Eucalrobusone C suppresses cell proliferation and induces ROS-dependent mitochondrial apoptosis via the p38 MAPK pathway in hepatocellular carcinoma cells

Phytomedicine 25 (2017) 71–82

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Original article

Eucalrobusone C suppresses cell proliferation and induces ROS-dependent mitochondrial apoptosis via the p38 MAPK pathway in hepatocellular carcinoma cells Kai-Li Jian, Chao Zhang, Zhi-Chun Shang, Lei Yang∗, Ling-Yi Kong∗ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 26 October 2016 Revised 17 December 2016 Accepted 23 December 2016

Keywords: Eucalrobusone C Human hepatocellular carcinoma Mitochondrial pathway ROS p38 MAPK

a b s t r a c t Background: Eucalyptus extracts have anti-cancer activity against various cancer cells. Formylphloroglucinol meroterpenoids (FPMs), which are typical secondary metabolites of the genera Eucalyptus, have many important pharmacological activities. Purpose: Eucalrobusone C (EC), a new bioactive phytochemical, was first isolated from the leaves of Eucalyptus robusta in our laboratory. EC is a FPM, and our previous research revealed that EC showed strongest cytotoxicity in three cancer models than other compounds isolated from the leaves of E. robusta. This study investigated its anti-tumor effects on human hepatocellular carcinoma (HCC) and its underlying mechanisms. Methods: Cell viability was measured by MTT assay. Cell cycle, apoptosis and mitochondrial transmembrane potential were determined by flow cytometry. Immunofluorescence was determined by a laser scanning confocal microscope. Protein levels were analyzed by Western blotting. Results: Our results showed that EC exerted strong anti-proliferative activity against HCC cells in a concentration- and time-dependent manner. EC markedly induced apoptosis through the caspasedependent mitochondrial pathway, and the cell cycle was arrested at S phase. SB203580, a p38 MAPK inhibitor, effectively decreased cell death caused by EC. Moreover, the ROS scavenger N-acetyl cysteine (NAC) significantly attenuated apoptosis induced by EC and reversed EC-induced p38 MAPK activation. Conclusion: Our findings indicate that EC induces mitochondrial-dependent apoptosis in HCC cells through ROS generation and p38 MAPK activation, making EC a promising candidate for further development as an anticancer agent for HCC cells. © 2016 Elsevier GmbH. All rights reserved.

Introduction Human hepatocellular carcinoma (HCC) is currently the sixth most common cancer and the third most frequent cause of cancer death worldwide (Diaz-Gonzalez et al., 2016). At early stages, the tumor might be curable by resection, liver transplantation, or ablation and 5-year survival rates greater than 50% can be achieved. At advanced stages, patients with the disease have a dismal prognosis and almost no intervention will be effective. Sorafenib have Abbreviations: DCFH-DA, 2 , 7 - dichlorofluorescein-diacetate; DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2 -deoxyuridine; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal protein kinases; MAPK, mitogen-activated protein kinase; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; ROS, reactive oxygen species. ∗ Corresponding authors. E-mail addresses: [email protected] (L. Yang), [email protected] (L.-Y. Kong). http://dx.doi.org/10.1016/j.phymed.2016.12.014 0944-7113/© 2016 Elsevier GmbH. All rights reserved.

shown survival benefits for individuals with advanced HCC (Forner et al., 2012). However, HCC is also resistant to sorafenib after three months treatment (Wysocki, 2010). To reduce the mortality of HCC, further exploration and validation of therapeutic reagents for HCC are urgently required. Apoptosis is one of the most clearly characterized cell death processes. There are two main apoptotic pathways: the intrinsic or mitochondrial pathway and the extrinsic or death receptor pathway (Liu et al., 2011). The control and regulation of apoptotic mitochondrial events occurs through members of the Bcl-2 family proteins. Moreover, numerous natural compounds induce cell death through the mitochondrial apoptotic pathway. Zhao et al. found that DHB-induced apoptosis was mediated through mitochondrial intrinsic pathway evidenced by the release of cytochrome c into cytosol in human lung cancer cells (Zhao et al., 2016). Wang et al. proved that Peperomin E induced apoptosis by reducing the mitochondrial membrane potential and reducing the ratio of Bcl-2/Bax

