The Study Of The Effect And Mechanism Of Norcantharidin(NCTD) On Human Multiple Myeloma Cell Line U266

Background: The incidence rate of multiple myeloma increased and patient's age tend to more younger with the coming of society ageing and the improving of clinical technology, more and more haematologist pay close attention to the treatment of multiple myeloma. Multiple myeloma is hematological malignant tumor origining from plasmacytic system in over 60 years population, involving pathological changes of blood, immunity, bones, kidney and so on. Traditional chemotherapy has a considerable side-effect and lowing remission rate. Moreover the endurance and survival time of patient is poor.At present, norcantharidin (NCTD)had been widely used in adjunctive therapy of solid tumors such as liver cancer, esophageal cancer, and attracting lots of researcher to study the anti-tumor mechanisms and getting perfect result. At the same time, these studies offer theoretical evidence to clinical medication for cancer therapy, on the other side, it also extend the method of therapy on malignant tumor. The active substance in cantharides is Cantharidin (CA), which can inhibit protein synthesis in cells, impact growth and division of cells. However, because of intense irritant action on urinary system, it can not be used extensive in clinic. NCTD, which remove 1,2-methyls from CA, had effective activtion on anti-cancer and lower side-effect, besides, it is the only anti-tumor drug that have the function enhancing white blood cells. Both vivo and vitro, NCTD have significant inhibit on cells of hematological malignant tumor such as acute promylocytic leukemia(APL), acute T-lymphoblastic leukemia and solide tumours, including liver cancers, esophageal, colorectal, lung, gallbladder, ovarian, as well as melanoma. It demonstrated that NCTD played a significant role in anti-cancer, including cytotoxicity, induced apoptosis, anti-adhesion and anti-metastasis and inhibit the generating of vascular tumor.Objective: Researched the effect and mechanism of NCTD on proliferation and apoptosis in multiple myeloma cell line U266, achieving to decrease complications, improve the remission rate, enhance endurance, extend platform period and life span of the patient.Method: Different doses of NCTD were used on human multiple myeloma cell line U266 to study its effect. Such indexes were discovered: 1.Counting the rate of trypan blue dye exclusion to identify NCTD anti-proliferative effect on the U266 cell line, in order to find the best dose and work time. 2.Inverted microscope and optical microscope were used to observe the density, morphous and structure in cells. 3. Apoptosis ratio and cell cycle were detected by flow cytometer to investigate U266 cell line apoptosis and it's mechanism. 4.The gene and protein expressions of Survivin, VEGF were detected by RT-PCR and immunohistochemistry to further study the mechanism of inducement apoptosis in multiple myeloma cell line by NCTD.Result:1 NCTD can perfect inhibit the proliferation of multiple myeloma cell line U266, and it increased anti-proliferation with the rising of dose and time. there was dependence between inhibition ratio and dose and time of the drug, it will get the summit at 40μmol/L in 48 h.2 NCTD can induce apoptosis in multiple myeloma cell line U266, by optical microscope we observed typical changes of apoptosis including chromatin margination, nucleus shrinkage, nucleus fragmentation and nucleusdecompose. With the increase of dose and time, the apoptosis ratio increased gradually, and have significant different between different group.3 Detecting by flow cytometer we found U266 cell line blocked in G2 /M after NCTD treated, The cell become dumbbell observing by inverted microscope , to hint that the cell was blocked at division stage.4 The mRNA and protein expression of Survivin and Vascular endothelial growth factor( VEGF )are reduced after NCTD treated.Conclusion:1 NCTD can inhibit proliferation of U266 cell line and in- duce apoptosis and the cell was blocked at division stage.2 The apoptosis mechanism induced by NCTD may relative to regulate of Survivin and VEGF.