Prisconnatanones A, a cytotoxic naphthoquinone from Prismatomeris connata, suppresses the proliferation of human laryngocarcinoma HEp-2 cells in vitro

Abstract Prisconnatanones A (Priscon-A) is a rare tetrahydroanthraquinone isolated from herbal Prismatomeris connate. In this study, we examine its anti-tumour activity on human laryngocarcinoma HEp-2 cells in vitro. The CCK-8 assay was performed to evaluate its cytotoxicity. Cell cycle and apoptosis were analysed using flow cytometric analysis. Here, we showed Priscon-A inhibited the proliferation of HEp-2 cells in a dose-dependent manner, and at 5 μM it almost completely inhibited cell growth. Its cytotoxicity was associated with the cell cycle arrest at G2/M phase. The Annexin V-FITC/PI binding assay showed that the cell death induced by Priscon-A was associated with apoptosis. And, western blot analysis revealed that the levels of the apoptosis protein, cleaved caspase-3, PARP, p21 and Bax protein increased, while the level of anti-apoptosis protein Bcl-2 decreased.. These data demonstrated that Priscon-A significantly inhibited HEp-2 cell growth, induced the cell cycle arrest at the G2/M phase and efficiently induced cell apoptosis. Prisconnatanones A, a cytotoxic naphthoquinone from Prismatomerisconnata, suppresses the proliferation of human laryngocarcinoma HEp-2 cells in vitro


Introduction
Naphthoquinones are a class of anti-cancer candidates with promising cytotoxicity and which attracted the attention of many researchers (Wellington 2015). Recently, they have been reported to have cytotoxic activity, by inducint apoptosis and blocking the cell cycle, such as, shikonin, anthracycline, lapachol and β-lapachone. (Tuntiwachwuttikul et al. 2008;Júnior et al. 2009;Chen et al. 2012). However, as of today only a few of naphthoquinones are used clinically as anti-cancer drugs with certain toxic and side effects, more new naphthoquinone agents need to be explored along with further investigation of their potential for clinical use (Mallavadhani et al. 2014).

Priscon-A reduced cell viability and inhibited cellular proliferation in cancer cells
To evaluate the cytotoxicity of Priscon-A, human cancer cell line, HEp-2 was treated with various concentrations of Priscon-A for 48 h, and the CCK-8 assay was performed. As Figure     shows, Priscon-A significantly reduced the survival of HEp-2 cells in a dose-dependent manner. There is no significant inhibition of HEp-2 cells in cultures treated with 0.5-2 μM; however, when the drug concentration reached 5 μM, the HEp-2 cell survival rate was below 50%. To exactly analyse the effect of Priscon-A on laryngocarcinoma cells, we performed growth curve assay. The result showed that Priscon-A inhibits the proliferation of HEp-2 cells in a dose-dependent manner, and exhibited significant cell growth inhibition at the concentration of 5 μM (Figure 2(B)).

Priscon-A arrested cell cycle in G2/M phase and induced apoptosis in HEp-2 cells
To further investigate the cytotoxic mechanism, the HEp-2 cells were treated with Priscon-A and the cell cycle was analysed using FACS. The result showed that Priscon-A induced HEp-2 cells to accumulate in G2/M phase in a dose-dependent manner (Figure 3(A)). To assess whether the cell death induced by Priscon-A was associated with apoptosis, Annexin V-FITC/ PI binding assay was performed. In Figure 3(B), it is clear that apoptosis was induced in HEp-2 cells after treatment with Priscon-A in a dose-dependent manner. Apoptotic cells increased from 1.14 to 35.73% upon treatment of Priscon-A in HEp-2 cells. These results indicated that Priscon-A efficiently induced HEp-2 cells apoptosis.

Priscon-A induced apoptosis through inhibition of Bcl-2 and increase of p21 in HEP-2 cells
In order to further determine the apoptosis pathway, cells were treated with Priscon-A and the apoptosis protein levels were examined by western blot. ProCaspase-9 plays an important role in apoptosis and is involved in extrinsic apoptotic pathway. Activated procaspase-9 cleaves the effector caspase-3 to promote cell apoptosis. Meanwhile, PARP was cleaved by the caspase-3 (Zhao et al. 2013). As shown in Figure S5, the levels of cleaved caspase-3 and PARP, increased with Priscon-A treatment. p21 is a universal inhibitor of cyclin kinases, which is located in the lower reaches of the p53 gene. And p21 promotes p53-dependent cell cycle arrest and apoptosis (Zhao et al. 2013). Figure S5 shows that the p21 protein level was increased. Bcl-2 family proteins, which are subdivided into three groups on the basis of their pro-or anti-apoptotic action and the Bcl-2 Homology (BH) domains they possess, play an important role in the process of apoptosis (Martinou & Youle 2011). Among them, Bcl-2 is the crucial anti-apoptotic protein and Bax is the important pro-apoptosis protein. In this study, the level of anti-apoptosis protein Bcl-2 was decreased by Priscon-A in a dose-dependent manner, while the level of pro-apoptosis protein Bax was increased. These results further showed that Priscon-A efficiently induced HEp-2cell apoptosis.

Conclusion
Our study revealed the cytotoxicity of Priscon-A on human laryngocarcinoma HEp-2 cells, which dramatically inhibited HEp-2 cell growth, induced the cell cycle arrest at the G2/M phase and efficiently induced cell apoptosis. This preliminary work would be helpful for further investigation of the anti-cancer property of Naphthoquinones, as well as providing a basic work for the development of Priscon-A as a leading compound of anti-cancer drugs.

Disclosure statement
No potential conflict of interest was reported by the authors.