Experimental Study Of The Effect Of Antitumor And The Molecular Mechanism Of Honokiol

BACKGROUND:Pancreatic cancer is one of the most malignant tumors in digestive system,with the eighth leading cause of ten cancer-related death.There are 227,000 people who died from pancreatic cancer with the incidence of 0.007%-0.009%in men and 0.0045%-0.006%in women and its morbidity is on the increase all over the world. Operative resection is the only way to cure this desease.But only 10%of these patients has the chance of operative resection due to the low rates of diagnosis and rapid progression.The overall 5-year survival rate of patients with pancreatic cancer is lower than 1%owing to recurrence and metastasis.Chemotherapy is one of the most important adjuvant treatment strategies.But the insensitivity of pancreatic cancer to existing chemotherapy resulted in 20%reaction rate.Therefore,it is urgent and necessary to go on seeking new durgs.Honokiol,a major phenolic constituent isolated from the root and stem bark of magnolia species,has been reported to have several pharmacological effects, including anti-inflammatory effects,anti- thrombosis,antioxidant and anti-anxiety.Recently,honokiol has been shown to induce apoptosis in several cancer cell lines.And honokiol was shown to overcome conventional drug resistance in human multiple myeloma by induction of tumor cell apoptosis,there are data to support the view that tumor cells are more susceptible to honokiol-iduced apoptosia compared with normal PBMCs.In such circumstance,honokiol may be promising and valuable in the treatment of pancreatic cancer.And as far as we know, there is no report about the effect of honokiol on the treatment of pancreatic cancer until now.It is both very promising and of great importance to explore the effect of honokiol and its action mechanisms in the treatment of pancreatic cancer.Part 1 Effects of honokiol on proliferation and apoptosis of human pancreatic cancer PANC-1 cellsObjective:To decide whether honokiol can inhibit the growth of PANC-1 cells in vitro;and to examine the effect of honokiol on cell cycle and cell apoptosis in PANC-1 cells.Methods:1,MTT colorimetric assay was used to determine the effect of honokiol on the growth of PANC-1 cells.2,Flow cytometric analysis was used to detect cell cycle changing after honokiol intervention. 3,Flow cytometric analysis was used to detect the percentage of apoptotic cells.Results:1,Honokiol inhibited PANC-1 cells proliferationPANC-1 cells were treated with 2.5,5,10,15,20,30,40,50μg/ml honokiol, and cell viability was assayed at 12,24 and 48h using MTT assay.Proliferation inhibition rate with honokiol treatment at concentrations of 5-40μg/ml after 12 h ranged from 0.88%to 59.97%,whereas after 24 and 48 h ranged from 3.13%to 79.91%and 7.66%to 92.97%,respectively.These findings demonstrated that honokiol inhibited cell viability in a dose- and time-dependent manner.2,Honokiol induced cell cycle blockFlow cytometric analysis was used to detect cell cycle changing after honokiol (10-40μg/ml)treated PANC-1 cells for 24h.The percentage of cells in the G1,S and G2 phases of the cell cycle in untreated PANC-1 cells were 47.33%±2.52%,36.82%±0.89%and 15.85%±1.41%,respectively,after exposed to 10,20 and 40μg/ml honokiol for 24 hours,the rates of cells in the G1 phase were 57.87%±1.43%,72.95%±1.96%and 77.08%±1.58%,respectively;the rates of cells in the S phase were 27.62%±1.04%,13.10%±1.81%and 15.07%±0.99%, respectively;the rates of cells in the G2/M phase were 14.51%±2.21%,13.95%±1.06%and 7.85%±0.46%,respectively.These data showed that honokiol induced the accumulation of PANC-1 cells in the G1 phase of the cell cycle in a dose-dependent manner.3,Effect of honokiol on apoptosis of PANC-1 cellsFlow cytometric analysis was used to detect cell apoptosis after honokiol (10-40μg/ml)treated PANC-1 