| Hepatocellular carcinoma (HCC) is the sixth most prevalent malignant neoplasm worldwide. In earliest stages, HCC is frequently asymptomatic and goes undetected, leading to almost 85% of patients being diagnosed at intermediate or advanced stages. Although liver transplantation, resection and local ablation are potentially curative treatments, it is available for only a small fraction of patients with early-stage disease. For the majority of patients who are often diagnosed at an advanced stage, limited treatment options are available. In addition, even if patients receive potentially curative therapies, the long-term prognosis of this disease remains dismal, due to a high rate of recurrence.In recent years, the understanding of molecular pathogenesis and subsequent identification of multiple signaling pathways involved in the development and progression of HCC have spurred efforts to develop the molecular targeted therapy for this malignant disease. Currently, the only molecular targeted agent approved for the treatment of HCC is sorafenib. In recent randomized, placebo-controlled trial, the median overall survival increased by sorafenib was 2 to 3 months, as compared with placebo in patients with advanced HCC. These results not only established the crucial role of molecular targeted therapy in advanced HCC treatment, but also triggered the search for novel molecular targeted agents to further expand patient survival.The mammalian target of rapamycin (mTOR) is a conserved serine-threonine protein kinase that functions as a central controller of cell proliferation, differentiation, apoptosis, angiogenesis, and autophagy. Depending on their binding partners, mTOR resides in at least two distinct complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The mTOR pathway (PI3K\AKT\mTOR) is aberrantly activated in about 50% of human HCC cases. Therefore, targeting mTOR is being investigated as a promising therapy for this malignant disease.Most studies targeting the mTOR pathway in cancer therapy mainly focus on conventional mTOR inhibitors, rapamycin and its derivatives, which are inhibitors of mTORCl but not of mTORC2. Based on existing data, it appears that the efficacy of rapamycin or its derivatives monotherapy for HCC is highly limited. This may be due to the fact that rapamycin and its derivatives do not completely block mTORCl and lack effective inhibition of mTORC2 and result in feedback activation of AKT signaling that attenuate their antitumor activity. In contrast to conventional mTOR inhibitors, recently developed novel dual mTOR inhibitors, such as PP244,INK126, Torin 1, WYE-125132, AZD8055 and AZD2014 are able to inhibit both mTORCl and mTORC2. Of these inhibitors, AZD8055 has gained intense research interest, where its rapid metabolism and poor pharmacokinetics have been reported. In contrast to AZD8055, AZD2014 with improved aqueous solubility and pharmacokinetics display a better feasibility in clinical application. However, the antitumor potential of this dual mTORCl/2 inhibitor AZD2014 in HCC has not been investigated.Liver transplantation is one of the important choice for HCC treatment, which has unparalleled advantages over other therapies for this malignant disease. Selection of patients with HCC who meet the Milan criteria for liver transplantation offers 4-year overall survival rate of 85% and a recurrence rates below 15%. In theory, immunosuppressant required after liver transplantation may accelerate tumor recurrence. If immunosuppressant possesses antitumor effect, perhaps it can reduce tumor recurrence when used as immunosuppression after liver transplantation for HCC. mTOR pathway also plays a pivotal role in immune regulation and is targeted clinically by conventional mTOR inhibitor rapamycin to inhibit graft rejection following liver transplantation. However, the antirejection potential of this novel dual mTORCl/2 inhibitor AZD2014 in liver transplantation has not been reported.In this study, we investigated the biochemical activity of AZD2014 in blocking mTORCl and mTORC2 signaling, and the