| Objective: Multiple myeloma (MM) is a neoplasm of plasma cells. Multiple myeloma accounts for approximately 1% of all the malignant diseases and 10% of hematologic malignancies. Most of the patients are aged people. The median age at diagnosis for MM patients is approximately 69 years old for male and 71 years old for female; less than 5% of the patients are younger than 40 year-old. In the past 30 years, the mortality rate in multiple myeloma has increased, because the number of multiple myeloma cases who are more than 85-year-old increased (Compared with that in 1968, the multiple myeloma patients who are more than 85-year-old have increased by 45%.). In view of the actual situation of our country at present, majority of patients can not receive the better treatment of autologous hematopoietic stem cell transplantation (HSCT) because of economic reasons and concepts about HSCT and many patients can not afford the high cost of HSCT. Artesunate is one product of the antimalarial drugs. Recently, many scholars have found that artesunate also has anti-tumor activity. However, whether artesunate has anti-human myeloma effect is not clear. Therefore, in the present study, we made a study on the proliferation inhibition, apoptosis induction and invasion inhibition effect of artesunate on human myeloma cell line RPMI 8226 cells to explore the anti-myeloma mechanisms of artesunate. By doing these, we tried to provide more effect treatment options for the aged patients with multiple myeloma.Methods:1.1 Cell Culture: RPMI8226 cells were cultivated in RPMI 1640 medium conventionally. Culture medium contained 10% viviparous bovine serum (FBS), 100μg/mL penicillin, 100U/mL streptomycin and 2mmol / L glutamine. Cells were cultured and passaged at 37℃and in 5% CO2 saturated environment. The culture medium were changed when the cells were 70% to 80% confluent. The experiments were performed when cells entered the logarithmic phase.1.2 Inhibition of cell proliferation assay: the growth inhibition rate was measured with MMT assay method, after the RPMI 8226 cells were treated with artesunate, at concentrations of 2.5μg/ml, 12.5μg/ml, 25μg/ml and 50μg/ml, for 48 hours. The cells cultured in RPMI 1640 alone were used for blank control group. Inhibition rate was calculated according to the following formula: Inhibition rate (%) = (the optical density(OD) value of control group- the OD value of experimental group) / the OD value of control group×100%.1.3 Observation of cell morphology change: RPMI8226 cells were treated with artesunate at final concentrations of 25μg/ml, 50μg/ml or without artesunate for 48h, and then the number of cells and the change of cell morphology was measured or observed under an optical microscope separately.1.4 AnnexinV / PI double staining was performed to detect cell apoptosis rate: RPMI 8226 cells were collected after the cells treated with artesunate at final concentrations of 0μg/ml, 25μg/ml, 50μg/ml for 24 hours, washed 2 times with 1×PBS. Cells were re-suspended with 500μL Binding Buffer, stained with 10μL Annexin V-FITC and 5μL PI. The reaction was developed for 15min at room temperature in dark, then, the apoptosis rate was detected by flow cytometry.1.5 Measurement of cell cycle distribution by flow cytometry: logarithmic phase RPMI8226 cells were treated with artesunate at final concentrations of 0μg/ml, 25μg/ml and 50μg/ml for 48h, collected and washed with PBS and fixed with 70% ethanol at 4℃for 15 min. The cells were then treated with RNA enzyme and followed by two washes with PBS. Finally the cells were stained with PI at a final concentration of 50μg/ml for 30 min. Cell cycle was detected immediately by flow cytometry.1.6 Transwell assay was performed to measure the effect of artesunate on the invasion ability to RPMI8226 cells in vitro.1.7 The activity of caspase3 / 7 enzyme was detected by Microplate reader: The RPMI8226 cells were seeded into 96-well plates. The cells were treated with artesunate at different final concentrations of 0μg/ml, 25μg/ml and 50μg/ml for 12h at 37℃, 5% CO2. 100μl caspase3 / 7 enzyme detecting reagent was added to each well, and the optical density value of each well was detected at 405nm wave length with a microplate reader.Results:2.1 Artesunate inhibited the proliferation of RPMI 8226 cells in a dose-dependent manner of which P<0.05. The inhibition rate of RPMI8226 cells increased from 33.55% to 74.71% when the concentration of artesunate increased from 2.5μg/ml to 50μg/ml. The IC50 of artesunate was 36.47μg/ml (calculated by LOGIT METHOD).2.2 Effect of Artesunate on RMI8226 cells'morphological changesRPMI8226 cells were treated with different final concentration of artesunate (0μg/ml, 25μg/ml, and 50μg/ml) for 48h. The morphological changes induced by artesunate treatment were observed under an inverted microscope. The results showed that the number of living cells decreased significantly, cell volume shrinked, and cell fragmentation appeared after artesunate treatment. While in the control group, the number of living cells was much larger, the volume of the cell and nucleus did not change. Each cell in the control group had abundant cytoplasm.2.3 Effect of Artesunate on cell apoptosis in RPMI8226 cells Cell apoptosis rate was detected by Annexin V / PI double staining assay. The result showed that the cell apoptotic rate was increased in a dose-dependent manner after cells treated with different concentrations of artesunate for 24h. When RPMI8226 cells were treated with 50μg/ml artesunate, the apoptotic rate was 68.1%±5.9, which was significantly higher than that of the control group.2.4 Effect of Artesunate on cell cycle change of RPMI8226 cellsRPMI8226 cells were treated with 0μg/ml,25μg/ml,50μg/ml of artesunate for 48 hours. Then, cell cycle distribution was detected by flow cytometry. The results showed that with the increasing of artesunate concentration, the proportion of G0/G1 phase cells decreased gradually, and the proportion of G2 / M phase cells increased gradually. When the cells were treated with artesunate at a final concentration of 50μg/ml, the proportion of G0/G1 cells reduced from (52.4±3.7)% of control group to (21.4±2.5)% of the artesunate treated group (P <0.05), and the proportion of G2 / M phase was increased from (28.9±2.9)% of the control group to (54.3±4.2)% of the artesunate treated group (P <0.05).2.5 The result of Transwell chamber assayIn our preliminary experiment, it was proved that there was not a statistically significant difference in cell growth inhibition rate between control group and the group of cells treated with 12.5μg/ml artesunate for 12h. Therefore, 12-hour was chosen as time point for artesunate treatment. An equal number of living cells from the control group or 12.5μg/ml artesunate treatment group was prepared for the transwell chamber assay. The assay was duplicated and repeated for 3 times. The results showed that the number of cells adhered to the surface membrane of the lower chamber plus the number of cells leaked into the lower chamber was significantly smaller in the 12.5μg/ml artesunate treatment group than that in the control group: (34.6±6.8) versus (77.6±8.7) (P <0.01).2.6 Caspase-3 / 7 activity assayThe experimental results showed that the optical density value of caspase-3 / 7 in the untreated group (control group) was 0.336±0.041 and was 0.451±0.042 in 25μg/ml artesunate treatment group, and was 0.612±0.051 in 50μg/ml artesunate treatment group. The difference between the OD value of artesunate treatment group and control group was statistically significant, (P <0.05), indicating that artesunate could enhance the activity of caspase-3 / 7, thereby promoted apoptosis.Conclusion: Artesunate could significantly inhibit growth and migration of RPMI8226 cells and induce apoptosis in RPMI8226 cells. It probably plays a role in suppressing myeloma progression via activation of caspase family.
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