Diethyldithiocarbamate induces apoptosis in HHV-8-infected primary effusion lymphoma cells via inhibition of the NF-κB pathway

Abstract

Primary effusion lymphoma (PEL) is a subtype of B-cell lymphoma caused by human herpes virus 8/Kaposi sarcoma-associated herpes virus (HHV-8/KSHV), which is mostly found in patients with AIDS and has poor prognosis. Nuclear factor (NF)-κB pathway is constitutively activated in HHV-8-infected PEL cells and plays a crucial role in tumorigenesis. Recently, it has been shown that diethyldithiocarbamate (DDTC), an active metabolite of disulfiram, has apoptotic activity in cancer cells. Here, we investigated the effect of DDTC on PEL using a PEL mouse model generated by intraperitoneal injection of BC-3 cells, a PEL cell line. DDTC ameliorated the symptoms of PEL in these mice, such as development of ascites, splenomegaly and increase of body weight, in comparison with PBS-treated controls. Moreover, we determined in vitro that DDTC suppressed the constitutively activated NF-κB pathway in BC-3 cells. Methylthiotetrazole assay revealed that the cell proliferation of various PEL cell lines was significantly suppressed by the treatment of DDTC. DDTC also induced the expression of cleaved caspase-3, an apoptosis marker, whereas the addition of Q-VD-OPh, a pan-caspase inhibitor, inhibited cell apoptosis induced by DDTC treatment. Together, our results indicated that DDTC induces apoptosis via inhibition of the NF-κB signaling pathway in HHV-8-infected PEL cells. This study suggests the potential use of DDTC as a therapeutic approach for PEL.

Figure 1

Effects of DDTC on immunodeficient mice inoculated with BC-3 cells. (A) Schematic outline of the time of BC-3 cell inoculation and DDTC treatment. (B) Representative photographs of DDTC-treated and non-treated ascites-bearing mice 6 weeks after inoculation with BC-3 cells intraperitoneally. The abdominal distention prominent in control mice was relieved by DDTC treatment. (C-E) The body weight (C), weight of ascites (D) and spleen weight (E) of the mice inoculated with BC-3 cells, untreated or treated with DDTC, was measured after the last treatment with DDTC. (F) Representative photographs of livers from DDTC-treated and non-treated mice 6 weeks after inoculation with BC-3 cells intraperitoneally. The tumor burden of livers was decreased by DDTC treatment. Data are presented as means ± SE, n=4–7 mice/group. ^*P<0.05, ^**P<0.01 vs. control [BC-3-inoculated, PBS(−)-treated mice], assessed by one-way ANOVA.

Figure 2

DDTC has proteasome inhibitory activity in HHV-8-infected and -uninfected cells. (A) BC-3 and K562 cells were treated with 3 μM DDTC and incubated at 37°C for 12 or 24 h. After incubation, cells were recovered and nuclear proteins were extracted. Proteins were immunoblotted using the indicated antibodies. Actin and γ-tubulin were used as protein loading control. Ub; ubiquitinated protein, Cyt; cytosolic, Nuc; nuclear. (B) BC-3 cells were treated with 3 μM DDTC and incubated at 37°C for 12 h. After incubation, the mRNA expression of IL-6 was measured by quantitative real-time PCR. IL-6 mRNA levels were normalized to the levels of β-actin. Data are presented as means ± SE (n=3). ^***P<0.001 vs. control, assessed by one-way ANOVA.

Figure 3

DDTC suppresses the NF-κB pathway in HHV-8-infected PEL cells. (A and B) DDTC treatment increases sub-G1 (apoptosis) populations only in HHV-8-infected cells. BC-3 (A) and K562 (B) cells were treated with 3 μM DDTC for 48 h, then cells were washed and stained with PI and analyzed for DNA content by flow cytometry. (C) BC-3 and K562 cells were treated with 3 μM DDTC and incubated at 37°C for the indicated time. After incubation, cells were recovered and the levels of cleaved caspase-3 were determined by immunoblotting. Actin was used as loading control. (D) BC-3 cells were pre-treated with caspase inhibitor Q-VD-OPh at the indicated dose for 1 h and subsequently treated with 3 μM DDTC for 24 h. After incubation, cells were recovered and subjected to MTT assay. Data are presented as mean ± SE (n=3 per group). ^*P<0.05, ^***P<0.001 vs. DDTC-treated cells, assessed by one-way ANOVA.

Figure 4

DDTC induces caspase-3-dependent apoptosis in HHV-8-infected cells. (A) HHV-8-infected cells (BCBL-1, BC-1 and BC-3) were treated with DDTC at the indicated dose and incubated at 37°C for 24 h. After incubation, cells were recovered and subjected to MTT assay. To confirm the specificity of DDTC treatment for HHV-8 infected cells, we tested the effect of DDTC on chronic myelogenous leukemia cell line K562 (HHV-8-uninfected cells). (B) Apoptosis was evaluated after treating HHV-8-infected and HHV-8-uninfected cells with 3 μM DDTC, and staining with Annexin V at 48 h. Flow cytometry profile represents Annexin V-FITC staining in x axis and PI in y axis. The number represents the percentage of early apoptotic cells (lower right quadrant) and late apoptotic cells (upper right quadrant) in each condition.