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in human gastric carcinoma cells (Wang et al., 2016). Hence, natural compounds are very promising to become candidates for cancer therapy. Eucalyptus extracts was reported to have anti-cancer activity against various cancer cells, such as cancers of the colon, lung, prostate, ovary, cervix, pancreatic and liver (Bhagat et al., 2012; Islam et al., 2012; Vuong et al., 2015). Formyl-phloroglucinol meroterpenoids (FPMs) are typical secondary metabolites of the genera Eucalyptus. FPMs have many important pharmacological activities such as antimicrobial, anticancer and anti-HIV effect (Nishizawa et al., 1992; Shou et al., 2014; Yang et al., 2012). Eucalrobusone C (EC), a new bioactive phytochemical, was first isolated from the leaves of E. robusta in our laboratory. Furthermore, EC possesses significant growth inhibitory effects against three cancer cell lines as reported in our previous study. The anti-proliferation effect of EC towards HepG2 cells was stronger than that of MCF-7 cells, and EC induces apoptosis in MCF-7 cells (Shang et al., 2016). However, whether EC induces apoptosis in HCC cells and the anticancer mechanism against HCC cells are not fully understood. In this work, the inhibitory effects of EC on cell growth in two HCC cells lines, HepG2 and Bel-7402, were assessed. The mechanism of EC was also analyzed regarding cell cycle arrest, cell apoptosis induction, cytochrome c release and the mitochondrial membrane permeability (MMP) loss. Importantly, we demonstrated that EC-induced mitochondria-dependent apoptosis was regulated by an increase in ROS production and the activation of p38 MAPK pathways. Our results suggest that EC is a promising compound for the treatment of HCC. Materials and methods Reagents and chemicals EC (Fig. 1A) was first isolated from leaves of E. robusta (Shang et al., 2016); Sorafenib was purchased from Aladdin (Shanghai, China). Both compounds were more than 98% pure and dissolved in DMSO (Sigma Aldrich, St. Louis, MO) at a stock concentration of 50 mM. Control groups were treated with the same amount of DMSO (0.1%) in the corresponding experiments. NAC, the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB203580) were purchased from Beyotime Biotechnology (Shanghai, China). MTT (5 mg/ml) was dissolved in PBS and filter-sterilized. Cleaved caspase-3, cleaved caspase-9, cleaved caspase-7, cleaved-PARP, caspase-3, caspase-9, caspase-7, PARP antibody, Bax (D2E11), Bcl2 (D55G8), phospho-p44/42 MAPK (Thr202/Thr204), phosphop38 MAPK (Thr180/Thr182), phospho-SAPK/JNK (Thr183/Thr185), p44/p42 MAP Kinase, SAPK/JNK, p38 MAP Kinase antibody, GAPDH and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The pan-caspase inhibitor Z-VADFMK was purchased from Tocris Bioscience (Tocris Cookson Limited, Bristol, UK). Cell culture HCC HepG2 and BEL-7402 cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HepG2 and BEL-7402 cells were cultured in DMEM and RPMI-1640 media, respectively, which was supplemented with 10% FBS. Cell lines were grown in an incubator at 37 ºC with 5.0% CO2 in a humidified atmosphere. Cell viability assay and tumor colony formation assay Cell viability and tumor colony formation assays were performed as previously described (Ma et al., 2016).

CFDA-SE cell tracer assay Cell proliferation was determined using the CFDA-SE cell tracer kit (Beyotime, China) in accordance with the manufacturer’s instructions. HepG2 and BEL-7402 cells were labeled with CFDA-SE and then plated on six-well plates. After a 24 h incubation, the medium was replaced with fresh medium containing different concentrations of EC. The cells were harvested after 48 h, and the fluorescence intensity was measured by flow cytometry (BD Biosciences, San Jose, CA).

EdU incorporation assay The EdU incorporation assay was performed using an EdU labeling/detection kit (Ribobio, Guangzhou, China). The operating methods were performed as previously described (Geng et al., 2014).

Cell cycle distribution assay Cell cycle distribution assay was determined using KeyGEN DNA Content Quantitation Assay (Cell Cycle) kit (KeyGen Biotech Co., Ltd., Nanjing, China). HepG2 and BEL-7402 cells were plated in 6-well culture plates (2.5 × 105 cells per well). After incubation overnight, various concentrations of EC (5, 10 or 15 μM) or 0.1% DMSO were added. Cells were harvested after a 48 h incubation and then fixed in ice chilled 70% ethanol over night at 4 ºC. After that, cells were incubated for 30 min at 37 ºC in a PBS (0.01 M) solution containing 100 μl RNase A. Propidium iodide (PI, 50 μg/ml) was added into the solution for 30 min incubation at 4 ºC with protection from light. DNA content analysis was performed using flow cytometry.

APC Annexin V and 7-AAD double staining After incubation with EC for 48 h, the cells were trypsinized and then suspended in 100 μl of binding buffer containing 5 μl of APC Annexin V (BD Biosciences, San Jose, CA, USA) and 5 μl of 7-AAD (KeyGen Biotech Co., Ltd., Nanjing, China). After incubation at room temperature and protection from light for 20 min, the cells were subjected to flow cytometry for analysis.

Measurement of the mitochondrial membrane potential The MMP was determined using a JC-1 assay kit (KeyGen Biotech Co., Ltd., Nanjing, China). Following collection, the HepG2 and BEL-7402 cells were supplemented with 500 μl of JC-1 dye staining solution, and then incubated in the dark at 37 ºC for 25 min. After incubation, the cells were centrifuged at 20 0 0 rpm × 5 min and washed twice with 1 × incubation buffer. The fluorescence was then detected using flow cytometry (488 nm excitation and 525 nm emission filters) after the cells were resuspended in 500 μl of 1 × incubation buffer.

