Ebracteolatain A and Ebracteolatain B Induce Apoptosis of Human Hepatoma Cell Line (HepG2)

Purpose: To evaluate the effects of ebracteolatain A (EA) and ebracteolatain B (EB) from Euphorbia ebracteolata Hyata (Euphorbiaceae) on the proliferation of HepG2 cells and the possible mechanisms. Methods: EA and EB from E. ebracteolata were obtained by column chromatography. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays were used to study the cytotoxic and pro-apoptotic activities of EA and EB against HepG2 cells. Western blot assay was used to investigate the possible mechanisms of action. Results: EA and EB were successfully isolated from E. ebracteolata by column chromatography. The results of MTT assay indicate that EA and EB have significant anti-proliferative activities against HepG2 cells in dose- and time-dependent manners with half maximal inhibitory concentration (IC 50 ) of 28.48 and 31.72 μg/mL. respectively. The results of flow cytometry assay suggest that EA and EB significantly (p < 0.01) induced the apoptosis of HepG2 cells at the levels of 47.45 and 42.26 %, respectively. Western blot data indicate that EA and EB significantly (p < 0.05 or 0.01) down-regulated the expression levels of anti-apoptotic proteins (survivin and Bcl-2) and up-regulated the expression levels of pro-apoptotic proteins (Smac, Bax, c-caspase-3 and c-caspase-9) in mitochondria-mediated apoptotic pathway. Conclusion: EA and EB inhibit the proliferation of HepG2 cells, the probable mechanisms being associated with mitochondria-mediated apoptosis.

Ebracteolatain A (EA) and ebracteolatain B (EB) from E. ebracteolata are phloroglucinol derivatives.Phloroglucinol derivatives such as dryofragin and 2,4-bis(2-fluorophenylacetyl) phloroglucinol exhibits anti-cancer effects [9][10][11].Based on these reports, we supposed that EA and EB may have anti-cancer effects.In this work, we used 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and flow cytometry assays to investigate the effect of EA and EB on the proliferation and apoptosis of HepG2 cells in vitro by determining their inhibition rates and apoptosis rates.Moreover, the Western blot assay was used to explore the possible mechanisms.

EXPERIMENTAL Materials
The roots of E. ebracteolata were obtained from Zhong Yao Cai Tian Di Internet (www.zyctd.com) in March 2013, which were identified by Gang Peng from Suizhou Hospital, Hubei University of Medicine.A voucher specimen (Herbarium No. 201303154) of this plant is stored in Hubei University of Medicine for future reference.

Isolation of EA and EB
The air-dried roots (10 kg) of E. ebracteolata were smashed and extracted thrice with 95 % 60 L of ethanol.After filtering, the combined ethanol solution was evaporated by rotary evaporator under high vacuum to obtain ethanol extract (1357 g), which was suspended in water and subsequently partitioned with petroleum, chloroform and n-butyl alcohol.The chloroform fraction (234 g) was subjected to 200 -300 mesh silica gel column chromatography eluting with chloroform-methanol (19:1, 9:1, 7:3, 5:5) to obtain 7 fractions.Fraction 2 (17.5

Cell culture
HepG2 cells were provided by American Type Culture Collection (ATCC, Manassas, VA, USA).According to the existing literature [12], HepG2 cells were cultured in RPMI-1640 medium supplemented with 10 % FBS, penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 °C in 5 % CO 2 /95 % air.HepG2 cells were sub-cultured to logarithmic phase of growth, and then assays were carried out on the re-cultured HepG2 cells.

