Inhibition of IκBα phosphorylation potentiates regulated cell death induced by azidothymidine in HTLV-1 infected cells

Abstract

Adult T cell leukemia/lymphoma (ATL) can be susceptible, at least transiently, to treatments with azidothymidine (AZT) plus IFNα and/or arsenic trioxide. However, the real role of AZT in this effect is still unclear. In fact, while reverse transcriptase (RT) inhibition could explain reduction of clonal expansion and of renewal of HTLV-1 infected cells during ATL progression, this effect alone seems insufficient to justify the evident and prompt decrease of the pro-viral load in treated patients. We have previously demonstrated that AZT is endowed with an intrinsic pro-apoptotic potential towards both peripheral blood mononuclear cells from healthy donors or some tumor cell lines, but this cytotoxic potential cannot be fully achieved unless IκBα phosphorylation is inhibited. Since the constitutive activation of NF-kappa B (NF-κB) appears a common biological basis of HTLV-1-infected cells, a pharmacological inhibition of IκBα phosphorylation seems a potential strategy for treating and preventing HTLV-1 related pathologies. In this study, we have demonstrated that a combination treatment with the IκBα phosphorylation inhibitor Bay 11-7085 and AZT induced increased levels of regulated cell death (RCD) by apoptosis compared to the single treatments in HTLV-1 infected cells of different origin. Importantly, levels of RCD were considerably higher in infected cells in comparison with the uninfected ones. Inhibition of NF-κB activation following the combined treatment was confirmed by analysis of both gel-shift and functional activity of the NF-κB complex proteins, p65/p52. Moreover, a transcriptional analysis revealed that the addition of Bay 11-7085 to AZT treatment in HTLV-1-infected cells modified their transcriptional profile, by inducing the upregulation of some pro-apoptotic genes together with the downregulation of some anti-apoptotic genes. Our data suggest that addition of adequate concentrations of IκBα phosphorylation inhibitor to therapeutic regimens including AZT could be a promising strategy in ATL.

Keywords: Cancer therapy; Preclinical research.

Fig. 1. Apoptotic RCD induced by AZT towards PBMC from healthy donors and MT-2 cells.

Percentages of hypodiploid nuclei, detected by flow cytometry, in AZT treated PBMC (a) and MT-2 cells (b). The cells were treated with medium as a vehicle (CTR) or treated with 8, 32, and 128 µM AZT for 72 h. The results are expressed as mean values ± S.D. obtained from three independent experiments. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences between treated and control cells.

Fig. 2. Apoptotic RCD induced by pharmacological inhibition of IκBα phosphorylation towards PBMC from healthy donors and MT-2 cells.

Percentages of hypodiploid nuclei, detected by flow cytometry analysis, were assessed in PBMC (a) and MT-2 (b) cells either treated with medium as a vehicle (CTR) or treated with medium plus DMSO at the higher concentration utilized for diluting Bay 11-7085 (DMSO), or treated with 1, 2.5, 5, 10, and 20 μM Bay 11-7085, for 24 h. The results are expressed as mean values ± S.D. obtained from three independent experiments. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences between treated and CTR cells.

Fig. 3. Apoptotic RCD induced by a combination treatment with AZT and an inhibitor of IκBα phosphorylation towards HTLV-1 chronically infected cell lines.

Percentages of hypodiploid nuclei, detected by flow cytometry, in MT-2 (a), C5/MJ (b), and C91/PL (c) cells, or PBMC from healthy donors (d), after treatment with vehicle (CTR), with 128 µM AZT (AZT), with 1 µM Bay 11-7085 (BAY), or pre-treatment with 1 µM Bay 11-7085 for 2 h and subsequent treatment with 128 µM AZT (AZT+BAY) for 72 h, i.e. after a total of 3 days in culture (day 3) or following a second retreatment with the same protocol for another 72 h, i.e. after a total of 6 days in culture (day 6). The results are expressed as mean values ± S.D. obtained from three independent experiments. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences between groups (connection bars).

Fig. 4. Apoptotic RCD induced by a combination treatment with AZT and an inhibitor of IκBα phosphorylation towards not-yet-transformed, in vitro HTLV-1 infected cells.

