Experimental Effect Of Phorbol Ester On Leukemia DA Chemotherapy Regimen In Vitro And In Vivo

Acute myeloid leukemia?AML?is a hematological malignancy in which myeloid hematopoietic stem/progenitor cells proliferate abnormally.As the incidence of AML has increased by 20% over the past two decades,it has become a serious health problem and needs rigorous monitor and innovative treatment strategies.In the past 40 years,the basic chemotherapy regimen for the treatment of acute leukemia is still DA?Daunorubicin,Ara-C,DA??3+7?regimen-3 days intravenous infusion of daunorubicin combined with 7 days intravenous infusion of cytarabine,the complete response rate of this therapeutic regimen for young patients is 60-80%,older patients is 40-60%.Only 20-30% of patients can survive without disease for a long time,Higher dose of the DA chemotherapy regimen get higer remission rate but with the greater side effects of cardiotoxicity and myelosuppression.Therefore,it is important to find drugs which increase the efficacy of DA and reduce its adverse reactions.12-O-tetradecanoylphorbol-13-acetate?TPA?is a compound extracted from croton oil which is similar to the second messenger diacylglycerol.It can activate protein kinase C,stimulate the cascade of downstream signaling pathways,affect cell proliferation,differentiation,migration,invasion and so on.Many researches find that it can induce tumor cell differentiation,especially myeloid leukemia cell differentiation,and promote the differentiation and apoptosis of human primary leukemia cells and leukemia cell lines such as THP-1,K562,HL-60,U937 and MEG-01 at lower concentrations.And obtain the marker and functions of mature cells.TPA can enhance the activity of chemotherapy drugs and reduce toxicity,suggesting that it has potential therapeutic value,but in vivo studies have rarely been reported.TPA also regulates the expression of cytokines and the response of cells to cytokines and other ligands.Pharmacokinetics and biological factors of the body may also result in differences in the effects of TPA in vivo and in vitro.Therefore,we hopes that on the basis of in vitro studies,TPA still has a therapeutic effect in leukemia animal models and enhance the effect of DA chemotherapy regimen.Methods1 In vitro synergistic effect of TPA on DA chemotherapy regimen1.1 Cell cultureHuman leukemia cell line THP-1 was cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibiotic in a constant temperature sterile incubator at 5% CO₂ saturated humidity,and subcultured once every other day;WEHI-3 was cultured in a DMEM high glucose medium containing 10% fetal bovine serum and 1% double antibiotic in a constant temperature sterile incubator at 5% CO₂ saturated humidity,and subcultured every other day.1.2 MTT assay for cell proliferationMTT assay was used to detect proliferation effect of TPA combined with DA chemotherapy on THP-1 and WEHI-3 cell,and Prism drew dose-response curve,and SPSS anylased the data.2 Effect of TPA combined with DA chemotherapy on leukemia model mice2.1 Effect of TPA combined with DA chemotherapy on mortality and survival time in leukemia miceThe mice were divided into eight groups,normal group,model group,TPA low and high dose group,DA low and high dose group,DA low dose and TPA combined group?100?g/kg?,DA high dose and TPA combined group?100?g/kg?.All mice of group were intravenously injected with 1×10⁶ WEHI-3 cells except for normal group,Ten days after WEHI-3 cells injection,mice of TPA low and high dose group respectively intravenously were injected with TPA 50?g/kg and 100?g/kg for three days.Mice of DA high-dose group intravenously injected with daunorubicin 5mg/kg and cytarabine 75mg/kg for three consecutive days,mice of DA low-dose group intravenously injected with daunorubicin 3 mg/kg and cytarabine 50 mg/kg for three consecutive days.One week later,mice of low and high dose of DA and TPA combined group were intravenously injected with TPA 100 ?g/kg for three consecutive days,the mortality and survival time of each mice of group were observed,and the body weight of the animals was recorded and the state of the mice was observed.2.2 Effect of TPA combined with DA chemotherapy on the proportion of primitive and immature cells in leukemia miceMice were divided into 5 groups: normal group,model group,TPA group,DA group,TPA combined with DA group.Except for the normal group,mice of the other groups were intravenously injected with 1×10⁵ cells.25 days after WEHI-3 cells injection,mice of the TPA group intravenously injected with TPA 100 ?g/kg for five consecutive days.Mice of the DA group intravenously were injected with daunorubicin 4 mg/kg and cytarabine 50 mg/kg for three consecutive days.Mice of DA combined with TPA group was injected with the same dose of daunorubicin and cytarabine as the DA group,followed by intravenous injection of TPA 100 ?g/kg for five consecutive days.On the thirty-seventh day,all mice were sacrificed,the femur was washed with phosphate buffer,centrifuged,the supernatant was discarded,smears were stained with Wright's Giemsa stain,and the ratio of primitive and immature cells were counted under a microscope for three times.Three fields of the microscope were randomly selected,and the count was repeated three times.2.3 Effect of TPA on myelosuppression of DA chemotherapy regimenAnimal grouping and administration were the same as 2.2.After the 25 th day of injection of WEHI-3 cells,blood was collected from the eyelids of the mice,and the number of peripheral blood cells was detected.Mice of the DA group,DA+TPA group and normal group were detected after the completion of the DA protocol.Blood was collected from the eye of the mouse and bone marrow suppression was detected.After the administration of TPA finished,blood was also collected to detect whether TPA has an effect of raising white