Compound

K-1 ( Figure 1 ) was synthesized according to the methods as described earlier.

Chemical structure of compound K-1

Chandra. Inhibition of hyperplasia formation in rat uterus by benzopyran derivative. Am J Obstet Gynecol 2011.

Animal preparation and treatment schedule

Young adult rats (Sprague Dawley strain) of body weight of 150 g of the institute colony were used in this study. Animals were housed under uniform animal husbandry conditions (24 ± 1°C) with free access to pelleted food and water.

All animal procedures were carried out as per the guidelines provided by the institute’s Animal Ethics, Use, and Care Committee. Prior approval was obtained from the Institutional Animal Ethics Committee for animal experimentation.

Rats were ovariectomized bilaterally under ether anesthesia and given a rest period of 2 weeks. Following the rest period, rats were divided into various groups (6-8 rats per group): group I received olive oil and gum acacia as vehicle, group II received estradiol (20 μg/kg body weight, in olive oil, subcutaneously), groups III and IV received, in addition to estradiol, K-1 at 100 μg and at 200 μg/kg body weight doses, respectively (in gum acacia, orally), and groups V and VI received only K-1 at 100 μg/kg body weight and 200 μg /kg body weight, respectively. All treatments were given for 14 days. Animals were killed 24 h after the last treatment.

Uteri were collected, weighed, and stored at –80°C until analysis. For ribonucleic acid (RNA) isolation, tissue was collected in RNA later at room temperature for 24 h and then stored at –80°C. A midportion of a single horn of each uteri was preserved in 4% paraformaldehyde for histological and histomorphometric analysis.

Histomorphometric analysis

Formalin-fixed uterine tissues were sectioned, stained with hematoxylin/eosin, and examined under light microscope (Nikon 80i; Nikon, Tokyo, Japan). Images were captured with NIS-Elements F 3.0 camera (Nikon) and analyzed using Leica QWin software (Wetzlar, Germany). Endometrial area (EA), luminal area (LA), luminal epithelial cell height (LEH), gland number, glandular area (GA), stromal area, and glandular/stromal area ratio (G/S) as an indicator for cellular hypertrophy were calculated by averaging the measurements at 3 locations in 3 different hematoxylin and eosin–stained uterine tissue sections for each individual animal.

Western blot analysis

Uterine tissue was homogenized in an ice-cold phosphate buffer (50 mM) containing 10 mM sodium molybdate, 50 mM sodium fluoride, 1 mM EDTA, 400 mM sodium chloride, 12 mM monothioglycerol, 2 mM phenylmethylsulfonyl fluoride, leupeptin (2 μg/mL), and protease inhibitor cocktail (50 μg/g tissue) (Sigma Aldrich, St Louis, MO) using ultraturrax homogenizer. The homogenate was incubated on ice for 1 hour with occasional shaking and centrifuged at 16,000 × g for 15 minutes at 4°C.

Protein concentration in supernatant was determined by Bradford assay. Sample containing 35 μg protein was boiled for 10 minutes in denaturing sample buffer consisting of 10% glycerol, 1% sodium dodecyl sulfate, 1% β-mercaptoethanol, 10 mM Tris-HCl (pH 6.8), and 0.01% bromophenol blue; separated on 12% acrylamide gels; and transferred to Immuno-Blot polyvinyl difluoride membrane (Millipore, Bedford, MA).

Nonspecific sites were blocked with 5% skimmed milk for 2 hours at room temperature and then incubated overnight at 4°C with primary antibody at 1:5000 dilution ER, progesterone receptor (PR), proliferating cell nuclear antigen (PCNA), and cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA) or at 1:1000 dilution for cleaved caspase-3, cleaved caspase-9, cleaved poly(adenosine diphosphate–ribose) polymerase (PARP), S437p-Akt, Akt, X-linked inhibitor of apoptosis protein (XIAP), c-Fos, c-Jun, c-erbB2, c-myc, β-catenin, Bax, and Bcl-2 (Cell Signaling Technology, Beverly, MA). Subsequently the blots were washed 3 times in 0.1% Tween 20 in Tris-buffered saline (TBS) and then incubated with 1:10,000 dilution of secondary antibody (horseradish peroxidase conjugate) for 1 hour at 25°C.

After extensive washing with 0.1% Tween 20 in TBS, substrate solution was added to the membrane, incubated for 5-15 seconds, and exposed at room temperature. The membranes were developed with an enhanced chemiluminescence kit, following the manufacturer’s instructions (GE Healthcare, Indianapolis, IN). For normalization, the membranes were stripped using buffer containing 62.5 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, and 100 mM β-mercaptoethanol and then reprobed with anti-β-actin antibody (Santa Cruz Biotechnology). Quantitation of band intensity was performed by using Quantity One software (version 4.5.1; Bio-Rad Laboratories, Hercules, CA).

Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)

TUNEL staining was performed by using an in situ cell death detection kit (Roche, Stockholm, Sweden) to demonstrate the apoptosis in uterine sections. The manufacturer’s protocol was followed for paraffin-embedded sections. The slides were mounted in 3:1 Vectashield (Vector Laboratories, Burlingame, CA): 4′,6-diamino-2-phenylindole (Invitrogen, Carlsbad, CA) and sealed. The sections were examined under light microscope (Nikon 80i), and images were captured using an NIS-Elements F 3.0 camera (Nikon).

Caspase-3 colorimetric assay

One of the major steps in apoptotic cell death is the activation of caspases. Caspase-3 activity was measured using colorimetric caspase-3 assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, whole uterine extract (approximately 50 μg protein) was incubated for 2 hours at 37°C in the presence of 1 mM caspase-3 substrate (DEVD-pNA), and the optical density was measured at 405 nm and activity was expressed as fold changes.

Statistical analysis

Results are expressed as mean ± SEM for the number of experiments indicated. Statistical analysis was performed using analysis of variance followed by Newman Keul’s test. Differences were considered significant at P < .05.