The selenium analog of the chemopreventive compound S,S'-(1,4-phenylenebis[1,2-ethanediyl])bisisothiourea is a remarkable inducer of apoptosis and inhibitor of cell growth in human non-small cell lung cancer

Abstract

Lung cancer continues to be the leading cause of cancer deaths throughout the world and conventional therapy remains largely unsuccessful. Although, chemoprevention is a plausible alternative approach to curb the lung cancer epidemic, clinically there are no effective chemopreventive agents. Thus, development of novel compounds that can target cellular and molecular pathways involved in the multistep carcinogenesis process is urgently needed. Previous studies have suggested that substitution of sulfur by selenium in established cancer chemopreventive agents may result in more effective analogs. Thus in the present study we selected the chemopreventive agent S,S'-(1,4-phenylenebis[1,2-ethanediyl])bisisothiourea (PBIT), also known to inhibit inducible nitric oxide synthase (iNOS), synthesized its selenium analog (Se-PBIT) and compared both compounds in preclinical model systems using non-small cell lung cancer (NSCLC) cell lines (NCI-H460 and A549); NSCLC is the most common histologic type of all lung cancer cases. Se-PBIT was found to be superior to PBIT as an inducer of apoptosis and inhibitor of cell growth. Se-PBIT arrested cell cycles at G1 and G2-M stage in both A549 and H460 cell lines. Although both compounds are weakly but equally effective inhibitors of iNOS protein expression and activity, only Se-PBIT significantly enhanced the levels of p53, p38, p27 and p21 protein expression, reduced levels of phospholipase A2 (PLA2) but had no effect on cyclooxygenase-2 (COX-2) protein levels; such molecular targets are involved in cell growth inhibition, induction of apoptosis and cell cycle regulation. The results indicate that Se-PBIT altered molecular targets that are involved in the development of human lung cancer. Although, the mechanisms that can fully account for these effects remain to be determined, the results are encouraging to further evaluate the chemopreventive efficacy of Se-PBIT against the development of NSCLC in a well-defined animal model.

Fig. 1

Schematic presentation of the synthesis of PBIT and Se-PBIT.

Fig. 2

The effect of PBIT and Se-PBIT on iNOS protein expression and nitric oxide production in A549 cells. (A) A representative Western blot protein expression of iNOS at 5.0, 10.0 and 20 µM doses of PBIT and Se-PBIT. Cells were pretreated with cytomix (10 ng/ml IL-1β, 10 ng/ml TNF-α and 100 ng/ml IFN-γ) and starved of FBS for 24 h before treatment. The control band in A is iNOS protein expression from total cell protein lysate from A549 cells that had not been treated with any compound. Actin was used as a housekeeping gene for protein loading control and was further used to normalize iNOS protein expression across samples. Relative band densities after actin normalization were used to construct the bar graph. (B) Bar diagram of the effect of PBIT and Se-PBIT on iNOS protein expression from densitometric measurement of three Western blots from three separate treatments. (*) Statistically significant from the control treatment, P < 0.05. (C) Bar diagram of the effect of PBIT and Se-PBIT on the total nitrite production from calorimetric measurement of three separate treatments. (*) Statistically significant (P < 0.05, t-test) from control treatments.

Fig. 3

The effect of PBIT and Se-PBIT on cell growth using NCI-H460, A549 and normal lung fibroblast cells as determined by MTT assay. Percentage of control was quantitated from three independent experiments performed in triplicate. Mean of three values was determined and the results are expressed as percent of control. Data are presented as mean ± S.E. (*) Statistically significant (P < 0.05, t-test) as compared with untreated controls.

Fig. 4

The effect of PBIT and Se-PBIT on the induction of apoptosis using NCI-H460 and A549 as determined by cell-death-ELISA and protein expression of cleaved PARP analysis. Actin was used as a housekeeping gene for protein loading control. Percentages of apoptotic cells were quantitated from three independent experiments. The assay was performed in triplicate and the results are presented as enrichment factor (ratio of treatment over control). Data are presented as mean ± S.E. (*) Statistically significant (P < 0.05, t-test) as compared with untreated controls.

Fig. 5

Bar diagram of the effect of PBIT and Se-PBIT on cell cycle analysis using H460 and A549 cells from flow cytometric measurements at doses 1.25, 2.5 and 5.0 µM. In each triplet of bars the 1st bar represents the percentage of cells in stage G1, the 2nd bar represents the percentage cells in stage S and the 3rd bar represents the percentage cells in G2–M stage.

Fig. 6

The effect of PBIT and Se-PBIT, using Western blot analysis on cPLA2, COX-2, p53, p38, p27 and p21 in A549 cells. Similar results were obtained in H460 cells.