Determination of cellular reactive oxygen species (ROS) For quantitative detection of cellular ROS levels, HepG2 and BEL-7402 cells were pretreated with 0.1% DMSO or various concentrations of EC (5, 10 or 15 μM) for 48 h; and then incubated with 10 μM oxidation-sensitive fluorescent probe DCFH-DA (Beyotime, Jiangsu, China) in the dark for 20 min at 37 ºC. DCFH-DA was cleaved by intracellular esterase to liberate free DCFH. The relative ROS levels of the cells were measured using flow cytometry.

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Fig. 1. Effect of EC on cell viability in various HCC cells and normal hepatocyte (L02) cells. (A) Chemical structure and molecular weight of EC. (B) HCC BEL-7402, Hep3B, HCCLM3, HepG2, QGY-7701, SMMC-7721 and normal hepatocyte (L02) cells were treated with different EC concentrations for 48 h, and the IC50 values were quantified using the MTT assay. (C) HepG2 and (D) BEL-7402 cells were treated with different EC concentrations (0, 1.875, 3.75, 7.5, 15 or 30 μM) for the indicated times, and cell viability was determined by the MTT assay. (E) Sorafenib was used as a positive control in cell viability assay of HepG2 cells. The data are represented as the mean ± SEM from three independent experiments. ∗ P < 0.05 and ∗ ∗ P < 0.01, compared with control.

Immunofluorescence

Statistical analysis

HepG2 cells in 6 cm dish were treated with or without EC for 48 h. Prior to the end of treatment, cells were incubated live with MitoTracker® Deep Red FM (YESEN, Shanghai, China) for 30 min. Cells were then fixed with 4% paraformaldehyde for 15 min and blocked with 5% BSA for 1 h. Cells were then incubated with primary antibody (anti-cytochrome c, 1:100) at 4 ºC overnight. Next, cells were incubated with Alexa Flour 488-conjugated secondary antibody (1:300) for 1.5 h at room temperature in the dark. The nuclei were counterstained with DAPI (Beyotime, Haimen, China) for 10 min before imaging. A laser scanning confocal microscope LSM 700 (Carl Zeiss, Oberkochen, Germany) was used for colocalization analysis.

All results were calculated as the mean ± SEM for at least three independent experiments. Experimental data were analyzed by one-way or two-way ANOVAs using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical significance was considered at P < 0.05.

Western blot analysis Proteins were extracted and immunoblotted as previously described (Ma et al., 2016).

Results EC inhibits proliferation of HCC cells in a time- and dose-dependent manner The structure of EC is depicted in Fig. 1A. We first investigated the inhibitory effects of EC on HCC BEL-7402 (IC50 = 8.38 μM), Hep3B (IC50 = 15.94 μM), HCCLM3 (IC50 = 21.70 μM), HepG2 (IC50 = 7.41 μM), QGY-7701 (IC50 = 8.74 μM), SMMC-7721 (IC50 = 23.79 μM) and normal hepatocyte (L02) cells (IC50 = 28.89 μM) by MTT assay after 48 h co-incubation with

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EC (Fig. 1B). The inhibition effects of EC against HepG2 and BEL7402 cells were more effective than other HCC cells. However, L02 cells were less sensitive to EC than HepG2 and BEL-7402 cells, while IC50 at 48 h was 3.90-fold and 3.45-fold higher than that of HepG2 and BEL-7402 cells, respectively. Next, HepG2 and BEL-7402 cells were treated with 0, 1.875, 3.75, 7.5, 15 or 30 μM of EC for 24, 48 and 72 h and were then subjected to cell viability assay. EC dose- and time-dependently inhibited the proliferation of HepG2 and BEL-7402 cells. Sorafenib was used as a positive control. As shown in Fig. 1C–E, EC exhibited more potent cytotoxicity than sorafenib. The IC50 values at 24 h, 48 h and 72 h were 18.61, 7.41, 2.93 μM and 31.59, 21.84, 15.26 μM for EC and sorafenib, respectively. Therefore, EC exhibits proliferation inhibition on HCC cells and stronger cytotoxicity compared with sorafenib. EC inhibits colony formation, cell division and DNA synthesis in HCC cells The anti-proliferation effect of EC on HepG2 and BEL-7402 cells was confirmed by CFDA-SE labeling assay, colony formation assay and EdU incorporation assay. To monitor whether cells divided after EC treatment, HepG2 and BEL-7402 cells were labeled with CFDA-SE before the 48 h incubation. As shown in Fig. 2A and B, the treated cells exhibited increased CFDA-SE fluorescence intensity in a dose-dependent manner, suggesting that EC inhibited cell division. Then, EC treatment for 48 h significantly reduced the number of colonies in a dose-dependent manner compared with untreated cells. The results indicated a significant reduction in the ability of colony formation at 5 μM and a complete cessation of colony formation at 10 μM and 15 μM (Fig. 2C). Furthermore, we used the EdU incorporation assay to detect the effects of EC on DNA synthesis. EC dramatically decreased the EdU fluorescence intensities of HepG2 cells compared with untreated cells, further suggesting that EC dose-dependently inhibited cell proliferation (Fig. 2D). In conclusion, EC treatment markedly inhibited colony formation, cell division and DNA synthesis in HCC cells. EC induces S phase cell cycle arrest and caspase-dependent apoptosis in HCC cells To determine whether the growth-inhibitory effect of EC was mediated by cell cycle arrest, cell cycle distribution in HepG2 and BEL-7402 cells was examined by flow cytometry. As shown in Fig. 3A, EC treatment for 48 h significantly increased in the proportion of cells at S phase. The appearance of sub-diploid DNA content (Sub-G1 population) indicated that EC induced apoptosis in HCC cells. The percentages of HepG2 cells at S phase after incubation with EC were increased to 43.35% (10 μM) and 46.37% (15 μM), compared with 28.55% in the control. In addition, the percentages of sub-G1 HepG2 cells were increased to 19.98% (10 μM) and 29.09% (15 μM), compared with 5.49% in the control. Similar results were observed in BEL-7402 cells. To find out whether the anti-proliferative effect of EC is related to apoptosis, HepG2 and BEL-7402 cells were treated with EC for 48 h, and then apoptotic cells were determined by APC Annexin V/7-AAD double staining. Following a 48 h treatment of HepG2 cells, the percentages of early apoptotic cells were dosedependently increased to 22.6% (15 μM) and 25.9% (20 μM), compared with 3.2% in the control (Fig. 3B). Similar results were obtained in BEL-7402 cells. Overall, these data indicated that EC induces S phase cell cycle arrest and apoptosis in HCC cells. To determine whether EC induced caspase-dependent or independent apoptosis, HCC cells were co-treated with EC and Z-VADFMK. When cells were pre-treated with Z-VAD-FMK (10 μM) for 1 h, the cell viability increased, and the percentages of apoptotic