potential therapeutic value of AZD2014 by determining its effects on the proliferation, apoptosis, cell cycle, autophagy, migration, invasion and EMT progression in human HCC cell lines, in vitro. Then, we evaluated the tumor-suppressive activity in HCC-transplanted mice, in vivo. Finally, we examined the ability of AZD2014 to inhibit acute graft rejection in allogeneic rat liver transplantation model. Aim to provide theoretical basis for further investigation of AZD2014 as a clinical agent for HCC treatment and liver transplantation.Chapter 1 The effects of the dual mTORCl/2 inhibitor AZD2014 on human HCC cell lines in vitroObjectiveTo investigate the biochemical activity of AZD2014 in blocking mTORCl and mTORC2 signaling, and the potential therapeutic value of AZD2014 by determining its effects on the proliferation, apoptosis, cell cycle, autophagy, migration, invasion and EMT progression in human HCC cell lines, in vitro.Methods1. Activation status of mTORC1 and mTORC2 in human HCC cells and liver cellsWestern blot analysis was carried out to detect mTORCl and mTORC2 activities in HL-7702, HCCLM3, Huh-7, SMMC-7721, and HepG2 cells. mTORCl activity was reflected by p-S6K Thr 389 and p-4EBP1 Thr 37/46, and mTORC2 activity was indicated by p-AKT Ser 473.2. Effects of AZD2014 on mTORCl and mTORC2 signaling in human HCC cellsHuman HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2 were treated with DMSO or increasing concentrations (from 0 to 1,000 nM) of AZD2014 or rapamycin for 1 hour. Then, total protein was extracted from these cells and western blot analysis was carried out to detect the expression of p-S6K Thr 389, P-4EBP1 Thr 37/46 and p-AKT Ser 473.3. Effects of AZD2014 on cell proliferation of human HCC cellsHuman HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2 were seeded in 96-well plates at 4,000 to 8,000 cells per well for 12 hours, treated with DMSO or various concentrations (from 0 to 20,000 nM) of AZD2014 or rapamycin. Cell number was determined 72 hours later by a Cell Counting Kit-8 (Dojindo, Tokyo, Japan) following the kit assay protocol. The half maximal inhibitory concentration (IC50) was generated for all cell lines and used in further experiments.4. Effects of AZD2014 on cell apoptosis of human HCC cellsHuman HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2 were collected at 48 hours after incubation with or without AZD2014 or rapamycin. In the first place, cell apoptosis was quantitated using the Annexin V-FITC/PI Apoptosis Detection Kit (Sigma, MO, USA) as detailed in the manufacturer’s instructions. HCC cells were washed in cold PBS and incubated with Annexin V-FITC and propidium iodide (PI) in the dark, and then analyzed for apoptosis (acquired 10,000 cells/test) by flow cyto meter (BD Bioscience). In the second place, apoptosis was also assessed by the TUNEL technique, according to the manufacturer’s protocol (Roche Applied Science, Mannhelm, Germany). The images were obtained by a fluorescent microscopy (Nikon, Tokyo, Japan) and accompanying NIS-Elements BR3.1 software. Cells exhibiting green fluorescence were defined as TUNEL-positive apoptotic cells. In the end, total protein was extracted from these cells and western blot analysis was carried out to detect the expression of apoptosis-related proteins, bax, cleaved PARP and cleaved caspase-3.5. Effects of AZD2014 on cell cycle of human HCC cellsHuman HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2 were collected at 48 hours after incubation with or without AZD2014 or rapamycin. In the first place, for cell cycle analysis, cells were fixed overnight at 4℃ in 70% ethanol, washed and stained with PI solution (Sigma, St Louis, MO) containing RNase in the dark, and analyzed for cell cycle distribution (acquired 10,000 cells/test) on flow cytometer (BD Biosciences) and Modfit LT Software (Verity Software House, Tosham, ME). In the second place, total protein was extracted from these cells and western blot analysis was carried out to detect the expression of cell cycle-related proteins, Cyclin D1 and CDK4.6. Effects of