Cell proliferation assay
The effects of EA and EB on the proliferation of HepG2 cells were evaluated using MTT assay according to the existing literature [13].Briefly, HepG2 cells were seeded onto 96 well cell culture plates at 5000/well and incubated for 24 h at 37 °C [14].Then HepG2 cells were treated with EA or EB at different concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL for 48 h or at same concentrations of 30 μg/mL for 0, 12, 24, 36 and 48 h.After treatments, 20 μL MTT solution (5 mg/mL, dissolved in PBS) was added into each well incubated at 37 °C in 5 % CO 2 /95 % air for another 3 h to generate purpleblue formazan precipitate.Subsequently, DMSO (200 μL) was added into each well to dissolve the purple-blue formazan precipitate and the optical density (OD) of the DMSO solution was measured at 570 nm in a microplate reader (Bio Rad, Model 680).The inhibition rates were used to assess the effects of EA and EB on the proliferation of HepG2 cells and calculated using the following equation: Inhibition rate (%) = (OD control -OD treatment)/OD control × 100.

Flow cytometry analysis
After treatment with EA or EB at concentration of 30 μg/mL for 48 h, HepG2 cells were harvested and washed thrice with PBS.Subsequently, the washed HepG2 cells were re-suspended and stained with Annexin V-FITC/PI, and then analyzed by flow cytometer according to the manufacturer's instructions.The early apoptotic cells were a kind of Annexin V-FITC-positivity and PI-negativity cells, and the late apoptotic cells were a kind of Annexin V-FITC-positivity and PI-positivity cells [15].The numbers of apoptotic cells were calculated as the total numbers of the early and late apoptotic cells in flow cytometry.

Western blot analysis
HepG2 cells were collected to extract total proteins after treatment with EA or EB at different concentrations of 15, 30 and 45 μg/mL for 48 h.The Enhanced BCA Protein Assay Kit was used to determine the concentration of total proteins.Then equal amounts of total proteins (about 40 μg) were separated on 12 % sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and blotted on PVDF membrane.After blocking with 5 % fat-free milk in Tris buffered saline-Tween (TBS-T), the PVDF membranes were incubated with primary antibodies including anti-β-actin, anti-Survivin, anti-Bcl-xl, anti-Bcl-2, anti-Smac, anti-Bax, anti-ccaspase-3, anti-cleaved c-caspase-9 overnight at 4 °C, followed by incubation with HRPconjugated secondary antibody for 1 h at room temperature.Chemiluminescence detection was used to detect all proteins.To assess the proteins loading, β-actin was selected as the internal control, and the expression levels of other proteins were expressed as proteins level/β-actin level.

Statistical analysis
The results are presented as mean ± standard deviation (SD) (n=3).Differences between different groups were analyzed by one-way ANOVA (Dunnett test) on SPSS 19.0.P-value less than 0.05 or 0.01 was taken as statistically significant.

Identifications and purities analysis of EA and EB
The target analytes were identified as EA and EB by comparing their NMR data with the literature [1] and their chemical structures are shown in Figure 1.The results of HPLC area normalization method indicated that the purity levels of EA and EB were 93.0 % and 92.8 %, respectively.

Effects of EA and EB on the proliferation of HepG2 cells
As shown in Figure 2, after treatment with EA or EB at different concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL for 48 h, the proliferation of HepG2 cells was significantly inhibited in dose-dependent manner and their half maximal inhibitory concentration (IC 50 ) values were 28.48 and 31.72 μg/mL, respectively.After treatment with EA or EB at same concentration of 30 μg/mL for 0, 12, 24, 36 and 48 h, the proliferation of HepG2 cells was significantly inhibited in time-dependent manner.

HepG2 cells apoptosis induced by EA and EB
The results of flow cytometry analysis (Figure 3) suggested that after treatment with EA or EB at concentration of 30 μg/mL for 48 h, the apoptosis of HepG2 cells were significantly (p < 0.01) induced, compared with the control, and their apoptosis rates were 47.45 and 42.26 %, respectively.

