This observational cohort study used the cancer registry dataset of the ‘2010 Longitudinal Generation Tracking Database (LGTD 2010),’ established by the Health and Welfare Data Science Center (HWDC), Ministry of Health and Welfare (MOHW) in Taiwan. The Taiwan National Health Insurance (NHI) program has been implemented since 1995 and offers comprehensive medical care coverage to >99% of the country's 23 million inhabitants (26).

The present study collected data from the LGTD 2010, which were randomly selected from a cohort of 2 million individuals covered by the NHI Program, between January 2011 and December 2011. The LGTD 2010 contains all information on diagnostic codes, detailed prescriptions, dates of clinical visits, dates of admission and discharge, and expenditures for the enrollees of 2 million beneficiaries (26). No statistical differences was observed in the age and sex distributions between the cohorts in the LGTD and Taiwan NHI enrollees. All patients were regularly monitored after diagnosis until death or at the last follow-up date. Follow-up was completed on December 31, 2015 (27). From the Taiwanese National Health Insurance Research Database (https://www.mohw.gov.tw/mp-2.html), data from patients newly diagnosed with colon cancer were identified using the International Classification of Diseases for Oncology codes (C180-C189), treated with or without itraconazole, according to the Anatomical Therapeutic Chemical code: J02AC02, between January 2011 and December 2015. A total of 5,221 patients newly diagnosed with colon cancer were included in the present study, and confounding factors, such as age, sex, clinical stage, surgery, radiation therapy and chemotherapy were adjusted accordingly. Patients were pathologically diagnosed with primary colon cancer based on Taiwan Cancer Registry, a population-based cancer registry enrolled. The exclusion criteria for this study were patients' age at diagnosis was unknown; incomplete data of tumor stage, pathology and treatment history; lacking clear records on survival status; previous history of cancer; incomplete data on the American Joint Committee on Cancer (AJCC) stage and cause of death. All staging was performed according to the AJCC Staging System (7th edition) (28). To protect the privacy of individuals included in the database, the NHI only released data with encoded identification numbers so that personnel without authorization would not be able to associate any direct information to the enrollees. The Bureau of NHI approved the application after reviewing all the required medical documents. The present study was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (IRB no. KSVGH18-CT10-07) and performed in accordance with the tenets of the Declaration of Helsinki (1975) and its later amendments (2013). The requirement for written informed consent was waived.

The human colon adenocarcinoma cell line, COLO 205, was purchased from the Food Industry Research and Development Institute, and the colorectal cell line, HCT 116, was kindly gifted by Dr Tzu-Ming Jao from Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). Both cell lines were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA), 2 mM L-glutamine (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), at 37°C with 5% CO₂.

To determine the role of autophagy on itraconazole induced apoptosis in COLO 205 cells, 5 mM 3-MA (3-methyladenine, Sigma-Aldrich; Merck KGaA) was co-cultured with 50 µM itraconazole (Sigma-Aldrich; Merck KGaA) at 37°C for 24 h.

The MTT assay (Sigma-Aldrich; Merck KGaA) was performed to assess the cytotoxic effect of itraconazole on COLO-205 and HCT 116 cells. A total of 1×10⁴ cells were seeded into 96-well plates and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA), 2 mM L-glutamine (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), at 37°C with 5% CO₂ for overnight. Following incubation, 1, 2, 5, 10, 25, 30, 50, 60, 100, 125, 250 and 500 µM itraconazole were added into each well and cells were incubated for an additional 24 h at 37°C. The culture medium was subsequently replaced with complete medium (DMEM, Gibco; Thermo Fisher Scientific, Inc.) containing 5 mg/ml MTT and incubated for 2 h at 37°C. The purple formazan crystals were dissolved using DMSO and data were subsequently measured at a wavelength of 570 nm, using an ELISA reader (BioTek Instruments, Inc.).

