Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 12  |  Issue : 2  |  Page : 963--968

Chemotherapy effectively suppresses interleukin-20, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin levels in patients with lung adenocarcinoma and bone metastasis


Mingyang Yu1, Yun Su1, Daping Cui1, Qiang Sun1, Bowu Luan2, Dewei Zhao3,  
1 Department of Traumatic Orthopedics, Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning, China
2 Department Chemistry, Stony Brook University, NY, USA
3 Department of Orthopaedics, Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning, China

Correspondence Address:
Dewei Zhao
Department of Orthopaedics, Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning
China

Abstract

Background: Bone metastasis (BM) is common in patients with lung cancer. Osteolysis is caused by increased osteoclast activity. Interleukin-20 (IL-20) and receptor activator of nuclear factor kappa-B ligand (RANKL) are crucial for osteoclast formation. Osteoprotegerin (OPG) inhibits a receptor activator of RANKL/RANK signaling. The aims of this study were to analyze the serum levels of IL-20, OPG, and RANKL in patients with and without BM and to observe the effect of chemotherapy on these cytokines. Patients and Methods: A total 54 cases of pathologically confirmed lung adenocarcinoma (ADC) and 18 healthy individuals (Control) were enrolled in this study. Eligible patients were divided into three groups (18 patients per group): ADC without BM (ADC), ADC with BM (ADC + BM), and ADC with BM treated with chemotherapy (ADC + BM + Chemo). Serum IL-20, RANKL, and OPG levels were analyzed by enzyme-linked immunosorbent assay. Results: Serum IL-20, RANKL, and OPG levels in ADC + BM patients were significantly elevated compared with that in the Control or ADC groups (both P < 0.001). The serum cytokine levels were significantly lower following chemotherapy compared with that in patients who did not receive chemotherapy (P < 0.001). Conclusions: Serum IL-20, RANKL, and OPG levels increase in patients with lung cancer and BMs. Chemotherapy suppresses the elevation of these cytokines.



How to cite this article:
Yu M, Su Y, Cui D, Sun Q, Luan B, Zhao D. Chemotherapy effectively suppresses interleukin-20, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin levels in patients with lung adenocarcinoma and bone metastasis.J Can Res Ther 2016;12:963-968


How to cite this URL:
Yu M, Su Y, Cui D, Sun Q, Luan B, Zhao D. Chemotherapy effectively suppresses interleukin-20, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin levels in patients with lung adenocarcinoma and bone metastasis. J Can Res Ther [serial online] 2016 [cited 2020 Apr 1 ];12:963-968
Available from: http://www.cancerjournal.net/text.asp?2016/12/2/963/179085


Full Text



 Introduction



Bone metastasis (BM) commonly occurs in patients with lung cancer.[1],[2] It is the most common cause of morbidity in many cancers including breast, lung, kidney, and prostate cancer, and multiple myeloma.[3],[4] In addition, osteolytic metastases often cause severe pain, pathologic fractures, hypercalcemia, spinal cord compression, and other nerve compression syndromes.[5],[6] Bone remodeling is well-controlled by the balance between bone formation and resorption.[7],[8] Excessive bone resorption by osteoclasts is an important cause of osteolysis in cancer-related osteolysis.[9]

Interleukin-20 (IL-20) is a proinflammatory cytokine belonging to the IL-10 family. It is expressed in various cell types including monocytes, epithelial cells, and endothelial cells.[10],[11],[12] IL-20 activates a heterodimer receptor complex of either IL-20 receptor 1 (IL-20R1)/IL-20R2 or IL-22R1/IL-20R2.[13],[14],[15] IL-20 also mediates osteoclastogenesis by upregulating the expression of receptor activator of nuclear factor kappa-B (RANK) in osteoclast precursor cells and RANK ligand (RANKL) in osteoblasts.[16] RANKL, a tumor necrosis factor family member, is expressed in osteoblasts and stromal cells; it regulates osteoclastogenesis differentiation, fusion, survival, and activation.[17],[18],[19] Osteoprotegerin (OPG) inhibits a receptor activator of RANKL/RANK signaling. OPG inhibits osteoclast formation and activity in vitro and in vivo.[20] OPG overexpression in transgenic mice resulted in osteopetrosis due to impaired osteoclast formation, an effect readily apparent in osteoblasts cocultured with hematopoietic cells in vitro in the presence of OPG.[21],[22] The RANKL–OPG balance may affect osteoclast activity, which is why it has been suggested that the serum RANKL/OPG ratio is a critical factor for determining osteoclast activation.[23],[24]

