Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

Abstract

Background: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood.

Methods: In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (DeltaPsim) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis.

Results: Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of DeltaPsim and the activation of caspase-3.

Conclusion: These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

Figure 1

Cell viability was measured by CCK-8 assay via concentration- and time-dependent manners in vitro. a ASTC-a-1cells were seeded into 96-well flatbottomed microplate and incubated with various concentrations of DHA for 48 h. b Cells were seeded into 96-well flatbottomed microplate and incubated with 20 μg/ml of DHA for the indicated times. Data analyzed with SPSS10.0 software are expressed as mean ± SD. n = 4 wells for all CCK-8 assay. *P < 0.05, **P < 0.01, compared with control.

Figure 2

Apoptosis induced by DHA in ASTC-a-1cells. The cells were treated with 20 μg/ml of DHA for 48 h. Nuclear morphology was detected by Hoechst33258 staining and examined by fluorescence microscope. a Control cells with dispersive light blue nuclei and intact structure. c With 20 μg/ml of DHA treated, cell shrinkage, chromatin condensation and margination in the nucleus were visualized as well as STS treatment for 6 h (b: positive control). d The same cells as panel c showing that the plasma membrane remained well defined. e PI didn't penetrate into DHA-treated cells, demonstrating that DHA caused cell apoptosis but not necrosis. f DIC morphology of control cells was determined and photographed by the confocal microscope. (h, i) 10, 20 μg/ml DHA treated respectively: cell shrinkage, ovalisation, membrane frill, blebbing and the boundary between nucleus and cytoplasm became blurred.a-d: Magnification × 400; e-i: Scale Bar: 20 μm.

Figure 3

After 20 μg/ml DHA treatment for 24 and 48 h, control cells, STS-treated cells and DHA-treated cells were stained with 5 μl of annexinV-FITC and 10 μl propidium iodide (10 μg/ml) for 10 min before being subjected to flow cytometry analysis. a Untreated cells displayed a low background of staining with either Annexin V or PI. b Positive control (1 μM STS-treated cells for 6 h). (c, d) In contrast, Annexin V- and PI-positive cells were dramatically increased in a time-dependent manner under the coincubation with 20 μg/ml DHA for 24 h (c) and 48 h (d).

Figure 4

DHA-induced apoptosis was attenuated by Z-VAD.fmk and Caspase-3 activation was determined using FRET receptor photobleaching technique in single living cells. a The effect of DHA and STS (as a positive control) on apoptosis attenuated by Z-VAD.fmk was determined using CCK-8 assay. The ASTC-a-1cells were treated with 20 μg/ml DHA and with or without 10 μM of Z-VAD.fmk for 24 and 48 h, respectively. The result was expressed as mean ± SD of three determinations. n = 3 wells for CCK-8 assay. *P < 0.05, **P < 0.01, compared with control. (b, c, d) Fluorescence images of cells stably expressing SCAT3. The circled parts were the bleaching area. (e, f, g) FRET receptor photobleaching assay determined the activation of caspase-3 during DHA- and STS-induced apoptosis in single cells. e Control. f Positive control (1 μM STS-treated cells for 6 h). g After the treatment of 20 μg/ml DHA for 48 h, fluorescence intensity of CFP channel was nearly invariable during the Venus photobleaching, implying that SCAT3 had been cleaved by the activated caspase-3.b-d: Scale Bar: 10 μm.

Figure 5

DHA induces dose- and time-dependent activation of caspase-3 in living cells which was attenuated by Z-VAD.fmk (10 μM). a The emission spectral analysis of the SCAT3 in living cells stably expressing SCAT3 after 0, 5, 10, 20 μg/ml DHA treatment for 48 h. The emission peak at 524 nm gradually decreased after 5 and 10 μg/ml DHA treatment, and completely disappeared after 20 μg/ml DHA treatment, while the peak at 481 nm increased to the maximum, implying that DHA activated caspase-3 which cleaved SCAT3. b 20 μg/ml DHA was adopted to determine the time-dependent activation of caspase-3 for indicated time (0, 12, 24, 48 h). The Fluorescence spectrum of SCAT3 didn't have an obvious change at 12 h, but the emission peak at 524 nm decreased at 24 h and completely disappeared after 48 h DHA treatment. d The inhibition of DHA-induced caspase-3 activation was triggered by Z-VAD.fmk. The reoccurrence of FRET effect was indicative of the inhibition of DHA-mediated caspase-3 activation under the co-treatment of DHA and Z-VAD.fmk as well as 1 μM STS treatment (c: as a positive control) for 6 h.

Figure 6

DHA induced caspase-3 activation in ASTC-a-1 cells. Cells were incubated with 1 μM STS for 12 h, 20 μg/m1 DHA for 48 h and co-treatment with 20 μg/m1 DHA and 10 μM Z-VAD.fmk for 48 h, and then were analyzed by caspase-3 activity and Western Blot assay. a Caspase-3 activity was measured using the fluorescence substrate Ac-DEVD-AFC as described in Materials and Methods. The activation index was determined as the ratio between the activity in treated cells and untreated cells. Data analyzed with SPSS10.0 software is representative of three identical experiments. **P < 0.01, compared with control cells; ^##P < 0.01, compared with only DHA-treated cells. b The cells were treated with STS as a positive control showed the decreased proform and the increased cleavage of caspase-3. DHA-treated cells also showed the loss of the proform and appearance of cleaved caspase-3 compared with control. Moreover, DHA-induced caspase-3 activation was blocked under the co-treatment with DHA and Z-VAD.fmk.

Figure 7

Treating ASTC-a-1 cells with DHA induced alteration of mitochondrial morphology. Cells were loaded with Mito-tracker-red and then treated with 20 μg/ml DHA for analysis.a The mitochondria within the untreated control cells showed small-defined mitochondria. b The mitochondria of DHA-treated cells were obviously swollen. The right-hand panels represent enalrged views of the boxed regions within the left-hand panels, as indicated. (c, d) The spatial distribution of mitochondrial sizes indicated as the white line in a and b, respectively. e For every condition tested, the width of 100–200 mitochondria in at least 50 different cells was determined. Data analyzed with SPSS10.0 software are expressed as mean ± SD. *P < 0.05, **P < 0.01, compared with control. Scale Bar: 10 μm

Figure 8

Loss of mitochondrial membrane potential (ΔΨ_m) was involved in DHA-triggered apoptosis. a The dynamics of the loss of the mitochondrial membrane potential were monitored by the potential-sensitive dye Rho123 under confocal microscope in DHA-treated ASTC-a-1 cells for 48 h. Scale Bar: 10 μm. b Individual traces of two typical cells treated with DHA. c Mean trace of five cells calculated from single cell kinetics by setting the time of onset of the decrease of fluorescence intensity to zero. d The precentage of cells with rodamine 123 fluorescence were 94%, 74%, 62.1% and 28.9% at 0, 12, 24 and 48 h after 20 μg/ml DHA treatment, respectively, implying that DHA induced a time-dependent loss of ΔΨ_m.