The role of Capon in multiple myeloma

Abstract

Capon is a ligand protein of nitric oxide synthase 1. Recently, studies have shown that Capon is involved in the development of tumors. It is independent of the regulation of nitric oxide synthase 1 in this process. At the same time, studies have found that nitric oxide synthase 1 is expressed in multiple myeloma, but its role in the development and progression of myeloma remains unclear. In this study, we found that there was a different expression of Capon between the normal multiple myeloma cells and the adherent multiple myeloma cells. In the process of myeloma cell proliferation, the reduced expression of Capon reduces the arrest of the cell cycle in the G1 phase and promotes the proliferation of myeloma cells. Cell adhesion–mediated drug resistance is one of the most important factors, which affect the chemotherapy effect of multiple myeloma. If the expression of Capon is decreased, myeloma cells are adhered to fibronectin or bone marrow stromal cells (bone marrow mesenchymal stem cells). In addition, the sensitivity of the cell line to chemotherapeutic agents was reduced after silencing Capon in the myeloma cell line which was adhered to bone marrow mesenchymal stem cells. We also found that reduced expression of Capon resulted in the activation of the AKT signaling pathway. In conclusion, these results may be helpful in studying the role of Capon in multiple myeloma.

Introduction

Multiple myeloma (MM) is a common and incurable malignant blood disease. ¹ It is characterized that the clonal proliferation of malignant plasma cells in the entire bone marrow (bone marrow) increased the risk of infection, eventually leading to the occurrence of multiple system lesions. ² At present, chemotherapeutic agents such as bortezomib (Bort) and doxorubicin for the treatment of MM often lead to disease recurrence because of the “cell adhesion-mediated drug resistance” (CAM-DR).^3–6 Therefore, it is necessary for CAM-DR to find new treatment goals, in order to improve the treatment of MM.

Capon, as the ligand protein for nitric oxide synthase 1 (NOS1), was first discovered by the presence of Capon in the brain tissue of mice in 1998, which was further found to be widely present in the myocardium and pancreas.^7–10 Studies have found that NOS1 is expressed in MM, but its role in the development and progression of myeloma remains unclear. ¹¹ Some studies have shown that Capon may be involved in tumorigenesis. Capon is linked to scribble and then binds to the ternary complex CAPON-scribble-Yes-related protein (YAP), which is composed of YAPs. ¹² Scribble is related to cell differentiation and migration, YAP plays an important role in the regulation of cell proliferation in the Hippo signaling pathway.^13–16 Scribble and YAP play a role in cell proliferation, suggesting that Capon may also have a similar role in cell proliferation. At the same time, studies have shown that breast cancer, low expression of Capon could promote the proliferation of breast cancer cell.^12,17 In gliomas, low expression of Capon can regulate cell proliferation by regulating AKT signaling pathways.^18,19 These evidences suggest that Capon may be a potential cancer suppressor.

In this study, we found that the expression of Capon was significantly decreased after suspension of MM cells adhered to bone marrow stromal cells (HS-5) or fibronectin (FN). It is well known that the interaction between the extracellular matrix of bone marrow and MM cells leads to the emergence of anti-apoptotic and cell-cycle arrest signals, resulting in drug resistance.^20–22 Therefore, it is necessary to study the role of Capon in cell adhesion–mediated (CAM) MM cells. In the subsequent experiments, we found that the expression of Capon was gradually decreased with the development of cell proliferation. Interfering Capon can inhibit the cell-cycle G1-phase arrest. The sensitivity of the cells to chemotherapy drugs was decreased in the adhesion of myeloma cells after interfering the expression of Capon. In summary, Capon may play a role in the adhesion of myeloma cells, which provides new vision for the treatment of MM.

Results

The expression of Capon is related to the adhesion of MM cells

In order to clarify the relationship between Capon’s expression and MM cell adhesion, cell adhesion culture was used to study the expression of Capon. Western blot and messenger RNA (mRNA) analysis showed that the expression of Capon was significantly decreased after cell adhesion (Figure 1). Since cell adhesion is closely related to cell growth, we hypothesize that Capon may have a role in the proliferation of MM cells. In addition, cell adhesion can make the cell sensitive to change in chemotherapy drugs, so we speculate that Capon may be associated with MM CAM resistance.

Figure 1.