The commercial siRNA gene silencer (LC3B) and scrambled siRNA oligo were purchased from Cell Signaling Technology, Inc. (cat. nos. 6212S and 6568S). Briefly, 1×10⁶ COLO 205 and HCT 116 cells were seeded into 6-well plates, respectively, and cultured in complete medium overnight at 37°C. siRNA (100 nM) and scrambled siRNA (100 nM) were transfected into cells using Lipofectamine^® RNAiMAX (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following incubation at 37°C for 24 h, the supernatant was removed, washed with PBS and incubated with different concentrations of itraconazole in complete medium for an additional 24 h at 37°C. Representative images were obtained from three independent experiments.

Following transfection with LC3B or scrambled siRNA, COLO 205 and HCT 116 cells were treated with different concentrations of itraconazole in triplicate wells at 37°C for 24 h. Cell viability was assessed via the CCK-8 assay. Briefly, 100 µl of cell suspension was seeded into a 96-well plate, and 10 µl of CCK-8 reagent (Sigma-Aldrich; Merck KGaA) was added to each well and incubated for 1 h. Optical density was measured at a wavelength of 450 nm, using an enzyme microplate reader (Synergy HTX; BioTek Instruments, Inc.).

Following transfection with LC3B or scrambled siRNA, COLO 205 and HCT 116 cells were treated with different concentrations of itraconazole in triplicate wells for 24 h. Cells were collected and resuspended in the culture medium. Subsequently, cells were mixed with trypan blue at a ratio of 1:1 (Gibco; Thermo Fisher Scientific, Inc.) and counted using a hemocytometer (Yung Yuen Scientific Instrument Co., Ltd.).

Itraconazole-induced cell apoptosis was detected via Annexin V/PI staining. A total of 1×10⁶ cells were seeded into 6-well plates and incubated in complete medium overnight at 37°C. Following attachment to the plate, cells were treated with different concentrations of itraconazole for 24 h. Annexin V/PI staining was performed according to the manufacturer's instructions (AAT Bioquest, Inc.). The percentage of apoptotic cells was detected using FACSCalibor (BD Biosciences). Representative images are shown for three independent experiments.

A total of 1×10⁶ COLO 205 and HCT 116 cells were seeded into 6-well plates and treated with different concentrations of itraconazole for 24 h. Following treatment, cells were fixed with 100% ethanol overnight at −20°C. DNA in the nuclei was stained with PI (50 µg/ml) and RNase A (0.5 µg/ml) for 30 min at 4°C. Cell cycle distribution was analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.). Representative images were obtained from three independent experiments.

Following treatment with itraconazole for 24 h, mRNA was extracted from COLO 205 and HCT 116 cells using the GENEzol™ TriRNA Pure kit (Geneaid, http://www.geneaid.com/Tri-RNA/GZXD), according to the manufacturer's instructions. A total of 1 µg RNA was reverse transcribed into cDNA using the cDNA reverse transcription kit (Morrebio, http://www.light-biotech.com/pcr), according to the manufacturer's instructions. The temperature protocol for RT was as follows: 50°C for 15 min followed by 85°C for 5 sec, and the cDNA products were used for the gene expression assay. The PCR primer and the TaqMan probe were purchased from Thermo Fisher Scientific, Inc. The primer and probe sequences used in the present study were as follows: LC3B (Gene ID: Hs00797944_s1), p62 (Gene ID: Hs02621445_s1) and GAPDH (Gene ID: Hs02786624_g1). Gene expression was detected via qPCR (StepOnePlus™; Thermo Fisher Scientific, Inc.) and the protocol was as follows: 10 µl 2× PCR master mix, 2 µl cDNA, 7 µl dd H₂O and 1 µl probe were mixed in each reaction. The following thermocycling conditions were used for qPCR: 50°C for 2 min, 95°C for 2 min followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. LC3B and p62 expression levels were calculated using the 2^−ΔΔCq method and normalized to β-actin (29).

