Research ArticleExperimental Studies

Quantitative Structure–Cytotoxicity Relationship of Pyrano[4,3-b]chromones

JUNKO NAGAI, HAIXIA SHI, YUKA KUBOTA, KENJIRO BANDOW, NORIYUKI OKUDAIRA, YOSHIHIRO UESAWA, HIROSHI SAKAGAMI, MINEKO TOMOMURA, AKITO TOMOMURA, KOICHI TAKAO and YOSHIAKI SUGITA
Anticancer Research August 2018, 38 (8) 4449-4457; DOI: https://doi.org/10.21873/anticanres.12747

Abstract

Background/Aim: 4H-1-Benzopyran-4-one (chromone) provides a backbone structure for the chemical synthesis of potent anticancer drugs. Since studies of the biological activity of pyrano[4,3-b]chromones are limited, we investigated a total of 20 pyrano[4,3-b]chromones (10 sets of diastereomers) for their cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and human normal oral cells, and then carried out a quantitative structure–activity relationship (QSAR) analysis. Materials and Methods: Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Tumor-specificity (TS) was evaluated by the ratio of mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human OSCC cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by the CC50 against tumor cells. Apoptosis induction was evaluated by morphological observation, western blot analysis and cell-cycle analysis. For QSAR analysis, a total of 3,072 physicochemical, structural and quantum chemical features were calculated from the most stabilized structure optimized using CORINA. Results: 8-Chloro-4,4a-dihydro-3-methoxy-3-methyl-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (16) and 3-ethoxy-4,4a-dihydro-8-methoxy-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (17) had the highest TS, higher than that of 5-flurouracil and melphalan, without induction of apoptosis. Compound 16 induced cytostatic growth inhibition and much lower cytotoxicity against human normal oral keratinocytes compared to doxorubicin. TS of 20 pyrano[4,3-b]chromones was correlated with 3D structure, polarity, ionic potential and electric state. Conclusion: Chemical modification of 16 may be a potential choice for designing a new type of anticancer drug.

Using 4H-1-Benzopyran-4-one (chromone), found ubiquitously in the plant kingdom (1), as a backbone structure, we synthesized 3-styrylchromones (2), 3-styryl-2H-chromenes (3) and 2-azolylchromones (4), and found them to have much higher cytotoxicity against human oral squamous cell carcinoma (OSCC) cell lines than against human normal oral mesenchymal cells (gingival fibroblast, periodontal ligament fibroblast, pulp cells). These compounds were relatively less cytotoxic against human oral keratinocytes as compared with common anticancer drugs (5).

As far as we know, studies of the biological activity of pyrano[4,3-b]chromones have been limited to the identification of new compounds from marine fungus (6), binding affinity to human opioid receptors (subtypes δ, κ, and μ) and cannabinoid receptors (CB1 and CB2) (7), and their antimicrobial activity (8). In continuation of discovering new biological activities of chromone derivatives, we investigated a total of 20 pyrano[4,3-b]chromones (10 pairs of diastereomers) (A-series 1-10 and B-series 11-20 in Figure 1) for their cytotoxicity against four human OSCC cell lines and three human normal oral cell types, and then subjected them to quantitative structure–activity relationship (QSAR) analysis.

Figure 1.
Figure 1.

Structure of 20 pyrano[4,3-b]chromones investigated in this study.

Materials and Methods

Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) from GIBCO BRL (Grand Island, NY, USA); fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), melphalan, doxorubicin, ribonuclease (RNase) A from Sigma-Aldrich Inc. (St. Louis, MO, USA); 5-fluorouracil (5-FU) from Kyowa (Tokyo, Japan); propidium iodide (PI), dimethyl sulfoxide (DMSO), actinomycin D, 4% paraformaldehyde phosphate buffer solution from Wako Pure Chem. Ind. (Osaka, Japan); Nonidet-P40 (NP-40) from Nakalai Tesque Inc. (Kyoto, Japan); and culture plastic dishes and 96-well plates from Techno Plastic Products AG (Trasadingen, Switzerland).

Synthesis of pyrano[4,3-b]chromone derivatives. Diastereomer pairs of 3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (1, 11), 3-butoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (2, 12), 4,4a-dihydro-3-methoxy-3-methyl-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (3, 13), 8-chloro-3-ethoxy-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (4, 14), 3-butoxy-8-chloro-4,4a-dihydro-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (5, 15), 8-chloro-4,4a-dihydro-3-methoxy-3-methyl-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (6, 16), 3-ethoxy-4,4a-dihydro-8-methoxy-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (7, 17), 3-butoxy-4,4a-dihydro-8-methoxy-3H, 10H-pyrano[4,3-b][1]benzopyran-10-one (8, 18), 4,4a-dihydro-3,8-dimethoxy-3-methyl-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (9, 19), and 2,3,12a,12b-tetrahydro-1H,4aH,7H-pyrano[3’,2’:5,6]pyrano[4,3-b][1]benzopyran-7-one (10, 20) were synthesized by the cycloaddition reactions of 3-formylchromones with selected enol ethers, according to previous methods (9). All compounds were dissolved in DMSO at 40 mM and stored at −20°C before use.

