Research ArticleExperimental Studies

Behavior-selective Apoptotic Capacity of 4-(3,4,5-Trimethoxyphenoxy) Benzoic Acid and its Methyl Derivatives on Two Breast Cancer Cell Lines

KUAN-HAN LEE, WEN-YUEH HO, SHU-JING WU, TSUNG-LIN CHENG, PO-JUI HUANG, CLAY C.C. WANG and JUI-HSIANG HUNG
Anticancer Research April 2014, 34 (4) 1801-1809;

Abstract

Breast cancer is one of the most common tumors in females. The therapeutic resistance of breast cancer has motivated the development of new agents for prevention and treatment. For the present study, several compounds were designed and analyzed for their antitumor activity in many cancer cell lines. 4-(3,4,5-Trimethoxyphenoxy) benzoic acid (compound 1) and its derivatives were selected for studying the anti-proliferative and cytotoxic effects on five human cancer cell lines. Results indicated that compounds 1 and 2 significantly suppressed the cell viability of MCF-7 and MDA-MB-468 cancer cells. However, compounds 1 and 2 had only minor effects on HepG2, Huh-7, and Hela cells. Moreover, compounds 1 and 2 exhibited a novel anti-tumor activity through the induction of cell-cycle arrest at G2/M and apoptosis in MCF-7 and MDA-MB-486 breast cancer cells. Both compounds reduced colony-forming ability in MCF-7 cells. Flow cytometric analysis indicated that caspase-3 activity was increased in response to treatment with compounds 1 and 2. Taken together, these findings suggest that the novel compounds 1 and 2 are potential anticancer agents with clinical promise for breast cancer therapy.

Breast cancer is the most frequently-diagnosed type of cancer. It is one of the most common causes of death among females in North America and Europe (1, 2). Many specific factors are associated with increased breast cancer risk, such as gene mutation, radiation, lifestyle, weight, and alcohol intake (3-8). Several drugs have been used for the prevention and the treatment of breast cancer, such as Herceptin and tamoxifen (9, 10). Overexpression of human epidermal growth factor receptor-2 (HER2) gene occurs in approximately 25-30% of breast cancer cases (11, 12). Herceptin, a humanized monoclonal antibody with high affinity for the HER2 protein, has been used in combination chemotherapy for the treatment of HER2-overexpressing metastatic breast cancer (13, 14). Tamoxifen, an antagonist of the estrogen receptor, has been used extensively in the treatment of breast cancer and was approved as a chemopreventive agent for women at-risk for breast cancer (15, 16). However, even with these strategies, the death rate remains unacceptably high. One of the major challenges in the systemic treatment of breast cancer is cellular resistance to conventional cytotoxic agents (17, 18). Therefore, there is a current need for the development of novel chemotherapeutic agents and therapeutic strategies for breast cancer.

In order to develop potent, selective and effective drugs for breast cancer therapy, a series of compounds (compounds 1 to 7) was synthesized for this study. 4-(3,4,5-Trimethoxyphenoxy)benzoic acid, compound 1, was used as a scaffold. 4-(3,4,5-Trimethoxyphenoxy)benzoate derivatives mono-substituted with methyl, ethyl, propyl, butyl, pentyl, or hexyl group were designated as compounds 2-7, respectively. To define their biological activities, these seven structural analogs of compound 1 were prepared, and their anti-tumor activities were analyzed. The present study investigated the inhibitory effects of these compounds 1 on the proliferation and cell death phenotype of five cancer cell lines, HepG2 (hepatocellular carcinoma), Huh-7 (hepatocellular carcinoma), Hela (cervical cancer), MCF-7 (breast cancer) and MDA-MB-468 (breast cancer).

Materials and Methods

Cell culture and chemicals. Huh-7, HepG2, Hela, MCF-7, and MDA-MB-468 cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and these cell lines were maintained at 37°C in a 5% CO2 incubator in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Grand Island, NY, USA). Caspase-3 activity and cell-cycle assay kit were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell viability. Cell viability was assessed using the MTT assay in three replicates. Huh-7, HepG2, Hela, MCF-7, and MDA-MB-468 cells were seeded at 5×103 per well in 96-well flat-bottomed plates and incubated in 10% FBS-supplemented DMEM for 16 h. Cells were treated with 1-5 μM concentrations of different compounds. Controls received the vehicle, dimethyl sulfoxide (DMSO), at a concentration same as that in drug-treated cells. After 72 h, the drug-containing medium was replaced with 20 μl of 10% FBS-supplemented DMEM containing 0.5 mg/ml MTT, and cells were incubated in CO2 incubator at 37°C for 4 h. After the medium was removed, the reduced MTT was solubilized in 100 μl of DMSO per well, and the contents of each well were transferred to 96-well plates for absorbance measurement at 570 nm.

