Bortezomib could down-regulate the expression of RANKL,inhibit cell proliferation and induce cell apoptosis in the human myeloma cell line RPMI 8226 by activating casepase-3

Abstract

OBJECTIVE:

In spite of bortezomib being developed and demonstrated as a safe drug therapy for multiple myeloma (MM), the role of bortezomib-induced receptor activator of nuclear factor (NF)- $\kappa$ B ligand (RANKL) in the MM cell lines remains to be understood. Thus the present study aims to explore the impact of bortezomib on RANKL expression, cell growth and apoptosis in human myeloma cell line RPMI 8226.

METHODS:

Four experiment groups were set according to different concentrations of bortezomib, namely blank group (treated with DMEM solution free of other drugs), low-dose group (treated with 10 nmol/L bortezomib), middle-dose group (treated with 20 nmol/L bortezomib) and high-dose group (treated with 40 nmol/L bortezomib). Western blotting was adopted to detect RANKL protein expression. MTT assay was performed to detect cell proliferation. Flow cytometry was used to analyze cell cycle and apoptosis. Spectrophotometry was applied to determine caspases-3 activity.

RESULTS:

Compared with the blank group, the RANKL protein expression, cell number at the S stage was reduced while cell inhibition rate, cell apoptosis rate and caspase-3 activity enhanced remarkably in the low-dose, middle-dose and high-dose groups with dose-dependent manner. Compared with those treated with bortezomib (20 nmol/L and 40 nmol/L) for 6 h, the RANKL expression was down-regulated, cell inhibition rate was increased, cells at the S stage were reduced, cell apoptosis rate was enhanced, and caspase-3 activity elevated in the RPMI 8226 cells as treated with bortezomib for 24 h, with a dose- and time-dependent manner.

CONCLUSIONS:

Bortezomib could reduce the RANKL expression, inhibit cell proliferation and activate caspase-3 activity to induce cell apoptosis in RPMI 8266 cells.

1. Introduction

Multiple myeloma (MM), characterized by an asymptomatic premalignant proliferation of bone marrow plasma cells and osteolytic bone lesions, has been recognized as the second most prevalent malignancy of the blood in America, resulting in approximately 1% of neocplastic diseases as well as 13% of haematological malignancies [1]. It is reported that MM accounts for 0.8% of new cancer cases and 0.9% of cancer-related deaths annually on a worldwide scale, with an estimated 86,000 new cases diagnosed and 63,000 cases died from this disease [2]. As a study shows, myeloma bone disease (MBD) is a hallmark of MM, resulting from lytic bone lesions, and the pathogenesis of MBD implicates the factors in the tumor cells themselves, the release of activated growth factors from bone and the secretion by bone marrow stromal cells, as well as osteoblasts of growth factors and cytokines that induce bone destruction and enhance the growth of the tumor [3]. Unfortunately, MBD is not triggered by direct invasion of tumor cells to bone mass, thus the pathogenesis of MBD fails to be well-understood.

Receptor activator of nuclear factor (NF)- $\kappa$ B ligand (RANKL), receptor activator of NF- $\kappa$ B (RANK), and osteoprotegerin (OPG) have been commonly involved in the normal osteoclast development [4]. Currently, multiple hormones and local cytokines including glucocorticoid, parathyroid hormone (PTH), prostaglandin (PGE2), interleukin (IL)-6, IL-1 $\beta$ , and tumor necrosis factor (TNF)- $\alpha$ are able to regulate osteoclast formation and the function of osteoclasts by adjusting the expression and ratio of OPG, RANKL, and RANK, indicating that OPG/RANK/RANKL system is the main way to affect osteoclast formation, development and function [5]. Additionally, several researches support that MM cells have the ability of secreting RANKL that is positively related to the degree of bone dissolution [6, 7, 8]. Bortezomib is the first proteasome inhibitor for clinical trials that approved by the US Food and Drug Administration (FDA) in 2003, which is also a new anti-cancer drug that mainly used for the treatment of relapsed and refractory MM patients [9]. Though bortezomib as a drug therapy of MM can induce the apoptosis of tumor cells, its role in bone metabolism is unclear. Therefore, the study aims to demonstrate the effects of bortezomib on the RANKL expression, cell proliferation and apoptosis in human myeloma cell line RPMI 8226.

