MDA19, a novel CB2 agonist, inhibits hepatocellular carcinoma partly through inactivation of AKT signaling pathway

Background CB2 (cannabinoid receptor 2) agonists have been shown to exert anti-tumor activities in different tumor types. However, there is no study exploring the role of MDA19 (a novel CB2 agonist) in tumors. In this study we aimed to investigate the effects of MDA19 treatment on HCC cell lines, Hep3B and HepG2 and determine the relevant mechanisms. Results Cell proliferation analysis, including CCK8 and colony formation assays, indicated that MDA19 treatment inhibited HCC cell proliferation in a dose- and time-dependent manner. Flow cytometry suggested that MDA19 induced cell apoptosis and activation of mitochondrial apoptosis pathway. Transwell assay indicated that HCC cell migration and invasion were significantly inhibited by MDA19 treatment. Mechanism investigation suggested that MDA19 induced inactivation of AKT signaling pathway in HCC cells. In addition, we investigated the function of CB2receptor in HCC and its role in the anti-tumor activity of MDA19. By searching on Kaplan-Meier plotter (http://kmplot.com/analysis/), we found that HCC patients with high CB2 expression had a better survival and CB2 expression was significantly associated with gender, clinical stages and race of HCC patients (P < 0.05). CB2 inhibited the progression of HCC cells and its knockdown could rescue the growth inhibition induced by MDA19 in HCC. Moreover, the inhibitory effect of MDA19 on AKT signaling pathway was also reversed by CB2 knockdown. Conclusion Our data suggest that MDA-19 exerts an anti-tumor activity at least partly through inactivation of AKT signaling pathway in HCC. CB2 functions as a tumor suppressor gene in HCC, and MDA19-induced growth inhibition of HCC cells depends on its binding to CB2 to activate it. MDA-19 treatment may be a promising strategy for HCC therapy. Reviewer This article was reviewed by Tito Cali, Mohamed Naguib and Bo Chen.


Background
Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide and ranks as the third leading cause of cancer-related deaths, killing more than 600,000 people each year [1,2]. Previous reports have identified many risk factors, including alcohol use, liver cirrhosis, hepatitis. B/C virus (HBV/HCV) infection and environmental pollution [3]. Current therapies for HCC mainly include liver resection and transplantation, percutaneous ablation, and chemotherapy [1,3]. However, the treatment status of HCC patients still remains unsatisfactory and the The five-year survival rate for HCC patients is only 3-5% [4][5][6]. Therefore, it is increasingly important to explore the regulatory mechanisms of HCC carcinogenesis and identify effective therapeutic targets.
In recent years it has been found that Cannabinoids exert anti-tumor activities by inducing tumor cell death, cell cycle arrest or inhibiting tumor angiogenesis [7,8]. The function of Cannabinoids depends on its interaction with the endocannabinoid system (ECS), including CB 1 or CB 2 . CB 1 is mainly expressed in the central nervous system whereas CB2 is expressed in peripheral or immune tissues [9]. N′-[(3Z)-1-(1-hexyl)-2-oxo-1, 2-dihydro-3H-indol-3-ylidene] benzohydrazide (MDA19), as a selective agonist for CB 1 and CB 2 , has been demonstrated to play an important role in relieving neuropathic pain without a potential adverse effect on the central nervous system [10]. According to a previous study, MDA19 displayed 4-fold-higher affinity at the human CB 2 than at the human CB 1 receptor and nearly 70-fold-higher affinity at the rat CB 2 than at the rat CB 1 receptor [11]. At present, some agonists of CB 2 or CB 1 have been shown to exert anti-tumor activity in some tumor models [12][13][14]. However, there is currently no study on the role of MDA19 in tumors.
In this study, we investigated the effects of MDA19 on tumor-related phenotypes in Hep3B and HepG2 HCC cells. AKT signaling pathway was investigated to explain the action mechanism of MDA19 in HCC. In addition, we investigated the prognostic value of CB 2 in HCC and its correlation with clinical pathological parameters of patients. The functions of CB 2 in HCC were also investigated by using siRNA technology. Finally, we evaluated the role of CB 2 in the anti-tumor activity of MDA19 in HCC.

