Oridonin, An Ent-Kaurane Diterpenoid, Shows Antileukemic Effect and Enhances the Effect of Indarubicin on Human T-Cell Leukemia Cell Line
Authors: Wu Kangni , Zhao Yanmin , Xiao Haowen , Zhang Lifei , Liu Lizhen , Wang Yingjia , Yu Xiaohong , Huang He 
Published: October 2019

Abstract
Abstract 4933

Introduction
Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in young children, and also affects adults, especially those aged 65 years or older. Although the outcome of children with ALL has been improved dramatically with current chemotherapy strategy, adult patients still have a worse outcome and the incidence of relapse remains high either in children or adult patients. Furthermore, there has been very little progress in improving the outcome of patients who experience relapse. Oridonin, an ent-kaurane diterpenoid derived from the herbal Rabdosia rubescens, has been proved to have significant antitumor effect on a large variety of cancer cells. In this study, we aimed to assess whether oridonin had antileukemic effection and the possibility of combination effect with indarubicin (IDA), an important component commonly used in T-ALL induction regimen.

Methods
Human T-cell leukemia cell line Jurkat cells in culture medium were treated with different concentrations of oridonin (1umol/L~ 20umol/L) for 24h and 48h. Cell Counting Kit-8 (CCK8) assay was used to detect the cell growth inhibitory rate. Apoptosis was measured with flow cytometric (FCM) by staining cells with Annexin V-fluorescein-isothiocyanate(FITC) and propidium iodide (PI). Hoechst 33258 staining was used to observe the apoptotic morphology. The expression of caspase-3, caspase-8 and other apoptosis modulators, including Bcl-2 family members, was analyzed by western blotting. When the combination effect of oridonin and indarubicin was investigated, oridonin was given one hour before indarubicin. The calculation of combined effect was used Isobologram Analysis.

Results
CCK8 assay revealed that oridonin could significantly inhibit the growth of Jurkat cells in vitro. The suppression was both time- and dose-dependent. The concentration of oridonin lower than 5 umol/L showed little inhibitory effect, but it presented significant inhibition of the proliferation at a higher concentration, especially when the concentration was higher than 10 umol/L. The 50% inhibitory concentration (IC50) values for 24h and 48h were 9.44 and 9.06 umol/L respectively. After Jurkat cells were treated with higher than 5 umol/L of oridonin for 24–48h., the percentage of Annexin VFITC positive cells was gradually increased in a time and dose-dependent manner and the percentage of Annexin VFITC positive cells could reach 40% to 50% after 10 umol/L of oridonin treatment for 48h. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed using Hoechst 33258 stain after 48h. Western blotting analysis showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 20-kDa subunit when apoptosis occurred; Expression of Bcl-2 was downregulated significantly after the cells were treated with oridonin for 24 h. The expression of caspase-8 remained constant before and after apoptosis occurred. We therefore concluded that oridonin has significant antiproliferation effects on Jurkat cells by activation of caspase-3 as well as down-regulation of anti-apoptotic protein Bcl-2. The 24h IC50 of IDA for Jurkat cells was 25.85nmol/L. When combined with 1umol/L of oridonin, the IC50 of IDA decreased to 4.93nmol/L. The combine index was < 1, indicating the synergistic antitumor effect of IDA and oridonin. The data from FCM showed that 2.5 umol/L of oridonin could augment the apoptosis effect of 2.5nmol/L of IDA from 5.1% to 12.7%. During the experiment, we found that oridonin given one hour before IDA could make a better combination effect than given simultaneously. We also discovered that low dose oridonin (lower than 5 umol/L) had little inhibitory effect on normal bone marrow mononuclear cells.

Conclusions
The results showed oridonin could effectively induce ALL cells apoptosis, and in low dose showed a significant synergistic effect with IDA, indicating oridonin may serve as a potential and secure antileukemia reagent for ALL, especially in the combination chemotherapy. The underlying machanisms of the combination effect with anthracycline should be explored.

Disclosures:
No relevant conflicts of interest to declare.