2. Materials and methods

2.1 Cells culture and medication

Human myeloma cell line (RPMI 8826) was provided by Professor Jin Jie of the Medical School Blood Research Institute of Zhejiang University (ATCC No. CCL-155). The RPMI 8826 cell line was cultured by 10% fetal bovine serum dulbecco’s modified eagle medium (FBS-DMEM) in a 37 ${}^{\circ }$ C incubator with 5% CO ${}_2$ . According to the literature before [10, 11, 12] and pre-experiment data, the RPMI 8226 cell line were randomly assigned into blank group (treated with DMEM solution free of other drugs), low-dose group (treated with 10 nmol/L bortezomib), middle-dose group (treated with 20 nmol/L bortezomib) and high-dose group (treated with 40 nmol/L bortezomib) and the reaction lasted for 6 h and 24 h in each group. The cells at the logarithmic phase were adjusted with a concentration of 2 $\times$ 10 ${}^5$ /mL and seeded into a 6-well plate (5 mL/well). Different concentrations of bortezomib were added into the corresponding groups and reactions lasted for 6 h and 24 h. Cells were collected after centrifugation at 200 $\times$ g for 5 min and the supernatant was eliminated. Then, the cells were re-suspended in 4% formaldehyde or phosphate buffer solution (PBS) and these cells (1 $\times$ 10 ${}^6$ /mL) kept standing at a room temperature for 10 min. Subsequently, cells were collected after centrifugation at 300 $\times$ g for 5 min and the supernatant was also abandoned. The collected cells was slightly shaken with the addition of 80% pre-cooled ethyl alcohol, and stored at 4 ${}^{\circ }$ C. The samples can be persevered for a month.

2.2 Cell proliferation detected by MTT

The obtained cells were cultured at 37 ${}^{\circ }$ C with 5% CO ${}_2$ , and 48 h later, 10 $\mu$ L of MTT (0.5 mg/mL) was added for incubation for 4 h. The the supernatant was aspirated after centrifugation and 200 $\mu$ L of dimethylsulfoxide (DMSO) was added into each well. The reaction was stopped after a few minutes vibration and automatic enzyme-linked immunosorbent assay system was applied to testify the absorbance (A) value at the wavelength of 490 nm. And the proliferation inhibition rate was calculated. The above processes were conducted repeatedly for 3 times.

