Albendazole sensitizes cancer cells to ionizing radiation
© Patel et al; licensee BioMed Central Ltd. 2011
Received: 19 September 2011
Accepted: 17 November 2011
Published: 17 November 2011
Brain metastases afflict approximately half of patients with metastatic melanoma (MM) and small cell lung cancer (SCLC) and represent the direct cause of death in 60 to 70% of those affected. Standard of care remains ineffective in both types of cancer with the challenge of overcoming the blood brain barrier (BBB) exacerbating the clinical problem. Our purpose is to determine and characterize the potential of albendazole (ABZ) as a cytotoxic and radiosensitizing agent against MM and SCLC cells.
Here, ABZ's mechanism of action as a DNA damaging and microtubule disrupting agent is assessed through analysis of histone H2AX phosphorylation and cell cyle progression. The cytotoxicity of ABZ alone and in combination with radiation therapy is determined though clonogenic cell survival assays in a panel of MM and SCLC cell lines. We further establish ABZ's ability to act synergistically as a radio-sensitizer through combination index calculations and apoptotic measurements of poly (ADP-ribose) polymerase (PARP) cleavage.
ABZ induces DNA damage as measured by increased H2AX phosphorylation. ABZ inhibits the growth of MM and SCLC at clinically achievable plasma concentrations. At these concentrations, ABZ arrests MM and SCLC cells in the G2/M phase of the cell cycle after 12 hours of treatment. Exploiting the notion that cells in the G2/M phase are the most sensitive to radiation therapy, we show that treatment of MM and SCLC cells treated with ABZ renders them more sensitive to radiation in a synergistic fashion. Additionally, MM and SCLC cells co-treated with ABZ and radiation exhibit increased apoptosis at 72 hours.
Our study suggests that the orally available antihelminthic ABZ acts as a potent radiosensitizer in MM and SCLC cell lines. Further evaluation of ABZ in combination with radiation as a potential treatment for MM and SCLC brain metastases is warranted.
Melanoma and small cell lung cancer (SCLC) have a high propensity for metastasizing to the brain, accounting for the most common and third most common cause of brain metastasis, respectively . With no currently FDA-approved agents that cross the blood brain barrier (BBB) to target SCLC, conventional therapy for brain metastasis is limited to whole brain radiation therapy. Even though SCLC is radiosensitive, patients receiving prophylactic cranial irradiation after a complete response to chemotherapy still have a 33% 3-year brain metastasis rate and only a 21% 3 year-overall survival rate . While the standard of care for melanoma brain metastases is temozolomide (TMZ) and whole brain radiation therapy (WBRT), this combination therapy does not improve overall survival with this radioresistant tumor [3, 4]. The poor prognoses of SCLC and melanoma brain metastases highlight the need for an effective radiosensitizer that can cross the BBB and offer more effective systemic therapy.
Recently, we have shown that mebendazole (MBZ), a marketed benzimidazole (BZ) antihelminthic, is an effective anti-melanoma agent given its ability to disrupt microtubule stability at clinically achievable concentrations, thereby inducing apoptosis . Albendazole (ABZ) is another marketed antihelminthic that is structurally related to MBZ. ABZ, however, has the unique advantage of crossing the BBB, a characteristic that is used to treat parasitic infections of the central nervous system and may be harnessed to potentially target brain metastasis . Although ABZ is structurally similar to MBZ, our data suggests that ABZ also possesses DNA damaging capabilities.
With both metastatic melanoma (MM) and SCLC having a high propensity of brain metastases, we hypothesized that ABZ would be a potent chemotherapeutic and radiosensitizing agent for melanoma and SCLC brain metastases. We show here that at clinically achievable plasma concentrations, ABZ decreases proliferation, which correlates with arrest of the cancer cells in the G2/M phase of the cell cycle. We establish that ABZ radiosensitizes MM and SCLC and that this effect is synergistic. Radiosensitization by ABZ is characterized by enhanced radiation induced apoptosis.