Osteoclasts play a key role in BMs of lung cancer. Considering IL-20 and RANKL/OPG regulate osteoclast formation and osteolysis after BMs, we hypothesized that the IL-20/RANKL/OPG axis may be important to BMs in lung cancer and that this axis could potentially be a chemotherapy target. The aims of this study were to analyze the serum levels of IL-20, OPG, and RANKL in patients with and without BM and to observe the effect of chemotherapy on these cytokines. The serum levels of IL-20, OPG, and RANKL were detected by enzyme-linked immunosorbent assay (ELISA). Our study showed that IL-20, RANKL, and OPG levels in lung adenocarcinoma (ADC) patients with BM were elevated. Following chemotherapy, the cytokine levels were inhibited.

 Patients and Methods



Study design

The protocol was reviewed and approved by the local Ethics Committee and performed according to Good Clinical Practice and the Helsinki Declaration. All participants provided written informed consent to participate in the study. This prospective study was carried out at our hospital, China, from March 2012 to December 2013. A total 54 cases of pathologically confirmed ADC and 18 healthy individuals (Control) were enrolled in this study. Healthy individuals were asymptomatic volunteers who had undergone a routine medical examination at our institution. Eligible patients were divided into three groups (18 patients per group): ADC without BM (ADC), ADC with BM (ADC + BM), ADC with BM treated with chemotherapy (ADC + BM + Chemo). The inclusion criteria for patients with ADC were: (1) Age ≥18 years; (2) chest solid tumor revealed by computed tomography or radiographic examination; (3) pathologically confirmed ADC; (4) BM diagnosed by bone scintigraphy; (5) Eastern Cooperative Oncology Group performance status ≤2; life expectancy >6 months; (6) normal renal and hepatic function with total bilirubin <2 mg/dl and serum creatinine <2 mg/dl. The exclusion criteria for patients with ADC were: (1) Treatment with bisphosphonates, surgery, or radiotherapy; (2) history of postmenopausal osteoporosis.

Variables

”Nonsmoking” refers to smoked cigarettes <100. “Smoking” included both current and previous smoking. “Previous smoking” refers to previously smoked cigarettes >100 and smoking cessation >6 months. “Current smoking” refers to currently smoked cigarettes >100. Nonsmall cell lung cancer (NSCLC) stage was defined according to the 2009 International Association for the study of lung cancer guidelines.[25] Lung cancer with BM was diagnosed by bone scintigraphy. Chemotherapy was defined as patients receiving intravenous infusion of chemotherapeutic agents. Three different chemotherapy regimens were used for treating the 18 patients in the ADC + BM + Chemo group: (1) 1000 mg/m 2 gemcitabine on days 1 and 8 each time + 75 mg/m 2 cisplatin on days 1, 2, and 3 each time; (2) 75 mg/m 2 docetaxel on day 1 + 75 mg/m 2 cisplatin on days 1, 2, and 3 each time; (3) 500 mg/m 2 pemetrexed on day 1 + 75 mg/m 2 cisplatin on days 1, 2, and 3 each time. One course spanned 21 days. The detailed courses and treated cases are summarized in [Table 1].{Table 1}

Peripheral venous blood samples were obtained from all enrolled participants. Blood samples were collected from patients in the ADC and ADC + BM groups when lung cancer was diagnosed and before any treatments. Blood samples were collected from patients in the ADC + BM + Chemo group at the end of chemotherapy. Serum IL-20, OPG, and RANKL levels were detected using specific ELISA kits (Abgent, San Diego, CA, USA) according to the manufacturer's instructions.

