Albendazole regulates radiosensitivity of human pancreatic cancer cells by inhibiting HIF-1α and basic fibroblast growth factor

Background Albendazole, a clinical antiparasitic drug, has been shown to have antitumor activity and supress expression of hypoxia-inducible factor 1-alpha. While hypoxia, the most prominent feature of tumor microenvironment, is associated with radiotherapy tolerance. Herein, we aimed to identify Albendazole as a candidates that improves tumor microenvironment and enhances the radiosensitivity of human pancreatic cancer cells. Methods MTT assay, clone formation and flow cytometry were performed to assess the effect of ABZ and radiation on PC cell line proliferation and apoptosis induction. In addition, the expression levels of hypoxia-inducible factor 1-alpha (HIF-1α) and basic fibroblast growth factor (bFGF) were assessed using western blotting. Finally, the effects of ABZ on tumor growth and radiosensitivity were examined using nude mice xenograft model. Results ABZ significantly improved hypoxia-induced radiation resistance in PC cell line PATU8988 and SW1990 as evidenced by decreased absorbance of MTT, reduced colony number, and increased apoptotic cell ratio. Furthermore, the in vivo results confirmed that ABZ suppressed tumor growth. On mechanisms, treatment with ABZ decreased HIF-1α and bFGF expression levels, which correlated with radioresistance in cells exposed to hypoxia in vitro and tumor to radiation in vivo. Conclusion Taken together, our datas show that HIF-1α and bFGF regulate radiation sensitivity in PC cells under hypoxic conditions. And ABZ enhances radiosensitivity of pancreatic cancer by suppression of HIF-1α and bFGF expression. study, we investigated the effects of ABZ on HIF-1α and bFGF expression levels, the key factors for tumor microenvironment, and explored the link between the two factors.


Background
Pancreatic cancer is a highly malignant tumor with high invasiveness [1,2]. Due to late diagnosis and poor prognosis, most PC patients are fit for surgical resection at the time of diagnosis. This leads to high mortality rate in PC, making it the fourth leading cause of cancerrelated deaths. Of note, the incidence of PC has been on the rise globally [3] [4] [5].
Radiotherapy, chemotherapy and surgical resection are the main treatment approaches for pancreatic cancer patients [3]. Radiation therapy plays a key role in treatment of unresectable locally advanced and intolerable surgical resection pancreatic cancer patients [6]. The emergence of stereotactic body radiotherapy has accelerated the application of radiotherapy in cancer treatment [7].
However, tumor resistance caused by the tumor microenvironment compromises the efficacy of radiotherapy. Hypoxia is the most prominent feature in many solid tumors and is associated with poor prognosis and clinical resistance to radiotherapy in pancreatic cancer patients [8]. Elevated expression of HIF-1α was found in tumor tissue under hypoxic microenvironment and is associated with tumor radiotherapy resistance, development and invasion [9,10]. In addition, hypoxia up-regulates bFGF expression, which enhances the cancer cell growth and increases the radiation resistance of cancer cells [11,12]. Further, bFGF promotes the expression of hypoxiainducible factor 1a by modifying the tumor matrix environment, an effect that aggravates the tumor hypoxia [13]. Therefore, we explored the effects of ABZ on the expression of HIF-1α and bFGF in tumors, and tumor radiotherapy resistance.
ABZ a benzimidazole carbamate (methyl 5-propylthio-1H-benzimidazole-2-yl carbamate) is clinically used to treat human and animal helminth parasites infections [14]. The antitumor effect of ABZ on various cancers has been reported in several tumors [15,16]. However, the effect of ABZ on the tumor microenvironment has not been studied in depth, especially tumor fiber microenvironment. In this study, we investigated the effects of ABZ on HIF-1α and bFGF expression levels, the key factors for tumor microenvironment, and explored the link between the two factors.

Western bolt analysis
PATU8988 and SW1990 proteins were used for western blot analysis as described previously [17]. Equal amounts of protein were separated using SDS-PAGE and then transferred onto PVDF membrane. The protein was blocked with 5% skim milk and incubated with the primary antibody (HIF-1α, 1:2000; Abcam, Cambridge, UK and FGF-2, 1:1000; Abcam, Cambridge, UK) in PBST for 2h at room temperature. The membrane was incubated with secondary antibody (Goat Anti-Rabbit lgG HRP, Bioworld Technology, Lot NO: AB21171, Dilution:1:10000;) in PBST for 1 hrs. GAPDH was used as an internal control. Proteins were visualized by ECL kit (Beyotime).