cells for 24h.The percentage of early apoptotic cells and late apoptotic cells in untreated PANC-1 cells were 2.38%±1.65%and 1.26%±0.23%,respectively.After exposed to 10,20 and 40μg/ml honokiol for 24 hours,the percentage of early apoptotic cells were 7.8%±1.56%,9.0%±0.64%and 19.4%±1.09%,respectively;the percentage of late apoptotic cells were 11.34%±0.76%,31.23%±1.76%and 40.98%±2.31%,respectively.Conclusion:Honokiol can significantly inhibit the growth of PANC-1 cells via cell apoptosis and G1 cell cycle arrest.Part 2 Effects of honokiol on invasion and metastasis of human pancreatic cancer PANC-1 cellsObjective:To examine the effect of honokiol on invasion and metastasis of human pancreatic cancer PANC-1 cells.Methods:1,Cell-base adhesion assay was used to determine the effect of honnokiol on the adhesion of PANC-1 cells to fibronectin(Fn).2,Wound healing assay was used to determine the effect of honokiol on the migration of PANC-1 cells. 3,Invasion assay was used to determine the effect of honokiol on the invasion of PANC-1 cells.Results:1,The effect of honnokiol on the adhesion of PANC-1 cells to fibronectinAfter treatment by honokiol for 24 hours,the ability of adhesion was attenuated between PANC-1 cells and fibronection in a dose-dependent manner.After exposed to 10,20 and 40μg/ml honokiol for 24 hours,the adhension inhibition rate were 20.01±1.56%,42.78±3.89%and 65.34±2.67%,respectively.2,The effect of honokiol on the migration of PANC-1 cellsWound healing assay showed that the cellular motility was evidently inhibited in dose-dependent manner.3,The effect of honokiol on the invasion of PANC-1 cellsThe results of the in vitro invasion assay displayed that honokiol was able to inhibit invasion ability of PANC-1 cells dose-dependently.After treatment by honokiol(10-40μg/ml),the percentage of invasion were 76.34%,52.72%,26.54%, respectively.Conclusions:Honokiol elicited significant inhibition of cell adhesion,migration,and invasion in human pancreatic cancer PANC-1 cells in vitro. Part3 Molecular mechanisms of honokiol-induced apoptosis,cell cycle arrest and inhibition of invasion abilityObjective:To identify the molecular mechanisms of honokiol-induced apoptosis,cell cycle arrest and inhibition of invasion ability.Methods:1,Western blot assay was used to determine the protein level of caspase-8,procaspase-9,cytochrome C,procaspase-3 and PARP.2,Western blot assay was used to determine the protein level of Bcl-X_L,Bcl-2,Bad and Bax.3,Western blot assay was used to determine the protein level of CyclinD1 and CDK4.4,Western blot assay was used to determine the protein level of MMP9.5,Western blot assay was used to determine the effect of honokiol on MAPK and PI3K/Akt pathways.6,MTT colorimetric assay was used to determine the effect of p3gMAPK pathway inhibitor(SB 203580)and ERK pathway inhibitor(PD 98059)on the growth inhibition of honokiol.7,Western blot assay was used to examine the protein level of cleaved-caspase-3,Cyclin D1 and CDK4 after treatment by honokiol and SB203580. Results:1,The effect of honokiol on the expression of caspase-8,procaspase-9,cytochrome C,procaspase-3 and PARPWestern blot analysis indicated that treatment of PANC-1 cells with honokiol resulted in a decreased expression of pro-caspase-3 and -9,the release of mitochondrial cytochrome C,the cleavage of poly(ADP-ribose)polymerase(PARP), but did not lead to the activation of caspase-8.These data showed that honokiol triggers apeptotic cell death in PANC-1 cells largely through the intrinsic pathway.2,The effect of honokiol on Bcl-X_L,Bcl-2,Bad and Bax expressionHonokiol caused a decrease in Bcl-X_L and Bcl-2 protein levels,and an increase in Bad protein level,but Bax protein was not significantly altered by honokiol in PANC-1 cells.3,The effect of honokiol on Cyclin D1 and CDK4 expressionHonokiol treatment(10-40μg/ml for 