EA and EB regulate the expression levels of apoptotic proteins
As depicted in Figures 4 and 5, EA and EB had similar effects on the expression levels of these apoptotic proteins (Survivin, Bcl-xl, Bcl-2, Smac, Bax, c-caspase-3 and c-caspase-9).After treatment with EA or EB at different concentrations of 15, 30 and 45μg/mL for 48 h, the expression levels of anti-apoptotic proteins (Survivin and Bcl-2) were significantly (p < 0.01) down-regulated and the expression levels of proapoptotic proteins (Smac, Bax, c-caspase-3 and c-caspase-9) were significantly (p < 0.01 or 0.05) up-regulated, compared with the control, whereas the expression level of anti-apoptotic protein (Bcl-xl) was not affected by the treatment.

DISCUSSION
Many study reports [3][4][5][6] have confirmed that E. ebracteolata can be used to treat cancer.In the present study, we studied the effects of EA and EB from E. ebracteolata on the proliferation of HepG2 cells and the possible mechanisms in vitro by MTT, flow cytometry and Western blot assays.The results showed that EA and EB inhibited the proliferation of HepG2 cells by inducing apoptosis via mitochondria-mediated apoptotic pathway.
MTT, a yellow dye, interacts with succinodehydrogenase (SDH) derived from mitochondria of living cells to generate the purple-blue formazan precipitate, but dead cells do not have the function [13].Then formazan precipitate was dissolved in DMSO and the OD value of the DMSO solution was used to evaluate cell viability.The smaller the OD value is, the lower the cell viability is.Based on the theory, MTT assay is usually used to evaluate the effects of anticancer agents on proliferation of cancer cells [16].The results of MTT assay (Figure 2) suggested that EA and EB significantly inhibited the proliferation of HepG2 cells.
The externalization of phosphatidylserine (PS) is observed in early apoptotic cells and the Annexin V can combine with the external PS.PI can combine with cell nucleus through the cell membrane in late apoptotic cells.After early or late apoptotic cells were stained with Annexin V-FITC/PI, they can be analyzed by flow cytometry [17].The results of flow cytometry analysis (Figure 3) indicated that EA and EB induced the apoptosis of HepG2 cells.The results of MTT assay and flow cytometry analysis indicated that the anti-proliferative activities of EA and EB are associated with apoptosis.Mitochondria-mediated apoptosis is an important pathway that is used to induce the apoptosis of cells, and the apoptotic proteins (Bid, Bax, Bcl-2, Smac, survivin, cytochrome c, caspase-9 and caspase-3) play an important role in mitochondria-mediated apoptotic pathway [18,19].When cells are stimulated by apoptotic stimuli, mitochondria will make a series of responses to reply the apoptotic stimuli.Firstly, the releases of Smac and cytochrome c from mitochondria to cytoplasm are up-regulated [20], but the releases are inhibited by Bcl-2, whose function can be suppressed by Bax and Bid [21].Secondly, cytochrome c along with dATP, Apaf-1 and pro-caspase-9 form apotosome and then procaspase-9 is activated to generate c-caspase-9 [22].Subsequently, caspase-3 is activated by c-caspase-9 to generate c-caspase-3 which induces the apoptosis of cells [23].Meanwhile, survivin inhibits the generation and function of ccaspase-3 [24].However, the inhibitory effect of survivin is eliminated by Smac [25].The results of western blot assay (Figures 4 and 5) indicated that EA and EB significantly down-regulated the expression levels of anti-apoptotic proteins (Survivin and Bcl-2) and up-regulated the expression levels of pro-apoptotic proteins (Smac, Bax, c-caspase-3 and c-caspase-9) without effect on the expression level of antiapoptotic protein (Bcl-xl), indicating that the proapoptotic mechanisms of EA and EB in HepG2 cells were related to mitochondria-mediated apoptosis.

CONCLUSION
The findings of this study indicate that EA and EB inhibit the proliferation of HepG2 cells and the possible mechanisms are associated with mitochondria-mediated apoptosis.The results also provide evidence to support the ethnomedical use of E. ebracteolata for the treatment of cancer and as a potential source of lead compounds for the development of antihepatoma drugs.

Figure 2 :
Figure 2: Inhibitory effects of ebracteolatain A (EA) and ebracteolatain B (EB) on the proliferation of HepG2 cells