Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK, p38 MAPKs and Akt signaling pathways

Abstract

Lung cancer is the leading cause of cancer death worldwide. Recently, two plant-derived drugs triptolide (TP) and hydroxycamptothecin (HCPT) both have shown broad-spectrum anticancer activities. Our previous study documented that combination treatment with these two drugs acted more effectively than mono-therapy, however, the molecular basis underlying the synergistic cytotoxicity remains poorly understood. In this study, we aimed to clarify the molecular mechanism of TP/HCPT anticancer effect in A549 lung adenocarcinoma cells, by investigating the involvement of phosphatase 2A (PP2A) and PP2A-regulated mitogen-activated protein kinases (MAPKs) and Akt signaling pathways. The results showed that TP and HCPT synergistically exerted cytotoxicity in the growth of A549 cells. Combinatorial TP/HCPT treatment significantly enhanced the activation of caspase-3 and -9, Bax/Bcl-2 ratio, release of cytochrome c from mitochondrial and subsequent apoptosis. While the Akt survival pathway was inhibited, ERK and p38 MAPKs were dramatically activated. Furthermore, the activity of PP2A was significantly augmented. Regulation of p38, ERK and Akt by PP2A was demonstrated, by using a specific PP2A inhibitor okadaic acid (OA). Finally, pharmacological inhibitors OA, SB203580, SP600125 and PD98059 confirm the role of PP2A and its substrates ERK, p38 MAPK and Akt in mediating TP/HCPT-induced apoptosis. Taken together, this study provides the first evidence for a synergistic TP/HCPT anticancer activity in A549 cells and also supports a critical role of PP2A and PP2A-regulated signaling pathways, providing new insight into the mode of action of TP/HCPT in cancer therapy.

Figure 1

Chemical structures of the compounds. (A) Triptolide (TP). (B) 10-hydroxycamptothecin (HCPT).

Figure 2

Combinatorial effects of TP and HCPT on A549 cells. (A) Individual effect of TP or HCPT on cellular growth. A549 cells were treated with graded concentrations of TP (6.25, 12.5, 25, 50, 100, 200 and 400 ng/ml, respectively) or HCPT (0.25, 0.5, 1, 2, 4, 8 and 16 μg/ml, respectively) for 24 h. Cellular growth was measured by MTS assay. Data are expressed as the means ± standard error (SE) (n=4). (B) The combined effects of TP and HCPT on cellular growth. A549 cells were treated with HCPT (0–8 μg/ml) combined with or without TP (25 ng/ml) for 24 h, and the cell proliferation was monitored. Data are expressed as the means ± SE (n=4).

Figure 3

Effect of TP and HCPT combination treatment on the induction of apoptosis. (A) Flow cytometry analyses of A549 cells under various treatments using double staining with Annexin V (horizontal line) and propidium iodide (PI, vertical line); TP, 25 ng/ml TP; HCPT(H), 4 μg/ml HCPT; TP/HCPT(L), 25 ng/ml TP + 1 μg/ml HCPT; TP/HCPT(M), 25 ng/ml TP + 2 μg/ml HCPT; TP/HCPT(H), 25 ng/ml TP + 4 μg/ml HCPT. (B) Bar graphs representing the proportion of early stage apoptosis cells (Annexin V-positive/PI-negative). Data are expressed as the means ± SE (n=3). ^*p<0.05 and ^**p<0.01 vs. TP group; ^#p<0.05 and ^##p<0.01 vs. HCPT group.

Figure 4

Effect of TP/HCPT treatment on the apoptosis-associated proteins in A549 cells. A549 cells under various treatments were harvested for examining the associated protein levels. (A) Mitochondrial apoptotic pathway detected by antibodies against caspase-3, -9, cytochrome c, Bax and Bcl-2. (B) ER stress apoptotic pathway detected by antibodies against caspase-12, GRP78 and CHOP. β-actin was used as an internal control.

Figure 5

Effect of TP/HCPT treatment on the MAPKs and Akt signaling pathways as well as PP2A activity in A549 cells. (A) The cell lysates from A549 cells under various treatments were subjected to immunoblot analysis with antibodies that detect either the specified protein or the specified protein when it is phosphorylated at a designated site, which are representative of at least 3 separate experiments. (B) Phosphatase activity of PP2A was determined with phosphatase assay system (see Materials and methods). The values are expressed as a percentage of the control (untreated cells; 100% PP2A activity). a, ^*p<0.05 vs. the control group; b, ^*p<0.05 vs. TP group; c, ^*p<0.05 vs. HCPT group (n=4). (C) Alterations of various PP2A subunit levels detected by antibodies against the PP2A-A subunit, PP2A-C subunit and phospho-PP2A Cα (Tyr307). β-actin was used as an internal control.

Figure 6

Effect of PP2A inhibition on ERK, p38 and Akt signaling pathways, as well as TP/HCPT-induced apoptosis. (A) TP/HCPT-enhanced PP2A activity was inhibited by OA. A549 cells were also treated with OA (50 nM) for 2 h prior to 24 h of combinatorial treatment with TP (25 ng/ml) and HCPT (4 μg/ml). Cell lysates were prepared and assayed for phosphatase activity of PP2A. The values are expressed as a percentage of the control (untreated cells; 100% PP2A activity). ^**p<0.01 vs. the TP/HCPT group (n=3). (B) Effect of PP2A inhibition on ERK, p38 and Akt signaling pathways. Cell lysates after different treatments were prepared and subjected to western blot analysis. The results are representative of three independent experiments, and the corresponding densitometric analyses for relative protein expression were shown in the right hand panels. (C) Effect of inhibitors of pp2A, p38, ERK and Akt on TP/HCPT-induced apoptosis. The cells were treated with OA (50 nM), p38 inhibitor-SB 203580 (10 μM), ERK inhibitor-PD 98059 (10 μM) and Akt inhibitor-LY294002 (25 μM) for 2 h prior to 24 h of TP/HCPT treatment. Then the cells were collected and analyzed using Annexin V/PI double staining, and bar graphs shown in the right hand panel representing the apoptotic rates. ^*p<0.05 and ^**p<0.01 vs. the TP/HCPT group (n=3).

Figure 7

Schematic diagram of the mechanism by which TP together with HCPT induce apoptosis in A549 lung adenocarcinoma cells. TP and HCPT synergistically stimulate the activation of tumor suppressor PP2A, and modulate PP2A-regulated intracellular signaling pathways, involving activation of ERK1/2 and p38 MAPKs cascades, and inhibition of Akt survival pathway. Finally, combinatorial TP/HCPT drug regimens cause Bax- and Bcl-2-mediated mitochondrial apoptotic pathways, resulting in caspase-9 and -3 triggered cell apoptosis.