Statistical analysis

Statistical analyses were performed using SPSS 18.0 (IBM, Chicago, IL, USA). Serum IL-20 or OPG or RANKL levels are expressed as the mean ± standard deviation. One-way analysis of variance and the post hoc Tukey test were used to analyze differences among four groups. A P < 0.05 was considered statistically significant.

 Results



Baseline characteristics of subjects

A total 18 healthy controls (Control) were enrolled in this study, comprising nine men and nine women; the mean age was 51 years (range, 37–61 years). Seven (39%) were nonsmokers, and 11 (61%) were smokers. A total 54 cases of ADC were divided into three groups according to BM and chemotherapy. There were 12 men and six women in the ADC group; their mean age was 55 years (range, 41–63 years). Of these patients, 3 (17%) were stage IIIA, 6 (33%) were stage IIIB, and 9 (50%) were stage IV. There were nine men and nine women in the ADC + BM group; their mean age was 57 years (range, 37–76 years). All of them had advanced-stage disease (IIIB: 22%; IV: 78%). There were nine men and nine women in the ADC + BM + Chemo group; their mean age was 58 years (range, 51–67 years). All of them had advanced-stage disease (IIIB: 17%; IV: 83%) [Table 2].{Table 2}

Comparison of serum interleukin-20 levels among different groups

To determine the changes in serum IL-20 levels in ADC patients following chemotherapy, we detected the serum IL-20 levels in all enrolled subjects. Serum IL-20 levels were 19.61 ± 5.72 pg/ml (Control), 25.35 ± 7.94 pg/ml (ADC), 43.26 ± 11.84 pg/ml (ADC + BM), and 30.58 ± 9.38 pg/ml (ADC + BM + Chemo). Serum IL-20 levels in ADC + BM patients were significantly elevated compared with that in the Control or ADC groups (both P < 0.001) and were significantly lower following chemotherapy compared to that in patients who did not receive chemotherapy (P < 0.001) [Figure 1].{Figure 1}

Comparison of serum receptor activator of nuclear factor kappa-B ligand levels among different groups

To determine the effect of chemotherapy on serum RANKL levels in patients with ADC and BM, we examined the serum RANKL levels in all enrolled subjects, which were 168.96 ± 76.00 pg/ml (Control), 182.48 ± 15.30 pg/ml (ADC), 347.58 ± 102.26 pg/ml (ADC + BM), and 119.52 ± 12.99 pg/ml (ADC + BM + Chemo). Serum RANKL levels in ADC + BM patients were significantly elevated compared with that in the Control or ADC groups (both P < 0.001) and were significantly lower following chemotherapy compared to that in patients who did not receive chemotherapy (P < 0.001) [Figure 2].{Figure 2}

Comparison of serum osteoprotegerin levels among different groups

Serum OPG levels in the ADC (78.01 ± 14.08 pg/ml) or ADC + BM group (104.81 ± 19.34 pg/ml) were significantly increased compared with that in the Control group (58.69 ± 18.50 pg/ml) (all P < 0.01). Serum OPG levels in the ADC + BM + Chemo group (92.65 ± 18.93 pg/ml) were significantly lower compared that in patients who did not receive chemotherapy (P < 0.05) but remained higher than in patients with ADC or the normal controls (P < 0.05, P < 0.001, respectively) [Figure 3].{Figure 3}

Comparison of receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio among different groups

The RANKL/OPG ratio was significantly lower in the ADC group compared with the Control group (2.44 ± 0.61 vs. 3.20 ± 1.76, P < 0.05). The ratio of the ADC group was also lower than that of the ADC + BM group (3.40 ± 1.10) (P < 0.05), while that of the ADC + BM + Chemo group was even lower (0.96 ± 0.22), which was significantly different compared to the other three groups (all P < 0.05) [Figure 4].{Figure 4}

 Discussion



We have shown that serum levels of IL-20, RANKL, and OPG are elevated in patients with ADC and BM. Meanwhile, chemotherapy suppressed the increases in these cytokines.

