Haprolid Inhibits Tumor Growth of Hepatocellular Carcinoma through Rb/E2F and Akt/mTOR Inhibition.

cancers Article Haprolid Inhibits Tumor Growth of Hepatocellular Carcinoma through Rb/E2F and Akt/mTOR Inhibition Jun Xing 1,2 , Vikas Bhuria 1, Khac Cuong Bui 1, Mai Ly Thi Nguyen 1, Zexi Hu 3, Chih-Jen Hsieh 1, Kathrin Wittstein 4, Marc Stadler 4, Ludwig Wilkens 5, Jun Li 6,7, Markus Kalesse 6,7, Przemyslaw Bozko 1,* and Ruben R. Plentz 1,8,* 1Department of Internal Medicine I, Medical University Hospital, Eberhard Karls Universität Tübingen, 72076 Tubingen, Germany; [email protected] (J.X.); [email protected] (V.B.); [email protected] (K.C.B.); dr[email protected] (M.L.T.N.); [email protected] (C.-J.H.) 2The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230001, China 3Department of Internal Medicine VIII, Medical University Hospital, 72076 Tübingen, Germany; [email protected] 4Department Microbial Drugs, Helmholtz Centre for Infection Research GmbH (HZI), 38124 Braunschweig, Germany; [email protected] (K.W.); mar[email protected] (M.S.) 5Institute of Pathology, Hannover Regional Hospital, 30043 Hannover, Germany; [email protected] 6Institute of Organic Chemistry, Leibniz University of Hannover, 30167 Hannover, Germany; [email protected] (J.L.); [email protected].de (M.K.) 7Centre of Biomolecular Drug Research, Helmholtz Centre for Infection Research GmbH (HZI), 38124 Braunschweig, Germany 8Department of Internal Medicine II, Klinikum Bremen Nord, 28755 Bremen, Germany *Correspondence: przemyslaw[email protected] (P.B.); [email protected] (R.R.P.); Tel.: +49-07071/29-83137 (P.B.); +49-0421/6606-1304 (R.R.P.); Fax: +49-07071/29-4680 (P.B.); +49-0421/6606-1720 (R.R.P.) Received: 1 December 2019; Accepted: 5 February 2020; Published: 6 March 2020   Abstract: Background: Hepatocellular carcinoma (HCC) represents a major health burden with limited curative treatment options. There is a substantial unmet need to develop innovative approaches to impact the progression of advanced HCC. Haprolid is a novel natural component isolated from myxobacteria. Haprolid has been reported as a potent selective cytotoxin against a panel of tumor cells in recent studies including HCC cells. The aims of this study are to evaluate the antitumor effect of haprolid in HCC and to understand its underlying molecular mechanisms. Methods: The efficacy of haprolid was evaluated in human HCC cell lines (Huh-7, Hep3B and HepG2) and xenograft tumors (NMRI-Foxn1 nu mice with injection of Hep3B cells). Cytotoxic activity of haprolid was determined by the WST-1 and crystal violet assay. Wound healing, transwell and tumorsphere assays were performed to investigate migration and invasion of HCC cells. Apoptosis and cell-cycle distribution were measured by flow cytometry. The effects of haprolid on the Rb/E2F and Akt/mTOR pathway were examined by immunoblotting and immunohistochemistry. Results: haprolid treatment significantly inhibited cell proliferation, migration and invasion in vitro . The epithelial–mesenchymal transition (EMT) was impaired by haprolid treatment and the expression level of N-cadherin, vimentin and Snail was downregulated. Moreover, growth of HCC cells in vitro was suppressed by inhibition of G1/S transition, and partially by induction of apoptosis. The drug induced downregulation of cell cycle regulatory proteins cyclin A, cyclin B and CDK2 and induced upregulation of p21 and p27. Further evidence showed that these effects of haprolid were associated with Rb/E2F downregulation and Akt/mTOR inhibition. Finally, in vivo nude mice experiments demonstrated significant inhibition of tumor growth upon haprolid treatment. Conclusion: Our results show that haprolid inhibits the growth of HCC through dual inhibition of Rb/E2F and Akt/mTOR pathways. Therefore, haprolid might be considered as a new and promising candidate for the palliative therapy of HCC. Cancers 2020,12, 615; doi:10.3390/cancers12030615 www.mdpi.com/journal/cancers Cancers 2020,12, 615 2 of 17 Keywords: HCC; haprolid; Rb/E2F; Akt/mTOR; EMT; cell cycle; apoptosis 