MTT analysis
Cells were seeded on 96-well plates at the concentration of 8×10 3 cells/well. After 24 hours, the cells were treated with serial concentrations of ABZ or CoCl 2 for 24 and 48 hours, followed by incubation with 20 μl MTT solution (0.5 mg/mL) for 4 hrs at 37°C, washed in solution, and dissolved in 200 μL DMSO. The absorbance value was measured at a wavelength of 490 nm.

Colony formation analysis
Pancreatic cancer cells (200 cells per well) were seeded into six-well plates. After 24 hours, the cells were exposed to normoxia and Cobalt Chloride (CoCl 2 , 50 µM) to induce hypoxia in the presence or absence of ABZ (200 nM) for 24 hrs. Cells were then treated with 4 Gy of X-ray irradiation.
After incubation for 20 days, the colonies were fixed and stained with Giemsa dye for 15 min, followed by 3 times' PBS wash and colonies (>50 cells/colony) counting under a microscopy.

X-ray irradiation
Cell irradiation was performed using X-RAD320iX (Precision X-Ray, North Branford, CT, USA) at a dose of 4Gy with 8.15-mA X-ray, 1.5Gy/min. Prior to irradiation, the cells were treated in normoxia and Cobalt Chloride (CoCl 2 , 50 µM) to induce hypoxia in the presence or absence of ABZ (200 nM). Before each experiment, the mice were anesthetized by intraperitoneal injection of chloral hydrate, fixed on a special plate, exposed at the right front leg with the other body parts covered with a lead plate, and the mouse was placed in an irradiation apparatus.

Flow cytometric analysis of apoptosis
To detect apoptosis effects, Annexin V-FITC was used to stain PATU8988 and SW1990 cells treated in normoxia and CoCl 2 (50 µM) -induced hypoxia conditions 6 with ABZ (200 nM) or control for 24 hrs. Cells were, then, treated with 4Gy of X-ray irradiation. After 24 hours of incubation, cells were harvested and resuspended with 100 µL 1x binding buffer, followed by Annexin V-FITC and PI Staining before subjection to flow cytometry analysis.

Xenograft nude mice model
Six-week-old male BALB/c nude mice were obtained from Beijing Vitallihua. This study protocol was approved by the Institutional Animal Care and Usage Committee of Soochow University. PATU8988 cell line (5×10 6 ) were subcutaneously inoculated into the right flank of nude mice. When tumors size reached 5-10 mm in diameter, the mice were randomly divided into four different groups (n=6): Vehicle group (sesame oil), ABZ treatment group, Irradiation treatment group and ABZ and irradiation co-treatment group. As described by TC Hardin [18], the second and fourth groups received 300 mg/kg twice daily oral gavage ABZ suspended in sesame oil, whereas the control group was treated with the vehicle control (sesame oil) for 20 days. Tumor irradiation was performed using X-RAD320iX (Precision X-Ray, North Branford, CT, USA) at a dose of 4Gy with 8.15-mA X-ray, 1.5Gy/min on days 8, 15,22. The tumor volumes were measured every day with a vernier calipers on day 8 and were calculated using the formula: tumor volume=ab 2 /2, where "a" was the longest tumor diameter and "b" was the shortest tumor diameter measured. The PATU8988 derived tumors were harvested on the 27 th day from the mice and fixed in 4% formaldehyde solution for pathological analyses or lysed in RIPA lysis buffer for western bolt analyses.

Immunohistochemical analysis
The fixed tumors were placed in paraffin blocks. Paraffin sections were examined for 7 the expression of ki67 and HIF-1α antigen by immunohistochemistry. As described by Krajewski S [19], tumor tissue paraffin section were deparaffinized in xylene, hydrated in gradient alcohol, followed by antigen retrieval in boiling 0.1% citrate buffer (pH 6.0) for 30 min and endogenous peroxidase removal by 3% H 2 O 2 . Further, sections were washed three times in phosphate-buffered saline, blocked with 10% fetal bovine serum (Bisharp) containing 1% Tween 20 (TBST) at room temperature for 1 hr and exposed to mice anti-ki67 (1/100) primary antibody overnight at 4 °C.
Finally, the expression of the antigen was visualized by HRP conjugated secondary antibody and DAB substrate exposure.

