Culture Supernatants Of Toxoplasma Gondii 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. The treatment of acute myelogenous leukemia is still chemotherapy. But the chemotherapy drugs have side effects, long-term use of patients with high-dose hematopoietic system and immune system, and a lot of damage to human body. It is very necessary to find medicines with less side effects, high selectivity and clinical efficacy. Researches showed that culture supernatants of Toxoplasma gondii could inhibit breast cancer, liver cancer and stomach cancer, leukemia tumor cell growth. This study is to focus on the anti-tumor effect of the culture supernatants of T.gondii mainly by inhibiting tumor cell proliferation and apoptosis-oriented, affect the cell cycle, reduce cancer predisposition. Methods:1. Inverted microscope to observe the use of changes in cell morphology. The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay.2. Flow cytemetry was used to observe the effect of culture supernatants of T. gondii on apoptosis and cell cycle distribution of THP-1 cells;after the treatment of THP-1 cells with culture supernatants of T. gondii, morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined with Annexin V- FITC/PI double staining by flow cytometry.3. The expression and distribution change of the phosphorylation P-p38MAPK (P-p38MAPK) Fas/FasL proteins were detected by immunohistochemical staining. The changes of P-p38 MAPK and Fas/FasL proteins were detected by Western blot.Results:1. The proliferations of leukemia cell line THP-1 were inhibited by culture supernatants of T. gondii. MTT assay showed that culture supernatants of T. gondii inhibited the proliferation of the THP-1 cell, and the inhibition was dependent on both dose and time.The IC50 of 72 hours was 6.4×107/ml. The percentage of G0/Gl phase cells was increased, but the S phase cell was decreased when treated with the culture supernatants of T. gondii for 72 hours. At time point of 72 hours after exposure to 3.2×107/ml,6.4×107/ml culture supernatants of T. gondii, the apoptotic rate of THP-1 cells was (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). The cells could be classificated into early apoptotic cells, late apoptotic cells, alive cells and dead cells under the fluorescent microscope.2. Annexin V-FITC / PI double staining showed that apoptosis rates were (27.8267±1.1667)% and(45.283 3±1.3372)% after treated with culture supernatants of T. gondii for 72 hours. They were also dose dependence, and statistically significant when comparing with the controls (p<0.01).3. The immunohistochemical staining test showed that, 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 culture supernatants of T. gondii to THP-1 cell. Fas/FasL was located in the cytoplasm and cytolemma. Western blot test showed that the P-p38MAPK proteins and the Fas/FasL protein expression were proportional to the concentration of culture supernatants of T. gondii 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).Conclusions:1. The proliferations of leukemia cell line THP-1 are inhibited by culture supernatants of T. gondii, and the inhibition was dependent on both dose and time.2. Culture supernatants of T. gondii can affect THP-1 cell cycle distribution, inhibiting cell proliferation and promote apoptosis.3. Culture supernatants of T. gondii could induce apoptosis in THP-1 cells, and its mechanism of induction of apoptosis may be achieved by activating P-p38MAPK/Fas/FasL.