1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancers globally and its incidence and mortality rate have still been increasing over the past decades [ 1 ]. More than half of all HCC patients are diagnosed at advanced stage when curative treatments are not applicable [ 2 ]. Currently, palliative first-line treatments with tyrosine kinase inhibitors like sorafenib or lenvantinib are available [ 3 ]. Data about immune checkpoint inhibitors are also promising, but new treatment approaches with less side-effects and good response are still needed to overcome this deadly disease [3,4]. Haprolid was isolated from the myxobacterium Byssovorax cruenta in 2000. Haprolid has been reported as an effective cytotoxic compound against many tumor cell lines including HCC cells and its IC50 values are within the nanomolar range [ 5 , 6 ]. In this study, we aimed to assess the efficacy of haprolid in HCC and to elucidate its underlying molecular mechanisms of function, thereby providing a rationale for testing it as a novel anticancer drug in clinical trials. This study is the first report for the application of haprolid in an in vitro and in vivo model of HCC. Many primary pathways altered in human HCC have been described. The retinoblastoma tumor suppressor (Rb) pathway is frequently disrupted in HCC [ 7 , 8 ]. Classically, Rb functions as a central player in cell cycle regulation [ 9 , 10 ]. Mitogenic signals are conveyed through cyclin-dependent kinases (CDKs) which subsequently regulate Rb phosphorylation. Phosphorylated Rb disassociates with E2F transcription factors, a family of cell-cycle-regulated transcription factors which stimulate proliferation, and afterwards initiates the transcription of E2F responsive genes. Rb, p107 and p130 are homologous proteins collectively known as the ‘Rb pocket protein family’ which contributes to the regulation of E2F-responsive genes [ 11 , 12 ]. The key role of the Rb/E2F pathway in regulating cell cycle progression and controlling proliferation offers tremendous potential for the development of therapeutics. The PI3K/Akt/mTOR signaling cascade is another major signaling pathway implicated in HCC carcinogenesis and plays a central role in driving tumor cell proliferation [ 8 , 13 ]. The PI3K/Akt/mTOR signaling pathway is a prototypal survival pathway which is constitutively activated in a broad range of cancers. This pathway is involved in many types of cellular processes including survival, proliferation, metastasis, metabolism and angiogenesis [ 13 – 15 ]. Given the frequent hyperactivation or deregulation of Akt/mTOR pathway in HCC [ 15 – 17 ], several small molecule inhibitors targeting this pathway have been developed and are currently undergoing preclinical or clinical trials [8,18]. Our data suggest that haprolid treatment has great antitumor activity in HCC through dual inhibition of Rb/E2F and Akt/mTOR pathways; thus, it may be a new and promising candidate for systemic treatment of HCC. 2. Materials and Methods 2.1. Cell Culture Human HCC cell line Huh-7 was obtained from the laboratory of Lars Zender, University Hospital Tübingen, Tübingen, Germany. HepG2 was obtained from the laboratory of Michael Bitzer, University Hospital Tübingen, Germany. Hep3B was purchased from Leibniz Institute DSMZ—German collection of microorganisms and cell cultures. Cell lines were cultured at 37 ◦ C under a 5% CO 2 environment in DMEM (Thermo Scientific, Darmstadt, Germany) enriched with 10% fetal bovine serum (FBS) (Biochrom, Berlin, Germany) and antibiotics of penicillin/streptomycin (50 units/mL) (Lonza, Verviers, Belgium). Cancers 2020,12, 615 3 of 17 2.2. Drug Preparation and In Vitro Treatment Haprolid was isolated from the culture broth of Byssovorax cruenta, using the chromatographic procedure described by Steinmetz et al. [ 6 ], and prepared as a 10 mg/mL stock in dimethyl sulfoxide (DMSO) (AppliChem, Darmstadt, Germany). The drug was divided in aliquots, stored at 4 ◦ C and used for in vitro and in vivo experiments. Cells were treated with DMSO as a control or with haprolid in different concentrations (from 0.006 to 6 µ g/mL) and were analyzed after 24 to 96 h for certain experiments, namely cell proliferation assay, crystal violet staining, migration assay, invasion assay, tumor spheroid assay, cell cycle analysis and apoptosis assay. The protocols used in this study are described in detail in Supplementary Materials and Methods. 