6,8-Diprenylorobol Induces Apoptosis in Human Hepatocellular Carcinoma Cells via Activation of FOXO3 and Inhibition of CYP2J2

6,8-Diprenylorobol is a phytochemical derived from the roots of Glycyrrhiza uralensis Fisch. 6,8-Diprenylorobol exhibits several biological activities, but the effects of 6,8-diprenylorobol on cancers have been hardly investigated. This study is aimed at elucidating the anticancer effect and working mechanism of 6,8-diprenylorobol in HepG2 and Huh-7, two kinds of human hepatocellular carcinoma (HCC) cell lines. WST-1, cell counting, and colony formation assays and morphological change analysis showed that 6,8-diprenylorobol treatment decreased the cell viability and proliferation rate. Cell cycle analysis indicated that 6,8-diprenylorobol treatment increased the population of the G1/0 stage. Annexin V/PI double staining and TUNEL analysis showed that 6,8-diprenylorobol treatment increased the apoptotic cell population and DNA fragmentation. Western blot analysis showed that 6,8-diprenylorobol treatment increased the expression of cleaved PARP1, cleaved caspase-3, FOXO3, Bax, Bim, p21, and p27 but decreased the expression of Bcl2 and BclXL. Interestingly, 6,8-diprenylorobol inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities with Ki values of 9.46 and 2.61 μM, respectively. CYP2J2 siRNA transfection enhanced the anticancer effect of 6,8-diprenylorobol in HepG2 and Huh-7 cells through the downregulation of CYP2J2 protein expression and upregulation of FOXO3. Taken together, this study proposes that 6,8-diprenylorobol treatment may be a useful therapeutic option against HCC by targeting CYP2J2 and FOXO3.


Introduction
In 2000, liver cancer accounts for the ninth leading cause of cancer death but increased to sixth in 2016 [1]. Liver cancer has been recognized as highly fatal, and death rates are increasing much faster than those for any other cancers in the United States [2,3]. Globally, liver cancer is the second leading cause of death related to cancer [4]. Furthermore, according to recent cancer statistics, liver cancer incidence has increased much faster than any other cancers in both sexes [5]. So far, liver transplantation and resection have been recognized as the most effective treatment for hepatocellular carcinoma (HCC), which is one of the most common liver cancers [6]. However, there are several side effects of these therapeutic methods. After liver transplantation, patients should take medications for the rest of their life to help prevent their body from rejecting the donor's liver [7]. These antirejection medications can cause a variety of side effects, such as bone thinning, diabetes, high blood pressure, and high cholesterol level [8]. Therefore, it is necessary to develop alternative therapeutic strategies to treat HCC.
Phytochemicals are natural compounds produced by many kinds of plants, and the function is generally to support them thrive or thwart predators or pathogens [9]. It is reported that there are several beneficial effects of phytochemicals to health, such as reducing reactive oxygen species (ROS) in the human body [10]. Furthermore, phytochemicals are potential modulators of immunological processes related to anticancer, antioxidant, and anti-inflammatory [11]. For the last ten years, phytochemicals have been widely investigated to develop effective medicine for cancer treatment because phytochemicals have a potential to be developed as an anticancer agents with high efficacy and few side effects [12]. Recent studies showed that phytochemicals are potent modulators of autophagy for cancer treatment [13,14]. Furthermore, phytochemicals suppressed migration of metastatic breast cancer cells [15].
Cytochrome P450 2J2 (CYP2J2) is a member of the cytochrome P450 enzyme superfamily [16]. CYP2J2 is expressed in the vascular endothelium and is a prominent enzyme modulating metabolism of endogenous polyunsaturated fatty acids [17]. CYP2J2 has been recognized as a crucial biomarker of the disease. According to a recent study, CYP2J2 is a key enzyme in bioactivation of cyclophosphamide and a promising biomarker for hematological malignancies [18]. Interestingly, CYP2J2 is found to be upregulated in various cancers, and it plays a crucial role in cancer cell proliferation and human cancer metastasis [19][20][21]. Upregulation of let-7b suppressed the expression of CYP2J2 protein in cancerous tissues, which causes the inhibition of tumor phenotypes [22]. Furthermore, CYP2J2 has a protective effect in breast cancer MDA-MB-468 cells against cell death mediated by reactive oxygen species (ROS) [23]. Therefore, CYP2J2 may be an important biomarker to develop anticancer drugs.
Forkhead box O3 (FOXO3) is a member of the O subclass of the forkhead family, and it functions as a transcription factor regulating multiple physiological processes such as programmed cell death, cell cycle, and oxidative stress response [24]. FOXO3 is associated with various diseases, particularly in malignancy of various cancers such as breast, liver, colon, and prostate cancer [25][26][27]. Specifically, previous studies showed that FOXO3 plays an important role in the regulation of the cancer proliferation and apoptosis process [28,29]. It is reported that activation of FOXO3 activity inhibits the proliferation of colon cancer HT-29 cells [30]. Moreover, overexpression of FOXO3 induces apoptosis in the human prostate cancer cell line [28]. In addition, activation of FOXO3 displays an anticancer effect on human ovarian cancer SKOV3 cells [31]. Therefore, FOXO3 may be an important therapeutic target of various cancers.

Cell
2.11. siRNA Transfection. siRNA against CYP2J2 and control siRNA were purchased from Santa Cruz Biotechnology. For transfection with siRNA, cells were transfected with CYP2J2 siRNA or control siRNA using the Lipofectamine 2000 transfection reagent (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol.          2.13. Statistical Analysis. Results are expressed as arithmetic mean ± SEM (the standard error of the mean). To compare the statistical meaning between the groups, two-sided unpaired Student's t-test was used. All experiments were repeated three times, and the representative data were shown. Statistical analyses were performed using SPSS software (version 19.0, SPSS Inc., Chicago, IL, USA). Mean differences with p values less than 0.05 were considered statistically significant.     (Figures 3(a) and 3(b)).

