Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65

Abstract

Background: In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins.

Results: The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated.

Conclusions: The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm antileukemic potential.

Figures

Figure 1

Evaluation of viability in U937 human leukemia cells treated with PTX, MG132 and PTX + MG132. U937 cells were incubated in the presence of PTX, MG132, or PTX + MG132 for 18, 24, 36, and 48 hours as previously indicated. After incubation, WST-1 was added and 3 hours later, viability was assessed by spectrophotometry at 450 nm. The results represent mean ± standard deviation of three independent experiments performed in triplicate. Statistical analysis Mann-Whitney U test. ♦ p <0.05 PTX or PTX + MG132 vs untreated control group or MG132 group. ● p <0.05 PTX + MG132 vs all groups. * p < 0.05 PTX, MG132 or PTX + MG132 vs untreated control group.

Figure 2

PTX and MG132 modulate the cell cycle in U937 cells. U937 cells were incubated alone or were treated with 8 mM PTX, 1 μM MG132, or PTX + MG132 for 24 hours as previously indicated. After incubation the cell cycle was analyzed by flow cytometry. The results represent mean ± standard deviation of three independent experiments performed in triplicate. Statistical analysis Mann-Whitney U test * p <0.05 PTX, MG132 or PTX + MG132 vs untreated control group. ● p <0.05 MG132 or PTX + MG132 groups vs PTX or untreated control group.

Figure 3

Induction of apoptosis in U937 cells treated with PTX, MG132 and PTX + MG132. U937 cells were incubated exclusively in RPMI-S culture medium or were treated with PTX, MG132, or PTX + MG132 for 24 hours. After incubation apoptosis was assessed using Annexin V-FITC/PI. The results represent mean ± standard deviation of three independent experiments performed in triplicate. Mann-Whitney U test. *p <0.05 all groups vs untreated control group; ●p <0.05 PTX + MG132 vs all groups.

Figure 4

PTX + MG132 induces loss of the mitochondrial membrane potential (ΔΨm). U937 cells were cultured and treated with 8 mM PTX, 1 μM MG132, or PTX + MG132. After 24 hours, the cells were harvested and the ΔΨm was assessment by flow cytometry using DIOC6 staining. Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control. The results represent mean ± standard deviation of three independent experiments performed in triplicate. Statistical analysis Mann-Whitney U test. * p <0.05 all groups vs untreated control group (UCG); ●  p <0.05 PTX + MG132 vs all groups.

Figure 5

Western blot analysis of caspases -3,-8,-9 and cytochrome c in U937 cells treated with PTX, MG132 or PTX + MG132. U937 cells were cultured and treated with 8 mM PTX, 1 μM MG132, or PTX + MG132. After 24 hours, the cells were harvested and lysed. Equivalent amounts of individual lysates were placed on 10% SDS gradient polyacrylamide gels for electrophoresis and then were electrotransferred to Immobilon-P PVDF membranes. A representative study is shown and two additional experiments yielded similar results.

Figure 6

Determination of phosphorylated p65 (NF-κB subunit) in U937 cells treated with PTX, MG132 and PTX + MG132. U937 cells were incubated either alone or treated with 8 mM PTX, 1 μM MG132, or PTX + MG132. After 1 hour, the phosphorylated p65 protein was determined by flow cytometry. For each sample at, least 20,000 events were acquired. The results represent mean ± standard deviation of the Mean Fluorescence Intensity (MFI) of phosphorylated p65 of three independent experiments carried out in triplicate. *p <0.01 vs the untreated control group.

Figure 7

PTX and MG132 reduce the expression of Bcl-2 and Bcl-XL antiapoptotic proteins in U937 cells. U937 human leukemia cells were incubated alone or were treated with 8 mM PTX, 1 μM MG132, or PTX + MG132. After 24 hours the Bcl-2 (A) and Bcl-XL (B)antiapoptotic proteins were determined by flow cytometry. For each sample, at least 20,000 events were acquired. The results represent mean ± standard deviation of the Mean Fluorescence Intensity (MFI) of Bcl-2 or of Bcl-XL of three independent experiments carried out in triplicate. *p <0.01 vs the untreated control group.

Figure 8

Expression of pro- and antiapoptotic genes in U937 cells treated with PTX, MG132 and PTX + MG132. U937 human leukemia cells were incubated alone or treated with 8 mM PTX, 1 μM MG132, or PTX + MG132. After 3 hours gene expression was assessed by quantitative Real-Time PCR. The data are expressed as messenger (mRNA) fold change in relative normalized quantities employing the RPL32 gene expression. In all cases, standard deviation was not >0.08. Arbitrary was considered as significant upregulation or downregulation when the change was ≥ 30% in relation to constitutive gene expression.