Changes in the expression of Capon in suspended and adherent myeloma cells. (a and b) Western blot and mRNA levels were used to measure the changes in expression of Capon after normal myeloma cells (RPMI 8226) adhered to bone marrow stromal cells (HS-5) or fibronectin (FN). (c, d, and e) Detecting the change in expression of Capon in other myeloma cells (NCI-H929) after adhesion culture. The ratio of Capon to β-actin by densitometry in different cells is shown in the bar chart in (b) and (d). The data are expressed as the mean ± standard deviation of the three independent experiments (*p < 0.05).

Capon’s expression changes with the proliferation of MM cells

In order to clarify the relationship between Capon and MM cell growth, we did experiments on starvation. Through the passage of detection, it was found that the G1 phase of the block is constantly reduced with the proliferation of cells (Figure 2(c) and (d)). Western blot analysis showed that the expression of Capon decreased gradually and the expression of cyclinE increased gradually (Figure 2(a) and (b)) during cell proliferation. According to the above experiment, we speculate that Capon may play a role in the inhibition of proliferation in MM cells.

Figure 2.

To detect the role of Capon in the proliferation of multiple myeloma cells. (a and b) Western blot analysis of the changes in expression of Capon, PCNA, cyclinE, and CDK2 at different time in cell proliferation, which were serum starved (S) for 72 h and then refed (R) with RPMI 1640 medium containing 10% fetal bovine serum for 0, 6, 12, 24, and 48 h. (c and d) The distribution of cell cycle in myeloma cells was measured by flow cytometry at different time (S72h, R6h, R12h, R24h, and R48h). The ratio of Capon/PCNA/cyclinE/CDK2 to β-actin by densitometry at different time in cell proliferation and the distribution of cell cycle at different time is shown in the bar chart in (b) and (d). The data are expressed as the mean ± standard deviation of the three independent experiments (*p < 0.05; #p < 0.05; ^p < 0.05).

Interference with Capon expression promotes the proliferation of MM cells

In order to further clarify the effect of Capon on the proliferation of MM cells, first we transfected shCapon-1 and shCapon-2 in MM cells. We found that shCapon-2 had better interference with Capon expression, so we used shCapon-2 as the shCapon to interfere with Capon expression in the following experiments (Figure 3(a) and (b)). The Cell Counting Kit-8 (CCK-8) reagent was used to detect the proliferation of bone marrow cells. We found that the expression of Capon could promote cell proliferation (Figure 3(c)). Western blot analysis showed that the expression of proliferating-cell nuclear antigen (PCNA), cyclinE, and cyclin-dependent kinase 2 (CDK2) was upregulated after interference with Capon expression (Figure 3(d)). We used cell-cycle detection to find out whether the G1 phase of cells transfected with Capon was significantly higher, compared with which were transfected with shCapon and the normal myeloma cells (Figure 3(e)). In summary, Capon may play a role in inhibiting cell proliferation in MM.

Figure 3.

The effect of low expression of Capon on the proliferation of myeloma cells. (a and b) Western blot analysis of shCapon-1 and shCapon-2 to downregulate the expression of Capon at protein level in myeloma cells. (c) The expression of Capon could promote cell proliferation detected by the CCK-8 reagent. (d) Western blot was used to detect the change in the proliferation of myeloma cells transfected with shCapon. (e) Flow cytometry to detect the cell-cycle distribution of myeloma cells transfected with shCapon or Capon plasmids and normal myeloma cells. All data related to western blot and cell cycle in the experiment were shown by the bar chart in (a), (b), (d), and (e). The data are expressed as the mean ± standard deviation of the three independent experiments (*p < 0.05; #p < 0.05).

Capon may adjust the AKT signal path in MM

Based on the previous results, we hypothesize that low expression of Capon can promote cell proliferation in MM. In gliomas, Capon can affect cell proliferation by regulation of the AKT signaling pathway. But we are uncertain whether Capon also adjusts the AKT signal pathway which affects the proliferation of cells in MM. By comparing the western blotting of the suspension- and adherent-cultured MM cells, we found a decrease in the expression level of Capon and increase in the expression of AKT and p-AKT in adherent-cultured cells (Figure 4(a) and (b)). In a further trial, we found that the expression of AKT and p-AKT increased with the reduction of Capon’s expression after transfection of shCapon in suspension- and adherent-cultured MM cells (Figure 4(c) and (d)). From these experimental results, we speculate that Capon may have the same effect in MM as it does in the glioma, promotes the proliferation of cells by regulating the AKT signaling pathway.

Figure 4.