AZD2014 on autophagy of human HCC cells The autofluorescent dye monodansylcadaverine (MDC) is commonly used tomonitor autophagy. Human HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2 were cultured in the presence or absence of AZD2014 or rapamycin for 48 hours, and then incubated with 0.05 mM MDC in PBS at 37℃ for 10 minutes, and washed for four times with PBS. Cells were immediately observed under a confocal laser scanning microscope (FV10i-w, Olympus, Japan) and accompanying FVlOi-ASW 3.0 software. In some experiments, HCC cells were pre-incubated for 3 hours with 10 mM 3-MA (autophagy inhibitor), before AZD2014 or rapamycin treatment. In addition, total protein was extracted from these cells and western blot analysis was carried out to detect the expression of autophagy-related proteins, LC3B-Ⅱand Beclin-1.7. Effects of AZD2014 on migration, invasion and EMT progression of human HCC cellsAfter incubation with or without AZD2014 or rapamycin for 48 hours, human HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2 were trypsinized and resuspended in serum-free medium, and 50,000 cells were added to the top well (insert) of each migration chamber with 8 um pores (Corning). The top well was loaded with 200 μl of cell suspension with or without AZD2014 or rapamycin. The migration was induced by the presence of 20% FBS medium in the bottom chambers. After 48 hours, cells in upper chambers were removed, and then inserts were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. The numbers of migrated cells were counted in three different fields with an inverted microscope (Nikon, Tokyo, Japan). For the invasion assay, all conditions were as described in the migration assays, except that 50,000 cells were added to the matrigel-coated inserts. For immunoblot analysis, total protein was extracted from these cells and western blot was carried out to detect the expression of EMT-related proteins, E-cadherin, N-cadherin, Vimentin, Snail and MMP2.Results1. The activation status of mTOR pathway was examined in human HCC cell lines, HCCLM3, Huh-7, SMMC-7721, and HepG2 cells. HL-7702 cell line served as a normal control. mTORCl activity was reflected by p-S6K and p-4EBP1, while mTORC2 activation was indicated by phosphorylation of AKT at Ser 473. The expression of p-S6K Thr 389 and p-4EBPl Thr37/46 was higher in the four HCC cell lines than that in HL-7702 cell line. In contrast, only SMMC-7721 cells displayed an increased level of p-AKT Ser 473 compared with HL-7702 cells.2. The biochemical activity of AZD2014 in blocking mTORCl and mTORC2 signaling was investigated in HCCLM3, Huh-7, SMMC-7721, and HepG2 cells.Concentration-dependent experiments showed that low concentrations (10-100 nM) of AZD2014 effectively inhibited p-S6K Thr 389and p-4EBP1 Thr37/46. Rapamycin, in contrast, potently inhibited p-S6K Thr389 but did not result in the inhibition of 4EBP1 phosphorylation at Thr 37/46 even at a high dose of 1,000 nM. The direct mTORC2 substrate p-AKT Ser 473 was markedly inhibited by AZD2014. In contrast, rapamycin treatment resulted in a significant increase in p-AKT Ser 473.3. AZD2014 exhibited a more profound antiproliferative activity than rapamycin in a dose-dependent manner in the four HCC cell lines, HCCLM3, Huh-7, SMMC-7721, and HepG2. Rapamycin, however, in general elicited a flat, modest suppression of cell proliferation and had a much higher IC50 than AZD2014. Interestingly, we found that SMMC-7721 cells were resistant to rapamycin, as shown by no significant decrease in cell proliferation even at a high concentration of 20 μM. HCCLM3, Huh-7, SMMC-7721, and HepG2 cells were found to be sensitive to AZD2014 at IC50 101.6,441.6,141, and 600 nM, respectively.4. The Annexin V-FITC/PI FACS assay was conducted as described in Materials and Methods. In HCCLM3 and Huh-7 cells, both drugs induced an increase in apoptotic cells from 1.6% (control) to 19.0% and 49.0%, and 7.9% (control) to 29.0% and 43.2% with rapamycin and AZD2014, respectively. Strangely enough, both rapamycin and AZD2014 did not have significant effect on