Abstract
Background/Aim: Quinazolinone is a privileged chemical structure employed for targeting various types of cancer. This study aimed to demonstrate the antitumor activity of synthesized 6,7-disubstituted-2-(3-fluorophenyl) quinazolines (HoLu-11 to HoLu-14). Materials and Methods: The cytotoxicity was assessed by the sulforhodamine B (SRB) assay. The cell cycle was examined by flow cytometry. The expression levels of cell cycle- and apoptosis-related proteins were estimated by western blotting. A xenograft animal model was used to explore the antitumor effects of HoLu-12. Results: Among four synthetic quinazolinone derivatives, HoLu-12 significantly reduced the viability of oral squamous cell carcinoma (OSCC) cells. HoLu-12 induced G2/M arrest and increased the expression of cyclin B, histone H3 (Ser10) phosphorylation, and cleaved PARP, indicating that HoLu-12 could induce mitotic arrest and then apoptosis. Moreover, the combination of HoLu-12 and 5-fluorouracil (5-FU) displayed synergistic toxic effect on OSCC cells. HoLu-12 significantly inhibited tumor growth in vivo. Conclusion: HoLu-12 induces mitotic arrest and leads to apoptosis of OSCC cells. Furthermore, HoLu-12 alone or in combination with 5-FU is a potential therapeutic agent for OSCC.
Head and neck squamous cell carcinomas arise in the oral cavity, oropharynx, hypopharynx and larynx (1). Among them, oral squamous cell carcinoma (OSCC) is the most common disease site (1, 2). Advances in surgical techniques and preoperative management have improved the survival rate to some extent; however, the overall 5-year survival rate for OSCC patients remains at 50% without significant improvement over the past three decades (2). Although chemotherapy, the main pharmaceutical strategy, has been developed for decades, drug resistance and serious adverse effects often lead to treatment failure (3). Therefore, the development of agents that can increase OSCC sensitivity is a critical challenge.
Quinazolinone derivatives have been studied for more than 30 years and have been found to be toxic to a variety of cancers (4). Possible anticancer mechanisms of quinazolinones include the inhibition of DNA repair and transcription in cancer cells by inhibiting the DNA repair enzyme system, EGFR and human epidermal growth factor receptor 2 (HER2) (5-7), and thymidylate synthase (TS) activity, thus making cancer cells unable to replicate DNA and leading to cell death (8). Quinazolinone derivatives can inhibit the polymerization of tubulin and prevent cancer cells from undergoing normal mitosis (9). Based on the various mechanisms of action, the quinazoline scaffold has received substantial consideration for targeting various types of cancer, including OSCC.
Promotion of apoptosis has been considered a strategy for anticancer drug discovery. Compounds based on quinazolines have been shown to be useful scaffolds for the development of both apoptosis inducers and inhibitors (10). Based on the pharmacological indication, the quinazoline apoptosis inducers can be divided into EGF inhibitors, inhibitors of apoptosis proteins (TAPS), activators of the caspase cascade, activators of the Akt pathway, propylpeptidase inhibitors, activators of the p53 protein, inhibitors of tyrosine kinases, and anti-apoptosis protein inhibitors (10). Understanding the apoptosis mechanism induced by quinazolinone scaffold compounds may provide guidance for their clinical use.
5-FU is a commonly used chemotherapeutic drug against different cancer types, including OSCC (11). However, resistance to 5-FU is commonly found during treatment. Therefore, 5-FU is often used in combination with other drugs to improve drug efficacy. For example, 5-FU has been used in combination with the antifolate antagonist leucovorin (LV) in the treatment of colorectal cancer (12). The synergistic effects are due to LV forming a stable complex with 5-FU and TS, resulting in an increase in the inhibitory effect of 5-FU on TS (12). Another example is the combination treatment of 5-FU and oxaliplatin (12, 13). Oxaliplatin forms a large complex with DNA, especially guanine, which in turn makes the cells unable to divide normally, leading to an enhancement in the induction of apoptosis after combination with 5-FU (12).
