Research ArticleExperimental Studies

Quantitative Structure–Cytotoxicity Relationship of Cinnamic Acid Phenetyl Esters

YOSHIHIRO UESAWA, HIROSHI SAKAGAMI, NORIYUKI OKUDAIRA, KAZUHIRO TODA, KOICHI TAKAO, HAJIME KAGAYA and YOSHIAKI SUGITA
Anticancer Research February 2018, 38 (2) 817-823;

Abstract

Background/Aim: Many phenolic acid phenethyl esters possess diverse biological effects including antioxidant, cytoprotective, anti-inflammation and anti-tumor activities. However, most previous antitumor studies have not considered the cytotoxicity against normal cells. Ten cinnamic acid phenetyl esters were subjected to quantitative structure–activity relationship (QSAR) analysis, based on their cytotoxicity and tumor-specificity, in order to find their new biological activities. Materials and Methods: Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor specificity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by CC50 against tumor cells. Apoptosis markers were detected by western blot analysis. Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by force-field minimization. Results: Western blot analysis demonstrated that [9] stimulated the cleavage of caspase-3, suggesting the induction of apoptosis. QSAR analysis demonstrated that TS values were correlated with shape, size and ionization potential. Conclusion: Chemical modification of the lead compound may be a potential choice for designing a new type of anticancer drugs.

Many phenolic acid phenethyl esters possess diverse biological effects including antioxidant activity (evaluated by radical scavenging activity) (1-4), cytoprotective activity (against oxidative stress) (5-7), anti-inflammation activity (evaluated by inhibition of 5-lipoxygenase and leukotriene biosynthesis) (8) and anti-tumor activity against implanted tumors (9) in mice and various tumor cell lines (10-12). However, most previous antitumor studies in vitro have not assayed the cytotoxicity against control normal cells. We have established the simple in vitro assay system of antitumor potential, using four human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) and three human normal oral cells (gingival fibroblast, HGF; periodontal ligament fibroblast, HPLF; pulp cell, HPC) (13, 14). Using this system, we have demonstrated that many anticancer drugs (camptothecin, SN-38, etoposide, doxorubicin, daunorubicin, mitomycin C, methotrexate, 5-fluorouracil, docetaxel, melphalan, gefitinib) showed excellent tumor-specificity (15). Among a total of 133 compounds, (E)-3-[2-(4-hydroxyphenyl)ethenyl]-6-methoxy-4H-1-benzopyran-4-one (classified as 3-styrylchromone) (16, 17), (E)-3-[2-(4-chlorophenyl)ethenyl]-7-methoxy-2H-1-benzopyran (classified as 3-styryl-2H-chromenes) (18) showed the highest tumor specificity with the least keratinocyte toxicity (14).

In continuation of discovering new biological activities of phenylpropanoid derivatives, a total of ten cinnamic acid phenetyl esters (Figure 1) (4) were investigated for their cytotoxicity against four human OSCC cell lines and four human normal oral cells, and then subjected to quantitative structure–activity relationship (QSAR) analysis.

Figure 1.
Figure 1.

Structure of ten cinnamic acid esters.

