Increased ceramide accumulation correlates with downregulation of the autophagy protein ATG-7 in MCF-7 cells sensitized to photodamage

Abstract

The purpose of this study was to determine the sphingolipid (SL) profile in cells defective in autophagy protein ATG-7 and overall cell death after photodynamic therapy (PDT) with the photosensitizer Pc 4. MCF-7 human breast cancer cells with downregulated ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to increased cell killing. PDT evoked an early (2h) greater global increase in ceramides in ATG-7 defective cells compared to Scr cells. The total increases in dihydroceramide (DHceramide) were significant at 2 and 24h in both cell types post-PDT. The levels of sphingosine-1-phosphate (S1P) and sphingosine were decreased below resting levels at both time points irrespective of the cell type. The data imply that ceramide might be a marker of ATG-7 deficiency in cells sensitized to PDT.

Figure 1

De novo ceramide biosynthesis and metabolism.

Figure 2

ATG-7 is downregulated in ATG-7 knockdown cells. Western blot analysis of ATG-7 expression in Scr and ATG-7 knockdown cells is shown. Untreated cells were collected and processed for Western. The protein loading was 20 μg per well in this and other Western blots. Scr, scrambled control.

Figure 3

LC3 processing in two cell types after PDT. Cells were incubated overnight with Pc 4 prior to irradiation, incubated for 24 h, collected, and processed for Western blot analysis.

Figure 4

Global ceramide accumulation is enhanced in ATG-7 cells at 2 h post-PDT. The levels of DHCeramides and S1P in two cell types at 2 h post-PDT. The levels of ceramides in two cell types at 24 h past-PDT. The levels of DHCeramides nad S1P in two cell types at 24 h post-PDT. Global increase in ceramides in ATG-7 cells at 2 h post-PDT. Changes in the levels of ceramides and DHCeramides in two cell types at 24 h post-PDT. Mass spectrometric analysis reveals signature effects on the SL profile after PDT in the two cell types. Controls include untreated and Pc 4-treated cells. Cells were incubated overnight with Pc 4 (100 nM) prior to irradiation (400 mJ/cm²), incubated for 2 or 24 h, collected, and processed for MS. In the plots, for Y axis log scale is used. The boxes contain 50% of the data and the median value is shown as the horizontal thick line within the box. The whiskers extend to the most extreme data point (minimum and maximum value) which is no more than 1.5 times the interquartile range (i.e., remaining 50% of the data) from the box. Outliers are shown as empty circles. The horizontal line at a value of 1.0 indicates no change. (A and B) Show the ratios of PDT vs. controls for ceramides and DHceramides at 2 h, respectively. (C and D) Show the ratios of PDT vs. controls for ceramides and DHceramides at 24 h, respectively. Corresponding ceramide and DHceramide species are designated as C14, C16, etc. (E and F) Show the ratios of ATG-7 vs. Scr for all ceramides and DHceramides after PDT. The average ratio ATG-7/Scr at 2 h post-PDT is significantly different from 1 (p<0.004) and is indicated by an asterisk.

Figure 4

Global ceramide accumulation is enhanced in ATG-7 cells at 2 h post-PDT. The levels of DHCeramides and S1P in two cell types at 2 h post-PDT. The levels of ceramides in two cell types at 24 h past-PDT. The levels of DHCeramides nad S1P in two cell types at 24 h post-PDT. Global increase in ceramides in ATG-7 cells at 2 h post-PDT. Changes in the levels of ceramides and DHCeramides in two cell types at 24 h post-PDT. Mass spectrometric analysis reveals signature effects on the SL profile after PDT in the two cell types. Controls include untreated and Pc 4-treated cells. Cells were incubated overnight with Pc 4 (100 nM) prior to irradiation (400 mJ/cm²), incubated for 2 or 24 h, collected, and processed for MS. In the plots, for Y axis log scale is used. The boxes contain 50% of the data and the median value is shown as the horizontal thick line within the box. The whiskers extend to the most extreme data point (minimum and maximum value) which is no more than 1.5 times the interquartile range (i.e., remaining 50% of the data) from the box. Outliers are shown as empty circles. The horizontal line at a value of 1.0 indicates no change. (A and B) Show the ratios of PDT vs. controls for ceramides and DHceramides at 2 h, respectively. (C and D) Show the ratios of PDT vs. controls for ceramides and DHceramides at 24 h, respectively. Corresponding ceramide and DHceramide species are designated as C14, C16, etc. (E and F) Show the ratios of ATG-7 vs. Scr for all ceramides and DHceramides after PDT. The average ratio ATG-7/Scr at 2 h post-PDT is significantly different from 1 (p<0.004) and is indicated by an asterisk.

Figure 4

Global ceramide accumulation is enhanced in ATG-7 cells at 2 h post-PDT. The levels of DHCeramides and S1P in two cell types at 2 h post-PDT. The levels of ceramides in two cell types at 24 h past-PDT. The levels of DHCeramides nad S1P in two cell types at 24 h post-PDT. Global increase in ceramides in ATG-7 cells at 2 h post-PDT. Changes in the levels of ceramides and DHCeramides in two cell types at 24 h post-PDT. Mass spectrometric analysis reveals signature effects on the SL profile after PDT in the two cell types. Controls include untreated and Pc 4-treated cells. Cells were incubated overnight with Pc 4 (100 nM) prior to irradiation (400 mJ/cm²), incubated for 2 or 24 h, collected, and processed for MS. In the plots, for Y axis log scale is used. The boxes contain 50% of the data and the median value is shown as the horizontal thick line within the box. The whiskers extend to the most extreme data point (minimum and maximum value) which is no more than 1.5 times the interquartile range (i.e., remaining 50% of the data) from the box. Outliers are shown as empty circles. The horizontal line at a value of 1.0 indicates no change. (A and B) Show the ratios of PDT vs. controls for ceramides and DHceramides at 2 h, respectively. (C and D) Show the ratios of PDT vs. controls for ceramides and DHceramides at 24 h, respectively. Corresponding ceramide and DHceramide species are designated as C14, C16, etc. (E and F) Show the ratios of ATG-7 vs. Scr for all ceramides and DHceramides after PDT. The average ratio ATG-7/Scr at 2 h post-PDT is significantly different from 1 (p<0.004) and is indicated by an asterisk.

