• PARTHASARADHI MATHI^1*,
• et al.: Comparing Anticancer Properties Piceatannol¹,
• Resveratrol Resveratrol¹,
• Biochanin A¹

1. ¹ Pharmacognosy Magazine, Volume 15, Issue 66, October-December 2019 (Supplement 3) S411 INTRODUCTION Biochanin A is one of the dietary constituents found in soy, peanut, and chick pea.[1] In in vivo, this compound can be metabolized to genistein (angiogenesis inhibitor).[2] Piceatannol is a metabolic derivative of well‑known anti‑angiogenic compound resveratrol. Both (biochanin A and piceatannol) compounds were isolated from Sophora interrupta.[3] Biochanin A has shown to possess anticarcinogenic, anti‑proliferative, and anti‑inflammatory activity in different types of cancers such as the breast, pancreas, lung, and melanoma. Biochanin A has shown to possess chemopreventive efficiency against breast cancer[4] and to increase the tumor latency period, decreased the promotion of tumors, and decreased tumor multiplicity in rodents with chemically‑induced mammary carcinogenesis.[5] It was found that biochanin A selectively targets human epidermal growth factor receptor 2‑positive SK‑BR‑3 breast cancer cells without affecting normal breast epithelial cells (Michigan Cancer Foundation [MCF]‑10A), and fibroblast cells (NIH‑3T3).[6] Apart from breast cancer, this compound has also shown to play a potential role in the chemoprevention of prostate cancer through the enhancement of tumor necrosis factor‑related inducing ligand‑mediated apoptosis in LNCaP and DU145 prostate cancer cells,[7,8] shown to inhibit the activity of Protein Kinase-B (AKT) and MAPK pathways in pancreatic cancer cells, inhibition of nuclear factor‑kappa B (NF‑κB) and MAPK signaling pathway[9] in SK‑Mel‑28 melanoma cancer cell lines. Piceatannol is a type of phenolic compound and belongs to the class of stilbenes.[10] Stilbenes (C6–C2–C6) are derived from the common phenylpropene (C6–C3) skeleton building block. Piceatannol has been found in various plants, including grapes, passion fruit, white tea, and Japanese knotweed.[11] Piceatannol is a metabolite of resveratrol and has shown to possess antitumor, antioxidant, and anti‑inflammatory activities.[12] The key difference between resveratrol and piceatannol is the presence of an extra hydroxyl group at the C3 position of the aromatic rings.[13] Identical compound (s) were found in Salvia yunnanensis[14] and led to suppress the expression of vascular endothelial growth factors (VEGF) in the ECV304 cell line, it suggests that compounds from these family members possibly inhibit angiogenesis.[10] Piceatannol can inhibit cell proliferation by arresting the cell cycle in G0 and G1 phases in liver cancer cells and leukemic cells.[15] Piceatannol suppresses breast cancer cell invasion through the inhibition of Matrix metallopeptidase 9 (MMP-9); involvement of Phosphoinositide 3-kinase (P13K)/AKT, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)[16] Resveratrol is a dietary polyphenol with nutraceutical properties toward suppressing cancer cell growth in tumor microenvironment.[17] Resveratrol is a well‑known anticancer compound with fewer side effects. Resveratrol has shown to suppress the proliferation of uterine cancer cells by inhibiting the WNT signaling pathway, breast cancer by inhibiting estrogen metabolism, pancreatic cancer by sensitizing DNA repair pathways, by upregulating cellular apoptotic missionary through PI3k/AKT signaling pathway, ovarian cancer cells by downregulation of Notch/PTEN/AKT signaling, gastric cancer cells by the inhibition of metastasis‑associated lung adenocarcinoma transcript 1‑mediated epithelial to mesenchymal transition (EMT).[18‑22] EMT is the hallmark for cancer metastasis.[23] Metastatic cells have shown to undergo an altered metabolic pathway for procuring energy requirements for cell growth. Resveratrol has shown to alter the metabolic transformation by attenuating autophagy in cancer cells.