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.

Findings

Diffuse large B cell lymphoma (DLBCL) is the most common aggressive form of non-Hodgkin’s lymphoma (NHL) in adults, and it can be distinguished into two major groups, the germinal center B cell (GCB) subtype and the non-germinal center B cell-like (non-GCB) subtype [1, 2]. The non-germinal center B cell-like subtype of diffuse large B cell lymphoma (non-GCB DLBCL) presents aggressive clinical courses and poor prognosis [3, 4]. Targeting key pathways may raise the possibility of improving clinical outcomes.

Our previous studies have indicated that oridonin and NVP-BEZ235 have some antitumor effects in DLBCL cells [5, 6]. The aim of this study was to determine whether oridonin combined with NVP-BEZ235 could achieve a more significant antitumor effect on the non-GCB DLBCL, and to further investigate the underlying mechanism. The materials and methods used in this study are detailed in Additional file 1.

Our results demonstrate that oridonin and NVP-BEZ235 exhibit a synergistic effect on non-GCB DLBCL cell lines (OCI-Ly3 and SU-DHL-2), and the co-treatment was more effective on cell proliferation inhibition compared with single-agent therapy (Additional file 2). And then cytotoxic effect of oridonin and NVP-BEZ235 alone or in combination were evaluated in nude mice bearing SU-DHL-2 tumors. Compared with the control group or single-agent group, the co-treatment group exhibited more significant DLBCL cell growth inhibition in terms of tumor size (Fig. 1a) and weight (Fig. 1d) and prolonged the mice survival (Fig. 1b). H&E staining and TUNEL assay showed that co-treatment with oridonin and NVP-BEZ235 obviously increased apoptosis (Fig. 1c, e).

Fig. 1

Oridonin combined with NVP-BEZ235 dramatically inhibited tumor growth and prolonged the survival in a non-GCB DLBCL xenograft mouse model (SU-DHL-2). a, d Mice in each cohort were treated with oridonin (5 mg/kg) and NVP-BEZ235 (20 mg/kg) alone or in combination every other day. Tumor volumes were measured once every 4 days. After 32 days, the mice were sacrificed, and the tumors were removed and weighed. b Overall survival was prolonged by oridonin and NVP-BEZ235 combination therapy compared with the control group and single-agent group (p < 0.005). c HE staining and TUNEL assay was performed to examine the apoptosis in tumor tissues. e Bar graph illustrate the proportion of positive cells showed in TUNEL assay. *p < 0.05, **p <0.01, ***p < 0.001 compared with the control group; # p < 0. 05, ## p < 0.01 compared with single-agent group

To explore the mechanism underlying the synergistic antitumor effect, both cell lines were exposed to 2 μM oridonin and 25 nM NVP-BEZ235 alone or in combination for 24 and 48 h. The results showed that the co-treatment induced higher apoptosis in non-GCB DLBCL cell lines (Fig. 2a, b). Meanwhile, the apoptosis induced by the drug combination was further confirmed by assessment of caspase family and Bcl-2 family protein expression (Additional file 3). However, co-treatment does not further enhance cell-cycle arrest in G0/G1 phase (Additional file 4).

Fig. 2

The mechanism of synergistic antitumor effect of oridonin and NVP-BEZ235 on non-GCB DLBCL. a, b Cell lines were treated with oridonin (2 μM) and NVP-BEZ235 (25 nM) alone or in combination for 24 and 48 h, analyzing apoptosis by Annexin-V/PI staining. c Cell lines were treated with oridonin (2 μM) and NVP-BEZ235 (25 nM) alone or in combination for 48 h. Western blot analysis was performed to identify the expression of total AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, and p-mTOR. d Western blot for NF-kB, p-NF-kB, IkBα, and p-IkBα. e The expression of γH2AX and H2AX was analyzed by western blotting. f Cell lines were simultaneously treated with oridonin (2 μM) and NVP-BEZ235 (25 nM) for 48 h, and FACS quantitative analysis of DCF-DA was used to detect the expression of ROS. g Pretreatment of co-treatment group cells with NAC (5 mM) and Z-DEVD-FMK (10 μM), respectively, for 48 h, analyzing apoptosis by Annexin-V/PI staining with t test statistic assay. (Mean ± SD, n = 3, *p < 0.05, **p < 0.01 compared with Ori + BEZ group. Z: Z-DEVD-FMK)

To further investigate the mechanism of synergistic drug effects, the expression of AKT/mTOR and NF-kB pathway was assessed by western blotting. The data suggest that the simultaneous inhibition of the PI3K/AKT/mTOR and NF-kB pathways abrogated the key survival signals for non-GCB DLBCL, which might indicate the mechanism for the synergistic pro-apoptotic effect of oridonin and NVP-BEZ235 in combination (Fig. 2c, d). We then found that oridonin and NVP-BEZ235 in combination markedly induced the expression of γH2AX, a marker of DNA damage. Flow cytometry also demonstrated a significant increase in reactive oxygen species (ROS) (Fig. 2e, f). Addition of N-acetyl-l-cysteine (NAC) largely reversed co-treatment-induced apoptosis, while treating the cells with Z-DEVD-FMK (caspase 3 inhibitor) had little effect (Fig. 2g, Additional file 5).

Taken together, our findings demonstrated the synergistic antitumor effect of oridonin combined with NVP-BEZ235 in non-GCB DLBCL cell lines. The potential molecular mechanism might be multifunctional, involving apoptosis, AKT/mTOR and NF-kB inactivation, and ROS-mediated DNA damage response. Moreover, co-treatment was also effective in a non-GCB DLBCL xenograft model. Therefore, our study provides a theoretical basis and preclinical evidence for this novel strategy and suggests that the combination of oridonin and NVP-BEZ235 might have promising therapeutic application in non-GCB DLBCL patients.