Mol. Cells 2015; 38(2): 138-144
Published online December 24, 2014
https://doi.org/10.14348/molcells.2015.2193
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: jjhwang@amc.seoul.kr (JJH); cskim@amc.seoul.kr (CSK)
Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting
Keywords AMPK, ATP, autophagy, breast cancer, raloxifene
Macroautophagy (autophagy) is a self-digestion mechanism for degrading damaged organelles and misfolded proteins in the lysosomal compartments. Autophagy starts with the formation of double-membraned vesicles, or autophagosomes, which undergo maturation by fusion with lysosomes in order to create autolysosomes. In autolysosomes, the inner membrane of the autophagosome and its contents are degraded by lysosomal enzymes (Eskelinen, 2008; Maiuri et al., 2007). Under metabolic stress, autophagy maintains a balance between synthesis, degradation, and the subsequent recycling of macromolecules and organelles in order to continue survival. On the other hand, the overactivation of autophagy can promote cell death during persistent stress (Eskelinen, 2008; Levine, 2007; Levine and Kroemer, 2008; Morselli et al., 2009).
The paradox that autophagy plays a role in both survival and death is more complicated in cancer cells. The first specific link between autophagy and cancer was reported in 1999 by Levine et al. They reported that
Previously, we reported that tamoxifen induces autophagy-dependent cell death in MCF-7 cells via the accumulation of intracellular zinc ions and reactive oxygen species (ROS), which finally leads to lysosomal membrane permeabilization (LMP) (Hwang et al., 2010). Tamoxifen is a selective estrogen receptor modulator (SERMs) that binds to the estrogen receptor (ER) and exhibits selective agonistic or antagonistic effects against target tissue (Fabian and Kimler, 2005). Tamoxifen is the first SERM to be used to treat and prevent ER-positive breast cancer (Fisher et al., 1998). Raloxifene has been used to prevent and treat osteoporosis in 2001, since it has an estrogenic activity in bone (Gizzo et al., 2013). In contrast, since it had and antiestrogenic activity in breast, U.S. Food and Drug Administration (FDA) approved raloxifene for reduction the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer in 2007 (Powles, 2011). In breast cancer cells, many studies demonstrated that
In our current study, we evaluated whether raloxifene induces autophagy-dependent mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and autophagy, and is accordingly responsible for the anti-proliferative effects of raloxifene on MCF-7 breast cancer cells.
MCF-7 human breast cancer cells expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) and red fluorescent protein (mRFP)-GFP tandem fluorescent-tagged LC3 (mRFP-GFP-LC3-MCF-7) were established as previously described (Hwang et al., 2010). These cells were pre-treated with various concentrations of raloxifene (Cayman, USA) in RPMI1640 medium containing 10% charcoal-stripped FBS (Thermo Scientific, Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, USA). Pancaspase inhibitor, caspase-9 inhibitor (R&D Systems, USA), 3-methyladenine (3-MA) (Sigma, USA), siRNA control, and siRNA
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay) reagent (Promega, USA) was added to each well containing cells that had been treated with various drugs according to the manufacturer’s instructions. Cell viability was determined by measuring absorbance at 490 nm using a Sunrise microplate reader (TECAN, Switzerland).
Cells were stained with 0.1% trypan blue solution (Invitrogen) for 1 min and counted using a homocytometer under a light microscope. The percentage and total number of stained dead cells were calculated.
The CellTiter-Glo Luminescent Assay reagent (Promega) was added to each well according to the manufacturer’s instructions. The level of ATP was determined using an EnVision Multilabel Reader (Perkin-Elmer, USA) by measuring the luminescent signal.
Western blot analysis was performed, as previously described (Hwang et al., 2010), using antibodies against BECN1, phospho-AMPK (Thr172), AMPK, phospho-ULK1 (Ser317), phospho-ULK1 (Ser757), ULK1, phospho-mTOR (Ser2448), mTOR, phospho-AKT (Ser473), AKT, tubulin (Cell Signaling, USA), ATG5 (Abcam, UK), LC3 (NOVUS Biologicals, USA), caspase-7, caspase-9, PARP (Santa Cruz Biochemicals, USA), and actin (Sigma). Actin or tubulin was used as the loading control.
