doi: 10.1111/jop.12205
J Oral Pathol Med (2015) 44: 103–108 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Zoledronic acid and irradiation in oral squamous cell carcinoma Pia Lopez Jornet, Sanchez Carrillo Susana, Tudela Mulero Rosario, Pons-Fuster Alvaro Department of Oral Medicine, University of Murcia, Murcia, Spain
OBJECTIVE: This in vitro study evaluated cytotoxicity and cell migration effects of zoledronic acid and irradiation upon oral squamous cell carcinoma. MATERIALS AND METHODS: Zoledronic acid was administrated at doses of 10, 25, 50, and 100 lM to PE/ CA-PJ15 oral squamous cell carcinoma cultures, irradiated with different doses (0, 5, 15, and 30 Gy), followed by evaluation of the effects on cell viability. Cell migration capacity was studied after 24- and 72-h incubation. RESULTS: At 24 h, the 100 lM concentration of zoledronic acid combined with 15 Gy irradiation caused the greatest decrease in cell viability. At 72 h, statistically significant decreases in cell viability were found with all concentrations of zoledronic acid with or without irradiation: 0 Gy (P < 0.001), 5 Gy (P < 0.001), 15 Gy (P < 0.001), and 30 Gy (P < 0.001). 50 lM and 100 lM doses of zoledronic acid combined with 5 Gy irradiation yielded the greatest decrease in cell migration capacity. CONCLUSIONS: Zoledronic acid increases cytotoxic activity in the PE/CA-PJ15 cell line and reduces cell migration capacity. These findings suggest that combination therapy using biphosphates and radiation may offer a promising therapy. J Oral Pathol Med (2015) 44: 103–108 Keywords: in vitro cell line; irradiation; squamous cell carcinoma; zoledronic acid
Introduction Oral squamous cell carcinoma (OSCC) is a growing global public health problem. Standard therapeutic strategies have failed to make significant improvements to survival rates, which have remained around 50% over the past three decades (1–6). Cancers of the oral cavity and oropharynx Correspondence: Pia Lopez Jornet, PhD, MD, DDS, Clınica Odontologica Universitaria, Unidad Docente de Medicina Bucal, Hospital Morales Meseguer (2 planta), Avda. Marques de los Velez s/n, 30008 – Murcia, Spain. Tel: +34868888588, Fax: +34868888576, E-mail: [email protected] Accepted for publication April 21, 2014
represent approximately 3% of all malignancies in men and 2% in women. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, and it has been found that oral cancer invasion of the mandible, especially OSCC, has a poor prognosis (5–7). Rao et al. (8) state that mandibular invasion by oral carcinoma presents a frequency of between 12% and 56%. Bone destruction in OSCC causes serious problems in terms of both function and prognosis (6), and for this reason, there is a pressing need for new treatments against bone invasion by oral cancers. The standard treatment for localized OSCC is surgical excision of the primary tumor. Treatment options for inoperable OSCC cases are palliative and include chemotherapy or radiotherapy or the fractional combination of both (2–10). Radiotherapy (RT) is an indispensable therapy for controlling surgically unresectable metastases, particularly in bone (10). Bone is one of the most common metastasis sites, and the bone lesions are predominantly osteolitic and cause a considerable number of skeletal-related events (SRE), including pathological fracture and compression of the spinal cord that significantly deplete patient quality of life (2, 10). RT for bone metastasis often relieves pain, but rarely delivers results in objective radiological responses or in reducing the risk of SRE. Zoledronic acid (ZOL; 2-(imidazol-1-yl)-hydroxy-ethylidene-1,1-bisphosphonic acid), is a powerful inhibitor of osteoclast activity and has been widely used to treat bone metastasis (11–19). It is a potent antiresorptive agent that reduces the frequency and severity of malignant skeletal complications in humans. As an antiresorptive agent, ZOL exerts its biologic effects on osteoclasts by inhibiting the mevalonate pathway, which is necessary for protein prenylation. Algur et al. (20) studied the effect on the synergistic cytotoxicity of a combination of zoledronic acid (ZOL) and radiation in prostate and myeloma cell lines in vitro. It was found that combining ZOL with radiation showed a more prominent antiproliferative effect than either of the two treatments alone. This novel effect of ZOL might be described as radiosensitization, although the article does not explore the possible mechanisms of this action (20). Recently, biphosphates (BP) have displayed direct antitumoral activity, both in vitro and in vivo, for a variety
Zoledronic acid and irradiation in oral cancer Lopez Jornet et al.
