Flubendazole demonstrates valid antitumor effects by inhibiting STAT3 and activating autophagy
Signal transducer and activator of transcription 3 (STAT3) is an oncogene, which upregulates in approximately 70% of human cancers. Autophagy is an evolutionarily conserved process which maintains cellular homeostasis and eliminates damaged cellular components. Moreover, the STAT3 signaling pathway, which may be triggered by cancer cells, has been implicated in the autophagic process.
In this study, we found that the anthelmintic flubendazole exerts potent antitumor activity in three human colorectal cancer (CRC) cell lines and in the nude mouse model. The inhibition of cell proliferation in vitro by flubendazole was evaluated using a clonogenic assay and the MTT assay. Western blot analysis, flow cytometry analysis, siRNA growth experiment and cytoplasmic and nuclear protein extraction were used to investigate the mechanisms of inhibiting STAT3 signaling and activation of autophagy induced by flubendazole. Additionally, the expression of STAT3 and mTOR was analyzed in paired colorectal cancer and normal tissues collected from clinical patients.
Flubendazole blocked the IL6-induced nuclear translocation of STAT3, which led to inhibition of the transcription of STAT3 target genes, such as MCL1, VEGF and BIRC5. In addition, flubendazole also reduced the expression of P-mTOR, P62, BCL2, and upregulated Beclin1 and LC3-I/II, which are major autophagy-related genes. These processes induced potent cell apoptosis in CRC cells. In addition, flubendazole displayed a synergistic effect with the chemotherapeutic agent 5-fluorouracil in the treatment of CRC.
Taken together, these results indicate that flubendazole exerts antitumor activities by blocking STAT3 signaling and inevitably affects the autophagy pathway. Flubendazole maybe a novel anticancer drug and offers a distinctive therapeutic strategy in neoadjuvant chemotherapy of CRC.
KeywordsFlubendazole Colorectal cancer STAT3 Autophagy Apoptosis
BCL2-associated X protein
B-cell lymphoma 2
Baculoviral IAP Repeat Containing 5 (Survivin)
Dulbecco’s Modified Eagle Medium
Fetal bovine serum
Food and Drug Administration
Glyceraldehyde 3-phosphate dehydrogenase
The half maximal inhibitory concentrations
c-Jun N-terminal kinase
microtubule-associated proteins light chain 3
myeloid cell leukemia sequence 1
mechanistic target of rapamycin kinase
Ubiquitin-binding protein p62
Roswell Park Memorial Institute
Sodium dodecyl sulfate-polyacrylamidegel
Standard error of mean
Small interfering RNA
Signal transducer and activator of transcription 3
Tris-Buffered Saline and Tween 20
Vascular endothelial growth factor
Colorectal cancer (CRC) is one of the most common digestive tract malignancies worldwide, which is among the leading causes of cancer death. Meanwhile, the high incidence rate of CRC maintains a significant increase [1, 2]. Despite the many advances in CRC research, including screening and treatment, the overall survival rate of patients with CRC is still low and the rate of tumor recurrence remains currently invariable . Therefore, researchers have intense interest in finding new strategies aimed at improving treatment effectiveness and prognosis .
The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is overactive in most of human cancers including CRC . Compelling evidence has demonstrated the crucial role of STAT3 in promoting tumor cell proliferation, angiogenesis, metastasis and resistance to therapies by regulating the expression of correlative genes such as vascular endothelial growth factor (VEGF) [6, 7, 8]. In addition, STAT3 is associated with tumor cell apoptosis through the regulation of BCL2, BAX, MCL1 and survivin (BIRC5) [9, 10, 11]. Hence, inhibiting the STAT3 signaling axis has gradually emerged as an important strategy for the treatment of cancer .
Autophagy, a form of programmed cell death, is involved in cellular growth, autoimmunity and tumor progression as a conserved intracellular degradation system . Increasing evidence demonstrates that numerous therapeutic strategies induce cell apoptosis through upregulation of autophagy . However, the specific molecular mechanisms connecting apoptosis and autophagy are still not deciphered. Thus, the study of autophagy activation should provide new insights into the antitumor effect mechanism mediated through autophagy .
