|
Objective: multiple myeloma (Multiple myeloma, MM) is a malignant plasma cell disease remains incurable malignancy. IL-6 plays an important role in promoting the proliferation of myeloma cells. Boron is a proteasome inhibitor Bortezomib (Bortezomib, BZ), made significant efficacy in the treatment of MM disease, about single drug overall response rate of approximately 40%. Sodium Valproate (Valproic acid, VPA) is a histone deacetylase inhibitor (histone deacetylase inhibitor, HDACI), clinical commonly used in anti-epileptic treatment, the attention of potential anti-tumor effect. Foreign studies have shown that VPA can promote apoptosis of MM cell lines. Pim-2 kinase is a serine / threonine protein kinase family members, a tumor related proto-oncogene activation can be stimulated by a variety of mitogenic signals, resulting in the malignant transformation of hematopoietic cells. Bcl-2 gene is a known anti-apoptotic genes that direct the synthesis of proteins control mitochondrial major adjustment factor to promote the release of apoptotic factors. Bax and Bcl-2 belong to the same members of the Bcl-2 family of proteins, Bax gene is in connection with the relative pro-apoptotic gene, Bax and Bcl-2 balance between regulating apoptosis. In this experiment, non-dependent of IL-6 MM cell lines PRMI8226 and dependent on IL-6 MM cell line U266 cell growth and proliferation, apoptosis and Bcl-2, Bax and Pim-2 gene expression and other cell biology behavior perspective explore of BZ joint VPA MM cell proliferation and apoptosis and Bcl-2, Bax and Pim-2 gene expression changes, looking for new BZ combination regimens provide a theoretical basis for clinical. Method: 1 cell line culture: the conventional cultured human MM cell lines RPMI8226 and U266. The RPMI8226 cells passaged 1 time every 48 hours, U266 cells were passaged every 72 hours, experimental use of the logarithmic growth phase cells. MTT assay Cell Proliferation: RPMI8226 cells to adjust the cell density of 4 × 10 4 / ml in U266 cells was 2 × 10 5 / ml in were inoculated in 96-well sterile culture plates, each hole 180 μl, BZ monotherapy respectively acting on the concentration of the RPMI8226 and U266 cells GT; 0.1 × 10 -6 mol / L, in both cell proliferation inhibition rate increased significantly . Therefore, the concentration of the BZ selected as 0.1 × 10 -6 mol / l. VPA single role of the drug concentration in RPMI8226 and U266 cells were 2,4,8,16,32,64 mmol / l, with VPA concentration increased cell growth inhibition rate is gradually increased, and 16mmol / l and 32mmol / l inhibition rate increasing trend is obvious. The experiment used the final concentration of VPA 2mmol / l, 16mmol / l, 32mmol / l experiment. Were divided into five groups: A / A 'group: control group; B / B' group: BZ 0.1 × 10 -6 mol / l; C / C 'group: BZ 0.1 × 10 < sup> -6 mol / l VPA 2mmol / l; D / D 'group: BZ 0.1 × 10 -6 mol / l VPA 16mmol / l; E / E' group: BZ 0.1 × 10 -6 mol / l VPA 32mmol / l. Based on the growth cycle of RPMI8226 and U266 cells, RPMI8226 cells (A, B, C, D, E group) were taken 12h, 24h, 36h time point, U266 cells (A ', B', C ', D', E ' group) were taken 12h, 24h, 48h time point, cell proliferation was detected by MTT assay. Measuring the absorbance value of each hole, at the OD570nm of the enzyme-linked immunosorbent detector indirectly reflects the number of surviving cells to the OD value, and calculating the cell growth inhibition rate = [(control group OD-experimental group OD) / control group OD] x 100%. Each group set up four wells. 3 AnnexinV / PI assay cell apoptosis: adjust RPMI8226 and U266 density of 2-5 × 105/ml application prepared by the MTT assay five groups of drug concentration, the same role time point to collect cells in each group, AnnexinV / PI double staining detect apoptosis. 