Molecular and cellular mechanisms for zoledronic acid-loaded magnesium-strontium alloys to inhibit giant cell tumors of bone

Abstract

Giant Cell Tumors of Bone (GCTB) are benign but aggressive and metastatic tumors. Surgical removal cannot eradicate GCTB due to the subsequent recurrence and osteolysis. Here we developed Zoledronic acid (ZA)-loaded magnesium-strontium (Mg-Sr) alloys that can inhibit GCTB and studied the molecular and cellular mechanisms of such inhibition. We first formed a calcium phosphate (CaP) coating on the Mg-1.5 wt%Sr implants by coprecipitation and then loaded ZA on the CaP coating. We examined the response of GCTB cells to the ZA-loaded alloys. At the cellular level, the alloys not only induced apoptosis and oxidative stress of GCTB cells, and suppressed their resultant pre-osteoclast recruitment, but also inhibited their migration. At the molecular level, the alloys could significantly activate the mitochondrial pathway and inhibit the NF-κB pathway in the GCTB cells. These collectively enable the ZA-loaded alloys to suppress GCTB cell growth and osteolysis, and thus improve our understanding of the materials-induced tumor inhibition. Our study shows that ZA-loaded alloys could be a potential implant in repairing the bone defects after tumor removal in GCTB therapy.

Statement of significance: In clinics, giant cell tumors of bone (GCTB) are removed by surgery. However, the resultant defects in bone still contain aggressive and metastatic GCTB cells that can recruit osteoclasts to damage bone, leading to new GCTB tumor growth and bone damage after tumor surgery. Hence, it is of high demand in developing a material that can not only fill the bone defects as an implant but also inhibit GCTB in the defect area as a therapeutic agent. More importantly, the molecular and cellular mechanism by which such a material inhibits GCTB growth has never been explored. To solve these two problems, we prepared a new biomaterial, the Mg-Sr alloys that were first coated with calcium phosphate and then loaded with a tumor-inhibiting molecule (Zoledronic acid, ZA). Then, by using a variety of molecular and cellular biological assays, we studied how the ZA-loaded alloys induced the death of GCTB cells (derived from patients) and inhibited their growth at the molecular and cellular level. At the cellular level, our results showed that ZA-loaded Mg-Sr alloys not only induced apoptosis and oxidative stress of GCTB cells, and suppressed their induced pre-osteoclast recruitment, but also inhibited their migration. At the molecular level, our data showed that ZA released from the ZA-loaded Mg-Sr alloys could significantly activate the mitochondrial pathway and inhibit the NF-κB pathway in the GCTB cells. Both mechanisms collectively induced GCTB cell death and inhibited GCTB cell growth. This work showed how a biomaterial inhibit tumor growth at the molecular and cellular level, increasing our understanding in the fundamental principle of materials-induced cancer therapy. This work will be interesting to readers in the fields of metallic materials, inorganic materials, biomaterials and cancer therapy.

Keywords: CaP coating; Giant cell tumors of bone; Magnesium-strontium alloys; Mechanisms; Zoledronic acid.

Fig. 1.

Chemical structure of zoledronic acid.

Fig. 2.

Coating of ZA-loaded CaP on Mg-based alloys that can serve as a potential implant to repair the defects after the GCTB tumor removal (top) and the possible signaling pathway (bottom) for the alloys to cause GCTB cell death and inhibit GCTB cell growth.

Fig. 3.

Characterization of the ZA-loaded CaP coated Mg-Sr alloys. (A) SEM micrographs performed on the CaP coating grown on the surface of Mg-Sr alloys (a) before and (b, c, d) after the treatment with 10^‒2, 10^‒3, and 10^‒4 M of ZA. (B) The corresponding elemental compositions of coatings by EDS.

Fig. 4.

Characterization of the ZA-loaded CaP coating on the Mg-Sr alloys. (A) Cross-section morphologies of coatings on the surface of Mg-Sr alloys (a) before and (b, c, d) after the treatment with 10^‒2, 10^‒3, and 10^‒4 M of ZA. (B) XRD spectra of different concentrations of ZA loaded CaP coating. ZA powder was set as reference. (C) FTIR-ATR spectra of ZA loaded CaP coating. ZA powder was set as reference.

Fig. 5.

Characterization of the corrosion rate and ZA release of the Mg-Sr alloys. (A) pH change during the immersion of the Mg-Sr alloys in Hank’s solution. (B, C) Weight loss of the Ca-P coatings before (B) and after (C) incorporating ZA during immersion in Hank’s solution for 14 days. (D) Cumulative amount of ZA released from Mg-Sr alloys after 1 to 7 days and determined by HPLC.

Fig. 6.