Capon regulates AKT signaling pathway in multiple myeloma cells. (a and b) The changes in expression of Capon, AKT, and p-AKT were detected by western blot in normal MM cells and MM cells adhered to FN. (c) Detection of changes in the expression levels of Capon, AKT, and p-AKT in myeloma cells after transfection with shCapon. (d) Western blot was used to detect the changes in expression of Capon, AKT, and p-AKT in myeloma cells adhered to FN after transfection with shCapon. All data related to western blot in the experiment are shown in a histogram. The data are expressed as the mean ± standard deviation of the three independent experiments (*p < 0.05; #p < 0.05).

The low expression of Capon can reduce the sensitivity of MM cells to chemotherapeutic drugs

Cell adhesion–mediated drug resistance (CAM-DR) is one of the reasons why MM cells are less susceptible to chemotherapeutic drugs. At the same time, AKT signaling pathway is closely related to the development of MM.^11,23,24 Therefore, we speculate that Capon has a role in CAM-DR. After using the Bort to stimulate the suspension-cultured myelomacells and adherent-cultured myeloma cells, the expressionof Capon in adherent-cultured cells was significantly lower than that in suspended cells, and the expression of caspase-3 (cleaved) and poly(ADP-ribose) polymerase (PARP; cleaved) decreased significantly (Figure 5(a) and (b)).After Bort stimulated the adherent myeloma cells, which were transfected with shCapon, we found that the expression of caspase-3 (cleaved) and PARP (cleaved) was reducedin the adherent myeloma cells transfected with shCapon (Figure 5(c)). Further detection of apoptotic cells showed that the apoptosis of cells was significantly reduced after the expression of Capon was disrupted in both normal myeloma cells and adherent-cultured myeloma cells (Figure 5(d)). In summary, the low expression of Capon may have the effect of reducing the sensitivity of cells to chemotherapeutic drugs in MM.

Figure 5.

The effects of low expression of Capon on the drug sensitivity of MM cells. (a and b) Western blot was used to detect the expression of Capon/caspase-3 (cleaved)/PARP (cleaved) in normal MM cells and adherent MM cells after Bort stimulation. (c) Analysis of changes in the expression of Capon /caspase-3 (cleaved)/PARP (cleaved) after myeloma cells were transfected of shCapon by western blot. (d) Flow cytometry was used to analyze the apoptotic changes after myeloma cells were transfected with shCapon and the apoptotic changes of adherent myeloma cells transfected with shCapon. All data related to western blot are represented by a histogram in (a), (b), and (c). The data are expressed as the mean ± standard deviation of the three independent experiments (*p < 0.05; ^p < 0.05).

Discussion

In this study, we found that the expression of Capon was significantly decreased after myeloma cells were adhered to bone marrow stromal cells or FN, which was decreased with the proliferation of myeloma cells. Subsequently, protein immunoblotting, cell-cycle detection, and apoptosis detection experiments are also confirmed: Capon low expression can activate the AKT signaling pathway, reduce cell-cycle G1-phase arrest, and reduce the sensitivity of the cell to chemotherapy drugs. These findings suggest that Capon may play a role in suppressing the AKT signaling pathway in MM.

Studies have shown that low expression of Capon in breast and gliomas can promote tumor cell proliferation. In this study, we found that Capon inhibited the proliferation of myeloma cells. Therefore, we further study whether Capon’s inhibition of proliferation of myeloma cells is associated with cell cycle. Flow cytometry analysis showed that cell accumulation in G1 phase was significantly consistent with cell-cycle factor E and CDK2 after Capon silencing. Then, we check whether DTX3L affects CAM-DR. The study found that silencing the Capon could reduce the sensitivity of chemotherapy drugs in myeloma cells. In addition, we also found that Capon may regulate the AKT signal pathway in the MM as it regulates AKT in gliomas. However, the reason for the low expression of Capon in adhesion-mediated drug resistance, the low expression of Capon leads to a decrease in cell cycle G1 phase arrest, the specific mechanism which CAM-DR cells are less sensitivity to chemotherapeutic agents are still unclear.

CAM-DR is one of the causes of multiple drug resistance in the late stages of chemotherapy. Our study shows that low expression of Capon plays an important role in cell proliferation and CAM-DR. Interference with Capon expression can promote cell proliferation and reduce the sensitivity of CAM-DR cells to chemotherapeutic drugs. In conclusion, our study may help to further improve the treatment of MM.

Experimental section

Cell culture

Bone marrow stromal cell line HS-5 and the human MM cell lines RPMI 8226 and NCI-H929 were purchased from Cell Library, China Academy of Science. The cell lines NCI-H929 and RPMI 8226 were grown in RPMI 1640 (Gibco BRL, Grand Island, NY, USA) and the HS-5 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco BRL). The cells were cultured in a cell incubator containing 5% CO₂ under the saturated humidity at 37°C.