Cell culture. Human normal oral mesenchymal cells (human gingival fibroblast, HGF; human periodontal ligament fibroblast, HPLF) were established from the first premolar tooth extracted from the lower jaw of a 12-year-old girl (10), and cells at 10-18 population doubling levels were used in this study. Human oral OSCC cell lines (Ca9-22, derived from gingival tissue); HSC-2, derived from tongue) were purchased from Riken Cell Bank (Tsukuba, Japan). All of these cells were cultured at 37°C in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml, penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO2 atmosphere. Human oral keratinocyte (HOK) cells (purchased from Cosmo Bio Co. Ltd., Tokyo, Japan) were cultured in keratinocyte growth supplement (OKGS, Cat, No. 2652; CliniSciences, Nanterre, France) and cells at 7-11 population doubling levels were used in the present study. Cell morphology was checked periodically under a light microscope (EVOS FL; Thermo Fisher Scientific, Waltham, MA, USA).

Assay for cytotoxic activity. Cells were inoculated at 2×103 cells/0.1 ml in a 96-microwell plate. After 48 h, the medium was replaced with 0.1 ml of fresh medium containing different concentrations of single test compounds. Cells were incubated for a further 48 h and the relative viable cell number was then determined by the MTT method (2-5). The relative viable cell number was determined from the absorbance of the cell lysate at 560 nm, using a microplate reader (Infinite F50R; TECAN, Männedorf, Switzerland). Control cells were treated with the same amounts of DMSO and the cell damage induced by DMSO was subtracted from that induced by test agents. The concentration of compound that reduced the viable cell number by 50% (CC50) was determined from the dose–response curve and the mean value of CC50 for each cell type was calculated from triplicate assays.

Calculation of tumor-specificity index (TS). TS was calculated using the following equation: TS=mean CC50 against normal oral cell types/mean CC50 against OSCC cell lines. Since both Ca9-22 and HGF cells were derived from gingival tissue (11), the relative sensitivity of these cells was also compared (as: mean CC50 against HGF/mean CC50 against Ca9-22).

View this table:
Table I.

Cytotoxic activity of 20 pyrano[4,3-b]chromones against oral malignant and non-malignant cells. Each value represents the mean of triplicate determinations. Two sets of tumor-specificity index (TS) and potency-selectivity expression (PSE) values were determined using all oral squamous cell carcinoma (OSCC) compared with non-malignant cells, and paired cells derived from the same (gingival) tissue.

Calculation of potency-selectivity expression (PSE). PSE was calculated by the following equation: PSE=mean CC50 against normal oral cell types/(CC50 against OSCC cell lines)2 ×100 (HGF, HPLF vs. Ca9-22, HSC-2); and as mean CC50 against HGF/(CC50 against Ca9-22)2 ×100 using the pair of cell types from the same tissue (gingiva) (see Table I).

Western blot analysis. Cells were washed with phosphate-buffered saline (PBS) and re-suspended in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 1% NP-40 and protease inhibitors (RIPA buffer). After ultrasonication using Bioruptor (UCD-250; Cosmo Bio) for 12.5 min (10 s on, 20 s off) at 250 W at 4°C, the soluble cellular extracts were recovered after centrifugation for 10 min at 16,000 × g. The protein concentration of each sample was determined using BCA Protein Assay Reagent Kit (Thermo Fisher Scientific) and cell extracts were subjected to western blot analysis. The blots were probed with anti-poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology Inc., Beverly, MD, USA), anti-caspase 3 (Cell Signaling Technology Inc.), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA), followed by a horseradish peroxidase-conjugated anti-α-rabbit IgG secondary antibody (DAKO, Glostrup, Denmark). The immune complexes were visualized using Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific). Western blotting results were documented and quantified using ImageQuant LAS 500 (GE Healthcare, Tokyo, Japan) (12).