Cells culture and treatment
HCC cell lines, Hep3B and HepG2, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM (HyClone, Logan, UT) medium supplemented with 10% fetal bovine serum (FBS, GibcoBRL; Grand Island, NY, USA) at 37°C with 5% CO 2 . MDA19 (Cat# HY-15451, Med-ChemExpress, USA) was dissolved in DMSO and then diluted with DMEM to a specific concentration to incubate with HCC cells. siRNA targeting CB 2 and a negative control siRNA (siNC) were synthesized by Guangzhou RiboBio Co., Ltd. and transfected into HCC cells by using Lipofectamine2000 liposomes (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The sequences of siRNAs were as follows: CB 2 siRNA1: 5′-CCAGGTCAAGAAGGCCTTT-3′; CB 2 siRNA2: 5′-GCTTGGATTCCAACCCTAT-3′; CB 2 siRNA3: 5′-CCTGGCCAGTGTGGTCTTT-3′; siNC: 5′-UUCUCCGAACGUGUCACGUTT-3. Clone formation assay: HCC cells were treated with MDA19 or siRNA transfection for 48 h. Then cells were collected and seeded in a 6-cm dish at a total number of 200~300. Cells were continually cultured until clones could be observed in naked eyes. After stained with 0.1% crystal violet for 20 min and washed with PBS twice, HCC cells were imaged and counted.

Migration and invasion analysisBDI90241
For invasion assay, Matrigel-coated transwell inserts (BD Biosciences, San Jose, CA, USA) were prepared. Then, HCC cells treated with MDA19 or siRNA transfection were seeded into the upper chambers at a number of 1 × 10 4 and the lower chambers were added with serum-free medium. After incubation for 48 h, non-invaded cells on the upper surface of transwell membrane were removed by a cotton swab and invaded cells were fixed with 4% paraformaldehyde for 30 min, stained with crystal violet for 30 min, and counted under a microscope. Migration assay was similar to the invasion assay except that transwell chambers weren't pre-coated with Matrigel and the number of seeded cells was 5 × 10 3 .

Apoptosis analysis
Cell apoptosis was analyzed by a typical PI-AnnexinV-FITC assay. Briefly, after treated with MDA19 for 48 h or siRNA transfection for 48 h, HCC cells were starved in serum-free medium for 24 h. The cells in control group were also starved in serum-free medium for 24 h. After that, cells were washed with PBS and then mixed with PI-AnnexinV-FITC for 15 min in the dark. Cell apoptosis was detected by a flow cytometer (BD FACSC anto II, BD Biosciences, USA). The data were analyzed using FlowJo software (Tree Star).

Western blot assay
Total proteins of cells were extracted by using ice-cold RIPA Lysis Buffer (CWBIO, Beijing, China) added with 1% protease inhibitor. Protein concentrations were determined by using BCA Protein Assay Kit (Beyotime, China). Equal amounts of proteins (20 μg) from each sample were subjected to a 10% SDS-PAGE electrophoresis and transferred onto a PVDF membrane (Millipore, Bedford, USA). After blocking in 5% non-fat milk for 1 h, protein bands were successively incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 h at room temperature. After being washed three times, protein bands were developed by using chemiluminescence detection kit (Thermo Scientific, USA). The primary antibodies against Bcl2

Statistical analysis
All statistical analysis was performed by using GraphPad Prism 7.0 software (GraphPad Software Inc., La Jolla, CA, USA). Comparison of two groups was subjected to unpaired Student's t-test, indicated by t value. One-way ANOVA was performed to compare three or more groups followed by Turkey's post hoc test. P < 0.05 was considered statistically significant.

