Effect Of9-cis Retinoic Acid And Cytotoxic Agents On Proliferation Apoptosis And MycN And MDR1mRNA Expression In MycN Amplification Neuroblastoma

BackgroundsNeuroblastoma (NB) is the most common pediatric extracranial solid tumor of neural crest origin, which accounts for8%to10%of all childhood cancers. The incidence of NB per year is10.5per million children (age<15years). It is the third of the children’s malignant tumor. NB is characterized by high malignant, strong aggressivity and rapid progress. At the time of diagnosis approximately70%of patients with NB present with metastatic disease. Metastatic spread of NB can happen by both lymphatic and hematogenous routes.The bone marrow, bone, liver, lymph nodes and skin are common target of spread. So far, patients with stage3-4NB present with poor prognosis in spite of using multimodality therapy. The5year-event free survival was still less than40%and approximately15%of cancer-related deaths happened in high-risk NB groups.At present, the treatment of NB is mainly based on the risk of tumor grading and staging. The treatment model of high-risk groups is multi-disciplinary comprehensive treatment, which mainly consists of large doses induction chemotherapy, surgery, radiotherapy, bone marrow transplants after myeloablative Conditioning, biological immunotherapy and so on. Systemic chemotherapy with cisplatin, VP16, vincristine, doxorubicin and cyclophosphamide has been always in the core status. The aims of chemotherapy are killing cancer cells directly and inhibiting tumor cell growth. But at the same time cytotoxic drugs also damage normal tissue cells, whose side effects have seriously affect the children’s quality of life and growth and development. In addition, chemotherapy drug resistance of tumor has attracted more and more concern. Studies have shown that more than50%of the children in the high risk group of NB relapse resulting from chemotherapy drug resistance. In general, single-agent chemotherapy present with the bigger drug dosage, the more obvious side effects and the easier resistance. However, combination therapy can reduce or avoid the above incidence. Therefore, it is very important in exploring the way and mechanism of combination therapy.NB has the unique biological characteristics with self-differentiation and natural regression. Spontaneous degradation is found in the stage of newborns and infants. For those with4s period of International Neuroblastoma Staging System (INSS), they have eusemia and high healing rate although long distant metastasis have been observed. So, people continue to focus on studying differentiation treatment of NB. The study in vitro have found that some material, such as retinoic acid (RA), nerve growth factor (NGF), gamma interferon (y-IFN), interleukin-2(IL-2), phenylacetic acid sodium and so on, can induce NB to differentiate. Among these induced agents, the RA is studied mostly. Studies suggest that RA play a crucial role in the anti-tumor by induction of neuroblastoma cells apoptosis and (or) induction of cell differentiation. In the nature, RA exist three isomers:all-transretinoic acid (atRA),9-cis retinoic acid (9-cis RA),13-cis retinoic acid (13-cis RA). A long-term, randomized clinical trial in Children’s Oncology Group (COG) showed that13-cis RA administrated after intensive chemotherapy resulted in significantly improvement in5-year overall survival (OS). NB cell lines experiments in vitro demonstrate that the9-cis RA can induce expression of RA receptors (RAR), bind RA X receptor (RXR) with high affinity, and has the role of antiproliferation and provoking cell apoptosis. In addition, differentiation induced by9-cis RA is not reversed. Research of Chu, etc suggested9-cis RA is more effective in antiproliferation than13-cis RA. Therefore,9-cis RA has the most popular future. But research about NB treated with9-cis RA is very little. Domestic peers mainly engage in atRA study, and most of researchers abroad engage in the study of13-cis RA.In recent years, the molecular research of NB also enhances unceasingly in the wake of the development of molecular biology techniques. Many molecular biology index has been widely used in the diagnosis and treatment of NB. Currently NB molecular studies have shown that DNA and RNA ploidy,MycN gene amplification and expression, multiple drug resistance gene (multidrug hold genel, MDR1) and TrkA gene expression have close relation with occurrence, development, metastasis, regression and prognosis of NB. Especially amplification of MYCN is acknowledged as poor prognosis factor of NB. Data showed that20%of NB and40%of advanced stage cases exhibit amplification of the Myc-N proto-oncogene. MYCN amplification strongly correlated with advanced disease, rapid disease progression and poor outcome. MycN amplification mostly accompany high expression of MycN, which promotes the growth and proliferation of tumor cells and inhibits differentiation and apoptosis of tumor cells. So how to down-regulated MYCN expression of MYCN-amplificated NB is the focus of clinical research. Drug resistance of NB is an important reason of chemotherapy failure and tumor relapse, which seriously affects the prognosis. The drug resistance mechanism about tumor cells is very complex. The enhancement expression of MDR1gene which produces drug-resistant tumor cells is one of the most important reasons for the drug