Reactive oxygen species dependent phosphorylation of the liver kinase B1/AMP activated protein kinase/ acetyl-CoA carboxylase signaling is critically involved in apoptotic effect of lambertianic acid in hepatocellular carcinoma cells

Though lambertianic acid (LA) is reported to have hypolipidemic activity in liver, its underlying anticancer mechanism is poorly understood so far. Thus, in the present study, apoptotic mechanism of LA was elucidated in HepG2 and SK-Hep1 hepatocellular carcinoma (HCC) cells. Here LA increased cytotoxicity, sub-G1 population and Annexin V/PI positive cells in two HCC cells. Also, LA cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP), activated phosphorylation of liver kinase B1 (LKB1)/AMP activated protein kinase (AMPK)/ acetyl-CoA carboxylase (ACC) pathway and also suppressed antiapoptotic proteins such as phosphorylation of Akt/ mammalian target of rapamycin (mTOR) and the expression of B cell lymphoma-2 (Bcl-2)/ B-cell lymphoma-extra large (Bcl-xL) and cyclooxygenase-2 (COX-2) in two HCC cells. Furthermore, LA generated reactive oxygen species (ROS) in HepG2 cells and AMPK inhibitor compound C or ROS inhibitor N-acetyl-L-cysteine (NAC) blocked the apoptotic ability of LA to cleave PARP or increase sub G1 population in HepG2 cells. Consistently, cleavages of PARP and caspase-3 were induced by LA only in AMPK+/+ MEF cells, but not in AMPK-/- MEF cells. Also, immunoprecipitation (IP) revealed that phosphorylation of LKB1/AMPK through their binding was enhanced in LA treated HepG2 cells. Overall, these findings suggest that ROS dependent phosphorylation of LKB1/AMPK/ACC signaling is critically involved in LA induced apoptosis in HCCs.


INTRODUCTION
Hepatocellular carcinoma (HCC) is approximately 90% of all cases of primary liver cancer, the 5th most common cancer worldwide and the third leading cause of cancer-related mortality [1,2]. The main risk factors include hepatitis B and C virus infection, alcoholrelated liver cirrhosis, non-alcoholic steatohepatitis and ingestion of aflatoxin B1 [3]. Patients with early-stage of HCC are amenable to therapies such as resection, liver transplantation or local ablation [4]. Nevertheless, less than 20% of patients are eligible for curative treatment including chemoembolization or sorafenib because of its late-stage, multiple comorbidities and hepatic dysfunction [5]. Also, even sorafenib has not been widely used because of its high cost and toxicity [6]. Thus, recently natural compunds are attractive for combination therapy or novel target therapy for HCC.
AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism [7] by activating the synthesis of fatty acid, cholesterol, protein through phosphorylation of metabolic enzymes and ATPgenerating processes, including glucose uptake [8]. A tumor suppressor liver kinase B1 (LKB1) is its upstream kinase that phosphorylates and activates AMPK signaling by encoding serine/threonine kinase [9,10]. Also, many www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 41), pp: 70116-70129 Research Paper www.impactjournals.com/oncotarget studies revealed that AMPK suppresses cell proliferation by the inhibition of cell cycle progression and regulation of mitosis [11,12]. Thus, AMPK activation is a therapeutic target for the prevention and treatment of cancer by metformin and other anticancer agents.
In the same line, though labmertianic acid (LA) is known to have anti-obesity [40], stress-protective [41], anti-allergic [42] and neurotropic [43,44] activities, its other anti-cancer studies have not been reported except our group's report on anticancer effect of LA via androgen receptor(AR) ablation [45] inhibition until now. Thus, in the current study, the underlying apoptotic mechanism of LA was elucidated in association with reactive oxygen species(ROS) and liver kinase B1 (LKB1)/AMP activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling pathway in HCCs.

Cytotoxic effect of LA in human hepatocellular carcinoma (HCC) cells
The cytotoxicity of LA in HepG2 and SK-Hep1 and Hep3B hepatocellular carcinoma (HCC) cells and Chang hepatocytes was evaluated by using MTT assay. Cells were treated with indicated concentrations of LA (0, 10, 20, 40, 80 μM) for 24 h. As shown in Figure 1, LA significantly suppressed the viability of HepG2, SK-Hep1 and Hep3B cells in a concentration dependent fashion, but not Chang normal hepatocyte cells. However, we used noninvasive and lipidemic HepG2 cells and metastatic SK-Hep1 cells origianted from hepatoblastoma of white male rather than invasive Hep 3B cells originated from HCC of black male in next experiments [46], though the susceptibility of Hep 3 B cells to LA was almost similar to that of SK-Hep1 cells. Next, cell proliferation assay was conducted in HepG2 and SK-Hep1 cells using crystal violet staining. After exposure to LA for 24 h, 48 h and 72 h, LA significantly inhibited proliferation of two HCC cells in a concentration and time dependent manner ( Figure 1C).