cells decreased markedly (Fig. 3C and E). Moreover, the efficient activation of cleaved PARP and cleaved caspase-3 was rescued in the presence of Z-VAD-FMK (Fig. 3D), suggesting that Z-VAD-FMK could rescue cell death caused by EC. These data indicate that the inhibition of cell proliferation by EC may be partly due to the activation of the caspase-dependent pathway. EC triggers apoptosis through mitochondrial pathway It is generally accepted that MMP disruption irreversibly leads to cell death via the mitochondrial apoptosis pathway (Ly et al., 2003). Therefore, we investigated disruption of MMP using JC1 straining of cells treated or untreated with EC as assessed by flow cytometry. Compared with the control groups, EC dramatically decreased mitochondria membrane potential dose-dependently in HepG2 and BEL-7402 cells (Fig. 4A), indicating that EC induced mitochondrial dysfunction, which contributes to HCC cell apoptosis. An imbalance between pro-apoptotic protein Bax and antiapoptotic protein Bcl-2 results in the release of cytochrome c from mitochondria, caspase-3 activation and subsequent apoptosis (Zhang et al., 2015). Therefore, we next examined Bax and Bcl-2 levels by Western blotting in EC-treated cells. EC down-regulated the expression of Bcl-2 in a dose-dependent manner, increasing the Bax/Bcl-2 ratio to 7.5 (10 μM) and 11.4 (20 μM) compared with untreated HepG2 cells (Fig. 4B and C). The caspase-cascade system plays crucial roles in cell apoptosis, and caspases are the executors of apoptosis (Boatright and Salvesen, 2003). Thus, we examined the expression of caspaserelated proteins. Fig. 4D and E showed that the expression levels of pro-caspase-3, pro-caspase-9 and pro-caspase-7 were decreased after treatment with different concentration of EC for 48 h. Moreover, remarkable cleavage of caspases-3, caspase-9 and caspase-7 was observed compared with control cells. The intact 116 kDa moieties of PARP were decreased, and that of the cleaved forms (89 kDa) were increased in HCC cells treated with EC. In mitochondrial death pathway, cytochrome c is released from mitochondria to cytosol (Cory and Adams, 2002). Therefore, the release of mitochondrial cytochrome c in HepG2 cells were investigated by immunofluorescence. As shown in Fig. 4F, cytochrome c was mainly localized in the mitochondria in control cells. Moreover, because MitoTracker red and cytochrome c-associated green fluorescence merge together, it shows a yellow-orange staining in untreated cells. While the cells treated by EC (5, 10 and 15 μM) exhibiting green fluorescence indicated release of cytochrome c from mitochondria into cytosol. Together, these data indicate that EC efficiently induces apoptosis through the mitochondrial pathway in HCC cells. EC activates the p38 MAPK/mitochondrial apoptotic pathway Many anticancer compounds activate MAPK signaling, and ultimately cause apoptosis in cancer cells (Zhang et al., 2015; Zou et al., 2015). Hence, we next examined the phosphorylation status of the p38, ERK and JNK proteins. HepG2 and BEL-7402 cells were exposed to different EC concentrations for 48 h by Western blotting. The levels of phospho-p38 and JNK gradually increased after the EC treatment, indicating the activation of p38 and JNK (Fig. 5A and B). We then examined whether activation of p38 and JNK pathways is necessary for EC-induced apoptosis in HepG2 cells. HepG2 cells were first pretreated with the JNK inhibitor SP600125 and the p38 inhibitor SB203580. SB203580 partially reversed the inhibiting effect induced by EC, whereas SP600125 did not (Fig. 5C). In addition, SB203580 also partially protected HepG2 cells from ECinduced apoptosis whereas SP600125 did not. The apoptosis rates decreased from 22.6% to 13.3% when cells were treated with EC and SB203580 (Fig. 5D). To confirm the role of p38 activation in