resistance. Therefore, we choose the expression of both MycN and MDR1as drug evaluation indicators.This experiment makes use of9-cis RA combined with the cytotoxic drugs on NB cells, and the research datas of which is a little at home and abroad. The following research reports is the basis of the experimental design:9-cis RA and cytotoxic drugs have a good synergistic effect on antiproliferation and cell apoptosis in SH-SY5Y NB cells in vitro, but their molecular mechanisms are not detailed.9-cis RA and gamma interferon can reduce MycN and MDR1mRNA expression through complementary action. Foreign studies reported that13-cis RA and cytotoxic drugs can down-regulate MYCN protein expression in vitro in Kelly NB cells. IMR32cells is applied in our experiment, as known MycN amplification cell lines model, which has many molecular biology characteristics of malignant NB with a small amount of S type cells. And domestic research mostly use SK-N-SH or SH-SY5Y cell lines without MycN amplification. Thus, our result of IMR32cells experiment is more representative and persuasive. In addition, this experiment utilizated the most advanced molecular biological technology and methods, containning Cell Counting Kit-8(CCK-8), Flow Cytometer (FCM), real-time Fluorescent Quantitative Polymerase Chain Reaction (FQ-PCR) in the world today. Our experiment design is rigorous, scientific and feasible. The research result has high reliability and certain clinical guidance value.ObjectiveTo study the effect of the combination of9-cis retinoic acid (RA) and cytotoxic agents on proliferation, apoptosis and MycN and MDR1mRNA expression in MYCN amplification neuroblastoma, which will provide theoretical basis for clinical rational use of drugs.MethodsExperiment was divided into the control group, single drug group (9-cis RA and four kinds of cytotoxic drugs) and double drug group (9-cis RA and each cytotoxic drug). The treated concentration of9-cis RA was15μmol/L. Concentrations of cytotoxic drugs were determined with IC(40±2) doses. The treated concentration of Vincristine (Vcr), cis-dichlorodiammine platinum (cDDP), etoposide (VP16) and adriamycin(Adr) was500μg/L,2000μg/L,500μg/L,10000μg/L respectively.IMR32cells were cultured in MEM medium containing10%fetal bovine serum,50units/mL penicillin G, and50mg/mL streptomycin sulfate. Cells were grown in a CO2incubator at37℃, with5%CO2and95%filtered air. Cell culture medium was renewal every2days. Cells were subcultured once every4-5days. When cells were in the logarithmic phase, cell cultures with1%serum were seeded into the96-well plates (2.5x104cells/100μl/well) for CCK-8assay, the12-well plates (2.5x105cells/lml/well) for FCM assay, or the6-well plates (5.0x105cells/2ml/well) for PCR assay. After24hour of incubation, the drugs were administrated into culture plates according to the above grouping and concentration. After24hour again, some experiments were done as follows:1) The cell morphological changes were observed by inverted microscope.2) The cell grow inhibition rate of various groups was assessed by CCK-8assay.3) The cell apoptosis index of various groups was assessed by flow cytometry.4) The gene expression of MYCN and MDR1of various groups was detected by real time Fluorescent Quantitative PCR.Results1) The cell morphological differentiation appeared in IMR32cells treated by9-cis RA alone. The cell became more round. The dendrite cells increased, and the cell neurites extended from the cell body. The degree of differentiation was more obvious than the control group (untreated by any drug). But there were no significant differences on cells morphological differentiation among other groups. Most of cells treated by cytotoxic drugs losed the adhesion and their dendrite became short or disappeared.2) The cell inhibition ratio of double drug groups was higher than the mono-drug groups (P<0.01). That of9-cis RA+Adr group was higher than the9-cis RA+cDDP group and9-cis RA+VP16group (P<0.05)3) The apoptosis index of double drug groups was higher than the control and mono-drug groups (P<0.01). That of9-cis RA+cDDP group was higher than other double drug groups.4) MycN-mRNA expression level of any drug groups was lower than the control group (P<0.05), that of double drug groups was lower than the mono-drug groups (P<0.05), and that of9-cis+Vcr group was lower than other double drug groups(P <0.05). MDR1-mRNA expression level of each groups except9-cis RA mono-drug group was higher than the control group (P<0.05), that of double drug groups was lower than the cytotoxic mono-drug groups (P<0.01), that of9-cis RA+Adr group was higher than other double drug groups (P<0.01)Conclusions9-cis RA can induce IMR32cells differentiation alone, but the differentiation is not observed obviously after using cytotoxic agents.9-cis retinoic acid and cytotoxic agents may have a synergistic effect on proliferation, apoptosis and down-regulated MycN expression in neuroblastoma cells. The combination of the9-cis RA with other drug groups can up-regulate the MDR1expression. By contrast,9-cis RA+cDDP has the better synergistic effect on apoptosis,9-cis RA+Vcr is more effective to reduce MycN expression.9-cis RA+Adr have higher growth inhibition rate, but leading high expression levels of MDR1. Therefore, the combination of9-cis RA with cisplatin or vincristine may be the better option as low-dose maintenance therapy for neuroblastoma.