LA increased sub G1 population and regulated apoptosis related proteins in HCC cells
To test whether the cytotoxic effect of LA is due to apoptosis induction, cell cycle analysis and Western blotting were performed. LA increased sub-G1 in a dosedependent fashion. Especially, LA at 40 μM elevated the sub-G1 population to 22.14% compared to untreated control (2.06%) ( Figure 2A). Also, Western blotting showed that LA cleaved caspase-3, PARP and suppressed Bcl-2, Bcl-xL ( Figure 2B) in HepG2 and SK-Hep1 cells. However, pan-caspase inhibitor Z-VAD-fmk reversed the cytotoxicity induced by LA in HepG2 cells ( Figure 2C).

LA activated phosphorylation of LKB1/AMPK/ ACC signaling in HCC cells
To confirm whether the apoptotic effect of LA is related to AMPK signaling, Western blotting was performed in LA treated HCC cells with antibodies of p-LKB1, p-AMPK and p-ACC. Here, phosphorylation of LKB1, AMPK and ACC was increased in a dose dependent manner in LA treated HepG2 and SK-Hep1 cells ( Figure  3A). As shown in Figure 3B, the cytotoxic effect of LA was significantly reversed by AMPK inhibitor compound C treatment. In addition, Western blotting showed that LA suppressed the expression of PI3K, p-AKT, p-mTOR and COX-2 in HepG2 and SK-Hep1 cells ( Figure 3C). Also, as shown in Figure 3D, LA activated phosphorylation of ERK and p38 and attenuated phosphorylation of JNK in HepG2 cells. In contrast, LA decreased phosphorylation of p38 and JNK and increased phosphorylation of ERK in SK-Hep1 cells.

LA generated ROS production in HepG2 cells
To test whether ROS play a crictial role in LA induced apoptosis, DCFH-DA staining was used to measure the ROS generation by FACS Calibur. As shown in Figure 4, LA induced ROS production in HepG2 and SK-Hep1 cells in a time dependent manner compared to untreated control.

ROS dependent AMPK activation is critically involved in LA induced apoptosis in HepG2 and SK-Hep1 cells
Given that ROS generation is related to AMPK mediated apoptosis [47,48], cell cycle anaylsis and Western blotting were conducted with AMPK inhibitor compound C or ROS inhibitor NAC in LA treated HepG2 cells. The increased sub-G1 population by LA was significantly attenuated in HepG2 and SK-Hep1 cells by compound C and/or NAC ( Figure 5A). Consistently, Cell apoptosis assay using Annexin-V/PI double staining revealed that LA increased the percentage of early apoptotic cells (Annexin V+/PI− staining: 30.1 %) for 24 h and late apoptotic or necrosis cells (Annexin V+/PI+ staining: 22.9%) for 48h. Conversely, increased early/late apoptosis by LA was significantly reversed by compound C or NAC in HepG2 cells ( Figure 5B). Likewise, the increased phosphorylation of AMPK and ACC, PARP www.impactjournals.com/oncotarget   cleavage and decreased expression of Bcl-2 and Cox-2 by LA were reversed in HepG2 and SK-Hep1 cells by compound C (Figure 5C) or in HepG2 cells by NAC ( Figure 5D).
To further confirm the critical role of AMPK in LA induced apoptosis, AMPK α wild type and knockout (KO) mouse embryonic fibroblast (MEF) cells were used in this study. Herein LA increased sub-G1 population to 10.24% in AMPK α wild type MEF cells compard to untreated control (4.77%), while there were no significant changes of subG1 population in AMPK KO MEF cells ( Figure 6A). Consitently, Western blotting showed that cleavages of PARP and caspase-3 and phosphorylation of AMPK and ACC were induced in AMPK α wild type MEF cells by LA, but not in AMPK KO MEF cells ( Figure  6B). Of note, STRING database showed that Protein-   Protein Interaction(PPI) scores between AMPK and other proteins such as ACC, LKB1, mTOR and AKT1 were found 0.998, 0.995, 0.981 and 0.480, respectively ( Figure  6C). Next, immunoprecipitation (IP) was performed with lysates from HepG2 cells using anti-AMPK antibody and Western-blot analysis was also conducted to confirm the binding of AMPK and LKB1. Here IP revealed that AMPK was directly bound to LKB1 in LA treated HepG2 cells ( Figure 6D).

