| Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors in China. And it is in our country that more than half of the new cases happen throughout the world every year. Most of HCC patients died in one year after being diagnosed. Because most cases are not found until in very late stage and lose the chance of radical resection. So they have to be treated in nonoperative way such as chemotherapy, radiotherapy or interventional therapy. However, the consequences are very poor, the 5-year survival rate is only 7%. And to the patients who have undergone radical resection, it is necessary to administrator active and effective adjuvant drug therapy for a better clinical reward.But the progress of drug therapy in HCC is very slow, single drug got low efficiency but toxic side effect. In recent years, some new Pharmaceuticals such as capecitabine, a precursor drug of 5-FU, and the third-generation platinum drug oxaliplatin have been widely used in clinical cancer treatment, and they shed new light on HCC chemotherapy. Following the molecular targeted type drugs such as sorafenib appearing, drug therapy for HCC presents a new situation. But in fact, formal random group design of clinical research demonstrated that these medicines can only prolong very limited lifetime of advanced HCC patients. So, evolving new drugs or pharmaceutical strategies is very urgent to improve curative effect for HCC patients.A hallmark feature of aggressive cancers is a general increase in protein synthesis and protein degradation, both of which are required to increase tumour growth. There are two major protein degradation systems in eukaryotic cells, the ubiquitin-proteasome system and the autophagy-lysosomal systems.The former is responsible for degradation of signal molecules, tumor-inhibiting factors, cell cycle proteins, apoptosis-suppressing factors, etc. Proteasome inhibitors may repress the function of ubiquitin-proteasome system, and then disturb the cell process of proliferation, differentiation, and apoptosis. When they were found to induce programmed cell death preferentially in transformed cells, proteasome inhibitors began to be thought of as potential drug candidates. In recent years, there is a rapid development in this field. The proteasome inhibitor bortezomib is currently being used for the treatment of multiple myeloma. But limited progress with these agents have thus far been conducted in solid tumors, and very little preclinical studies was done on HCC. Apoptosis resistance to chemotherapy is a hallmark feature of cancer cells, and it is also the major reason of drug resistance, recurrence or metastasis in cancer therapy. Some findings show that autophagy-lysosomal protein degradation pathway (autophagy pathway or autophagy-lysosomal pathway) may be activated to play an important role for cancer cell to defense against injuring during chemotherapy, Apoptosis signaling pathway and autophagy signaling pathway intersect mutually and inter depend with each other to determine the final fate of cancer cells. In morphology, autophagy-lysosomal pathway is characterized by forming double or multiple lipid membrane structure to pack cytoplasm and organelle to form autophagy vesicles, then fusing with lysosomes for degradation. Besides ubiquitin-proteasome pathway, autophagy-lysosomal pathway is the other most important degradation system in organism. Long live proteins, olden organelles, RNA and some macromolecules were degraded by autophagy to keep metabolic balance as well as homeostasis for internal environment. So some people regard that autophagy will help tumor cells to survive under the nutrition deficiency or medical stress environment through degradation of old organelles and long live proteins. Conversely, disruption of autophagy pathway may result in generous tumor cell death. Other study also show that over-activated autophagy pathway may lead to "autophagic cell death" or "typeâ…¡programmed cell death" due to excessive protein degradation. So, autophagy regulation may be a new method to enhance drug efficiency in cancer therapy.In this study, we make a deep research in the phenomenon and mechanism of autophagy activation during drug therapy from three levels of HCC cell lines, nude mouse HCC models and clinical HCC tissue samples.And we explored the intersection and molecular mechanisms of autophagy, autophagic cell death and apoptosis during this process. Preliminary, we researched for the mutual influence of two protein degradation pathway in organisms. Then, we may offer a new choice in HCC treatment, supplied a theoretical basis for combination drug therapy, and provide a new idea to improve chemotherapy of HCC patients.Part oneStudy of autophagy-lysosomal pathway in Proteasome Inhibitors against HCC in vitroVariation of the ubiquitin-proteasome pathway occures in many malignant tumors including HCC. As a new method, targeting ubiquitin proteasome pathway may acquire clinical reward in HCC treatment. Some findings also indicated that the ubiquitin proteasome system and autophagy lysosome system may be mechanistically linked. In this part, we will preliminary study the anti-cancer effect of proteasome inhibitors on HCC in vitro and understand the objective relationship between ubiquitin proteasome pathway and autophagy lysosomal pathway, further identify the precise role of autophagy during the treatment