Figure 4

Global ceramide accumulation is enhanced in ATG-7 cells at 2 h post-PDT. The levels of DHCeramides and S1P in two cell types at 2 h post-PDT. The levels of ceramides in two cell types at 24 h past-PDT. The levels of DHCeramides nad S1P in two cell types at 24 h post-PDT. Global increase in ceramides in ATG-7 cells at 2 h post-PDT. Changes in the levels of ceramides and DHCeramides in two cell types at 24 h post-PDT. Mass spectrometric analysis reveals signature effects on the SL profile after PDT in the two cell types. Controls include untreated and Pc 4-treated cells. Cells were incubated overnight with Pc 4 (100 nM) prior to irradiation (400 mJ/cm²), incubated for 2 or 24 h, collected, and processed for MS. In the plots, for Y axis log scale is used. The boxes contain 50% of the data and the median value is shown as the horizontal thick line within the box. The whiskers extend to the most extreme data point (minimum and maximum value) which is no more than 1.5 times the interquartile range (i.e., remaining 50% of the data) from the box. Outliers are shown as empty circles. The horizontal line at a value of 1.0 indicates no change. (A and B) Show the ratios of PDT vs. controls for ceramides and DHceramides at 2 h, respectively. (C and D) Show the ratios of PDT vs. controls for ceramides and DHceramides at 24 h, respectively. Corresponding ceramide and DHceramide species are designated as C14, C16, etc. (E and F) Show the ratios of ATG-7 vs. Scr for all ceramides and DHceramides after PDT. The average ratio ATG-7/Scr at 2 h post-PDT is significantly different from 1 (p<0.004) and is indicated by an asterisk.

Figure 4

Global ceramide accumulation is enhanced in ATG-7 cells at 2 h post-PDT. The levels of DHCeramides and S1P in two cell types at 2 h post-PDT. The levels of ceramides in two cell types at 24 h past-PDT. The levels of DHCeramides nad S1P in two cell types at 24 h post-PDT. Global increase in ceramides in ATG-7 cells at 2 h post-PDT. Changes in the levels of ceramides and DHCeramides in two cell types at 24 h post-PDT. Mass spectrometric analysis reveals signature effects on the SL profile after PDT in the two cell types. Controls include untreated and Pc 4-treated cells. Cells were incubated overnight with Pc 4 (100 nM) prior to irradiation (400 mJ/cm²), incubated for 2 or 24 h, collected, and processed for MS. In the plots, for Y axis log scale is used. The boxes contain 50% of the data and the median value is shown as the horizontal thick line within the box. The whiskers extend to the most extreme data point (minimum and maximum value) which is no more than 1.5 times the interquartile range (i.e., remaining 50% of the data) from the box. Outliers are shown as empty circles. The horizontal line at a value of 1.0 indicates no change. (A and B) Show the ratios of PDT vs. controls for ceramides and DHceramides at 2 h, respectively. (C and D) Show the ratios of PDT vs. controls for ceramides and DHceramides at 24 h, respectively. Corresponding ceramide and DHceramide species are designated as C14, C16, etc. (E and F) Show the ratios of ATG-7 vs. Scr for all ceramides and DHceramides after PDT. The average ratio ATG-7/Scr at 2 h post-PDT is significantly different from 1 (p<0.004) and is indicated by an asterisk.

Figure 4

Global ceramide accumulation is enhanced in ATG-7 cells at 2 h post-PDT. The levels of DHCeramides and S1P in two cell types at 2 h post-PDT. The levels of ceramides in two cell types at 24 h past-PDT. The levels of DHCeramides nad S1P in two cell types at 24 h post-PDT. Global increase in ceramides in ATG-7 cells at 2 h post-PDT. Changes in the levels of ceramides and DHCeramides in two cell types at 24 h post-PDT. Mass spectrometric analysis reveals signature effects on the SL profile after PDT in the two cell types. Controls include untreated and Pc 4-treated cells. Cells were incubated overnight with Pc 4 (100 nM) prior to irradiation (400 mJ/cm²), incubated for 2 or 24 h, collected, and processed for MS. In the plots, for Y axis log scale is used. The boxes contain 50% of the data and the median value is shown as the horizontal thick line within the box. The whiskers extend to the most extreme data point (minimum and maximum value) which is no more than 1.5 times the interquartile range (i.e., remaining 50% of the data) from the box. Outliers are shown as empty circles. The horizontal line at a value of 1.0 indicates no change. (A and B) Show the ratios of PDT vs. controls for ceramides and DHceramides at 2 h, respectively. (C and D) Show the ratios of PDT vs. controls for ceramides and DHceramides at 24 h, respectively. Corresponding ceramide and DHceramide species are designated as C14, C16, etc. (E and F) Show the ratios of ATG-7 vs. Scr for all ceramides and DHceramides after PDT. The average ratio ATG-7/Scr at 2 h post-PDT is significantly different from 1 (p<0.004) and is indicated by an asterisk.