher than the control group,that of the combination group was not only higher than the control group but also distinctly higher than Tan IIA or ATO monotherapy group,the difference was of statistical significance(P <0.05).Conclusion(1)Tan IIA had an anti-proliferation and growth inhibition effect on human APL cell line NB4 in a time and dose dependent manner;Tan IIA could synergistically enhance the anti-proliferation effect of ATO on NB4 cells,thus possessed a chemotherapy sensibility effect;(2)Tan IIA could induce NB4 cells apoptosis and autophagy in a time and dose dependent manner,this effect of combination group was greater than monotherapy group;(3)Tan IIA combined with ATO upregulated the expression level of apoptotic protein Caspase-3,thus induced NB4 cell apoptosis and exerted anti-leukemia activity;This effect of combination group was greater than ATO and Tan IIA monotherapy group,which may be one of the synergistic mechanism of chemotherapy sensitizing;(4)Tan IIA combined with ATO upregulated the expression level of autophagy-related protein Beclin-1and LC3-II,thus induced autophagic cell death in NB4 cells and exerted anti-leukemia activity;This effect of combination group was greater than ATO and Tan IIA monotherapy group,which may be another synergistic mechanism of chemotherapy sensitizing;(5)Tan IIA combined with ATO inhibited the singal pathway of PI3K-I /Akt/m TOR through decreasing the phosphorylation modification level of these proteins,and regulated the signaling pathways of Bcl-2 family protein by up-regulating the transcription level of beclin-1 m RNA and down-regulating the transcription level of bcl-2 m RNA,thus promoting the apoptotic and autophagic activity of NB4cells;this effect of Tan IIA combined with ATO group was greater than ATO and Tan IIA monotherapy group,that maybe one of the molecular regulation mechanisms of chemotherapy sensitization;(6)Tan IIA combined with ATO had synergisticly inhibiting effect on NB4 xenograft growth and this therapeutic project treated human APL xenografts safely and effectively;(7)The effect of upregulating Caspase-3 and LC3-II protein exptession level of combination group was greater than ATO and Tan IIA monotherapy group,indicating that the synergistic mechanism of chemotherapy sensitization of Tan IIA combined with ATO was also related to promoting cell death through inducing apoptosis and autophagic cell death in vivo.(8)Inducing both apoptosis and autophagic cell death and promoting cell death was the possible mechanism of chemotherapy sensitization of Tan IIA combined with ATO in vitro and in vivo.