Colony-formation assay. For colony formation, MCF-7 cells were seeded at 3,000 per well in 6-well flat-bottomed plates and incubated in 10% FBS-supplemented DMEM for 16 h. The cells were treated with 1, 2.5 and 5 μM of the different compounds for 24 h. The culture medium was replenished, and cells were maintained at 37°C for 10 days, with the medium changed every other day. Grown colonies were fixed with 3.7% formaldehyde and stained with crystal violet. The number of cell colonies was determined directly in each well.

Cell-cycle assay. To determine cell-cycle distribution, 5×105 cells in 6-cm dishes were treated with different concentrations of compounds for 24 h. After incubation, the supernatant was removed, and the cells were then fixed in 70% ethanol/ phosphate buffered saline (PBS), pelleted, and resuspended in buffer containing RNase A and propidium iodide. Cell-cycle distribution was determined by flow cytometry, and the percentages of cells were determined using WinMDI software (designed by Joe Trotter; La Jolla, CA, USA).

Analysis of caspase-3 activity. Caspase-3 activity was determined using phycoerythrin (PE) active caspase-3 apoptosis kit (BD Pharmingen). Briefly, HepG2 (5×105) cells in 6-cm dishes were subjected to different drug treatments as indicated for 24 h and were re-suspended in 0.5 ml Cytofix/Cytoperm solution for 20 min on ice. Furthermore, cells were incubated in 100 μl of Perm/Wash buffer containing 20 μl caspase-3 antibody for 30 min at room temperature. Each sample was then added to 400 μl Perm/Wash buffer, and caspase-3 activity signals were analyzed by flow cytometry.

Figure 1.
Figure 1.

Compound 1 and its derivatives. The compound number, structure and molecular weight are shown.

Statistical analysis. Results are presented as the mean±SD of three independent experiments in triplicates and analyzed by Student's t-test. Differences were considered significant at p<0.05.

Results

Compounds 1-7 were synthesized and provided by Dr. Wen-Yueh Ho, Dr. Kuan-Han Lee and Dr. Po-Jui Huang (patent application number in Taiwan: TW 100128744; application researchers: Clay C.C. Wang, Kuan-Han Lee, Wen-Yeuh Ho and Jui-Hsiang Hung). Figure 1 lists the name, structure and molecular weight of the synthesized compounds.

To identify the in vitro anti-tumor efficacy of compounds 1-7, proliferative effects on Huh-7, HepG2, Hela, MCF-7, and MDA-MB-468 cells were examined. These five human cancer cell lines were treated with test compounds at the indicated doses. The cell viability was determined by MTT assay. As shown in Figure 2, the 72-h treatment with different compounds resulted in a dose-dependent anti-proliferative effect. The cancer cells showed differential susceptibility to the anti-proliferative effect of those compounds. As shown in Figure 2, HepG2, Huh-7, and Hela cancer cell lines were more resistant to these compounds. However, breast cancer cell lines, MCF-7 and MDA-MB-468, were very sensitive to compounds 1 and 2 (Figure 2A-E). The results indicated that compounds 1 and 2 significantly induced cell death in MCF-7 and MDA-MB-468 cells. Furthermore, compound 2 exhibited better anticancer effect compared to compound 1. The half-maximal inhibitory concentration (IC50) values of compound 1 in MCF-7 and MDA-MB-468 cells were 5.9±3 and 8.7±0.1 μM, respectively, and the IC50 values of compound 2 in both cell lines were 1.4±0.5 and 3.7±0.1 μM, respectively (Table I).

View this table:
Table I.

The IC50 values of compounds 1-7 in five different cell lines. Each value represents the mean (IC50)±SD of three independent experiments.

In order to determine whether compounds affect the colony-forming ability of MCF-7 cells, the number of colonies of MCF-7 cells cultured for 14 days was determined. MCF-7 cells were exposed to 5 μM of compounds 1-7. After incubation, the number of colonies was determined by staining with crystal violet. The data indicated that incubation at 5 μM, compounds 1 and 2 significantly reduced colony-formation ability in MCF-7 cells (Figure 3A). It is noteworthy that compound 2 caused a greater reduction in the number of colonies compared to compound 1 (Figure 3B).