2.3 Cell cycle and apoptosis analyzed by flow cytometry

The obtained cells were centrifuged slowly at 4 ${}^{\circ }$ C, washed by PBS for 2 times, mixed with 2 mL 80% ethyl alcohol ( $-$ 20 ${}^{\circ }$ C) at 4 ${}^{\circ }$ C overnight, washed by PBS for 2 times and dyed by 10 g/L polyimide (PI) for 30 min. The FACSCallibur flow cytometry (Becton, Dickinson and Company, New York, USA) was applied for the detection and the CellQuest for the analysis of the cell cycle and calculation of the proportions of each cell cycle. The above processes were conducted repeatedly for 3 times. The cells (1 mL, 1 $\times$ 10 ${}^6$ /mL) were centrifuged at 300 $\times$ g for 5 min in microcentrifuge tube for cell collection again and 80% ethyl alcohol was aspirated, after which the collected cells were re-suspended in 200 $\mu$ L of 1 $\times$ tert-butyldimethylsilyl (TBS) for incubation at room temperature for 10–15 min. Again, cells were collected after centrifugation at 300 $\times$ g for 5 min and the supernatant was aspirated. The proteinase K was diluted to 20 $\mu$ g/mL by 10 mmol/L tris. The cells were re-suspended in 100 $\mu$ L of proteinase K diluent for incubation at room temperature for 5 min (excessive incubation should be avoided). Cells were collected after centrifugation at 300 $\times$ g for 5 min and the supernatant was aspirated. In 80 $\mu$ L of dH ${}_2$ O, the 20 $\mu$ L of 5 $\times$ terminal deoxynucleotidyl transferase (TdT) equilibration was diluted to a final concentration of 1 $\times$ TdT equilibration. The cells were then re-suspended in 100 $\mu$ L of 1 $\times$ TdT equilibration for incubation at room temperature for 10–15 min. Fluorescain-Frag EL ${}^{\text{TM}}$ (Merck KGaA, Darmstadt, Germany) TdT (57.0 $\mu$ L) and 3.0 $\mu$ L of TdT enzyme were slightly mixed in a clean microcentrifuge tube on ice. Continually, cells were collected after centrifugation at 300 $\times$ g for 5 min and the supernatant was aspirated. The cells were re-suspended in 60.0 $\mu$ L of Fluorescain-Frag EL ${}^{\text{TM}}$ TdT for incubation in darkness at 37 ${}^{\circ }$ C for 1.5 h. Cells were collected after centrifugation at 300 $\times$ g for 5 min and the supernatant was aspirated. The 1 $\times$ TBS was used to wash cells for 2 times (centrifuged at 300 $\times$ g for 5 min) after which the cells were resuspended in 200 $\mu$ L of 1 $\times$ TBS. The final cells were added into the flow cytometry (FCM) at wavelength of 488 nm and the CellQuest software was applied for data collection and analysis. All the processes were conducted repeatedly for 3 times.

2.4 Caspase-3 activity detected by spectrophotometer method

Cells of all groups were collected, washed by PBS 2 times with 1 $\sim$ 5 $\times$ 10 ${}^6$ cells were obtained PBS aspirated as much as possible. The ice-cold lysis buffer (50 $\mu$ L) was added into collected cell deposition, after which the cells undergone splitting decomposition on the ice for 20 min were centrifuged at 4 ${}^{\circ }$ C, 12000 $\times$ g for 10 min. The supernatant was transferred into a new microcentrifuge tube and stored on the ice. The bicinchoninic acid (BCA) method was conducted to testify protein concentration in each group and the BCA protein assay kit was purchased from the Beyotime Biotechnology Corporation (Shanghai, China). The supernatant (50 $\mu$ L), containing 40 $\mu$ g of protein, was collected, and the lysis buffer was used as complement until the volume of the supernatant was less than 50 $\mu$ L. After the addition of 2 $\times$ reaction buffer (50 $\mu$ L) (0.5 $\mu$ L of dithiothreitol (DTT) was added with every 50 $\mu$ L of 2 $\times$ reaction buffer) and 5 $\mu$ L of caspase-3 substrate was incubated away from light at 37 ${}^{\circ }$ C for 1.5 h. The spectrophotometer (100 $\mu$ L of cuvette) was adopted to testify the light absorption value at $\lambda$ $=$ 405 nm. The caspase-3 activity was confirmed by the times of optical density (OD) of experimental group/OD of control group. The spectrophotometry detection kit was obtained from the Nanjing KeyGen Biotech Co. Ltd (Jiangsu, China). Notice: 50 $\mu$ L of lysis buffer $+$ 50 $\mu$ L of 2 $\times$ reaction buffers were used for zero setting.

Figure 1.

The comparisons of RANKL expressions in the RPMI 8226 cells among four groups. Note: A, Western blotting images for the RANKL expressions in the RPMI 8226 cells among four groups; B, The histogram for the RANKL expressions in the RPMI 8226 cells among four groups in 6 and 12 h after bortezomib treatment; RANKL, receptor activator of nuclear factor (NF)- $\kappa$ B ligand; ${}^{*}$ , $P<$ 0.05, compared with the blank group; ${}^{\mathrm{\#}}$ , $P<$ 0.05 compared with the low-dose group; ${}^{\&}$ , $P<$ 0.05, compared with the middle-dose group. Each experiment was repeated for three times.

Figure 2.

