3-Deazaneplanocin A Elicits Hepatoma Cellular Senescence Through DNA Damage Response

Enhancer of zeste homolog 2(EZH2) is a key componet of the polycomb repressive complex 2(PRC2), which has histone methyltransferase activity and can participate tri-methylation of H3K27 status. Epigenetic silencing induced by histone modification can be regarded as a risk factor for the inactivation of tumor-resistant genes. Corresponding to this, EZH2 can be recognized as a therapeutic target for cancer due to the phenomenon of the overexpression of EZH2 protein is quit commonly in various cancers. 3-deazaneplanocin(DZNep) is an inhibitor of S-adenosylhomocysteine hydrolase(SAHH), which could reduce the level of histone methylation and the status of methionine metabolism by indirectly inhibiting numberous methyltransferases activity. By now, DZNep has been employed as an anti-cancer drug which has proven effective in treatment for tumor suffers whose EZH2 protein is abnormal elevation. However, from essence, DZNep is not an inhibitor for EZH2 specifically; it is an inhibitor of SAHH as mentioned above. Hence, the mechiansm of the DZNep's anti-cancer effects is controversial. Due to inhibiting SAHH is the most direct biochemical action of DZNep which resulte in the accumulating of the S-adenosylhomocysteine(SAH) and then inhibite many transmethylases activity including EZH2. The effects of the intracellular changes may influence various biologicale effects, i.e, methyl donor availability and the metabolic stress reaction. Therefore, we offer a hypothesis that the inhibiting of EZH2 should not be the only pathway for the mechanism of DZNep's anti-cancer effects. Objective:We diucuss that the inhibiting of EZH2 should not be the only pathway for the mechanism of DZNep's anti-cancer effects and whether DZNep could enhance the sensitivity to DNA-damaging stimuli by transforming the status of chromatin and then induces the cellular senescence. Methods:DZNep and GSK343 are different inhibitor of EZH2 molecule which is indirect and direct inhibitating EZH2, seperatly. Fistly, based on HepG2 cell model, cell growth curve was assayed after DZNep and GSK343 treatment by cell proliferation assays. Secondly, accoding to response to two drugs treatment, we analyzed the cell proliferation, death and morphology changes conditions, we hypothesized that the reason for DZNep can suppress tumor cell proliferation was by promoting cellular sescence. Thirdly, the senescence-like phenomena was analyzed by staining of senescence-associated ?-galactosidasel. Forthly, based on analyzed cell cycle arrest, the expression of the cyclin-dependent kinase inhibitors, we recognized the signaling pathway which was influnced by DZNep treatment. Fifthly, employing cellular immune fluorescence analysis, the expression of p53 protein and its nuclear transfer ststus was identified to determine the activity of the p53. Finally, a penal of upstream signaling(?H2AX ? ATM ? CHK1 ? CHK2) in DNA damage response and senescence-associated inflammatory cytokine or chemokine secretion(IL6?IL8?IGFBP7) were assayed by RT-PCR, western blot and immunofluorescence analysis. Results:1. As an inhibitor of EZH2 directly, the death of HepG2 cell relied on GSK343 dose. However, the effect of DZNep inhibiting cell growth was not dose-dependent and DZNep could suppress the cell growth. Meanwhile, the accumulation of SAH could be found by use of HPLC qualitative analysis.2. The morphology changes of HepG2 cell after DZNep treatment was from round to flat. Besides, the intracellular particulate matter increased with DZNep treatment. Meanwhile, the staining of senescence-associated ?-galactosidasel was green and this implied that the result of staining of SA--galAfter was positive. After all, the senescence-associated changes could be found in the HepG2 cell after DZNep treatment.3. The proportion of cells at G0/G1 phase was depressed and the proportion of cells increased from 11.01% to 28.72% after DZNep-treatment.DZNep retained the cell cycle at G2/M phase. With the corresponding, the expression of p21 which is a kind of cell cycle inhibitor was increased significantly.4. DZNep promoted the stabilization of p53 protein in a mild and chronic manner and induced p53 protein transfer into nuclear. Combined with our previous results, HepG2 cell senescence caused by DZNep treatment was mediated through p53-p21 pathway.5. A penal of upstream signaling in DDR, such as the expression of ?H2AX?ATM?pCHK1 and pCHK2 was increased. Among this, the expression of ATM and pCHK1 at the day 24 was the highest and then began to attenuate gradually after 72 hours. The results implied that DNA damage response induced by DZNep treatment was early-effective of the drug.6. With HepG2 cell senescence induced by DZNep-treatment, inflammatory factors CXCR2, IGFBP7 and IL8 were dramatically increased. This implied that DDR promoted the cell senescence and these senescent cells further strengthen the effect of senescence by senescence-associated secretory phenotype.7. The results of microarray data confirmed that the top 10 pathways-related to the effect of DZNep treatment were histone isomer-associted, the result suggested that the effect of DZNep were relevant to chromatin state disturbance and remodelin. Meanwhile,the microarray data also pointed out that at least 3 known genes(ALDH3A, TRIM29, and TP53I3) closely associated with DNA repair were indentified to upregulated significantly in DZNep-treated cells. Conclusions:Aiming to explore the mechanism of DZNep anti-cancer effect, we found that the inhibiting of EZH2 should not be the only pathway for the mechanism of DZNep's anti-cancer effects. HepG2 cellular senescence induced by DZNep was mediated through p53-p21 pathway and triggered by enhanced ATM activation related to chromatin changes. Our studies could provide some meaningful information as shown following:Firstly, our results suggested that similar with promote apoptosis of tumor cells, promoting tumor cell senescence was also a promising method to develop anti-tumor therapeutic;Secondly, employing the feature of DNA damage response induced by DZNep, along with in combination with radiotherapy and chemotherapy, seemed logical to improve the effectiveness of these traditional methods of cancer treatment.Finally, our studies implied that ATM could be activated induced by DZNep. However, which pathways of activation function was mediated by, i.e DNA breakage, ROS increase, chromatin state changes, etc., should be subject to further study.