Bisphosphonates (BPs) are the keystone to treat bone disorders. Despite the great benefits of BPs, medication-related osteonecrosis of the jaw (MRONJ) arouse as a potential side effect. Nitrogen-containing BPs (N-BPs) as zoledronate (ZA) act by the inhibition of specific enzymes of the mevalonate pathway resulting in altering protein prenylation which is required for the posttranslational maturation of the small GTP-binding proteins. Geranylgeraniol (GGOH) is an intermediate product in the mevalonate pathway having positive effects on different cell types treated with BPs by salvaging protein prenylation improving cell viability and proliferation in tissue regeneration, thus overcoming N-BP-induced apoptosis. Here, the effect of different concentrations of zoledronate (ZA) on the bone cells has been investigated by cell viability assay, live/dead staining, and western blot to understand if GGOH was able to rescue bone cells and levels of statistical significance were indicated at
Bisphosphonates (BPs) are considered the keystone to treat bone disorders as osteoporosis, osteogenesis imperfecta, and Paget’s disease as well as bone metastases from various malignancies as multiple myeloma or breast/prostate cancer. Despite the great benefits of BPs, medication-related osteonecrosis of the jaw (MRONJ) arouse as a potential side effect of two pharmacological agents: antiresorptives (including bisphosphonates (BPs) and receptor activator of nuclear factor kappa-B ligand inhibitors) and antiangiogenics. MRONJ pathogenesis has been widely investigated, yet not fully understood. Lately, various factors have been formulated to discuss the possible mechanism as interaction between bone turnover, impairment of angiogenesis, infection, local trauma, oral mucosal toxicity, or immunomodulation [
BPs are stable analogues of natural inorganic pyrophosphates [
At the molecular level, ZA inhibits specific enzymes of the MVP resulting in the loss of isoprenoid intermediates altering protein prenylation which is required for the posttranslational maturation of the small GTP-binding proteins which are divided into at least five families, including Ras, Rho, Rab, Arf, and Ran [
Isoprenoid compounds as farnesol (FOH) and geranylgeraniol (GGOH) are intermediate products in the MVP essential for cell proliferation [
Thus, the aims of this study were to (1) investigate the effect of different concentrations of ZA on the bone cells and (2) understand if isoprenoids as GGOH was able to rescue bone cells which could be proposed as a future local therapy for the treatment of MRONJ.
Zoledronate (ZA) obtained as a gift from Chemos (Regenstauf, Germany) was chosen due to its high relative potency increasing the probability of osteonecrosis. A stock solution of 20 mM ZA was prepared by dissolving the powder in physiological saline (0.9% NaCl), sterile filtered before use, and stored at -20°C. The drug was diluted in appropriate culture media to the given concentration to be used in the experiments. The dose range was chosen to be 0.1, 25, and 100
Geranylgeraniol (GGOH) was purchased from Sigma (G3278-100MG; Munich, Germany). A stock solution of 5 mM was prepared by dissolving GGOH in pure ethanol, sterile-filtered, and stored at -20°C. Different concentrations of GGOH (10, 20, 40, and 80
Cells were seeded at the required density in the cell culture plates and incubated before treatment for 24 hours. For use in experiments, different concentrations of GGOH and ZA were diluted in the culture media and used throughout the whole experiment. The drugs were administered individually or simultaneously in combination for 7 days. Cells cultured without ZA/GGOH drugs served as the negative control while cells cultured with ZA or GGOH served as positive controls.
To determine whether ZA, GGOH, or ZA/GGOH affected cell growth in culture, cellular proliferation experiments were performed via WST-1 assay kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions on hOBs and hOCs. In brief, cells were seeded at the required density in complete culture medium for 24 h. Next day, the cells were treated with ZA, GGOH, or ZA/GGOH for 7 days. For measurements, the medium was replaced by fresh medium supplemented with WST-1 reagent added directly into the incubation media (diluted 1 : 10 with culture media). After 4 hours of incubating the cells at 37°C in 5% CO2 to form purple formazan crystals, the optical absorbance of the supernatants was determined at 450 nm against a reference wavelength of 620 nm using Multiskan FC microplate reader (Thermo Scientific, Massachusetts, USA). All WST-1 experiments were performed in triplicate and repeated at least 3 times to obtain the mean values. Cytotoxicity of the compounds was expressed as percentage cell viability compared to control. The absorbance of cells exposed to normal culture media (negative controls) was considered to be 100% cell viability. Positive controls were reading obtained from cells treated with ZA or GGOH.