The relationship between RANKL expressions and reaction time. Note: A, relationship between RANKL expressions in the RPMI 8226 cells and reaction time detected by Western blotting; B, The histogram for the relationship between RANKL expressions and reaction time; RANKL, receptor activator of nuclear factor (NF)- $\kappa$ B ligand; ${}^{*}$ , $P<$ 0.05, compared with the blank group; ${}^{\mathrm{\#}}$ , $P<$ 0.05, compared with the low-dose group; ${}^{\&}$ , $P<$ 0.05, compared with the middle-dose group. Each experiment was repeated for three times.

2.5 RANKL expression in the RPMI 8226 cells line detected by Western blotting

Cells were collected in 1.5 mL eppendorf (EP) tube after centrifugation to remove nutrient solution and washing by PBS for 2 times, into which 100 $\mu$ L Western blotting and immunol precipitation (IP) cell lysis buffer (Beyotime Biotechnology Co., Shanghai, China) was added. And then a pipette was applied for repeated suction, after which the cells were fully cracked on the ice for 30 min and centrifuged at 12000 $\times$ g for 10 min. Then the supernatant was collected. The albuminous degeneration processes were as follows: 20 $\mu$ L of protein lysate containing 30 $\mu$ g of protein was obtained. And PBS was used as complement until the volume of the lysate reached 20 $\mu$ L. The samples were mixed with the 6 $\times$ sodium dodecyl sulfonate (SDS) loading buffer at ratio of 1: 5 and boiled at 100 ${}^{\circ }$ C for 5 min, after which the samples were stored on the ice until they returned to the room temperature and stored at $-$ 20 ${}^{\circ }$ C. The protein concentration was detected by the BCA protein assay kit. The polyacrylamideg (PAGE) elelectrophoresis (consistent 100 V, 50 min) was performed on each well, after which the proteins were transferred onto the nitrocellulose (NC) membrane (Applygen Technologies Inc. Beijing, China). And the membrane was slowly shaken in tris buffered saline tween (TBST) containing 5% skimmed milk powder for 1.5 h. After mouse anti-human RANKL monoclonal antibody (1 $\mu$ g/mL, 1: 500, MAB6261, R&D Systems Inc., Minneapolis, Minnesota, USA) and rabbit anti-human $\beta$ -actin monoclonal antibody (Cell Signaling Technologies (CST), Beverly, MA, USA; 1: 1000) were added respectively, and then the samples were incubated at room temperature for 1 h or kept standing at 4 ${}^{\circ }$ C in a wet box overnight. Then the TBST was used to wash the NC membrane for 3 times (10 min/time). Both the goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG (# 7074, Cell Signaling Technologies (CST), Beverly, MA, USA) labeled by horse radish peroxidase (HRP) were diluted with confining liquid at 1: 10000, after which the NC membrane was added into the mixed liquid with protein surface that kept upwards and then incubated at room temperature for 1 h. The TBST was used to wash the NC membrane for 3 times (10 min/time). The Quantity One software was selected to calculate the OD ratio of the target protein and $\beta$ -actin, representing as the relative expression quantity of RANKL.

Table 1

The ratio of RPMI 8226 cell proliferation after bortezomib treatment among four groups

Drug concentration (nmol/L)	6 h				24 h
	A value		Inhibition rate/%		A value		Inhibition rate/%
Blank group (0 nmol/L)	1.385	$\pm$ 0.228	0		1.294	$\pm$ 0.307	0
Low-dose group (10 nmol/L)	0.963	$\pm$ 0.335 ${}^{*}$	31	.07 ${}^{*}$	0.826	$\pm$ 0.318 ${}^{*}$	34	.51 ${}^{*}$
Middle-dose group (20 nmol/L)	0.811	$\pm$ 0.362 ${}^{*\mathrm{\#}}$	42	.26 ${}^{*\mathrm{\#}}$	0.605	$\pm$ 0.261 ${}^{*\mathrm{\#}}$	49	.19 ${}^{*\mathrm{\#}}$
High-dose group (40 nmol/L)	0.627	$\pm$ 0.281 ${}^{*\mathrm{\#}\&}$	55	.41 ${}^{*\mathrm{\#}\&}$	0.399	$\pm$ 0.197 ${}^{*\mathrm{\#}\&}$	64	.84 ${}^{*\mathrm{\#}\&}$

Note: ${}^{*}$ , $P<$ 0.05, compared with the blank group; ${}^{\mathrm{\#}}$ , $P<$ 0.05 compared with the low-dose group; ${}^{\&}$ , $P<$ 0.05, compared with the middle-dose group. Each experiment was repeated for three times.