DISCUSSION
Hepatocellular carcinoma (HCC) as one of the most malignant human cancers [49] is known to be caused by risk factors including hepatitis B and C virus infection, alcohol abuse, non-alcoholic fatty liver disease, aflatoxins, diabetes, obesity, and genetic factors [50]. Nevertheless, the evident therapeutics for HCC still remain unclear so far [51,52]. Hence, the development of novel therapies for HCC has been requested all over the world.
Apoptosis is well defined as a programmed cell death; there are several types of cell death including apoptosis, pyroptosis, necrosis, or autophagy [53][54][55]. It is characterized by cell shrinkage, nuclear condensation and fragmentation, loss of adhesion to extracellular matrix and membrane blebbing in the cells [56]. Nowadays, apoptosis induction is recognized as one of the important strategies for cancer prevention or treatment [57,58].
In the current study, the apoptotic mechanism of LA was elucidated in HCCs. LA suppressed the viability of three HCC cells in a dose-dependent manner. To confirm the cytotoxicity of LA was due to apoptosis induction, cell cycle analysis was conducted. LA significantly increased sub-G1 population and the number of Annexin V/PI stained cells in HepG2 and SK-Hep1 cells, indicating that cytotoxicity of LA is induced by apoptotic effect of LA [59].
The cysteine-dependent aspartate-specific proteases as caspases have pivotal roles in apoptosis induction. Caspases consist of two groups; initiator caspases (caspase-2, -8, -9 and -10) and effector caspases (caspase-3 and -7) [60]. Once the process of intrinsic apoptosis pathway is initiated, cytochrome c is released from the mitochondria, which is a peripheral protein of the mitochondrial inner membrane [61]. The released cytochrome c attaches to apoptosis-activating factor-1 (Apaf-1) and procaspase-9 to form apoptosome in cytosol [62]. After procaspase-9 is activated by apoptosome, which in turn activates procaspase-3 to caspase-3 leading to apoptosis [60]. Here, LA cleaved PARP, caspase-3 and inhibited antiapoptotic proteins such as Bcl-2, Bcl-xL and also suppressed the expression of COX-2, PI3K, p-AKT and p-mTOR as survival pathway proteins in two HCC cells, implying LA induces apoptosis via inhibition of antiapoptotic and survival proteins. Also, the apoptotic effect of LA was blocked by pan-caspase inhibitor, Z-VADfmk, indicating that apoptosis induced by LA is mainly via caspase activation. Also, It was well documented that MAPKs including ERK, p38 and JNK regulate multicellular functions such as proliferation, differentiation, mitosis, gene expression, cell survival and apoptosis [63]. As shown in Figure 3D, LA activated phosphorylation of ERK and p38 and attenuated phosphorylation of JNK in HepG2 cells, while it decreased phosphorylation of p38 and JNK and increased phosphorylation of ERK in SK-Hep1 cells, implying cell specific effect of LA on MAPKs and further mechanistic study in the near future. AMPK is a serine/threonine protein kinase, a key regulator of cellular metabolism and plays an important role in apoptosis [64,65]. Several reports have revealed that AMPK can be a potential therapeutic target for cancer treatment [66,67]. Here, LA induced phosphorylation of AMPK, its upstream LKB1 and downstream ACC and also decreased protein expression of COX-2 in HCC cells, indicating the important role of LKB1/AMPK/ACC signaling in LA induced anticancer effect, since AMPK related signaling is critically involved in survival and proliferation of cancer cells through Warburg effect during lipid metabolism including lipogenesis [68,69].
It is well documented that the increased level of ROS is often observed during apoptosis induction in several cells [70]. ROS are ubiquitous among biological activities and excessive generation of ROS has been shown to induce damage in a variety of cancer cells via disruption of lipid membranes, proteins and DNA [71]. The current study revealed that LA increased ROS production in a time dependent manner. Conversely, the increase of sub G1 population and Annexin V/PI stained cells by LA was reversed by AMPK inhibitor compound C or ROS scavenger NAC in two HCC cells. Consistently, compound C blocked the ability of LA to induce phosphorylation of AMPK/ACC, PARP cleavage and decreased expression of Bcl-2 and COX-2 in two HCC cells. Also, NAC reversed phosphorylation of AMPK, PARP cleavage and decreased expression of Bcl-2 and COX-2 in HepG2 cells, demonstrating that LA generates ROS and subsequently induces AMPK phosphorylation, PARP cleavage and inhibits antiapoptotic proteins such as Bcl-2 and COX-2 leading to apoptosis in HCC cells (Figure 7).
Notably, LA increased sub-G1 population only in AMPK α wild type MEF cells, but not in AMPK KO MEF cells. Similarly, LA induced cleavages of PARP and caspase-3 and phosphorylation of AMPK/ACC in AMPK α wild type MEF cells, but not in AMPK KO MEF cells, indicating the pivotal role of LKB1/AMPK/ACC signaling in LA induced apoptosis. Overall, our findings support scientific evidence that LA can be a potent chemopreventive candidate for HCC treatment via ROS dependent phosphorylation of LKB1/AMPK/ACC signaling.