In this study, we explored the anticancer effects of quinazolinone derivatives (HoLu-11 to HoLu-14) in human OSCC cells. Among these quinazolinone derivatives, HoLu-12 was found to have superior therapeutic specificity for OSCC cells over other types of cancer cells and normal noncancerous cells. Furthermore, we investigated the mechanisms involved in the anticancer potential of HoLu-12 and the efficacy of the combinatorial HoLu-12 with 5-FU in OSCC cells.
Materials and Methods
General procedure for the synthesis of 6,7-disubstituted-2-(3-fluorophenyl)quinazolines (5-8, HoLu-11 to HoLu-14). Compounds 1-4 prepared according to the published method (14), were reacted with 3-fluorobenzaldehyde to obtain compounds 5-8 (HoLu-11 to HoLu-14), respectively.
Cell lines and culture conditions. Human OSCC lines including CAL27, OECM-1, and SAS, and human noncancerous lines, including keratinocyte cells (HaCaT), fetal colon epithelial cells (FHC), and oral mucosa fibroblasts (OMF), were used in this study. CAL27 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). OECM-1, SAS, HaCaT, FHC, and OMF cells were obtained from Dr. Te-Chang Lee (Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, ROC). In addition, we used CAL27-meta cells, which were established using the CAL27 and cultured as previously described (15, 16). All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), penicillin, and streptomycin. Cells were grown in an incubator with 5% CO2 at 37°C.
In vitro cytotoxicity assays. Cell viability was determined utilizing the sulforhodamine B (SRB)-based toxicity assay as described previously (16). Briefly, logarithmically growing cells were plated at a density of 5×103 cells/well into 96-well plates in four replicates and treated with 5-FU and compounds HoLu-11 to HoLu-14 at 10 μM for 72 h or dimethyl sulfoxide (DMSO) as a vehicle control. After treatment, the cells were fixed with 10% trichloroacetic acid and then, stained with 0.4% (w/v) SRB in 10% acetic acid for 30 min. The absorbance of the dissolved SRB granules within the cells was determined at 510 nm using a microplate reader (Bio-Rad model 550; Bio-Rad, Hercules, CA, USA) to calculate viability (% of control). In addition, OSCC cells were plated and treated with different concentrations (2-80 μM) of HoLu-12 alone or in combination with 5-FU or DMSO alone as a vehicle control for 72 h. The values of the 50% inhibition concentration (IC50) and combination index (CI) for combination treatment were calculated by the CompuSyn software (CompuSyn Inc., Paramus, NJ, USA) (17).
Analysis of DNA content by flow cytometric analysis. CAL27 cells at a density of 2×105 cells/well were subcultured into 6-well plates and treated with HoLu-12 at various concentrations (5-40 μM) for 6, 12, 48 and 72 h. Following incubation, the cells were fixed with ice-cold 70% ethanol and then, stained with 4 μg/ml propidium iodide in PBS containing 1% Triton X-100 and 0.1 mg/ml RNase A and subjected to flow cytometric analysis (Beckman Coulter Gallios, Brea, CA, USA) as previously described (18). The cell cycle distribution was determined using ModFit LT 3.0 software (Verity Software House, Topsham, ME, USA).
Immunofluorescence staining and confocal microscopy. Immunofluorescence staining was performed as described previously (19). Antibodies against α-tubulin (Sigma, ST. Louis, MO, USA) and centrin (Millipore, Billerica, MA, USA) were used. Alexa Fluor 488- or 633-conjugated goat anti-mouse and rabbit IgG were obtained from Invitrogen. The nuclei or chromosomes were counterstained with DAPI. The stained cells were observed and images were obtained by z projection with an upright confocal microscope (Leica TCS-SP5, Leica Microsystems, Wetzlar, Germany). The percentage of mitotic cells containing abnormal mitotic spindles was determined using at least 250 mitotic cells from three independent experiments.