blood cells.Results1 Synergistic effect of TPA on DA chemotherapy regimen in vitro1.1 Effects of TPA and DA on the proliferation of THP-1 and WEHI-3 cellsTPA inhibited the proliferation of THP-1 cells and WEHI-3 cells at a lower concentration.DA inhibited proliferation of THP-1 cells with the DNR/Ara-C IC₅₀ is 15.02/75.1 nmol/L.And DA inhibited proliferation of WEHI-3 cells with DNR/Ara-C IC₅₀ 2.25/11.27 nmol/L.Both TPA and DA could inhibit the proliferation of THP-1 and WEHI-3 cells.1.2 The effect of TPA combined with different concentrations of DA chemotherapy regimen on the proliferation of THP-1 and WEHI-3 cellsTPA?1.62 nmol/L?showed no significant increase in the inhibition of THP-1 by the DA regimen within the study concentration range?13.80 nmol/L-55.20 nmol/L?.TPA?1.62 nmol/L?significantly increased the proliferation inhibition of WEHI-3 by the DA regimen?2.75 nmol/L-11.04 nmol/L?within the study range.2 Effect of TPA combined with DA chemotherapy on leukemia model mice2.1 Establishment of a mouse model of leukemiaAfter the mice were injected with 1×10⁶ WEHI-3 leukemia cells,the mental state of the mice began to deteriorate on 10 th day.The mice in the late stage of the disease had lower limb paralysis,hepatosplenomegaly,and peripheral blood leukocyte?45.91±24.00?×10⁹/L was significantly higher than the pre-injected leukocyte count?7.02±0.83?×10⁹/L?P<0.05?,and the peripheral blood smear could find immature cells.The proportion of primitive cell in the bone marrow was more than 20%,indicating that a mouse model of leukemia can be established after injection of 1×10⁶ WEHI-3 cells.2.2 Effect of TPA combined with DA chemotherapy on mortality and survival time in leukemia miceAfter injection of WEHI-3 cells in mice,the mortality of the model group was 90%,and the mortality was significantly higher than that of the normal group?P<0.001?.Mortality was 90% in the high-dose TPA group and 90% in the low-dose group.Mortality was 100% in the low-dose DA group and 50% in the low-dose DA-TPA group.The mortality of low-dose DA-TPA group was significantly lower compared with the model group?P<0.05?,and was significantly lower compared with the low-dose DA group?P<0.05?.Mortality was 60% in the high-dose DA group and 20% in the high-dose DA-TPA group.The mortality of DA-TPA group was significantly lower compared with the model group?P<0.05?,and was significantly lower compared with the high-dose DA group?P<0.01?.On the deadline of day 41,the median survival time of the model group was 25.5 days,the median survival time of the TPA high and low dose group was 27.5 days,and the median survival time of the low dose DA group was 37.5 days,and was significantly higher than that of model group?P<0.01?,low-dose DA-TPA group died less than 50%?no median survival?,and the median survival of low-dose DA-TPA group was significantly higher than that of model group?P<0.0001?,The median survival of the low-dose DA-TPA group was significantly higher than that of the lowdose DA group?37.5 days??P<0.01?.The high-dose DA group and the high-dose DA-TPA group died less than 50%.The median survival time of the high-dose DA group was significantly higher than that of the model group?P<0.0001?.Compared with the high-dose DA group,median survival of high-dose DA-TPA group was significantly prolonged?P<0.05?.2.3 The effect of TPA combined with DA chemotherapy on the proportion of primitive and immature cells in leukemia miceCompared with the normal group?5.58±0.89?%,the proportion of primitive and immature cells in the model group?43.70±24.08?% was significantly higher?P<0.05?.After chemotherapy,the bone marrow of the mice was relieved.Compared with model group?43.70±24.08?%,The primitive and immature cells of DA combined with TPA group?6.82±3.13?% were significantly lower?P<0.05?.2.4 Effect of TPA combined with DA chemotherapy on liver and spleen pathology in leukemia miceA large amount of leukemia infiltrated in the spleen and liver of the model group,the spleen was disorderly arranged,and the cells were partially necrotic.The structure of the liver lobule is basically normal,the central vein is congested,and the hepatocytes are swollen with some necrosis.The other groups of mice had no obvious leukemia cell infiltration in the liver and spleen,and the spleen white pulp proliferated in the TPA group.2.5 Effect of TPA combined with DA chemotherapy on organ index in leukemia miceThere was no significant difference in the spleen index?1.19±1.11?between the model group and the normal control group?0.39±0.02?.There were no statistical differences between the other groups including liver,heart and lung index.2.6 Effect of TPA on myelosuppression of DA chemotherapy regimenOn the second day after chemotherapy,the white blood cell count?3.73±1.37?×10⁹/L in the DA group and the white blood cell count?3.69±1.43?×10⁹/L in the combined group all decreased,but there was no statistical difference.White blood cell count?12.73±4.56?×10⁹/L after TPA administration in the combination group was significantly higher than that in the DA group?5.54±2.12?×10⁹/L?P<0.05?,indicating that TPA may accelerate leukocyte recovery and reduce myelosuppression by DA.2.7 Effect of TPA and DA chemotherapy on body weight of leukemia miceOn the 24 th day after injection of WEHI-3 cells,the body weight of the model group?19.81±2.65?g was statistically different from the normal control group?23.41±1.57?g?P<0.05?.There was no significant difference between the weight of the TPA low dose group?21.13±0.55?g and the TPA high dose group weight?20.84±2.03?g compared with the weight of model group.And TPA had no significant effect on the body weight of the DA chemotherapy regimen.Conclusions1 TPA synergizes with DA to inhibit proliferation of WEHI-3 cells.2 TPA can enhance the therapeutic effect of DA chemotherapy in leukemia animals.