[24] MATERIALS AND METHODS Cell viability and cell culture assay Cells were cultured using established protocol.[25] In brief, 5 × 106 cells of MCF‑7 cells were seeded in a 96 well plate and were maintained in Dulbecco’s modified eagle’s medium and F12K medium, respectively. Both the cell lines were supplemented with 10% fetal bovine serum (heat‑inactivated) and 1% antibiotic (100 U/ml of penicillin and 100 µg/mL streptomycin) gently mixed and placed in a 5% CO2‑humidified incubator at 37°C. To study the anticancer activity of biochanin A, 5000 cells of breast (MCF‑7) origin were seeded in a 96 well plate, cells were treated with increasing concentrations of biochanin A (1, 10, 50, 100, 250, 300, 500, and 1000 µg/ml) and dimethyl sulfoxide (DMSO) as a control for 24 h. Following incubation, 15 µl of 3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazo‑ lium bromide (MTT) (5 mg/ml) reagent was added to the culture media and further incubated for 4 h at 37°C in CO2 incubator. After an incubation period, MTT containing supernatant was aspirated, 200 µl of DMSO and 25 µl of Sorenson glycine buffer (0.1 M glycine and 0.1 M NaCl, pH 10.5) were added to lyse the cells and solubilize the water‑insoluble formazan crystals. Absorbance values of the lysates were determined on a Fluostar Optima microplate reader (BMG Labtech, Germany) at 570 nm. The percentage of inhibition was calculated as: % cell viability = Mean OD of vehicle‑treated cells Mean OD of drug‑treated cells Mean OD of vehicle‑treated cells 100 The inhibitory concentration (IC50) values were calculated using a GraphPad prism, version 5.02 software (GraphPad Software Inc., CA, U.S.A). Negative controls were maintained with DMSO. In a separate experiment, the effects of three extracts on cells were confirmed to observe the morphological changes such as cell shape. The size was captured using a phase contrast microscope (Zeiss, Axiovert 25, Germany). Acridine orange/ethidium bromide staining To validate the cellular permeability of the dyes, 0.5 × 106 cells of MCF‑7 and PC‑3 cells in a 6 well plate were seeded and cultured. Following 24 h of incubation, the media was replaced with fresh media consisting of biochanin‑A and further allowed for incubation of 24 h at 37°C in a 5% CO2 incubator. The cells were washed with phosphate buffer saline (PBS), added 100 µl of acridine orange (AO) and ethidium bromide (EtBr) (50 µg/ml each), respectively, to each well, and incubated for 15 min in CO2 incubator. Following incubation, the medium was aspirated and washed thrice with PBS. The intensity of fluorescent staining was observed, and the images were captured with the help of a fluorescent microscope (Zeiss, Axiovert 25, Germany) using appropriate color filters. In vitro scratch assay To measure the cell proliferation/motility, performed in vitro scratch assay using MCF‑7 cells. In brief, MCF‑7 cells were seeded in a 6 well microtiter plate until about 90% confluent. The media was then removed, and equal size “scratch” was created using a pipette tip and then rinsed with PBS (phosphate buffered saline) to remove detached cells. The medium with the indicated concentrations of biochanin‑A was then added for 24 h incubation in the presence of resveratrol as standard to control alteration in cell proliferation. The microscopic observations of the cells were recorded at 0, 6, 12, and 24 h after treatment. The images were captured using a fluorescent microscope (Zeiss, Axio‑vert 25, Germany) under ×10 and analyzed using T‑Scratch software v 7.8. Fourier‑transform infrared spectrometer The data were collected and processed by analyst ChemStation software, and also Fourier‑transform infrared (FT‑IR) spectrometer equipped with (Lithium tantalite detector). The sample was introduced in Hygroscopic KBr glass windows were exactly 100 µL sample. FT‑IR spectra were obtained by collecting 100 scans with spectra was collected.

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