2.3. Animals and Treatment NMRI-Foxn1 nu female mice were bought from Charles River Laboratories International (Sulzfeld, Germany). Eight mice were treated intraperitoneally with haprolid (2 mg/kg body weight, on days 1 to 3 each week); six mice were treated with vehicle (DMSO). Treatment was started around 10 weeks of age and mice were sacrificed after 5 weeks of treatment. All treatment solutions were prepared freshly on the day of delivery. Health status of mice was monitored every day. Sizes of visible tumors were measured by caliper and organ tissues were harvested. Tumor volume was calculated using the following formula: Tumor volume V =((Width) 2× (Length)/2). Tumor tissues were fixed in 4% formalin for histology. 2.4. Histology and Immunoblotting Detailed information about antibodies and protocols can be found in the Supplementary Materials and Methods. 2.5. Statistics All the experiments were repeated 2–3 times. The results were analyzed using GraphPad Prism version 7.03 (San Diego, CA, USA) and ImageJ 1.47 (National Institutes of Health, Bethesda, MD, USA) software. The tests included Student’s t-test (paired and unpaired) and two-way ANOVA. Differences were considered as statistically significant when the p-value was <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****) or as not significant (ns). 2.6. Study Approval Mice used in this study were maintained in the animal care of University Hospital Tübingen, Tübingen, Germany. All experimental protocols were reviewed and approved by institutional guidelines for animal care of University Hospital Tübingen (protocol No. M7/17), and all studies were performed according to the methods approved in the protocol. 3. Results 3.1. Haprolid Treatment Inhibits Proliferation and Metastasis in Human HCC Cells It was shown before that haprolid (Figure 1A) has antitumor effects against various tumor cells [ 6 ]. However, data about HCC and haprolid are limited. Therefore, we determined the cytotoxic effect of haprolid on three human HCC cell lines (Hep 3B, Huh-7 and HepG2) by WST-1 assay. In addition, human fibroblasts, as noncancer cells, were similarly tested to display differential cytotoxicity (Figure S1). The concentrations for 50% of maximal inhibition of cell proliferation (IC50) for Hep3B, Huh-7 and HepG2 cells after 96 h exposure were 0.1533, 0.0313 and 0.0618 µ g/mL, respectively (Figure 1B). We continued with concentrations ranging from 0.006 to 6 µ g/mL for our further in vitro experiments. Next, we performed cell proliferation and crystal violet assays. As depicted in Figure 1C,D and Cancers 2020,12, 615 4 of 17 Figure S1, Haprolid treatment suppressed cell proliferation of Hep3B, Huh-7 and HepG2 cell lines in a doseand time-dependent manner. Cancers 2020, 12, x FOR PEER REVIEW 4 of 16 S1, Haprolid treatment suppressed cell proliferation of Hep3B, Huh-7 and HepG2 cell lines in a doseand time-dependent manner. Figure 1. Haprolid treatment inhibits proliferation of human hepatocellular carcinoma (HCC) cells. (A) Chemical structure of haprolid. (B) Cytotoxic effect of haprolid (IC50): Hep3B, Huh-7 and HepG2 cells were treated for 96 h with increasing concentrations of haprolid (0.001 to 18 µg/mL). WST-1 assay was performed to analyze cellular viability, DMSO was used as negative control. (C) Cell proliferation of HCC cells treated with the indicated concentrations of haprolid or DMSO measured by WST-1 assay. The relative cell number was normalized with the control. (D) HCC cells were fixed and stained with crystal violet 48 and 96 h after haprolid treatment. Data represent means ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure 1. Haprolid treatment inhibits proliferation of human hepatocellular