Effects of Arsenic Trioxide Alone and in Combination with Bortezomib in Multiple Myeloma RPMI 8266 Cells

The aim of this study was to detect the efficiency of arsenic trioxide (ATO) alone or together with bortezomib to inhibit proliferation and induce apoptosis in a multiple myeloma (MM) RPMI 8266 cells. Mechanisms of action were also investigated. RPMI 8266 cells were treated with ATO alone and in combination with bortezomib for 24 hours, and cell viability was assessed by modified MTT. Annexin V-F1TC and PI staining was used to detect the apoptosis rate and cell cycling was investigated by flow cytometry, along with expression of cell surface death receptor-4(DR4) and death receptor-5 (DR5). Western blotting was applied to detect the expression of bcl-2, caspase-3, caspase-8, and caspase-9. As a result, the ATO combined with bortezomib group showed more inhibition of RPMI 8266 cell viability than theATO group. Expression of DR4 and DR5 on the cell surfaces, and the apoptosis rate were increased after treatment by ATO alone or combined with bortezomib. The cells appeared to arrest in G2/M phase after treatment. Expression of bcl-2 was more significantly decreased in the combination group, and that of caspase-3, caspase-8 and caspase-9 was significantly increased as well. Therefore, bortezomib can enhance ATO actions to induce apoptosis in RPMI 8266 cells, with decrease in expression of bcl-2 and increase of caspase-3, caspase-8 and caspase-9 proteins.


Introduction
Multiple myeloma (MM) is hematological malignant disorder characterized by proliferation and accumulation of clonal abnormal plasma cells in the bone marrow.It accounts for 1% of neoplastic diseases and 13% of hematologic cancers (Mousa et al., 2007;Palumbo and Anderson, 2011;Sanaat et al., 2011).MM is an incurable plasma cell malignancy by far.Drug resistance and relapse is the main reason for its refractory feature (Badros et al., 2005;Hou et al., 2005;Morales et al., 2008).
Arsenic trioxide (ATO) is an effective chemotherapeutic agent in treating some kind of tumors including MM (Wang et al., 2008;Yedjou et al., 2010).The mechanisms of action derivatives in this disease and other malignancies include induction of apoptosis, inhibition of proliferation and inhibition of angiogenesis (Mousa et al., 2007).Moreover, ATO as a single agent induced clinical responses in 21-33% of patients with relapsed/refractory MM.Bortezomib, a highly selective, reversible inhibitor of the 26S subunit of the proteasome complex, offers 50% of responses in refractory/relapsed patients affected by MM (Canestraro et al., 2010).
Although the two drugs showed effective in MM, there

Materials and Methods
Cell culture and drug preparation MM cell line RPMI 8266 cells were cultured in 25 cm2 tissue culture flask under standard culture medium RPMI-1640 (GiBco, USA) supplemented with 10% fetal calf serum (FCS) (Beijing Ding States Biological Technology Co., Ltd, China).The medium was changed twice a week.ATO (Harbin Yida Pharmaceutical Co., Ltd, China), packaged and stored at room temperature before being used to the required concentration, was diluted with normal saline to 1000 μmol/L solution.Bortezomib (Millennium, USA) stored at -20℃ before being used to the required concentration was diluted with saline to 1000 nmol/L.

Cell viability assay
The effect of the drugs on RPMI 8266 cell viability was assessed by modified MTT assay (purchased from Beijing Ding States Biological Technology Co., Ltd.China).The cells were seeded into three 96-well tissue culture plates at a density of 5×10 3 cells/well, and treated with ATO alone (0.1, 1, 5, 10, 50 μmol/ L), bortezomib alone (0.1, 1, 5, 10, 50 nmol/L)and combination of the two drugs, 10 nmol/L of bortezomib was added to each concentration of ATO and each group in a plate should be in a final volume of 100 μl culture medium, and incubated for 24 hours.For MTT assay, 10 μl (5 mg/ml) MTT solution was added to each well and incubated for 4 hours.100μl of the solution was added into each well and incubated overnight at 37°C, 5% CO 2 .The absorbance was measured in microplate reader (BIO-TEK Inc) at wave length 570 nm.Each experiment was repeated in triplicate format, and results were expressed as mean ±SD.