Table 2

The ratio of RPMI 8226 cell apoptosis after bortezomib treatment among four groups

Drug concentration (nmol/L)	Cells apoptosis rate (%)
	6 h		24 h
Blank group (0 nmol/L)	1.52	$\pm$ 0.76	2.53	$\pm$ 0.93
Low-dose group (10 nmol/L)	5.16	$\pm$ 2.23 ${}^{*}$	55.19	$\pm$ 4.90 ${}^{*}$
Middle-dose group (20 nmol/L)	16.35	$\pm$ 5.68 ${}^{*\mathrm{\#}}$	73.41	$\pm$ 5.81 ${}^{*\mathrm{\#}}$
High-dose group (40 nmol/L)	39.73	$\pm$ 3.08 ${}^{*\mathrm{\#}\&}$	89.50	$\pm$ 4.39 ${}^{*\mathrm{\#}\&}$

Note: ${}^{*}$ , $P<$ 0.05, compared with the blank group; ${}^{\mathrm{\#}}$ , $P<$ 0.05 compared with the low-dose group; ${}^{\&}$ , $P<$ 0.05, compared with the middle-dose group. Each experiment was repeated for three times.

Figure 3.

The influence of bortezomib on cell cycle of the RPMI 8226 cells among four groups. Note: ${}^{\text{a}}$ , $P<$ 0.05, compared with the blank group; ${}^{\text{b}}$ , $P<$ 0.05 compared with the low-dose group; ${}^{\text{c}}$ , $P<$ 0.05, compared with the middle-dose group; ${}^{\mathrm{\#}}$ , $P<$ 0.05, compared with 6-h of bortezomib treatment. Each experiment was repeated for three times.

2.6 Statistical analysis

Data were analyzed using the statistical package for the social sciences (SPSS) version 21.0 (SPSS Inc.; Chicago, IL, USA). All data performed normality test, which was displayed as mean $\pm$ standard deviation, and the differences between two groups were analyzed by $t$ test and among more than two groups by one-way analysis of variance (ANOVA). If the variance was equal, the least significant difference (LSD) test was applied, or Tamhane’s T2 was adopted. $P$ $<$ 0.05 was regarded as significant difference.

3. Results

3.1 RANKL expression in the RPMI 8226 cell line

The RANKL was significantly down-expressed in the low-dose, middle-dose and high-dose groups in comparison to the blank group (all $P<$ 0.05). With the increase of the drug concentration and reaction time, the RANKL protein was less expressed. The association between the RANKL relative expression and bortezomib concentration and reaction time was presented in Figs 1 and 2.

3.2 Bortezomib could inhibit the proliferation of the RPMI 8226 cells

Compared with those in the blank group, the inhibition rates of the RPMI 8226 cells were significantly increased in the low-dose, middle-dose and high-dose groups at the same time point (all $P<$ 0.05). And with the increase of the bortezomib concentration, the cell inhibition rate was also elevated ( $P<$ 0.05). In those groups treated with the 20 nmol/L and 40 nmol/L bortezomib (the middle-dose and high-dose groups), the cell inhibition rate in the 24 h were significant higher than those in the 6 h (all $P<$ 0.05) (Table 1).