MDA19 inhibited proliferation of HCC cells in a dose-and time-dependent manner
As shown in Fig. 1a, cell proliferation of Hep3B and HepG2 was inhibited by MDA19 in a dose-dependent manner. The significant cytotoxic effect of MDA19 was starting at concentration of 10 μM for both HCC cell lines. At 50 μM, cell viability of Hep3B and HepG2 cells was significantly inhibited by 49 and 41% respectively.
The calculated IC50 values were 56.69 μM and 71.13 μM for Hep3B and HepG2 cells, respectively. 30 μM for Hep3B cells and 40 μM for HepG2 cells were used for the following experiments. The result in Fig. 1b indicated that MDA19 inhibited the proliferation of Hep3B and HepG2 cells in a time-dependent manner. At 72 h, cell viability was respectively inhibited to 58 and 50% in Hep3B and HepG2 cells. Colony formation ability of HCC cells was also significantly inhibited by MDA19. Colony number of MDA19 treatment group was decreased to 37 and 18% in Hep3B and HepG2 cells respectively (Fig. 1c). The results suggested that MDA19 inhibited HCC cell proliferation in a dose-and timedependent manner.

MDA19 induced apoptosis of HCC cells by activating mitochondrial apoptosis pathway
In order to determine whether MDA19-induced growth inhibition was mediated by cell apoptosis, a PI-Annex-inV-FITC assay was performed. As shown in Fig. 2a, the percentage of apoptotic cells was increased from 11.36% of control group to 26.1% of MDA19 group for Hep3B cells and 12.78 to 41.5% for HepG2 cells. Next, we investigated whether mitochondrial apoptosis pathway was affected by MDA19 treatment. The results indicated that MDA19 treatment significantly increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl2 in both Hep3B and HepG2 cells (P < 0.05, Fig. 2b). Moreover, Caspase3, the apoptotic executioner, was also significantly down-regulated (P < 0.05, Fig. 2b). Taken together, these data suggested that MDA19 promoted HCC cell apoptosis by activating mitochondrial apoptosis pathway.

MDA19 inhibited migration and invasion of HCC cells
Invasion and migration are important features of cancer cells, which frequently initiate tumor metastasis in vivo. To further investigate whether MDA19 affected cell mobility of HCC, a transwell assay was performed. As shown in Fig. 3a, HCC cell migration was significantly inhibited by MDA19, inhibitory rate reaching 76.6% for Hep3B cells and 27.5% for HepG2 cells (P < 0.05). Figure 3b revealed that MDA19 inhibited Hep3B cell invasion by 79.8% and HepG2 by 30.5%. Thus, it was suggested that MDA19 restrained the invasive and migratory abilities of human HCC cells.

MDA19 inactivated AKT signaling pathway in HCC cells
We next explored the mechanism underlying the cellular phenotype alterations induced by MDA19 treatment in HCC. AKT signaling pathway is widely reported to participate in tumor cell proliferation, survival and malignant development [15][16][17]. As shown in Fig. 3c, phosphorylation level of AKT was significantly decreased in MDA19 treated HCC cells (P < 0.05). Cyclin D1 and CDK4/6 are downstream proteins of p-AKT, which form a complex to control cell cycle progression [18]. It was suggested that MDA19 treatment inhibited the expression of Cyclin D1 and CDK4/6 significantly (P < 0.05, Fig. 3). Taken together, these data suggested Then the clone formation ability of HCC cells was detected. All experiments were performed at 3 times. *P < 0.05 that MDA19 treatment inhibited HCC progression at least partly through inactivation of AKT signaling pathway.
High expression of MDA19 receptor CB 2 predicted a better prognosis in HCC MDA19 was reported as an agonist for CB 2 , so we further investigated the function of CB 2 in HCC. The Kaplan-Meier plotter (http://kmplot.com/analysis/) is an online analyzing tool for evaluating the survival effect of a gene or combination of genes in breast, ovarian, lung, liver and gastric cancers [19]. The Kaplan-Meier survival curves for HCC patients with low and high CB 2 expression were shown in Fig. 4a, which illustrated better prognosis for those HCC patients with high CB 2 expression. As shown in Fig. 4b, the expression of CB 2 was significantly correlated with gender, clinical stages and race of HCC patients (P < 0.05). Taken together, these observations suggested that CB 2 expression was associated with a better prognosis of HCC patients.

