The Effect Of Arsenic Trioxide, Paclitaxel And Both To Multiple Myeloma RPMI8226 Cells

Objective: Multiple myeloma(MM) is a kind of malignant clonal and proliferative plasma cell disease, which clinical features are bone pain, osteolytic lesions, pathological fractures and anemia. Although there are many kinds of treatment methods for MM, such as chemotherapy, immune therapy, autologous hematopoietic stem cell transplantation(ASCT) and more, but still cannot be cured completely. The vast majority of patients will killed by palindromia, drug resistant and some beyond. The patients’ median survival time is generally 3-5 years.At present, the ideal state of the patients with multiple myeloma are controlling disease progression, improving the quality of patients’ life and prolong disease free survival by clinical treatment.M2,MP regimen(Melphalan+Prednisone),and VAD scheme( Vincristine + Doxorubicin + Dexamethasone) are commonly used to response to multiple myeloma rimary-care patients in clinical, but the methods above are not completely cure patients. In recent years, combined chemotherapy of MM has made new progress along with the continuously appear of various improvement initiatives, through in-depth study on many aspects of multiple myeloma pathogenesis and mechanism of drug resistance, new drug immune inhibitors, proteasome inhibitors and monoclonal antibody in the treatment for MM has made great achievements in clinic, so that the treatment effective rate, the clinical remission rate, disease-free survival rate obviously increased. But almost all patients will relapse, drug resistance is still a big problem, the huge medical expenses is also one of the reasons why a lot of patients and their families give up the best treatment. Therefore, the continuous development of new drugs is be imperative, and explore a chemotherapy regimen is worthy of attention.Arsenic trioxide(ATO) not only has obvious effect on a variety of solid tumors and hematological tumor cells for inhibiting proliferation and induction of apoptosis in vitro, is also widely applied in clinical. That gives full rein to its antitumor effect, particularly has a great contribution on the treatment of malignant hematological disease. Scholars studied the mechanism that how ATO effect on the human myeloma cells, the results show that: ATO inhibit proliferation and induce apoptosis of RPMI8226 cells obviously.RPMI8226 cells are arrested in G1 phase by ATO, The VEGF expression and secretion of the cells is less, and the Bcl-2 protein expression decreased after the effect of ATO. But it has no effect on the phosphorylation protein expression of ERK1/2 and itself. ATO has become one of the chemotherapy drugs choices because it can achieve the purpose of anti tumor through multiple actions of mechanisms. For example,it induce apoptosis and differentiation of tumor cells, inhibit the proliferation of tumor cells and the activity of telomerase and so on.Paclitaxel(PTX) is a common chemotherapy drugs in clinical, the main antitumor mechanisms is inhibiting the process of mitosis, and arresting the cells cycle in the G2/M phase, so as to achieve the purpose of inhibiting the proliferation of tumor cells. The drug is widely used in lung cancer, esophageal cancer, ovarian cancer and other solid tumors, and its adverse reactions including allergic reaction, bone marrow suppression, cardiac toxicity, neurotoxicity reflect and grand mal epilepsy, the serious patients have severe clinical manifestation, those has aroused the attention of clinical workers. So use paclitaxel to anti tumors safely and effectively is the goal of scholars constant pursuit. Application of paclitaxel to malignant tumor cells of hematological system in clinical trials have definite conclusion, confirmed that it has a significant role in the malignant hematologic diseases to induce apoptosis obviously.Methods: Use different concentrations of ATO, paclitaxel and combination of the two, treat human myeloma cells RPMI8226 cell line. In the pre experiment, treat RPMI8826 cells by drug of paclitaxel alone in different concentrations(Drug,concentrations,are0,0.01,0.02,0.04,0.08,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3μmol/L). The experiment is divided into the experimental group and the control group(group A),the experimental group divided into drug of ATO alone(Drug concentrations are 1,2,4μmol/L as group B,C and D respectively), drug of paclitaxel alone(Drug concentration is 0.01μmol/L as E group) and the combination group(Drug concentration are ATO 1μmol/L + PTX0.01μmol/L 、 2μmol/L + PTX0.01μmol/L 、 4mol/L +PTX0.01μmol/L as group