To observe the changes in morphology caused by compounds 1 and 2, MCF-7 cells were treated with compounds 1 and 2 at the indicated doses. Compounds 1 and 2 caused progressive changes in MCF-7 cells from flat to round (Figure 4A). Furthermore, in order to determine the effect of compounds 1 and 2 on cell-cycle distribution in MCF-7 and MDA-MB-468 cell lines, both breast cancer cell lines were incubated with compounds 1 and 2 at the indicated doses. Figure 4B and C show the results of DNA flow cytometric analyses of MCF-7 and MDA-MB-468 cells treated with compound 1 or compound 2 at 0-5 μM for 24 h. As shown in the Figure, both cell lines responded with a dose-dependent accumulation of cells at G2/M phase. As the percentage of cells in G2/M increased, the percentage of cells in G1 phase decreased; the proportion of S-phase cells was not significantly altered by the treatments with compound 1 or 2 in either of the two cell lines. MCF-7 cells showed a dose-dependent accumulation of cells in G2/M phase after 24 h of incubation (Table II). Treatment of MDA-MB-468 cells with 5 μM compound 1 or compound 2 for 24 h resulted in an increase in the percentage of compared to the control cells (Table III). Quantification of cell-cycle phases and accumulation of cells in the G2/M phase in MCF-7 and MDA-MB-468 cells were clearly induced by compounds 1 and 2. Notably, compound 2 caused a higher percentage of G2/M cell accumulation than did compound 1.

View this table:
Table II.

Cell-cycle distribution of MCF-7 cell line using compounds 1 and 2.

View this table:
Table III.

Cell-cycle distribution of MDA-MB-468 cell line using compounds 1 and 2.

The induction of MCF-7 and MDA-MB-468 cell death by compounds 1 and 2 was observed. The effects of compounds 1 and 2 on cell apoptosis and caspase-3 activity were determined. Flow cytometric analysis of caspase-3 activity showed that the treatment with compounds 1 and 2 significantly increased caspase-3 activity in MCF-7 cells (Figure 5A). For example, compounds 1 and 2 caused 3.3-fold and 5.1-fold increases in caspase-3 activity respectively in MCF-7 cells after 5 μM treatment (Figure 5B). Furthermore, to test the effect of compounds 1 and 2 on the colony-forming ability of MCF-7 cell line, the number of colonies in MCF-7 cells cultured for 14 days was determined (Figure 5C). MCF-7 cells were exposed to 1, 2.5 and 5 μM of compounds 1 and 2. After incubation, the number of colonies was determined by crystal violet staining. The data showed that when incubated with compounds 1 and 2, the colony-formation ability of MCF-7 cells significantly decreased in a dose-dependent manner (Figure 5D).

Figure 2.
Figure 2.

Hela, HepG2, Huh-7, MCF-7 and MDA-MB-468 cells exhibited differential susceptibility to compound-induced apoptotic death. The effects of compounds on cell survival rate of Hela (A), HepG2 (B), Huh-7 (C), MCF-7 (D) and MDA-MB-468 (E) cells were determined and cells were maintained in FBS-supplemented DMEM for 3 days, and the number of cells was assessed by the MTT assay. Data are the mean; bars, SD (n=3).

Figure 3.
Figure 3.

Effect of compounds 1-7 on colony-formation ability in MCF-7 cells. A: MCF-7 cells were incubated with compounds 1-7, and colony formation was scored after 14 days. B: The number of colonies in the graphs is representative of three independent experiments. The data represent the mean±SD (n=3). Significant differences (**p<0.01; ***p<0.001) between the control and experimental group are marked with an asterisk.

Discussion

Despite advances in the diagnosis and treatment of breast cancer, more than 44,000 women in the United States will die this year from metastatic breast cancer (19). Development of potential drugs for breast cancer therapy is necessary and urgent. In the current work, several compounds were tested for their activity against different cancer cell lines. Compounds 1 and 2 demonstrated potential for further development into new chemotherapeutic agents for breast cancer. Both breast cancer cells, MCF-7 and MDA-MB-468, are obviously more sensitive to compounds 1 and 2. Moreover, cell-cycle G2/M arrest in MCF-7 and MDA-MB-468 cells were induced by the treatments with compounds 1 and 2. In addition, compounds 1 and 2 reduced the colony-formation ability and increased caspase-3 activity in MCF-7 cells. In addition, the cell death caused by compounds 1 and 2 was mediated, at least in part, via the induction of G2/M cell-cycle arrest, the inhibition of colony formation and the activation of caspase-3.

