Abstract
The level of histone deacetylation is closely associated with the genesis and development of tumors, but the antitumor effect and mechanism of the class I histone deacetylase inhibitor (HDACI) valproate acid sodium (VPA) on hepatocellular carcinoma cells has not been clearly demonstrated. In the present study, the antitumor effect and mechanism of VPA were measured in vitro. Firstly, it was found that, as an HDAC inhibitor, VPA could inhibit HDAC activity and HDAC1 gene expression in hepatocellular carcinoma cells and, as a result, an inhibition of cell proliferation was detected by MTT assay. Subsequently, the cell cycle and cell apoptosis profiles were analyzed using flow cytometry (FCM). The expression of the mRNA and protein of cyclins A, D1 and E and P21Waf/cip1 was measured by reverse transcription-polymerase chain reaction and FCM analysis to determine the molecular mechanism of VPA-induced cell cycle arrest. The activity and mRNA and protein expression of caspases 3, 8 and 9 were detected to determine the apoptotic pathway. Caspase expression was blocked by caspase inhibitors in order to observe whether the intrinsic or extrinsic pathway contributed to HepG2 cell apoptosis. The results revealed that the mRNA and protein expression of cyclins A and D1 was downregulated while the expression of P21Waf/cip1 was upregulated by VPA. The expression of cyclin E was only slightly affected by VPA. The mRNA and protein expression and activity of caspases 3 and 9 were upregulated by VPA. By contrast, inhibitors of caspases 3 and 9 could reverse cell apoptosis and there was no notable change in caspase 8 expression in any of these experiments. The intrinsic apoptosis pathway, but not the death receptor pathway, contributed to the induction of apoptosis in hepatocellular carcinoma cells. Furthermore, VPA could inhibit the proliferation of hepatocellular carcinoma cells by inducing G1 phase arrest and cell apoptosis. These effects were attributed to the change in the caspase level.
Highlights
Numerous histone deacetylase inhibitor (HDACI) that are currently used in the clinic, including trichostatin A (TSA), apicidin and suberoylanilide hydroxamic acid (SAHA), have been restricted due to toxicity and a short half‐life [7]
The experiment was repeated two times. (A) Apoptosis profile of HepG2 cells treated with Valproate acid sodium (VPA) for 24, 48, 72 and 96 h. (B) Caspase activity inhibited by VPA. *P0.05. (C) Caspase protein expression affected by VPA. *P0.05. (D) Effect of caspase inhibitors on HepG2 cell apoptosis. *P0.05
The present results revealed that, following treatment with VPA for 48 h, the cell cycle was arrested at the G0/G1 phase and the mRNA and protein expression of cyclin A and D1 was downregulated in HepG2 cells, while the expression of P21Waf/cip1 was upregulated
Summary
Histone acetylation is associated with the genesis and development of certain tumors and is regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) [1,2]. Suppressing HDAC can be used as a novel antitumor therapy [3,4]. HDAC inhibitors (HDACIs) are notable due to their antitumor function [5,6]. Valproate acid sodium (VPA), a short‐chain fatty acid with the chemical name 2‐sodium valproate, was demonstrated to be a specific HDAC inhibitor and has been used widely as an anticonvulsant drug with low toxicity and a long half‐life [8]. Classical therapy for hepatocellular carcinoma, a malignant tumor that exhibits a quick progression, poor prognosis and high mortality rate, is unsatisfactory and novel treatment methods are required [9]. VPA was used to reverse the malignant phenotypes of hepatocellular carcinoma
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