Cell co-culture

The dishes were mantled overnight at 37°C with 40 µg/mL FN (Sigma–Aldrich, Rehovot, Israel) in a final volume of 1 mL phosphate-buffered saline (PBS) or HS-5 cells. Then, MM cells (100,000 cells/mL) were adhered to pre-established monolayers of FN or HS-5 for 2–4 h. Finally, adhered cells were carefully removed for the following experiments, with the HS-5 monolayer kept intact.

Preparation of plasmid and shRNA and transient transfection

The human capon (accession number: NM_014697) was inserted into the pWPXLd plasmid at BamH I and Mlu I sites for increasing the expression of Capon in MM cells. Capon’s shRNAs were designed and synthesized by GeneChem (Shanghai, China). The shRNA oligos (target sequences: 5′-GCCAGCAATATCTTCAGATG-3′ for shCapon-1; 5′-GCCTCAGAGTATGAGTCCAA-3′ for shCapon-2) for silencing CAPON in MM cells. The shRNA oligos targeting on Capon gene and the negative control (NC) shRNA were designed and provided by GeneChem. According to the instructions of manufacturer, transfections were performed by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37°C in RPMI 1640 medium without serum for 6 h, and then, the medium was replaced with RPMI 1640 containing 10% fetal bovine serum. Transfected cells were harvested after transfection for 48 h.

Antibodies

The primary antibodies used are as follows: anti-Capon antibody (1:500), anti-cyclinE antibody (1:500), anti-PCNA antibody (1:500), anti-CDK2 antibody (1:500), anti-caspase3 antibody (cleaved1:1000), anti-AKT antibody (1:1000), anti-p-AKT antibody (1:1000), anti-PARP antibody (cleaved1:1000), and anti-β-actin antibody (1:1000). All antibodies used were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except anti-caspase3 antibody, anti-PARP antibody, anti-AKT antibody, and anti-p-AKT antibody (Cell Signaling Technology, Danvers, MA, USA). The experiments were carried out on three separate occasions.

Western blot analysis

Total protein assay was conducted as previously described. The proteins were transferred onto polyvinylidene difluoride (PVDF) filter membranes (Millipore, Bedford, MA, USA) after subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were incubated with the primary antibodies overnight at 4°C after blocking with PBS containing 0.1% Tween-20 and 5% non-fat milk. The filters were washed three times with PBS containing 0.1% Tween-20, each for 5 min, and incubated with horseradish peroxidase–conjugated secondary antibody for 2 h at room temperature. The membranes were developed using the enhanced chemiluminescence (ECL) detection system. The band density was quantified by the ImageJ analysis system (Wayne Rasband, National Institutes of Health, USA).

Cell-cycle analysis

The cell-cycle distribution flow instrument analysis. The cold cell was fixed in 70% ethanol at −20°C and collected after 24 h, and the cells were washed twice with PBS and incubated with 1 mg/mL RNase A for 20 min at 37°C. Then, these cells were stained with propidium iodide (PI, 50 µg/mL; Becton Dickinson, San Jose, CA, USA) in PBS and 1% Triton-X 100 and incubated for an additional 20 min at 4°C in dark. The data were analyzed by Becton–Dickinson flow cytometer BD FACScan.

Flow cytometry analysis of cell apoptosis

The drug-induced apoptosis was tested following exposure to bortezomib (Sigma) in MM cells. Flow cytometry analysis was conducted as previously described. ¹⁹ As per the manufacturer’s protocol, following 48 h exposure to chemotherapy agents, the apoptotic cells were detected with Annexin-V-FLUOS Staining Kit (Roche, Shanghai, China). The cells were washed three times with cold PBS and resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/mL. Then, 5 µL 7-ADD solution and 5 µL AnnexinV were added to each tube. Another 400 µL 1× binding buffer was added to each tube after incubating for 15 min at room temperature in the dark. The apoptosis assay was performed with Becton Dickinson flow cytometer BD FACScan according to the manufacturer’s instructions.

Statistical analysis

All experiments were repeated at least three times. Each value is reported as mean ± standard error of mean (SEM). The calculations were analyzed by the Statistical Package for the Social Science SPSS 19.0 software (SPSS Inc. (An IBM Company), Chicago, IL, USA). The probability of 0.05 or less was considered statistically significant.