Impact of calcium channel antagonists for estrogen action on the endometrial carcinoma HEC-1A cells

Abstract

To study the effects of nifedipine and mibefradil on the cell proliferation, apoptosis, migration on the HEC-1A in vitro and also study the mRNA and protein expression levels of the calcium channel alpha1D (Cav1.3) and calcium channel alpha1G (Cav3.1) to discuss the effects of the calcium antagonists on the mechanisms of the endometrial carcinoma. (1) Add 10 µmol/L nifedipine and mibefradil at 15 minutes before adding 10 µmol/L 17β-estradiol (17β-E(2)) and 100 µmol/L b-estradiol-6-(O-carboxymethyl)oxime (E(2)-BSA) to the HEC-1A in different time including 0, 5, 15, 30, 60, 120 minutes. Then the changes of mRNA and protein were detected by reverse transcripiton (RT)-PCR and western-blot. (2) Add 1.25, 2.5, 5, 10, 20, 40, 80, 100 µmol/L nifedipine and mibefradil to the HEC-1A at 24, 48, 96 hours to detect the cell proliferation by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. (3) Add 10 µmol/L nifedipine and mibefradil to the HEC-1A, then detect the apoptosis at 0 minute, 30 minutes, 1 hour, 6 hours, 24 hours and migration in vitro at 36 hours with transwell methods. (1) After the pretreated effect of the nifedipine before 17β-E(2), the mRNA express of Cav1.3 genes was lowest at 15 minutes, and returned to the control level after 30 minutes. The protein level didn't change very much in 30 minutes, but rose after 60 minutes. The Cav3.1 genes mRNA express was lowest at 5 minutes, rose at 30 minutes and returned to the 0 minute level gradually. (2) After the pretreated effect of the nifedipine before E(2)-BSA, the Cav1.3 genes mRNA was lowest at 5 minutes and returned at 15 minutes. The protein level rose gradually in 15 minutes but reduced after 15 minutes. The Cav3.1 mRNA and protein level were reduced at every time point. (3) After the pretreated effect of the mibefradil before 17β-E(2), there was no change of mRNA expression of Cav1.3 genes. The protein level rose at 15 and 60 minutes, there was no change in any other time. The Cav3.1 genes mRNA were gradually reduced and the protein level rose at 15 minutes, and there was no change in any other times. (4) After the pretreated effect of the mibefradil before E(2)-BSA, the mRNA and protein of Cav1.3 levels were reduced after 15 minutes. There was no mRNA expression of Cav3.1, while the protein level was lowest at 15 minutes. (5) Nifedipine and mibefradil affected HEC-1A proliferation depended on the different concentration and interval time points. There was significant difference than those in control group (P < 0.05). (6) There were statistical differences in apoptosis rate after adding nifedipine (P < 0.05), while rose at mibefradil treated the same time (24 hours: 8.41 ± 0.07, 0 minute: 3.74 ± 0.18; P < 0.05). (7) The numbers of stained cells after both nifedipine and mibefradil treated reduced more than control group. (1) Nifedipine and mibefradil could inhibit both the effect of the estrogen on the L-type and T-type calcium channel in short time, meanwhile the mibefradil effects last long time. (2) The inhibited effect of the mibefradil on the proliferation, apoptosis and migration of HEC-1A cells in vitro is more significant than that by nifedidipine.