Inhibition of protein kinase C by isojacareubin suppresses hepatocellular carcinoma metastasis and induces apoptosis in vitro and in vivo

Targeted inhibition of protein kinase C (PKC) inhibits hepatocellular carcinoma (HCC) proliferation and metastasis. We previously reported the cytotoxicity of a series of synthetic phenyl-substituted polyoxygenated xanthone derivatives against human HCC. In the current study, the most potent natural product, isojacareubin (ISJ), was synthesized, and its cellular-level antihepatoma activities were evaluated. ISJ significantly inhibited cell proliferation and was highly selective for HCC cells in comparison to nonmalignant QSG-7701 hepatocytes. Moreover, ISJ exhibited pro-apoptotic effects on HepG2 hepatoma cells, as well as impaired HepG2 cell migration and invasion. Furthermore, ISJ was a potent inhibitor of PKC, with differential actions against various PKC isotypes. ISJ selectively inhibited the expression of aPKC (PKCζ) in the cytosol and the translocation of cytosolic PKCζ to membrane site. ISJ also directly interacted with cPKC (PKCα) and nPKC (PKCδ, PKCε and PKCμ) and thereby inhibited the early response of major MAPK phosphorylation and the late response of HCC cell invasion and proliferation. In a hepatoma xenograft model, ISJ pretreatment resulted in significant antihepatoma activity in vivo. These findings identify ISJ as a promising lead compound for the development of new antihepatoma agents and may guide the search for additional selective PKC inhibitors.


Results
ISJ synthesis. For ISJ synthesis, the construction of the xanthone nucleus and the benzopyran ring were performed based on our previous work 33,34 . The retrosynthetic analysis of ISJ is outlined in Fig. 1a. With this approach, the important intermediate, compound 7, was synthesized in five steps from the commercially available starting materials, benzoic acid (compound 1), with an approximately 45% yield. Then, an efficient method to remove the dibenzyl group completed the total synthesis of ISJ. The synthetic route of ISJ is shown in Fig. 1b.
First, compound 1 was treated with thionyl chloride, affording compound 2. A Friedel-Crafts acylation reaction and removal of the 2-methyl group produced the methanone 4 in good yield (65%, over two steps). Converting compound 4 to 5 was conveniently achieved by base-catalyzed cyclization with an 83% yield. Compound 6 was obtained by removing all the methyl groups of compound 5 (refluxing in HBr-HAc, 17 h) with a yield of 97%. Dichlorodiphenylmethane protected the O-hydroxy group of compound 6, and target compound 7 was obtained with an 85% yield. As the 1-OH of compound 7 can form a hydrogen bond with the adjacent carbonyl group, the propargylic ether, compound 8, was selectively obtained in the presence of KI and K 2 CO 3 with a catalytic amount of CuI at a yield of 63%. A p-tosyl group was attached to the free 1-OH of compound 8 to provide the sulfonate, compound 9, which underwent thermal cyclization to selectively furnish the angular isomer compound 10 in DMF at 150 °C.
Because of its existing olefinic bond, the benzyl group cannot be removed by means of the conventional catalytic hydrogenation method (Pd/C, H 2 ). Hydrogenation of the double bond is avoided by refluxing in HAc-H 2 O 35 . However, the yield with this approach is approximately 20%. Therefore, other organic acids were screened in different solvents, including p-toluenesulfonic acid and camphorsulfonic acid (CAS). The optimal reaction was then obtained by refluxing in MeOH-THF (1:1) for 7 h, with a yield of more than 80%. Finally, removing the dibenzyl and p-toluenesulfonyl groups of compound 10 gave the natural product ISJ an overall yield of 19% for ten steps.
In vitro antiproliferative activity. A preliminary screen of ISJ and its structural analogs (compounds 5-17) against HepG2 and QGY-7703 cells was performed at an initial concentration of 50 μ M. The results are shown in Table 1. Among these derivatives, compound 7 and ISJ showed greater efficacy than the other compounds against HCC cells. As a result, a second screen was performed, the IC 50  Effects of compound 7 and ISJ on HCC cell apoptosis. To further explore the potential mechanisms of the antiproliferative effects induced by compound 7 and ISJ, Annexin V-FITC/PI double  staining was performed to quantify cellular apoptosis by flow cytometry. HepG2 cells were cultured for 48 h in the absence or presence of compound 7 or ISJ at different concentrations (5 or 15 μ M). Treatment with a low concentration (5 μ M) of compound 7 or ISJ did not alter the number of apoptotic cells compared to the control group. In contrast, the percentage of apoptotic cells increased significantly after treatment with higher concentrations of compound 7 or ISJ (15 μ M) (Fig. 2a). These results demonstrate that compound 7 and ISJ induce apoptosis in HepG2 cells, which at least partially contributes to their antiproliferative activity.