Materials and methods
A375 and A2058 metastatic melanoma cells lines were obtained from ATCC (Manassas, VA). H153 and H446 SCLC lines were generously provided by Drs. J. Donnington and H. Sauthoff (New York University School of Medicine, New York, NY), respectively. All cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5 units/mL penicillin, and 5% glutamate. All cells were incubated at a humidified atmosphere of 5% CO2 at 37°C. ABZ was purchased from Sigma (St Louis, MO).
Cell Proliferation Assay
All cells were seeded in 96-well plates at a density of 5000 cells per well. After overnight incubation, cells were treated with ABZ at concentrations ranging from 100 nM to 1000 nM. After 12 or 72 hr incubation periods, proliferation was assessed using a tetrazolium dye reduction assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI) . Absorbance was recorded on a microplate reader at 495 nm. Cellular proliferation was expressed as a percentage with vehicle-treated cells set at 100%. Each assay was performed in triplicate with mean values reported.
Cell Cycle Analysis
A2058 and H153 cells were seeded at 50,000 cells/ml in a 10 cm2 tissue culture dish and incubated overnight. Cells were treated with ABZ at 100 nM and 500 nM. After 6, 12, 18, and 24 hrs, cells and media were collected, washed with 1× PBS, and fixed in 70% ethanol at 4°C. Following incubation in ethanol, cells were washed in phosphate citrate buffer (0.1 M citric acid, 0.2 M Na2HPO4, pH 7.8), treated with 5 μg Rnase A and incubated in 10 μg propidium iodide solution (Sigma, St Louis, MO). Cell-cycle distribution was determined by flow cytometery of at least 10,000 gated cells using a FACScan flow cytometer. All cell cycle analyses were performed at least twice.
Clonogenic Survival Assay
Cells were seeded in 6 well plates at a density of 200-400 cells per well. After an 8 hr attachment period, cells were treated with ABZ at 100 nM or 500 nM for 12 hrs. The cells were then unirradiated (control) or irradiated with 6 MV photons (0.300 Gy/min) for total doses ranging from 1 to 8 Gy. Immediately after radiation, media containing ABZ was exchanged for fresh media. Cells were subsequently cultured for approximately 2 weeks, with media changed every third day. Next, colonies were stained with 0.1% crystal violet and counted. The percent plating efficiency and surviving fraction were calculated based on the survival of non ABZ treated, nonirradiated cells treated. Each assay was performed in doublet with the mean reported.
Calculation of Radiation-ABZ Interactions
The results from the clonogenic assay measurements were analyzed using Calcusyn software (Biosoft, Cambridge, UK). The program calculates the "Combination Index"  based upon the methods of Chou and Talalay . CI < 0.90 is indicative of synergistic interactions, CI of 0.90 to 1.0 indicates additive interactions, and CI > 1.0 indicates antagonistic interactions.
Western Blot Analysis
A2058 and H153 cells were harvested after treatment with 0, 100, or 500 nM ABZ for 12 hrs. Cells were then irradiated at 2 or 10 Gy or left unirradiated as a control. Immediately after radiation, media was changed and cells were incubated for 72 hrs. Cells were then collected, washed in ice cold 1× PBS, and harvested in an extraction buffer (1% Triton X-100, 50 mmol/L Tris, 2 mmol/L EDTA, 150 mmol/L NaCl, pH 7.5) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor cocktail (Sigma, St Louis, MO). The lysates were centrifuged at 14,000 × g, 4°C for 20 min in a microcentrifuge. The protein concentrations of supernatants were measured with a protein assay kit (Bio-Rad, Hercules, CA). Proteins were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Polyscreen, Perkin-Elmer). For apoptotic studies, antibodies against cleaved forms of poly (ADP-ribose) polymerase (PARP) and caspase 9 were obtained from Cell Signaling Technology (Danvers, MA). For pH2AX studies, mouse monoclonal anti-pH2HAX IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used. For all blots, anti-actin (Sigma, St Louis, MO) was used as a loading control. Immunoreactive bands were visualized using enhanced chemiluminescence detection reagent (Perkin-Elmer, Waltham, MA) and X-OMAT processing.
Unless otherwise noted, experiments were performed in triplicate. All data are presented as the average ± standard error of the mean (SEM). P-values were determined by a two-sided Student's t-test with unequal variance, with P < 0.05 considered significant.
