The Apoptosis-Inducing Effect Of Gambogic Acid On Malignant Hematologic Cell Lines And Its Mechanism

[Objective]To investigate the apoptosis-inducing effect of Gambogic acid (GA) on malignant hematologic cell lines and its mechanisms, and the function-inhibiting effect of GA on normal human immunocytes and hematopoietic stem cells (HSCs).[Methods] The Jurkat,Raji,RPMI8226,U266,HL-60,K562,K562/A02 cell lines were treated with GA at different concentrations for 24h-72h, the inhibition rates were detected by MTT assay. Cell morphology and apoptosis were measured by Annexin V/PI staining assay. Mitochondrial membrane potential (ΔΨm) was measured by JC assay. Activated Caspase 3,Caspase 8 and Caspase 9 level in above living cells were measured by Caspase-GloTM fluorescein staining kit. The GA effect on lymphocyte proliferation function was measured by MTT assay when lymphocytes were induced by PHA or LPS. Phagocytotic function of normal monocytes was measured by the intake dose of Neutral Red Dye. CFU-GM was assessed by semi-solid culture assay for determination of colony formation capacity of HSCs.[Results]1. Jurkat,Raji,RPMI8226,U266,HL-60,K562,562/A02 cells'growth were inhibited in a concentration dependent manner. K562/A02 cells needed higher GA concentration (>2μg/ml) to show antiproliferative effect compared with k562 cells (>0.5μg/ml). The IC50 of GA on K562 was 2.23μg/ml, Rajil.86μg/ml, Jurkatl.35μg/ml, HL-60 0.68μg/ml, RPMI8226 0.52μg/ml, U266 0.44μg/ml respectively。GA did not significantly influence the cell cycle of K562,urkat,Raji cell lines at the concentration of 0,0.5,1.0,2.0,4.0μg/ml for 24h-72h.2. The typical apoptosis morphology change could be visualized after Jurkat,Raji,RPMI8226. U266. HL-60,K562 cell lines were incubated with GA. GA decreased the Mitochondrial membrane potential(ΔΨm) of all above cell lines in a concentration dependent manner. GA increased the apoptosis rate of all above cell lines and increased activated caspase 3, caspase 8, caspase 9 positive cell number for different degree in 24h and 48h.3. After treatment with GA, the BCR-ABL protein level in K562 cells was decreased by Cytometric beads array.4. GA could inhibit the proliferation of normal lymphocytes treated with PHA or LPS in concentration dependent manner. GA had negative effect on monocytes to ingest Neutral Red Dye, GA could inhibit the CFU-GM colony formation capacity of normal bone marrow/peripheral HSCs but at low concentration (<1μg/ml) the effect on that of normal bone marrow HSCs was not significant.[Conclusion]1. GA has inhibitive effect on various malignant hematologic cell lines'growth in vitro. The cell cycles are not disturbed obviously in some malignant hematologic cell lines.2. GA induces apoptosis on some lymphoma, leukemia and multiple myeloma cell lines through both intrinsic and extrinsic pathways.3. GA has negative effect on the proliferation of immunocytes and HSCs in vitro. The cytotoxic effect on the function of normal bone marrow HSCs is not found at low GA concentration.