doi: 10.1038/s41598-018-30158-6.
Fenbendazole acts as a moderate microtubule destabilizing agent and causes cancer cell death by modulating multiple cellular pathways
Affiliations
- PMID: 30093705
- PMCID: PMC6085345
- DOI: 10.1038/s41598-018-30158-6
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Fenbendazole acts as a moderate microtubule destabilizing agent and causes cancer cell death by modulating multiple cellular pathways
Sci Rep.
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Abstract
Drugs that are already clinically approved or experimentally tested for conditions other than cancer, but are found to possess previously unrecognized cytotoxicity towards malignant cells, may serve as fitting anti-cancer candidates. Methyl N-(6-phenylsulfanyl-1H benzimidazol-2-yl) carbamate [Fenbendazole, FZ], a benzimidazole compound, is a safe and inexpensive anthelmintic drug possessing an efficient anti-proliferative activity. In our earlier work, we reported a potent growth-inhibitory activity of FZ caused partially by impairment of proteasomal function. Here, we show that FZ demonstrates moderate affinity for mammalian tubulin and exerts cytotoxicity to human cancer cells at micromolar concentrations. Simultaneously, it caused mitochondrial translocation of p53 and effectively inhibited glucose uptake, expression of GLUT transporters as well as hexokinase (HK II) - a key glycolytic enzyme that most cancer cells thrive on. It blocked the growth of human xenografts in nu/nu mice model when mice were fed with the drug orally. The results, in conjunction with our earlier data, suggest that FZ is a new microtubule interfering agent that displays anti-neoplastic activity and may be evaluated as a potential therapeutic agent because of its effect on multiple cellular pathways leading to effective elimination of cancer cells.
Conflict of interest statement
The authors declare no competing interests.
Figures
FZ treatment alters tubulin network of human cancer cells. (a) A549 cells were treated with 1 uM FZ or 50 ng/ml colchicine for 24 h. Following treatment, the cells were processed for immunofluorescence using anti α-tubulin primary and FITC conjugated secondary antibodies. (Nuclei were counter stained with propidium iodide) (b) bovine tubulin (1.8 mg/mL) was incubated with DMSO (control), FZ (10 uM) or colchicine (100 nM) and the effect on polymerization was monitored spectrophotometrically by measuring turbidity at 340 nm as described under “Methods.” (c) Cells were treated with FZ, nocodazole, taxol or colchicine for 24 h and then lysed and fractionated into soluble (S) and polymerized (P) extracts. The extracts were separated with SDS-PAGE, transferred onto PVDF membranes and probed with both anti-α-tubulin and anti-β-actin antibodies. A representative immunoblot analysis in A549 cells is shown. (d) Intensity of each band of the immunoblot was measured by the NIH ImageJ program, and the ratios of soluble and polymerized tubulin and β-actin in each treatment were calculated. (e) Cells were treated with different MTAs as indicated for 24 h and western blotting was then performed using Ac-α-tubulin (6–11B-1) specific and β-actin antibodies. (Full-length uncropped blots are included in Supplementary Fig. S6).
P-gp inhibition has no effect on FZ mediated cell death. (a) A549 cells were left untreated or treated with 10 uM FZ or 10 uM Verapamil for 6 h. Rh123 was then added and fluorescence images were acquired after washings with PBS as described under “Methods”. (b) The cells were treated as before and the fluorescence was measured at Ex507/Em529 using a Tecan multimode plate reader. (c) A549 and H460 cells were treated with 1 uM FZ in the absence or presence of 10 uM verapamil for 24 h. Cell proliferation was then measured by MTT assay.
FZ causes early elevation of cyclin B1 levels and induces mitotic arrest. a(i) & a(ii) A549 cells were synchronized by serum starvation for 48 h and then left untreated or treated with 1 uM FZ or 50 nM colchicine for the indicated time intervals. The cell extracts were then processed for western immunoblotting using cyclin B1, cdk1 and β-actin antibodies. b(i) & b(ii) A549 cells were treated with 1 uM FZ for the indicated time intervals and the extracts were then processed for western blotting using pH3 and β-actin antibodies. The bands were quantitated using ImageJ software. (Full-length uncropped blots are included in Supplementary Fig. S6).
Mitotic arrest followed by cell death in response to FZ in human NSCLC cells. (a and b) Cells were treated after synchronization as before and FACS analysis was done to detect the fraction of populations in different phases of cell cycle, as well as apoptotic cells. (c) H460 cells were treated with 1 uM FZ for 24 h and cell morphology was observed under phase contrast microscope (i & ii) or after Hoechst 33342 staining under fluorescence microscope (iii & iv). TdT staining was done to detect apoptotic nuclei (v & vi).