Flubendazole is a well-known anthelmintic drug that is widely used to treat infections of worm and intestinal parasites in clinical practice. The anthelmintic action of flubendazole is based on altering microtubule structure, inhibition of tubulin polymerization and disruption of microtubule function . Notably, other research groups have suggested that flubendazole is a potential antitumor agent . However, although several studies have reported various mechanisms and pathways mediating the antitumor effect of flubendazole, the precise mechanism remains unclear [18, 19]. In this study, we confirmed that flubendazole effectively inhibits cell proliferation and induces apoptosis in human CRC by blocking the STAT3 signaling axis and activation of autophagy. Meanwhile, flubendazole demonstrated synergistic effect with 5-Fluorouracil in human CRC cells. These findings suggest that flubendazole may be a novel antitumor therapeutic candidate drug in CRC treatment strategy in clinical practice.
Materials and methods
Antibodies and reagents
Flubendazole and 5-Fluorouracil were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the cell culture reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies of GAPDH(#5174), STAT3 (#12640), Survivin (#2808), MCL1 (#94296), P-mTOR (#5536), mTOR (#2983), Beclin 1 (#3495) and LC3-I/II (#4108) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies of P-STAT3 (ab76315), VEGF (ab52917), Lamin B1 (ab16048), BAX (ab32503) and Ki67(ab156956) were purchased from Abcam Co. (Cambridge, MA, USA). Antibody of BCL2 (sc-7382) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Total Protein Extraction Kit was purchased from Boster Biological Technology, (Wuhan, China). The siRNAs against STAT3 (si-STAT3), corresponding negative control siRNA (si-NC) were designed and synthesized by GenePharma (Shanghai, China). Lipofectamine 3000 Transfection Kit was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The Caspase3 Colorimetric Assay Kit was purchased from Abcam Co. (Cambridge, MA, USA). FITC Annexin V Apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ). TUNEL Apoptosis Assay Kit was purchased from Beyotime Institute of Biotechnology (China).
Cell lines and culture
The human colorectal cancer cell lines HCT116, RKO, SW480, the normal human liver cell LO2 and cardiomyocytes H9C2 were purchased from Cell Resources Center of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). The short tandem repeat (STR) DNA profiles for HCT116, SW480 and RKO are shown in Additional files 2, 3 and 4. HCT116 cell line was maintained in Mc Coy’s 5A medium supplemented with 10% fetal bovine serum (FBS). RKO and SW480 cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS. Human liver cell LO2 was cultured in RPMI 1640 medium supplemented with 15% FBS. Human normal cardiomyocytes H9C2 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. Routinely, cells were incubated at 37 °C in an atmosphere of 5% CO2.
Methyl thiazolyl tetrazolium (MTT) assay
The colorectal cancer cells and normal human cells were seeded into 96-well plate at a density of 3000–5000 cells per well in different medium. The cells were incubated at 37 °C in 5% CO2. Test compounds (flubendazole or 5-fluorouracil) were dissolved in DMSO. Cells were maintained with test compounds for 48 h before the MTT assay. The plate was then incubated in a CO2 incubator for 4 h, crystals dissolved with 150 μL DMSO, and analyzed in a Microplate Reader at 490 nm. The half maximal inhibitory concentrations (IC50) value was calculated by Graphpad Prism 7.0 software.
Cell clonogenic assay was conducted as previously described . Cancer cells were seeded into 6-well plate (500 cells per well). After cancer cell attachment, different concentrations of test compounds were added and incubated for 24 h. After 7 days, colonies were fixed by 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet at room temperature.
Western blot analysis
Tissue and cell protein were extracted using tissue or cell protein lysate buffer (Total Protein Extraction Kit). Cancer cells were seeded in 6-well plate and treated with different concentrations of flubendazole for 24 h, after that lysed with ice-cold lysate buffer. Proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamidegel (SDS-PAGE). And then proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skim milk for 1.5 h. The blots were incubated with specific primary antibody in Tris-Buffered Saline and Tween 20 (TBST) overnight at 4 °C. Then, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies and washed three times with 1 × TBST. The density of the immunoreactive bands was analyzed using Image J computer software.
Transient transfection of small interfering RNA (siRNA)
The siRNAs against STAT3 (si-STAT3), corresponding negative control siRNA (si-NC) were designed and synthesized by GenePharma (Shanghai, China). The process of transient cell transfection was conducted standardly according to manufacturers’ instructions. Sequences of siRNAs were shown as follows: STAT3 siRNA STAT3-Homo-398 5′-CCACUUUGGUGUUUCAUAATT-3′; STAT3 siRNA STAT3-Homo-978 5′-GCAACAGAUUGCCUGCAUUTT-3′; STAT3 siRNA STAT3-Homo-1070 5′-CCCGUCAACAAAUUAAGAATT-3′.