Cells were transfected with 0.17 μM
mRFP-GFP-LC3-MCF-7 cells were fixed with 4% paraformaldehyde (PFA, Sigma) and stained with 10 μM Hoechst33342 (Sigma) after treatment with raloxifene or rapamycin (Sigma). Images of the cells were obtained from the Operetta High Content Imaging System (Perkin-Elmer) and analyzed using the Harmony Analysis Software (Perkin-Elmer). Cells were detected with green (GFP) or red (mRFP) fluorescence. Autophagosomes are yellow puncta and autolysosomes are only red puncta in merged images. Autophagic flux was determined by increased percent of only red puncta in the merged images.
Data were obtained from ≥ 3 independent experiments and are presented as the mean ± standard deviation (SD). Statistical evaluations of the results were performed using one-way ANOVA. Data were considered significant at
Raloxifene has
To monitor autophagic vacuoles (AVs), we used MCF-7 cells that constitutively expressed GFP-tagged LC3 (GFP-LC3-MCF-7). GFP-LC3 diffuses into the cytoplasm and nucleus under normal conditions, but conjugates with phosphatidylethanolamine (PE) and is incorporated into the AV membrane upon the induction of autophagy. These GFP-positive vacuoles can be visualized using fluorescent microscopy (Dorsey et al., 2009). When we exposed GFP-LC3-MCF-7 cells to raloxifene for 8 h, GFP-positive AVs were obviously apparent in comparison with the sham-washed control cells (Fig. 2A). We also detected autophagic marker proteins using Western blot analysis. Raloxifene augmented the level of BECN1 required for early autophagophore formation, in addition to the ATG12-ATG5 conjugates and LC3-II needed to elongate the autophagosomal membrane in a time-dependent manner (Figs. 2B and 2C). Pretreatment with 3-MA, which blocks autophagy by inhibiting class III phosphatidylinositol 3-kinase (PI3K), decreased GFP-positive AVs (Fig. 2A) and the level of LC3-II increased following raloxifene treatment (Figs. 2D and 2E), thereby confirming the activation of autophagy by raloxifene.
LC3-II is increased when the production of autophagophores or autophagosomes are increased or the fusion of autophagosomes with lysosomes are inhibited. So it is important to know whether raloxifene activates the whole process of autophagy. This process is called as autophagic flux which includes degradation of the contents of AVs after formation of autolysosome. It was reported that lysosomal hydrolases cleaved GFP-LC3-II and increased free-GFP proteins during the autophagic flux (Balgi et al., 2009). Raloxifene induced a time-dependent increase in free-GFP (Fig. 3A). Besides, we used MCF-7 cells that constitutively expressed mRFP-GFP tandem fluorescent-tagged LC3 (mRFP-GFP-LC3-MCF-7) to monitor autophagic flux. Because GFP fluorescence is unstable in the low pH, it will be quenched in the autolysosomes. But acidic insensitive-mRFP fluorescence will not be quenched (Mizushima et al., 2010). Therefore, while the yellow puncta represent the autophagosomes, the red puncta indicate autolysosomes in the merged fluorescent image, representing autophagic flux. Raloxifene increased the yellow and red puncta (Figs. 3B and 3C), indicating that raloxifene stimulates autophagic flux as well as the formation of AVs in MCF-7 cells.
Because MCF-7 cells are ER-positive breast cancer cells, we also examined if raloxifene induces autophagy in ER-negative SKBr-3 breast cancer cells and HCT116 colon cancer cells. In accordance with our previous study on tamoxifen (Hwang et al., 2010), raloxifene increased the level of LC3-II in these cell lines (data not shown). These results indicate that either raloxifene or tamoxifen activates autophagy regardless of the ER status in breast cancer and even colon cancer cells.