A total of 1×10⁶ COLO 205 and HCT 116 cells, were seeded into 6-well plates and treated with different concentrations of itraconazole for 24 h. Following treatment, total protein was extracted using RIPA buffer (Biomed, http://www.bio-protech.com.tw/products_detailed.php?id=347) and quantified using the BCA kit (Invitrogen; Thermo Fisher Scientific, Inc.). Equal amounts (25 µg/lane) of protein extracts were subjected in 12% polyacrylamide gel and separated by electrophoresis then transferred onto PVDF membranes and blocked with 5% skim milk for 1 h at room temperature. Membranes were washed three times with PBST (PBS with 0.05% Tween-20) and incubated with primary antibodies against TKT (1:1,000; cat. no. SC-390179; Santa Cruz Biotechnology, Inc.), Cleaved caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.), Caspase-3 (1:1,000; cat. no. E-AB-30756; Elabscience, http://www.elabscience.com/PDF/Cate81/E-AB-30756-Elabscience.pdf), Bax (1:1,000; cat. no. 2772; Cell Signaling Technology, Inc.), LC3B (1:1,000; cat. no. 83506; Cell Signaling Technology, Inc.), p62 (1:1,000; cat. no. 5114; Cell Signaling Technology, Inc.), Beclin-1 (1:1,000; cat. no. 3738; Cell Signaling Technology, Inc.) and β-actin (1:5,000; cat. no. E-AB-20034; Elabscience) overnight at 4°C. The membranes were washed three times with PBST and subsequently incubated with anti-mouse IgG, HRP-linked antibody (1:5,000; cat. no. 7076P2; Cell Signaling Technology, Inc.) or goat anti-rabbit IgG antibody (1:5,000; cat. no. A0545; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Protein bands were visualized using the ECL western blot detection kit (Thermo Fisher Scientific, Inc.) and analyzed using Alliance Q9 software (V18.11; UVITEC; http://www.uvitec.co.uk/alliance-q9-advanced). Representative images and band quantification were obtained from three independent experiments.

To detect autophagosome formation, 1×10⁵ COLO 205 and HCT 116 cells were seeded onto cover glasses in a 6-well plate. Following treatment with itraconazole for 24 h, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) in PBS for 15 min at room temperature. Cells were subsequently blocked with 1% BSA for 1 h at room temperature. Cells were incubated with LC3B primary antibody (Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. DAPI was used for nuclear staining. Cells were observed under a Zeiss LSM 5 Pascal confocal microscope (Carl Zeiss). Number of LC3 puncta per cell was further quantified using ImageJ software (Version 1.50i; National Institutes of Health) (30). Representative images were obtained from three independent experiments.

Statistical analysis was performed using SAS software (version 9.3; SAS Institute, Inc.) and SPSS software (version 20; IBM Corp.). Descriptive statistics were used to analyze baseline demographic data and the distribution of each variable among the study population. Continuous variables are presented as the mean ± SD. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Univariate Cox regression analysis was performed to assess the association between survival and the impact of itraconazole. Hazard ratios (HRs) and their 95% confidence intervals (CIs) from the Cox regression analyses were used to estimate the relative risk. Then stratified survival analyses were also performed for different groups based on multivariate Cox regression analysis. The results of the control and treatment with different concentrations of itraconazole were analyzed via one-way ANOVA followed by Tukey's post hoc test, and performed using GraphPad Prism software (version 6.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

In the present study, a total of 5,221 patients with colon cancer were identified using ICD-O3 codes between 2011 and 2015, and data were analyzed. The demographic characteristics of all patients, including age, sex, primary tumor location, AJCC clinical stage, treatment, itraconazole use and mortality are presented in Table I. The average age of all patients was 65.6±13.7 years (age range, 21–85 years). The proportions of stage I+II and stage III+IV were 42 and 58%, respectively. Most patients underwent surgery, and the proportions of patients who received radiation therapy and chemotherapy were 2 and 38%, respectively. Notably, only 1% of all patients received itraconazole treatment. The mortality rate of all patients with colon cancer was 34%.

