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Influence of Dendritic Cells Combined with Docetaxel to Biological Behavior of Liver Cancer

Author: ZhangHongChao
Tutor: LiangJian
School: China Medical University
Course: Surgery
Keywords: dendritic cells Granulocyte-macrophage colony-stimulating factor (GM-CSF) liver cancer cells Docetaxel
CLC: R735.7
Type: PhD thesis
Year: 2008
Downloads: 138
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Abstract


IntroductionPaul Langerhans first saw DCs in 1868 within the skin epithelium.In 1973 Ralph Steinman,identified a rare cell type from mouse spleen that is involved in the induction of immune responses.For nearly 20 years,Dendritic cells(DC)derive from CD34~+ bone marrow stem cells.They are the most potent antigen-presenting cells in vivo which can take up,process and present antigens.DC display an extraordinary capacity to stimulate naive T cells,initiate primary immune responses and play unique roles in the induction of immune responses.Animal experiments confirmed mature DC pulsed with antigens elicited antigen-specific cytotoxic T lymphocytes to kill tumor cells.Since the first report on the successful clinical trials in B-cells lymphoma using DC pulsed with tumor antigen peptides in 1996,DC-based anti-tumor immunotherapy has raised a great deal of interest.The clinical and fundamental studies of DC-based vaccines need high quality and sufficient numbers of DC.DC extensively distribute in body,but they are present in such minute numbers in tissues and lack specificantigens.In recent years, people have been exploring how to get DC efficiently.The methods include isolating DC by density gradient centrifugation from tissues containing DC,inducing DC with GM-CSF(granulocyte-macrophage colony-stimulating factor)and IL-4 (interleukin-4)、low concentrations of GM-CSF、GM-CSF and IL-4 and IL-10(interleukin-10).All of the methods have both advantages and disadvantages.The quality and quantity of the obtained DC differ for these different methods In this study, our aim is to establish the method of inducing DC in vitro,to evaluate objectively characteristics of DC from different sources and to generate sufficient numbers of well-characterized cells to prepare for active immunotherapy.Primary liver cancer(PLC)is a common malignancy,and approximately 90%of PLC cases are hepatocellular carcinoma(HCC).Without any treatment,HCC would have a very poor prognosis with a median survival of 3 to 6 months.Surgical resection and liver transplantation should be the first choice in the therapy of HCC.However,it is difficult to detect HCC in an early stage for its clinical manifestations are highly nonspecific;and in patients with terminal HCC,if good chance of surgery missed,their prognosis would have been extremely bad;even in patients who have received curative resection,forty to sixty percent of patients would have recurrences five years after surgery.The occurrence of tumor metastasis and high recurrence rate would yield disappointing results in HCC patients.Therefore,novel strategies to prevent Recu-rrences and metastasis after hepatic resection are urgently needed.The rapid increase in knowledge of molecular biology and immunology has sparked new hopes for patients who have suffered from tumors.Most tumor biotherapies especially tumor vaccine strategies have focused on dendritic cells(DCs), which were considered to be the most important professional antigen presenting cells. Des express high levels of major histocompatibility complex molecules,costimulatory molecules,which can make the difference between tuining off the CTL precuesor and activating it.DCs also secrete critical cytokines such as IL-12 that contribute to CTL activation and memory.There have been several different strategies applied in animal experiments or clinical trials to deliver antigens to DCs,such as pulsing synthetic or eluted peptides,Transfec-tion with cDNA or RNA encoding known TAA,loading whole tumor lysate or tumor cell- derived RNA.However,these approaches are currently limited for clinical application because few human tumor specific antigens have been identified,and their immunogenicity is uncertain in most malignant tumors. The high polymorphism of the HLA system has also made it difficult to identify tumor-associated peptides as a vaccine for cancer therapy.In addition,tumor cell lysates or toal RNA loading creates the risk of inducing immune responses against numerous self-antigens shared with normal cells.An attractive approach to the enhancement of antitumor activity is to generate the fusions of whole tumor cells with Des.The use of whole-tumor as antigenic