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), doxorubicin from Sigma-Aldrich Inc., St. Louis, MO, USA; dimethyl sulfoxide (DMSO). Culture plastic dishes and plates (96-well) were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Synthesis of test compounds. (2E)-3-(4-Hydroxy-3-methoxyphenyl)-2-propenoic acid 2-(3,4-dihydroxyphenyl)ethyl ester [1], (2E)-3-(4-hydroxyphenyl)-2-propenoic acid 2-(3,4-dihydroxyphenyl)ethyl ester [2], (2E)-3-(3,4-dihydroxyphenyl)-2-propenoic acid 2-(3,4-dihydroxyphenyl)ethyl ester [3], (2E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 2-(4-hydroxyphenyl)ethyl ester [4], (2E)-3-(4-hydroxyphenyl)-2-propenoic acid 2-(4-hydroxyphenyl)ethyl ester [5], (2E)-3-(3,4-dihydroxyphenyl)-2-propenoic acid 2-(4-hydroxyphenyl)ethyl ester [6], (2E)-3-phenyl-2-propenoic acid 2-(4-hydroxyphenyl)ethyl ester [7], (2E)-3-(4-hydroxyphenyl)-2-propenoic acid 2-phenylethyl ester [8], (2E)-3-(3,4-dihydroxyphenyl)-2-propenoic acid 2-phenylethyl ester [9], (2E)-3-phenyl-2-propenoic acid 2-phenylethyl ester [10] were synthesized by the condensations of cinnamic acid derivatives with selected phenethylalcohol derivatives, according to previous methods (4). 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; human pulp cells, HPC) were established from the first premolar tooth extracted from the lower jaw of a 12-year-old girl (19), and cells at 10-18 population doubling levels were used in this study. Human oral squamous cell carcinoma (OSCC) cell lines [Ca9-22 (derived from gingival tissue); HSC-2, HSC-3, HSC-4 (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.

Assay for cytotoxic activity. Cells were inoculated at 2.5×103 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 further for 48 h and the relative viable cell number was then determined by the MTT method (15, 17, 18). The relative viable cell number was determined by the absorbance of the cell lysate at 560 nm, using a microplate reader (Infinite F 50 R, TECAN, Kawasaki, Japan). 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-selectivity index (TS). TS was calculated using the following equation: TS = mean CC50 against three normal cells/mean CC50 against for four OSCC cell lines [(D/B) in Table I]. Since both Ca9-22 and HGF cells were derived from the gingival tissue (20), the relative sensitivity of these cells was also compared [(C/A) in Table I]. We did not use human normal oral keratinocytes as controls, since many anticancer drugs showed potent cytotoxicity against normal keratinocytes by inducing apoptosis (15).

Calculation of potency-selectivity expression (PSE). PSE was calculated using the following equation: PSE=TS/CC50 against tumor cells ×100 (10) [that is, (D/B2) ×100 (HGF, HPLF, HPC vs. Ca9-22, HSC-2, HSC-3, HSC-4) and (C/A2) ×100 (HGF vs. Ca9-22 in Table I).

Estimation of CC50 values. Since the CC50 values had a distribution pattern close to a logarithmic normal distribution, we used the pCC50 (i.e., the −log CC50) for the comparison of the cytotoxicity between the compounds. The mean pCC50 values for normal cells and tumor cell lines were defined as N and T, respectively (18).

Calculation of chemical descriptors. The 3D-structure of each chemical structure (drawn by Marvin Sketch ver 16, ChemAxon, Budapest, Hungary, http://www.chemaxon.com) was optimized by CORINA Classic (Molecular Networks GmbH, Germany) and force-field calculations (amber-10: EHT) in Molecular Operating Environment (MOE) version 2015.1001 (Chemical Computing Group Inc., Quebec, Canada). The number of structural descriptors calculated from MOE and Dragon 7.0 (Kode srl., Pisa, Italy) after the elimination of overlapped descriptors were 284 and 2750, respectively.

View this table:
Table I.

Cytotoxic activity of ten cinnamic acid esters against oral malignant and non-malignant cells. Each value represents the mean of triplicate determinations.

The following 14 Dragon descriptors (21) and 2 MOE descriptors (22) listed in Table II were significantly correlated with T, N and T-N. Western blot analysis. The cells were washed with PBS and processed for western blot analysis, as described previously (15). Antibodies against cleaved caspase-3 (Cell Signaling Technology Inc., Beverly, MP, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Trevigen, Gaithersburg, MD, USA) were used as primary antibodies. As secondary antibodies, we used α-rabbit IgG (DAKO, Tokyo, Japan) antibodies which were conjugated with horseradish peroxidase.