Cell-cycle analysis. Treated and untreated cells (approximately 106 cells) were harvested, fixed with 1% paraformaldehyde in PBS without calcium and magnesium ions [PBS(−)]. Fixed cells were then washed twice with PBS(−), and treated for 30 min with 400 μl of 0.2 mg/ml RNase A (preheated for 10 min at 100°C to inactivate DNase) to degrade RNA. Cells were then washed twice with PBS(−) and stained for 15 min with 0.01% PI in the presence of 0.01% NP-40 in PBS(−) to prevent cell aggregation. After filtering through Falcon® cell strainers (40 μM) (Corning, NY, USA) to remove aggregated cells, PI-stained cells were subjected to cell sorting (SH800 Series; SONY Imaging Products and Solutions Inc., Kanagawa, Japan). Cell-cycle analysis was performed with Cell Sorter Software version 2.1.2. (SONY Imaging Products and Solution Inc.).

Estimation of CC50 values. Since the CC50 values had a distribution pattern close to a logarithmically normal distribution, we used the negative log CC50 (pCC50) values for the comparison of cytotoxicity between compounds. The mean pCC50 values for normal cells and tumor cell lines were defined as N and T, respectively (3).

Figure 2.
Figure 2.

Cytotoxicity of compounds 16 and 17 against human oral squamous cell carcinoma cell lines Ca9-22 and HSC-2, and human normal oral cells, human gingival fibroblast (HGF) and human periodontal ligament fibroblast (HPLF). Cells were incubated for 48 h without (control) or with the indicated concentrations of 16 or 17, and cell viability was determined by MTT method, and expressed as a percentage that of the control. Each value represents the mean±S.D. of triplicate assays.

Calculation of chemical descriptors. The 3D structure of each chemical structure (Marvin Sketch ver 16; ChemAxon, Budapest, Hungary, http://www.chemaxon.com) was optimized by CORINA Classic (Molecular Networks GmbH, Nürnberg, Germany) with forcefield calculations (amber-10: EHT) in Molecular Operating Environment (MOE) version 2018.0101 (Chemical Computing Group Inc., Quebec, Canada). The number of structural descriptors calculated from MOE (13) and Dragon 7.0 (14) (Kode srl., Pisa, Italy) was 344 and 5,255, respectively. Among them, the number of descriptors used for analysis was 290 and 2,782 (total 3,072), respectively.

Statistical treatment. The CC50 values were expressed as mean±S.D. of triplicate assays. The relation among cytotoxicity, TS and chemical descriptors were investigated using simple regression analyses by JMP Pro version 13.2.0 (SAS Institute Inc., Cary, NC, USA). The significance level was set at p<0.05.

Results

Cytotoxicity. A total of 20 pyrano[4,3-b]chromone derivatives, consisting of 10 pairs of diastereomers were synthesized (A-series 1-10 and B-series 11-20 in Figure 1).

Replacement of ethoxy group at the C-3 position with butoxy or methoxy and methyl group increased the cytotoxicity of most of these compounds, as evidenced by decreasing CC50 values: 282→146→30 (1-3), >357→155→37 (4-6), 158→153→14 (7-9), 109→58→20 (11-13), 73→32→5 μM (14-16) in OSCC cells (Table I), and >400→>394→257 (1-3), >400→>400→167 (4-6), >399→>396→95 μM (7-9) in human normal oral cells (Table I). The replacement effects were more pronounced against OSCC cell lines than normal oral cells.

View this table:
Table II.

Toxicity of compound 16 against human oral keratinocytes (HOK) and human oral squamous cell carcinoma (OSCC) cell lines as compared with doxorubicin.

The replacement of ethoxy with additional pyran moiety (10, 20), and the introduction of chlorine or methoxyl group (1-3 vs. 4-6 or 7-9; 11-13 vs. 14-16 or 17-19) only slightly affected cytotoxicity (Table I).

Tumor specificity. Among the 20 compounds, 16 had the highest TS (TS=47.8), followed by 17 (TS>28.2). The TS value of other compounds was below 10. TS value of 16 was slightly higher than that of 5-FU and melphalan, and comparable with that of doxorubicin (Table I).

Figure 3.
Figure 3.

Correlation of tumor-specificity (TS) and potency-selectivity expression (PSE) for diastereomer pairs from A (1-10) and B (11-20) series of compounds, plotted per pair: (1, 11), (2, 12), (3, 13), (4, 14), (5, 15), (6, 16), (7, 17), (8, 18), (9, 19) and (10, 20). Fitted curves or lines and r2 values were calculated by Microsoft Excel (Windows 10, Microsoft Corporation, Redmond, WA, USA). Correlations are shown for TS and PSE values for all oral squamous cell carcinoma (OSCC) cell lines versus non-malignant cells (left), and paired cells derived from the same (gingival) tissue (right).

