Itraconazole Combined with Daunorubicin Synergistically Inhibit Proliferation and Enhance Apoptosis on KG-1a Cell Line through the Suppression of Hedgehog Pathway
Authors: Zhou Ruiqing , Xu Xiaojun , Wang Jing , He Huiqing , Guo Ziwen , Li Weichao , Huang Guinian , Ren Zhijuan , Liu Qifa 
Published: October 2019

Abstract
Background:Itraconazole is widely used as antifungal drug. It was recently found to be a potent antagonist of the Hedgehog (Hh) signaling pathway. Due to its inhibition of Hedgehog singling, it may prove beneficial in the treatment of hematological malignancies when combined with chemotherapy.
Objective To explore the effect of single itraconazole and in combination with daunorubicin on the growth, apoptosis and expression of shh and Gli1 of acute myeloid leukemia cell line KG1α.
Methods Different concentrations of itraconazole (ITC), daunorubicin (DNR) and IC30 concentration of daunorubicin (0.73 μΜ) in combination with different concentrations of itraconazole (2, 6,15μΜ) were added to acute myeloid leukemia cell line KG1α. Cell proliferation activity were detected with CCK8 method after cultivating for 48h. Cell apoptosis rate, mitochondria injury and cell cycle arrest condition were detected 48 hours after the action with single drug or drug combination with flow cytometry. Clone forming experiment was performed to detect the clone forming ability of KG 1α continuously treated with the drug. 46 kinds of apoptosis related protein were detected by protein array. Expression of shh and Gli1 were detected by Western Blot.
Results: The detected KG1α cell proliferation inhibition rate increased with the increase of the concentration of daunorubicin and itraconazole. The combination of DNR 0.73μΜ and ITC 6μΜ showed synergistic effect; The apoptosis rate in two drug combination group was of statistical difference compared with that of ADM 0.73μM group and ITC 6μM group (P<0.01). Mitochondria injury showed that the P2/P3 ratio in single ADM and ITC group was significantly lower than that of the combination group (P<0.05). DNR arrested the cells in G2 phase, while ITC arrested cells in G1 phas; the two drugs combination significantly arrested cells in G2 phase, with statistical difference. Protein array showed significantly decrease of apoptosis inhibit factor Xiap and increase of apoptosis induce factor Smac in the combination group. ITC inhibited cell’s clone forming ability, and down-regulated the expression of shh and Gli1.
Conclusion: Itraconazole or in combination with daunorubicin may inhibit KG1α cell proliferation, induce mitochondria injury and apoptosis, which might be caused by the suppression of Hedgehog signal pathway related protein.

Disclosures
Liu: It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017);: Research Funding; It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1);: Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027).: Research Funding.