Statistical treatment. The relation among cytotoxicity, tumor specificity index and chemical descriptors was investigated using simple regression analyses by JMP Pro version 12.2.0 (SAS Institute Inc., Cary, NC, USA). The significance level was set at p<0.05.

Results

Cytotoxicity. We have synthesized a total of ten cinnamic acid phenetyl esters, without or with different numbers of substituted methoxy or hydroxyl group (Figure 1). Backbone structure is (2E)-3-phenyl-2-propenoic acid 2-phenylethyl ester [10], that showed the lowest cytotoxicity against both OSCC (Ca9-22, HSC-2, HSC-3, HSC-4) and normal oral cells (HGF, HPLF, HPC) (mean of CC50=196.6 and >399.3 μM) (Table I). Introduction of hydroxy group caused increased their cytotoxicity to varying degrees. Compounds [3, 6, 9], that have two hydroxy group at R1 and R2 positions of benzene ring, showed the highest cytotoxicity against OSCC cell lines (CC50=23.5, 25.1 and 8.5 μM) (Table I).

Figure 2.
Figure 2.

Apoptosis induction by [9] in HSC-2 human oral squamous cell carcinoma cell line. Act.D: Actinomycin D (1 μM).

Tumor-specificity. Tumor-specificity (TS) were calculated by dividing the mean CC50 value towards three normal cells by the mean CC50 value towards four OSCC cell lines (D/B, Table I). Since compounds [3, 6, 9] showed one order less cytotoxicity against human oral normal cells (CC50=339.6, 254.1 and 198.7 μM, respectively), they showed the highest tumor specificity (TS=14.4, 10, 23.4) (Table I).

Figure 3.
Figure 3.

Determination of coefficient between chemical descriptors and cytotoxicity of ten cinnamic acid esters against tumor cells (defined as T). The mean (pCC50 i.e., the −log CC50) values for tumor cell lines were defined as T.

View this table:
Table II.

Source and explanation of chemical descriptors that correlate with cytotoxicity to tumor cells, normal cells and tumor-specificity.

Considering that HGF is the normal cell corresponds to cancer cell Ca9-22 (both derived from gingival tissues), TS values were also generated by dividing the average CC50 value towards HGF cells by the CC50 value towards Ca9-22 cells (C/A, Table I). Compounds [3, 6, 9] showed TS value of 5.6, 8.5 and 11.4, respectively, comparable with doxorubicin, anthracycline-type anticancer drug (Table I).

PSE value. In order to identify the most promising compounds in terms of both good potencies and selective cytotoxicity, the potency-selectivity expression (PSE) values were calculated. Among [3, 6, 9], [9] having two hydroxyl groups in R1 and R2, but not in R3 and R4, showed the highest cytotoxicity (CC50=8.5 μM). As expected, [9] yielded the greatest PSE values: 275.1 and 85.2 (calculated as D/B2 ×100 and C/A2 ×100, respectively, in Table I), one or two orders higher than that of other compounds [1, 2, 4, 5, 7, 8, 10] (Table I).

Figure 4.
Figure 4.

Determination of coefficient between chemical descriptors and cytotoxicity of ten cinnamic acid esters against normal cells (defined as N). The mean (pCC50 i.e., the −log CC50) values for normal cells were defined as N.

Western blot analysis demonstrated that [9] stimulated the cleavage of caspase-3, suggesting the induction of apoptosis (Figure 2).

Computational analysis. We next performed the QSAR analysis of ten cinnamic acid phenetyl esters in regards to their cytotoxicity against tumor cells and normal cells. Among a total of 3,034 descriptors (dragon, 2,750 descriptors; MOE, 284 descriptors), 14 dragon descriptors and 2 MOE descriptors correlated well with cytotoxicity and tumor specificity (Table III).