Inhibitory effects of compound 7 and ISJ on HCC cell migration and invasion. The effects
of compound 7 and ISJ on cell mobility were investigated using wound healing and Matrigel invasion assays. In the wound healing assay, 24-h treatment with 10 μ M compound 7 or ISJ strikingly decreased HepG2 cells migration at the edge of the exposed regions by 76.4% and 77.8% (P < 0.05), respectively, when compared to the control cultures (Fig. 2b,d). In the invasion assay, the invasivenesses of compound 7-and ISJ-treated HepG2 cells (24 h, 10 μ M) were significantly inhibited by 69% and 76% (P < 0.05), respectively, when compared to the HepG2 control cells (Fig. 2c,e). Thus, treatment with compound 7 and ISJ effectively inhibited HepG2 cell migration and invasion.
Inhibitory effects of compound 7 and ISJ on PKC activity and expression. The activity of PKC in HepG2 cells was assessed 3 h after the addition of compound 7 or ISJ by measuring the phosphorylation of a PKC-specific target peptide. Compared to the basal control (in the absence of any compound), PKC activity was inhibited by compound 7 and ISJ. Both compound 7 and ISJ displayed greater inhibitory potencies at the higher concentration of 10 μ M compared to 5 μ M, which was particularly evident for ISJ at 10 μ M (Fig. 3a, lane 6). At concentrations of 10 μ M and 5 μ M, compound 7 inhibited PKC activity by 54.1% and 40.9%, respectively, compared to the basal control; the comparable inhibition rates for ISJ were 77.7% and 52.3%, respectively (Fig. 3b).
To further determine whether compound 7 and ISJ represent potential candidates for selective PKC expression inhibitors against hepatoma, the expression of PKC isoforms in HepG2 cells was characterized    Binding properties of ISJ to PKC isoforms and targeting effects. To identify whether ISJ and compound 7 target PKC in cells, we performed pull-down experiments using ISJ-and compound 7-coupled Sepharose beads with HepG2 cell lysates. Endogenous cPKC (PKCα ) and nPKC (PKCδ , PKCε and PKCμ ) in HepG2 lysates were pulled down by ISJ-coupled CNBr-activated Sepharose 4B beads (Fig. 5a). Additionally, the presence of free ISJ (20 μ M) resulted in an obvious decline in the amounts of cPKC and nPKC captured by the ISJ-coupled 4B beads (Fig. 5b), indicating that free ISJ competes with conjugated ISJ for binding to cPKC and nPKC, which suggests that the binding is saturated. Additional PKC targeting effects involved in the early response of major MAPK phosphorylation and the late response of HCC cell invasion and apoptosis were investigated. After 24 h of treatment with ISJ at the indicated concentrations, the total protein lysates from HCC cells were analyzed (Fig. 6). ISJ inhibited the phosphorylated, activated forms of c-Raf, MEK1/2 and ERK1/2 in the MAPK signaling pathway in both HepG2 and QGY-7703 cells (Fig. 6a). Furthermore, the expression levels of matrix metalloproteases 9 (MMP-9) and fibroblast growth factor receptor 1 (FGFR-1) were markedly reduced by ISJ. In contrast, E-cadherin expression was elevated, specifically at the highest concentration of ISJ (20 μ M) (Fig. 6b). In addition, we analyzed several representative apoptotic proteins, including cleaved PARP (c-PARP), p53 and p21. The expression levels of these proteins were increased, specifically 20 μ M ISJ strongly induced cellular apoptosis (Fig. 6c). These findings support our hypothesis that ISJ restrains HCC cell metastasis and induces HCC cell apoptosis by specifically inhibiting PKC.
In vivo antitumor activities of compound 7 and ISJ. To evaluate the antitumor activities of compound 7 and ISJ in vivo, human HCC xenografts were established by subcutaneous injection of HepG2 cells into the backs of nude mice. After the treatment, the body weights of the mice were monitored and recorded at the indicated days. Compared to the vehicle-treated controls, there was no significant change in body weight in the compound 7-and ISJ-treated groups. Compound 7 and ISJ treatment at a dose  (Fig. 7).
At the end of the animal experiment, the control and experimental mice were sacrificed, and the tumors were removed and histologically evaluated by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis. Apoptosis was induced by compound 7 and ISJ in xenograft tumors, and H&E staining revealed significant tissue death in compound 7-and ISJ-treated tumors compared to the tumors from mice treated with the vehicle controls (Fig. 8a). IHC analysis of the tumors revealed upregulated expression of c-PARP, cleaved caspase (c-Cas) 3 and c-Cas9 apoptotic protein levels in the tumors treated with compound 7 and ISJ, whereas the vehicle control tumors showed much weaker staining (Fig. 8b,c). To further validate these in vivo results, an immunoblotting assay was used to detect c-PARP, c-Cas3 and c-Cas9 in tumor tissue samples. Compared to the vehicle controls, the levels of these proteins were increased in compound 7-and ISJ-treated mice, and ISJ was a particularly strong inducer of apoptosis (P < 0.05, Fig. 8d,e).

