Progesterone increases apoptosis and inversely decreases autophagy in human hepatoma HA22T/VGH cells treated with epirubicin

Abstract

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. Epirubicin can induce intracellular reactive oxygen species and is widely used to treat unresectable HCC. Progesterone has been found to inhibit the proliferation of hepatoma cells. This study was designed to test the combined effects of epirubicin and progesterone on human hepatoma cell line, HA22T/VGH. These cells were treated with different concentrations of epirubicin with or without the coaddition of 30 μM progesterone and then analyzed for apoptosis, autophagy, and expressions of apoptotic-related proteins and multidrug-resistant gene. Epirubicin treatment dose-dependently inhibited the growth of HA22T/VGH cells. Addition of 30 μM progesterone, which was inactive alone, augmented the effect of epirubicin on the inhibition of growth of HA22T/VGH cells. Cotreatment with progesterone enhanced epirubicin-induced apoptosis, as evidenced by greater increase in caspase-3 activity and in the ratio of the apoptosis-regulating protein, Bax/Bcl-X(L). The combination also caused a decrease in autophagy and in the expression of multidrug resistance-related protein 1 mRNA compared to epirubicin alone. This study shows the epirubicin/progesterone combination was more effective in increasing apoptosis and inversely decreasing autophagy on HA22T/VGH cells treated with epirubicin alone, suggesting that this combination can potentially be used to treat HCC.

Figure 1

Effects of epirubicin on cell growth without or with progesterone addition. HA22T/VGH cells were treated with different concentrations of epirubicin without or with 30 μM progesterone for 24 h (a) or 48 h (b). Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. E: epirubicin; P30: 30 μM progesterone. *P < 0.05 compared to the corresponding untreated controls. ^# P < 0.05 compared to the corresponding epirubicin-treated group.

Figure 2

Apoptosis induction by epirubicin without or with progesterone addition. HA22T/VGH cells were treated with indicated concentrations of epirubicin without or with 30 μM progesterone for 24 h and evaluated by (a) TUNEL staining or (b) caspase-3 activity. Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.03: 0.03 μM epirubicin, E0.1: 0.1 μM epirubicin, E0.3: 0.3 μM epirubicin, and P30: 30 μM progesterone. *P < 0.05 compared to the untreated controls. ^# P < 0.05 compared to the corresponding epirubicin-treated group.

Figure 3

Western blotting shows Bax and Bcl-X_L expressions of HA22T/VGH cells after epirubicin and/or progesterone treatment for 24 h. Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.3: 0.3 μM epirubicin, and P30: 30 μM progesterone. *P < 0.05 compared to the untreated controls. ^# P < 0.05 compared to epirubicin-treated group.

Figure 4

Epirubicin and/or progesterone effect on autophagy induction. HA22T/VGH cells were treated with epirubicin and/or progesterone for 24 h then stained with acridine orange and followed by flow cytometric analysis of autophagy. Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.03: 0.03 μM epirubicin, E0.1: 0.1 μM epirubicin, E0.3: 0.3 μM epirubicin, and P30: 30 μM progesterone. *P < 0.05 compared to the untreated controls. ^# P < 0.05 compared to epirubicin-treated group.

Figure 5

Western blotting shows Beclin-1 and LC3-I expressions in HA22T/VGH cells after epirubicin and/or progesterone treatment for 24 h. Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.3: 0.3 μM epirubicin, and P30: 30 μM progesterone. *P < 0.05 compared to the untreated controls. ^# P < 0.05 compared to epirubicin-treated group.

Figure 6

Epirubicin and/or progesterone effect on MRP-1 expression. HA22T/VGH cells were treated with epirubicin and/or progesterone for 6, 12, or 24 h, and then MRP-1 mRNA was analyzed by RT-PCR. The MRP-1 mRNA expression was normalized to GAPDH mRNA. The density of band was expressed as the relative density compared to that in untreated cells (control), taken as 100%. Results are expressed as the mean ± standard deviation (S.D.) for three separate experiments. C: untreated cells, E0.3: 0.3 μM epirubicin, and P30: 30 μM progesterone. *P < 0.05 compared to the untreated controls. ^# P < 0.05 compared to epirubicin-treated group.