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Fig. 2. EC inhibited HCC cell proliferation. (A) HepG2 and (B) BEL-7402 cells were treated with different EC concentrations for 48 h, and the CFDA-SE staining assay was performed by flow cytometry. (C) The effect of EC on the clonogenic ability of HepG2 and BEL-7402 cells treated for 48 h. (D) HepG2 cells were incubated with various EC concentrations for 48 h. EdU staining was then performed, and the cells were observed using confocal microscopy. The data are presented as the mean ± SEM from three independent experiments. ∗ P < 0.05 and ∗ ∗ P < 0.01, compared with control.

EC-induced apoptosis, we detected the expression levels of PARP, cleaved PARP, Bax and Bcl-2 as shown in Fig. 5E. Notably, SB203580 reduced the levels of PARP cleavage and partially reversed the increase in the ratio of Bax/Bcl-2 induced by EC. The results suggested that the p38 pathway potentially mediates EC-induced mitochondrial apoptosis. ROS generation is the upstream regulator of EC-induced apoptosis Previous studies have reported that ROS generation could trigger cell apoptosis via activating mitochondrial pathways (Ma et al., 2014; Yu et al., 2011). To determine whether EC affects on ROS levels in HCC cells, we used the fluorescent probe (DCFH-DA) to de-

tect intracellular ROS levels in EC-treated and untreated cells by flow cytometry. As shown in Fig. 6A and B, treatment with EC caused a significant dose-dependent increase in ROS levels compared with control cells. To identify the role of ROS in mediating EC-induced anti-cancer effects in HepG2 cells, we used the ROS scavenger NAC, which is commonly used to inhibit ROS production. As expected, pretreatment of cells with 5 mM NAC for 1 h significantly inhibited EC-induced ROS production (Fig. 6C). Interestingly, NAC abolished HepG2 cells inhibition induced by EC (Fig. 6D). Cotreatment with NAC rescued EC-induced apoptosis as detected by flow cytometry (Fig. 6E). These results suggest that EC-induced cell death in HepG2 cells is potentially mediated by increased ROS accumulation.

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Fig. 3. EC induced caspase-dependent apoptosis and altered cell cycle distribution in HCC cells. HepG2 and BEL-7402 cells were treated with different concentrations of EC for 48 h. (A) Cells were stained with propidium iodide (PI), and cell cycle distribution was assessed by flow cytometry. (B) The APC Annexin V/7-AAD staining assay was performed to analyze apoptosis as detected by flow cytometry. HepG2 and BEL-7402 cells were pretreated with or without 10 μM Z-VAD-FMK for 1 h, and then followed by 15 μM EC for 48 h; (C) cell viability was determined by MTT assay. (D) The expression levels of pro-caspase-3, cleaved caspase-3, PARP and cleaved PARP were detected by Western blotting. (E) The APC Annexin V/7-AAD staining assay was performed to analyze apoptosis as detected by flow cytometry. Data are presented as the mean ± SEM from three independent experiments. ∗ P < 0.05 and ∗ ∗ P < 0.01, compared with control. # P < 0.05 and ## P < 0.01 related to EC alone.

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Fig. 4. EC triggered apoptosis through the mitochondrial death pathway. (A) The mitochondrial membrane potential of HepG2 and BEL-7402 cells treated with EC for 48 h, as measured via flow cytometry with JC-1 staining. (B and C) The expression levels of Bax and Bcl-2 in HCC cells incubated with EC for 48 h. (D and E) The expression levels of cleaved caspase-3, pro-caspase-3, cleaved caspase-7, pro-caspase-7, cleaved caspase-9, pro-caspase-9, PARP and cleaved PARP in HCC cells after treatment with various concentrations of EC for 48 h. (F) Effects of EC on cytochrome c release. Cells were incubated with EC at 5, 10 and 15 μM for 48 h, stained with anti-cytochrome c antibody and MitoTracker red, and viewed by laser scanning confocal microscope. Data are presented as the mean ± SEM from three independent experiments. ∗ P < 0.05 and ∗ ∗ P < 0.01, compared with control. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)

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Fig. 4. Continued

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Fig. 5. The activation of p38 MAPK is a crucial mediator of apoptosis induced by EC in HCC cells. (A and B) Western blot analysis of phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38 and GAPDH in HCC cells treated with EC at the indicated doses for 48 h. Cells were pre-incubated with the JNK inhibitor, SP600125 (10 μM) or the p38 MAPK inhibitor SB203580 (10 μM) for 1 h before treatment with 15 μM EC for 48 h; (C) cell survival was determined by the MTT assay. (D) Apoptosis was detected by APC Annexin V/7-AAD staining. (E) The expression levels of phospho-p38, p38, cleaved PARP, PARP, Bax and Bcl-2 in cells were assayed by Western blotting. Data are presented as the mean ± SEM from three independent experiments. ∗ P < 0.05 and ∗ ∗ P < 0.01, compared with control. # P < 0.05 and ## P < 0.01 related to EC alone.