carcinoma (HCC) cells. ( A ) Chemical structure of haprolid. ( B ) Cytotoxic effect of haprolid (IC50): Hep3B, Huh-7 and HepG2 cells were treated for 96 h with increasing concentrations of haprolid (0.001 to 18 µ g/mL). WST-1 assay was performed to analyze cellular viability, DMSO was used as negative control. ( C ) Cell proliferation of HCC cells treated with the indicated concentrations of haprolid or DMSO measured by WST-1 assay. The relative cell number was normalized with the control. ( D ) HCC cells were fixed and stained with crystal violet 48 and 96 h after haprolid treatment. Data represent means ± SEM of at least three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Furthermore, wound healing and Matrigel transwell chamber assays were conducted to examine the effect of haprolid on the cell metastasis. In all cell lines, haprolid treatment induced significant inhibition of wound closure (Figure 2A). Haprolid significantly reduced the number of invasive cells, as illustrated in Figure 2B. Thus, treatment with haprolid can effectively suppress the migration and invasion in human HCC cells. Furthermore, we explored an in vitro three-dimensional tumor spheroid model to mimic physiologic tissue’s microenvironment for investigating drug efficiency of haprolid. As we can see in Figure 3A, these results further confirmed that haprolid treatment significantly Cancers 2020,12, 615 5 of 17 inhibited the spheroid growth and invasion in Hep3B and Huh-7 cells. Our experiments showed that incubation of the Huh-7 cell line with haprolid for 10 days suppressed the growth of spheroid size but with no regression, whereas similar incubation of the Hep3B cell line readily resulted in reduction of tumor spheroid size at the concentration of 0.6 µ g/mL as early as 4 days. Taken together, these data suggest that haprolid can significantly inhibit proliferation, migration and invasion of human HCC cells. In general, Hep3B cells shows overall stronger inhibitory effect under haprolid treatment compared to Huh-7 and HepG2 cells. Cancers 2020, 12, x FOR PEER REVIEW 5 of 16 Furthermore, wound healing and Matrigel transwell chamber assays were conducted to examine the effect of haprolid on the cell metastasis. In all cell lines, haprolid treatment induced significant inhibition of wound closure (Figure 2A). Haprolid significantly reduced the number of invasive cells, as illustrated in Figure 2B. Thus, treatment with haprolid can effectively suppress the migration and invasion in human HCC cells. Furthermore, we explored an in vitro three-dimensional tumor spheroid model to mimic physiologic tissue’s microenvironment for investigating drug efficiency of haprolid. As we can see in Figure 3A, these results further confirmed that haprolid treatment significantly inhibited the spheroid growth and invasion in Hep3B and Huh-7 cells. Our experiments showed that incubation of the Huh-7 cell line with haprolid for 10 days suppressed the growth of spheroid size but with no regression, whereas similar incubation of the Hep3B cell line readily resulted in reduction of tumor spheroid size at the concentration of 0.6 µg/mL as early as 4 days. Taken together, these data suggest that haprolid can significantly inhibit proliferation, migration and invasion of human HCC cells. In general, Hep3B cells shows overall stronger inhibitory effect under haprolid treatment compared to Huh-7 and HepG2 cells. Figure 2. Inhibition of migration and invasion in haprolid-treated HCC cells. (A) Wound healing assay was performed for Hep3B, Huh-7 and HepG2 cells after haprolid (0.06, 0.6 and 6 µg/mL) Figure 2. Inhibition of migration and invasion in haprolid-treated HCC cells. ( A ) Wound healing assay was performed for Hep3B, Huh-7 and HepG2 cells after haprolid (0.06, 0.6 and 6 µ g/mL) treatment. The dotted lines represent edges of the wound. Photographs were taken at 0 and 24 h under light microscope (40 × magnification). The migration index was calculated as described in Section 2and plotted in bar graphs. ( B ) HCC cells were seeded in Matrigel transwell invasion chambers and treated with indicated concentrations of haprolid for 48 h to investigate the effect on invasiveness. The number of cells that invaded through the membrane was determined under light microscope (200 × magnification). Scale bars: 100 µ m. Invasion index was calculated as described in Section 2 and plotted in bar graphs. Data represent means ± SEM of at least three independent experiments. