Mechanism Of Allicin Induced Proliferation Inhibition And Apoptosis Of THP-1 Cell

Background and Objective: Acute myeloid leukemia (AML) is a heterogeneous group of diseases characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells and impaired production of normal hematopoiesis leading to neutropenia, anemia, and thrombocytopenia. If untreated, patients die of infection or bleeding usually in a matter of weeks Now for the treatment of acute myelogenous leukemia, treatment is still chemotherapy. But the chemotherapy drugs have side effects, long-term use of patients with high-dose hematopoietic system and immune system, do a lot of damage.So find side effects of small and high selectivity, is to improve the clinical efficacy of drugs. Garlic is liliaceous plant (allium sativum garlic bulbs) contained in an anti-cancer activity ingredients, chemical name for two allylic three sulfide (DADS).Research shows that allicin can inhibit breast cancer, liver cancer and stomach cancer, leukemia tumor cell growth. Anti-tumor effect mainly by inhibiting tumor cell proliferation and apoptosis-oriented, affect the cell cycle, reduce cancer predisposition.In recent years,many studies show that allicin role is to inhibit tumor cell proliferation and promote with apoptosis. At home and abroad, there is no study reported allium of THP-1 cells growth inhibition. If the allium to THP-1 cells still has anti-tumor effect,so as to further clinical for anti-tumor effect and adjuvant chemotherapy provides more powerful basis for elderly patients with leukemia, and it brings new hope with leukemia.Methods:1.Inverted microscope to observe the use of changes in cell morphology.2.The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay.3.Flow cytemetry was used to observe the effect of allicin on apoptosis and cell cycle distribution of THP-1 cells.4. After the treatment of THP-1 cells by allicin, morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope.5. Apoptosis rate was determined with Annexin V- FITC/PI double staining by flow cytometry.6. The expression and distribution change of the phosphorylation p38MAPK(P-p38MAPK) Fas/FasL proteins were detected by immunohistochemical staining.7. The changes of P-p38 MAPK and Fas/FasL proteins were detected by Western blot.Result: 1.The proliferations of leukemia cell line THP-1 are inhibited by Allicin. MTT assay showed that allicin can inhibit the proliferation of the THP-1 cell,and the inhibition was dependent on both dose and time.The IC50 of 72 hours was 12.8μg/mL. The percentage of G0/Gl phase cells was increased, but the S phase cell was decreased when treated with allicin for 72h. 72h after exposure to 6.4μg/mL, 12.8μg/mL allicin, the apoptotic rates of THP-1 cells were (22.93±2.93) % and (36.73±2.88) %, respectively. They were also dose dependence, and statistically significant when comparing with the controls (P<0.01).2. The cells could be classificated into:early apoptotic cells, late apoptotic cells, alive cells and dead cells under the fluorescent microscope.3 Annexin V-FITC / PI double staining showed that apoptosis rates were (27.8267±1.1667)% and(45.283 3±1.3372)% after treated with allicin for 72h. They were also dose dependence, and statistically significant when comparing with the controls (P<0.01).4. After the immunohistochemical staining test, the P-p38MAPK was located in the cell nucleus and plasma,showing deep brown, the expression of Fas/FasL increased in THP-1cells was induced when adding Allicin to THP-1 cell. Fas/FasL was located in the cytoplasm and cytolemma.5.Western blot test showed that the P-p38MAPK proteins and the Fas/ FasL protein expression were proportional to the concentration of Allicin and was also dose dependent. The levels of P-p38MAPK in negative control group, 1/2IC50 of 72 hours group and IC50 of 72 hours group were 0.2598±0.0132,0.3612±0.0083 and 0.5056±0.0055 respectively, the levels of Fas proteins were 0.2874±0.0089,0.4268±0.0079 and 0.5971±0.0109 and the levels of FasL proteins were 0.2124±0.0141, 0.2988±0.0280 and 0.4087±0.0266, respectively. The difference was statistically significant when compared with the negative control group (P<0.01).Conclusion:1.The proliferations of leukemia cell line THP-1 are inhibited by Allicin. Allicin can inhibit the proliferation of the THP-1 cell,and the inhibition was dependent on both dose and time.2. Allicin can affect THP-1 cell cycle distribution, inhibiting cell proliferation and promote apoptosis.3. Allicin could induce apoptosis in THP-1 cells, and its mechanism of induction of apoptosis may be achieved by activating P-p38MAPK/Fas/FasL.