Tumor Biol. (2015) 36:1173–1177 DOI 10.1007/s13277-014-2699-x
RESEARCH ARTICLE
Ammonium chloride inhibits autophagy of hepatocellular carcinoma cells through SMAD2 signaling Ranran Sun & Yonggang Luo & Juan Li & Qiongye Wang & Jingjing Li & Xiaolong Chen & Kelei Guan & Zujiang Yu
Received: 21 September 2014 / Accepted: 30 September 2014 / Published online: 24 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Autophagy is a cellular degradation process for the clearance of damaged or superfluous proteins and organelles, the recycling of which serves as an alternative energy source during periods of metabolic stress to maintain cell homeostasis and viability. The anti-necrotic function of autophagy is critical for tumorigenesis of many tumor cells, including hepatocellular carcinoma (HCC). However, the underlying mechanism is not clarified yet. Ammonium chloride (NH4Cl) is a well-known autophagy inhibitor, whereas its interaction with SMAD2 signaling pathway has not been reported previously. Here, we show that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through decreasing the levels of Beclin-1, autophagy-related protein 7 (ATG7), p62, and autophagosome marker LC3 and significantly decreased the level of phosphorylated SMAD2 in rapamycin-treated HCC cells. In order to find out whether NH4Cl may inhibit the autophagy in rapamycin-treated HCC cells through inhibition of SMAD2 signaling, we used transforming growth factor β1 (TGFβ1) to induce phosphorylation of SMAD2 in HCC cells. We found that induction of
Ranran Sun and Yonggang Luo have equal contribution. R. Sun : J. Li : X. Chen : Z. Yu (*) Department of Infectious Disease, The First Affiliated Hospital of Zhengzhou University, 1 Jianshendong Road, Zhengzhou 450000, China e-mail: [email protected] Z. Yu e-mail: [email protected] Y. Luo Department of Intensive Care Unit, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China Q. Wang : J. Li : K. Guan Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
SMAD2 in HCC cells completely abolished the inhibitory effect of NH4Cl on rapamycin-induced autophagy in HCC cells, suggesting that NH4Cl inhibits autophagy of HCC cells through inhibiting SMAD2 signaling. Keywords Ammonium chloride . Hepatocellular carcinoma . Autophagy . SMAD2
Introduction Hepatocellular carcinoma (HCC) is the most common liver cancer and has a very poor treatment outcome [1–3]. Nevertheless, although our understanding of the pathogenesis of HCC has significantly improved in the past years, the precise molecular basis underlying HCC cell growth and survival from chemotherapy remains not clarified [4]. Autophagy is a regulated catabolic pathway to degrade cellular compartments, allowing the recycling of cellular components [5]. Autophagy promotes survival in response to nutrient deprivation, but can also promote cell death, which appears to depend on the specific biological context [5]. In line with these paradoxical pro- and anti-survival effects on physiological cellular metabolism, the role of autophagy in cancer is also complex, which associates with both tumor suppression and therapeutic resistance [6, 7]. The underlying mechanism for this synergy appears to be that autophagy is low under normal conditions in most tumor types but is induced upon harsh environment as a survival mechanism [6, 7]. Therefore, it is critical to examine the contributions of autophagy in particular tumor types. Of note, the involvement of different signal transduction pathways in autophagy is critical for the cells in determining their undergoing autophagy, apoptosis, necrosis, or necroptosis. However, our knowledge on these contexts is rather limited. In HCC, recent studies have suggested that
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transforming growth factor β (TGFβ) receptor signaling may play a role in autophagy [8, 9]. TGFβ receptor signaling pathway is essential for various biological events [10–15] and specifically has a paradoxical effect during the tumorigenesis of various cancers, in which its tumor-inhibitory role at early stages turns into a tumor-promoting role at later stages [16]. When a ligand binds to a type II TGFβ receptor, it catalyzes the phosphorylation of a type I TGFβ receptor, which triggers phosphorylation of two intracellular proteins SMAD2 and SMAD3 to form heteromeric complexes with SMAD4. The activated SMAD complexes then translocate to the nucleus, where they regulate the transcription of target genes, e.g., p21, p27, or cell-cycle activators [10]. Here, we examined the role of SMAD2 signaling in HCC cells treated with rapamycin as an autophagy inducer and ammonium chloride (NH4Cl) as an autophagy inhibitor. We found that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through inhibition of SMAD2 signaling.