To determine if raloxifene induces autophagy-dependent cell death, cell viability was measured in MCF-7 cells that were treated with raloxifene after
To elucidate the molecular mechanisms that underlie raloxifene-induced autophagy, we examined the upstream signaling pathways. First, we examined the inhibition of AKT and mTOR, which are well-known mechanisms of autophagy activation (He and Klionsky, 2009; Jung et al., 2010; Ryter et al., 2013; Yang and Klionsky, 2010). In contrast to our expectations, Western blot analysis revealed that the phosphorylation of AKT and mTOR increased following raloxifene treatment. Moreover, raloxifene did not change the phosphorylation of ULK1 at serine 757, an inhibitory site phosphotylated by mTOR (Fig. 5A). These results indicate that raloxifene-activated autophagy is not related to mTOR signaling. We next examined the level of intracellular ATP, because decrease in ATP activates AMPK. Exposure to raloxifene decreased the level of intracellular ATP to 12% (Fig. 5B), thereby increasing the phosphorylation of threonine 172 on APMK and serine 317 on ULK1 which is required to initiate autophagy (Figs. 5A and 5C). (Alers et al., 2012; Egan et al., 2011; Kim et al., 2011; Lee et al., 2010). The addition of ATP, which raised the level of intracellular ATP to 36% (Fig. 5B), rescued the cell viability reduced by raloxifene (Fig. 5D) and decreased phospho-AMPK as well as LC3-II (Figs. 5C). Accordingly, nicotinamide adenine dinucleotide (NAD), which accelerates the production of ATP (Khan et al., 2007), recovered the viability of the raloxifene-exposed cells (Fig. 5D). Collectively, these results suggest that raloxifene-induced autophagy and death are mediated by the activation of AMPK, without the inhibition of AKT/mTOR pathway.
According to the 1996 study by Bursch et al. (1996) tamoxifen reportedly activates autophagy and induces type II cell death. We have also reported that tamoxifen increases the ROS- and zinc-mediated overactivation of autophagy, thereby leading to lysosomal membrane permeabilization (LMP) (Hwang et al., 2010). de Medina et al. (2009) reported that tamoxifen and other SERMs activate autophagy by modulating cholesterol metabolism. However, none of these studies described raloxifene in detail. Here, we show that raloxifene increases autophagy-mediated cell death by activating the AMPK pathway via decreases in intracellular ATP in MCF-7 breast cancer cells. The addition of ATP increased the intracellular level of ATP in this experiment, thereby protecting cells from raloxifene-induced cell death. However, we cannot rule out the possibility that extracellular role of ATP. Extracellular ATP binds to specific plasma membrane receptors (called P2 receptors) and initiates cellular signaling events such as growth, proliferation, and apoptosis (Deli and Csernoch, 2008). The anti-cancer activity of ATP has been reported by many groups, which have reported that ATP-activated P2 receptors decrease tumor growth and increase apoptosis in diverse types of cancers (Hopfner et al., 1998; Wang et al., 2004; White and Burnstock, 2006). Conversely, extracellular ATP activates P2 receptors followed by increases intracellular calcium and cell proliferation of MCF-7 cells, which is supported by ATP acting as a promoter of cell proliferation and growth (Dixon et al., 1997; Wagstaff et al., 2000). Therefore, further studies on the mechanisms by which raloxifene inhibits P2 receptor-mediated signaling are needed.
In this study, we suggest that raloxifene-induced decrease in ATP activates AMPK, leading to autophagy and autophagic flux. The overactivation of autophagy can lead to cell death, which can be one of the mechanisms of anti-cancer effect of raloxifene.
Mol. Cells 2015; 38(2): 138-144
Published online February 28, 2015 https://doi.org/10.14348/molcells.2015.2193
Copyright © The Korean Society for Molecular and Cellular Biology.