CB 2 knockdown reversed the inhibition of HCC cell proliferation induced by MDA19
To comprehensively evaluate the effect of the interaction between MDA19 and CB 2 on HCC cells, we used RNAi technology to reduce the expression of CB 2 in HCC cells, and combined it with MDA19 treatment. As shown in Fig. 4c, compared to NC group, the mRNA expression of CB 2 was significantly inhibited by siRNAs in both Hep3B and HepG2 cells. siRNA1 was used for the following experiments. The inference effects were also validated on protein level using western blot assay (Fig. 4d). Figure 4e indicated that CB 2 knockdown significantly promoted cell proliferation compared with NC group. Moreover, CB 2 knockdown reversed the inhibition of HCC cells by MDA19, which proved that the anti-tumor activity of MDA19 was mediated by activating CB 2 (Fig. 4e). These data suggested that CB 2 functions as a negative regulator in HCC proliferation and the anti-tumor activity of MDA19 was dependent on CB 2 expression.

CB 2 knockdown inhibited cell apoptosis of HCC
Cell apoptosis analysis was used to determine the effect of CB 2 knockdown on HCC cell survival. HCC cells were starved in serum-free medium for 24 h to induce cell apoptosis. As shown in Fig. 5a, compared with NC group, CB 2 knockdown significantly reduced apoptosis percentage in HCC cells (P < 0.05). Mitochondrial apoptosis pathway was also examined by western blot assay. As expected, the expression of anti-apoptotic =protein Bcl-2 was up-regulated in CB 2 -KD group, while pro-apoptotic proteins Caspase3 were down-regulated (P < 0.05, Fig. 5b). Furthermore, we found that CB 2 -KD could reverse the effects of MDA19 on the expression of apoptosis-related protein expression (Fig. 5b). These data suggested that CB 2 knockdown inhibited HCC cell apoptosis through inactivation of mitochondrial-dependent apoptosis pathway and the pro-apoptotic effects of MDA19 on HCC cells might be mediated by CB 2 .

CB 2 knockdown promoted cell mobility in HCC and activated AKT signaling pathway
The effect of CB 2 knockdown on HCC cell mobility was determined by a transwell assay. As shown in Fig. 6a, CB 2 knockdown significantly promoted cell migration in Hep3B and HepG2 cells. Figure 6b revealed that CB 2 knockdown also promoted Hep3B cell invasion by 2 fold and HepG2 by 2.5 fold. Thus, it was suggested that CB 2 knockdown increased the mobility of HCC cells.
We further investigated whether CB 2 was also involved in the regulation of AKT signaling pathway. As shown in Fig. 6c, it was suggested that p-AKT and Cyclin D1 were both up-regulated by CB 2 knockdown. Furthermore, CB 2 -KD reversed the inhibitory effect of MDA19 on AKT signaling pathway in both Hep3B and HepG2 cells (Fig. 6c). These data indicated that CB 2 knockdown could activate AKT signaling pathway and MDA19 functioned as a negative regulator of AKT pathway through interaction with CB 2 .
Cell proliferation analysis showed that MDA19 treatment inhibited cell viability in a dose-and time-dependent Apoptosis analysis showed that MDA19 treatment significantly increased the proportion of apoptosis in HCC cells, and the induction of apoptosis was mediated by activation of mitochondrialdependent apoptosis pathway, including increased Bax and Caspase3 and decreased Bcl2. When mitochondrialdependent apoptosis pathway is activated, increased Bax moves to the mitochondrial outer membrane and multimerizes, forming membrane channels that stimulate mitochondria to release cytochrome C (Cyt C) [23,24]. Cyt C triggers cell apoptosis through Caspase9/3 cascade reaction [23,24]. Bcl2 exerts anti-apoptosis effect by antagonizing Bax in this process [23,24]. In addition, some CB 2 agonists have also been reported to exert a suppressive effect on tumor metastasis [12][13][14]. Here by using transwell assay we reported that MDA19 significantly inhibited HCC cell migration and invasion.
Several mechanisms of the anti-tumor activity of CB 2 agonists have been reported, including PI3K/AKT inhibition [25], modification of metalloproteinases [21], induction of reactive oxygen species [26], MAPK modulation [20] and so on. Here, our data showed that MDA19 treatment led to inactivation of AKT signaling pathway in HCC cells. It is widely held that AKT signaling pathway plays a vital role in maintaining cell proliferation, survival and cell cycle progression [27,28]. And increasing studies demonstrate that inactivation of AKT signaling pathway inhibits tumor growth and enhances the sensitivity of tumor cells to chemotherapy drugs such as cisplatin and temozolomide [29][30][31], suggesting that inhibition of AKT pathway may be a promising strategy for HCC treatment.
Emerging evidences suggest that the cannabinoid receptor CB 2 can be an anti-tumor target in several types of cancers [32][33][34]. For example, Cianchi et al. report that CB 2 activation induces apoptosis through tumor necrosis factor alpha-mediated ceramide de novo synthesis in colon cancer cells [33]. Moreover, Khan et al. report that CB 2 is involved in inducing cell cycle arrest and apoptosis in renal cell carcinoma [35]. We would like to know whether MDA19, an agonist of CB 2 , exerts its anti-tumor activity by interacting with CB 2 . We investigated the relationship between CB 2 expression and the survival rate of HCC patients on Kaplan-Meier plotter (http://kmplot.com/analysis/), suggesting that the survival rate of HCC patients with high expression of CB 2 was significantly higher than that of patients with low expression of CB 2 . Moreover, cell viability of CB 2 silenced HCC cells was significantly higher than that of control group. These results indicated that CB 2 exerted an anti-tumor function in HCC. In addition, we examined the effect of CB 2 knockdown on proliferation, apoptosis, invasion, and migration in HCC cells. The results showed that knockdown of CB 2 could reverse MDA19-induced growth inhibition in HCC cells. Mechanism studies suggested that CB 2 knockdown also inhibited mitochondrial-dependent apoptosis pathway and The data were analyzed using FlowJo software.; (b) The expression of apoptosis related proteins Bcl2 and Caspase3 was detected by western blot and analyzed by Image J software. All experiments were performed at 3 times. *P < 0.05 activated AKT signaling pathway. These findings suggested that CB 2 played an anti-tumor role in HCC, which is consistent with the previous studies.

