Thymoquinone Promotes Pancreatic Cancer Cell Death and Reduction of Tumor Size through Combined Inhibition of Histone Deacetylation and Induction of Histone Acetylation

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is virtually therapy-resistant. As noninvasive lesions progress to malignancy, the precursor period provides a window for cancer therapies that can interfere with neoplastic progression. Thymoquinone (Tq), a major bioactive component of essential oil from Nigella sativa's seeds, has demonstrated antineoplastic activities in multiple cancers. In this study, we investigated antineoplastic potential of Tq in human PDAC cell lines, AsPC-1 and MiaPaCa-2. Tq (10-50 μM) inhibited cell viability and proliferation and caused partial G2 cycle arrest in dose-dependent manner in both cell lines. Cells accumulated in subG0/G1 phase, indicating apoptosis. This was associated with upregulation of p53 and downregulation of Bcl-2. Independently of p53, Tq increased p21 mRNA expression 12-fold. Tq also induced H4 acetylation (lysine 12) and downregulated HDACs activity, reducing expression of HDACs 1, 2, and 3 by 40-60%. In vivo, Tq significantly reduced tumor size in 67% of established tumor xenografts (P < 0.05), along with increased H4 acetylation and reduced HDACs expression. Our results showed that Tq mediated posttranslational modification of histone acetylation, inhibited HDACs expression, and induced proapoptotic signaling pathways. These molecular targets demonstrate rationale for using Tq as a promising antineoplastic agent to prevent postoperative cancer recurrence and to prolong survival of PDAC patients after surgical resection.

Figure 1

Effect of Tq on PDAC cell proliferation. (a) MiaPaCa-2 and (b) AsPc-1 cells were exposed to Tq (10–50 μM) during 24, 48, and 72 h. ^∗P < 0.05 versus control, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student's t-test.

Figure 2

Cell cycle profiles by flow cytometry: (a) MiaPaCa-2 and (b) AsPc-1 cells. Tq induced accumulation of the cells in preG1 phase. The data shown are typical of one of three independent experiments. DNA histograms show evident accumulation of hypodiploid cells at preG1 peak within 24 h indicating accumulation of dead and apoptotic cells. At 30 μM of Tq the percentage of apoptotic cells increased to 29% at 24 h and reached 49% at 48 h (a), while it increased to 15% in AsPC-1 cells after 24 h and reached 24% after 48 h (b). Data were quantified from three experiments, ^∗P < 0.05, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student's t-test.

Figure 3

Apoptosis in Tq-induced PreG1 fraction. (a) MiaPaCa-2 and (b) AsPc-1 cells were treated with Tq (30 and 50 μM) for 24 h, stained with Annexin V and PI, and apoptosis was analyzed by flow cytometry. The upper right quadrants represent apoptotic cells, Annexin V positive and PI negative. Quantification of cell numbers in histogram shows significant increase in the number of late apoptotic cells. Data were quantified from four experiments ^∗P < 0.05, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student's t-test.

Figure 4

Real time qPCR analysis shows that Tq (50 μM) significantly increased p53, p21, and Bax mRNA expression and downregulated Bcl-2 mRNA expression in MiaPaCa-2 cells. Cells were also pretreated with p53 inhibitor, PFT-α, for 1 h prior to addition of Tq. Values are expressed as mean ± SEM of three experiments. ^∗P < 0.05  ^#P < 0.02 versus control levels ^∗∗P < 0.05  ^##P < 0.02 versus Tq treated values, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student's t-test.

Figure 5

Real time qPCR analysis of MiaPaCa-2 cells showing significant reduction of HDACs 1, 2, and 3 levels upon treatment with Tq (50 μM). ^∗P < 0.05 versus control levels, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student's t-test.

Figure 6

MiaPaCa-2 cells were treated with or without Tq (50 μM) for 12–48 h. HDACs activity decreased significantly at 24 and 48 h. Values are expressed as mean ± standard deviation of three different observations. ^#P < 0.05  ^∗P < 0.002 versus control levels, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student's t-test.

Figure 7

Representative Western immunoblot of nuclear extracts from MiaPaCa-2 cells. H4 Ac K12 protein is expressed as 8 kDa band. Significant increase of H4 Ac K12 protein is seen in cells treated with Tq (30–50 μM) after 48 h. Data are means ± SEM. ^∗P < 0.05 versus control untreated cells.

Figure 8

Xenografts generated in 4-week-old male nude mice using AsPC-1 and Hs766T cells. When tumors reached 1 cm, animals were treated with Tq (30 mg/kg body weight i.p. for 5 weeks). Tumor size was significantly (P < 0.05) shrunken in 67% of the animals.

Figure 9

Real time qPCR of Tq treated xenografts showing significant reduction of HDACs 1, 2, and 3 mRNA expression in AsPC-1 cells. Data are means ± SEM. ^∗P < 0.05 versus control untreated tumors.

Figure 10

Tq actions as HDAC inhibitor. Combined inhibition of histone deacetylation and induction of histone acetylation.