Dong Eun Kim1, Yunha Kim1,10, Dong-Hyung Cho2, Seong-Yun Jeong1,3, Sung-Bae Kim4, Nayoung Suh3,5, Jung Shin Lee1,6, Eun Kyung Choi1,7, Jae-Young Koh8, Jung Jin Hwang1,3,*, and Choung-Soo Kim1,9,*
1Institute for Innovative Cancer Research, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 2Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea, 3Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 4Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 5Department of Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 6Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 7Department of Radiation Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea, 8Neural Injury Research Center and Department of Neurology, Asan Medical Center, Seoul, Korea, 9Department of Urology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea, 10Present address: Center for Neuro-Medicine, Brain Science Institute Korea Institute of Science and Technology, Seoul 110-799, Korea
Correspondence to:*Correspondence: jjhwang@amc.seoul.kr (JJH); cskim@amc.seoul.kr (CSK)
Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting
Keywords: AMPK, ATP, autophagy, breast cancer, raloxifene
Macroautophagy (autophagy) is a self-digestion mechanism for degrading damaged organelles and misfolded proteins in the lysosomal compartments. Autophagy starts with the formation of double-membraned vesicles, or autophagosomes, which undergo maturation by fusion with lysosomes in order to create autolysosomes. In autolysosomes, the inner membrane of the autophagosome and its contents are degraded by lysosomal enzymes (Eskelinen, 2008; Maiuri et al., 2007). Under metabolic stress, autophagy maintains a balance between synthesis, degradation, and the subsequent recycling of macromolecules and organelles in order to continue survival. On the other hand, the overactivation of autophagy can promote cell death during persistent stress (Eskelinen, 2008; Levine, 2007; Levine and Kroemer, 2008; Morselli et al., 2009).
The paradox that autophagy plays a role in both survival and death is more complicated in cancer cells. The first specific link between autophagy and cancer was reported in 1999 by Levine et al. They reported that
Previously, we reported that tamoxifen induces autophagy-dependent cell death in MCF-7 cells via the accumulation of intracellular zinc ions and reactive oxygen species (ROS), which finally leads to lysosomal membrane permeabilization (LMP) (Hwang et al., 2010). Tamoxifen is a selective estrogen receptor modulator (SERMs) that binds to the estrogen receptor (ER) and exhibits selective agonistic or antagonistic effects against target tissue (Fabian and Kimler, 2005). Tamoxifen is the first SERM to be used to treat and prevent ER-positive breast cancer (Fisher et al., 1998). Raloxifene has been used to prevent and treat osteoporosis in 2001, since it has an estrogenic activity in bone (Gizzo et al., 2013). In contrast, since it had and antiestrogenic activity in breast, U.S. Food and Drug Administration (FDA) approved raloxifene for reduction the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer in 2007 (Powles, 2011). In breast cancer cells, many studies demonstrated that
In our current study, we evaluated whether raloxifene induces autophagy-dependent mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and autophagy, and is accordingly responsible for the anti-proliferative effects of raloxifene on MCF-7 breast cancer cells.
MCF-7 human breast cancer cells expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) and red fluorescent protein (mRFP)-GFP tandem fluorescent-tagged LC3 (mRFP-GFP-LC3-MCF-7) were established as previously described (Hwang et al., 2010). These cells were pre-treated with various concentrations of raloxifene (Cayman, USA) in RPMI1640 medium containing 10% charcoal-stripped FBS (Thermo Scientific, Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, USA). Pancaspase inhibitor, caspase-9 inhibitor (R&D Systems, USA), 3-methyladenine (3-MA) (Sigma, USA), siRNA control, and siRNA
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay) reagent (Promega, USA) was added to each well containing cells that had been treated with various drugs according to the manufacturer’s instructions. Cell viability was determined by measuring absorbance at 490 nm using a Sunrise microplate reader (TECAN, Switzerland).
Cells were stained with 0.1% trypan blue solution (Invitrogen) for 1 min and counted using a homocytometer under a light microscope. The percentage and total number of stained dead cells were calculated.
The CellTiter-Glo Luminescent Assay reagent (Promega) was added to each well according to the manufacturer’s instructions. The level of ATP was determined using an EnVision Multilabel Reader (Perkin-Elmer, USA) by measuring the luminescent signal.