Discussion
Cancer is one of the leading causes of death in the United States as well as globally [36,37]. Recently, drug-targeted therapies have been developed and improved cancer patient care [38]. However, there are still several side effects of the current cancer therapies on the advanced metastasized cancer [39,40]. Therefore, searching for a more effective and less dangerous treatment is required to improve the efficiency of treatment and reduce the treatment cost for cancer care.
Recently, cancer chemoprevention with natural phytochemicals has been recognized as a promising strategy to prevent and treat cancer [41]. As a natural compound, we expect that 6,8-diprenylorobol plays a crucial role in cancer therapy. For the last fifteen years, several studies showed that 6,8-diprenylorobol has an anti-Helicobacter pylori effect and antiestrogenic activity [34,35]. However, the potential effects of 6,8-diprenylorobol on various diseases have not been investigated well. Specifically, there is only one study investigating the anticancer effect of 6,8-diprenylorobol on cancer cells [42]. According to them, 6,8-diprenylorobol showed potent cytotoxic effects toward HL-60 human leukemia cells with an IC 50 value of about 10 μM. Although it was not a direct study to show the anticancer activity of 6,8-diprenylorobol, Sun et al. reported that 6,8-diprenylorobol inhibited aromatase, one of the targetable enzymes for cancer therapy, with a K i value of 1.42 μM [43]. In this study, we focus on studying the effect of 6,8-diprenylorobol on human HCC Huh-7 and HepG2 cells.
Cell cycle arrest means that cells are no longer involved in the duplication and division process [44]. Many studies 14 Oxidative Medicine and Cellular Longevity showed that lots of phytochemicals could induce cell cycle arrest in various cancer cell lines. Phytochemical extracts from cranberry induce G0/1 phase cell cycle arrest and apoptosis in human breast cancer MCF-7 cells [45]. Furthermore, gallic acid induces G0/1 phase cell cycle arrest and apoptosis through inhibition of cyclins D and E and activating a mitochondria-dependent apoptotic pathway in human leukemia HL-60 cells [46]. As shown in Figures 2(a) and 2(b), our results suggested that 6,8-diprenylorobol induced G0/1 cell cycle arrest in Huh-7 and HepG2 cells. Previously, we reported that CYP2J2 downregulation by siRNA transfection combined with acetylshikonin or broussochalcone A treatment induced apoptosis in HCC cells via activation of FOXO3 and inhibition of CYP2J2 [47,48]. In this study, we observed that 6,8-diprenylorobol showed the similar results with our previous report. To identify which kinase is involved in 6,8-diprenylorobol-mediated activation of FOXO3 and inhibition of CYP2J2, we analyzed the phosphorylated status of AKT, ERK, JNK, and p38. FOXO3 has been known to participate in cell growth inhibition by upregulating cell cycle regulation and proapoptotic proteins transcriptionally [49]. As shown in Figure 5, the phosphorylated AKT was significantly decreased by 6,8diprenylorobol treatment, but the phosphorylated ERK was significantly increased by 6,8-diprenylorobol treatment. The phosphorylation of JNK and p38 was not critically changed. FOXO3 could be phosphorylated in Thr32, Ser253, and Ser315 by AKT, leading to faster protein degradation [49]. Thus, the inhibition of the phosphorylation of AKT by 6,8diprenylorobol might contribute to the enhancement of the tumor-suppressive transcriptional activity of FOXO3 to block Huh-7 and HepG2 cell growth.
The ERK cascade is one of the major signaling pathways of the mitogen-activated protein kinase (MAPK) sig-naling, and it plays a crucial role in the regulation of cell proliferation, differentiation and cell cycle, and apoptosis [50,51]. According to a previous research, ERK activation induced cell cycle arrest and DNA damage-induced apoptosis [52]. Furthermore, it is reported that activation of ERK plays an important role in quercetin-induced apoptosis in lung carcinoma A549 cells [53]. ERK is required for the activation of cisplatin-induced apoptosis by mediating the mitochondria-dependent apoptotic signaling in renal epithelial cells [54]. As shown in Figures 5(a) and 5(b), our results showed that 6,8-diprenylorobol activated ERK and it might modulate apoptotic signaling pathways in Huh-7 and HepG2 cells, which was consistent with the previous references.
CYP2J2 is an epoxygenase enzyme, and its role is to metabolize arachidonic acid to epoxyeicosatrienoic acids [55]. It is reported that CYP2J2 is highly upregulated in various human carcinoma cell lines and CYP2J2 could promote human cancer metastasis and tumor cell growth [56,57]. According to a previous study, the doxorubicin-induced reduction of viability was markedly attenuated by upregulation of CYP2J2 expression. The increase in the Bax/Bcl2 ratio and the decrease in procaspase-3 expression level were also recovered by CYP2J2 upregulation [20]. Therefore, CYP2J2 may be an important target to develop the effective therapeutic methods for cancers. In fact, we have reported that acetylshikonin or broussochalcone A has anticancer activity in HCC cells through the inhibition of CYP2J2 [47,48]. We observed that 6,8-diprenylorobol inhibited CYP2J2mediated astemizole O-demethylase and ebastine hydroxylase activity with an IC 50 value of 7.33 μM, which is comparable to the IC 50 values of decursin (IC 50 = 6:95 μM) [58], thelephoric acid (IC 50 = 3:23 μM) [59], and tanshinone IIA (IC 50 = 2:5 μM) [60], in a noncompetitive way. The