The univariate Cox proportional hazard model was used to assess the HR for patients with colon cancer, in the presence and absence of itraconazole treatment. Statistically significant HRs for the 5-year survival rate were observed for age (HR, 1.03; 95% CI, 1.01–1.04; P<0.001), AJCC clinical stage (HR, 3.08; 95% CI, 2.75–3.44; P<0.001), surgery (HR, 4.15; 95% CI, 3.77–4.57; P<0.001) and radiation therapy (HR, 0.43; 95% CI, 0.33–0.58; P<0.001) (Table II). Subgroup analysis was performed to assess the effect of itraconazole on patients with late-stage colon cancer who received chemotherapy. Following adjustment for age, sex, surgery and radiation therapy, the benefit of itraconazole treatment in reducing the risk of mortality was observed (HR, 0.27; 95% CI, 0.07–1.09; P=0.067; Table III). Although there was not statistically significant, however the trend in reducing the risk of mortality could still be noted. The 5-year survival rate also increased in patients treated with itraconazole group, according to Kaplan-Meier curve by log-rank test with significant statistical difference. (P<0.05; Fig. 1). Taken together, these results suggest that itraconazole can increase the 5-year survival rate in patients with late-stage colon cancer.

To further investigate the effects and underlying molecular mechanisms of itraconazole on colon cancer, the effect of itraconazole on the proliferation of COLO 205 and HCT 116 cells was assessed. Cells were treated with different concentrations of itraconazole for 24 h, and cell viability was assessed via the MTT assay. The results demonstrated that cell viability significantly decreased following treatment with itraconazole, in a dose-dependent manner (P<0.05; Fig. 2A). Apoptosis-related proteins and TKT expression were detected in COLO 205 and HCT 116 cells treated with itraconazole. The results demonstrated that the expression levels of cleaved caspase-3 and Bax increased after itraconazole treatment, in a dose-response manner for 24 h (P<0.05; Fig. 2B). TKT expression was decreased following treatment with itraconazole in a time-dependent manner (P<0.05; Fig. 2C). Annexin V-PI staining demonstrated that COLO 205 and HCT 116 cells treated with itraconazole significantly induced cell apoptosis (P<0.05; Fig. 2D). Furthermore, treatment with itraconazole significantly induced subG₁ phase, G₁ arrest and decreased the number of cells in the G₂/M phase (P<0.05; Fig. 2E). Collectively, these results suggest that itraconazole decreases colon cancer proliferation and TKT expression and induces apoptosis.

Autophagic cell death plays an important role in the treatment of cancer (31); therefore, the present study investigated the effect of itraconazole on inducing autophagy in colon cancer cells. The results demonstrated that the expression levels of the autophagy-related proteins, LC3B and p62, significantly increased in COLO 205 and HCT 116 cells following treatment with itraconazole for 24 h (P<0.05; Fig. 3A), and the mRNA expression levels of LC3B and p62 significantly increased following treatment with itraconazole, in a concentration-dependent manner (P<0.05; Fig. 3B). In addition, the number of LC3B puncta/cell increased following treatment with itraconazole (P<0.05; Fig. 3C). Taken together, these results suggest that itraconazole can induce autophagy in colon cancer cells.

A previous study has reported that itraconazole can induce both apoptosis and autophagy in colon cancer cells (32). The role of autophagy in itraconazole-induced cell death remains controversial; thus, the present study used autophagy siRNA targeting LC3B to determine the role of autophagy in itraconazole-induced cell death. The results demonstrated that the expression levels of the autophagy-related proteins, LC3B and p62, decreased following transfection of COLO 205 and HCT 116 cells with LC3B siRNA (P<0.05; Fig. 4A). Furthermore, the expression levels of the apoptosis-related proteins, cleaved caspase-3 and Bax, also decreased (P<0.05; Fig. 4A). The viability of cells transfected with LC3B siRNA was assessed via the CCK-8 and trypan blue exclusion assays. The results demonstrated that cell viability decreased following treatment with itraconazole and transfection with the scrambled siRNA. Notably, these effects were reversed following transfection with LC3B siRNA (P<0.05; Fig. 4B and C). Collectively, these results suggest that autophagy plays an important role in itraconazole-induced cell death.