Protein kinase C involvement in aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell

Abstract

1. This study demonstrated aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell lines CH27 (human lung squamous carcinoma cell) and H460 (human lung non-small cell carcinoma cell). Aloe-emodin- and emodin-induced apoptosis was characterized by nuclear morphological changes and DNA fragmentation. 2. During apoptosis, an increase in cytochrome c of cytosolic fraction and activation of caspase-3, identified by the cleavage of its proform, were observed. 3. To elucidate whether the expression of protein kinase C (PKC) isozymes are involved in aloe-emodin- and emodin-induced apoptosis, this study examined the changes of PKC isozymes by Western blotting techniques during aloe-emodin- and emodin-induced apoptosis. 4. The expression of PKC isozymes involved in aloe-emodin- and emodin-induced apoptosis of CH27 and H460 cells. In this study, aloe-emodin and emodin induced the changes of each of PKC isozymes in CH27 and H460 cells. 5. The decrease in the expression of PKC delta and epsilon may play a critical role in aloe-emodin- and emodin-induced apoptosis in CH27 and H460 cells. 6. The present study also demonstrated that PKC stimulation occurs at a site downstream of caspase-3 in the emodin-mediated apoptotic pathway.

Figure 1

Effects of aloe-emodin and emodin on cell death in CH27 and H460 cells. Cells were cultured 24 h before drug treatment in 12-well plates. CH27 (A) and H460 (B) cells were treated with 0.1% DMSO, aloe-emodin or emodin in the presence of 1% serum at 37°C for different times (2, 4, 8, 16, 24, 48 and 72 h), and cells were washed and counted by Trypan blue exclusion with hemocytometer. All determinations are expressed as the mean percentage of control±s.d.mean of triplicate from three independent experiments.

Figure 2

Aloe-emodin and emodin-induced phenotypic changes in CH27 and H460 cell nucleus. CH27 (A) and H460 (B) cells were cultured for 16 h in 1% serum medium with 0.1% DMSO, 40 μM aloe-emodin or 50 μM emodin. After treatment, cells were fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 and stained with 1 μg ml⁻¹ DAPI for 5 min at 37°C. The cells were then washed with PBS and examined by fluorescence microscopy. Results are representative of three independent experiments. Bar=50 μm.

Figure 3

Aloe-emodin and emodin-induced internucleosomal DNA fragmentation in CH27 and H460 cell nucleus. CH27 (A) and H460 (B) cells were incubated with 0.1% DMSO (lane 1), 40 μM aloe-emodin (lane 2), or 50 μM emodin (lane 3) for 24 h in 1% serum medium. Total DNA was extracted from cells and separated by electrophoresis on 1.5% agarose gel. The molecular weight marker lane (lane M) represents DNA base pairs. Results are representative of three independent experiments.

Figure 4

Aloe-emodin- and emodin-induced the appearance of a sub-G₁ peak in CH27 and H460 cells by flow cytometry assay. CH27 (A) and H460 (B) cells were treated with vehicle alone (0.1% DMSO), 40 μM aloe-emodin, or 50 μM emodin in the presence of 1% serum for 24 h. After treatment, cells were harvested and subjected to cytometric analysis. Apoptosis was measured by cell cycle analysis with propidium iodide staining and the percentage of hypodiploid cells (apoptotic population of cells) was calculated. Results are representative of three independent experiments.

Figure 5

Effects of aloe-emodin and emodin on the release of cytochrome c and the expression of caspase-3 and PARP in CH27 and H460 cells. The effect of aloe-emodin and emodin on cytochrome c (Cyt. c), caspase-3 (Cas-3) and PARP was detected by Western blot analysis in CH27 (A) and H460 (B) cells. Cells were incubated with 40 μM aloe-emodin or 50 μM emodin in the presence of 1% serum for 2, 4, 8, 16 and 24 h. Cell lysates were analysed by 8% (PARP), 12% (caspase-3) and 15% (cytochrome c) SDS – PAGE, and then probed with primary antibody as described in Materials and methods. Results are representative of three independent experiments.

Figure 6

Effects of aloe-emodin and emodin on the PKCδ and ε in CH27 and H460 Cells. The effect of aloe-emodin and emodin on PKCδ and ε was detected by Western blot analysis in CH27 (A) and H460 (B) cells. Cells were incubated with 40 μM aloe-emodin or 50 μM emodin in the presence of 1% serum for 2, 4, 8, 16 and 24 h. Cell lysates were analysed by 10% SDS – PAGE, and then probed with antibodies against peptides specific for PKCδ and ε. Western blot analysis with a mAb to detect PKCδ and ε was performed as described in Materials and methods. Results are representative of three independent experiments.

Figure 7

Effects of Ac-DEVD-CHO on aloe-emodin- and emodin-induced the expression of PKCδ in CH27 and H460 Cells. Cells were cultured for 3 h in medium that contained Ac-DEVD-CHO (100 μM, DEVD). Then, the cells were treated with 40 μM aloe-emodin (A) or 50 μM emodin (B) for 2, 8 and 16 h. Cell lysates were analysed by 10% SDS – PAGE, and then probed with antibody against PKCδ. Western blot analysis with a mAb to detect PKCδ was performed as described in Materials and methods. Results are representative of three independent experiments.