Western blotting assay. Western blotting was performed as described previously (15). Briefly, equal amounts of protein were electrophoretically separated and transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ, USA). The membrane was incubated with specific primary antibodies and then with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The membrane-bound antibodies were visualized by chemiluminescence using the Pierce SuperSignal West Pico chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA, USA) on the BioSpectrum AC® Imaging Systems (UVP, Upland, CA, USA). Primary antibodies for phosphor-cdc2 (Tyr15), cyclin A, cyclin B1, phospho-histone H3 (Ser10), and phospho-Wee1 (Ser642) were obtained from Cell Cycle Regulation Antibody Sampler Kit II, and cleaved PARP was obtained from Cell Signaling (Danvers, MA, USA). Primary antibody for β-Actin and secondary antibodies for mouse IgG (HRP) and rabbit IgG (HRP) were obtained from Abcam (Cambridge, MA, USA). Band intensities were quantified by ImageJ (NIH, Bethesda, MD, USA) and are shown normalized to the β-Actin lane.
Xenograft antitumor study. Animal experiments were approved by and performed according the guidelines of the Institutional Animal Care and Utilization Committee of Tzu-Chi University. NOD/SCID mice were obtained from the animal center of Tzu-Chi University (Hualien, Taiwan, ROC). CAL27 cells (1×107 cells/mouse) in 0.1 ml of PBS were subcutaneously injected into the flanks of 6- to -8-week old of NOD-SCID male mice to form a solid tumor as described previously (15). When the xenograft tumors reached a volume of approximately 90-120 mm3, twenty-four mice were randomly divided into three groups with eight mice in each group. The experimental treatments by intraperitoneal (i.p.) injection included HoLu-12 at dosages of 2.5 and 5.0 mg/kg body weight every other day for a total of twelve injections (dosing regimen: Q2D × 12, i.p.), whereas control mice intraperitoneally received DMSO. To monitor tumor growth, the longest and shortest diameters of the xenograft tumors were measured every 4 days using calipers. The tumor size (mm3) was calculated by the following formula: tumor volume=(length × width2)/2.
Statistical analysis. The results are expressed as the mean±standard error of the mean (SEM) from triplicate determinations. Statistical analysis was performed by Student’s t-test. Differences were considered significant at *p<0.05.
Results
Chemistry. The 6,7-disubstituted-2-(3-fluorophenyl)quinazolines (compounds 5-8, HoLu-11 to HoLu-14) were synthesized via the method presented in Figure 1. In the chemistry experiment, when sodium bisulfite, a cheap catalyst, was used with 2-fold molar of 2-amino-4,5-substituted benzamides (compounds 1-4) could achieve the best yield of products (compounds 5-8) within the shortest reaction time. The reaction time was decreased from 4-6 h to 2-3 h by the presence of sodium bisulfite. In addition, the reaction temperature couldn’t less than 150°C in order to thoroughly transfer the 2,3-dihydroquinazolinone intermediates to products 5-8 by dehydrogenation. The structures of HoLu-11 to HoLu-14 are shown in Figure 2A. The appearance, yields, melting points, and spectroscopic data are listed in Table I.
Synthesis of 6,7-disubstituted-2-(3-fluorophenyl)quinazolines (compounds 5-8, HoLu-11 to HoLu-14).
The physical and spectroscopic data of 6,7-disubstituted-2-(3-fluorophenyl)quinazolines.
The cytotoxic effects of quinazolinone derivatives. The cytotoxic potential of the quinazolinone derivatives was examined using different types of cell lines, as shown in Figure 2B. The cytotoxicity of 5-FU was also examined here. Treatment with 10 μΜ 5-FU resulted in viability values ranging from 33.3 to 67.7% for CAL27 and OECM-1 cells, respectively. Regarding HaCaT and FHC cells, as representative noncancerous lines, treatment with 5-FU showed viability values of 53.2 and 16.7%, respectively, indicating that 5-FU exhibits a nonspecific toxic effect on OSCC cells and noncancerous cells. Similar to 5-FU, treatment with 10 μΜ HoLu-11 or HoLu-12 resulted in viability values ranging from 31 to 52% for CAL27 and OECM-1 cells. Regarding HaCaT and FHC cells, treatment with 10 μΜ HoLu-11 showed viability values of 36.7 and 89.6%, respectively. Treatment with 10 μΜ HoLu-12 showed viability values of 83.1 and 93.5% for HaCaT and FHC cells, respectively, showing that HoLu-12 was less potent to the noncancerous cells. Other derivatives showed less toxic effects on the cancer and noncancerous lines. These results indicate that HoLu-12 may be a potential lead quinazolinone derivative against OSCC cells.