Albendazole treatment causes phosphorylation of H2AX
ABZ inhibits growth of MM and SCLC cells in vitro
Albendazole induces cell cycle arrest at G2/M
Albendazole arrests cell in the G2/M phase in a time and dose dependent fashion.
% G2/M cells
Albendazole radiosensitizes MM and SCLC cells
ABZ induces a lower SF2 (ie supra-additive) than the expected added SF2 (EAS) calculated by adding the effect of radiation alone and ABZ alone.
Albendazole acts synergistically with radiation in MM and SCLC cells.
Albendazole enhances radiation-induced apoptosis in MM and SCLC cells
Here, we demonstrate that ABZ, a BZ that effectively crosses the BBB, is cytotoxic to MM as well as SCLC (Figure 2). We found that this effect correlates with cellular arrest at the G2/M phase of the cell cycle (Table 1). We show ABZ's ability to act synergistically with radiation through combination index analysis (Table 3). ABZ achieves this effect in part by damaging DNA and increasing radiation-induced apoptosis (Figure 1 and 4).
Our study has potential clinical significance. ABZ is being explored as a potent in vitro and in vivo anti-neoplastic agent [12, 13], and here we demonstrate that it has further clinical potential as a radiosensitizer. The radiation dose of 2 Gy used in this study closely approximates the dose per fraction (2 to 3 Gy) used in patients receiving WBRT for metastatic melanoma  or prophylactic cranial irradiation for SCLC . Additionally, the concentration of ABZ employed in our studies is readily achievable by oral administration . A dose escalation phase 1 clinical trial of ABZ in 33 patients with a variety of metastatic cancers showed a maximum achievable plasma concentration after oral administration to be 10.25 μM . With other pharmacokinetic studies showing that average cerebrospinal fluid (CSF) concentration is approximately half the serum plasma concentration , our in vitro concentration of 500 nM is clinically achievable and well tolerated in patients with brain metastases. Furthermore, we observe radiosensitization at concentrations significantly lower than reported maximal CSF concentrations. At these lower concentrations, the incidence of side effects is expected to be reduced.
Like paclitaxel, ABZ is considered a spindle poison . At low clinical concentrations (less than 10% of the maximum plasma achievable concentration in humans), paclitaxel induces G2/M cell cycle arrest [16, 17]. With the G2/M phase being the most radiosensitive phase, paclitaxel has been show to sensitize melanoma to radiation, inducing a SER of 1.2 at 40 nM . Similarly, we show here that at low clinical concentrations, ABZ arrests cells in the G2/M phase and radiosensitizes melanoma, inducing a higher SER of 1.39. We hypothesize this difference in SER for comparable concentrations of ABZ and paclitaxel to be due to ABZ's ability to simultaneously induce DNA damage (Figure 1). Previous work has indicated that other BZ's possess DNA damaging capabilities [19, 20]. Moreover, additional studies have reported that paclitaxel-resistant cell lines are sensitive to ABZ, suggestive of a mechanism of action in addition to tubulin disruption [21, 22]. Indeed, data obtained from COMPARE suggesting that ABZ possesses a spectrum of activity most similar to the DNA damaging agent methyl-CCNU is consistent with this notion. Furthermore, ABZ is also a more suitable clinical agent for radiosensitization because unlike paclitaxel, it can cross the BBB to target melanoma or SCLC brain metastases. In conclusion, ABZ's ability to cross the BBB, radiosensitize cancer cells like melanoma and SCLC which carry a high propensity for metastasizing to the brain warrants further investigations of clinical application.
The marketed anti-parasitic drug albendazole possesses DNA damaging and microtubule disrupting capabilities. Based upon this unique spectrum of activity as well as its ability to cross the blood brain barrier, we examined albendazole's potential radiosensitization capabilities. At clinically achievable concentrations, albendazole in combination with irradiation causes enhanced apoptotic induction in melanoma and SCLC, which have an increased propensity to metastasize to the brain.
This work was supported in part by the NYU Center of Excellence in Cancers of the Skin. We would like to thank the NCI/NIH Developmental Therapeutics program (http://dtp.cancer.gov) for data provided as part of the NCI-60 DTP Human Tumor Cell Line Screen.
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