The Study Of Apoptotic HL-60 Cells Induced By As₂O₃ In Combination With TPA

Background and ObjectLeukemia is caused by the maglignant transformation of the hemopoietic stem cells. It is one of the most common cancer and its morbidity is increased in recent years.The stagging of the differentiation and the blocking of apoptosis of leukemic cells are the two main machanism of the leukemia development. So the drugs with differentiation induction or apoptosis promotion of leukemic cells are the powerful means in the treatment of leukemia.TEA (12-O- tetradecanoylphorbol-13-acetate) is a powerful tumor promoter, at the same time it's one of the most powerful inducers of differentiation. TPA can induce differentiative effects not only on many human and mouse leukemic cell lines, but also on human primary leukemiccells at low concentration (10"6mol/L~10"8mol/L) in vitro. In addition it has been demonstrated that the effect of the combination of TPA and RA on human primary acute leukemic cells was increased obviously. As2O3 is a deadly poison. And some enzymes including -SH will be inactive when combining with it. As2O3 could destroy the cell normal function, metabolism and the synthesis or reparation of DNA. It even can induce the cell to apoptosis. We have applied As2O3 to therapy for many years. In the 70s' of the 20th century, the doctors in China began to use As2O3 to treat APL, and obtained satisfied results. However more and more problems were found in the clinical application at the same time. So the way about how to improve the curative effect of As2O3 has been given more and more attention.Methods and SubjectsCell culture and grouping:The human leukemic cell lines HL-60 were grown in suspension in RPMI1640 medium in an atmosphere of 5% CO2 at 37°C.We collected the cells by centrifuge. Than the cell concentrations were modulated to 5-10 X105 cells per ml in RPMI-1640. Every subject was matched 8 groups (9ml every group): the control group (adding the solvent without TPA), TPA group (adding TPA only), As2O3 groups (adding As2O3 only), TPA+ As2O3 groups (adding both TPA and As2O3). The terminal concentration of TPA was 1.6X10"8mol/L, and the terminal concentrationof As2O3 was separately 0.2X 106mol/L, 2.0X 10'6mol/L, 20X 10"6mol/L in As2O3 groups and TPA+As2O3 groups. Then the cells of each group were plated in one 6- well culture plate and cultured in CO2 incubator, 3 wells for each group.The investigation index:(1) The growth states of M2 leukemic cells HL-60 cultured in vitro were observed by the invent microscope after cultured for 24, 48, 72 hours. (2) The count of live cell by trypan blue exclusion. (3) Estimating the cell growth by MTT method. After 72 hours'culture, each well was added 20 u 1 MTT and cultured 4 hours in CO2 incubator. Then detected OD values of each well by spectrophotometer. (4) Flow cytometer( FCM ) was used to detect the expression of surface antigen: CD33,CD64. (5) DNA ladder, the effect was observed by agarose gel electrophoresis. (6) The apoptosis was investigated by the TUNEL method.Results1. The morphology observation: After 24 hours culture, the cells in TPA group and TPA+As2O3 groups became adherent to the wallof the plates. The adherent cells took on shapes of shuttle, astroid, racket, and usually had long pseudopodia or filar stick-ups. After added As2O3, the adherent cells in TPA+ As2O3 groups decreased.2. The count of the live cells in TPA group grew slowly after 24 hours' culture. After 72 hours' culture, it was also found in TPA+As2O3 groupsand 20 X 10~6mol/LAs2O3 group .3. By the MTT assay, we found that: The cell growth and proliferation of all the experimental groups except the As2O3 groups ( 0.2X10"6mol/L, 2.0 X 10"6mol/L ) were inhibited in different extents. The inhibitions were significant differences in contrast with the control group (P<0.05), and the inhibition rate of TPA+As2O3 groups was higher than that of other experimental groups (P<0.05).4. The FCM results demonstrated that: the expression of CD64 in TPA group and TPA+ As2O3 groups increased, at the same time CD33 decreased and there were significant differences in contrast with the control group (P<0.05); CD33 positive cells in As2O3 group ( 20 X 10~6mol/L ) decreased significantly in contrast with the control group (P<0.05) ,but the CD64 didn't increase.5. DNA ladder could be found in As2O3 group (20 X 10"6mol/L ), TPA+ As2O3( 2.0X10'6mol/L, 20X10"6mol/L ) groups, but not in other groups.6. The results of TUNEL showed: the apoptotic rate in As2O3 groups ( 2.0 X 10"6mol/L, 20X10"6mol/L ), TPA+As2O3( 2.0 X 10"6mol/L, 20 X 10"6mol/L) groups had significant differences in contrast with the control group (P<0.05); and the rate of apoptotic cell of TPA group and As2O3 groups was lower than the counterpart TPA+As2O3 groups (P<0.05). There was no difference between other experimental groups and the controlgroup.Conclusion1. TPA and high concentration of As2O3(20X 10'6mol/L) could inhibit the growth and proliferation of human leukemic cells HL-60.2. TPA could induce human leukemic cells HL-60 to differentiate into mononuclear macrophage-like cells, and high concentration of AS2O3 ( 20 X 10"6mol/L ) could induce the cells to apoptosis. TPA combined with AS2O3 could enhance this effect of inducing cells to apoptosis.3. the combination of TPA and As2O3 may be a new powerful means to treat the refractory/relapse leukemia.