Anti-tumor effect in human lung cancer by a combination treatment of novel histone deacetylase inhibitors: SL142 or SL325 and retinoic acids

Abstract

Histone deacetylase (HDAC) inhibitors arrest cancer cell growth and cause apoptosis with low toxicity thereby constituting a promising treatment for cancer. In this study, we investigated the anti-tumor activity in lung cancer cells of the novel cyclic amide-bearing hydroxamic acid based HDAC inhibitors SL142 and SL325. In A549 and H441 lung cancer cells both SL142 and SL325 induced more cell growth inhibition and cell death than the hydroxamic acid-based HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Moreover, the combination treatment using retinoid drugs ATRA or 9-cis RA along with SL142 or SL325 significantly induced more apoptosis and suppressed colony formation than the single use of either. The expression of the retinoic acid receptors RARα, RARβ, RXRα and RXRβ were unchanged with the treatment. However a luciferase reporter construct (pGL4. RARE 7x) containing seven tandem repeats of the retinoic acid responsible element (RARE) generated significant transcriptional activity after the combination treatment of retinoic acids and SL142 or SL325 in H441 lung cancer cells. Moreover, apoptosis-promoting Bax expression and caspase-3 activity was increased after the combination treatment. These results suggest that the combination treatment of SL142 or SL325 with retinoic acids exerts significant anti-tumor activity and is a promising therapeutic candidate to treat human lung cancer.

Figure 1. SL142 and SL325 significantly suppressed cell viability in H441 and A549 lung cancer cells.

A. Chemical structure of SAHA, SL142 and SL325. B. Detection of H4 acetylation by immunoblot 24 hours after SAHA, SL142 or SL325 treatment (0.5 or 2.0 µM) in H441 lung cancer cells. β-actin is shown as control. C. Effect on cell viability induced by SAHA, SL142 or SL325. Cells were plated in 96-well plates at a density of 1×10³ cells/well 24 hours prior to treatment with SAHA, SL142 or SL325 (0.1 to 10 µM). Cell viability was evaluated at 96 hours following treatment by the WST1 assay (Roche, Basel, Switzerland) according to the manufacturer's protocol. **, significant difference from the cell viability treated with 0.1 µM of SAHA, SL142 or SL325 (p<0.01).

Figure 2. SL142 and SL325 suppressed cell growth and induced apoptosis in H441 and A549 lung cancer cells.

A. Analysis of caspase-3 activity in A549, H441 and H1299 lung cancer cells after SAHA, SL142 and SL325 treatment. Cells were treated with SAHA, SL142 or SL325 at a concentration of 2.5 µM for 36 hours. Triplicate experiments were performed; data represent the mean-fold increase ± S.E. Significant difference from cells treated with PBS and cells treated with HDAC inhibitors indicated was shown (*p<0.05, **<0.01). B. Flow cytometric analysis of apoptosis induced by SAHA, SL142 or SL325. Cells were infected with SAHA, SL142 or SL325 at the concentration of 2.5 µM for 72 hours and sub-G₀/G₁ DNA content was measured by propidium iodide staining and flow cytometric analysis.

Figure 3. Combination treatment of retinoic acids and SL142 or SL325 more significantly induced cell death in H441 lung cancer cells than those of single use.

A. Analysis of caspase-3 activity induced by ATRA or 9-cis RA (2.5 µM) and/or SAHA, SL142 or SL325 (0.5 µM) in H441 lung cancer cells. Cells were treated with SAHA, SL142 or SL325 at the concentration of 2.5 µM for 36 hours. After 36 hours of treatment, sub-G₀/G₁ DNA content was measured by propidium iodide staining and flow cytometric analysis. Triplicate experiments were performed; data represent the mean-fold increase ± S.E. *, significant difference from control cells (cells without treatments) (p<0.05). B. Flow cytometric analysis of apoptosis induced by ATRA or 9-cis RA (2.5 µM) and/or SAHA, SL142 or SL325 (0.5 µM) in H441 lung cancer cells. After 96 hours of treatment, sub-G₀/G₁ DNA content was measured by propidium iodide staining and flow cytometric analysis. C. Morphological analysis of H441 cells after ATRA or 9-cis RA (2.5 µM) and/or SL142 (0.5 µM) treatment.

Figure 4. Combination effects of ATRA or 9-cis RA and SAHA, SL142 or SL325 on suppression of colony formation in H441 lung cancer cells.

A. Colony formation of H441 cells treated with ATRA or 9-cis RA (2.5 µM) or/and SAHA, SL142 or SL325 (0.5 µM) for 36 hours. The assay was initiated by plating 2×10³ cells per 100-mm dishes. After 16 days, cells were fixed and stained with crystal violet. Representative pictures of experiments performed in triplicate are shown. B. Mean colony numbers were derived from quantitation of triplicate dishes for each treatment and percentage-specific cytotoxicity compared to colony formation in the DMSO group (control) was calculated. *, p<0.05 versus single agents.

Figure 5. The combination treatment of retinoic acids and/or SL142 or SL325 synergically increased the transcriptional activity of RARE in H441 lung cancer cells.

A. Transient transfection reporter assays in H441 cells with pGL4. or pGL4.RARE 7x (2 µg), plus pCMV. β-galactosidase (2 µg) after retinoic acids (2.5 µM) and/or SL142 or SL325 (0.5 µM) retinoic acids treatment. Results are presented as fold induction of relative light units normalized to β-galactosidase activity relative to that observed for control constructs. ^#, significant difference from the promoter activity generated by pGL4.RARE 7x after ATRA and SAHA treatment (p<0.05). *, significant difference from the promoter activity generated by pGL4.RARE 7x after 9-cis RA and SAHA treated cells (p<0.05). B. Immunoblot analysis of RARα, RARβ, RXRα and RXRβ expressions after retinoic acids and/or SL142 or SL325 treatment in H441 lung cancer cells. Human lung cancer cells were harvested 24 hours after treatment with ATRA or 9-cis RA (2.5 µM) or/and SAHA, SL142 or SL325 (0.5 µM). β-actin is shown as control.

Figure 6. The combination treatment of retinoic acids and/or SL142 or SL325 increased Bax protein expression and the promoter activity in H441 lung cancer cells.

A. Immunoblot analysis of RARα, RARβ, RXRα and RXRβ expressions after retinoic acids and/or SL142 or SL325 treatment in H441 lung cancer cells. Human lung cancer cells were harvested 24 hours after treatment with ATRA or 9-cis RA (2.5 µM) or/and SAHA, SL142 or SL325 (0.5 µM). β-actin is shown as control. B. Transient transfection reporter assays in H441 cells with pGL4. or pGL4.Bax (2 µg), plus pCMV. β-galactosidase (2 µg) after retinoic acids (2.5 µM) and/or SL142 or SL325 (0.5 µM) retinoic acids treatment. Results are indicated as Fig. 6A. *, significant difference from the promoter activity generated by pGL4.Bax after ATRA or 9-cis RA (2.5 µM) or SAHA, SL142 or SL325 (0.5 µM) (p<0.05).