Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

Abstract

Photodynamic therapy (PDT) uses a photosensitizer, light and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7(+) cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.

Figure 1

The effects of LC3 siRNA transfection on LC3-II levels and PDT-induced cell death. (A) MCF-7v and MCF-7c3 cells were transfected with LC3 siRNA, as described in Materials and Methods. Twenty-four hours later, nontransfected and transfected cells were loaded with 200 nM Pc 4 for 3 h, then irradiated with 200 mJ/cm² of red light and incubated for an additional 2 h or 22 h. Cells were collected, and protein from whole cell lysates was separated on SDS-PAGE gels, transferred to PVDF membranes, and probed with an anti-LC3 antibody. (B) MTT assay. MCF-7v and MCF-7c3 cells grown in 96-well plates were transfected with LC3 siRNA, as described above. After 24 h, the nontransfected and transfected cells were loaded with the indicated doses of Pc 4 for 4 h and then irradiated with 200 mJ/cm² red light and post incubated for 24 h before assay. The viability of cells transfected with siRNA alone was 70.6% and 77.2% of the untreated control cells for MCF-7v and MCF-7c3, respectively. Data are the mean ± standard deviation of results from 3 independent experiments (*p < 0.05, t-test).

Figure 2

Autophagy in PDT-treated Atg7⁺ and Atg7 ⁻ MCF-7 cells (from A. Kelekar). (A) The cells were untreated or treated with 200 nM Pc 4 and 200 mJ/cm² of red light and post-incubated for 20 h. Cells were collected and western blot analysis was performed with anti-Atg7 as well as anti-actin (for a loading control) antibodies. (B) Images of MDC-labeled control and PDT-treated cells. Cells were treated with 0, 25 or 150 nM Pc 4 and 200 mJ/cm² red light and post-incubated for 17 h. At the end of the incubation, cells were stained with MDC for 20 min and examined immediately. (C and D) PDT-induced autophagosome formation in MCF-7/Atg7⁺ and MCF-7/Atg7⁻ cells. Cells were transiently transfected with GFP-LC3 construct, then PDT-treated with 150 nM Pc 4 and 200 mJ/cm² red light and post-incubated for 6 or 24 hours. At the end of the incubation, GFp-positive cells were examined and photographed. Arrows in a representative fluorescence micrograph (C) indicate the autolysosomes/autophagosomes in PDT-treated cells. (D) The percentage of GFp-expressing cells with punctate GFP-LC3. Cells treated as described in (C) were visualized by fluorescence microscopy, and the GFP-expressing cells were counted as punctate or diffuse. The results are mean ± standard deviation of data from three independent experiments (*p < 0.01, t-test). (E) Expression of Beclin 1 in MCF-7 cells. Untreated cells of each MCF-7 sub-line were analyzed by western blot with anti-Beclin 1 and anti-actin (loading control) antibodies. Lane 1, MCF-7/Atg7⁺; lane 2, MCF-7/Atg7⁻; lane 3, MCF-7c3; lane 4, MCF-7v.

Figure 3

Clonogenic survival of PDT-treated Atg7⁺ and Atg7⁻ MCF-7 cells. Cells were incubated with various concentrations of Pc 4 for 18 h, as indicated on the abscissa, then irradiated with 200 mJ/cm² red light, collected and diluted to the appropriate concentrations for plating. Data for PDT-treated cells were normalized to the plating efficiency of the corresponding untreated controls (on average, 49.5% for Atg7⁺ cells and 38.4% for Atg7⁻ cells). Each datum is the mean ± standard deviation of at least triplicate results from three independent experiments (*p < 0.05, t-test).

Figure 4

Atg7 knockdown enhanced PARP cleavage in CPT- or STS-treated, but not in PDT-treated, MCF-7 cells. C: Control. MCF-7 cells (Atg7⁺ and Atg7⁻) were exposed to 200 nM Pc 4 and 200 mJ/cm² red light then further incubated for 24 h or treated with 2 µM CPT for 24 h (A). Cells were also incubated in 1 µM sTs for 6 h (B). At the end of the incubation, whole cell lysates were subjected to electrophoresis and analyzed by western blot for the presence of PARP cleavage, and the levels of LC3 and Atg7.