Radiation-induced AR in IMR-90 cells under 2-D condition.

<p>For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. <b>(A)</b> The changes of γ-H2AX and p53 (ser-15) expression at 1h after D2 radiation in IMR-90 cells by immunofluorescent staining. <b>(B)</b> The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell. <b>(C)</b> The statistical analysis of γ-H2AX and p53(ser-15) expression from immunofluorescent staining based on the percentage of positive cells. <b>(D)</b> The changes of γ-H2AX, p53 (ser-15) and p21 expression in radiation-induced AR by western blot. <b>(E)</b> The statistical analysis of γ-H2AX, p53 (ser-15) and p21 expression from Western blot. * <i>p</i><0.05 vs control; # <i>p</i><0.05 vs D2. The bar corresponds to 25 μm.</p

Radiation-induced AR in RMP-4 cells under 2-D condition.

<p>For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. <b>(A)</b> The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining after 2 Gy radiation in different time points. <b>(B)</b> The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining at 1h after different doses of radiation. <b>(C)</b> The radiation-induced AR based on γ-H2AX changes. <b>(D)</b> The changes of γ-H2AX, p53, and p21 expression in radiation-induced AR by western blot. <b>(E)</b> The statistical analysis of γ-H2AX, p53 and p21 expression from Western blot. <b>(F)</b> The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different doses of radiation. (G)The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different dose rate of radiation.* <i>p</i><0.05 vs control; # <i>p</i><0.05 vs D2. The bar corresponds to 25 μm.</p

Radiation-induced AR in MEF cells under 2-D condition.

<p>For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. <b>(A)</b> The changes of γ-H2AX and p53 (ser-15) expression after radiation in MEF cells by immunofluorescent staining. <b>(B)</b> The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell. <b>(C)</b> The statistical analysis of γ-H2AX and p53 (ser-15) expression from immunofluorescent staining based on the percentage of positive cells. <b>(D)</b> The changes of γ-H2AX, p53 (ser-15) and p21 expression in radiation-induced AR by western blot. <b>(E)</b> The statistical analysis of γ-H2AX, p53 (ser-15) and p21 expression from Western blot. * <i>p</i><0.05 vs control; # <i>p</i><0.05 vs D2. The bar corresponds to 25 μm.</p

Ferroptosis and autophagy induced cell death occur independently after siramesine and lapatinib treatment in breast cancer cells

<div><p>Ferroptosis is a cell death pathway characterized by iron-dependent accumulation of reactive oxygen species (ROS) within the cell. The combination of siramesine, a lysosome disruptor, and lapatinib, a dual tyrosine kinase inhibitor, has been shown to synergistically induce cell death in breast cancer cells mediated by ferroptosis. These treatments also induce autophagy but its role in this synergistic cell death is unclear. In this study, we determined that siramesine and lapatinib initially induced ferroptosis but changes to an autophagy induced cell death after 24 hours. Furthermore, we found that intracellular iron level increased in a time dependent manner following treatment accompanied by an increase in ROS. Using the iron chelator deferoxamine (DFO) or the ROS scavenger alpha-tocopherol decreased both autophagy flux and cell death. We further discovered that decreased expression of the iron storage protein, ferritin was partially dependent upon autophagy degradation. In contrast, the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. This indicates that ferroptosis and autophagy induced cell death occur independently but both are mediated by iron dependent ROS generation in breast cancer cells.</p></div

Siramesine and lapatinib induced ferropoptosis at 4h and autophagic cell death at 24 hours.

<p>(A, B) The effects of ferrostain-1 (5 microM) on cell death under siramesine and lapatinib treatment in MDA MB 231 and SKBR3 for 4 and 24 hours respectively. Cell death was quantified by flow cytometer. Before treatment under siramesine and lapatinib, cells were pretreated with ferrostain-1 for 1 hour. These results were representative of three independent experiments (n = 3). *p<0.05; **p<0.01.</p

Autophagy promotes ferritin degradation following siramesine and lapatinib treatment in MDA MB 231 cells.

<p>(A) MDA MB 231 cells were lysed after treatment with DMSO (D), siramesine (S), lapatinib (L) and siramesine and lapatinib (S + L) for 4 and 24 hour. Western blot determination of iron-related proteins ferritin (FTH1) transferrin, transferrin receptor (CD71), ferroportin (FPN) was performed. Actin was used as a loading control. (B) The effects of 3-MA (2mM) and MG132 (1 microM) on expression level of FTH1 following siramesine and lapatinib treatment in MDA MB 231cells for 24 hours respectively. Before treatment under siramesine and lapatinib, cells were pretreated with 3mM and MG132 for 1hour. (C) Effects of knockdown of <i>Atg5</i> on siramesine and lapatinib-induced FTH degradation at 24 hour in MDA MB 231 cells.</p

Autophagy flux increases following treatment of cells with siramesine and lapatinib.