of proteasome inhibitors on HCC.Two different kinds of proteasome inhibitors, MG-132 and bortezomib, two different autophagy inhibitors based on distinctive mechanism,3-methyladenine (3-MA) and chloroquine(CQ), and three HCC cell lines having different apoptosis resistance and metastasis potential, MHCC-97H, PLC/PRF/5 and Huh-7(MHCC-97H >PLC/PRF/5>Huh-7) were selected in this part. MTT tetrazolium salt formation was used to detect proliferation. Morphological change of apoptotic cells was observed by Hoechst 33342 staining under fluorescent microscope. Apoptosis index was determined by flow cytometry after Annexin V-FITC/PI double staining. The autophagic activity was determined by GFP-LC3 redistribution and Beclin-1, LC3 Western blot. Then the autophagic activity was inhibited by silencing Atg5,3-MA or low-dose CQ, and the apoptosis induced by MG-132 or bortezomib was assessed to verify the accurate role of autophagy in the system.We found that MG-132 and bortezomib had significant anti-proliferative and pro-apoptotic effect in all three HCC cell lines. Among them, Huh-7 was most sensitive, median lethal dose needs 36h, MHCC-97H was some resistant, median lethal dose needs 60h, And PLC/PRF/5 had a moderate reactivity (48h). Also, autophagy was activated in all three HCC cell lines after two proteasome inhibitors treatment. After autophagy pathway was disrupted by CQ or 3-MA, apoptosis induced by MG-132 or bortezomib increased significantly in all three HCC cell lines (P< 0.05). At last, autophagic activity was inhibited by silencing Atg5 through siRNA, after MG-132 or bortezomib interference, apoptosis index also increased (P<0.05).The preliminary evidence in vitro indicates that targeting ubiquitin proteasome pathway is a new method for HCC treatment. Autophagy-lysosomal pathway will be activated as a compensatory system after proteasome inhibition and exert a protective role for cancer cells. Autophagy inhibition will reinforce the anticancer effect of proteasome inhibitors in cancer therapy. Proteasome Inhibitor bortezomib has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with multiple myeloma and mantle cell lymphoma. In contrast to the results from hematological malignancies, many clinical trials reported that bortezomib got a poor response in many solid tumors, such as gastric cancer and pancreatic cancer. We have proved that bortezomib has anti-proliferative and pro-apoptotic effect indeed on HCC cell lines in vitro. And also, Autophagy-lysosomal pathway was activated to exert a protective role for cancer cells. So we consider that autophagy may be one of the drug resistance mechanisms of bortezomib on solid tumors. Next, we will focus on the two protein degradation pathway in vivo for deeper research.MHCC-97H which is resistant to bortezomib and Huh-7 which is sensitive were selected in this part. Then we set up orthotopic nude mouse HCC models for MHCC-97H and xenograft nude mouse HCC models for Huh-7. Three days later, two kinds of HCC mouse models were randomly assigned to four groups (6 mice in one group) and received vehicle, CQ, bortezomib and CQ plus bortezomib, respectively. Mice were euthanized by day 32 after treatment. The tumor volume was calculated. Tumors of each group were analyzed by histological staining:Tunel staining to detect apoptosis, Ki-67 staining to detect proliferation and P62 staining to detect autophagy. At last, electron microscopy was used to analyze apoptosis and autophagy directly.35 days later, single bortezomib on Huh-7 xenograft models which was sensitive in vitro didn't show distinguished antitumor activity, there was no significant difference in mean tumor volume of bortezomib group compared with control group (P=0.85). Yet in MHCC-97H orthotopic models which was resistant in vitro, bortezomib inhibited the tumor growth and reduced the tumor volume significantly (P <0.05). And when combined with CQ, mean tumor volume reduced significantly compared with bortezomib alone in both two tumor models. Tunel staining showed that bortezomib alone may induce apoptosis significantly in both HCC tumor models compared with each control group (P< 0.05). Ki-67 staining indicated that bortezomib alone decreased proliferation index in MHCC-97H orthotopic models but not in Huh-7 xenograft models, and CQ improved the anti-proliferative and pro-apoptotic effect of bortezomib in two HCC models significantly(P<0.05). P62 staining suggested that bortezomib activated autophagy in two HCC models and CQ may inhibit this effect. At last TEM showed the evidence of apoptosis induction, autophagy activation of bortezomib and autophagy inhibition by CQ.This part found that bortezomib may inhibit the MHCC-97H orthotopic model but not Huh-7 xenograft model. Combined with other results, we consider the major reason of opposite results in vitro and in vivo is due to different tumor perfusion based on different tumor model types. Drug exposure of bortezomib should be much better in MHCC97H orthotopic model than Huh-7 xenograft model through intraperitoneal injection. So we presume that bortezomib may exert it's anti-cancer activity in advanced HCC patients through hepatic arterial infusion administration. In the same way, we found that autophagy-lysosomal pathway was also activated in vivo to protect tumor cells. Combination with autophagy inhibitor CQ may improve the anti cancer effect of