ZA-loaded Mg-Sr alloy suppressed GCTB cells growth and hemocompatibility. (A) Hemolysis tests of the Mg-Sr alloys. The insets are the positive control, negative control, ZA2-, ZA3-, ZA4-, and CaP coated Mg-1.5%Sr alloys from the left to the right, respectively. The results of hemolysis ratio (HR) indicated that all of our alloys were not hemolytic to human red blood cells. (B) Cell proliferation measured by colorimetric CCK-8 assay. Tumor cells cultured in the extracts medium of ZA2 coating and ZA3 coating alloys had significantly lower absorbance than others on day 1, 3 and 7. (C) Cytotoxic response measured by LDH assay. Tumor cells in ZA coating alloys released more LDH into the media than others. (D) DNA contents measured with Cyquant cell proliferation assay kit. The DNA level was significantly lower in tumor cells treated with ZA2 and ZA3 coated alloys for 24 h. (E) Cell viability conducted by Live/Dead staining kit. A large number of dead tumor cells with red fluorescence were visible in ZA2 coated group. All data represent the mean ± standard deviation of three independent experiments. ^#: p < 0.05 and ^##: p < 0.01 compared with ZA3 coating. *: p < 0.05 and ^**: p < 0.01 compared with ZA2 coating. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

Vinculin (green), actin (red), and cell nucleus (blue) fluorescence images of GCTB cells cultured on Mg-Sr alloys. Little focal adhesion and poor cytoskeleton organization indicated unfavorable adhering and spreading properties of GCTB cells exposed to specified ZA-loaded Mg-Sr alloys. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8.

ZA-loaded Mg-Sr alloys enhanced ROS generation in GCTB. (A-B) Fluorescence microscopy images (A) and flow cytometry (B) of tumor cells stained with DCFH-DA. (C) mean fluorescence intensity of the flow cytometer results. Tumor cells exposed to ZA-loaded Mg-Sr alloy exhibited a dose-dependent increase in oxidative stress. Data were collected from three independent experiments. ^#: p < 0.05 and ^##: p < 0.01 compared with ZA3 coating. *: p < 0.05 and ^**: p < 0.01 compared with ZA2 coating.

Fig. 9.

ZA-loaded Mg-Sr alloys induced mitochondria-dependent apoptosis of GCTB. (A) ZA2- and ZA3-loaded Mg-Sr alloys induced tumor cells apoptosis and necrosis, which was verified by Annexin V-FITC/7-AAD double staining and flow cytometry. Quantification of apoptotic cells (lower right) was shown. (B) ZA2- and ZA3-loaded Mg-Sr alloys decreased mitochondrial membrane potential (Dwm) and induced mitochondrial dysfunction by JC-1 staining and flow cytometry. (C) ZA2- and ZA3-loaded Mg-Sr alloys increased the expression of Bax, p53 proteins, the cleavage of caspase-3, and the release of cytochrome c. All data represent the mean ± standard deviation of three independent experiments. ^#: p < 0.05 and ^##: p < 0.01 compared with ZA3 coating. *: p < 0.05 and ^**: p < 0.01 compared with ZA2 coating.

Fig. 10.

ZA-loaded Mg-Sr alloy reduced migration of GCTB. (A) Migration images of tumor cells mediated by ZA-loaded Mg-Sr alloy at 0 and 24 h. The scale bar represents 100 lm. (B) The migration ratio which represents the ratio of migration distance to the originally wounded distance. (C) mRNA expression level of MMP-9, MMP-13, and E-cadherin genes in GCTB cell lysates. These data indicated that ZA-loaded Mg-Sr alloys could arrest tumor cell migration by down-regulating the mRNA expression of metastasis-related genes. All data represent the mean ± standard deviation of three independent experiments. ^#: p < 0.05 and ^##: p < 0.01 compared with ZA3 coating. *: p < 0.05 and ^**: p < 0.01 compared with ZA2 coating.

Fig. 11.

Specific inhibitory effect of ZA-loaded Mg-Sr alloys on GCTB-induced pre-osteoclasts recruitment. (A) Schematic representation of the experiment. (B) Migrated pre-osteoclasts were stained, photographed, and the percent of invaded cells was calculated. The migration of pre-osteoclasts was specifically abrogated by ZA-loaded Mg-Sr alloys but ignored in CaP coating and blank control group. The scale bar represents 200 µm. (C) ZA-loaded Mg-Sr alloys suppressed osteoclastgenesis-related gene expression dose dependently. All data represent the mean ± standard deviation of three independent experiments. ^#: p < 0.05 and ^##: p < 0.01 compared with ZA3 coating. *: p < 0.05 and ^**: p < 0.01 compared with ZA2 coating.

Fig. 12.

The activation of NF-кB signaling was prevented by the pretreatment of ZA-loaded Mg-Sr alloys in GCTB cells. (A) Tumor Cells were incubated with p65 antibody and Alexa 488-conjugated anti-rabbit IgG, and nuclei were stained with DAPI. The nuclear translocation of p65 (indicated by cyan fluorescence and red arrow) was dose dependently abrogated by ZA-loaded Mg alloys. (B-C) Western blotting assay (B) of p65, p-p65 and IкB-α proteins and the corresponding histogram (C) of protein expression level. These data showed that ZA2- and ZA3-loaded Mg alloys significantly inhibited the degradation of IкB-α and the phosphorylation of p65. The bands of β-actin protein were loading controls. ^#: p < 0.05 and ^##: p < 0.01 compared with CaP coating. *: p < 0.05 and ^**: p < 0.01 compared with Blank control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)