Discussion
HCC is a leading cause of cancer-related mortality worldwide 36 . Chemotherapy has been an effective method of treating cancers for many years, especially for patients with unresectable tumors. However, the treatment options for HCC are limited, and conventional chemotherapeutic agents, including cyclophosphamide, cisplatin and fluorouracil, have severe side effects 37 . Therefore, the development of new chemotherapeutic drugs is of great significance. Plant-derived natural products and synthetic analogs are significant components of cancer chemotherapy and have been shown to suppress metastasis and induce apoptosis in malignant tumor cells while exerting low toxicity to normal cells and minimal adverse effects 38,39 .
In our previous studies, a new series of derivatives of phenyl-substituted polyoxygenated xanthone were synthesized and evaluated for in vitro cytotoxicity against HCC cells and hepatocytes 33,34 . In the present study, ISJ and compound 7 demonstrated potency at several cellular-level activities, thereby supporting the derivatives' antitumor effects. First, the natural product ISJ and its intermediate, compound 7, exhibited more efficacious antiproliferative activities for HCC cells and exerted no apparent toxicity to hepatocytes. Second, ISJ and compound 7 effectively induced HepG2 cell apoptosis, and third, they significantly suppressed the migration and invasion of HepG2 cells.
The PKC family of serine/threonine protein kinases contains multiple isoforms. PKC overexpression is correlated with the progression and prognosis of malignant diseases, including HCC [11][12][13] . To begin to address the regulatory mechanisms of ISJ and compound 7 on PKC, we examined their inhibitory effects using an in vitro kinase assay and found that ISJ exhibited stronger PKC inhibitory activity compared to compound 7. Different PKC isotypes are known to play distinct regulatory roles. Therefore, immunoblotting was conducted to characterize the expression of PKC isoforms in HepG2 cells and explore whether ISJ and compound 7 represent potential candidates for selective PKC expression inhibitors in hepatoma. Increased PKCζ expression in different types of human cancers supports the clinical correlation of PKCζ   as a tumor promoter, with high PKCζ activity predicting poor survival in several cancers [40][41][42] . Recently, an anti-apoptotic effect was attributed to PKCζ 43 , and PKCζ inhibition reduced tumor cell proliferation and increased pancreatic tumor necrosis 44 . Furthermore, specific inhibition of PKCζ through RNAi impaired breast cancer and glioblastoma cell invasion in vitro 45 and reduced prostate cancer cell invasion both in vitro and in vivo 46 . We observed a significant downregulation of PKCζ expression in the cytosolic and membrane fractions when HepG2 cells were exposed to ISJ. Therefore, ISJ likely acts as a selective inhibitor of PKC expression and further direct interaction investigations suggest that ISJ possess differential actions against different PKC isotypes. ISJ selectively inhibited the expression of aPKC (PKCζ ) in the cytosol and the translocation of cytosolic aPKC (PKCζ ) to the plasma membrane. Indeed, the translocation of PKC in response to agonists is an indicator of enzyme activation 47 . We also observed direct interactions between ISJ and cPKC (PKCα ) or nPKC (PKCδ , PKCε and PKCμ ).
The cascades downstream of PKC include Raf/MEK/ERK MAPK signaling pathways. The epithelial-to-mesenchymal transition (EMT), in which cells undergo transformation from an epithelial phenotype to a motile and invasive phenotype, is a critical step in the metastatic dissemination of malignant cells [48][49][50] . This phenomenon is connected with increased MMP activity and loss of E-cadherin expression 51 . Matrix degradation by MMP-9 is dependent on PKC activity 52,53 , and E-cadherin-mediated cell-cell adhesion plays important roles in cell polarity maintenance 54 . In HCC, the expression of E-cadherin is significantly downregulated, which is associated with tumor metastasis and prognosis 53 and the functional overexpression of E-cadherin in HCC cells significantly inhibits cell invasion 55,56 . In addition, FGFR-1 overexpression increases HCC cell proliferation and invasion, thereby facilitating the progression of HCC 57,58 . We investigated the PKC targeting effects of ISJ, specifically by examining proteins involved in the MAPK signaling pathways and those associated with invasion and apoptosis. Our results showed that ISJ inhibited the early response of major MAPK phosphorylation and the late response of HCC cell invasion and proliferation. Furthermore, in the hepatoma xenograft model, ISJ pretreatment showed potent antihepatoma activity in vivo.
Taken together, our current findings indicate that ISJ, the lead polyoxygenated xanthone-based PKC inhibitor, represents a novel and promising antitumor drug to target HCC.