TUNEL Apoptosis Assay
Colorimetric TUNEL Apoptosis Assay kit was used to measure tumor apoptosis.
Tumor tissue paraffin sections were deparaffinized in xylene, hydrated in gradient alcohol, followed by permeabilization of cell membrane with 20 µg/ml proteinase K

Sirius red assay
Sirius red assays were performed as described by Grimm PC [20]. Pancreatic xenograft tumor tissues fixed in 4% formaldehyde were embedded in paraffin for pathological analysis, and then stained with the Sirius Red Kit (Yuanye BioTechnology, Shanghai, China) for 1 hr. Subsequently, the sections were washed 8 with water for 5 min, counterstained with Mayer's hematoxylin, and visualization done under an inverted microscope.

Statistical analysis
All statistical analyses were performed with GraphPad Prism software (GraphPad Software, Inc, La Jolla, CA). All results were presented as mean± standard deviation (SD). Student t-test and one-way ANOVA assay was used for between group comparison and multiple group comparisons. All P values<0.05 was considered statistically significant.

Radiation dosage, ABZ concentration and hypoxia condition optimization
In order to observe evaluate the effect of ABZ against radiation resistance of PC cells exposed to hypoxia, PATU8988 and SW1990 human pancreatic cancer cell lines were firstly treated with series dosage of radiation and series concentration of ABZ.
As shown in Fig.1A&B, a significant inhibition on cell viability could be achieved at 4 Gy dosage in both cell lines at 48 h time points. Moreover, we also found that at the concentration of 200 nM ABZ, a significant inhibition on the PC cell viability could be achieved (Fig. 1C&D). In addition, cobalt chloride (CoCl 2 ) was employed to mimic hypoxia condition, and we observed a significant induction of the HIF-1α without affecting the cell viability at the concentration of 50 µM ( Fig.2A, B, C&D). Therefore, 4 Gy radiation and 200 nM ABZ were used as the treatment modality, whereas 50 µM CoCl 2 was employed as the hypoxia induction condition in the following experiment setting.

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The colony formation rate for cells exposed to normoxia condition significantly decreased in comparison to cells cultured in chemical hypoxia environment after Xray radiation, indicating their resistance to radiation. Interestingly, ABZ improved the radiosensitivity of human pancreatic cancer cells under hypoxia condition ( Fig.2A and B). Moreover, we explored the effects of ABZ on apoptosis of PC cells exposed to normoxia and hypoxia conditions in the presence of X-ray radiation. We observed that cell apoptosis rate was lower under hypoxia condition than under normoxia condition and ABZ significantly enhanced the apoptosis rate both in normoxia and hypoxia condition (Fig.2C). These results indicated that besides the killing effects by ABZ at normoxia condition, ABZ could also improve radiation resistance of hypoxia pancreatic cancer cells lines.

ABZ promotes the radiosensitivity of PATU8988 xenograft growth in vivo
To explore the potential radiosensitization effect of ABZ on human pancreatic cancer cells in vivo, we subcutaneously injected PATU8988 cells into the right armpit of BALB/c nude mice to establish a xenograft model. The mice were, then, treated with the vehicle (sesame oil), ABZ at 300 mg/kg (n = 6) by oral gavage every day or X-ray at a dose of 4Gy on day 8 and 15., After treatment for 21 days, mice were sacrificed, and tumor tissues harvested and photographed (Fig.3A). ABZ and irradiation co-treatment group was more effective in suppressing tumor growth compared to control group, ABZ-treated group, radiation-treated group (Fig.3B).
Moreover, ki-67 expression was evaluated histologically. The findings showed that ABZ and irradiation co-treatment induced significantly higher numbers of positive cells compared to vehicle (sesame oil), ABZ or X-ray irradiation treatment alone (Fig.3C). Furthermore, the number of TUNEL-positive cells was significantly higher in the ABZ and irradiation co-treatment groups than in vehicle (sesame oil), ABZ or X-ray irradiation treatment alone (Fig. 3C). bFGF promotes fiber collagen synthesis via stimulation of fibroblast differentiation, bFGF levels indirectly reflects the activity of basic fibroblast growth factor in tumor tissue. The Sirius Red Kit was used to stain the tissue to assess the amount of collagen fiber in tumor stroma. We demonstrated that the content of collagen fiber in ABZ-treated group was significantly higher than in control-treated group (Fig.3C). In summary, our results demonstrated that ABZ enhanced the radiosensitivity of human pancreatic cancer cells to X-ray irradiation treatment via affecting.