4 semi-quantitative RT-PCR method to detect Bcl-2, Bax mRNA expression: RPMI8226 cells and U266 cells were collected to extract total RNA, and RNA was detected OD260 value concentration and OD260/OD280 values, RNA electrophoresis RNA integrity. Reverse transcriptase synthesis of cDNA. Bcl-2 upstream primer: 5'-GACTTCGCCGAGATGTCCAGC-3 ', reverse primer: 5'-GCATCCCAGCCTCCGTTATCC -3', fragment length of 260 bp. Bax upstream primer: 5'-TTTGCTTCAGGGTTTCATCCAGG -3 ', reverse primer: 5'-GAGCTCCATGTTACTGTCCAGTTCG-3', fragment length 154bp. upstream of the β-actin primers: 5'-GTGACATTAAGGAGAAGCTG -3 ', reverse primer: 5'-CTAGAAGCATTTGCGGTGGAC-3', fragment length: 510bp. The PCR reaction was 25μl. The electrophoresis results read plastic instrument AlphaImager1200 scanning amplification products and semi-quantitative analysis of Bcl-2, the average absorbance values ??compare the mean absorbance value of Bax mRNA and β-actin mRNA, Gel pro software. Semi-quantitative RT-PCR assay pim-2 mRNA expression: the same experimental methods, Pim-2 upstream primer: 5'-CAGCCATCCAGCACTGCCATTC-3 ', reverse primer: 5'-AGTCTGGGGAGACATGGGCTGG-3', fragment length 330bp. 6 Statistical analysis SPSS 13.0 software analysis, grouped data are expressed as mean ± standard deviation, two sets of application T-test method, the multi-group two-factor analysis of variance, P lt; 0.05 was considered statistically significant. Results: 1-BZ joint VPA RPMI8226 cell proliferation affect VPA single-agent role RPMI8226 cells gradually increased as the concentration increased proliferation inhibition rate. 2mmol / l to 64mmol / l, the concentration of the VPA, growth inhibition rate of 7.9% to 34.7% (P = 0.005). BZ single drug concentration gt; 0.1 × 10 -6 mol / l of RPMI8226 proliferation inhibition rate significantly increased trend; RPMI8226 cells the BZ with VPA drug combination treatment, 12h growth inhibition rate B group to group E significantly increased (P = 0.000); 24h cell growth inhibition rate, respectively, for group B 0.2808 ± 0.0273 in group C ± .4109 0.0363 Group D 0.4504 ± 0.0581, Group E 0.5142 ± 0.0137, between groups exist significantly with differences ( P = 0.000); 36h of cell growth inhibition rate is also the same trend (P = 0.047). Tip BZ joint VPA RPMI8226 growth inhibition rate increase gradually as the VPA concentrations increase in a dose-dependent manner. 12h, 24h, 36h three different points in time under the same drug concentration, cell growth inhibition rate has a significant difference (P lt; 0.05), suggesting that increased gradually with time inhibits proliferation effect was time-dependent . The study found that the concentration of the VPA 32mmol / l joint BZ 0.1 × 10 -6 mol / L for RPMI8226 cells 36h, cell growth inhibitory effect, inhibition rate was 0.5142 ± 0.0137. 2 BZ joint VPA on U266 cell proliferation: VPA role of single-agent U266 cells with increasing concentrations of the growth inhibition rate gradually increased, but the the concentration inhibition rate is less than RPMI8226 cells. When the VPA concentration was 16mmol / l 64mmol / l when the difference more obvious, In RPMI8226 VS U266: 23.1% 3 4.7% VS 17.27% 2 8.6 %. BZ single concentration gt; 0.1 × 10 -6 mol / l inhibition rate of U266 cells have significantly higher trend. Joint medication at 12h and 24h, cell growth inhibition rate with increasing VPA concentration and significantly with increase group are exist significant differences (P values ??are = 0.000); 48h of cell growth inhibition rate, respectively, for B 'group 0.2927 ± 0.0232, C 'group 0.3194 ± 0.0428, D' group 0.6008 ± 0.0645, E 'group 0.7819 ± 0.0462, between groups there is also a significant difference (P = 0.000). Joint BZ VPA was also found a dose-dependent growth inhibition of U266 cells. 12h, 24h, 48h three different points in time when the rate of cell growth inhibition in the same drug concentration between the significant difference (P lt; 0.05), the combination of two drugs inhibit proliferation effect of U266 cells in a time-dependent manner. 2mmol / l and the concentration of the VPA joint the BZ role U266 cells, with inhibition rate than BZ no difference (P = 0.356). The inhibitory rate comparison between the two types of cells, suggesting that the inhibition rate of the value-added of the the BZ joint VPA U266 cells RPMI8226 cells was no significant difference (P = 0.599). 