Percentages of hypodiploid nuclei were assessed in IL-2 dependent, in vitro HTLV-1 infected BM24 (a) and BM7 (b) cell cultures, at 12 and 52 weeks in culture, respectively, from infection, following treatment with vehicle (CTR), with 128 µM AZT (AZT), with 1 µM Bay 11-7085 (BAY), or pre-treatment with 1 µM Bay 11-7085 for 2 h and subsequent treatment with 128 µM AZT (AZT+BAY) for 72 h. The results are expressed as mean values ± S.D. obtained from three independent experiments. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences referred to CTR cells (no connection bar) or between groups (connection bars).

Fig. 5. Detection of NF-κB activation in MT-2 cells treated with AZT and an inhibitor of IκBα phosphorylation.

MT-2 cells were either treated with vehicle (CTR) or treated with 1 µM Bay 11-7085 alone (BAY), 128 µM AZT alone (AZT), or with both (AZT+BAY), and then assayed at 1 and 24 h after the last treatment for NF-κB DNA-binding activity using non-radioactive electro-mobility shift assay (EMSA) (a), or detection of phosphorylated p65, p50 and p52 binding by an enzyme-linked immunosorbent assay (ELISA) (b). a A representative gel (upper side) and results of densitometry analysis (lower side) expressed as mean values ± S.D. obtained from three gels generated in independent experiments. T0 sample refers to cells assayed by immediately before starting any treatment. b The histograms represent the mean values ± S.D. from three independent experiments and are expressed as the ratio of the values obtained from samples of the experimental groups versus those obtained in samples of the CTR group at 24 h. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences referred to the CTR group at 1 h (no connection bar) or between groups (connection bars).

Fig. 6. RQ-PCR analysis of apoptosis-related gene expression in MT-2 cells treated with AZT and an inhibitor of IκBα phosphorylation.

MT-2 cells were either treated with vehicle (CTR) or treated with 128 µM AZT alone (AZT), with 1 µM Bay 11-7085 alone (BAY), or with both (AZT+BAY), and then assayed 24 h after the last treatment for gene-expression by real-time quantitative reverse transcription PCR (RQ-PCR). Normalization of crude values with the GUSB gene as a housekeeping gene, was performed. Relative gene expression of genes grouped as pro-apoptotic (a), anti-apoptotic (b), or multi-functional (c), was calculated versus time 0 control samples and is expressed as (log ₁₀). The histograms represent the mean values ± S.D. from three independent experiments. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences referred to time 24 h CTR samples.

Fig. 7. RQ-PCR analysis of apoptosis-related gene expression in C5/MJ cells treated with AZT and an inhibitor of IκBα phosphorylation.

C5/MJ cells were either treated with vehicle (CTR) or treated with 128 µM AZT alone (AZT), with 1 µM Bay 11-7085 alone (BAY), or with both (AZT+BAY), and then assayed 24 h after the last treatment for gene-expression by real-time quantitative reverse transcription PCR (RQ-PCR). Normalization of crude values with the GUSB gene as a housekeeping gene, was performed. Relative gene expression of genes grouped as pro-apoptotic (a), anti-apoptotic (b), or multi-functional (c), was calculated versus time 0 control samples and is expressed as (log ₁₀). The histograms represent the mean values ± S.D. from three independent experiments. Asterisks indicate significant (*p < 0.05) and highly significant (**p < 0.001) differences referred to time 24 h CTR samples.

Fig. 8. Effects of a combination treatment with AZT and an inhibitor of IκBα phosphorylation on the expression of the HTLV-1 doubly-spliced Tax/Rex transcripts in MT-2 cells.

MT-2 cells were treated with vehicle (CTR), with 1 µM Bay 11-7085 (BAY), with 128 µM AZT (AZT), or both (AZT+BAY) for a total of 3 days in culture (day 3) or, following a second retreatment with the same protocol, for a total of 6 days in culture (day 6). The histograms represent the mean values ± S.D. from four independent experiments. Asterisks indicate highly significant (**p < 0.001) differences referred to corresponding CTR samples.