Apoptosis detection by annexin V-FITC/PI
Apoptosis was also evaluated by Annexin V-FITC and propidium iodide (PI) kit (purchasedfrom Beyotime Institute of Biotechnology -China).Briefly, 7×10 5 cells/ well were seeded in 6-well plates and treated with ATO alone (1, 10, 50 μmol/L), bortezomib alone (1, 10, 50 nmol/L) and combination of the two drugs, 10 nmol/L of bortezomib was added to each concentration of ATO and incubated at 37°C and 5% CO 2 in humidified incubator for 24 hours.The cell was centrifuged for 5 minutes, supernatant was discarded and the cells was washed with PBS one time.195μl Annexin V-FITC liquid and 5μl Annexin V-FITC were added gently to the suspended cells, mixed gently and incubated in room temperature (20-25 ℃) at dark for 10 minutes (Aluminum foil can be used to avoid light).Centrifugation for 5 minutes, supernatant was discarded.190μl Annexin V-FITC liquid was added gently to the suspended cells.10μl of propidium iodide staining solution was added, gently mixed and incubated in ice bath at dark.Samples were analyzed by flow cytometry to determine the percentage of cells displaying annexin V positive/PI-negative (early apoptosis) or annexin V positive/PI-positive staining (late apoptosis and cell death).

Cell cycle detection
Cell cycle distribution was determined by staining DNA with PI using Cell cycle and apoptosis detection kit (purchased from Beyotime Institute of Biotechnology, China).The RPMI 8266 cells were seeded into 6-well tissue culture plates at a density of 7×10 5 cells/well and and divided into three group, ATO group (final concentration was 5.0 μmol/L), bortezomib group (final concentration was 10 nmol/L) and Combination group (final concentration was 5 μmol/L+10 nmol/L).All groups were incubated at 37°C and 5% CO 2 in humidified incubator for 24 hours.The cells were centrifuged for 5 minutes.Supernatant was discarded, and the cells were washed with pre-cooled PBS one time and transferred into 1.5 ml centrifuge tubes and fixed by 1ml ice-bathed pre-cooled 70% ethanol, mixed gently and incubated at 4°C for 12 hours, then the tubes were centrifuged for 5 minutes in 1000g, the supernatant discarded carefully and the cells were washed with pre-cooled PBS.0.5 ml of propidium iodide staining solution was added to each tube of cell samples and incubated in dark at 37 ℃ for 30 minutes.Flow cytometry was used to detect the red fluorescence, excitation wavelength at 488 nm and simultaneous detection of light scattering to analysis the cell cycle.

Death receptor detection by flow cytometry
Cell surface expression of DR4 and DR5 on RPMI 8266 cell was measured by flow cytometry.1×10 6 cells/ well was seeded in 6-well plates, was divided into ATO group (final concentration for 5.0 μmol/L), bortezomib group (final concentration as 10 nmol/L), combination group (final concentration as 5 μmol/L+10 nmol/L) and the control group (add the same volume of PBS) and incubated for 24 h.Washed twice with ice cold PBS and incubated for 30 min at 4°C with primary anti-human-DR4 (mouse IgG1) or anti-human-DR5 (mouse IgG1) monoclonal antibodies (Santa Cruz Company, USA), cells were washed with ice-cold PBS and incubated with FITC-labeled goat anti-mouse secondary antibodies for 30 min at 4°C in darkness.Sample was analyzed with a FACScan flow cytometer and CellQuest software (Beckton Dickinson, USA).Each experiment was repeated three times.

Statistical analysis
Each experimental value was expressed as mean ± standard deviation (SD), analyzed by using Origin7.0 software.The significance of differences between groups was considered as P≤0.05.All data represented the mean of triplicates.

Cell apoptosis was increased by treatment with ATO or ATO combined with bortezomib
The cells undergoing apoptosis were detected by Flow cytometry analysis using Annexin V and PI staining.Compared to the negative control, the apoptosis rates of ATO group (5, 10, 50 μmol/ L) and bortezomib group (5, 10, 50 nmol/ L) were increased gradually according to the drug concentrations.Figure 2.A showed the results of ATO group at different concentration, (13.25±1.60)%,(42.0±3.24)% and (70.3±4.21)%VS control (2.48±0.47)%.Figure 2B showed the results of bortezomib group at different concentration, (10.27±2.14)%,(35.78±3.68)%,and (67.25±3.20)%VS control (2.48±0.47)%.And in combination group (10 nmol/L of bortezomib was added to each concentration of ATO), the apoptosis rates of each concentration in the combination group were relatively higher than single treatment group (48.67±3.89,76.78±4.65 and 94.08±4.32%,respectively) as shown in Figure 2C.This indicated that the combination can further increase the apoptosis induced by ATO and bortezomib.

Cell cycle was arrested at G2/M phase in treated groups
Compared to control group and the treated groups of ATO and bortezomib showed increased number of cells at the G2/M phase, but induced a significant decrease in the number of cells at S phase.And in combination of ATO and bortezomib, the ratio of apoptosis and G2/M phase was relatively higher than single treatment group.It indicated that the RPMI 8266 cells were arrested further at G2/M phase in the combination group as shown in Figure 3.