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of malignant tumors but their effect on oral cancers are not so manifest. The aim of this study was to evaluate the effect of zoledronic acid in combination with irradiation on OSCC.
Material and methods Cell line The study examined PE/CA-PJ15 human oral squamous carcinoma cell lines (European Collection of Cell Cultures) cultured in Iscove’s modified Dulbecco’s modified Eagle medium (DMEM) (IMDM) with 10% fetal calf serum (FCS), 1% penicillin, and 1% streptomycin (full medium) at 37°C, in an atmosphere of 95% oxygen and 5% CO2. The medium (IMDM) was supplemented by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO). Drug preparation Zoledronic acid (Intatrade Chemical GmbH, Muldestausee, Germany) was dissolved in 0.5% DMSO, with 1 mg/ml of zoledronic acid used a stock solution. The DMSO concentration in the assay did not exceed 0.1%. Irradiation Cell irradiation was performed using a linear accelerator (Yxlon Smart, Krautkr€amer-Forster Espanola S.A, Madrid, Spain). The machine was calibrated for the field size of interest using special small ionization chambers and thermoluminescence dosimetry. Single irradiation doses of 0, 5, 15, and 30 Gy were administrated. Cells were irradiated in 96-microwell plates. Cell viability test (MTT) Cell viability was quantified using the technique described by Carmichael et al. (21, 22) adapted to the study’s specific culture conditions. The cells were cultured at a density of 5 9 103 cells/well in 96-microwell plates, after which
Figure 1 Cell migration measurement. J Oral Pathol Med
zoledronic acid was added at different concentrations (10, 25, 50, and 100 lM), 30 min after plate irradiation (0, 5, 15, and 30 Gy) (18–20). At different time-points after the start of treatment (24 and 72 h), the medium was eliminated and the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemistry, S.A., Madrid, Spain) (1 mg/ml) for 4 h, after which the non-metabolized MTT was discarded and 100 ll of DMSO was added to each well. Absorbance in each well was measured with an enzyme-linked immunosorbent assay (ELISA), using a Multiskan MCC/340P plate spectrophotometer at a reading wavelength of 570 nm and a reference wavelength of 690 nm. The assay was performed in triplicate. Migration (scratch wound healing) Scratch wounds were generated in confluent monolayers of cells using a sterile 200-ll pipette tip (23). After washing away suspended cells with phosphate buffered saline (PBS), the culture medium was changed and added at different concentrations (10, 25, 50, and 100 lM), 30 min after plate irradiation (0, 5, 15, and 30 Gy). Migration into the wound space was photographed using an inverted microscope equipped with a digital camera at the time of the initial wound and at time intervals of 4 and 8 h post-wounding. The relative distances between edges of the injured monolayer were obtained via pixel counts at a minimum of 10 sites per wound, using MIP-4â image software (CID, Barcelona, Spain) and applying the formula: migration distance = initial distance of free-of-cells space distance at 4 or 8 h of free-of-cells space (24). This assay was performed in triplicate (Fig. 1). Statistical analysis Data were analyzed using the SPSS version 12.0 statistical package (SPSSâ Inc., Chicago, IL, USA). A descriptive
Zoledronic acid and irradiation in oral cancer Lopez Jornet et al.