Considering that HGF is the normal cell corresponding to Ca9-22 OSCC cell line (since both derive from gingival tissues), TS values were also calculated by dividing the average CC50 value towards HGF cells by the CC50 value towards Ca9-22 cells (C/A, Table I). The TS values derived in this way for 16 (TS=52.6) and 17 (TS>34.9) were higher than that of melphalan but comparable with those of 5-FU and doxorubicin (Table I).

Compounds 16 and 17 showed cytostatic growth inhibition of OSCC cells (Figure 2). Cytotoxicity of 16 against human oral keratinocytes was approximately 14% of that of doxorubicin (Table II).

PSE. In order to identify the most promising compounds in terms of both good potency and selectively cytotoxicity, the PSE values were calculated. PSE values of 16 and 17 (953.0 and >225.9, respectively) against malignant cells were 125- and 30-fold higher, respectively, than that of 5-FU and 20- and 5-fold higher, respectively, than that of melphalan. For gingival tissue, PSE values of 16 and 17 (1,118.2 and >315.4) were 11-and 3-fold higher, respectively, than that of 5-FU, and 52 and 15-fold higher, respectively, than that of melphalan (Table I). However, PSE values of 16 and 17 was one order lower than that of doxorubicin (Table I).

There was weak correlation between TS for all malignant cells, and for gingival cells (r2=0.2362 and 0.0536, respectively) and PSE values (r2=0.2399 and 0.1451, respectively) between each pair of diastereomers (Figure 3).

Type of cell death induced by 16. When HSC-2 cells were incubated for 24 h with increasing concentrations (5, 10, 20, 40, 80 μM) of 16, cells became gradually enlarged. In contrast, actinomycin D treatment induced cell shrinkage, characteristic of apoptosis (Figure 4A). A shorter incubation time (24 h) was used to detect early changes in cellular metabolism. This caused the difference in the viable cell number between the control and treated cells detected by MTT method to be much smaller.

Figure 4.
Figure 4.

Effect of compound 16 on cell morphology (A), cell-cycle distribution (B) and expression of apoptosis-related proteins (C) in oral squamous cell carcinoma cell line HSC-2. Cells were incubated for 24 h with the indicated concentrations of 16 or 1 μM actinomycin D (Act D) as positive control and then assessed for morphology under light microscopy (EVOS FL; Thermo Fisher Scientific), cell-cycle distribution by cell sorting and apoptosis induction by western blot. Bar=100 μm. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, PARP: poly (ADP-ribose) polymerase.

Cell-cycle analysis demonstrated that actinomycin D, but not 16, produced a sub-G1 cell population that is characteristic of apoptotic cells (Figure 3B). The percentage of G2+M phase cells was gradually reduced (from 10.7% to as low as 6.7%, similar to the level with actinomycin D), as concentrations of 16 increased (Figure 4B).

Western blot analysis demonstrated that 16 did not lead to caspase-3 activation, as evidenced by lack of cleavage of PARP and capspase-3, in contrast to actinomycin D treatment (Figure 4C). These data suggest that 16 did not induce apoptosis.

Computational analysis. We next performed the QSAR analysis of 20 pyrano[4,3-b]chromones in regards to their cytotoxicity against tumor cells and normal cells. Since 554, 638 and 130 chemical descriptors were significantly (p<0.05) correlated with cytotoxicity against tumor cells, cytotoxicity against normal cells, and TS (data not shown), we chose the top six chemical descriptors for QSAR analysis (Figures 5, 6 and 7, and Table III).

The cytotoxicity of 20 pyrano[4,3-b]chromones derivatives against human OSCC cell lines was positively correlated with descriptors R8s (3D shape, size and electric state) (r2=0.661, p<0.0001), J_G (3D shape) (r2=0.607, p<0.0001), RDF055s (3D shape and electric state) (r2=0.595, p<0.0001), R7s (3D shape, size and electric state) (r2=0.570, p=0.0001), HATS7s (3D shape, size and electric state) (r2=0.556, p=0.0002) and RTs (3D shape, size and electric state) (r2=0.552, p=0.0002) (Figure 5).

The cytotoxicity of 20 pyrano[4,3-b]chromones derivatives against human normal oral mesenchymal cells was correlated positively with R6v+ (3D shape and size) (r2=0.768, p<0.0001), R1s (3D shape, size and electric state) (r2=0.658, p<0.0001), R4v (3D shape and size) (r2=0.656, p<0.0001), J_G (r2=0.651, p<0.0001), R4p (3D shape, size and polarizability) (r2=0.636, p<0.0001), and R3v+ (3D shape and size) (r2=0.633, p<0.0001) (Figure 6).

Figure 5.
Figure 5.