Table 3

Activity of caspase-3 in the bortezomib-treated RPMI 8226 cells among four groups

Drug concentration (nmol/L)	6 h				24 h
	Absorbency		Relative activity		Absorbency		Relative activity
Blank group (0 nmol/L)	0.494	$\pm$ 0.137	1.00	$\pm$ 0.00	0.388	$\pm$ 0.127	1.00	$\pm$ 0.00
Low-dose group (10 nmol/L)	0.890	$\pm$ 0.235	1.80	$\pm$ 0.02	1.978	$\pm$ 0.119	5.10	$\pm$ 0.09 ${}^{*}$
Middle-dose group (20 nmol/L)	1.581	$\pm$ 0.179 ${}^{*\mathrm{\#}}$	3.20	$\pm$ 0.06 ${}^{*\mathrm{\#}}$	2.521	$\pm$ 0.209 ${}^{*\mathrm{\#}}$	6.50	$\pm$ 0.11 ${}^{*\mathrm{\#}}$
High-dose group (40 nmol/L)	2.315	$\pm$ 0.277 ${}^{*\mathrm{\#}\&}$	4.69	$\pm$ 0.08 ${}^{*\mathrm{\#}\&}$	3.253	$\pm$ 0.228 ${}^{*\mathrm{\#}\&}$	8.38	$\pm$ 0.12 ${}^{*\mathrm{\#}\&}$

Note: ${}^{*}$ , $P<$ 0.05, compared with the blank group; ${}^{\mathrm{\#}}$ , $P<$ 0.05 compared with the low-dose group; ${}^{\&}$ , $P<$ 0.05, compared with the middle-dose group. Each experiment was repeated for three times.

Figure 4.

Effect of bortezomib on apoptosis of the RPMI 8226 cells among four groups. Note: Each experiment was repeated for three times.

3.3 Influence of bortezomib on cell cycle in the RPMI 8226 cells

Compared with the blank group, the RPMI 8226 cells at the S stage were remarkably reduced in the low-dose, middle-dose and high-dose groups. And with the increase of the bortezomib concentration, the cells at the S stage were also decreased (all $P<$ 0.05). In middle-dose and high-dose groups, the RPMI 8226 cells at the S stage in the 24 h were significantly reduced in comparison to those in the 6 h (all $P$ $<$ 0.05) (Fig. 3).

3.4 Bortezomib could induce apoptosis of the RPMI 8226 cells

Compared with the blank group, the cell apoptosis rates in the 6 h and 24 h were significantly increased in the low-dose, middle-dose and high-dose groups (all $P<$ 0.05). With the increase of the bortezomib concentration, the cell apoptosis rates were also elevated ( $P<$ 0.05). The cell apoptosis rate in the 24 h was significant higher than that of the 6 h in the same experimental groups (all $P<$ 0.05) (Table 2, Fig. 4).

3.5 Impact of bortezomib on activity of caspase-3 in the RPMI 8226 cells

The caspase-3 activity in the low-dose, middle- dose and high-dose groups for 6 h were 1.80, 3.20 and 4.69 times more active than those in the blank group, respectively. With bortezomib treatment 24 h, the caspase-3 in these three groups was 5.10, 6.50 and 8.38 times more active than those in the blank group, respectively. Compared with the blank group, an increases of the caspase-3 activity were noted in middle-dose and high-dose groups except the group treated with 10 nmol/L bortezomib (except the low-dose group) for 6 h (all $P<$ 0.05) and dose-dependent manner was also found (all $P<$ 0.05) (Table 3).

4. Discussion

Currently, there are few effective drugs for the treatment of MBD. Bortezomib, a new treatment of MM, can induce the apoptosis of tumor cells, but its role in bone metabolism is unclear. Terpos and his team conduct a study on 34 MM patients who have relapsed after previous treatment, and they are treated with bortezomib (single dose 1.3 mg/m ${}^2$ ) for 4–8 cycles, with findings show that the serum levels of RANKL is significantly decreased than those before the treatment with complete 4 cycles of $P<$ 0.01 and complete 8 cycles of $P<$ 0.001, which implies that bortezomib inhibits the expression of RANKL through directly killing myeloma cells [13]. Our experiment previously confirms that RPMI 8226 can express RANKL and possess the biological activity through promoting the differentiation of osteoclast precursor cells into osteoclasts, and yet whether bortezomib can directly inhibit RANKL expression of MM cells remains unknown. Thus we make an experiment in vitro to elucidate the impact of the bortezomib on the RANKL expression in RPMI 8226 cells.