24h)of the PANC-1 cells resulted in significantly dose-dependent down-regulation of the protein expression of Cyclin D1 and CDK4.4,The effect of honokiol on MMP9 expressionWestern blot analysis indicated that treatment of PANC-1 cells with honokiol resulted in a dose-dependent decrease in MMP-9 protein expression.5,The effect of honokiol on MAPK and PI3K/Akt pathwaysp38 MAP kinase was significantly activated by 40μg/ml honokiol,honokiol increased the amount of phosphorylated p38 MAP kinase with the maximal induction at 30 min,and the activation lasted for 6h.And honokiol decreased the amount of phosphorylated ERK in a dose-dependent manner.However,honokiol had little effect on JNK and PI₃K/Akt pathway.These data suggested that ERK and p38 MAPK pathway maybe have an important role in anti-pancreatic cancer effect of honokiol.6,The effect of p38 MAPK pathway inhibitor(SB 203580)and ERK pathway inhibitor(PD 98059)on honokiol-induced growth inhibition in PANC-1 cellsMTT colorimetric assay showed that SB203580 significantly reversed honokiol-induced decrease in cell viability whereas PD98059 had no effect on it.7,Honokiol increased p38 MAPK phosphorylation,and SB203580,an p38 MAPK inhibitor,attenuated caspase-3 activation and increased the expression of cyclin D1 and CDK4Western blot assay showed that the cleaved caspase-3 was clearly apparent after treatment with honokiol whereas SB203580 attenuated caspase-3 activation.And SB203580 also reversed honokiol-induced decrease in the expression of cyclin D1 and CDK4.Conclusion:Honokiol-induced apoptotic cell death via the intrinsic pathway and cell cycle block are associated with p38 MAPK pathway.Honokiol induced the apoptosis in PANC-1 cells via activation of p38 MAPK leading to the downregulation of Bcl-X_L and Bcl-2,the upregulation of Bad,and then caspase-3 activation and cleavage of PARP.And honokiol induced cell cycle arrest via activation of p38 MAPK leading to the downregulation of cyclin D1 and CDK4. Part 4 The anti-cancer effect of honokiolin PANC-1 mice tumor models in vivoObjective:To evaluate the therapeutical effect of honokiol on pancreatic cancer in the experimental cacer models,and to investigate its potential mechanism.Methods:1,Athymic mouse tumor model was made and observed the effect of honokiol on tumor growth.2,The tissue and cell morphologic change were observed after treatment with honokiol.3,Immunohistological technique was used to detect the expression of vascular endothelial growth factor(VEGF).Results:1,The effect of honokiol on tumor growthCompared with the saline control group and DMSO control group,the tumor size of the groups treated with honokiol were significantly reduced,volumes of three groups were 4.456±1.93cm³,4.765±1.67 cm³ and 2.641±1.03 cm³,respectively. and the tumor weight of the groups treated with honokiol were also significantly reduced,weight of three groups were 2.354±0.634g,2.198±0.567g and 1.095±0.302g,respectively. 2,Tumor morphology was observed with HE stainingIt can be observed by light microscopy in HE staining sections that tumor cells in tumor tissue is not uniform in shape and size.Heteromorphism of cell nucleus was obvious,pathologic karyokinesis can be seen.Obvious necrosis and cell apoptosis were found in tissues of honokiol-treated group.3,The effect of honokiol on the expression of VEGF in tumor tissuesVEGF was expressed largely in the tumor cells,some vascular endothelial cell also have the expression of VEGF.Compared with the saline control group and DMSO control group,the expression of VEGF in the groups treated with honokiol was significantly reduced.These data showed that honokiol could reduce the expression of VEGF in tumor tissue.Conclusion:Honokiol can significantly inhibit the growth of pancreatic cancer in nude mice, which may be associated with decrease in VEGF expression in tumor tissue.