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Cancers 2020,12, 615 6 of 17 3.2. Haprolid Treatment Impairs EMT in HCC Cells Moreover, based on the findings that haprolid treatment significantly inhibited the HCC cell motility, we continued to investigate the impact of haprolid in EMT. At the molecular level, EMT is frequently determined by the expression level of putative EMT markers: the upregulation of mesenchymal markers, such as cytoskeletal protein vimentin and cell surface protein N-cadherin, and the downregulation of epithelial markers, including the junction protein E-cadherin [ 19 , 20 ]. In order to check whether EMT is impaired after haprolid treatment, we treated HCC cells with increasing dosages of haprolid for 96 h and compared with negative control. In both Hep3B and Huh-7 cells, haprolid treatment resulted in decreased expression of the mesenchymal proteins N-cadherin and vimentin as assessed by Western blot (Figure 3B). However, the expression of epithelial protein E-cadherin was only slightly upregulated (Figure 3B). Thus, we next checked the expression of Snail, a zinc-finger EMT transcription factor that functions as a potent repressor of E-cadherin [ 21 ]. As shown in Figure 3B, the level of Snail was markedly diminished after haprolid treatment. These changes indicate that haprolid treatment partially impairs EMT in HCC cells. Cancers 2020, 12, x FOR PEER REVIEW 6 of 16 treatment. The dotted lines represent edges of the wound. Photographs were taken at 0 and 24 h under light microscope (40× magnification). The migration index was calculated as described in Section 2 and plotted in bar graphs. (B) HCC cells were seeded in Matrigel transwell invasion chambers and treated with indicated concentrations of haprolid for 48 h to investigate the effect on invasiveness. The number of cells that invaded through the membrane was determined under light microscope (200× magnification). Scale bars: 100 µm. Invasion index was calculated as described in Section 2 and plotted in bar graphs. Data represent means ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 3.2. Haprolid Treatment Impairs EMT in HCC Cells Moreover, based on the findings that haprolid treatment significantly inhibited the HCC cell motility, we continued to investigate the impact of haprolid in EMT. At the molecular level, EMT is frequently determined by the expression level of putative EMT markers: the upregulation of mesenchymal markers, such as cytoskeletal protein vimentin and cell surface protein N-cadherin, and the downregulation of epithelial markers, including the junction protein E-cadherin [19,20]. In order to check whether EMT is impaired after haprolid treatment, we treated HCC cells with increasing dosages of haprolid for 96 h and compared with negative control. In both Hep3B and Huh7 cells, haprolid treatment resulted in decreased expression of the mesenchymal proteins N-cadherin and vimentin as assessed by Western blot (Figure 3B). However, the expression of epithelial protein E-cadherin was only slightly upregulated (Figure 3B). Thus, we next checked the expression of Snail, a zinc-finger EMT transcription factor that functions as a potent repressor of E-cadherin [21]. As shown in Figure 3B, the level of Snail was markedly diminished after haprolid treatment. These changes indicate that haprolid treatment partially impairs EMT in HCC cells. Figure 3. Cont. Figure 3. Cont. Cancers 2020,12, 615 7 of 17 Cancers 2020, 12, x FOR PEER REVIEW 7 of 16 Figure 3. Haprolid treatment inhibits tumor spheroids growth and invasion and partially impairs epithelial–mesenchymal transition (EMT) in human HCC cells. (A) Three-dimensional