Cytotoxicity of 5-FU and quinazolinone derivatives on OSCC cells and noncancerous cells. (A) Chemical structures of HoLu-11 to HoLu-14. (B) Proliferation of OSCC cell lines treated with 10 μM 5-FU and HoLu-11 to HoLu-14 was determined by the SRB assay. (C) Cytotoxicity of HoLu-12 in OSCC and oral mucosa fibroblast (OMF) cells. The proliferation of OSCC and OMF cells treated with increasing concentrations of HoLu-12 was determined by the SRB assay. All experiments were repeated independently at least three times, and the data are expressed as the mean±the standard error of the mean (SEM). *p<0.05 compared to OMF cells.
Cytotoxic effect of HoLu-12 on the OSCC lines and OMF cells. The cytotoxicity of HoLu-12 on OSCC lines was further examined by the SRB assay. As shown in Figure 2C, HoLu-12 still exhibited no toxic effects on OMF cells at the high concentration of 80 μM. Moreover, HoLu-12 decreased viability OSCC cells in a concentration-dependent manner. Among these tested OSCC lines, CAL27-meta OSCC cells, which exhibited higher metastatic activity and resistance to 5-FU (16), were found to be more sensitive to HoLu-12. The IC50 values of HoLu-12 in CAL27 and CAL27-meta cells were 7.09±0.91 and 4.84±1.13 μM, respectively. In addition, we found that HoLu-12 showed much higher toxicity to OSCC lines compared to other types of cancer lines, including RKO, HCT116, MCF-7, PaCa-2, HepG2, HuH7, U87, and PC3 (all IC50 values of cancer lines were more than 20 μM; data not shown). These results indicate that HoLu-12 has therapeutic potential for OSCC cells.
The effect of HoLu-12 on the cell cycle and apoptosis of CAL27 cells. We further examined whether to the effects HoLu-12 on cell viability were associated with cell cycle arrest. As shown in Figure 3A, cell cycle analysis revealed S and G2/M phase arrest after treatment of CAL27 cells with different concentrations (5-40 μΜ) of HoLu-12 for 6 h. Significant S and G2/M phase arrest was indicated by a decreasing proportion of cells in the G0/G1 phase. At 12 h after treatment, G2/M phase arrest and the number of cells in the sub-G1 phase were significantly increased, which is an indicator of apoptosis (Figure 3B). Prolonging the duration of HoLu-12 treatment for 48 and 72 h, the apoptotic population was also significantly increased (Figure 3C).
Effect of HoLu-12 on cell cycle distribution. (A) Representative DNA histograms for CAL27 cells. (B) Statistical analysis of the differences in the percent of cells in the G1, S, G2/M, and sub-G1 phases. (C) Statistical analysis of the differences in the percent of cells in the sub-G1 phases at 6, 12, 48 and 72 h after HoLu-12 treatment. All experiments were repeated independently at least three times, and the data are expressed as the mean±standard error of the mean (SEM). *p<0.05 compared to untreated control cells.
To clarify whether HoLu-12 induced mitotic spindle abnormalities, immunofluorescence staining and confocal microscopy were used. As shown in Figure 4A and B, most untreated mitotic OSCC cells (68.84±1.65%) showed normal bipolar morphology. Few untreated mitotic cells were bipolar with misaligned chromosomes (23.48±1.47%) and multipolar (7.69±0.18%). However, the population of mitotic cells displaying bipolar morphology with misaligned chromosome was significantly increased in HoLu-12 treated cells. We further evaluated the cell cycle checkpoints in HoLu-12-treated CAL27 cells. As shown in Figure 4C, HoLu-12 enhanced the mitotic-phase markers cyclin B and phosphohistone H3 but not the G2-phase marker cyclin A. In addition, HoLu-12 did not change the expression of phospho-WEE1 or phospho-cdc2. Phosphorylation of histone H3 (Ser10) has been shown to correlate with chromosomal condensation, which is essential for the segregation of chromosomes during mitosis (20, 21). Cyclin B is a key regulator of the cell cycle that controls the G2/M transition (22). Consistent with other studies reporting that long-term arrest of the cells in the M-phase triggers apoptosis by activation of Bax, RAPR, and caspase-3 (23), we found that cleaved PARP protein levels were significantly increased in CAL27 cells treated with HoLu-12 for 24 h. These results suggest that HoLu-12 induced OSCC mitosis arrest and apoptosis.