Conclusions
In conclusion, these data suggest that MDA-19 exerts an anti-tumor activity at least partly through inhibiting AKT signaling pathway in HCC. CB 2 functions as a tumor suppressor gene in HCC, and the anti-tumor activity of MDA19 depends on its binding to CB 2 to activate it.

Reviewers' comments
Reviewer's report 1 Tito Cali Reviewer comments: Reviewer1: MDA19, a novel CB2 agonist, inhibits hepatocellular carcinoma through inactivation of AKT signalling pathway This manuscript investigates the inhibitory role of the cannabinoid receptor 2 (CB2) agonist MDA19 in cancer progression, by focusing on hepatocellular carcinoma (HCC) cell lines (Hep3B and HepG2).
Rao, M. and colleagues demonstrate that MDA19 treatment inhibited HCC cell proliferation (dose-and time dependently), migration and invasion. They further show that growth inhibition might be mediated by cell apoptosis and activation of mitochondrial dependent pathway. Mechanistically, MDA19 is proposed to perform its action through inactivation of the AKT pathway. The MDA19-induced phenotype changes observed in HCC cells, including activation of the AKT signalling pathway, could be reversed by CB2 knockdown. Lastly, and interestingly, the authors also found a direct and positive correlation between CB2 expression and the survival of HCC patients as well as association with clinical factors such as gender, clinical stages and race. The authors conclude that MDA-19 exerts an anti-tumor activity through inactivation of AKT signalling pathway in HCC, therefore MDA-19 treatment may be a promising strategy for HCC therapy. As a general comment, with few exceptions, the overall manuscript is of immediate understanding. Concepts and descriptions are explained in a satisfactory manner and the Results section, although sometimes too succinct, is somehow in line with the experimental data. The Discussion is pertinent with the Results and the topic, and takes into account the recent literature in the field. The experimental data provided seem to be sufficient to support some of the conclusions drawn by the authors, nevertheless, there are some major points to be addressed. Major Points.
1. The experiments shown in Fig. 1a are performed at 24 h (see methods section), under these conditions the authors state that "The significant cytotoxic effect of MDA19 was starting at concentration of 10 μM for both HCC cell lines…. 30 μM for Hep3B cells and 40 μM for HepG2 cells were used for the following experiments". At 24 h, 30 μM for Hep3B cells and 40 μM for HepG2 are sufficient to induce a strong cytotoxic effect as shown in Fig. 1a, nevertheless, this is not found in Fig. 1b under the same experimental conditions where the effect at 24 h with the above-mentioned concentrations of MDA19 is not statistically significant. How do the authors explain this apparent discrepancy?
2. Concerning the figure legends, they should all be carefully revised since many details are lacking and the experiments are not described in a satisfactory manner. Please include the important information such as the time of treatment, concentrations and also briefly describe the experiment since the results section is sometimes too descriptive or not enough exhaustive.
3. The dose of MDA looks quite high (30-40 micromolar). This is important when considering the compound as promising for therapy. The authors should perform additional experiments to exclude extra side effects by looking at signalling pathways and/or cellular processes supposed not to be affected by the compound. MDA19 is a selective agonist of the human peripheral cannabinoid (CB2) receptor, the EC50 value for activation is reported to be 63.4 nM, well below the micromolar concentration used in this study. I think that this cast doubts about the specificity of the observed phenotypes and the therapeutic potential of the compound. 4. Is the compound cell permeant? The molecule is lipophilic so I think that the authors should perform experiments with selective CB2 antagonist such as AM630 to assess the effectiveness and the specificity of this compound for the observed phenotypes.