Live/dead staining was used to analyse qualitative cell viability by staining the cells with Calcein-AM/EthD-III (Live/Dead fluorescent Cell Staining Kit II, PromoKine, PK-CA707-30002, PromoCell GmbH, Heidelberg, Germany) according to the manufacturer’s protocol. In brief, cells were cultured for 24 h to allow attachment then treated with ZA or ZA/GGOH for 7 days. The cells were washed twice with PBS and sufficient volume of Calcein-AM/EthD-III staining solution was added to cover the cell monolayer. The cells were incubated for 30-45 minutes at room temperature protected from light then observed under the fluorescence microscope (AxioObserver Z1; Zeiss, Oberkochen, Germany). The experiment was repeated three times from two different donors.
Osteoclasts (OCs) were treated with different concentrations of zoledronate (ZA) as well as GGOH for 7 days and incubated at 37°C in 5% CO2. To evaluate osteoclast formation in all groups, tartrate-resistant acid phosphatase (TRAP) staining was performed using Acid Phosphatase, Leukocyte (TRAP) Kit (Cat no.387A-1KT, Sigma Aldrich, Munich, Germany) according to the manufacturer’s instructions.
In brief, OCs were washed with phosphate buffered solution (PBS), fixed by combining 3 parts of citrate solution, 8 parts of acetone, and 1 part of 37% formaldehyde for 30 seconds at 25°C and fixed cells were washed three times with PBS. During this time, the commercially available TRAP stain was prepared by prewarming to 37°C and added to each well of the plate to be stained. The plates were placed in the water bath at 37°C for 1 h protected from light. Later, the TRAP stain was aspirated and the wells were washed 3 times with prewarmed deionized water. The wells were counterstained with Gill’s Hematoxylin for 1–2 min. The wells were washed with alkaline tap water until an adequate colour intensity of the stain is achieved, typically when nuclei appear blue. TRAP-positive multinucleated cells (>3 nuclei) were observed and counted under an Axiovert 40 CFL microscope (Zeiss, Oberkochen, Germany). The positive cells developed red colour of different intensity.
For protein analysis, hOBs and hOCs were seeded at the required density and were let to adhere in the 6-well plates for 24 h. Next day, the cells were treated with different concentrations of ZA or ZA/GGOH for 7 days. After treatment, the cells were washed with ice-cold PBS and lysed in 500
The Micro BCA Protein Assay Kit (Cat no.23235, Thermo Scientific, Massachusetts, USA) was used according to the manufacturer’s instructions. Bovine serum albumin (BSA, 2 mg/mL) was used to create the standard curve. Absorbance was measured at 562 nm on Multiskan FC microplate reader (Thermo Scientific, Massachusetts, USA). The amount of protein in each well was calculated by plotting against a standard curve.
Ten microgram (10
All experiments were performed in triplicates from two different cell lots and the results were presented as the mean ± standard deviation. The data were analysed by two-way analysis of variance (ANOVA) with Bonferroni posttests for absolute data between the positive control and treated cells and levels of statistical significance were indicated at
The effect of GGOH on rescuing cells treated with ZA was studied and the results of WST-1 assay are presented in Figure
WST-1 activity of bone cells (OBs and OCs). Effect of ZA (0.1, 25, and 100
The viability of the cells was considered to be 100% in all the control groups after 7 days in the osteoblasts and osteoclasts.
In human osteoblasts, the treatment of the cells with low to moderate concentration of GGOH alone (10, 20, and 40
In human osteoclasts, the treatment of the cells with low to moderate concentration of GGOH alone (10, 20, and 40
Live/dead staining was performed after 7 days in order to test whether the apoptosis of bone cells by ZA was reversed by the mevalonate pathway metabolite (GGOH). The fluorescent microscopic analysis of live/dead cells is shown in Figure
Live/dead staining of bone cells (OBs and OCs). Fluorescence microscopy of live and dead cells treated at different concentrations of ZA (0.1, 25, and 100
As shown in Figures
The simultaneous addition of lower concentrations of GGOH had reversed the negative effect of ZA on bone cells. The addition of 10 and 20
The OCs were treated with ZA alone or a combination of ZA and GGOH for 7 days.