Effect of HoLu-12 on mitotic spindle structure and apoptosis of OSCC cells. (A) Representative mitotic spindle abnormalities in CAL27 cells treated with 20 μM HoLu-12. Cells were stained for α-tubulin (red) or centrin (green) and DAPI (blue) was used to stain nuclei. (B) Statistical analysis of the differences in the percent of mitotic spindle abnormalities at 24 h after HoLu-12 treatment. (C) The cells were then harvested and lysed for the determination of the levels of phospho-histone H3, cyclin B, phospho-Wee1, cyclin A, phospho-cdc2, and cleaved PARP by western blotting. β-actin was used as a loading control. All experiments were repeated independently at least three times and the data are expressed as the mean±standard error of the mean (SEM). *p<0.05 compared to untreated control cells.
The combination of HoLu-12 and 5-FU synergistically suppresses cell growth. Our results indicated that HoLu-12 could significantly reduce the viability of OSCC cells, and we further examined the efficiency of this compound and its combination with 5-FU in OSCC cells. The IC50 values of 5-FU and HoLu-12 in CAL27 cells were 2.5±0.6 mM and 7.09±0.91 mM, respectively. The cytotoxic effect of the combination of HoLu-12 and 5-FU in the toxic dose range on CAL27 cells was examined using the SRB assay, and the CI values were calculated. As shown in Table II, a synergistic toxic effect was observed following treatment with the combination of these drugs at doses lower than their individual IC50 values. The IC50 dose of 5-FU (2.5 μM) combined with HoLu-12 (0.5-5.0 μM) had a synergistic toxic effect on CAL27 cells, especially at an equivalent dose of 2.5 μM (1:1). Intriguingly, treatment of CAL27 cells with the combination of HoLu-12 (2.5 μM) and 5-FU (0.5-2.5 μM) resulted in the highest synergistic toxic effect, with a CI value of 0.63±0.04. These results demonstrate that HoLu-12 can be used in combination with 5-FU for OSCC treatment.
Summary of the combination indexes (CIs) of HoLu-12 with 5-FU in CAL27 cells.
Antitumor activity of HoLu-12 against CAL27 cells in a xenograft model. To examine the in vivo therapeutic potential of HoLu-12, NOD/SCID mice were subcutaneously injected with CAL27 cells and then administered HoLu-12 intraperitoneally every 2 days (2.5 and 5.0 mg/kg), and the body weights of the mice were measured every 4 days. After the last injection (the twelfth dose), observation of the mouse tumor growth and body weights continued for another 24 days. All mice were anesthetized and scarified on the 50th day. As shown in Figure 5A, there was no significant difference in body weight change between the HoLu-12-treated and control groups, indicating that HoLu-12 had low systemic toxicity. As shown in Figure 5B, the tumor size of the 2.5 mg/kg drug group was significantly reduced, and was 30% lower than that of the control group on the 50th day. In the 5.0 mg/kg drug group, the tumor size was obviously diminished on the 12th day (the sixth dose), and was 41.7% smaller than that of the control group on the 50th day, showing that HoLu-12 could suppress tumor growth in vivo.
In vivo antitumor effect of HoLu-12. (A) Body weights were measured every 4 days as an indicator of the systemic toxicity of the treatment. (B) The tumor size (mm3) was calculated every 4 days by the following formula: tumor volume=(length × width2)/2. The results are expressed as the mean±standard error of the mean (SEM). *p<0.05 compared to the control group.