material offered considerable advantages.It eliminated the requirement to identify and characterize the specific antigens and could offer a broader repertoire of tumor-associated antigens,and thereby reduce the likelihood of tumor escape.The De-tumor fusions present a broad spectrum of tumor-associated antigens,which are processed endogenously and presented to cytotoxic T cell populations by the MHC classⅠpathways in the context of Cost-imulatory signals.Since human HCC,however, are usually weakly immunogenic tumors,it would be important to increase HCC immunogenicity to allow infiltration of antitumoral effector cells resulting in prolonged survival.In the present study,we constructed fusion cells from human hepatocellular carcinoma cell HepG-2 and Des from HCC patients,and investigated their biological characteristics and immune effects in vitro and in vivo.De-based immunotherapy,however,remains difficult to handle for scale up, definition of quality control parameters,and long-term storage.Furthermore DC culture is time-consuming,expensive and lack of criteria for standardization of final product.Docetaxel is a new taxoid structurally similar to paclitaxel,a semisynthetic product of a renewable resource,the needles of the European yew,Taxus baccata L.In comparison to paclitaxel,Docetaxel is more potent as an inhibitor of microtubule depolymerization.Docetaxel has shown an active effect against cancers.Docetaxel has been used in the treatment of many carcinomas,including mammary cancer, NSCLC.pancreatic cancer,soft tissue sarcoma,head and neck cancer,stomach cancer, ovary cancer,prostate cancer,etc.It’s antitumor activity is remarkable either in monotherapy or in combination therapy.Docetaxel and De have been proved to hold tumor activity.their properties are linked to their cytotoxic role.But we knew little about their mechanism as yet.We have not heard that there been combined.In this communication,we aim to explore the effects of the Docetaxel combined with DC on lethal effect of HepG-2 Cells in vitro and subcutaneous tumor in nude mice in vivo.ObjectiveBy inducing dendritic cells in vitro,Correlation between different culture conditions and Biological characteristics of DCs is analyzed.Feasibility on inducing specific DC in clinicalStudy is Discussed.Lethal effect of hepG-2 cells activated by Docetaxel combined with whole cell lysates pulsed dendritic cell(DC)in vitro.New methods were discussed.The difference In the Inhibition of liver cancer activated whole cell lysates pulsed dendritic cell combined with Docetaxel and the two separate applications in order to lay the foundation for the clinical application.MethodThere are three methods inducing DC.The first protocol:dendritic cells(GMlo DC) are generated with low concentrations(0.2 ng/ml)of granulocyte-macrophage colony-stimulating factor(GM-CSF)in the absence of the other cell factors in vitro; The second protocol:dendritic cells are generated with,conventional method,routine concentration(5ng/ml)of GM-CSF plus interleukin-4(5ng/L);The third protocols: dendritic cells are generated with routine concentration(5ng/ml)of GM-CSF、interleukin-4(5ng/L)plus interleukin-10;DC are induced simultaneously and then harvested on day 13.The phenotypic and functional properties of DC induced with three protocols are compared with each other.Interleukin-12 concentrations of supernatant and cell yield,in three protocols,are investigated.HepG-2 cells is divided into four groups According to the experimental treatment by different factors of liver cancer cells:DC group:To detect the anti-hepatic cancer ability of whole cell lysates pulsed dendritic cell(DC)acted as an adjunvant;DOC group:To detect the anti-hepatic cancer ability of Docetaxel acted as an adjunvant. The joint group:To detect the anti-hepatic cancer ability of whole cell lysates pulsed dendritic cell(DC)combined with Docetaxel acted as an adjunvant.The control group:There were not any treatment factors.Apoptosis of cells in four groups in the number were detected by FCM.cell apoptosis in the morphology was observed by fluorescent staining.4-methyl-tetrazolium Act(MTT)observed liver cancer cells in apoptosis,as well as Western blotting detection bcl-2 protein to reflect the changes in liver cancer cell apoptosis.According to different factors treated to liver cancer,the nice mice were divided into four groups.Each is six nude mice.Treatment factors were injected every seven days.tumor size was measured once every five days.the weight of tumors was measured at the last time.as follows:DC group:To detect liver cancer inhibition ability of whole cell lysates pulsed dendritic cell(DC)acted