Anti-Cancerous Effect of 4,4'-Dihydroxychalcone ((2 E ,2' E )-3,3'-(1,4-Phenylene) Bis (1-(4-hydroxyphenyl) Prop-2-en-1-one)) on T47D Breast Cancer Cell Line

Aims: The majority of human breast tumors are estrogen receptor α (ERα) positive. However, not all of the ERα+ breast cancers respond to anti-estrogens drugs for those women who do respond, initial positive responses can be of short duration. Thus, more effective drugs are needed to enhance the efficacy of anti-estrogens drugs or to be used separately in a period of time. In view of potential cytotoxicity associated with silybin as polyhydroxy compounds a synthetic 4-hydroxychalcones (bis-phenol) was considered to explore its anti-carcinogenic effects in Conclusion: Our results strongly suggests that this premade synthetic 4,4'-dihydroxychalcone can promote anti carcinogenic actions on T47D cell line. All 4,4'-dihydroxychalcone doses had a much larger inhibitory effect on cell viability than silybin doses in T47D cells. The ratio of the IC 50 of 4,4'-dihydroxychalcone to silybin after 24 and 48 hours was 1: 2.3 and 1: 2.8 respectively.


INTRODUCTION
Breast cancer is the leading cause of female cancer death worldwide [1]. Normal breast epithelial cells and estrogen receptors (ERs) expressing breast cancer cells require estrogens to stimulate proliferation. Estrogen can regulate the transcription of its responsive genes by acting through the ERs, which in turn direct cellular proliferation. Therefore, patients with ER-positive breast cancers often undergo adjuvant treatment with antiestrogen tamoxifen [2]. A wide variety of anti-cancer drugs, including fulvestrant, cisplatin, doxorubicin and vinblastine are available. Although treatments of breast cancer patients with these anti-cancer drugs have shown good success, tumor resistance remains a major obstacle [3]. Clearly, more effective compounds are needed to enhance the efficacy of anticancer drugs in resistant breast tumors [4].
From a chemical viewpoint, chalcones consist of two aromatic rings linked by a three-carbon unit forming an α,β-unsaturated carbonyl moiety. They are pharmacologically relevant because of their ability to exert anti-carcinogenic, antimicrobial, anti-diabetic and antiinflammatory activities. Among chalcones, synthetic and naturally hydroxychalcones are of particular interest as they display a wide range of biological properties and exert diverse pharmacological activities. 4-Hydroxychalcones have demonstrated their effectiveness as cytotoxic, anti-tumor, anti-oxidative, anti-bacterial, anti-fungal, anti-leishmanial and antinociceptive agents [5][6][7][8][9].
Studies have also indicated that the core structure of the chalcones, 1,3-diphenyl-2propenone is able to potently inhibit proliferation of MCF-7 and MDA-MB-231 on human breast cancer cell lines by inducing apoptosis and blocking cell cycle progression in the G2/M phase [10]. Many reports have documented that chalcones are biologically active. For example, xanthoangelol was reported to induce apoptosis and inhibit tumor promotion and metastasis in several cancer cell lines [11,12]. Licochalcone-A, isoliquiritigenin and flavokawain A also have been indicated to induce apoptosis and cell cycle arrest in various cancer cells [13,14,15].

Tumor Cell Line and Culture Conditions
T47D is a human breast ductal carcinoma cell line with ER and PR over expression [25,26]. A T47D cell line was purchased from the National Cell Bank, Pasteur Institute of Iran. The cell line was cultured in RPMI 1640 medium (Invitrogen, Darmstadt, Germany ) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (all from PAA, Pasching, Austia), 2 g/l sodium bicarbonate and 2.5 g/l HEPES (Sigma-Aldich, Missouri, USA). T47D cells were grown under standard culture conditions (37°C, 95% humidified air and 5% CO2). For cell harvesting, 0.25% solution of trypsin (Sigma-Aldich, Missouri, USA) in PBS was used.

Sodium salt of 4, 4'-dihydroxychalcone
To a 10 ml round bottom flask was added 1 ml 96% EtOH and 8 mg NaOH. To the resulting mixture was added 37 mg of 4,4'-dihydroxychalcone and stirred for 30 min. After that the solvent was removed and the sodium salt of 4,4'-dihydroxychalcone (38 mg) was recovered.

Chemical Treatments and MTT Assay
For the MTT assay, the cells were first seeded in three 96-well microplates. Each well contained 100 µl complete growth medium and 7×10 3 cells were seeded. The next day, the

Statistical Analysis
Data were analyzed using SPSS 18 software. One-way ANOVA and Dunnett-t two-sided post hoc tests were employed to evaluate the statistical significance of differences between the control and all treatments. The P values that were considered significant are displayed (***: P < 0.001 in Fig. 1).

Inhibitory Effects of 4,4'-dihydroxychalcone in T47D Cell Line and Comparing with Silybin
The cytotoxicity effects of 4,4'-dihydroxychalcone in five doses (25,50,75,100,150 µM) for 24, and 48 hours were evaluated by MTT assay on the T47D cell line (Fig. 2). Briefly, 7×10 3 cells were seeded in 96 well plates for 24 hour, and treated with different doses in a complete medium (no serum starvation). Cell viability graphs were depicted by SPSS 18 (clustered bar, summaries for group of case). The cell viability of the T47D breast cancer cells in 24 and 48 hours for four doses (50,75,100,150 µM) of 4,4'-dihydroxychalcone and silybin is presented in Table 1. All doses reduced cell viability and were considered statistically significant (P<0.001). Data is presented as percentage of viability in three independent experiments. Each experiment had three individual samples (Error bars: +/-1 SD). The IC50 of 4,4'-dihydroxychalcone on T47D cells after 24 and 48 hours (Fig. 3) was 160.88+/-1 µM, 62.20+/-1 µM and for silybin was 373.42+/-1 µM,176.98+/-1 µM respectively.  On the contrary in other efforts sodium salt of 4,4'-dihydroxychalcone showed no significant cell growth inhibitory by the MTT test. The sodium salt of 4,4'-dihydroxychalcone was not effective as the 4,4'-dihydroxychalcone. These results indicate that lipophilicity is an important factor making compounds more bioavailable to cell membrane. Thus, cell viability graph for sodium salt of 4,4'-dihydroxychalcone was ignored for the ineffectual effects on T47D cells. The MTT test results shown less IC50 and significantly higher cell growth inhibitory effects for 4,4'-dihydroxychalcone over silybin (Table 1).

CONCLUSION
The comparison of 4,4'-dihydroxychalcone and silybin by the MTT assay indicates that all 4,4'-dihydroxychalcone doses had a much larger inhibitory effect on cell viability than silybin doses in T47D cells. The ratio of the IC50 of 4,4'-dihydroxychalcone to silybin after 24 and 48 hours was 1:2.3 and 1:2.8, respectively.