3 AnnexinV / PI assay cell apoptosis in RPMI8226 cells, the drug combination of five different concentrations role 12h, the apoptosis rate were: A group of 26.6000 ± 1.1000 in group B 25.1667 ± 3.5232, C group 33.0333 ± 2.4583 , D group 37.8333 ± 0.6110, Group E 41.4000 ± 1.0392; 24h apoptosis rate are: A group of 36.2333 ± 3.1628, group B 39.8000 ± .4358, C group 47.1666 ± 2.2810, Group D 50.5333 ± 1.6258, Group E 52.0333 ± 1.4640 ; 36h apoptosis rates were: group A 54.0666 ± 3.4122, 61.8333 ± 1.2741, C group B group 64.4000 ± 1.2124 D group 66.2000 ± .9539, Group E 68.5000 ± 1.0392, all time points between groups, P value lt; 0.05, suggesting that the increased rate of apoptosis with the time or the concentration of the drug action is gradually increased. Drugs of different concentrations of effector cells 12h rate of apoptosis in U266 cells are: A 'group 24.0333 ± 2.5658, B' group 24.4666 ± 1.2741, C 'group 26.1000 ± 1.05356, D' group 32.1000 ± 1.2165, E 'group 32.7333 ± .6429; 24h of apoptosis: A 'group 22.3000 ± 0.8000, B' group 24.2666 ± 1.1150, C 'group 34.3333 ± 1.2662, D' group 47.4666 ± .9073 E 'group 54.6000 ± 1.05366; 48h the cells apoptosis rates were: A 'group 23.2666 ± 1.4011, B' group 25.0666 ± 2.4172, C 'group 49.3000 ± .5196 D' group 63.0333 ± 1.3868, E 'group 65.4000 ± 1.0817, suggesting that rate of apoptosis in U266 cells with the The concentration of drug action and prolonged duration of action gradually increased (P = lt; 0.05). Single BZ group (B 'group) no significant increase with the increase rate of apoptosis, and apoptosis rate of the combined treatment group (C', D ', E' group) obvious concentration and time dependent. Comparison between the two cell lines, in RPMI8226 cells, E groups cells after 36 hours, apoptosis of the most obvious. Maximum apoptosis rate was 68.5000 ± 1.0392, compared with the control group, an increase of 14.5%;, apoptosis in U266 cells, E 'cells 48 hours after the most obvious, the maximum apoptosis rate of 65.4000 ± 1.0817, an increase of 42.2% compared to the control group. Between the two cell apoptosis rate, suggesting BZ joint VPA induction of apoptosis in U266 cells than RPMI8226 cells was significantly (P = 0.003). 4 Bcl-2 mRNA, Bax mRNA expression in myeloma cell lines, Bcl-2 mRNA were expressed in myeloma cell lines RPMI8226 and U266 cells. RPMI8226 cells, 24h Bcl-2 mRNA expression levels as follows: A set of 1.280 ± 0.160; B group 0.607 ± 0.035; C group 0.410 ± 0.105; the D group 0.355 ± 0.029; Group E 0.275 ± 0.400, prompt with the drug action increasing concentrations of Bcl-2 mRNA expression levels decreased (P lt; 0.01). 24h Bcl-2 mRNA expression levels in U266 cells as follows: A 'group 0.656 ± 0.013; B' group 0.543 ± 0.025; C 'group 0.437 ± 0.121; D' group 0.259 ± .070; E 'group 0.136 ± 0.197 With increasing concentrations of VPA drug action of Bcl-2 mRNA expression levels also decreased (P lt; 0.01). Bax expression in U266 cells positive. The drug action 24h Bax mRNA expression levels as follows: A 'group 0.573 ± 0.011; B' group 0.656 ± 0.032; C 'group 0.146 ± 0.007; D' group 0.089 ± 0.017; E 'group 0.123 ± 0.006, each group numerical comparison all P lt; 0.05, but no significant decreased trend. Its significance is unknown. 5 Pim-2 mRNA in RPMI8226 and U266 expression of K562 leukemia cell lines as a positive control, U266 cells Pim-2 mRNA in rendering positive expression, but different drug concentrations Pim-2 mRNA expression without significantly with impact (P = 0.447). RPMI8226 cells not detected Pim-2 mRNA. Conclusion: 1-BZ joint VPA inhibits RPMI8226 and U266 myeloma cell lines proliferation and induce apoptosis, its role compared with single-BZ increase in a dose-and time-dependent manner. 2 BZ joint VPA proapoptotic role of IL-6-dependent U266 cells than non RPMI8226 cells significantly dependent IL-6. 3 in the BZ Joint VPA cases, Bcl-2 mRNA expression in RPMI8226 and U266 both cell lines with the increase in the concentration of VPA reduce. Bax mRNA expression levels in U266 cell lines with VPA concentration changes without a decreased trend. 4 U266 cells, Pim-2 mRNA expression was positive for different concentration of the drug had no significant effect on Pim-2 mRNA expression levels; the RPMI8226 cells do not express pim-2 mRNA.
|