In this study, we observed that IL-20 was highly expressed in the serum of ADC patients with BM. This result is in line with previous reports, where IL-20 was upregulated in tumor tissue from patients with NSCLC.[26],[27] Hsu et al.[16] reported that IL-20 promoted osteoclast differentiation by upregulating RANK levels in osteoclast precursor cells. In that study, IL-20 markedly induced RANKL production in synovial fibroblasts, T helper 17 cells, and osteoblast cells by elevating cathepsin G.[16] Cathepsin G is capable of shedding the extracellular domain of RANKL from osteoblasts to generate active soluble RANKL.[16] Upregulation of RANKL, RANK, and OPG as well as increased RANKL/OPG ratio were observed in NSCLC cell lines and in tumor tissues with BM and were positively correlated with higher metastatic potential of lung cancer. These findings suggest that IL-20 plays a role during BM of ADC. IL-20 is a possible novel target for treating lung cancer.

We further analyzed the serum levels of RANKL and OPG, two important biomarkers relating to bone turnover and found that RANKL and OPG were also elevated in patients with ADC and BM. A previous study showed that RANKL significantly enhanced the migration and invasion ability of NSCLC cells in vitro and in vivo via either recombinant RANKL or overexpressed RANKL. However, the effect was inhibited by OPG. The study also showed that the increased levels of RANKL and OPG were positively correlated with tumor stage, lymph node metastasis, and distant metastasis. Differential expression of RANKL, RANK, and OPG is associated with the metastatic potential of human NSCLC to the bone. It raises the possibility that the RANKL/RANK/OPG axis could be a therapeutic target for treating metastatic NSCLC patients.[28]

Cancer-related BM is a chronic process, in which the destruction of the surrounding bone matrix occurs gradually. Therefore, it is difficult to determine an exact time frame of BMs. Osteolysis is caused by a tumor indirectly stimulating osteoclast differentiation and activity rather than by direct effects on the skeleton.[29] The invasive capabilities of cancer cells are important for tumor progression; however, the critical characteristic of the metastatic cancer cell phenotype is the ability to ultimately stimulate bone resorption.[6],[30] This function is exclusively carried out by monocyte/macrophage-derived osteoclasts, which creates an environment that is receptive to transitioning cancer cells and allows them to survive and proliferate.[31] In fact, tumor stimulation of osteoclastic bone resorption at the bone marrow and bone interface is required for tumor establishment as BM within the strict confines of the mineralized structure of bone.[30] Osteoclasts are multinucleated giant cells with bone-resorbing capacity.[31],[32],[33] RANKL, IL-20, and macrophage colony-stimulating factor are essential for osteoclast formation, promoting the differentiation of osteoclast precursors into mature osteoclasts.

A large number of osteoclast exist in peripheral blood of patients with cancer BM.[3] Our study found that a lot of cytokines are in peripheral blood of patients with BM, and thus, promote osteoclast formation. We speculate that tumor cells lead to an increase of IL-20, RANKL, and OPG of patients with cancer BM, which further induce the multinucleation of osteoclast precursors in peripheral blood, resulting in the elevated number of osteoclast. In conclusion, peripheral blood might be one of the places where an excess number of osteoclast in tumor patients. After osteoclast is generated in the peripheral blood, these cells are then circulated into the bone; due to blood sinuses and gravity, the osteoclasts remain in the bone marrow cavity.