FZ mediated inhibition of cancer cells in vitro is affected by p53 status. (a) Human H460 or A549 cells were plated onto 96-well tissue culture plates. Cells were left untreated or treated with different doses of FZ for 48 h. Cell viability was measured by MTT assay. (b) H460, A549, H522 or H1299 cells were treated with 1 uM FZ for 48 h and cell viability was measured by MTT assay. (c) H1299 cells were transfected with p53 expression construct or with vector alone. After 16 h of transfection, cells were left untreated or treated with FZ for 24 h following which cell viability was measured by MTT assay. (d) H460 cells or primary lung epithelial cells derived from rat lung tissue were treated with 1 uM FZ for different time points as indicated. Cell viability was determined by MTT assay. The significance level was set at p < 0.05. (*p < 0.05)
Apoptosis and p53 induction in human NSCLC cell lines following FZ treatment. (a) Western blot was performed for p53 target genes after 24 h of FZ treatment in H460 cells. (b) H1299 cells were transiently transfected with p53 expression construct and left untreated or treated with FZ for 24 h. Western blot was then done for p53 and p21. (c) Quantitation of “b” using Image J software. (Full-length uncropped blots are included in Supplementary Fig. S6) (d) immunostaining for p53 in H460 cells after treatment with increasing concentrations of FZ for 24 h.
FZ treatment results in increased p53 translocation to mitochondria. (a) H1299 cells were transiently transfected with GFP-p53 expression construct and treated as before. They were then stained with red mitotracker dye (Molecular Probes) and fluorescent images were acquired using a Nikon fluorescence microscope. (b) H460 cells were treated with 1 uM FZ, 500 nM nocodazole or 100 nM taxol for 24 h. After treatment, mitochondria were isolated using a mitochondria isolation kit from Sigma. Whole cell lysates (WC), cytosolic (C) and mitochondrial (M) fractions were then resolved on SDS-PAGE and subjected to western blot analysis using anti p53, β-actin and COX IV antibodies. (c) The band intensities from “a” were quantified using Image J software and normalized with β-actin (WC and C) or COXIV (M) levels. (Full-length uncropped blots are included in Supplementary Fig. S6) (d) H460 cells were either left untreated or treated with 1 uM FZ for 24 h and then processed for JC-1 staining (i, control; ii, FZ).
FZ alters glucose uptake and impairs enzymatic activity of HKII in NSCLC cells. (a) A549 or H460 cells were treated with 1 uM FZ for 4 h and uptake of the fluorescent glucose derivative 2-NBDG was examined thereafter by fluorescence microscopy as described. Representative images of cells from three independent experiments are shown. (b) Human NSCLC H460 cells were exposed to increasing doses of FZ for 24 h. Culture supernatants were then used to assess glucose consumption by glucose oxidation assay using GO assay kit from Sigma. (c) H460 cells were left untreated or treated with 1 uM FZ and the lactate levels in culture supernatants were assessed after the indicated time points using Lactate Assay Kit from BioVision. (d) Human H460 cells were exposed to 1 uM FZ for 24 or 48 h as indicated, total RNA was isolated and RT-PCR was performed using primers specific for the indicated genes. (e) (i) H460 cells were left untreated or treated with 1 uM FZ for 24 h and the cell extracts were then processed for a spectophotometric assay for SDH (A630nm). (ii) A549 cells were left untreated or treated with 1 uM FZ for 20 h following which they were processed for histochemical assay to assess SDH activity. Cells were then observed under a microscope and images were acquired at 40X magnification. (f) H460 cells were left untreated or treated with 1 uM FZ for 24 h. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. (g) H460 and A549 cell lysates were incubated with DMSO or FZ for 15 min prior to initiation of reaction. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. (h) Purified HKII from S. cerevisae was incubated with increasing doses of FZ and HK activity was then measured spectrophotometrically. (*p < 0.05, **p < 0.01, ***p < 0.005).
FZ treatment results in reduced tumorigenicity in vitro and in vivo. (a) Colony formation assay for H460 and A549 cells following treatment with 1 uM FZ for 48 h. (b) Soft agar assay following FZ treatment. Cell colonies were counted after staining with 5% crystal violet (c). (d) Tumors were established in nu/nu mice by subcutaneous injection of 5 × 106 A549 cells. After the tumors were 2–4 mm in diameter, the mice were orally fed with FZ dissolved in olive oil (1 mg/mouse) every second day, whereas control animals received olive oil only. Tumor volumes were then calculated by measuring tumor dimensions using Vernier callipers. (e and f) Tumors were excised, photographed and weighed. (g) Tumor vascularity in vivo was quantified between control and FZ treated mice by measuring hemoglobin spectrophotometrically (A590nm). (h) Tumor sections from control untreated and FZ treated mice were processed for TdT staining to identify apoptotic cells (i & iv), sections were immunostained using p53 specific antibody (ii & v), and immunohistochemistry was performed on sections for CD31 (iii & vi). i, ii & iii are sections from control mock treated mice and iv, v & vi are sections from FZ treated mice (*p < 0.05).
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