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. 2015;16(12):1820-9.
doi: 10.1080/15384047.2015.1078026.

Antiproliferative and proapoptotic effects of a pyrrole containing arylthioindole in human Jurkat leukemia cell line and multidrug-resistant Jurkat/A4 cells

Alex A Philchenkov  1 , Michael P Zavelevich  1 , Volodymyr P Tryndyak  2 , Ludmila M Kuiava  1 , Dmitry Yu Blokhin  3 , Koh Miura  4 , Romano Silvestri  5 , Igor P Pogribny  2
Affiliations

Antiproliferative and proapoptotic effects of a pyrrole containing arylthioindole in human Jurkat leukemia cell line and multidrug-resistant Jurkat/A4 cells

Alex A Philchenkov et al. Cancer Biol Ther. 2015.

Abstract

Recently, a series of novel arylthioindole compounds, potent inhibitors of tubulin polymerization and cancer cell growth, were synthesized. In the present study the effects of 2-(1H-pyrrol-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (ATI5 compound) on cell proliferation, cell cycle progression, and induction of apoptosis in human T-cell acute leukemia Jurkat cells and their multidrug resistant Jurkat/A4 subline were investigated. Treatment of the Jurkat cells with the ATI5 compound for 48 hrs resulted in a strong G2/M cell cycle arrest and p53-independent apoptotic cell death accompanied by the induction of the active form of caspase-3 and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage. ATI5 treatment also caused non-cell death related mitotic arrest in multidrug resistant Jurkat/A4 cells after 48 hrs of treatment suggesting promising opportunities for the further design of pyrrole-containing ATI compounds as anticancer agents. Cell death resistance of Jurkat/A4 cells to ATI5 compound was associated with alterations in the expression of pro-survival and anti-apoptotic protein-coding and microRNA genes. More importantly, findings showing that ATI5 treatment induced p53-independent apoptosis are of great importance from a therapeutic point of view since p53 mutations are common genetic alterations in human neoplasms.

Keywords: G2/M arrest; Jurkat leukemia cells; apoptosis; arylthioindoles; drug resistance; gene expression; microRNA.

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Figures

Figure 1.
Figure 1.
Effect of ATI5 treatment on growth inhibition and cell cycle progression in the Jurkat and Jurkat/A4 cells. (A) Chemical structure of ATI5 and combretastatin A-4 (CA4P). (B) Growth inhibition of the Jurkat and Jurkat/A4 cells treated with ATI5 or CA4P for 48 hrs. The results are presented as means ± SD (n = 3 ). (C) Cell cycle distribution in the Jurkat and Jurkat/A4 cells treated with ATI5 for 48 hrs. After staining with propidium iodide, cells were analyzed by flow cytometry. The percentage of cells in G0/G1 (white bars), S (gray bars) or G2/M phase (hatched bars) is shown.
Figure 2.
Figure 2.
Reversibility of ATI5 treatment effect on the cell cycle progression in the Jurkat cells. The cell cycle distribution of the Jurkat cells was analyzed after 3 hrs of treatment with ATI5 (B), 45 hrs after the cessation of ATI5 treatment (C), and after 48 hrs of ATI5 exposure (D). Untreated cells served as a control (A). The cell cycle distribution was analyzed by flow cytometry after the propidium iodide staining. Representative flow cytometry histograms are shown.
Figure 3.
Figure 3.
Effect of ATI5 on morphology of the Jurkat (A) and (B) and Jurkat/A4 (C) and (D) cells. Untreated cells (A) and (C) were used as control. Cells were stained using May–Grünwald–Giemsa technique. Representative images are shown (magnification 100x).
Figure 4.
Figure 4.
Effect of ATI5 on the induction of apoptosis in the Jurkat and Jurkat/A4 cells. Representative flow cytometry histograms showing the percentage of hypodiploid cells in the Jurkat (A) and Jurkat/A4 cells (B). Effect of pan-caspase inhibitor z-VAD-fmk on ATI5-induced apoptosis in the Jurkat (C) and Jurkat/A4 cells (D). Effect of caspase-8 z-LEHD-fmk inhibitor (E) or caspase-9 inhibitor Ac-IETD-cho (F) on ATI5-induced apoptosis in the Jurkat cells. Effect of caspase-8 z-LEHD-fmk inhibitor on agonistic anti-human Fas (CD95) mAbs-induced apoptotic cell death in the Jurkat cells (G). The percentage of hypodiploid cells was assessed by flow cytometry after the staining with propidium iodide. The results are presented as means ± SD (n = 3 ). * - Significantly different (p < 0.05) as compared to ATI5- or anti-human Fas mAbs-treated cells.
Figure 5.
Figure 5.
Effect of ATI5 on caspase-3 activation in the Jurkat (A) or Jurkat/A4 (B) cells. The percentage of cells with active form of caspase-3 in Jurkat and Jurkat/A4 cells treated with ATI5 for 48 hrs was assessed by flow cytometry as described in the “Materials and Methods.” (C) Analysis of PARP-1 cleavage in the Jurkat and Jurkat/A4 cells treated with ATI5. Representative Western blot PARP-1 and β-actin images are shown.
Figure 6.
Figure 6.
Delayed effect of ATI5 treatment on the induction of apoptosis, activation of caspase-3, and G2/M arrest in the Jurkat/A4 cells. The cells were treated with ATI5 at 100 nM or 1000 nM. At 72 hrs of treatment, ATI5-containing medium was replaced with drug-free medium, and cells were incubated for additional 48 hrs. The control cells were passaged in 72 hrs as usual and incubated for further 48 hrs. The percentage of hypodiploid cells (A), cells with active form of caspase-3 (B), and the cell cycle distribution (C) was analyzed by flow cytometry.
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