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Fig. 6. ROS generation is the upstream regulator of EC-induced apoptosis. (A and B) ROS generation was measured using the DCFH-DA by flow cytometry after treated with EC for 48 h. HepG2 cells were pretreated with or without 5 mM NAC for 1 h, followed by 15 μM EC for 48 h, and (C) cells were subjected to flow cytometry analysis of intracellular ROS. (D) Cell viability was determined by the MTT assay. (E) Apoptosis was detected by APC Annexin V/7-AAD staining. (F) Western blotting was performed for phospho-p38, p38, cleaved PAPR, PARP, Bax and Bcl-2. Data are presented as the mean ± SEM from three independent experiments. ∗ P < 0.05 and ∗ ∗ P < 0.01, compared with control. # P < 0.05 and ## P < 0.01 related to EC alone.

Given that MAPK pathways are activated by ROS (Liu et al., 2012), we further evaluated the effect of NAC on the activation of p38 MAPK induced by EC. EC-induced changes in p38 MAPK phosphorylation were reversed by NAC pretreatment, suggesting that ROS potentially mediates the activation of p38 induced by EC. Importantly, blocking ROS generation by NAC resulted in a remarkable decline in the Bax/Bcl-2 ratio and PARP cleavage (Fig. 6F). Taken together, these results suggest that ROS induction mediates EC-

activated mitochondria apoptotic pathways and might be a critical upstream regulator of the anti-cancer activity induced by EC. Discussion EC-induced cell death in MCF-7 cells associated with apoptosis (a marked increase in sub-G1 phase accumulation and early apoptosis signals) (Shang et al., 2016). However, the molecular

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mechanisms underlying the anticancer activity of EC, particularly in HCC, remain unclear to date. This study has demonstrated for the first time that EC is a promising agent to induce cell apoptosis in HCC cells. Mitochondria play an important role in the control of apoptosis (Desagher and Martinou, 20 0 0). The Bcl-2 family of proteins governs MMP and can be either pro-apoptotic or anti-apoptotic (Elmore, 2007). The up-regulation of Bax/Bcl-2 protein expression ratios promotes the permeabilization of mitochondrial membrane which is the leading cause of apoptosis (Belzacq et al., 2003). Our results clearly demonstrated that EC enhanced the loss of MMP, increased Bax protein levels and decreased Bcl-2 levels, leading to the increased Bax/Bcl-2 ratio (Fig. 4A–C). Release of cytochrome c from mitochondrion is a key step for mitochondrial apoptosis. Treating HepG2 cells with EC results in the cytochrome c release from the mitochondria into the cytosol in our results (Fig. 4F). The continuous activation of caspase family proteins is considered a requirement of apoptosis. After mitochondrial function is destroyed, apoptosomes form. The apoptosomes recruit multiple caspase-9 molecules and promotes their cleavage to an active form (Wang, 2001). After initiation, caspase-9 binding to the apoptosome acts as a cleavage factor of caspase-3. Caspase 3 is known as an effector caspase in the apoptotic cell death pathway. Our data demonstrated that EC treatment induced S phase cell cycle arrest, and dramatic increases in the sub-G1 phase and the proportion of Annexin-V-positive and 7-AAD-negative (indicative of early apoptosis) HCC cells (Fig. 3A and B), indicating the activation of apoptosis. Moreover, the cleavage of caspase-3, caspase-7, caspase-9 and PARP increased markedly (Fig. 4D and E). These results demonstrate that the cytotoxic effects of EC in HCC cells were caused by the activation of the mitochondrial apoptotic pathway. Given that caspase-dependent mitochondrial apoptotic pathways were activated by EC and only partly mediated the anticancer action of EC (Fig. 3C–E), we assessed the upstream ROS production. A slight increase in ROS levels can stimulate cell growth and proliferation (Boonstra and Post, 2004), whereas excessive ROS leads to cell death through several mechanisms, including apoptosis, autophagy and necrosis (Trachootham et al., 2008). Interestingly, cancer cells are more sensitive to rapid increases in ROS levels compared with normal cells (Trachootham et al., 2009). What’s more, ROS overproduction induces the depolarization of the mitochondrial membrane, which eventually results in an increase in the level of other pro-apoptotic molecules in the cytosol (Vrablic et al., 2001). In this study, we found that blockage of ROS by the NAC abolished the cytotoxicity, apoptosis and PARP cleavage induced by EC in HepG2 cells (Fig. 6D–F), suggesting the activation of ROS in EC-induced HCC cell apoptosis. These data validate that EC induces HCC cell death through activating ROS production. Several studies have demonstrated that apoptotic cell death induced by ROS is mediated by p38 MAPK and JNK activation (Zhang et al., 2015; Zou et al., 2015). MAPKs are serine-threonine protein kinases that play an important role in signal transduction from the cell surface to the nucleus. In HepG2 and BEL-7402 cells, treatment with EC increased p38 MAPK and JNK levels. Surprisingly, only p38 activation is involved in the events of EC-mediated HepG2 cell death, which was confirmed by the use of the p38 inhibitor (SB203580). Fig. 6F indicated that ROS blockage inhibited EC-induced p38 phosphorylation. From these data, we concluded that EC-induced apoptosis through the ROS-dependent p38 MAPK apoptotic pathway. Activation of the p38 pathway induces mitochondria-dependent cell apoptosis via activating mitochondrial Bcl-2 family proteins and caspase-3 (Liu et al., 2012). EC stimulation significantly increased the level of pro-apoptotic Bax and decreased the level of anti-apoptotic Bcl-2 in HepG2 and BEL-7402 cells, whereas pretreatment with SB203580 reversed these changes (Fig. 5E). In addition, we also found that EC-induced activation of

