Mechanism Of Propranolol Hydrochloride In Inhabiting The Growth Of Liver Cancer Cells

Liver cancer(LC)has the second highest annual mortality rate of malignant tumors and the fifth highest incidence rate in the world.In 2012,there were 782,500 new cases of liver cancer and 745,500 cases of liver cancer deaths.China account for about 50%(1,2).The clinical features of primary liver cancer are occult onset,rapid progress and extremely poor prognosis,which seriously threatens the health of people in the world.Therefore,actively seeking new therapeutic directions,it is an important task in the diagnosis and treatment of liver cancer in China and even in the world.Recent studies have found that ?-adrenergic receptor(?-AR)signaling pathway is closely related to the occurrence and development of many kinds of tumors,especially ?2 adrenergic receptor signaling pathway.?2-AR singling pathway also are involved in multiple processes of tumorigenesis and progression,including inflammation,angiogenesis,apoptosis,anoikis,cellular immune responses,cell movement and autophagy.It is closely related to the occurrence,development and metastasis of tumors(3).?-AR signaling pathway is also involved in the occurrence and development of liver cancer.In China,most of liver cancers develop on the basis of liver cirrhosis,especially hepatitis B related liver cirrhosis.In patients with cirrhosis,norepinephrine and epinephrine are significantly increased duing to the activity of sympathetic nervous system,and there is a disorder of catecholamine metabolism.Non-selective beta blocker(NSBB)may be unique in anti-cirrhosis patients.Inflammation promotes tumorigenesis,and non-selective beta-blockers reduce bacterial translocation,then reducing pro-inflammatory cytokines from the intestine into the liver.NSBB inhibit catecholamines which drive tumor cell migration,tumor angiogenesis,invasion,and proliferation.NSBB is anti-angiogenic and therefore can inhibit the growth of liver cancer.A meta-analysis also showed that NSBB reduce mortality in patients with cirrhosis,not only by reducing the rate of gastrointestinal bleeding,but also by reducing the incidence of liver cancer.Propranolol hydrochloride(5)is a primary preventive medicine to reduce portal vein pressure and prevent esophageal and gastric variceal hemorrhage in the "Clinical Guidelines for cirrhosis patients with esophageal and gastric varices bleeding" in China.In addition to the traditional treatment of hypertension,arrhythmia,ischemic heart disease and other diseases,recently studies found that propranolol inhibits angiogenesis and induces apoptosis of microvascular endothelial cells.PPL have significant curative effect on proliferation and remission of infantile hemangiomas.So it has been recommended for the treatment of infantile hemangiomas first-line drugs by many guidelines(4).For more than 40 years of clinical practice,PPL has been proven to be safe and well tolerated.Because of its blockade effect on ?-AR,especially ?2-AR,PPL should be further development in the treatment of tumors.In our study,PPL was given to of LC cells to observe whether it can inhibit cell proliferation in LC cells.The normal human liver cell line was used as a control to select the most appropriate concentration of PPL: this concentration can inhibit the proliferation of LC cells,while not affecting the proliferation of normal hepatocytes.At the same time,we test whether the expression level of ?-receptor on the surface of LC cells is higher than that of normal liver cells,in order to determine whether PPL regulates the growth of LC cells through the action of ?-receptor signaling pathway.We also study the effect of PPL on the migration and invasion of LC cells.Next,we will focus on whether the death of liver cancer cells caused by PPL is achieved through apoptosis or autophagy,and explore the mechanism of action of PPL in apoptosis and autophagy.This study can preliminarily explore the role of ?-receptor signaling pathway in the proliferation and death of LC cells,and explore the mechanism of PPL,a widely used clinical drug,to provide a direct basis for clinical experiments.In order to provide new methods for the treatment of LC,there are few reports in the world.Methods: 1.Immunofluorescence,Western Blot and q RT-PCR techniques were used to observe the distribution of ?