Cytotoxicity of ten cinnamic acid phenetyl esters against OSCC cell lines was correlated with SpMin2_Bh(s) (Electronic state) (r2=0.765, p=0.0009), R7s+ (Shape & electoric state (r2=0.740, p=0.0014), TDB07m (Shape & size) (r2=0.739, p=0.0014), RDF070v (Shape & Size) (r2=0.735, p=0.0015), h_pKb (pKb) (r2=0.730, p=0.0016) and CATS3D_06_DL (H bond & lipophilicity) (r2=0.729, p=0.0017) (Figure 3).

View this table:
Table III.

Properties of descriptors that significantly affects the cytotoxicity against tumor cells (T), normal cells (N) and tumor-specificity (T-N).

Cytotoxicity of ten cinnamic acid phenetyl esters against normal oral mesenchymal cells was correlated with CIC3 (Symmetry) (r2=0.609, p=0.0077), CIC2 (Symmetry) (r2=0.608, p=0.0078), SIC3 (Symmetry) (r2=0.596, p=0.0088), BIC3 (Symmetry) (r2=0.562, p=0.0125), VE2_B(s) (Shape & electoric state) (r2=0.531, p=0.0169) and VE1sign_Dt (Shape) (r2=0.492, p=0.0239) (Figure 4).

Figure 5.
Figure 5.

Determination of coefficient between chemical descriptors and tumor specificity of ten cinnamic acid esters (defined as T-N).

Tumor specificity of ten cinnamic acid phenetyl esters was correlated with R7s+ (Shape & electoric state) (r2=0.840, p=0.0002), PEOE_VSA+1 (Size & partial charge) (r2=0.784, p=0.0007), TDB10i (Shape & ionization potential) (r2=0.774, p=0.0008), h_pKb (pKb) (r2=0.729, p=0.0017), E2m (Shape & size) (r2=0.719, p=0.0019) and H7s (Shape & electoric state) (r2=0.716, p=0.002) (Figure 5).

Discussion

The present study demonstrated that among ten cinnamic acid phenetyl esters, compounds [3, 6, 9], that have two hydroxy group at R1 and R2 positions of benzene ring, showed the highest cytotoxicity against OSCC cell lines (CC50=23.5, 25.1 and 8.5 μM) (Table I). We found that when hydroxyl group in R1 of these compounds was replaced with methoxy group, their cytotoxic activity was reduced by 2.8-fold (66.6/23.5), 4.2-fold (104.8/25.1) and 12.0-fold (102.4/8.5), respectively (Table I). This suggests the importance of hydroxyl group at R1 of benzene in expressing higher cytotoxicity induction against OSCC cell lines.

Especially, [9], having two hydroxyl groups in R1 and R2, but not in R3 and R4, showed the highest tumor-specific cytotoxicity, as evidenced by the greatest TS and PSE values (Table I), and also apoptosis-inducing activity against OSCC cell line (Figure 2). Since a wide range of anticancer drugs (camptothecin, SN-38, etoposide, doxorubicin, daunorubicin, mitomycin C, methotrexate, 5-fluorouracil, docetaxel, melphalan, gefitinib) induced potent keratinocyte toxicity, albeit their much lower cytotoxicity against human normal oral mesenchymal cells (15), it is urgent to investigate whether [9] shows such keratinocyte toxicity before going into further mechanistic study.

QSAR analysis demonstrated that tumor-specificity of ten cinnamic acid phenetyl esters was correlated with shape, size and ionization potential. Chemical modification of [9] may be a potential choice for designing a new type of anticancer drugs.

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 November 20, 2017.
  • Revision received December 4, 2017.
  • Accepted December 5, 2017.

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Quantitative Structure–Cytotoxicity Relationship of Cinnamic Acid Phenetyl Esters
YOSHIHIRO UESAWA, HIROSHI SAKAGAMI, NORIYUKI OKUDAIRA, KAZUHIRO TODA, KOICHI TAKAO, HAJIME KAGAYA, YOSHIAKI SUGITA
Anticancer Research Feb 2018, 38 (2) 817-823;
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