tumor spheroid assay was performed to evaluate effects of haprolid on HCC cell proliferative and invasive ability in extracellular-matrix-like environment. The representative images of spheroids are shown. The changes of spheroids size were monitored and quantified up to 10 days and plotted in bar graphs. (B) Hep3B and Huh-7 cells were treated with DMSO or haprolid (0.06, 0.6 and 6 µg/mL) for 96 h. The expressions of EMT markers (N-cadherin, E-cadherin, vimentin and Snail) were analyzed by immunoblotting. Western blotting bands were quantified by Image J and normalized to their respective β-actin then compared with vehicle-treated controls. Data represent means ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 3.3. Haprolid Inhibits G1/S Transition and Partially Induces Apoptosis of HCC Cells To figure out the underlying mechanisms for the antiproliferative effect of haprolid, cell cycle and cell apoptosis were analyzed. Fluorescence-activated cell sorting (FACS) assay was carried out to determine the cell cycle profiles upon exposure to different concentrations of haprolid after 48 and 96. Figure 4A shows the distribution of cell cycle profiles after haprolid treatment in all three HCC cell lines. The percentages of the sub-G1-, G1-, Sand G2-M population are listed in Table S1. In Huh7 and HepG2 cells, haprolid treatment resulted in a significant accumulation of cells in G1 phase accompanied with a decrease of the cell numbers in S and G2-M phase, which indicated a block of G1/S progression (Figure 4A). Although Hep3B cells also accumulated in the G1 phase at lower concentration (0.06 µg/mL), at higher concentrations of the compound treatment, a large proportion of the cells accumulated in the sub-G1 phase, suggesting an induction of apoptosis (Figure 4A), which was also evidenced by following experiments. Overall, haprolid treatment inhibits the G1/S transition and results in marked cell cycle arrest in all tested cell lines. Moreover, haprolid treatment leads to growth arrest, later on inducing considerable apoptosis in Hep3B cells. To further substantiate cell cycle analysis results, we next investigated the kinetic expression of cell cycle checkpoint proteins via Western blot (Figure 4B). Consistent with the blocking of G1-to-S phase transition, we found that the expression levels of both cyclin A and cyclin B dynamically decreased. Figure 3. Haprolid treatment inhibits tumor spheroids growth and invasion and partially impairs epithelial–mesenchymal transition (EMT) in human HCC cells. ( A ) Three-dimensional tumor spheroid assay was performed to evaluate effects of haprolid on HCC cell proliferative and invasive ability in extracellular-matrix-like environment. The representative images of spheroids are shown. The changes of spheroids size were monitored and quantified up to 10 days and plotted in bar graphs. ( B ) Hep3B and Huh-7 cells were treated with DMSO or haprolid (0.06, 0.6 and 6 µ g/mL) for 96 h. The expressions of EMT markers (N-cadherin, E-cadherin, vimentin and Snail) were analyzed by immunoblotting. Western blotting bands were quantified by Image J and normalized to their respective β -actin then compared with vehicle-treated controls. Data represent means ± SEM of at least three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. 3.3. Haprolid Inhibits G1/S Transition and Partially Induces Apoptosis of HCC Cells To figure out the underlying mechanisms for the antiproliferative effect of haprolid, cell cycle and cell apoptosis were analyzed. Fluorescence-activated cell sorting (FACS) assay was carried out to determine the cell cycle profiles upon exposure to different concentrations of haprolid after 48 and 96. Figure 4A shows the distribution of cell cycle profiles after haprolid treatment in all three HCC cell lines. The percentages of the sub-G1-, G1-, Sand G2-M population are listed in Table S1. In Huh-7 and HepG2 cells, haprolid treatment resulted in a significant accumulation of cells in G1 phase accompanied with a decrease of the cell numbers in S and G2-M phase, which indicated a