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Table 1 Effects of zoledronic acid and irradiation on PE/CA-PJ15 cell viability (One-factor ANOVA at 24 h) 0 Gy zo. 10 lM zo. 25 lM zo. 50 lM zo 100 lM P-value
105.23 102.17 89.56 100.46
J Oral Pathol Med (2015) 44: 103–108 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Zoledronic acid and irradiation in oral squamous cell carcinoma Pia Lopez Jornet, Sanchez Carrillo Susana, Tudela Mulero Rosario, Pons-Fuster Alvaro Department of Oral Medicine, University of Murcia, Murcia, Spain
OBJECTIVE: This in vitro study evaluated cytotoxicity and cell migration effects of zoledronic acid and irradiation upon oral squamous cell carcinoma. MATERIALS AND METHODS: Zoledronic acid was administrated at doses of 10, 25, 50, and 100 lM to PE/ CA-PJ15 oral squamous cell carcinoma cultures, irradiated with different doses (0, 5, 15, and 30 Gy), followed by evaluation of the effects on cell viability. Cell migration capacity was studied after 24- and 72-h incubation. RESULTS: At 24 h, the 100 lM concentration of zoledronic acid combined with 15 Gy irradiation caused the greatest decrease in cell viability. At 72 h, statistically significant decreases in cell viability were found with all concentrations of zoledronic acid with or without irradiation: 0 Gy (P < 0.001), 5 Gy (P < 0.001), 15 Gy (P < 0.001), and 30 Gy (P < 0.001). 50 lM and 100 lM doses of zoledronic acid combined with 5 Gy irradiation yielded the greatest decrease in cell migration capacity. CONCLUSIONS: Zoledronic acid increases cytotoxic activity in the PE/CA-PJ15 cell line and reduces cell migration capacity. These findings suggest that combination therapy using biphosphates and radiation may offer a promising therapy. J Oral Pathol Med (2015) 44: 103–108 Keywords: in vitro cell line; irradiation; squamous cell carcinoma; zoledronic acid
Introduction Oral squamous cell carcinoma (OSCC) is a growing global public health problem. Standard therapeutic strategies have failed to make significant improvements to survival rates, which have remained around 50% over the past three decades (1–6). Cancers of the oral cavity and oropharynx Correspondence: Pia Lopez Jornet, PhD, MD, DDS, Clınica Odontologica Universitaria, Unidad Docente de Medicina Bucal, Hospital Morales Meseguer (2 planta), Avda. Marques de los Velez s/n, 30008 – Murcia, Spain. Tel: +34868888588, Fax: +34868888576, E-mail: [email protected] Accepted for publication April 21, 2014
represent approximately 3% of all malignancies in men and 2% in women. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, and it has been found that oral cancer invasion of the mandible, especially OSCC, has a poor prognosis (5–7). Rao et al. (8) state that mandibular invasion by oral carcinoma presents a frequency of between 12% and 56%. Bone destruction in OSCC causes serious problems in terms of both function and prognosis (6), and for this reason, there is a pressing need for new treatments against bone invasion by oral cancers. The standard treatment for localized OSCC is surgical excision of the primary tumor. Treatment options for inoperable OSCC cases are palliative and include chemotherapy or radiotherapy or the fractional combination of both (2–10). Radiotherapy (RT) is an indispensable therapy for controlling surgically unresectable metastases, particularly in bone (10). Bone is one of the most common metastasis sites, and the bone lesions are predominantly osteolitic and cause a considerable number of skeletal-related events (SRE), including pathological fracture and compression of the spinal cord that significantly deplete patient quality of life (2, 10). RT for bone metastasis often relieves pain, but rarely delivers results in objective radiological responses or in reducing the risk of SRE. Zoledronic acid (ZOL; 2-(imidazol-1-yl)-hydroxy-ethylidene-1,1-bisphosphonic acid), is a powerful inhibitor of osteoclast activity and has been widely used to treat bone metastasis (11–19). It is a potent antiresorptive agent that reduces the frequency and severity of malignant skeletal complications in humans. As an antiresorptive agent, ZOL exerts its biologic effects on osteoclasts by inhibiting the mevalonate pathway, which is necessary for protein prenylation. Algur et al. (20) studied the effect on the synergistic cytotoxicity of a combination of zoledronic acid (ZOL) and radiation in prostate and myeloma cell lines in vitro. It was found that combining ZOL with radiation showed a more prominent antiproliferative effect than either of the two treatments alone. This novel effect of ZOL might be described as radiosensitization, although the article does not explore the possible mechanisms of this action (20). Recently, biphosphates (BP) have displayed direct antitumoral activity, both in vitro and in vivo, for a variety
Zoledronic acid and irradiation in oral cancer Lopez Jornet et al.