Isolation of LA
Pinus koraiensis leaves (3 kg) were pulverized, immersed in 50% MeOH (10 L) for 3 days and distilled to be concentrated for 10 h by using Rotary Evaporator(IKA Korea Limited, Seoul, Korea). Then the MeOH extracts were partitioned with EtOAc / distilled water (1:1) and the water layer was suspended and partitioned with n-butanol/ distilled water. A part of EtOAc fraction was subjected to a celite column chromatography and eluted with CHCl 3 -MeOH (3:1) to yield 15 fractions. Among these fractions, a distinct and vivid red-purple spot from fr. 6 was isolated, purified and identified as lambetianic acid (LA) with over 98% purity based on spectroscopic analyses such as NMR, MS, and IR as well as the comparison of the data with those reported in the literature [72].

Cytotoxicity assay
The cytotoxicity of LA was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, HepG2, SK-Hep1 and Chang cells (1×10 4 cells/well) were seeded onto 96-well culture plate and exposed to various concentrations of LA for 24 h. The cells were incubated with MTT (1 mg/mL) (Sigma Chemical) for 2 h and then treated with MTT lysis solution overnight. Optical density (OD) was measured using a microplate reader (Molecular Devices Co., USA) at 570 nm. Cell viability was calculated as a percentage of viable cells in LA treated group versus untreated control.

Crystal violet assay
For viability and proliferation, crystal violet assay was performed inHepG2 and SK-Hep1 cells. The cells (1×10 5 cells/well) were seeded onto 6-well culture plate and exposed to various concentrations of LA for 24 h, 48h and 72h. The cells were fixed (4% paraformaldehyde) and stained with crystal violet solution (40% ethanol, 60% PBS and 0.5% crystal violet). Fifteen min later, 1 ml of 10% acetic acid was added to each well, and the absorbance was read at 590 nm using a microplate reader (Molecular Devices Co., USA).

Cell cycle analysis
HepG2 and SK-Hep1 cells (2 × 10 5 cells/ml) were treated with LA (0, 10, 20 or 40 μM) for 24 h, washed twice with cold PBS and fixed in 75% ethanol at −20 °C for 24 h. The cells were incubated with RNase A (10 mg/ml) for 1 h at 37°C and stained with PI (50 μg/ml) for 30 min at room temperature in dark. The stained cells were analyzed for the DNA content by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) using CellQuest Software.

Annexin V/propidium iodide apoptosis assay
Cell apoptosis assay was performed using the double-staining method of the Annexin-V/PI apoptosis detection kit (BD Pharmingen, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. HepG2 cells (2 × 10 5 cells/ml) were treated with LA (0, 20 or 40 μM) for 24 h and 48 h. Cells were stained with Annexin V-FITC/PI dye and analyzed immediately by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Apoptotic cells were identified as either Annexin V+/PI− staining (early apoptosis) or Annexin V+/PI+ staining (late apoptosis or necrosis cells).

Statistical analyses
Statistical analysis was performed by Graphpad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). The statistical significance was determined by using one-way ANOVA and Tukey's test or Student's t-test.
All data were expressed as means ± standard deviation (SD). Statistically significant difference (P<0.05).