ABZ suppresses HIF-1α and bFGF expression in hypoxic pancreatic cancer cells in vitro and in vivo.
We further explored the effect of ABZ on HIF-1α and bFGF expression in hypoxia pancreatic cancer cells in vitro and in PATU8988 xenograft in vivo. Decreased HIF-1α and bFGF expression levels were observed in ABZ-treated hypoxic PC cells compared to those hypoxic PC cells without treatment (Fig.5A&B). Furthermore, decreased HIF-1α and bFGF expression levels were observed in ABZ-treated mice compared to the vehicle-treated (sesame oil) mice (Fig.5C). We demonstrated that the content of collagen fiber in ABZ-treated group was significantly higher than in control-treated group (Fig.5C). discussion Pancreatic cancer, the most malignant tumor among the gastrointestinal tumors, is known as a ' silent killer' due to its poor prognosis [21]. Advanced pancreatic cancer could be presented at the time of diagnosis and occupy 80% of PC diseases, whereas radiotherapy is the mainstay treatment for PC [22]. Unfortunately, its therapeutic efficacy is limited by high tumor radioresistance and high-dose radiotherapy-induced side effect to patients. The tumor microenvironment (TME), especially that with high hypoxia is considered as the main cause of tumor radiotherapy resistance, and is closely related to hypoxia-inducible factor 1α {Wachters, #49; Green, 2007 #50}[23, 24]. In our study, we used cobalt chloride (CoCl 2 ) to mimic hypoxia condition and found that CoCl 2 increased HIF-1α expression in a dose-dependent manner. Moreover, the upregulation expression of HIF-1α was closely associated with the increased colony formation efficiency under radiation condition which is consistent with a previous study [25]. Futhermore, we found the expression of bFGF protein increases with increase in expression of hypoxia-induced hif-1a protein. These findings show that HIF-1α and bFGF activities are linked. On the one hand, Friedman et al and Houghton et al [26,27], reported that hypoxia induces the secretion of bFGF and consequent enhancement of tumor resistance to irradiation. Further, studies have reported that bFGF contributes to tumor vessel dysfunction, hypoperfusion and increased tumor hypoxia by promoting the formation of cancer associated fibroblasts (CAFs) from fibroblasts [28]. CAFs may directly increase the radiotherapy resistance of tumors [29]. In addition, bFGF affects the secretion of HIF-1α-dependent VEGF closely related to radiation resistance [30].
ABZ, an anti-intestinal parasite drug, has been used in clinical treatment since 1987 [31]. Its safety has been approved in several clinical applications. Therefore, we used ABZ to explore its effect on tumor radiosensitivity and explore the underlying mechanisms. The effect of ABZ on HIF-1α and VEGF expression levels has been reported in in studies [32,33]. This study reports that ABZ could simultaneously inhibit HIF-1α and bFGF expression induced by hypoxia. This property increases the sensitivity of tumor cells to radiation in vitro and significantly inhibits human pancreatic cancer growth in vivo. These findings indicate that ABZ is a potential radiotherapy sensitization agent.
In conclusion, we demonstrate that ABZ enhances radiosensitivity of pancreatic cancer and its mechanism of action is by suppression of HIF-1α and bFGF expression. Our experimental data supports that ABZ is a promising drug to improve the efficacy of radiation therapy for its antihypoxia effect.

conclusion
In conclusion, we demonstrate that ABZ enhances radiosensitivity of pancreatic cancer and its mechanism of action is by suppression of HIF-1α and bFGF expression. Our experimental data supports that ABZ is a promising drug to improve the efficacy of radiation therapy for its antihypoxia effect.

Ethics approval and consent to participate
All animal experiments were performed in strict accordance with the principles and procedures of Guide for the Care and Use of Laboratory Animal by the National Institutes of Health.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the 13 corresponging author on reasonable request.

Competing interests
There are no conflicts of interest.