The Study Of 9-Cis Retinoic Acid And Gamma-Interferon Synergize In Inducing Differentiation, Apoptosis, Growth Inhibition And Its Molecular Mechanisms Of Human Neuroblastoma Cell In Vitro

BACKGROUND:Neuroblastoma (NB) is the most common extracranial solid tumour origining from sympathetic nervous system in children. It is derivated from neuroblast migrating from growing spinal cord uter layer and primitive neural crest cell with clinical characteristic of high malignancy, strong invasiveness, more advanced stage cases and bad therapeutic efficacy. It is the third of the children's malignant tumor with the incidence rate of 1/100,000 and account 15% of case fatality ratio. It is characterized by a diversity of clinical behaviour. The patients less than 1 year old are usually own to better stge, sensitive chems and high cure rate. Some tumor may undergo spontaneous regression. For the children older than 1 year, the tumors can transfer to the distant locatin such as bone, liver, skin, marrow and lymph node in 75% cases. These tumors have high malignancy and permanent recurrence despite of combined therapy with surgical intervention, chemotherapy and bone marrow transplantation et al. It is becoming a common cause threating children's life in the morden society in which the level of diagnosis and treatment of congenital malformation and infectious diseases is obviously elevated. So how to improve the level of clinical research, diagnosis and treatment of NB has caused widespread attention in domestic and foreign scholars.In the last 20 years, even though the molecule mechanism and therapy method of NB has progressed greatly, there are still poor therapeutic effects. So the peculiar phenomenon of spontaneous degeneration of NB attracts people's interest. Spontaneous degeneration means tumor shrinks automatically even disappears in the circumstance of rare or no systematic treatment. NB is the most commen tumor which degenerated spontaneously in human being. In autopsy of mature fetus or newborn that died for other causes, nodule residua found under microscope are called normal position NB in some individual. It is same to typical NB in morphology and cytology but smaller in volume and no metastases. The detection rate of this kind of in situ neuroblastoma is 40 times higher than newborn NB found in clinic. In situ neuroblastomas are not found in infants older than 3 months, which indicates that some cases degenerate spontaneously within several months after birth. It is key characteristic that a few NB can differentiate to ganglioneuroblastoma naturally. Natural degeneration is found in the stage of newborn and infant. For those momingsickness at the 4 period of international staging, they have eusemia and high rate of natural degeneration although long distant diversion (liver, bone marrow et al) have taken place. So, people assumed that inducing cell differentiation and apoptosis is more consistent with nature than killing and wounding cell directly. NB is considered as the most hopeful tumor which can reveal retroconversion spontaneous process of cancer and conduce to restrain its malignant process by taking measures. The unique bionomics of natural regression and reversion of NB provides theoretical foundation for the therapy of differentiation and induction. How to induce differentiation, retroconversion and regression of NB cell by anthropogenic method is the issue of tumor research nowadays.Induced differentiation refers to the phenomenon that malignant tumor turn to normal direction under the action of in vivo and in vitro antileptic drugs. The study in vitro found that some material, such as ratinoic acid (RA), interferon (IFN), 2-dibutyl orthophosphoric acid gland and sodium phenylacetate, can induce NB to differentiate. Among these drugs the RA is studied mostly. RA and its receptor possess the ability of controling cell differentiation, proliferation and apoptosis. It is the syudy focus that using RA to treat NB and to make it to deteriorate. 9-cis RA, 13-cis RA and at RA are 3 kind of analog isoms of RA. NB cell line study indicates that 9-cis RA is more effective in the aspect of inducting expression of RA receptor, preventing NB proliferation, producing consecutive morphology differentiation and provoking cell apoptosis. So RA has the most popular future. Antileptic needs large dose when used alone. So it has obvious side effect and occurrence of persister also influence curative effect. The research found that known antileptics have different mechanism on inducing tumor differentiate, such as receptor mechanism, enzymology mechanism, immune enhancement mechanism, gene expression and regulation mechanism and apoptosis mechanism. The original marked change of NB differentiation induced by RA is down regulation genetic transcription of MYCN. Using MYCN gene complementation oligonucleotide to seal MYCN and unable its expression can cut down NB proliferation and induce its differentiation. It indicates that regulating MYCN gene is the significance component element of inducing differentiation by RA. Similar to RA,γ-IFN can down regulate MYCN expression in vivo and in vitro experiment and has synergistic reaction with RA. But they have different mechanism of priming MYCN. It provides the proof of combination INF with RA. RA and IFN belong to the exogenous and endogenous differentiation antileptic respectively. Combination of these drugs can elevate sensitivity of tumor, decrease dosage, avoid side effect and improve therapeutic efficacy.The last few years, profound investigation to molecule factor which affects prognosis of NB have carried