-AR on the surface of different hepatocellular carcinoma cell lines.At the same time,the changes of ?-receptors in LC lines after different concerntration of PPL were observed,to elucidate whether PPL exerts an effect on LC cells through ?-AR;2.The long-term dynamic imaging and analysis system of Incu Cyte Zoom was used to dynamically observe the proliferation of LC cells and normal hepatocytes after different concentrations of propranolol hydrochloride were administered;the proportion of dead cells was measured by flow cytometry,and isoproterenol hydrochloride was used to study in vitro.The effect of blocking ?-AR signaling pathway on the proliferation and survival of LC cells;3.ICI 118,551(the specific ?2 receptor inhibitor)and formoterol(specific ?2 receptor agonist)were administrated to LC cells in order to observing cell proliferation and studying whether the inhibition effect of PPL was mainly through inhabiting ?2-AR or not.4.The effect of PPL on the migration and invasion of LC cells was observed by scratch experiments using the Incu Cyte Zoom long-time dynamic imaging and analysis system.5.Morphological effects of PPL on apoptosis of LC cells were observed by DAPI staining and Hoechst33342/PI staining;quantitative detection of PPL on LC cells by flow cytometry Annexin V/PI double staining;6.The effect of PPL on the cell cycle of LC cells was detected by flow cytometry.7.The effect of PPL on the mitochondrial membrane potential of apoptotic cells was detected by immunofluorescence JC-1;Western Blot technique was used to study the mechanism of PPL blockade on the apoptosis of LC cells after ?-receptor signaling pathway.Explore the effects of ?2-AR signaling pathway on the apoptosis of LC cells induced by PPL were observed by ICI 118,551 and formoterol.9.Immunofluorescence and Western Blot were used to observe the induction of autophagy in LC cells by PPL;Lentivirus transfected with stub RFP-sens GFP-LC3 was transfected into LC cells to detect the changes of autophagy flux by PPL.10.By administration ICI 118,551 and formoterol to LC cells in order to observing whether ?2-AR signaling pathway was involved in PPL induced autophagy.11.Using WB technology to study the changes of autophagy related proteins in PPL and explore the possible mechanism.12.Incubate LC cells using apoptosis inhibitor(Z-VAD),autophagy inhibitor(3-MA)and autophagy activator(rapamycin)to study the relationship of between PPL-induced cell apoptosis and autophagy by WB,IF and so on.Result 1.?-AR expression is increased in LC cells,mainly ?2-AR.PPL inhibits proliferation of LC cells by decreasing ?-AR.2.PPL mainly inhibits the proliferation of LC cells by blocking ?2-AR.3.PPL can inhibit the migration and invasion of LC cells.4.PPL can induce apoptosis of LC cells,mainly by blocking the ?2-AR signaling pathway.5.PPL can induce LC cell apoptosis by mitochondrial pathway.6.PPL can induce autophagy in LC cells and increase autophagic flow production.7.PPL negatively regulates cell autophagy by blocking ?-AR signaling pathway.8.It was negative correlation between PPL-induced apoptosis and autophagy in LC cells.Conclusion In this study,LC cells were the research object.We demonstrated that PPL can control the growth of LC cells through the block of ?-AR.We have found that ?-AR are indeed increased in LC cells and the use of NSBB,PPL,can be effected through ?-AR.PPL can inhibit the proliferation,the migration and invasion of LC cells.On the other hand,PPL can induce apoptosis and autophagy in LC cells and the two antagonistic each other.Our study for comprehensively analyzed the effects of PPL on proliferation,invasion and migration,apoptosis and autophagy of LC cells,and preliminary explored its mechanism.This provides clues for the basic research on the mechanism of ?-AR signaling pathway in hepatocarcinogenesis,and has also found a new direction for the clinical exploration of LC drug assisted therapy(anti-hepatoma effect of PPL).Creative results: PPL can inhibit the proliferation,migration and invasion and induce apoptosis and autophagy of LC cells.PPL has anti-tumor effect.New insights: PPL has anti-hepatoma effects.