Western blot analysis was performed, as previously described (Hwang et al., 2010), using antibodies against BECN1, phospho-AMPK (Thr172), AMPK, phospho-ULK1 (Ser317), phospho-ULK1 (Ser757), ULK1, phospho-mTOR (Ser2448), mTOR, phospho-AKT (Ser473), AKT, tubulin (Cell Signaling, USA), ATG5 (Abcam, UK), LC3 (NOVUS Biologicals, USA), caspase-7, caspase-9, PARP (Santa Cruz Biochemicals, USA), and actin (Sigma). Actin or tubulin was used as the loading control.
Cells were transfected with 0.17 μM
mRFP-GFP-LC3-MCF-7 cells were fixed with 4% paraformaldehyde (PFA, Sigma) and stained with 10 μM Hoechst33342 (Sigma) after treatment with raloxifene or rapamycin (Sigma). Images of the cells were obtained from the Operetta High Content Imaging System (Perkin-Elmer) and analyzed using the Harmony Analysis Software (Perkin-Elmer). Cells were detected with green (GFP) or red (mRFP) fluorescence. Autophagosomes are yellow puncta and autolysosomes are only red puncta in merged images. Autophagic flux was determined by increased percent of only red puncta in the merged images.
Data were obtained from ≥ 3 independent experiments and are presented as the mean ± standard deviation (SD). Statistical evaluations of the results were performed using one-way ANOVA. Data were considered significant at
Raloxifene has
To monitor autophagic vacuoles (AVs), we used MCF-7 cells that constitutively expressed GFP-tagged LC3 (GFP-LC3-MCF-7). GFP-LC3 diffuses into the cytoplasm and nucleus under normal conditions, but conjugates with phosphatidylethanolamine (PE) and is incorporated into the AV membrane upon the induction of autophagy. These GFP-positive vacuoles can be visualized using fluorescent microscopy (Dorsey et al., 2009). When we exposed GFP-LC3-MCF-7 cells to raloxifene for 8 h, GFP-positive AVs were obviously apparent in comparison with the sham-washed control cells (Fig. 2A). We also detected autophagic marker proteins using Western blot analysis. Raloxifene augmented the level of BECN1 required for early autophagophore formation, in addition to the ATG12-ATG5 conjugates and LC3-II needed to elongate the autophagosomal membrane in a time-dependent manner (Figs. 2B and 2C). Pretreatment with 3-MA, which blocks autophagy by inhibiting class III phosphatidylinositol 3-kinase (PI3K), decreased GFP-positive AVs (Fig. 2A) and the level of LC3-II increased following raloxifene treatment (Figs. 2D and 2E), thereby confirming the activation of autophagy by raloxifene.
LC3-II is increased when the production of autophagophores or autophagosomes are increased or the fusion of autophagosomes with lysosomes are inhibited. So it is important to know whether raloxifene activates the whole process of autophagy. This process is called as autophagic flux which includes degradation of the contents of AVs after formation of autolysosome. It was reported that lysosomal hydrolases cleaved GFP-LC3-II and increased free-GFP proteins during the autophagic flux (Balgi et al., 2009). Raloxifene induced a time-dependent increase in free-GFP (Fig. 3A). Besides, we used MCF-7 cells that constitutively expressed mRFP-GFP tandem fluorescent-tagged LC3 (mRFP-GFP-LC3-MCF-7) to monitor autophagic flux. Because GFP fluorescence is unstable in the low pH, it will be quenched in the autolysosomes. But acidic insensitive-mRFP fluorescence will not be quenched (Mizushima et al., 2010). Therefore, while the yellow puncta represent the autophagosomes, the red puncta indicate autolysosomes in the merged fluorescent image, representing autophagic flux. Raloxifene increased the yellow and red puncta (Figs. 3B and 3C), indicating that raloxifene stimulates autophagic flux as well as the formation of AVs in MCF-7 cells.