As demonstrated in Figure
TRAP staining assay of osteoclast. Osteoclasts were cultured at different concentrations of ZA (0.1, 25, and 100
In contrast, multinucleated osteoclasts expressing TRAP were detected in ZA cultures combined with low concentration of GGOH (10
Immunoblot analysis was performed in order to confirm the impairment of geranylgeranylation by ZA in bone cells and to understand the mechanism of GGOH rescue of cellular viability and metabolic activity in proliferating cells. The prenylation status of Rap1A/B, which is a member of the GTPase superfamily of proteins known to modulate cellular activity, is shown in Figure
Western blot with anti-Rap1A/B antibody in bone cells (OBs and OCs). Effect of ZA (0.1, 25, and 100
In hOBs, Rap1A/B was detected in the negative controls, positive controls of ZA (0.1 and 25
The aim of this study was to reveal the great role of GGOH as mevalonic acid metabolite on reversing the profound cytotoxic effect of zoledronate (N-BP) on the function of bone cells suggesting that it could be a future local therapy for the treatment of medication-related osteonecrosis of the jaw (MRONJ). Moreover, we had shown that ZA induced cessation of mevalonate pathway and stopped protein prenylation and consequently induced cell death.
MRONJ is a well-known serious complication of antiresorptive therapy with denosumab or N-BPs [
Zoledronic acid (ZA) is the strongest inhibitor of farnesyl pyrophosphate synthase compared to other N-BPs [
In the present study, it was observed that high concentrations of ZA resulted in significant decrease in the metabolic activity and cell viability of bone cells. However, low concentrations of ZA appeared to have no effect on metabolic activity or cell viability. This was consistent with previous studies [
Geranylgeraniol (GGOH) is an isoprenoid playing different roles in various physiological processes in animals and plants. It was selected according to its reliance on mevalonate input where it has the ability not only to improve the side effects of bisphosphonate therapy by regulating the mevalonate pathway but also acts as anti-inflammatory, antitumorigenic, and neuroprotective [
The management of MRONJ is controversial with no current gold standard treatment. Several local treatment options have been described starting with local application of antibiotics, surgical debridement or hyperbaric oxygen (HBO) therapy, and using growth and differentiation factors [
Several studies have revealed the effects of increased viability and migration that GGOH caused in different cells previously treated with ZA. These cells included oral fibroblasts [
Although GGOH reverses the effects of BPs in the mevalonate pathway, it acts as a double weapon in the treatment of MRONJ in which the systemic administration would lead to faster and easier transport of GGOH to the cells, especially to the basal mucosal layers. However, systemic administration of GGOH may be problematic as it decreases the pharmacological action of the BPs with special concern to malignant patients facing the risk of the spread of the malignancy and high morbidity rate. To overcome these complications, it would be of great benefit to apply GGOH locally [
Protein prenylation is mediated by three protein prenyltransferase enzymes: farnesyltransferase, for farnesylation of proteins such as Ras and nuclear lamins; geranylgeranyltransferase type I, for geranylgeranylation of proteins such as Rho, Rac, and Rap1 (Ras-associated protein); and geranylgeranyltransferase type II, for geranylgeranylation of Rab [
Ras-associated protein (Rap) is the prototype for a large superfamily of GTPases belonging to the Ras superfamily that regulates multiple cellular processes and includes 5 members, Rap1A, Rap1B, Rap2A, Rap2B , and Rap2C, which are grouped into 2 subfamilies, Rap1 and Rap2, based on their sequence homology. Rap1A and Rap1B were shown to be essential for integrin activation and cell-cell adhesion of various cell types, such as leukocytes, platelets, fibroblasts, and progenitor cells [
In our experiments, we have examined the expression of Rap1A proteins not only in osteoclasts but also in osteoblasts and found that the levels of Rap1A expression were also changed according to the conditions of our experiments in osteoblast cultures indicating that Rap1A plays a role in these cell lines. Rap1A was used as a convenient biomarker for the impairment of posttranslational modification by geranylgeranylation of proteins in tissues. As determined by the characterization of Rap1A, ZA inhibited the mevalonate pathway and consequently inhibited the protein prenylation in bone cells in a dose-dependent manner. The combination of GGOH with ZA has been reported to reverse the inhibitory effects of ZA in some cell lines [
A new finding in our experiments was that ZA not only has shown potential for synergistic interaction with GGOH at very high concentrations of both drugs inducing apoptosis and cell death but also that GGOH alone is cytotoxic at very high concentrations. The protective role of GGOH is not generalized to all cell types. In some studies, GGOH did not protect rat hepatocytes from apoptosis and was toxic [
Although GGOH had a detectable effect on cell viability fully antagonizing the inhibition of cell growth induced by ZA, higher concentration of GGOH had enhanced the effects of ZA. GGOH had rescued the bone cells by acting on the protein prenylation. Thus, GGOH could be applied as a future local therapy to MRONJ. However, more experiments are required to be performed on GGOH in in vivo animal models to ensure its positive effects.
The data used to support the findings of this study are available from the corresponding author upon request.
This project is part of Saleh A. Entekhabi’s doctoral thesis.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This work was supported by the AO Foundation (grant number AOCMF-64-21O).