I-kappa-kinase-2 (IKK-2) inhibition potentiates vincristine cytotoxicity in non-Hodgkin's lymphoma

Abstract

Background: IKK-2 is an important regulator of the nuclear factor-κB (NF-κB) which has been implicated in survival, proliferation and apoptosis resistance of lymphoma cells. In this study, we investigated whether inhibition of IKK-2 impacts cell growth or cytotoxicity of selected conventional chemotherapeutic agents in non-Hodgkin's lymphoma.Two established model systems were used; Follicular (WSU-FSCCL) and Diffuse Large Cell (WSU-DLCL2) Lymphoma, both of which constitutively express p-IκB. A novel, selective small molecule inhibitor of IKK-2, ML120B (N-[6-chloro-7-methoxy-9H-β-carbolin-8-yl]-2-methylnicotinamide) was used to perturb NF-κB in lymphoma cells. The growth inhibitory effect of ML120B (M) alone and in combination with cyclophosphamide monohydrate (C), doxorubicin (H) or vincristine (V) was evaluated in vitro using short-term culture assay. We also determined efficacy of the combination in vivo using the SCID mouse xenografts.

Results: ML120B down-regulated p-IκBα protein expression in a concentration dependent manner, caused growth inhibition, increased G0/G1 cells, but did not induce apoptosis. There was no significant enhancement of cell kill in the M/C or M/H combination. However, there was strong synergy in the M/V combination where the vincristine concentration can be lowered by a hundred fold in the combination for comparable G2/M arrest and apoptosis. ML120B prevented vincristine-induced nuclear translocation of p65 subunit of NF-κB. In vivo, ML120B was effective by itself and enhanced CHOP anti-tumor activity significantly (P = 0.001) in the WSU-DLCL2-SCID model but did not prevent CNS lymphoma in the WSU-FSCCL-SCID model.

Conclusions: For the first time, this study demonstrates that perturbation of IKK-2 by ML120B leads to synergistic enhancement of vincristine cytotoxicity in lymphoma. These results suggest that disruption of the NF-κB pathway is a useful adjunct to cytotoxic chemotherapy in lymphoma.

Figure 1

ML120B inhibits cell growth, cell cycle progression and phosphorylation of IκBα in Lymphoma cell lines. A. IC₅₀ was calculated by trypan blue exclusion assay for each cell line after 48 h incubation. Cell number (vertical axis) = Mean and SEM [standard error of mean]. B. DNA content was analyzed by PI staining using flow cytometry after 48 h for each cell line. C. Lymphoma cell lines were cultured for 60 minutes with ML120B at the indicated concentrations and whole cell lysates were subjected to Western blot for detection of p-IκBα, IκBα and actin (Arrows indicate total IκBα).

Figure 2

ML120B synergizes with the microtubule inhibitor, vincristine. Cell viability was measured by trypan blue exclusion assay over a 72 h incubation period, Mean and standard deviation (SD). WSU-FSCCL cells were exposed to each agent at the indicated concentrations for 72 h or pretreated with ML120B (20 μM) for 60 minutes followed by (A) cyclophosphamide (5.84 pM), (B) doxorubicin (1.5 pM), or (C) vincristine (260 pM, or 520 pM). D. Student t-test was used to calculate the values for combinational treatments. E. Synergy was calculated using CalcuSyn V2; CI < 0.9 indicates synergy. ML120B:vincristine synergized at all concentrations used. F. Combinational index (CI) curve according to Chou-Talalay method (reference 24) used to determine synergy between ML120B and vincristine. A ratio of <0.9 indicates synergy. Abbreviations used: M = ML120B, C = cyclophosphamide monohydrate, H = doxorubicin, V = vincristine, CTR = control (vehicle-treated cells).

