Histone Deacetylase Inhibitor SAHA as Potential Targeted Therapy Agent for Larynx Cancer Cells

Abstract

Objective: Laryngeal squamous cell carcinoma is one of the most common malignant tumors in the head and neck region. Due to the poor response to chemotherapeutics in patients and low survival rate, successful treatment of larynx cancer still remains a challenge. Therefore, the identification of novel treatment options is needed. We investigated the anticancer effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on two different laryngeal cancer cell lines RK33 and RK45. We also studied the antiproliferative action of SAHA in combination with cisplatin and defined the type of pharmacological interaction between these drugs. Materials and Methods: Viability and proliferation of larynx cancer cell lines were studied by methylthiazolyldiphenyl-tetrazolium bromide method and 5-bromo-2-deoxyuridine incorporation assay, respectively. The type of interaction between SAHA and cisplatin was determined by an isobolographic analysis. Western blotting, flow cytometry and quantitative polymerase chain reaction method were used to determine acetylation of histone H3, cell cycle progression and genes expression, respectively. Apoptosis was assessed by means of nucleosomes released to cytosol. Results: SAHA alone or in combination with cisplatin inhibited larynx cancer cells proliferation, whereas displayed relatively low toxicity against normal cells - primary cultures of human skin fibroblasts. The mixture of SAHA with cisplatin exerted additive and synergistic interaction in RK33 and RK45 cells, respectively. We showed that SAHA induced hyperacetylation of histone H3 K9, K14 and K23 and triggered apoptosis. SAHA also caused cell cycle arrest by upregulation of CDKN1A and downregulation of CCND1 encoding p21WAF1/CIP1 and cyclin D1 proteins, respectively. Conclusion: Our studies demonstrated that SAHA may be considered as a potential therapeutic agent against larynx tumors.

Keywords: cisplatin (CDDP); histone deacetylase inhibitors; isobolography.; larynx cancer; suberoylanilide hydroxamic acid (SAHA).

Figure 1

Intracellular HDACs activity in larynx cancer cell lines after treatment with either culture medium alone (control), or SAHA (1-10 µM) for 6 hours. Data are presented as mean ± SEM at each concentration. (**p<0.01; ***p<0.001 by Student's t-test); (***p<0.001 by analysis of variance ANOVA test); n=9 per concentration from three independent experiments.

Figure 2

Detection of the histone H3 acetylation level at K9/14, K18 and K23 in RK33 (A) and RK45 (B) cells after treatment with either culture medium alone (control), or SAHA (1-10 µM) for 6 hours by Western blotting analysis. The membranes were re-probed with anti-H3 antibody of following incubation of larynx cancer cells with.

Figure 3

Antiproliferative activity of SAHA in larynx cancer cell lines (RK33 and RK45) and normal human skin fibroblasts (HSF). Larynx cancer cell lines or fibroblasts cells were treated with either culture medium alone (control), or SAHA for 72 hours. The viability and proliferation of cancer cells were measured by the MTT assay (A) and by means of BrdU incorporation (B), respectively. Results are presented as mean ± SEM at each concentration. (*p<0.05; ***p<0.001 by Student's t-test); (***p<0.001 by analysis of variance ANOVA test), n=40 per concentration from five independent experiments.

Figure 4

Log-probit analysis of dose-response curves of cisplatin (CDDP) and suberoylanilide hydroxamic acid (SAHA), administered alone and in combination at the fixed-ratio of 1:1, illustrating the anti-proliferative effects of the drugs in the RK33 (A) and RK45 (B) larynx cancer cell lines measured in vitro by the MTT assay. Doses of CDDP and SAHA were transformed into logarithms and the anti-proliferative effects produced by the drugs into probits according to Litchfield and Wilcoxon . Linear regression equations of dose-response curves are presented on the graph, where: y - is the probit of response, and x - is the logarithm (to the base 10) of a drug dose, R² - coefficient of determination. According to the test for parallelism, the experimentally determined dose-response curves for CDDP and SAHA were not parallel to one another.

Figure 5

Isobolograms illustrating types of interactions between cisplatin (CDDP) and suberoylanilide hydroxamic acid (SAHA) with respect to their anti-proliferative effects in the RK33 (A) and RK45 (B) larynx cancer cell lines measured in vitro by the MTT assay. The IC₅₀ ± S.E.M. of CDDP and SAHA are plotted graphically on the X- and Y-axes, respectively. The lower and upper isoboles of additivity represent the curves connecting the IC₅₀ values. The dotted line originating from the point (0, 0) corresponds to the fixed-ratio of 1:1 for the combination of CDDP with SAHA. Points A' and A” correspond to the theoretically calculated IC_{50 add} values for both, lower and upper isoboles of additivity. The point M illustrates the experimentally determined IC_{50 mix} value producing a 50% anti-proliferative effect in two cancer cell lines; RK33 (A) and RK45 (B). The S.E.M. values are presented on each graph as horizontal and vertical error bars for every IC₅₀ value. The experimentally-derived IC_{50 mix} value is placed close to the point A', indicating additive interaction between CDDP and SAHA in the cancer cell line RK33 (A), whereas in the cancer cell line RK45 (B), the IC_{50 mix} value is placed significantly (P<0.05) below the point A”, indicating supra-additive (synergistic) interaction between CDDP and SAHA.

Figure 6

Effects of SAHA on apoptosis in RK33 and RK45 cells. SAHA induces a concentration-dependent increase in apoptotic cells as measured using the Cell Death Detection ELISA^PLUS kit (Roche). The rate of apoptosis was expressed as an enrichment factor of oligonucleosome fragments released into the cytoplasm in the absence (control) and after treatment with SAHA (0.5-5 µM) for 24 hours. N=9 per concentration from three independent experiments, **p<0.01; ***p<0.001 versus control, Student's t-test.

Figure 7

Analysis of the cell cycle in RK33 (A) and RK45 (B) cells treated with SAHA. Larynx cancer cell lines were exposed to either culture medium alone (controls) or SAHA (0.5-5 µM) for 24 hours and cell cycle was assessed by propidium iodide-staining. (*p<0.05; **p<0.01; ***p<0.001; Student's t-test, n=9 per concentration from three independent experiments).

Figure 8

Changes in expression of genes encoding proteins engaged in G1/S transition in larynx cancer cell lines after treatment with SAHA. Expression of CCND1 (A) and CDKN1A (B) was analyzed by qPCR method in RK33 and RK45 cells exposed to either culture medium alone (controls) or SAHA (1-5 µM) for 24 hours. (*p<0.05, **p<0.01, ***p<0.001, n=9 measurements from three separate experiments, Student's t-test).