<p>(A, B) Western blot determination of autophagy protein LC3-II in the absence and presence of NH₄Cl when MDA MB 231 cells were treated with siramesine and lapatinib for 4and 24 hours. Actin (beta-actin) was used as a loading control (and same hereafter). (C, D) Autophagy was measured by quantifying the number of puncta of a fusion protein of red fluorescent protein LC3 (mRFP-LC3) per cell in MDA MB 231 cells. (E) Western blot determination of autophagy protein LC3-II when SKBR3 cells were treated with siramesine and lapatinib for 4 and 24 hours. (F) Autophagy was measured by quantifying the number of puncta of a fusion protein of red fluorescent protein LC3 (mRFP-LC3) per cell in SKBR3 cells. *p<0.05; **p<0.01.</p

Autophagy inhibitors increase cell death at an early time of siramesine and lapatinib treatment but inhibit cell death at a later time.

<p><b>(A)</b> Inhibition of autophagy by autophagy inhibitors 3-methyladenine (3-MA, 2 mM, and same hereafter) and spautin-1 (3 microM, and same hereafter) under siramesine and lapatinib treatment as demonstrated by LC3-II western blot in MDA MB 231 cells. <b>(B)</b> The effects of 3-MA on cell death under siramesine and lapatinib treatment in MDA MB 231 cells for 4 and 24 hours. <b>(C)</b> The effects of spautin-1 on cell death under siramesine and lapatinib treatment in MDA MB 231 for 4 and 24 hours. Cell death was quantified by flow cytometry as described in the Materials and Methods section (and same hereafter). Before treatment with siramesine and lapatinib, cells were pretreated with 3-MA or spautin-1 for 1 hour (and same hereafter). (D) Inhibition of autophagy by autophagy inhibitors 3-methyladenine (3-MA, 2 mM) and spautin-1 (3 microM) under siramesine and lapatinib treatment as demonstrated by LC3-II western blot in SKBR3 cells. (E) The effects of 3-MA on cell death under siramesine and lapatinib treatment in SKBR3 cells for 4 and 24 hours. These results were representative of three independent experiments (n = 3). *p<0.05; **p<0.01.</p

Knockdown of autophagy genes increases cell death during early treatment times with siramesine and lapatinib but inhibits cell death at a later times.

<p>(A) Knockdown of autophagy genes <i>Atg5</i> and <i>Becn1</i> by siRNAs as demonstrated by western blot of Atg5 and Beclin-1, respectively, in MDA MB 231 cells. The protein level of Atg5 was represented by the Atg5-Atg12 complex since the conjugation between these two proteins is an essential step during a functional autophagy process (and same hereafter). (B) Inhibition of autophagy by knockdown of <i>Atg5</i> as demonstrated by western blot of LC3I/LC3-II in MDA MB 231 cells. <b>(</b>C, D) Effects of knockdown of <i>Atg5</i> or <i>Becn1</i> on siramesine and lapatinib-induced cell death at 4 and 24 hours in MDA MB 231 cells. These results were representative of three independent experiments (n = 3), *p<0.05.</p

Siramesine and lapatinib increase iron level and ROS generation.

<p>(A, B) The effects of DFO (0.5mM) and ferrostain-1 (5 microM) on intracellular chelatable iron under siramesine and lapatinib treatment in MDA MB 231 and SKBR3 cells for 4 and 24 hours respectively. Intracellular chelatable iron was determined using the fluorescent indicator phen green SK, the fluorescence of which is quenched by iron. Samples were examined using a BD FACS Calibur. <b>(</b>C) Effects of knockdown of <i>Atg5</i> on siramesine and lapatinib-induced intracellular chelatable iron at 24 hour in MDA MB 231 cells. (D) Effects of knockdown of <i>Atg5</i> or <i>Becn1</i> on siramesine and lapatinib-induced ROS generation at 24 hour in MDA MB 231 cells. (E) The effect of siramesine and lapatinib on mitochondrial ROS generation. Mitochondrial ROS was determined using the fluorescent indicator mitoSOX, samples were examined using a BD FACSCalibur. These results were representative of three independent experiments (n = 3), *p<0.05; **p<0.01.</p