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Fig. 7. Proposed signal transduction pathways by which EC induces apoptosis of HCC cells. Anticancer activity of EC can be attributed to its apoptosis induction of HCC cells via ROS-dependent mitochondrial apoptosis pathways by suppressing Bcl-2 expression, inducing mitochondrial membrane potential loss, inducing cytochrome c release and inducing p38 MAPK and PARP activation.

Bax/Bcl-2 was almost completely inhibited by NAC (Fig. 6F), indicating that ROS acts as an upstream signaling molecule involved in EC-induced activation of the p38 MAPK/mitochondrial pathway. Fig. 7 showed a proposed signaling model leading to development of ROS-induced cell apoptosis induced by EC. In conclusion, EC suppressed the proliferation of HCC cells by inducing apoptosis via mitochondrial apoptotic pathways. The activation of this pathway was associated with decreased Bcl-2 expression and increased Bax expression, resulting in a loss of mitochondrial membrane potential. Furthermore, cell death was partly dependent on caspase and p38 MAPK activation. We also found that excessive ROS activated p38-mitochondrial apoptotic pathways. The suppression of apoptosis and p38 MAPK activation by NAC validates the critical role of ROS in EC-induced cell death. These results indicate that EC possesses great potential as a promising candidate for the treatment of HCC. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (NO.81673554 and 81503211), the Project Funded by the Priority Academic Program Development of Jiangsu Higher

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Education Institutions (PAPD) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63). We thank the Cellular and Molecular Biology Center of China Pharmaceutical University for confocal microscopy characterizations. We are grateful to Xiaonan Ma for her technical help. References Belzacq, A.S., Vieira, H.L.A., Verrier, F., Vandecasteele, G., Cohen, I., Prevost, M.C., Larquet, E., Pariselli, F., Petit, P.X., Kahn, A., Rizzuto, R., Brenner, C., Kroemer, G., 2003. Bcl-2 and bax modulate adenine nucleotide translocase activity. Cancer Res. 63, 541–546. Bhagat, M., Sharma, V., Saxena, A.K., 2012. Anti-proliferative effect of leaf extracts of Eucalyptus citriodora against human cancer cells in vitro and in vivo. Indian J. Biochem. Biophys. 49, 451–457. Boatright, K.M., Salvesen, G.S., 2003. Mechanisms of caspase activation. Curr. Opin. Cell Biol. 15, 725–731. Boonstra, J., Post, J.A., 2004. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 337, 1–13. Cory, S., Adams, J.M., 2002. The BCL2 family: regulators of the cellular life-or-death switch. Nat. Rev. Cancer 2, 647–656. Desagher, S., Martinou, J.C., 20 0 0. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10, 369–377. Diaz-Gonzalez, A., Forner, A., Rodriguez de Lope, C., Varela, M., 2016. New challenges in clinical research on hepatocellular carcinoma. Rev. Esp. Enferm. Dig. 108, 485–493. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516. Forner, A., Llovet, J.M., Bruix, J., 2012. Hepatocellular carcinoma. Lancet 379, 1245–1255. Geng, Y.D., Yang, L., Zhang, C., Kong, L.Y., 2014. Blockade of epidermal growth factor receptor/mammalian target of rapamycin pathway by Icariside II results in reduced cell proliferation of osteosarcoma cells. Food Chem. Toxicol. 73, 7–16. Islam, F., Khatun, H., Ghosh, S., Ali, M.M., Khanam, J.A., 2012. Bioassay of Eucalyptus extracts for anticancer activity against Ehrlich ascites carcinoma (eac) cells in Swiss albino mice. Asian Pac. J. Trop. Biomed. 2, 394–398. Liu, C., Gong, K., Mao, X., Li, W., 2011. Tetrandrine induces apoptosis by activating reactive oxygen species and repressing Akt activity in human hepatocellular carcinoma. Int. J. Cancer 129, 1519–1531. Liu, L., Fu, J., Li, T., Cui, R., Ling, J., Yu, X., Ji, H., Zhang, Y., 2012. NG, a novel PABA/NO-based oleanolic acid derivative, induces human hepatoma cell apoptosis via a ROS/MAPK-dependent mitochondrial pathway. Eur. J. Pharmacol. 691, 61–68. Ly, J.D., Grubb, D.R., Lawen, A., 2003. The mitochondrial membrane potential (ψ m) in apoptosis; an update. Apoptosis 8, 115–128. Ma, T., Fan, B.Y., Zhang, C., Zhao, H.J., Han, C., Gao, C.Y., Luo, J.G., Kong, L.Y., 2016. Metabonomics applied in exploring the antitumour mechanism of physapubenolide on hepatocellular carcinoma cells by targeting glycolysis through the Akt-p53 pathway. Sci. Rep. 6, 29926.