block of G1/S progression (Figure 4A). Although Hep3B cells also accumulated in the G1 phase at lower concentration (0.06 µ g/mL), at higher concentrations of the compound treatment, a large proportion of the cells accumulated in the sub-G1 phase, suggesting an induction of apoptosis (Figure 4A), which was also evidenced by following experiments. Overall, haprolid treatment inhibits the G1/S transition and results in marked cell cycle arrest in all tested cell lines. Moreover, haprolid treatment leads to growth arrest, later on inducing considerable apoptosis in Hep3B cells. Cancers 2020,12, 615 8 of 17 Cancers 2020, 12, x FOR PEER REVIEW 8 of 16 Figure 4. Haprolid treatment inhibits G1/S transition and induces cell cycle arrest in HCC cells. (A) HCC cells were treated with vehicle (DMSO) or increasing concentrations of haprolid for 48 and 96 h, fixed and stained with propidium iodide (PI) and subjected to flow cytometric analysis. Representative cell cycle histograms are presented (left panel). Cell cycle distribution in various phases is determined and shown (right panel). (B) Cyclin A and B expression levels were measured by Western blotting. Images representative of at least three independent experiments are shown. Additionally, annexin V and PI staining was performed on live cells to determine whether the cell-cycle-arrest phenotype was associated with the induction of apoptosis. We noticed that exposure to haprolid dose-dependently increased apoptotic cells in Hep3B cell line (Figure 5A,B). Moreover, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) as an indicative marker of apoptosis was evaluated by quantitative measurement [22]. Immunoblotting revealed distinct PARP cleavage after haprolid treatment in Hep3B cells (Figure 5C,D). Interestingly, Huh-7 and HepG2 cells are not sensitive to the apoptosis-inducing effects of haprolid (Figures S2 and S3). These results confirm our previous studies and suggest that Huh-7 and HepG2 cells are relatively resistant to apoptosis and mainly undergo cell cycle arrest under haprolid treatment, while haprolid induces noticeable apoptotic cell death in Hep3B cells following cytostatic effect. Figure 4. Haprolid treatment inhibits G1/S transition and induces cell cycle arrest in HCC cells. ( A ) HCC cells were treated with vehicle (DMSO) or increasing concentrations of haprolid for 48 and 96 h, fixed and stained with propidium iodide (PI) and subjected to flow cytometric analysis. Representative cell cycle histograms are presented (left panel). Cell cycle distribution in various phases is determined and shown (right panel). ( B ) Cyclin A and B expression levels were measured by Western blotting. Images representative of at least three independent experiments are shown. To further substantiate cell cycle analysis results, we next investigated the kinetic expression of cell cycle checkpoint proteins via Western blot (Figure 4B). Consistent with the blocking of G1-to-S phase transition, we found that the expression levels of both cyclin A and cyclin B dynamically decreased. Additionally, annexin V and PI staining was performed on live cells to determine whether the cell-cycle-arrest phenotype was associated with the induction of apoptosis. We noticed that exposure to haprolid dose-dependently increased apoptotic cells in Hep3B cell line (Figure 5A,B). Moreover, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) as an indicative marker of apoptosis was evaluated by quantitative measurement [ 22 ]. Immunoblotting revealed distinct PARP cleavage after haprolid treatment in Hep3B cells (Figure 5C,D). Interestingly, Huh-7 and HepG2 cells are not sensitive to the apoptosis-inducing effects of haprolid (Figures S2 and S3). These results confirm our Cancers 2020,12, 615 9 of 17 previous studies and suggest that Huh-7 and HepG2 cells are relatively resistant to apoptosis and mainly undergo cell cycle arrest under haprolid treatment, while haprolid induces noticeable apoptotic cell death in Hep3B cells following cytostatic effect. Cancers 2020, 12, x FOR PEER REVIEW 9 of 16 