Synergistic effect of oridonin and a PI3K/mTOR inhibitor on the non-germinal center B cell-like subtype of diffuse large B cell lymphoma

We demonstrate the synergistic antitumor effect of oridonin and the PI3K/mTOR inhibitor NVP-BEZ235 on the non-germinal center B cell-like subtype of diffuse large B cell lymphoma (non-GCB DLBCL) both in vitro and in vivo. The underlying mechanism may be multifunctional, involving apoptosis, AKT/mTOR and NF-kB inactivation, and ROS-mediated DNA damage response. Our findings pave the way for a new potential treatment option for non-GCB DLBCL with the combination of oridonin and NVP-BEZ235. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0303-0) contains supplementary material, which is available to authorized users.

NVP-BEZ235 was purchased from Selleck (Huston, TX, USA), and both were dissolved in DMSO. Ori treatment was administered at 0.25 μM, 0.5 μM, 1 μM, 2 μM and 4 μM, and the working concentration of BEZ235 was 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. The cells were seeded in a 96-well plate at a concentration of 1×10 5 /mL. After 48 h, 10 μL Cell Counting Kit-8 reagent was added to each well and incubated for 2-3 h. The absorbance at 450 nm was measured using a microplate reader.
Growth inhibition was calculated with the formula (OD absorbance of treatment group-OD absorbance of blank)/(OD absorbance of control group-OD absorbance of blank)×100%. The synergetic effect of two drugs was measured using the combination index (CI) using CalcuSyn software (Version 2.1). CI<1 indicates the synergetic effect, CI=1 indicates an additive effect, and CI>1 suggests antagonism.

Apoptosis assays
Apoptosis was assessed using flow cytometry according to the FITC Annexin V Apoptosis Detection Kit I protocol (BD Bioscience). Cells were collected and washed in cold phosphate-buffered saline (PBS), then resuspended in Annexin-binding buffer and stained with propidium iodide (PI) and FITC Annexin V. After incubation in the dark at room temperature for 15 min, the cell suspensions were diluted in Annexinbinding buffer and analyzed using BD LSRFORTESSA flow cytometry immediately.
The data were acquired using FlowJo software (Tree Star).

Cell cycle assay
Each cell line was seeded into a 12-well plate and exposed to drugs for 48 h. Then, the cells were collected, centrifuged, washed in ice-cold PBS and resuspended with 70% ethanol. After fixation at -20°C for 12 h, the cells were resuspended in PBS containing 20 μg/mL Ki67 and 20 μg/mL Hoechest33342 30 min prior to analysis. Cell cycle data were assessed using BD LSRFORTESSA flow cytometry and analyzed with FlowJo software (Tree Star). G0%, G1% and S/G2/M% were calculated to determine the distribution of cells in these phases.

Flow cytometry assays to detect reactive oxygen species (ROS)
Measurements of ROS production were performed using ROS Detection Reagents for 32 days and were calculated as V=a×b 2 /2 (a, the length of tumor; b, the width of tumor). At the end of the observation period, the mice were sacrificed, and the tumors were harvested. Then, the samples were fixed in 4% formalin and processed for frozen sectioning. The animal protocol was approved by the Animal Care and Use Committee of Ruijin Hospital affiliated to the School of Medicine, Shanghai Jiao Tong University.

Hematoxylin and eosin staining
Tumor tissues were immersed in 4% paraformaldehyde for 4 h, then transferred to 70% ethanol. Individual lobes from tumor tissue biopsies were placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in paraffin wax blocks.
Before immunostaining, 5-μm-thick tumor tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. Next, the samples were stained with hematoxylin and eosin (H&E). After staining, the sections were dehydrated through increasing concentrations of ethanol and xylene. Finally, one or two drops of mounting medium was added, and a coverslip was placed on top.

Terminal deoxytransferase-catalyzed DNA nick-end labeling (Tunel) assay
Frozen sections were stained with a terminal transferase recombinant kit (Roche, Indianapolis, IN, USA) using the manufacturer's protocol. Cells with green fluorescence were classified as apoptotic. Image-Pro plus 6.0 (Media Cybernetics, USA) was used to identify the positive cells on each slide.

Statistical analysis
All of the data in this study were analyzed with SPSS 17.0 software (Chicago, USA) and presented as the mean ± standard deviation. Independent t-tests were used to identify significant differences between groups. IC50 values were analyzed using a probit assay in SPSS. P<0.05 was considered statistically significant. Overall survival was measured with the Kaplan-Meier method. The drug interactions were stated according to the Chou-Talalay method and isobologram analysis.