bortezomib in vivo.Part threeStudy of autophagy-lysosomal pathway in oxaliplatin against HCC in vitro and in vivoIn the clinical treatment of HCC, some localized unresectable large HCCs may be converted to resectable small HCCs to undergo radical resection. Loco-regional chemotherapy such as hepatic arterial infusion chemotherapy is commonly used as a non-surgical approach for downstaging. However, it does not have the desired effect in most treated HCCs. We have confirmed that autophagy-lysosomal pathway was activated to play a protective role for HCC when proteasome inhibiton in vitro and in vivo. So, the same rule may also work for HCC during other chemotherapies. And it is worth exploring the possible mechanism for autophagy to protect cancer cells against apoptosis. Also autophagy status in HCCs after clinical therapeutic stress is need to be defined.The routine chemotherapy drug oxaliplatin, two autophagy inhibitors,3-MA (3-methyladenine) and CQ (chloroquine), antioxidant NAC (N-acetyl-cysteine) and two HCC cell lines, SMMC-7721 and Huh-7 were selected in this part. The autophagic activity was determined by GFP-LC3 redistribution, LC3 Western blot and TEM (transmission electron microscope). Apoptosis index was determined by flow cytometry after Annexin V-FITC/PI double staining. The intracellular levels of ROS in HCC cells were quantified by flow cytometry after DCF-DA (2',7'-dichlorofluorescein diacetate) staining. Xenograft Huh-7 HCC models were set up. Three days later, they were randomly assigned to four groups (6 mice in one group) and received vehicle, CQ, oxaliplatin and CQ plus oxaliplatin, respectively. Mice were euthanized by day 32 after treatment. The tumor volume was calculated. Tumors of each group were analyzed by histological staining:Tunel staining to detect apoptosis, LC3 staining to detect autophagy. At last, paraffin-embedded samples of 40 HCC cases (20 cases recieved locoregional chemotherapy and 20 were not) and 10 normal livers (as a baseline of autophagy in liver) were supplied to evaluate autophagy status of each group by LC3 immunohistochemical analysis.We found that autophagy-lysosomal pathway was also activated and played a protective role during apoptosis induction by oxaliplatin in HCC. Pretreatment with antioxidant NAC displayed significantly reduced apoptosis induction in response to oxaliplatin plus autophagy inhibition. Therefore, we conclude that the generation of ROS has an important role in apoptosis induction in this situation.This part further reveals that it is a common rule for autophagy-lysosomal pathway to be activated and exert a protective role during chemotherapies. Autophagy inhibition may improve drug effection. Increasing apoptosis caused by autophagy inhibition may be due to defects in autophagy result in accumulation of protein aggregates and defective mitochondria in the cytoplasm, then leading to increasing ROS production. Autophagy status in HCCs recieved locoregional chemotherapy is increased, so autophagy activation may be the reason for failure of downstaging, Autophagy inhibitors such as CQ is deserved to be used in clinic as adjuvant therapy.Conclusions1. Targeting ubiquitin proteasome pathway is a new method for HCC treatment. Autophagy-lysosomal pathway acts as a compensatory system and exerts a protective role for cancer cells. The anticancer effect of proteasome inhibitors will be reinforced through autophagy inhibition.2. The utilization of proteasome inhibitor bortezomib should not be limited to as adjuvant therapy with other chemotherapy drugs. It's anti-cancer validity depends on plenty tumor perfusion and effective drug exposure. To some extent, bortezomib does have anti-HCC activity, locoregional chemotherapy such as hepatic arterial infusion may exerts it's anti-cancer activity in advanced HCC patients to a best extent.3. It is a common rule for autophagy-lysosomal pathway to be activated and exerts a protective role during chemotherapies. Autophagy inhibition may improve actual drug effection. Increasing apoptosis caused by autophagy inhibition may be due to defects in autophagy result in accumulation of protein aggregates and defective mitochondria in the cytoplasm, leading to increasing ROS production.Novelty1. For the first time, we investigated the feasibility of targeting ubiquitin proteasome system as a method for HCC treatment,supplied an alternative way for HCC therapy. And we studyed the validity of proteasome inhibitor bortezomib in HCC treatment on the level of nude mouse HCC model, identified it's application conditions, widened it's utilization extent.2. For the first time, we clarified that it is a common rule for autophagy-lysosomal pathway to be activated and exerts a protective role during chemotherapies and the mechanism in it, supplied theoretical basis for combination therapy in clinic.Potential application1. Our research will be beneficial for further investigating the application of ubiquitin proteasome system in HCC treatment.2. Our research paved a way for proteasome inhibitor bortezomib application in clinical HCC treatment.3. It is a common rule to get better therapy reward through autophagy inhibition during chemotherapies. Combination with autophagy inhibitors such as CQ with other drugs on HCC or other tumors has a potential clinical significance.
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