The expression of death receptor increased in treated groups
Cell surface expression of DR4 and DR5 on RPMI 8266 cell was measured by flow cytometry.RPMI 8266 cells were treated with single-agent ATO (5.0 μmol/L), bortezomib (10 nmol/L), and combination of the two drugs (5 μmol/L ATO+10 nmol/L bortezomib).Figure 4 showed the changes in the expression of DR4 and DR5.Compared with the control group, the change in the combination group increased more significantly than in the single group (P <0.01).

The expression of Bcl-2, caspase-3, caspase-8 and caspase -9 in treated groups
The result showed that both ATO and bortezomib induced down-regulation of Bcl-2 expression and up-regulation of caspase-3, caspase-8 and caspase-9 activation.More increased expression of caspase-3, caspase-8 and caspase-9 proteins and more decreased expression of Bcl-2 protein in combination group as shown in Figure 5.

Discussion
ATO as a single agent or bortezomib offers response in refractory/relapsed patients with MM (Canestraro et al., 2010).However, resistance to each drug is still a big issue.We postulated that ATO and bortezomib have different mechanisms, and the combination of the two drugs will have synergistic effects.
In this study, the results showed that ATO and bortezomib have direct effects on MM cell line RPMI 8266 cells.It revealed that ATO and bortezomib inhibited the proliferation, promoted apoptosis of cells and that the combination group showed synergistic effects.Moreover, flow cytometric analysis showed that as compared with the control group, arrested cell cycle were arrested at G2/M phase in all the treated groups, and the ratio of G2/M phase increased gradually and reduced S-phase cells with the increase of the drug concentration.And the combination of the two drugs enhanced these effects.
The mechanism involved in the apoptosis was also investigated in this study.The apoptosis was induced mainly through the two major pathways, intrinsic (mitochondrial) pathway and the (death-receptor) extrinsic pathway.Where the death receptor was initiated by ligation of transmembrane death receptors including tumor necrosis factor (TNF) family receptors and their ligands, and activated caspase-8.Intrinsic pathway included the activation of the release of mitochondrial constituents and  doi.org/10.7314/APJCP.2013.14.11.6469 Arsenic Trioxide Alone and in Combination with Bortezomib in Multiple Myeloma RPMI 8266 Cells activation of caspase-9.Then, the two pathways activated caspase-3 and induced apoptosis (Johnstone et al., 2002;Tardy et al., 2006).
The results of this study showed that, the ATO and bortezomib induced DR4, DR5 up-regulation on RPMI 8266 cells, and the synergy raised to increase TRAIL receptor binding probability, in turn increase the activity of the TRAIL to promote apoptosis.
For the intrinsic pathway, we focused on detection of Bcl-2, active caspase-3 and 9 proteins, because Bcl-2 family proteins regulate mitochondria dependent apoptosis with the balance of the anti-and proapoptotic members (Canestraro et al., 2010).The release of cytochrome c and AIF is controlled by Bcl-2 family proteins, Bcl-2 and Bcl-xL inhibit the release of cytochrome c and AIF, whereas the release of cytochrome c and AIF enhanced by Bax (Gupta et al., 2003).It was shown that both ATO and bortezomib can induce apoptosis.They induced apoptosis by downregulation of Bcl-2 gene expression and up-regulation of caspase-9 and caspase-3 as shown in figure 5.
In conclusion, ATO and bortezomib induced apoptosis in RPMI 8266 cells and the mechanism related to decrease of bcl-2 protein (endogenous apoptotic signaling pathway) and they also induced apoptosis through the mechanism related to the change of DR4 and DR5 (extrinsic apoptotic pathway).ATO and bortezomib have synergistic inhibition and apoptosis in RPMI 8266 cells through the two pathways.The combination of ATO and bortezomib provided a novel strategy for the treatment of MM.And it is worthwhile to investigate in the clinical settings.

Figure 1 .
Figure 1.Cell Viability in ATO, Bortezomib and Combined Group.The result showed that the cell survival decreased gradually with the increase of drug concentration, in ATO, bortezomib and combined group (*P< 0.05, # P< 0.001)

Figure 2 .
Figure 2. The Apoptosis of RPMI 8266 Cells in Different treated Groups.A: ATO group, B: Bortezomib group, C: ATO+Bortezomib group.Cells in the lower right quadrant (LR) represented early apoptosis and in the upper right quadrant (UR) represented the late apoptosis

Figure 4 .
Figure 4.The Effect of ATO, Bortezomib and Combination of the Combined Drugs on Expression of DR4 and DR5 on RPMI 8266 Cells.A: Control without treatment, B: ATO group, C:bortezomib group, D:combination group DOI:http://dx.doi.org/10.7314/APJCP.2013.14.11.6469Arsenic Trioxide Alone and in Combination with Bortezomib in Multiple Myeloma RPMI 8266 Cells