104
of malignant tumors but their effect on oral cancers are not so manifest. The aim of this study was to evaluate the effect of zoledronic acid in combination with irradiation on OSCC.
Material and methods Cell line The study examined PE/CA-PJ15 human oral squamous carcinoma cell lines (European Collection of Cell Cultures) cultured in Iscove’s modified Dulbecco’s modified Eagle medium (DMEM) (IMDM) with 10% fetal calf serum (FCS), 1% penicillin, and 1% streptomycin (full medium) at 37°C, in an atmosphere of 95% oxygen and 5% CO2. The medium (IMDM) was supplemented by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO). Drug preparation Zoledronic acid (Intatrade Chemical GmbH, Muldestausee, Germany) was dissolved in 0.5% DMSO, with 1 mg/ml of zoledronic acid used a stock solution. The DMSO concentration in the assay did not exceed 0.1%. Irradiation Cell irradiation was performed using a linear accelerator (Yxlon Smart, Krautkr€amer-Forster Espanola S.A, Madrid, Spain). The machine was calibrated for the field size of interest using special small ionization chambers and thermoluminescence dosimetry. Single irradiation doses of 0, 5, 15, and 30 Gy were administrated. Cells were irradiated in 96-microwell plates. Cell viability test (MTT) Cell viability was quantified using the technique described by Carmichael et al. (21, 22) adapted to the study’s specific culture conditions. The cells were cultured at a density of 5 9 103 cells/well in 96-microwell plates, after which
Figure 1 Cell migration measurement. J Oral Pathol Med
zoledronic acid was added at different concentrations (10, 25, 50, and 100 lM), 30 min after plate irradiation (0, 5, 15, and 30 Gy) (18–20). At different time-points after the start of treatment (24 and 72 h), the medium was eliminated and the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemistry, S.A., Madrid, Spain) (1 mg/ml) for 4 h, after which the non-metabolized MTT was discarded and 100 ll of DMSO was added to each well. Absorbance in each well was measured with an enzyme-linked immunosorbent assay (ELISA), using a Multiskan MCC/340P plate spectrophotometer at a reading wavelength of 570 nm and a reference wavelength of 690 nm. The assay was performed in triplicate. Migration (scratch wound healing) Scratch wounds were generated in confluent monolayers of cells using a sterile 200-ll pipette tip (23). After washing away suspended cells with phosphate buffered saline (PBS), the culture medium was changed and added at different concentrations (10, 25, 50, and 100 lM), 30 min after plate irradiation (0, 5, 15, and 30 Gy). Migration into the wound space was photographed using an inverted microscope equipped with a digital camera at the time of the initial wound and at time intervals of 4 and 8 h post-wounding. The relative distances between edges of the injured monolayer were obtained via pixel counts at a minimum of 10 sites per wound, using MIP-4â image software (CID, Barcelona, Spain) and applying the formula: migration distance = initial distance of free-of-cells space distance at 4 or 8 h of free-of-cells space (24). This assay was performed in triplicate (Fig. 1). Statistical analysis Data were analyzed using the SPSS version 12.0 statistical package (SPSSâ Inc., Chicago, IL, USA). A descriptive
Zoledronic acid and irradiation in oral cancer Lopez Jornet et al.
105
Table 1 Effects of zoledronic acid and irradiation on PE/CA-PJ15 cell viability (One-factor ANOVA at 24 h) 0 Gy zo. 10 lM zo. 25 lM zo. 50 lM zo 100 lM P-value
105.23 102.17 89.56 100.46