Discussion
The present study is the first to explore whether the quinazolinone derivative HoLu-12 had an effective toxic effect on OSCC, while having a less toxic effect on noncancerous cells. The ability of HoLu-12 to increase the expression of cyclin B and phosphorylated histone H3 (Ser10) leads to mitotic arrest and the induction of apoptosis, thereby affecting the survival of CAL27 cells. HoLu-12 exhibited a synergistic toxic effect when combined with 5-FU. The anticancer potential of HoLu-12 was also demonstrated in an in vivo xenograft model. Thus, these results may provide valuable evidence for the application of HoLu-12 for OSCC therapy.
Several studies have demonstrated the potential of quinazolinone derivatives in oral cancer therapy. MJ-29 (6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolin-one) has been shown to inhibit oral cancer cell invasion and metastasis through suppression of MMP-2/9 expression and the down-regulation of both MAPK and Akt signaling (24). HMJ-38 can induce apoptosis of oral cancer cells through mitochondrial dysfunction (25). 2-Aryl-6-substituted quinazolinones (MJ compounds, MJ65-70) were designed and synthesized by introducing substituents at the C-2 and C-6 positions of HMJ-38, and was found that MJ65-70 strongly inhibited proliferation of OSCC cells (26). HoLu-12 was synthesized by adding a morpholino and a benzene ring with an F branch to the parent quinazoline molecule, which is similar in structure to the above quinazolinone derivative HMJ-38. We found that HoLu-12 was highly toxic to OSCC lines compared to other types of cancer lines. Therefore, HoLu-12 is a potential scaffold for OSCC drug development.
The effect of quinazolinone derivatives on G2/M arrest has been reported. MJ-29 has been shown to inhibit growth and induce the death of leukemia cells via mitotic arrest but did not obviously impair the viability of normal cells (27). HMJ-38 has been shown to induce mitotic arrest and apoptosis in OSCC cells but does not obviously affect the viability of normal skin fibroblast cells (25). Similarly, we found that HoLu-12 induced mitotic arrest and apoptosis but did not obviously affect the viability of normal OMF cells. In fact, cancer cells exhibiting a faulty G1 checkpoint due to loss of p53, pRb, or p21 may show greater sensitivity to G2/M checkpoint inhibitors (28, 29). In this case, cells with damaged DNA enter aberrant mitosis and undergo apoptosis (30). Thus, efforts to enhance G2/M arrest have been associated with enhanced apoptosis and increased cytotoxicity of chemotherapeutics (30). These results demonstrate that quinazolinone derivatives acting as G2/M regulators, including HoLu-12 may enhance the effect of conventional chemotherapy agents.
Our results showed that HoLu-12 is a cell cycle inhibitor and exhibits a synergistic toxic effect on OSCC cells in combination with 5-FU. More importantly, the synergistic effect of HoLu-12 could decrease the dose of 5-FU in OSCC cells. 5-FU is equally cytotoxic against OSCC and normal OMF cells. However, HoLu-12 exhibited effective cytotoxicity in OSCC cells but had no obvious cytotoxic effect on normal OMF cells. Therefore, HoLu-12 may be used in combination with 5-FU to enhance its therapeutic effects. It is essential to investigate the efficacy of co-treatments against other OSCC cell lines and to further investigate the antitumor effects on in vivo models.
Acknowledgements
This study was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 108-2320-B-320-004 and MOST 109-2320-B-182-044) to Kuo-Chu Lai and China Medical University, Taichung, Taiwan (CMU109-MF-104) to Mann-Jen Hour. We thank Miss HSU LI-MEI of the Instrument Center of National Chung Hsing University for help with measurements of High Resolution Mass Spectrometer.
Footnotes
Authors’ Contributions
Kuo-Chu Lai: Conceptualization, funding acquisition, and writing original draft. Yi-Ting Chia: Methodology, data analysis and curation. Ling-Huei Yih: Methodology and data analysis. Yi-Liang Lu: Data curation. Shih-Ting Chang: Data curation. Zi-Xuan Hong: Methodology. Tai-Lin Chen: Conceptualization and data curation. Mann-Jen Hour: Conceptualization and data curation. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
None of the Authors have any conflicts of interest to declare relative to this study.
- Received November 13, 2020.
- Revision received December 9, 2020.
- Accepted December 10, 2020.
- Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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