Tumor Biol. (2015) 36:1173–1177
and α-tubulin (all purchased from Cell Signaling, USA). α-Tubulin was used as a protein loading control. Statistical analysis All statistical analyses were carried out using the SPSS 16.0 statistical software package. All values are depicted as mean± standard deviation from five individuals and are considered significant if p
RESEARCH ARTICLE
Ammonium chloride inhibits autophagy of hepatocellular carcinoma cells through SMAD2 signaling Ranran Sun & Yonggang Luo & Juan Li & Qiongye Wang & Jingjing Li & Xiaolong Chen & Kelei Guan & Zujiang Yu
Received: 21 September 2014 / Accepted: 30 September 2014 / Published online: 24 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Autophagy is a cellular degradation process for the clearance of damaged or superfluous proteins and organelles, the recycling of which serves as an alternative energy source during periods of metabolic stress to maintain cell homeostasis and viability. The anti-necrotic function of autophagy is critical for tumorigenesis of many tumor cells, including hepatocellular carcinoma (HCC). However, the underlying mechanism is not clarified yet. Ammonium chloride (NH4Cl) is a well-known autophagy inhibitor, whereas its interaction with SMAD2 signaling pathway has not been reported previously. Here, we show that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through decreasing the levels of Beclin-1, autophagy-related protein 7 (ATG7), p62, and autophagosome marker LC3 and significantly decreased the level of phosphorylated SMAD2 in rapamycin-treated HCC cells. In order to find out whether NH4Cl may inhibit the autophagy in rapamycin-treated HCC cells through inhibition of SMAD2 signaling, we used transforming growth factor β1 (TGFβ1) to induce phosphorylation of SMAD2 in HCC cells. We found that induction of
Ranran Sun and Yonggang Luo have equal contribution. R. Sun : J. Li : X. Chen : Z. Yu (*) Department of Infectious Disease, The First Affiliated Hospital of Zhengzhou University, 1 Jianshendong Road, Zhengzhou 450000, China e-mail: [email protected] Z. Yu e-mail: [email protected] Y. Luo Department of Intensive Care Unit, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China Q. Wang : J. Li : K. Guan Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
SMAD2 in HCC cells completely abolished the inhibitory effect of NH4Cl on rapamycin-induced autophagy in HCC cells, suggesting that NH4Cl inhibits autophagy of HCC cells through inhibiting SMAD2 signaling. Keywords Ammonium chloride . Hepatocellular carcinoma . Autophagy . SMAD2
Introduction Hepatocellular carcinoma (HCC) is the most common liver cancer and has a very poor treatment outcome [1–3]. Nevertheless, although our understanding of the pathogenesis of HCC has significantly improved in the past years, the precise molecular basis underlying HCC cell growth and survival from chemotherapy remains not clarified [4]. Autophagy is a regulated catabolic pathway to degrade cellular compartments, allowing the recycling of cellular components [5]. Autophagy promotes survival in response to nutrient deprivation, but can also promote cell death, which appears to depend on the specific biological context [5]. In line with these paradoxical pro- and anti-survival effects on physiological cellular metabolism, the role of autophagy in cancer is also complex, which associates with both tumor suppression and therapeutic resistance [6, 7]. The underlying mechanism for this synergy appears to be that autophagy is low under normal conditions in most tumor types but is induced upon harsh environment as a survival mechanism [6, 7]. Therefore, it is critical to examine the contributions of autophagy in particular tumor types. Of note, the involvement of different signal transduction pathways in autophagy is critical for the cells in determining their undergoing autophagy, apoptosis, necrosis, or necroptosis. However, our knowledge on these contexts is rather limited. In HCC, recent studies have suggested that
1174
transforming growth factor β (TGFβ) receptor signaling may play a role in autophagy [8, 9]. TGFβ receptor signaling pathway is essential for various biological events [10–15] and specifically has a paradoxical effect during the tumorigenesis of various cancers, in which its tumor-inhibitory role at early stages turns into a tumor-promoting role at later stages [16]. When a ligand binds to a type II TGFβ receptor, it catalyzes the phosphorylation of a type I TGFβ receptor, which triggers phosphorylation of two intracellular proteins SMAD2 and SMAD3 to form heteromeric complexes with SMAD4. The activated SMAD complexes then translocate to the nucleus, where they regulate the transcription of target genes, e.g., p21, p27, or cell-cycle activators [10]. Here, we examined the role of SMAD2 signaling in HCC cells treated with rapamycin as an autophagy inducer and ammonium chloride (NH4Cl) as an autophagy inhibitor. We found that NH4Cl significantly inhibited rapamycin-induced autophagy in HCC cells through inhibition of SMAD2 signaling.
Tumor Biol. (2015) 36:1173–1177
and α-tubulin (all purchased from Cell Signaling, USA). α-Tubulin was used as a protein loading control. Statistical analysis All statistical analyses were carried out using the SPSS 16.0 statistical software package. All values are depicted as mean± standard deviation from five individuals and are considered significant if p
