Low dose of lenalidmide and PI3K/mTOR inhibitor trigger synergistic cytoxicity in activated B cell-like subtype of diffuse large B cell lymphoma

Abstract

Background: Activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL) presents aggressive clinical courses and poor prognosis. Targeting key pathways may raise the possibility of improving clinical outcomes.

Methods: The synergetic effects were assessed by CCK-8 assay and measured by isobologram analysis. The NVP-Bez235 and lenalidomide cytotoxicity were measured by flow cytometry, Western Blot and si-RNA transfection. The combined treatment inducing tumor regression in vivo was performed in nude mice of OCI-Ly10 xenograft mouse model.

Results: Low dose of two agents represented significant inhibition of proliferation with CI value < 1. NVP-Bez235 combined with lenalidomide remarkably increased apoptosis through intrinsic pathway by upregulating Bim, Bax and downregulating Bcl-xL. Akt, especially NF-κB, played an important role in the synergetic effects. Cotreatment also induced the cell cycle to be arrested in G0/G1 phase, and decreased S phase by increasing p21 expression, downregulating cyclinA and diminishing CDK2 phosphorylation in Su-DHL2 and OCI-Ly3 but not in OCI-Ly10. Mice treated with NVP-Bez235/lenalidomide represented obvious tumor growth regression and prolonged overall survival.

Conclusions: Our findings demonstrated the synergistic effect of low dose of NVP-Bez235 and lenalidomide in ABC-DLBCL, the underlying mechanism may be multifunctional, involving apoptosis, Akt and NF-κB inactivation and cell cycle arrest. Cotreatment was also effective in vivo. These data pave the way for potential treatment of ABC-DLBCL with combination of NVP-Bez235 and lenalidomide.

Keywords: Apoptosis; Cell cycle; Diffuse large B cell lymphoma; Lenalidomide; NF-κB; PI3K/mTOR inhibitor.

Fig. 1

Combination effects of NVP-Bez235 and lenalidomide in proliferation inhibition. a, b Cell viability of Su-DHL2, OCI-Ly3, and OCI-Ly10 was measured by CCK-8 assay after treated with NVP-Bez235 and lenalidomide alone or in combination for 72 h. Drugs were conducted simultaneously at the indicated concentrations. c Isobologram analysis of agent combination at ED50 and CI values were calculated as well. d CI values of all cell lines at ED25, ED50, ED75 and ED90. The lower CI values indicated the stronger synergism when CI < 1, and higher CI values means the stronger antagonism when CI >1. Additive effect was designated with CI = 1

Fig. 2

NVP-Bez235 combined with lenalidomide significantly increased the apoptosis in all cell lines. a, b Cell lines were simultaneously treated with NVP-BEZ235 (20nM) and lenalidomide (2 μM) for 72 h, analyzing apoptosis by Annexin-V/PI staining with t test statistic assay. (Mean ± S.D., n = 3, *, +, # p < 0.05, **, ++, ## p < 0.01, ***, +++, ### p < 0.005 compared with control group) (c) Cell lines were subjected to indicated treatments and protein lysates were performed with immunoblotting, incubating with PARP, caspase3, caspase9, caspase8 and their cleavage formate antibodies. d Cell extractions were conducted with Western Blot to detect Bcl-2 family members including Bax, Bad, p-Bad, Bim, Bcl-2 and Bcl-xL

Fig. 3

Synergistic inhibition of AKT and NF-κB pathway. a Cell lines were treated with NVP-Bez235 20nM and lenalidomide 2 μM alone or in combination for 72 h. Western blot analysis was performed to identify the expression of total Akt, p-Akt (Ser 473) and p-Akt (Thr308). b Western blot for Ikkα, Ikkβ, p-Ikkα/β, IΚBα, p-IΚBα, NF-κB and p-NF-κB

Fig. 4

The role of NF-κB in apoptosis induced by NVP-Bez235 and lenalidomide. a Cell lines were exposed to 5nM bortezomib for 24 h and 48 h, respectively. Cell lysis were conducted with immunoblotting for p-NF-κB and NF-κB. b Bar graph was represented as mean ± S.D (*p < 0.05) to show the cell viability of cell lines treated with 5nM bortezomib and concomitant treatmemt of NVP-Bez235 (20nM) and lenalidomide (2 μM). c All cell lines were transiently transfected with siRNA against NF-κB (si-NF-κB) and nonsilencing siRNA (si-NS) respectively for 24 h and then exposed to NVP-Bez235 (20nM) plus lenalidomide (2 μM) for 48 h. Then cells were collected for Western blot. d Cells were transfected as (c) for 24 h and treated with compounds for another 48 h. Then CCK-8 assay was performed to calculate the cell viability. *p < 0.05, **p < 0.01, ***p < 0.005 compared with si-NS group

Fig. 5

The effect of NVP-Bez235 and lenalidomide in cell cycle. a Cell lines were treated with components for 72 h, collected and fixed by 70 % ethanol for 24 h. Cell cycle assay was assessed with cytometry and Modfit LT. b The Bar graph illustrated the proportion of G0/G1 phase, S phase, G2/M phase of cell lines treated as mentioned above. Era bars indicated the standard deviation (n = 3). *, # p < 0.05, **, ## p < 0.01, ***, ### p < 0.005 compared with control group. c Western blot for cell lysis collected after treated with single agent or combination agents for 72 h

Fig. 6

Combination treatment affected mTOR pathway. All cell lines were treated with NVP-Bez235 and lenalidomide for 72 h. Cell lysis was determined by immunoblotting to identify the protein mTOR, p-mTOR, p70S6K, p-p70S6K, 4EBP1, and p-4EBP1

Fig. 7

NVP-Bez235 plus lenalidomide dramatically inhibited tumor growth and prolonged the survival in a DLBCL xenograft mouse model (OCI-Ly10). a Mice in each cohort were treated with lenalidomide (10 mg/kg) every other day and NVP-Bez235 (20 mg/kg/day) alone or in combination. Tumor volumes were measured every other day for 28 days. At the end of observation, tumor growth was significantly delayed by combination treatment compared with control group (***p = 0.0003) and NVP-Bez235 treatment group (**p = 0.006). b Overall survival was prolonged by NVP-Bez235 and lenalidomide combination therapy compared with the control group and single agent group (p < 0.0001). c IHC to identify expression of Ki67, p- NF-κB of tumor specimen Tunnel assay was performed to examine the apoptosis in tissues. d, e, f Bar graph illustrate the proportion of positive cells showed in panel C. *p < 0.05, **p <0.01, *** p < 0.005 compared with the control group