The most compelling evidence supporting RANKL-independent effects on tumor-induced osteoclastogenesis and osteolysis was produced in clinical trials of the fully human RANKL antibody (denosumab) in cancer patients with BM and elevated bone resorption.[34] The present prospective study highlights a clear trend toward a decreased RANKL/OPG ratio and N-telopeptide levels after 12-month treatment with zoledronic acid. The decreased RANKL/OPG ratio raises the interesting hypothesis that zoledronic acid may also influence osteoclast behavior during differentiation, perhaps via an indirect mechanism that leads to the blocking of the vicious cycle of BM.[35] Following chemotherapy, the concentration of serum RANKL in the patients was lower than even normal levels. The serum RANKL/OPG ratio following chemotherapy was significantly lower than that in the Controls or ADC patients without or with BM. A likely explanation is that: (a) Cisplatin, pemetrexed, or gemcitabine inhibited tumor cell activity or killed them directly, thus indirectly reducing serum RANKL levels; (b) cisplatin, pemetrexed, or gemcitabine had an inhibitory effect on RANKL-secreting cells. Therefore, our study suggests that chemotherapy inhibits osteolysis.

Serum OPG levels in ADC patients are higher than that in healthy individuals and are even higher in cancer patients with BM. This is possibly a protective mechanism of the human body when excessive bone resorption occurs. This study yields two interesting conclusions: (a) Serum IL-20/RANKL/OPG in the ADC + BM group is higher; (b) Chemotherapy can reduce RANKL and the RANKL/OPG ratio.

Although we observed that chemotherapy suppressed serum RANKL and OPG levels in cancer patients and reversed the RANKL/OPG ratio, we were unable to determine which chemotherapeutic agent played a role in this effect. We were also unable to determine whether the effect was achieved by cancer cell killing or growth inhibition, or by suppression of the cells that secrete RANKL and other cytokines.

