Study Of The Effects Of Carnosic Acid,Garlic Oil On Differentiation And Apoptosis Of Leukemia Cells And Carnosic Acid On Reversing Resistance To Retinoic Acid

Background Leukemia belonged to the hematologic malignant disease. The mortality in adolescent tumors was high. With the introduction of cellular differentiation and apoptosis therapy, the complete remission rate of leukemia increased and the mortality decreased. Now, the most often used agents were all-trans retinoid acid (ATRA) and arsenic trioxide (As₂O₃),which had different effects and adverse effects and resulted in the failure of therapy in some patients. So, it was needed urgently to explore new agents to induce leukemia cells differentiation and apoptosis, increase sensitivity and reverse resistance.Part â…  Study of the effects of carnosic acid on differentiation and apoptosis of leukemia cells and reversing resistance to retinoic acidObjective First, to investigate the effects of carnosic acid (CA) itself and combined with ATRA and As₂O₃ on HL-60 cells. Second, to explore the effects of CA on the retinoic acid resistant leukemia cells.Methods The APL cell line HL-60 was chosen as model. The effects of CA and combined with ATRA or As₂O₃ were evaluated according to cellular proliferation byMTT method, cellular morphology under the microscope, cellular apoptosis and differentiation rate and changes of cell cycle distribution by flow cytometry. Meanwhile, RT-PCR was used to detect the expression of bcl-2/bax, c-myc/mad, hTERT, and mpo genes in HL-60 cells before and after treatment. Second, the ATRA resistant APL cell line MR-2 was used to evaluate the effect of reversing resistance. Cellular proliferation rate, apoptosis rate, differentiation rate and the changes of cellular morphology and cell cycle distribution were observed. Western blot and RT-PCR were used to detect the changes of STAT1 and CYP2E-1 expression.Results 5|JM CA almost had no inhibition on the HL-60 cells, but synergized with 5|JM ATRA and 5|JM AS2O3 in inhibiting cells proliferation and apoptosis.After incubated with CA for 72 hours, some cells showed differentiation to the mature granulocytes, few cells showed apoptosis, other still had no changes. While, after treated with ATRA for 72 hours, most cells differentiated to the mature granulocytes. For the cells treated with AS2O3, most cells were apoptosis. The effects of ATRA and AS2O3 were enhanced after combined with CA.After incubated with ATRA/AS2O3 for 72 hours, the cellular apoptosis rate was 30.34%/67.98%, and the differentiation rate was 76.13%/13.44%. While the cellular apoptosis rate and differentiation rate were 66.12%/97.54%, 95.53%/89.95% respectively after HL-60 cells were treated with CA combined with ATRA/AS2O3. About 60.11%/71.21% cells were blocked in GO/Gl phase after cells incubated with CA and ATRA/ AS2O3. Compared with those of CA, ATRA and AS2O3 alone, all these data had significant statistic meanings.CA, ATRA and AS2O3 all could inhibit the expression of bcl-2,c-myc,and hTERT genes, and promote the expression of bax and mad genes. After combination with CA, the effects of ATRA and AS2O3 were synergized, especially in promoting the expression of bax and mad genes.CA could reverse the ATRA resistance of MR-2 cells. The combination of 5[JM CA and lpM ATRA showed inhibition to MR-2 cells, and the inhibition rate had time-effect relationship. Most cells were induced to differentiate to granulocytes observed under microscope. The apoptosis and differentiation rates were 46.12% and87.43% respectively, which had significant meanings compared with that of ATRA alone.The combination of CA and ATRA promoted the expression of STAT1 and inhibited the expression of CYP2E1. It may partly explain the resistance reversing effect.Conclusion 1. CA itself partly induced leukemia cell differentiation. Its effect on inducing cell apoptosis was weak. It almost had no inhibition on cell proliferation.2. CA synergized the effects of ATRA and AS2O3 on promoting cell differentiation and apoptosis. It also enhanced the influence on expression of bcl-2/bax, c-myc/mad and hTERT.3. CA reversed the retinoic acid resistance of MR-2 cells. It inhibited the expression of P450, and increased the cellular concentration of ATRA. Meanwhile, it promoted the expression of STAT1,which may active the downstream genes of RARq.All in a word, C A would be introduced to clinical treatment and act as a new agent in increasing leukemia cells sensitivity to ATRA and AS2O3, and reversing the ATRA resistance.Part II Study of the effects of garlic oil on differentiation and apoptosis ofleukemia cellsObjective First, to explore the effects of garlic oil on HL-60 cells, and compare the effects with that of ATRA and AS2O3. Second, try to explain the molecular mechanism.Methods The APL cell line HL-60 was chosen as model. The effects of 12.5mg/L garlic oil, 5|JM ATRA and 5|JM AS2O3 were evaluated according to cellular proliferation by MTT method, cellular morphology under the microscope, cellular apoptosis and differentiation rate and changes of cell cycle distribution by flow cytometry. Meanwhile, RT-PCR was used to detect the expression of bcl-2/bax, c-myc/mad, hTERT, and mpo genes in HL-60 cells before and after treatment. Finally, the comparison was made among them.Results 12.5mg/L garlic oil had great inhibition on the HL-60 cells. The inhibition rate was greatest and the effective time was earliest among the three agents. The comparison was statistic meaningful.After incubated with garlic oil for 72 hours, most cells showed differentiation to the mature granulocytes, and cell apoptosis was also easy to see. The cells had classic apoptosis changes, such as nuclear shrinkage, chromosome condensation and formation of apoptosis body. While after treated with ATRA, most cells differentiate. Apoptosis was easy to see after cells were incubated with AS2O3.After incubated with garlic oil for 72 hours, the cellular apoptosis rate was 89.75%, and the differentiation rate was 94.38%. About 75.08% cells were blocked in G0/G1 phase. The ability of garlic oil to induce cell differentiation was similar as that of ATRA. While, garlic oil was capable to induce cell apoptosis and inhibit cell proliferation and stronger than ATRA and AS2O3.Garlic oil, ATRA and AS2O3 all could suppress the expression of bcl-2,c-myc,and hTERT genes, and promote the expression of bax and mad genes. Among them, the changes of cells treated with garlic oil were most significant.Conclusion 1. Garlic oil not only inhibited HL-60 cells proliferation, but also induce cells differentiation and apoptosis.2. The ability of garlic oil to induce cell differentiation was similar as that of ATRA. While, garlic oil was capable to induce cell apoptosis and inhibit cell proliferation and stronger than ATRA and AS2O3.3.Garlic oil exerted its effects through bcl-2/bax, c-myc/mad and hTERT pathway. Its influence to the expression of these genes was significant.All in all, CA would be introduced to leukemia treatment and act as a new agent in inhibiting proliferation, inducing differentiation and apoptosis, besides ATRA and As2O3.