Figure 4.
Figure 4.

Induction of cell morphology changes and G2/M arrest by compounds 1 and 2. A: Morphological changes in MCF-7 cells after a 24-h treatment with compounds 1 and 2. Cells were imaged by photography under phase-contrast microscopy. B-E: G2/M arrest was induced by compounds 1 and 2 in MCF-7 and MDA-MB-468 cells. FACS analysis of cell-cycle distribution of MCF-7 and MDA-MB-468 cells after treatment with compounds at the dose indicated for 24 h. Untreated cells as controls. F: Comparison of mean proportions of cells in the G2/M phase of the cell cycle after 24 h of exposure to compounds 1 and 2 at thedose indicated. The data represent the mean±SD (n=3). *Significant differences between the control and experimental groups at p<0.05.

Figure 5.
Figure 5.

Induction of cell apoptosis by compounds 1 and 2. A: Flow cytometric analysis of dose-dependent effect of compounds 1 and 2 on caspase-3 activity in MCF-7 cells. B: Relative caspase-3 activity, normalized to that of the DMSO-treated control, at the indicated concentrations of compounds 1 and 2. Columns, mean of three independent experiments; bars, SD (n=3). C: Effect of compounds 1 and 2 on colony formation of MCF-7 scored after 14 days. The number of colonies in the graphs is representative of three independent experiments (lower panel). The data represent the mean±SD (n=3). Significant differences between the control and experimental groups at *p<0.05 ,**p<0.01, and ***p<0.001.

In the current study, various cancer cell lines were used to analyze the effect of compounds 1-7 on cell proliferation. Compounds 1 and 2 exhibited a unique ability to induce apoptosis in MCF-7 and MDA-MB-486 breast cancer cells. Untreated MCF-7 cells exhibited normal proliferation and the cell morphology was flat and broad. On treatment with compounds 1 and 2, the morphology of cells changed significantly, as revealed by the phase-contrast images. Cells treated with compounds 1 and 2 showed cell shrinkage; and a few cells detached from the plate into the medium and became floating cells. In addition, an increase in caspase-3 activity pointed to the occurrence of apoptosis (19, 20). Significant caspase-3 activity observed in MCF-7-treated cells compared with the untreated control suggests that the mechanism of cell death involved the induction of apoptosis. However, the detailed mechanisms of apoptosis by these compounds merit further investigation.

This study used compound 1 as a scaffold to synthesize this series of derivatives. However, after elongation of carbon numbers on 4-(3,4,5-trimethoxyphenoxy)benzoic acid, only compounds 1 and 2 exhibited potential antitumor activity against MCF-7 and MDA-MB-468 cells. The results suggested that extension of more than three carbons on 4-(3,4,5-trimethoxyphenoxy)benzoic acid resulted in the loss of antitumor activity, possibly due to target protein interaction or signal pathway activation/inhibition. On the other hand, in cell survival analysis, breast cancer cells were highly sensitive to compounds 1 and 2, which had only minor effects on Huh-7, HepG2 and Hela cells. Previous studies have indicated that breast carcinogenesis presents multiple processes involving mutations or amplification of oncogenes and inactivation of tumor suppressors, leading to increased invasiveness and dysregulation of cell cycle and apoptosis (21, 22). However, the mechanisms involved in apoptosis of human breast cancer cells after treatment with compounds 1 and 2 require more in-depth exploration. Taken together, the results show that these compounds are particularly active against breast cancer cells, possibly offering a novel and specific chemotherapeutic strategy for breast cancer.

Acknowledgements

This work was supported by a research grant from the National Science Council of Taiwan (NSC 101-2320-B-041-004), and with financial support from the Chia-Nan University of Pharmacy and Science, Tainan, Taiwan to Dr. Jui-Hsiang Hung.

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Received November 10, 2013.
  • Revision received December 3, 2013.
  • Accepted December 5, 2013.

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Behavior-selective Apoptotic Capacity of 4-(3,4,5-Trimethoxyphenoxy) Benzoic Acid and its Methyl Derivatives on Two Breast Cancer Cell Lines
KUAN-HAN LEE, WEN-YUEH HO, SHU-JING WU, TSUNG-LIN CHENG, PO-JUI HUANG, CLAY C.C. WANG, JUI-HSIANG HUNG
Anticancer Research Apr 2014, 34 (4) 1801-1809;
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