out by extensive scientific researchers. They found that DNA, RNA ploid and multiplication cycle of tumor cell, determination of MYCN and MDR1 gene et al. have close relation with occurrence, development, diversion, regression and prognosis of NB. So monitoring these indexes can reveal molecular biology mechanism that induction agent induce NB cell differentiate and apoptosis, evaluate curative effect of combination RA and IFN objectively, provide theory foundation and instruction for further clinical application.Although the orthodox therapeutic tools, such as operation, chems and radiotherapy, have a great effect on elevating survival rate of patients suffering malignant tumor in children, these methods can destroy normal tissue or organs, change normal physiological functions, kill and wound normal cells in a a great quantity, bring about serious toxin and side effects or interfere with endocrine function of human body at the same time of removing and ruinning tumors. So they influence quality of life and growth and development of patients in a different extent, even restrict curative effect improvemet further more in some extent. The differentiation therapy opens up a brand-new way to cure malignant tumor for its fundamental feature. Its purpose of treating tumours is to induce malignant tumor cells differentiate into normal or close to normal cells. So it has no toxic reactions of bone marrow and immune suppression for not killing and wounding normal cell of human body. Because the radiotherapy and chems have the side effects of affecting skeleton growth and reproduction function which can influence quality of life directly, so the differentiation therapy has specific clinical superiority for theses children patients who are at the stage of growth and development.This experiment utilizated the most advanced molecular biology experiment technique, methods and facilities, Flow Cytometer (FCM) and real-time fluorescent quantitation polymerase chain reaction (FQ-PCR) et al, in the world fully. Experimental design is rigorous, scientific, novel, and unique. It has high feasibility and value of clinical guidance. We haven't found same empirical study yet through consulting domestic and foreign literature widespreadly. In the condition of gene therapy faces a variety of barrier and is going to standstill, induction differentiation therapy of tumor is becoming a newly rising study issue as a brand-new approach for treating malignant tumor in children. It provid a powerful method and become an important ingredient of combined therapy which involves in multiple subjects. It has a positive significance on raising survival rate especially life quality of children patients. Differentiation therapy brings bright prospect for curing malignant tumor thoroughly as the development of induction study on tumour cells.PURPOSE:We have observed the effects of 9-cis retinoic acid (RA), gamma-interferon (γ-IFN), and the association of both agents on the cell morphology, cell circle, amplification of MYCN and MDR1 mRNA in human neuroblastoma (NB) SK-N-SH cell line with the aim of investigating the molecular mechanisms when using these agents alone or in combination to induce cell differentiation, apoptosis and growth inhibition. METHODS:The SK-N-SH cells were divided into four groups and treated with 9-cis RA,γ-IFN alone or in combination. At the desired times after treatment, the neuronal cell differentiation was counted by evaluation of morphological changes with neurite elongation, the cell apoptosis and death was observed by Hoechst-PI fluorescent staining, the growth inhibition rate was measured by MTT, the cell cycle and apoptosis was detected through Flow cytometry, the expressions of MYCN and MDR1 mRNA were assessed by RT-PCR and real time quantitative PCR.RESULTS:At the third day of combined treatment, the differentiation of cell morphology appeared. The cell body became smaller and more round. The neurites extended from the cell bodies, elongated gradually and interweaved into a net at last. The rate of neuronal cell differentiation was higher than other groups. MTT indicated that anti-proliferative effects were more evident when the two agents were used in combination as compared to the effects of the drugs given alone. Hoechst-PI and Annexin V/PI staining confirmed the significant synergistic effects of the combination on inducing cell apoptosis and death congruously. The increased ratio of G0-G1 stage cell and induced ratio of S stage cell ratio were found by cell cycle analysis using flow cytometry in combined treatment group. RT-PCR and real time quantitative PCR showed that the expressions of MYCN and MDR1 mRNA were down-regulated by combined therapy.CONCLUSION:Taken together, these data confirm the synergistic effects of a combination of 9-cis RA andγ-IFN on inducing differentiation, apoptosis and growth inhibition of human NB cells. It has outstanding therapeutic efficacy in low dose and outstrip used alone significantly. It suggests that its probable molecular mechanism is the reduction of MYCN and MDR1 mRNA expressions through complementary action. On one hand, they can inhibite the malignant biology display of proto-oncogene. On the other hand, they can prevent multidrug resistance. These results provide a rational basis and guidance for establishing a combination therapeutic approach for clinical use in the treatment of NB resistant to single inducers. Differentiation therapeusis of NB is a new method to tumor and provids favourable aid for clinical surgery. It has bright future owe to high efficiency, low toxicity and convenient application.