Figure 3

ML120B:vincristine combination induces cell cycle arrest and apoptosis. Flow cytometric analysis of cell cycle was done following PI staining of WSU-FSCCL cells. A. WSU-FSCCL cells were exposed to vincristine (V) for the indicated times and concentrations. B. WSU-FSCCL cells were pretreated with ML120B (M) for 60 minutes followed by vincristine (V) at the indicated concentrations and times. C. Apoptotic cells (Sub G0) analyzed by PI staining using flow cytometry in WSU-FSCCL for combinational treatments, Mean and standard error of mean (SEM). D. Apoptotic cells analyzed as in (C) in WSU-FSCCL cells treated with single agents.

Figure 4

ML120B:vincristine combination induces apoptosis in G2/M phase cells. WSU-FSCCL cells were pretreated with ML120B (M) for 60 minutes followed by vincristine (V) at indicated concentrations in Panel B then analyzed for Terminal transferase dUTP Nick End Labeling (TUNEL) staining using flow cytometry. A. Representative histograms using the higher concentrations of M and V; upper panel: untreated, control cells; lower panel: treated with ML120B (40 μM) and vincristine (520 pM). B. Full data of FITC positive cells by cell cycle phase (sub G0, G0/G1, S, G2/M) according to PI staining of DNA. The largest FITC positive population in treated cells was in G2/M followed by sub G0.

Figure 5

7-AAD flow cytometric analysis of apoptosis. Representative scattergrams generated from 7-AAd staining of WSU-FSCCL treated with vincristine at 520 pm (VCR), ML120B at 40 uM (ML) or the combination (ML + VCR) for 72 h (bottom panel left to right). Top panel left (+ control) are heat-treated dead cells; top left (untreated) are live WSU-FSCCL cells from control culture. Within each scattergram, 7-AAD (vertical axis) separates cells into dead (A, top), apoptotic (B, middle) or live (C, bottom).

Figure 6

IKKβ inhibition in combination with Vincristine reduces p65 nuclear translocation and induces apoptosis. WSU-FSCCL cells were incubated with ML120B or Vincristine alone, or pretreated with ML120B (60 minutes) then cultured with Vincristine at indicated concentrations for 24 h. Total cell lysates were subjected to Western blot for detection of indicated protein using actin as a loading control. Nuclear and cytosolic p65 protein fractions were extracted from total cell lysates. GAPDH is used as a cytosolic loading control.

Figure 7

Immunofluorescence microscopy images of WSU-FSCCL cells. Top panel represents control cells after 48 h of culture; top left, tubulin staining; top center, NF-κB p65; top right, overlay of tubulin and p65. Bottom panel represents images of WSU-FSCCL cells after 48 h of treatment with ML120B (40 μM) and vincristine (520 pM). ML120B was added 60 minutes prior to vincristine: Bottom left are vincristine-alone treated cells showing disruption of microtubules and translocation of p65 to the nucleus as indicated by yellow-orange color. Bottom center: ML120B plus vincristine treatment showing sequestration of p65 in the cytoplasm in most cells. Bottom right: is same as bottom center except with DAPI counterstain to demarcate the nuclei and confirm absence of p65 from nuclei in most cells.

Figure 8

In vivo activity of ML120B alone, and with CHOP in WSU-DLCL2-SCID xenograft mouse model, tumor weights represent Mean and standard error of mean (SEM). A. Activity of ML120B as single agent: WSU-DLCL₂ tumors were xenografted s.c. into 10 ICR SCID mice/group on day 0 and dosing was initiated on day 7. ML120B was administered p.o. 120 mg/kg one dose on day 7 (× 1), 60 mg/kg twice (BID) on day 7 (× 2), and 60 mg/kg BID for 28 days starting on day 7 (× 28). Each point represents mean tumor weight of animal in each group ± SEM. B. ML120B plus CHOP treatment: Xenografts were developed as in A and treatment started on day 7. CHOP was administered at maximum tolerated doses (MTD) as previously defined (see Material and Methods), ML120B was administered p.o. 60 mg/kg BID for 28 days (as in A), ML120B in combination with CHOP at MTD.