Cytoplasmic and nuclear protein extraction
Cytoplasmic and nuclear proteins of HCT116 cells were detached through the NE-PER Nuclear &Cytoplasmic extraction kit (Thermo, Waltham, MA, USA). HCT116 cells were treated with flubendazole for 24 h, stimulating with IL6 for 30 min before been lysed. Cells were lysed using nuclear and cytoplasmic protein extraction Kit according to the manufacturer’s protocol. Protein expression of the cytoplasmic and nuclear extractions was determined by immunoblot analysis respectively.
HCT116, RKO and SW480 cells were treated with different concentrations of flubendazole for approximately 48 h. Apoptotic cells were measured by flow cytometry using Apoptosis Detection Kit I. All samples were analyzed on a flowcytometer (BD Biosciences) and data was evaluated using FlowJo software.
Determination of caspase 3 activity
Caspase3 activity of cell lysates was measured by a caspase 3 assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. 1–5 × 10 6 CRC cells were collected after treatment with different concentrations of flubendazole (0, 0.3, 0.6 or 1.2 μM) about 24–48 h. Activity of caspase3 was normalized by the protein concentration of the corresponding cell lysate. The activity was measured at 405 nm through SpectraMAX iD3 (Molecular Devices, San Jose, CA, USA).
Hoechst 33342 staining
HCT116 and RKO cells were seeded in 6-well cell culture plate and treated with different concentrations of flubendazole for 12 h. Following fixation with 4% paraformaldelyde for 15 min at room temperature, cells were washed by PBS and stained with Hoechst 33342 for 20 min. Finally, cells were observed by fluorescence microscope (Nikon, Tokyo, Japan) using appropriate filters for blue fluorescence.
All animal care and experimental studies were performed according to the guidelines and approval of the Wenzhou Medical College Animal Policy and Welfare Committee. Female BALB/c athymic nude mice (6–8 weeks) were bred and maintained at the animal experimental center in Wenzhou Medical University. For colorectal cancer xenograft model, HCT116 cancer cells were harvested and subcutaneously implanted (5 × 10 6 cells in 100 μL of PBS) into the right flank of mice. Once tumor volumes reached ~ 100 mm3, mice were divided into three different groups which were no obvious differences in mean body weights or tumor volumes. The treatment groups (6 mice per group) were treated with flubendazole 10 mg/kg or 30 mg/kg by intraperitoneal (i.p.) injection every other day. The tumor volumes were measured length (l), width (w) and calculating volume (V = 0.5 × l × w2) before every injection. On day 14 and 2 h after the last treatment of flubendazole, all mice were executed. The tumors were removed and prepared for western blot analysis. Tumor weight was measured. Hearts, livers, kidneys and lungs were fixed immediately and paraffin-embedded. The sections were subjected to H&E staining.
Immunohistochemistry (IHC) analysis
Tumor tissue sections (4 μm) were deparaffinized, rehydrated and incubated with primarily Ki67 and P-STAT3 antibodies. HRP-conjugated secondary antibodies were used for detection. Images were obtained with Leica microscope.
Apoptosis assay (TUNEL staining)
A One Step TUNEL Apoptosis Assay Kit (Beyotime, China) was used to detect apoptosis of tumor tissues sections (4 μm). The process of Tunel apoptosis assay was conducted standardly according to manufacturers’ instructions. Cell nuclei was stained with 4′,6-diamidino-2-phenylindole (DAPI), and fluorescence was evaluated by fluorescence microscopy.
This study was approved by the Institutional Research Human Ethical Committee of the Wenzhou Medical University for the use of clinical biopsy specimens and informed consent was obtained from the patients. A total of 12 CRC patients biopsy samples were obtained. Clinical diagnosis was performed at the First Affiliated Hospital of Wenzhou Medical University. CRC tissues and matched tumor-adjacent morphologically normal CRC tissues were frozen and stored in liquid nitrogen until further analyses.
All experiments were assayed at least three times except animal models. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, CA, USA). Data are expressed as mean ± Standard deviation (SD). Students t-test was used to compare two groups (P-value < 0.05 was considered statistically significant).