Ma, W.D., Zou, Y.P., Wang, P., Yao, X.H., Sun, Y., Duan, M.H., Fu, Y.J., Yu, B., 2014. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. 70, 1–8. Nishizawa, M., Emura, M., Kan, Y., Yamada, H., Ogawa, K., Hamanaka, N., 1992. Macrocarpals - HIV-RTase inhibitors of eucalyptus-globulus. Tetrahedron Lett. 33, 2983–2986. Shang, Z.C., Yang, M.H., Jian, K.L., Wang, X.B., Kong, L.Y., 2016. (1) H NMR-guided isolation of formyl-phloroglucinol meroterpenoids from the leaves of Eucalyptus robusta. Chem. Eur. J. 22, 11778–11784. Shou, Q.Y., Smith, J.E., Mon, H., Brkljaca, Z., Smith, A.S., Smith, D.M., Griesser, H.J., Wohlmuth, H., 2014. Rhodomyrtals A-D, four unusual phloroglucinol-sesquiterpene adducts from Rhodomyrtus psidioides. Rsc. Adv. 4, 13514–13517. Trachootham, D., Alexandre, J., Huang, P., 2009. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discovery 8, 579–591. Trachootham, D., Lu, W., Ogasawara, M.A., Nilsa, R.D., Huang, P., 2008. Redox regulation of cell survival. Antioxid. Redox Signal. 10, 1343–1374. Vrablic, A.S., Albright, C.D., Craciunescu, C.N., Salganik, R.I., Zeisel, S.H., 2001. Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes. FASEB J 15, 1739–1744. Vuong, Q.V., Hirun, S., Chuen, T.L.K., Goldsmith, C.D., Munro, B., Bowyer, M.C., Chalmers, A.C., Sakoff, J.A., Phillips, P.A., Scarlett, C.J., 2015. Physicochemical, antioxidant and anti-cancer activity of a Eucalyptus robusta (Sm.) leaf aqueous extract. Ind. Crop. Prod. 64, 167–174. Wang, X.D., 2001. The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922–2933. Wang, X.Z., Cheng, Y., Wu, H., Li, N., Liu, R., Yang, X.L., Qiu, Y.Y., Wen, H.M., Liang, J.Y., 2016. The natural secolignan peperomin E induces apoptosis of human gastric carcinoma cells via the mitochondrial and PI3K/Akt signaling pathways in vitro and in vivo. Phytomedicine 23, 818–827. Wysocki, P.J., 2010. Targeted therapy of hepatocellular cancer. Expert Opin. Invest. Drugs 19, 265–274. Yang, S.P., Zhang, X.W., Ai, J., Gan, L.S., Xu, J.B., Wang, Y., Su, Z.S., Wang, L., Ding, J., Geng, M.Y., Yue, J.M., 2012. Potent HGF/c-Met axis inhibitors from Eucalyptus globulus: the coupling of phloroglucinol and sesquiterpenoid is essential for the activity. J. Med. Chem. 55, 8183–8187. Yu, H., Zhang, T., Cai, L., Qu, Y., Hu, S., Dong, G., Guan, R., Xu, X., Xing, L., 2011. Chamaejasmine induces apoptosis in human lung adenocarcinoma A549 cells through a Ros-mediated mitochondrial pathway. Molecules 16, 8165–8180. Zhang, X., Wang, X., Wu, T., Li, B., Liu, T., Wang, R., Liu, Q., Liu, Z., Gong, Y., Shao, C., 2015. Isoliensinine induces apoptosis in triple-negative human breast cancer cells through ROS generation and p38 MAPK/JNK activation. Sci Rep 5, 12579. Zhao, L., Wen, Q., Yang, G., Huang, Z., Shen, T., Li, H., Ren, D., 2016. Apoptosis induction of dehydrobruceine B on two kinds of human lung cancer cell lines through mitochondrial-dependent pathway. phytomedicine 23, 114–122. Zou, P., Zhang, J.R., Xia, Y.Q., Kanchana, K., Guo, G.L., Chen, W.B., Huang, Y., Wang, Z., Yang, S.L., Liang, G., 2015. ROS generation mediates the anti-cancer effects of WZ35 via activating JNK and ER stress apoptotic pathways in gastric cancer. Oncotarget 6, 5860–5876.