Figure 5. Haprolid treatment induces considerable apoptosis in Hep3B cells. (A) Hep3B cells were treated with DMSO or haprolid (0.06, 0.6 and 6 µg/mL) for 48 and 96 h, and the level of apoptosis was measured by staining with annexin V and PI using flow cytometry. Representative FACS measurements are presented. (B) The apoptotic positive cells were calculated and plotted in bar graph. (C) Expression of PARP and cleaved PARP (a marker for apoptosis) was measured by Western blotting. (D) Kinetic analysis of PARP cleavage was performed by Western blotting. Data represent means ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 3.4. Dual Inhibition of Rb/E2F and Akt/mTOR Pathways by Haprolid Based on the effect of haprolid on cell cycle, we next examined the expression levels of cell cycle checkpoint proteins and cell cycle modulation factors by Western blot analysis. First, we checked the expression level of Rb, which plays a central role in cell cycle regulation, particularly in initiating DNA replication and division [9]. Figure 6A demonstrated that—aside from the Rb-deficient Hep3B cell line—after haprolid treatment the level of p-Rb expression decreased and Rb changed from the hyperphosphorylated form to the hypophosphorylated form in both Huh-7 and HepG2 cell lines. Moreover, the protein level of E2F-1, the downstream target of Rb, which is the essential transcription factor that regulates cell cycle progression and stimulates proliferation [23], was considerably inhibited after exposure to haprolid in all three HCC cells. Inhibition of Rb phosphorylation and reduction in levels of free E2F-1 appear to play an important role in HCC growth arrest. Haprolid treatment also resulted in a noticeable downregulation of cyclin-dependent kinase 2 (CDK2) in all three HCC cells and upregulation of cyclin-dependent kinase inhibitors (CDKIs) p21 and p27 in both Hep3B and HepG2 cells. We also found upregulation of p21 but not p27 in Huh-7 cells (Figure 6A). Next the phospho-Ser10-histone H3 was checked as a specific marker of mitosis. As expected, in all tested HCC cells, p-histone H3 expression was remarkably downregulated (Figure 6A). Taken Figure 5. Haprolid treatment induces considerable apoptosis in Hep3B cells. ( A ) Hep3B cells were treated with DMSO or haprolid (0.06, 0.6 and 6 µ g/mL) for 48 and 96 h, and the level of apoptosis was measured by staining with annexin V and PI using flow cytometry. Representative FACS measurements are presented. ( B ) The apoptotic positive cells were calculated and plotted in bar graph. ( C ) Expression of PARP and cleaved PARP (a marker for apoptosis) was measured by Western blotting. ( D ) Kinetic analysis of PARP cleavage was performed by Western blotting. Data represent means ± SEM of at least three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. 3.4. Dual Inhibition of Rb/E2F and Akt/mTOR Pathways by Haprolid Based on the effect of haprolid on cell cycle, we next examined the expression levels of cell cycle checkpoint proteins and cell cycle modulation factors by Western blot analysis. First, we checked the expression level of Rb, which plays a central role in cell cycle regulation, particularly in initiating DNA replication and division [ 9 ]. Figure 6A demonstrated that—aside from the Rb-deficient Hep3B cell line—after haprolid treatment the level of p-Rb expression decreased and Rb changed from the hyperphosphorylated form to the hypophosphorylated form in both Huh-7 and HepG2 cell lines. Moreover, the protein level of E2F-1, the downstream target of Rb, which is the essential transcription factor that regulates cell cycle progression and stimulates proliferation [ 23 ], was considerably inhibited after exposure to haprolid in all three HCC cells. Inhibition of Rb phosphorylation and reduction in levels of free E2F-1 appear to play an important role in HCC growth arrest. Haprolid treatment also resulted in a noticeable downregulation of cyclin-dependent kinase 2 (CDK2) in all three HCC Cancers 2020,12, 615 16 of 17 24. 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