Study of anti-leukemia properties of cordyceps militaris extracts on FMS-Like Tyrosine Kinase 3 (FLT3) protein overexpressing leukemic cell lines

Abstract

Leukemia is a type of blood cancer, which is characterized by the clonal proliferation of malignant leukocytes resulting in production of abnormal blood cells that arise initially in the bone marrow before disseminating to the peripheral blood, lymph nodes, and other organs. Mutations in the FMS-like tyrosine kinase 3 (FLT3) genes are one of the most frequently identified genetic alterations that affect downstream intracellular signaling pathways thereby enhancing leukemogenesis. Although chemotherapy is the most common and effective treatment, this method still have limitations due to the therapeutic resistance and relapse in leukemia patients, as a consequence, patients with FLT3 gene mutations remain an ongoing challenge. Nowadays, drugs from herbal sources were selected as alternative drugs for studying their effectiveness on leukemia treatment. Cordyceps militaris (C. militaris) is a group of mushroom in a species of fungus in the family Clavicipitaceae, and the genus Cordyceps which has been widely used as an herbal remedy or crude drug in oriental medicine and folk tonic foods due to its numerous biological activities. In this study, C. militaris cultivated in the laboratory at Chiang Mai, was tested for a candidate anti-leukemic drug. This study aims (1) to evaluate the cytotoxicity of crude ethanolic extract and fractional extraction solvents including hexane, ethyl acetate, and ethanol fractions from C. militaris; (2) to examine their effects on FLT3 protein expression, cell cycle progression, and apoptosis induction in FLT3 overexpressing leukemic cell lines, including the wild-type FLT3 overexpressing leukemic cells (EoL-1) and FLT3-ITD mutant leukemic cells (MV4-11). MTT assay was used to determine the cytotoxicity of all C. militaris extracts at various concentrations in leukemic cell lines. The results showed that all the extracts demonstrated high inhibitory effects on EoL-1 cells when compared to MV4-11 cells and control. The ethyl acetate and hexane extracts showed strong cytotoxic effects with inhibitory concentration at 50% growth (IC50) values of 11.0±2.9 and 11.7±0.6 g/mL in EoL-1 cells while, 45.9±0.4 and 36.8±15.3 g/mL in MV4-11 cells, respectively. The inhibitory effect of the extracts on FLT3 protein expression in both leukemic cell lines were assessed by Western blot analysis. To determine the effect of extract on FLT3 protein expression, leukemic cells were treated with the non-toxic doses (IC20) of the extracts at indicated time periods. The results showed that crude ethanolic extract exhibited the strongest inhibitory effect on FLT3 protein expression in EoL-1 cells, whereas ethyl acetate fractional extract showed the most effective extract in inhibiting FLT3 protein expression in MV4-11 cells by a dose- and time-dependent manner. Furthermore, the effects of C. militaris extracts on cell cycle progression and apoptosis induction in leukemic cells were investigated using PI and Annexin V-PI stainings respectively. The results showed that both crude ethanolic extract and ethyl acetate fractional extract arrested the cell cycle at S phase in EoL-1 and MV4-11 cells in dose- and time-dependent manner. However, both extracts showed the apoptosis induction in both leukemic cells with not statistically significant when compared to the vehicle control group. Commercial grade cordycepin was used as a standard control in all experiments. This present study showed that anti-leukemia activities of C. militaris extracts in EoL-1 and MV4-11 cells may be involved in the inhibition of cell proliferation by inhibiting FLT3 protein expression and inducing cell cycle arrest at S phase. These findings suggested that C. militaris extracts may develop as an alternative anti-leukemic drug for treating leukemia, especially in FLT3 overexpressing acute myeloblastic leukemia.