Initially, as demonstrated in our study, three different concentrations of bortezomib reduced the RANKL expression in the RPMI 8226 cells with a dose- and time-dependent manner. A study performed by Silvestris et al. reflects that RANKL expression is up-regulated in myelomatous bones, which leads to an uncontrolled increase of osteoclast differentiation and then inhibits the bone formation, indicating that the inhibition of RANKL expression reduces bone destruction [14]. As Pennisi et al. reveal in their study that RANKL is critically implicated in MM bone disease, and bortezomib can reduce circulating levels of RANKL in the treatment of MM patients [15]. Roodman GD and his team also support the important role of RANKL expression in stimulating bone resorption, and they demonstrate that bortezomib decreases the serum levels of RANKL in MM patients [3]. All data above were in line with the findings of our study that RANKL expression could be decreased by bortezomib in the RPMI 8226 cells.

The key findings of this study are, when treated by bortezomib, increased cell inhibition rate, arrested cell cycle at the S stage, and increased cell apoptosis rate were shown in the the RPMI 8226 MM cells with a dose- and time-dependent manner. To our best knowledge, bortezomib potentially inhibits cell growth in 60 cell lines derived from multiple human tumors according to National Cancer Institute (NCI) and more significantly, it also plays a significant role in inhibiting the proliferation of the adherent MM cells [16]. Hideshima et al. also points out that a variety of MM cells are inhibited by bortezomib (PS-341) in a dose-dependent manner and 50% of growth inhibition of cells is found in different concentrations of bortezomib, and the study also proposes the apoptosis of RPMI 8226 cells is triggered by bortezomib that displays an irreversible progressive drug exposure time-dependent influence on RPMI 8226 cells [17]. Largely consistent with our study, as Que et al. and Elmahi et al. discover that, the cell viability of RPMI 8226 cells is declined and cell apoptosis and arrests the cell cycle increased with the treatment with bortezomib in a dose- and time-dependent manner, and it is believed that the combination of bortezomib and other inhibitors or agents acts synergistic inhibition and apoptosis in RPMI 8266 cells [18, 19]. Thus we concluded that bortezomib provided a novel strategy for the MM treatment by inhibiting the cell growth, inducing cell apoptosis, as well as blocking the cell cycle of human myeloma cell line.

In addition, our results stated the caspase-3 activity is prominently enhanced in the case group treated by bortezomib than that in the blank group, suggesting that bortezomib may increase the activity of caspase-3 in the RPMI 8226 cells. To our best knowledge, the caspase family produces significant effects on the regulation of apoptosis, and as a crucial enzyme and initiator of apoptosis, caspase-3 plays an essential role in the caspase cascade reaction [20]. According to Oca et al., low caspase-3 activity in tumor is a prognostic indicator of tumor recurrence in colon cancer, and cell apoptosis can be blocked by the inhibition of caspase-3 activity [21]. Thus we reached a conclusion that cell apoptosis is promoted with the enhancement of caspase-3 activity. A study performed by Zheng et al. reports that bortezomib results in an increase of the levels of active-caspase-3, which is responsible for the bortezomib-induced apoptosis [22]. More importantly, bortezomib has been confirmed to increase the caspase-3 activity with a dose-dependent fashion in MM cells [23], which is largely consistent with our study.

5. Conclusion

In conclusion, our study supported that bortezomib could down-regulate the RANKL expression, inhibit cell proliferation and induce cell apoptosis in the human myeloma cell line RPMI 8226 through activating casepase-3. These findings suggested the potential promise of bortezomib in the treatment of MM. However, our study has only one studied cell, thus it is better to select a large study subject size in association studies. Thus further in-depth researches will be performed to explore the mechanism of bortezomib on other MM cells to find more novel approaches for the therapy of the disease.

Conflict of interest

No conflict of interest was declared.