Materials and Methods
Materials. ISJ  Cell proliferation assay. The cell viability assay was performed as previously reported using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) 33,34 . The relative cell viability was presented as % inhibition and calculated using the following formula: Apoptosis assay. HepG2 cells were seeded in six-well plates (5 × 10 5 cells/mL) for 24 h and then treated with various amounts of the compounds or 1% DMSO (as control). After 48 h, the cells floating in the medium were collected, and the adherent cells were detached with 0.05% trypsin, washed twice with cold PBS, and centrifuged at 1000 rpm for 5 min at 4 °C. Subsequently, HepG2 cells were gently resuspended in the binding buffer. Thereafter, the cells were stained using the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA). After incubation at room temperature for 15 min in the dark, the apoptotic cells were immediately analyzed by flow cytometry (FACS Calibur).
Migration and invasion assay. Cell migration and invasion potencies were studied using wound healing and Matrigel invasion assays. The wound healing assay was performed by scratching the cell surface with a 10 μ L pipette tip. Then, the cells were treated with compound 7 and ISJ (10 μ M) and incubated for 24 h before being photographed under a microscope (DMI3000 B, Leica). Wound closure was evaluated using ImageJ software. Invasion through a Matrigel-coated filter was measured in transwell chambers (Corning Costa) as described previously 59 , with minor modifications. Briefly, HepG2 Cells (2 × 10 5 /well) were suspended in serum-free medium and added to the upper chamber of the Matrigel Scientific RepoRts | 5:12889 | DOi: 10.1038/srep12889 plate. FBS (10%) was used as the chemoattractant in the lower chamber. After incubation for 24 h, the non-invading cells were removed from the upper surface of the insert membrane with a cotton swab. The invading cells were fixed with methanol and stained with Giemsa (Sigma). Cells in five randomly selected fields were photographed, and the numbers of cells that crossed the membrane were counted. PKC activity assay. The PKC activity assay was carried out as previously reported 33 , according to the instructions of the PepTag non-radioactive PKC assay kit (Promega) 60 . The bands were photographed using the Syngene GBOX-iCHEMI-XR system, and the phosphorylated bands were cut out for quantification measurement at 570 nm.
Western blot. The total membrane and cytosolic proteins were extracted using a Membrane and Cytosol Protein Extraction Kit (Beyotime). Extracted proteins from cells or animal tissues were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked with 5% nonfat milk and incubated with primary antibodies overnight, followed by incubation with a donkey anti-rabbit or anti-mouse secondary antibody (IRDye 800, LI-COR, Biosciences). The images were captured using the Odyssey Infrared Imaging System (LI-COR, Biosciences), according to the manufacturer's instructions. The intensity was quantitative with Quantity One software (Bio-Rad). To ensure equal protein loading, each membrane was stripped and reprobed with an anti-β -actin antibody. All experiments were repeated independently no less than three times. HepG2 cells were harvested and resuspended in PBS. A total of 5 × 10 6 cells were subcutaneously injected into the right flank. Tumor volume was calculated using the following formula: V = (L × W 2 )/2, where L is the length and W is the width of the tumor nodules measured with vernier calipers. Once the volume of the tumors reached 75-100 mm 3 , the mice were randomly divided into three groups (n = 5). The mice were treated every other day for two weeks with tail vein injections of either vehicle (5% ethanol and 2.5% polyethylene glycol in saline), compound 7, or ISJ (5 mg/kg in the vehicle). Body weights were measured before each drug injection. After the 15th day, the mice were euthanized, and the tumors were isolated, weighed and photographed.

Antitumor activity in vivo.
To further confirm the antitumor activities of compound 7 and ISJ in vivo, histological analysis was performed on the hepatoma xenografts. Briefly, the samples were fixed in a 10% formalin solution, processed, embedded, sectioned and either H&E stained to reveal tumor tissue necrosis or IHC stained using antibodies against c-PARP, c-Cas3 or c-Cas9. The H&E and IHC images were obtained on a Leica DMI3000 B phase-contrast microscopy. The immunoreactive areas in the IHC images were quantified using ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). The integrated optical density (IOD) values were represented as the mean ± SEM.

Statistical analysis.
All experiments were performed in triplicate and repeated at least twice, with representative experiments selected for figures. The intergroup data were compared using Student's t-test, and multivariate analysis was determined by one-way analysis of variance (ANOVA). SPSS software (version 16.0) was used for statistical tests with a minimal level of significance at P < 0.05.