apoptosis in SMMC-7721 and HepG2 cells. Similar results were obtained by TUNEL assay. Western blot analysis detected increased Bax, and cleavage of poly ADP-ribose polymerase (PARP) and caspase-3 in HCCLM3 and Huh-7 cells treated with AZD2014 and rapamycin, but not in SMMC-7721 and HepG2 cells, which was consistent with the results of Annexin V/PI staining and TUNEL assay.5. Flow cytometric analysis of rapamycin or AZD2014-treated HCC cells indicated that AZD2014 led to a more profound increase in G1-phase cells than rapamycin in the four HCC cell lines, HCCLM3, Huh-7, SMMC-7721 and HepG2, suggesting that AZD2014 caused a strong G1 arrest in HCC cells. We also observed reduced Cyclin D1, CDK4 levels in AZD2014-treated HCC cells, which is consistent with its ability to induce G1 arrest. Notably, although rapamycin were able to induce a modest Gl arrest in HCCLM3 and Huh-7 cells, it had a minor effect on the cell cycle profile of SMMC-7721 and HepG2 cells.6. After exposing HCC cells to AZD2014 for 48 hours, autophagic vacuoles were detected by monodansylcadaverine (MDC) staining. AZD2014-treated HCC cells showed an increase in the number of MDC-labeled vacuoles as well as in their size. MDC staining could not be considered to be an indicator of autophagy until when the increased labeling of cellular compartments by this dye is prevented by treatment with autophagy inhibitors such as 3-MA. As expected, the formation of MDC-labeled vacuoles induced by AZD2014 was inhibited by 3-MA. LC3B is the most widely monitored autophagy-related protein. Thus, we detected the conversion of LC3B-I to LC3B-II by western blot. Consistent with the results of MDC staining, AZD2014 treatment resulted in an increase in the levels of LC3B-II, and this increase was more pronounced than rapamycin. Beclin-1 is one of the critical proteins for the initiation of autophagy. We examined the amount of Beclin-1 by western blot. AZD2014 treatment led to a significant upregulation of Beclin-1. It should be noted that rapamycin increased the number and size of MDC-labeled vacuoles in HCCLM3 and Huh-7 cells, but not in HepG2 and SMMC-7721 cells, and the result was confirmed by the expression of LC3B-II and Beclin-1.7. The ability of migration was assessed by Transwell assay. Treatment with AZD2014 significantly decreased the migration of HCCLM3, Huh-7, SMMC-7721, and HepG2 cells. Then, we evaluated the effect of AZD2014 on invasion using Matrigel-coated Transwell chambers. AZD2014 potently decreased the invasion ofHCCLM3, Huh-7, SMMC-7721, and HepG2 cells. Western blot analysis showed that AZD2014-treated HCC cells increased the expression of epithelial marker, E-cadherin, whereas decreased the expression of mesenchymal markers, N-cadherin, Vimentin, Snail and MMP2. Similar but modest results were obtained when HCC cells were treated with rapamycin.Conclusions1. Compared to immortalized liver cells (HL-7702), aberrant activation of mTORCl signaling exists in all HCC cell lines we tested. Whereas, only SMMC-7721 cells shows an increased activation of mTORC2 signaling when compared with HL-7702 cells.2. AZD2014 is a highly potent inhibitor of mTORCl and mTORC2 in human HCC cells. This dual mTORC1/2 inhibitor AZD2014 leads to a more complete inhibition of mTORCl than rapamycin, and the inhibition of mTORC2 prevented the feedback activation of AKT signaling.3. Dual mTORC1/2 inhibitor AZD2014 results in a profound antitumor effect in HCC cells, in vitro. AZD2014 is found to be more efficacious than rapamycin in inducing proliferation inhibition, apoptosis, cell cycle arrest and autophagy, and in inhibiting the migration, invasion and EMT progression of HCC cells.Chapter 2 The effects of the dual mTORCl/2 inhibitor AZD2014 on human HCC in vivoObjectiveTo investigate the antitumor effects of AZD2014 in human HCC in vivo.Cell line and nude miceHuman HCC cell line HCCLM3 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Male BALB/c athymic nude mice (4-6 wk) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China).MethodsApproximately 5×106 HCCLM3 cells in 100 