Determination of correlation coefficient between chemical descriptors and cytotoxicity of 20 pyrano[4,3-b]chromones against tumor cells. The mean values of the negative log of the concentration of compound that reduced the viable cell number by 50% (CC50) (T) against tumor cells were plotted. CC50: Concentration of compound that reduced the viable cell number by 50%. The following chemical descriptors were used: HATS7s, R7s, R8s, RTs: 3D shape, size and electric state; J_G: 3D shape; RDF055s: 3D shape and electric state.

Figure 6.
Figure 6.

Determination of correlation coefficient between chemical descriptors and cytotoxicity of 20 pyrano[4,3-b]chromones against normal cells. The mean values of the negative log of the concentration of compound that reduced the viable cell number by 50% (CC50) (N) against normal cells were plotted. The following chemical descriptors were used: R3v+, R4v, R6v+: 3D shape and size; R1s: 3D shape, size and electric state; J_G: 3D shape; and R4p: 3D shape, size and polarizability.

Figure 7.
Figure 7.

Determination of coefficient between chemical descriptors and tumor specificity of 20 pyrano[4,3-b]chromones [defined as: cytotoxicity against tumor cells–cytotoxicity against normal cells (T–N)]. The following chemical descriptors were used: R8s: 3D shape, size and electric state; HATS3i, HATS7i, 3D shape, size and ionization potential; HATS3u, HATS7u: 3D shape and size; Mor10i: 3D shape and ionitation potential.

View this table:
Table III.

Properties of descriptors that significantly affected cytotoxicity against tumor cells (T) and normal cells (N), and tumor specificity (T–N).

The TS of pyrano[4,3-b]chromones derivatives was positively correlated with R8s (r2=0.409, p=0.0024), and negatively with HATS7i (3D shape, size and ionization potential) (r2=0.383, p=0.0036), HATS3i (3D shape, size and ionization potential) (r2=0.373, p=0.0042), HATS3u (3D shape and size) (r2=0.353, p=0.0057), HATS7u (3D shape and size) (r2=0.341, p=0.0069), and Mor10i (3D shape and ionization potential) (r2=0.334, p=0.0076) (Figure 7).

Discussion

The present study demonstrated, for the first time, that among 20 pyrano[4,3-b]chromones derivatives, 16 and 17 had the highest tumor specificity (as shown by TS and PSE values), greater than that of 5-FU and melphalan, comparable to that of doxorubicin (Table I). Both 16 and 17 led to cytostatic growth inhibition (Figure 2). It is possible that the presence of the methoxy and methyl group at the C-3 position and chlorine at the C-8 position increased the cytotoxicity of 16 against OSCC cell lines. On the other hand, the presence of the methoxy group at the C-8 position may contribute to increasing the tumor specificity of 17. We confirmed our previous finding that doxorubicin showed potent cytotoxicity against human normal oral keratinocyte cells (5), and that 16 was much less cytotoxic against HOK than doxorubicin (Table II), suggesting that this compound may be an attractive compound for further research.

We found that 16 did not produce a G1 cell population nor did it induce caspase-3 activation, suggesting that 16 does not induce apoptotic cell death. This suggests that there may be no connection between the tumor specificity and apoptosis-inducing activity. There are a variety of types of cell death reported (15). Further study is needed to determine which type of cell death 16 induces in human OSCC cell lines.

QSAR analysis demonstrated that cytotoxicity of 20 pyrano[4,3-b]chromones derivatives against tumor cell lines was significantly positively correlated (p<0.002) with descriptors of 3D shape, size and electric state (Figure 5). Their tumor specificity was also significantly positively correlated (p=0.0024-0.0076) with 3D shape, size and electric state, and negatively correlated with 3D shape, size and ionization potential (Figure 7). Chemical modification using 16 as a lead compound may be a potential choice for designing a new type of anticancer drug.

Acknowledgements

This work was partially supported by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (15K08111, 16K11519).

Footnotes

  • This article is freely accessible online.

  • Conflicts of Interest

    The Authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

  • Received June 8, 2018.
  • Revision received July 1, 2018.
  • Accepted July 5, 2018.

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Quantitative Structure–Cytotoxicity Relationship of Pyrano[4,3-b]chromones
JUNKO NAGAI, HAIXIA SHI, YUKA KUBOTA, KENJIRO BANDOW, NORIYUKI OKUDAIRA, YOSHIHIRO UESAWA, HIROSHI SAKAGAMI, MINEKO TOMOMURA, AKITO TOMOMURA, KOICHI TAKAO, YOSHIAKI SUGITA
Anticancer Research Aug 2018, 38 (8) 4449-4457; DOI: 10.21873/anticanres.12747
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