Flubendazole inhibits CRC cells proliferation
Flubendazole exhibits pharmacological antitumor activity by blocking STAT3 signaling
Flubendazole disrupts nuclear translocation of STAT3
Flubendazole activates autophagy in CRC cells
Flubendazole induces apoptosis in CRC cells
Flubendazole inhibits growth of CRC tumor xenografts
Flubendazole exerts synergistic effect with 5-fluorouracil-based neoadjuvant chemotherapy in CRC
Consideration in terms of security and development costs, the exploration of novel anticancer agent by searching for existing drug repurposing has gradually emerged as a significant direction in preclinical research [14, 22, 23]. In this study, we found that the anthelmintic drug flubendazole could be repurposed as a potential anticancer drug. Our data shown that flubendazole clearly inhibits cell proliferation and induces apoptosis in CRC cells, while in normal human cells it only exhibits weak inhibition (Fig. 1). Additionally, we also revealed that flubendazole effectively blocks the growth of CRC tumor xenografts. Mechanistically, flubendazole exerted its antitumor activity by inhibiting STAT3 and thereby downregulating the expression of its key target genes, including BCL2, MCL1, survivin and VEGF. In addition, it was also determined that flubendazole prevents STAT3 translocation to the nucleus. Moreover, treatment with flubendazole disrupted STAT3 activation leading to cells apoptosis and activation of caspase-3 in CRC cells.
Recently studies have shown that flubendazole can inactivate mTOR signaling by displacing mTOR from lysosome and ultimately promote the maturation of autophagosome and autolysosome . The specific pharmacological action of flubendazole may be associated with disruption of microtubule function . Furthermore, treatment with flubendazole has been found to increase phosphorylation of JNK1 thereby decreasing the expression of BCL2 in BCL2-Beclin 1 complexes through acetylation of microtubules. Accordingly, flubendazole upregulates the release of Beclin 1 and initiation of autophagy. Additional studies indicate that flubendazole activates autophagy through the release of Beclin 1 from BCL2-Beclin 1 complexes and inactivating mTOR . Recent reports further indicate that different subcellular localization patterns of STAT3 affect autophagy in various ways . These recently studies of flubendazole suggest that it may be applicable in cancer treatment. Our results suggested that flubendazole may modulate expression of the STAT3 target gene BCL2 and lead to the initiation of autophagy and cell apoptosis. This represents a completely new idea in the preclinical research of anticancer therapy. In addition, Flubendazole can inhibit microtubule function and displays preclinical activity in leukemia and myeloma . Thus, these data indicated that STAT3 may not be the only target of flubendazole. Potential anti-cancer mechanisms of flubendazole may be more complicated.
We also found that the expression of STAT3 and mTOR was increasing in clinical samples of CRC tissues at the protein levels. Thus, these results suggest the potential application of STAT3 or mTOR as a prognostic and survival indicator in CRC patients. 5-fluorouracil is considered the classic chemotherapy strategy in the treatment of CRC, however, it can cause side effects [7, 25]. Combination therapy may enhance the curative effects and reduce the therapeutic concentration of chemotherapeutic drugs . Notably, we have demonstrated that administration of flubendazole and 5-fluorouracil results in synergistic antiproliferative effects in vitro. Flubendazole has a good inhibitory effect on CRC and can be used in combination with 5-fluorouracil to inhibit CRC. It may enhance the therapeutic effects and reduce the effective concentration. Thus, the precise mechanism-based rationale for the combination therapy will be a significant direction of our future research. Due to the safety and these preclinical studies of flubendazole, clinical trials may consider the application of flubendazole alone or in combination with chemotherapeutic drugs for the treatment of cancer, especially CRC.
SC L, LH Y, YL Y and LY X carried out the most experiments. SC L, YQ X and ZG Z provided and analysed clinical patient samples. SC L, LH Y, LX W and HY Z analyzed the data and prepared the Fig. CG Z, XY H and ZG Z conceived the idea and designed the research. CG Z, XY H and SC L wrote the manuscript. All authors read and approved final version of the manuscript.
This work was financially supported by the Zhejiang Provincial Natural Science Foundation of China (LY17H160055), Medical Scientific Research Fund of Zhejiang Province (2019322308) and Wenzhou science and technology project (Y20170280).
Ethics approval and consent to participate
All animal studies were performed with an approved protocol by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Patient samples study was approved by the Institutional Research Human Ethical Committee of the Wenzhou Medical University for the use of clinical biopsy specimens and informed consent was obtained from the patients.
Consent for publication
The authors declare that they agree to submit the article for publication.
The authors declare that they have no competing interests.
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