as an adjunvant;DOC group:To detect liver cancer inhibition ability of Docetaxel acted as an adjunvant.The joint group:To detect liver cancer inhibition ability of whole cell lysates pulsed dendritic cell(DC)combined with Docetaxel acted as an adjunvant.The control group:There were not any treatment factors.In vivo the toxic effects of whole cell lysates pulsed dendritic cell to the tumor cells was detected,the same study was done by two separate applications.By detecting the growth of tumor、apoptosis and proliferation by immunohistochemistry.Western blotting detected bcl-2 protein in liver cancer organizations to observe the expression of both the inhibition of liver cancer tumor.ResultsCompared to DC induced with the second protocol,There are small colony DC in the first and third protocol revealed by Inverted microscope in thirteenth day In a more decentralized.Electric microscope revealed that cells round and processes less.DCs showcharacteristics of immature DC.The second group show significant colonys by inverted microscope.There are burr-like processes around cells.It show rough surface under electric microscope.There seems to be a more the dendritic processes irregular-shaped protuberance on the surface of the show-intensive villous of cells.DCs show characteristics of mature DC.In thirteenth day the proportion of the surface expression of OX62 respectively is (79.4±5.7)%,(85.3±8.7)%and(79.6±4.6).there are not significant difference between the two groups(p>0.05).The ratio of expression of CD86 is respectively(13.5±2.1)%,78.3±11.6)%and 35.4±5.2)%.There is significant differences berween the first group、third group and the second group.So it is imDC in the first group and third group and mature DC in second group.MTT test show DC from the first group and third group can not effectively stimulate T-cell proliferation.But DC from the second group have a stronger ability to stimulate T-lymphocyte.Difference of SI of The third group and the first group compared with the second group is significant(P<0.05).Cell proliferation in the first group[(3.0±0.7)×106]was significantly lower than the second group[(13.0±1.1)×106]and the third group[(11.9±2.1)×106],there are significant differences(P<0.05).The lethal rate of target cells detected by MTT respectively in DC group were(12.1±1.9)%,(33.5±2.4)%,(70.0±3.4)%.It were(13.5±2.1)%,(18.0±1.7)%, (39.6±1.8%)in Doe group and(68.2±4.3)%,(51.9±3.2)%,(62.5±3.9)%in joint group.There are the highest rates of destruction in the Joint Group compared with the DC group and the DOC group of the highest rates of destruction.There are significant differences(P<0.01).In the DC group,HepG-2 liver cancer cells with PI-stained,and then detected by FCM,the apoptosis rate was 18.6±2.1%and in the DOC group 19.0±2.0%and in the group 32.6±2.3%.the control group without apoptosis performance.difference was significant between Joint group and the DC group、the DOC group.Application of Western blotting detected HepG-2 liver cancer cells in the bcl-2 protein expression.The Joint Group of bcl-2 expression rate than the DC group and the DOC group of bcl-2 expression was much lower,it is obvious differences.Detected by staining the number of apoptosis,PI alone with the joint group of apoptosis to significantly more than the number of DC Group and the DOC group of apoptosis in cells.Inhibition of the expression of bcl-2 in Joint Group is the most obvious in three groups.the test of the tumor size shows inhibition rate of DC group,DOC Group and the Joint Group were(40.6±3.4)%,(36.9±2.9)%and(60.0±3.0)%respectively. Differences were significant between the joint group and DC group,DOC Group (P<0.01).expression of bcl-2 protein in the Joint Group was lower significantly than that of the DC group and the DOC group.Conclusion1、DC induced with GM-CSF plus IL-4 plus IL-10 and the low concentration of GM-CSF is immature DC.DC induced with GM-CSF plus IL-4 is mature DC.the second and third protocol is better than the protocol with the low concentration of GM-CSF andthe surpassed the first protocol in the yield.2、Lethal effect of hepG-2 cells activated by whole cell lysates pulsed dendritic cell(DC)combined with Docetaxel in vitro is better than the two separate applications,which prompted the two factors in the anti-level mechanism to liver cancer exist to promote the role of each other.3、The inhibition to tumor from DC-HepG-2 hepatic cancer cell fusion vaccines combined with Docetaxel is better than the two separate applications,the promoting the role each other was proved further,so it may be a new protocol to the liver cancer.

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