5. For the CB2 knockdown, the results should be replicated by using at least three independent specific siRNA and a scramble siRNA as further control. Additionally, can the authors abolish (or at least strongly reduce) the effect of MDA19 treatment in the CB2-KD cells? The western blots shown in Figs. 5b and 6c should be performed by adding a CB2-KD treated group as done for the experiments shown in Fig. 4d. 6. On the same line, HCC cells also express CB1 receptor, do the authors observe the same effect by downregulating the CB1 receptor in the presence and/or in the absence of MDA19? 7. Along this line, can the authors phenocopy the effect of CB2 knockdown by treatment of the cells with specific CB2 antagonists (e.g., AM630 or others)?
8. In the Western blot shown in Fig. 2b, the intensity of the Bcl2 and Caspase3 bands in the control group is much higher than that reported in Fig. 5b for the same condition. This could be due to the fact that different exposure times are used. To increase the clarity of the phenotype and to better understand the relative levels of the markers taken into account between the two cell lines the western blots and the quantifications should be done by loading the Hep3B and the HepG2 lysates on the same membrane. This is true for Figs. 2, 3, 4 and 5. This is also due to the fact that Hep3B and HepG2 cells are also quite different in terms of aggressiveness, metabolic and growth rate, ability to form tumors in mice, presence absence of p53 mutations etc. 9. Last but not least there is an important concern to be addressed: the authors show an effect on mitochondrial targets (but not all of them) and AKT pathway, but there is an important control that should be performed, i.e., the lack of effect on unrelated pathways. For example, are markers of the extrinsic pathway unchanged? is there any ER stress ongoing? Are the markers (phosphorylation status) of pathways other than AKT unchanged? To state that the AKT pathway and/or that the mitochondria-mediated apoptosis are involved the authors should show that other signalling pathways are indeed unchanged. In conclusion, the potential of MDA19 in cancer therapy is interesting but some concerns regarding the experimental data should be carefully edited before to consider the paper of interest for publication in Biology Direct.
10. Page 7 end of line 13 Cone please correct with clone. Page 7 middle of line 10 "treatment with MDA inhibited Hep3B and HepG2,,," is not clear what is inhibited, growth? Colony formation?
Author's response: 1. We are very sorry that we made a mistake on the description of Dose-dependent assay method. The treatment time of MDA19 was 48 h, rather than 24 h.
2. The figure legends have been revised and added the necessary information such as experiment description.
3. We have read the article "Design and Synthesis of a Novel Series of N-Alkyl Isatin Acylhydrazone Derivatives that Act as Selective Cannabinoid Receptor 2 Agonists for the Treatment of Neuropathic Pain" reported by Diaz et al. MDA19 exhibited a low EC50 value for activation (63.4 nM) for CB2 receptor. It is very different from our study. As the reported experiments were based on Chinese hamster ovarian cells selectively expressing the human CB2 receptor, we believe that this difference may