 Conclusions



Our study demonstrates that serum IL-20, RANKL, and OPG levels are increased in patients with lung cancer and BM and that chemotherapy inhibits the increases in serum levels of these cytokines of such patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Coleman RE. Metastatic bone disease: Clinical features, pathophysiology and treatment strategies. Cancer Treat Rev 2001;27:165-76.
2Deberne M, Ropert S, Billemont B, Daniel C, Chapron J, Goldwasser F. Inaugural bone metastases in non-small cell lung cancer: A specific prognostic entity? BMC Cancer 2014;14:416.
3Roato I, Grano M, Brunetti G, Colucci S, Mussa A, Bertetto O, et al. Mechanisms of spontaneous osteoclastogenesis in cancer with bone involvement. FASEB J 2005;19:228-30.
4Robert J. Biology of cancer metastasis. Bull Cancer 2013;100:333-42.
5Lutz S, Berk L, Chang E, Chow E, Hahn C, Hoskin P, et al. Palliative radiotherapy for bone metastases: An ASTRO evidence-based guideline. Int J Radiat Oncol Biol Phys 2011;79:965-76.
6Mundy GR. Metastasis to bone: Causes, consequences and therapeutic opportunities. Nat Rev Cancer 2002;2:584-93.
7Bernhardt A, Thieme S, Domaschke H, Springer A, Rösen-Wolff A, Gelinsky M. Crosstalk of osteoblast and osteoclast precursors on mineralized collagen – Towards an in vitro model for bone remodeling. J Biomed Mater Res A 2010;95:848-56.
8Waning DL, Guise TA. Molecular mechanisms of bone metastasis and associated muscle weakness. Clin Cancer Res 2014;20:3071-7.
9Luis-Ravelo D, Antón I, Zandueta C, Valencia K, Ormazábal C, Martínez-Canarias S, et al. A gene signature of bone metastatic colonization sensitizes for tumor-induced osteolysis and predicts survival in lung cancer. Oncogene 2014;33:5090-9.
10Fickenscher H, Hör S, Küpers H, Knappe A, Wittmann S, Sticht H. The interleukin-10 family of cytokines. Trends Immunol 2002;23:89-96.
11Kotenko SV. The family of IL-10-related cytokines and their receptors: Related, but to what extent? Cytokine Growth Factor Rev 2002;13:223-40.
12Volk H, Asadullah K, Gallagher G, Sabat R, Grutz G. IL-10 and its homologs: Important immune mediators and emerging immunotherapeutic targets. Trends Immunol 2001;22:414-7.
13Blumberg H, Conklin D, Xu WF, Grossmann A, Brender T, Carollo S, et al. Interleukin 20: Discovery, receptor identification, and role in epidermal function. Cell 2001;104:9-19.
14Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld JC. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J Immunol 2001;167:3545-9.
15Parrish-Novak J, Xu W, Brender T, Yao L, Jones C, West J, et al. Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions. J Biol Chem 2002;277:47517-23.
16Hsu YH, Chen WY, Chan CH, Wu CH, Sun ZJ, Chang MS. Anti-IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss. J Exp Med 2011;208:1849-61.
17Dougall WC. RANKL signaling in bone physiology and cancer. Curr Opin Support Palliat Care 2007;1:317-22.
18Kartsogiannis V, Zhou H, Horwood NJ, Thomas RJ, Hards DK, Quinn JM, et al. Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues. Bone 1999;25:525-34.
19Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Goto M, et al. A novel molecular mechanism modulating osteoclast differentiation and function. Bone 1999;25:109-13.
20Blair JM, Zhou H, Seibel MJ, Dunstan CR. Mechanisms of disease: Roles of OPG, RANKL and RANK in the pathophysiology of skeletal metastasis. Nat Clin Pract Oncol 2006;3:41-9.
21Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Lüthy R, et al. Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 1997;89:309-19.
22Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, Fujise N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998;139:1329-37.
23Jin J, Wang L, Wang XK, Lai PL, Huang MJ, Jin DD, et al. Risedronate inhibits bone marrow mesenchymal stem cell adipogenesis and switches RANKL/OPG ratio to impair osteoclast differentiation. J Surg Res 2013;180:e21-9.
24Goranova-Marinova V, Goranov S, Pavlov P, Tzvetkova T. Serum levels of OPG, RANKL and RANKL/OPG ratio in newly-diagnosed patients with multiple myeloma. Clinical correlations. Haematologica 2007;92:1000-1.
25Mountain CF. Revisions in the international system for staging lung cancer. Chest 1997;111:1710-7.
26Heuzé-Vourc'h N, Liu M, Dalwadi H, Baratelli FE, Zhu L, Goodglick L, et al. IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression. Biochem Biophys Res Commun 2005;333:470-5.
27Baird AM, Gray SG, O'Byrne KJ. IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF. Eur J Cancer 2011;47:1908-18.
28Peng X, Guo W, Ren T, Lou Z, Lu X, Zhang S, et al. Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer. PLoS One 2013;8:e58361.
29Boyde A, Maconnachie E, Reid SA, Delling G, Mundy GR. Scanning electron microscopy in bone pathology: Review of methods, potential and applications. Scan Electron Microsc 1986;(Pt 4):1537-54.
30Martin TJ. Manipulating the environment of cancer cells in bone: A novel therapeutic approach. J Clin Invest 2002;110:1399-401.
31Suva LJ, Griffin RJ, Makhoul I. Mechanisms of bone metastases of breast cancer. Endocr Relat Cancer 2009;16:703-13.
32Kong YY, Boyle WJ, Penninger JM. Osteoprotegerin ligand: A common link between osteoclastogenesis, lymph node formation and lymphocyte development. Immunol Cell Biol 1999;77:188-93.
33Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315-23.
34Fizazi K, Lipton A, Mariette X, Body JJ, Rahim Y, Gralow JR, et al. Randomized phase II trial of denosumab in patients with bone metastases from prostate cancer, breast cancer, or other neoplasms after intravenous bisphosphonates. J Clin Oncol 2009;27:1564-71.
35Mercatali L, Ricci M, Scarpi E, Serra P, Fabbri F, Ricci R, et al. RANK/RANK-L/OPG in patients with bone metastases treated with anticancer agents and zoledronic acid: A prospective study. Int J Mol Sci 2013;14:10683-93.