Because MCF-7 cells are ER-positive breast cancer cells, we also examined if raloxifene induces autophagy in ER-negative SKBr-3 breast cancer cells and HCT116 colon cancer cells. In accordance with our previous study on tamoxifen (Hwang et al., 2010), raloxifene increased the level of LC3-II in these cell lines (data not shown). These results indicate that either raloxifene or tamoxifen activates autophagy regardless of the ER status in breast cancer and even colon cancer cells.
To determine if raloxifene induces autophagy-dependent cell death, cell viability was measured in MCF-7 cells that were treated with raloxifene after
To elucidate the molecular mechanisms that underlie raloxifene-induced autophagy, we examined the upstream signaling pathways. First, we examined the inhibition of AKT and mTOR, which are well-known mechanisms of autophagy activation (He and Klionsky, 2009; Jung et al., 2010; Ryter et al., 2013; Yang and Klionsky, 2010). In contrast to our expectations, Western blot analysis revealed that the phosphorylation of AKT and mTOR increased following raloxifene treatment. Moreover, raloxifene did not change the phosphorylation of ULK1 at serine 757, an inhibitory site phosphotylated by mTOR (Fig. 5A). These results indicate that raloxifene-activated autophagy is not related to mTOR signaling. We next examined the level of intracellular ATP, because decrease in ATP activates AMPK. Exposure to raloxifene decreased the level of intracellular ATP to 12% (Fig. 5B), thereby increasing the phosphorylation of threonine 172 on APMK and serine 317 on ULK1 which is required to initiate autophagy (Figs. 5A and 5C). (Alers et al., 2012; Egan et al., 2011; Kim et al., 2011; Lee et al., 2010). The addition of ATP, which raised the level of intracellular ATP to 36% (Fig. 5B), rescued the cell viability reduced by raloxifene (Fig. 5D) and decreased phospho-AMPK as well as LC3-II (Figs. 5C). Accordingly, nicotinamide adenine dinucleotide (NAD), which accelerates the production of ATP (Khan et al., 2007), recovered the viability of the raloxifene-exposed cells (Fig. 5D). Collectively, these results suggest that raloxifene-induced autophagy and death are mediated by the activation of AMPK, without the inhibition of AKT/mTOR pathway.
According to the 1996 study by Bursch et al. (1996) tamoxifen reportedly activates autophagy and induces type II cell death. We have also reported that tamoxifen increases the ROS- and zinc-mediated overactivation of autophagy, thereby leading to lysosomal membrane permeabilization (LMP) (Hwang et al., 2010). de Medina et al. (2009) reported that tamoxifen and other SERMs activate autophagy by modulating cholesterol metabolism. However, none of these studies described raloxifene in detail. Here, we show that raloxifene increases autophagy-mediated cell death by activating the AMPK pathway via decreases in intracellular ATP in MCF-7 breast cancer cells. The addition of ATP increased the intracellular level of ATP in this experiment, thereby protecting cells from raloxifene-induced cell death. However, we cannot rule out the possibility that extracellular role of ATP. Extracellular ATP binds to specific plasma membrane receptors (called P2 receptors) and initiates cellular signaling events such as growth, proliferation, and apoptosis (Deli and Csernoch, 2008). The anti-cancer activity of ATP has been reported by many groups, which have reported that ATP-activated P2 receptors decrease tumor growth and increase apoptosis in diverse types of cancers (Hopfner et al., 1998; Wang et al., 2004; White and Burnstock, 2006). Conversely, extracellular ATP activates P2 receptors followed by increases intracellular calcium and cell proliferation of MCF-7 cells, which is supported by ATP acting as a promoter of cell proliferation and growth (Dixon et al., 1997; Wagstaff et al., 2000). Therefore, further studies on the mechanisms by which raloxifene inhibits P2 receptor-mediated signaling are needed.
In this study, we suggest that raloxifene-induced decrease in ATP activates AMPK, leading to autophagy and autophagic flux. The overactivation of autophagy can lead to cell death, which can be one of the mechanisms of anti-cancer effect of raloxifene.
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