μL DMEM were subcutaneously injected in the right flank of each mouse. When tumors were macroscopic (10 days after injection), mice were randomized into 2 groups, and administered with AZD2014 (5 mg/kg/d, n=5) or vehicle (5 ml/kg/d, n=5) by intraperitoneal injection (i.p.) for 24 days. Tumor growth were measured with calipers every 4 days for the duration of the study. Tumor volume (V) was calculated, taking length to be the longest diameter across the tumor (a) and width to be the corresponding perpendicular diameter (b), using the following formula:V=a×b×2a×b xTT/6. Animals were euthanized at 34 days after tumor cell implantation. Tumor weight was recorded. Tumor tissues were excised and fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded blocks of tumors were sectioned and each sample was stained with hematoxylin and eosin (HE) for histopathologic analysis. Immunohistochemical staining was done using antibodies against Ki-67 for cell proliferation, cleaved caspase-3 for apoptosis, CD31 for angiogenesis, and EMT-related proteins, E-cadherin, N-cadherin, Vimentin.ResultsTreatment with AZD2014 daily at 5 mg/kg dramatically reduced HCC growth and weight relative to vehicle control group. Necrosis of tumor specimen was significantly increased in AZD2014-treated mice, confirmed by HE staining. We further demonstrated that AZD2014 inhibited tumor proliferation, angiogenesis and EMT progression, as shown by a decrease in Ki-67, CD31, N-cadherin,Vimentin staining and increase in E-cadherin staining, and induced apoptosis, as demonstrated by an increase in cleaved caspase-3 staining, in AZD2014-treated mice.ConclusionsDual mTORCl/2 inhibitor AZD2014 is a highly potent antitumor agent for HCC in a tumor xenografts model. AZD2014 effectively inhibit tumor proliferation, angiogenesis and EMT progression, and induce necrosis and apoptosis, in vivo.Chapter 3 Antirejection effects of the dual mTORC1/2 inhibitor AZD2014 in rat liver transplantationObjectiveTo investigate the ability of AZD2014 to inhibit acute graft rejection in allogeneic rat liver transplantation.RatsMale Brown Norway (BN), Lewis rats (7-9 wk) were purchased from the Beijing Vital River Experimental Animal Technical Co.Ltd. (Beijing, China).MethodsLiver transplantation from Lewis rat to BN recipient was performed according to Kamada’s two-cuff technique. This strain combination has previously been shown to induce acute graft rejection. BN recipients were randomized into 2 groups, and was administered with AZD2014 (5 mg/kg/d, n=4) or vehicle (2.5 ml/kg/d, n=4) by intraperitoneal injection (i.p.) for 14 days, starting from the first day after transplantation, we documented serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) 3 days before and 1,3,5,7,10,14 days after liver transplantation. Follow-up for survival after liver transplantation was 14 days. Graft fibrosis was assessed by sirius red. Immunohistochemical staining was done using antibodies against CD3 for T lymphocytes infiltration and Foxp3 for Treg cells infiltration in graft liver samples. Acute graft rejection was assessed on HE staining and based on portal inflammation, bile duct inflammation, and endothelial inflammation, also known as Banff schema.ResultsSevere acute rejection was observed in control group, as demonstrated by HE staining, massive T cells infiltration and little Foxp3+ cells infiltration, along with persistent high serum levels of ALT, AST and TBIL. Sirius red staining showed severe graft fibrosis in control group. Such degree of rejection caused death in 75% of the control rats within 14 days. However, recipient survival was 100% within 14 days after liver transplantation in AZD2014 group, and typical acute rejection was not observed in liver graft, as documented by HE staining, little T cells infiltration and increased Foxp3+ cells infiltration, along with low levels of ALT, AST and TBIL.ConclusionsDual mTORC1/2 inhibitor AZD2014 effectively inhibit acute graft rejection in an allogeneic rat liver transplantation model. |
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