F,G,H).Detect the inhibition of cell proliferation and cell growth rate of RPMI8226 cells by trypan blue staining and MTT colorimetric method at different time(24h,48 h,72h).Observe the apoptosis and the change of morphological of cells by inverted phase contrast microscope and AO fluorescent staining. Detect the change of cells apoptosis and the effect on cell cycle in the control group and the experimental group by flow cytometry. Check the effect on protein cbc-2 and bcl-2 that related to cell cycle with Western blot.Result:1 Observation of the morphology of RPMI8226 cells.Inverted phase contrast microscope: In the preliminary experiment, with prolonging the effect time of paclitaxel, obvious deformation occur in most RPMI8226 cells, as a rod shaped or spindle, The nucleus changes with the whole cell morphology and refractivity of cell will get worse gradually, finally the cell membrane is not complete, become cell debris. In the drug of ATO alone group, RPMI8226 cells shrink and pyknosis show as uniform dense with the increase of concentration and prolong the effect time. Cell membrane appear pseudopodia and cell refractivity get poor, until the membrane is not complete, becoming cell debris. The apoptosis and necrosis cells were increased in the combination group than the same concentration of drug alone group, and the cell morphology change similar to the drug of PTX alone group.2 Measure the inhibition rate of cell proliferation with MTT colorimetric method.2.1 The group of ATO: The inhibitory rate of cell proliferation increase depend on dose with increase the drug concentration of ATO and, the inhibitory rate of cell proliferation increase depend on ATO effect time with prolong the time in the same period. There are differences between each concentration in the same effect time group, so there are statistically significant(P<0.05).2.2 The group of PTX: When the concentration of PTX between 0.01μmol/L-1μmol/L, the inhibition rate of cell proliferation constant, no obvious dose dependent and, when the concentration between 0.01μmol/L-1μmol/L, the inhibition rate of cell proliferation increase with rising concentration in the same period. The cell proliferation inhibition rate increased with prolonging effect time when use the same concentration of PTX. The inhibition ratio are20.09±9.11% 、 39.77±1.85% 、 54.39±3.75% respectively when effect 24 h, 48 h and 72 h by PTX which concentration is 0.01μmol/L.2.3 The group of drug combination: Combine the PTX which keep the same concentration with different concentration ATO, the cell growth rate decreased with the increase of ATO concentration. For example, combine ATO(4μmol/L) with PTX(0.01μmol/L) and use it effect RPMI8226 cells 48 hours, the proliferation inhibition rate is 47.95±7.32%%. As a comparison, the proliferation inhibition rate are32.98±6.07% and39.77±1.85%( P<0.05) respectively when effect RPMI8226 cells 48 hours with ATO(4μmol/L) alone or PTX(0.01μmol/L) alone. So there are statistically significant. Drug combination with ATO and PTX effect on RPMI8226 cells and inhibit the proliferation of cells depend on concentration and time.3 Detect cell apoptosis by flow cytometry( Annexin V-FITC/PI double staining): Treat the RPMI8226 cells with single or combination drug, the survival cells decreased and the apoptotic cells increased gradually with prolonging effect time or increasing the drug concentration. The detect results of cells cycle show that the cells in the G0/G1 phase and S phase are 27.45%,68.50% respectively in the control group. And the cells in the G0/G1 phase and S phase are89.37%,8.48% respectively in H group. Compare the experimental group and control group, the difference has statistically significant( P<0.05).4 The detect result of Western blot suggests that the expression quantity of cdc2 protein had statistical significance in control group and the experimental group. Compare with control group, the expression quantity of Cyclin D1 protein reduce the difference has statistically significant( P<0.05).Conclusion:1 Either ATO or PTX can inhibit the cell proliferation and induce apoptosis of human myeloma RPMI8226 cells.2 The combination of the ATO and PTX can significantly increase the apoptosis rate of RPMI8226 cells, enhance the effect on inhibiting its growth.3 The proliferation inhibition depend on time when use the PTX treat RPMI8226 cells, the effect has no significant changes when drug concentration in certain range, but still can inhibit proliferation. With increasing drug concentration and reaches a certain point, PTX inhibit the proliferation of RPMI8226 cells in a dose-dependent.