Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Chemicals, Reagents, and Plasmids
2.3. Antibodies
2.4. Cell Viability Assay
2.5. Assays for the Detection of Caspase-3 Activity and Early Apoptotic Cells
2.6. Density-Based Membrane Flotation Technique
2.7. Isolation of ER and Cytosolic Fractions
2.8. Subcellular Fractionation
2.9. Western Blot and Co-Immunoprecipitation
2.10. Cell Surface Biotinylation
2.11. Measurement of Cell Surface or Intracellular GRP78 by Flow Cytometry
2.12. Rac1 Activation Assay
2.13. Determination of Cholesterol, Sphingomyelin, and Ceramide
2.14. Measurement ofMMP-2Promoter Activity
2.15. In Vitro Invasion Assay
2.16. Plasmid Transfection
2.17. NF-κB Promoter Activity
2.18. Statistical Analysis
3. Results
3.1. Delocalization of Cell Surface GRP78 by TSWU-BR4 Induces the Lipid Raft Membrane Localization of Unphosphorylated PTEN to Affect Cancer Cell Invasion
3.2. Recruitment of Unphosphorylated PTEN to the Lipid Raft Membranes to Form a Complex with p85α in TSWU-BR4-Treated Cells
3.3. TSWU-BR4-Induced Dissociation of GRP78 and p85α Leads to the Complex Formation of Lipid Raft Membrane-Associated p85α–Unphosphorylated PTEN and Subsequently Causes Increased ROS Accumulation and Decreased Levels of Cholesterol and Invaded Cells
3.4. Activation of ASM by TSWU-BR4 Deregulates Membrane Trafficking of GRP78, Causes the Lipid Raft Membrane-Associated p85α–Unphosphorylated PTEN Complex Formation, and Thereby Attenuates GRP78-Modulated Oxidative Stress Balance and Cell Invasion
3.5. TSWU-BR4-Induced Ceramide Generation Attenuates Cell Invasion by Suppressing the PI3K–Akt-Regulated NF-κB-Mediated MMP-2 Expression Pathway
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
Acid sphingomyelinase | ASM |
Endoplasmic reticulum | ER |
Glucose regulated protein 78 | GRP78 |
Matrix metalloproteinase-2 | MMP-2 |
Nasopharyngeal carcinoma | NPC |
Nuclear factor erythroid-2-related factor 2 | Nrf2 |
Nuclear factor-kappa B | NF-κB |
Phosphatase and tensin homolog deleted from chromosome 10 | PTEN |
Phosphatidylinositol 3-kinase | PI3K |
Phosphatidylinositol-4,5-bisphosphate | PIP2 |
Phosphatidylinositol-3,4,5-trisphosphate | PIP3 |
Protein kinase B | Akt |
Protein kinase RNA-like endoplasmic reticulum kinase | PERK |
Ras-related C3 botulinum toxin substrate 1 | Rac1 |
Reactive oxygen species | ROS |
Short hairpin RNA | shRNA |
Human pharyngeal squamous carcinoma | PSC |
References
- Beloribi-Djefaflia, S.; Vasseur, S.; Guillaumond, F. Lipid metabolic reprogramming in cancer cells. Oncogenesis 2016, 5, e189. [Google Scholar] [CrossRef] [PubMed]
- Brown, D.A.; London, E. Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 1998, 14, 111–136. [Google Scholar] [CrossRef] [PubMed]
- Drevot, P.; Langlet, C.; Guo, X.J.; Bernard, A.M.; Colard, O.; Chauvin, J.P.; Lasserre, R.; He, H.T. TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts. EMBO J. 2002, 21, 1899–1908. [Google Scholar] [CrossRef] [PubMed]
- Gao, X.; Lowry, P.R.; Zhou, X.; Depry, C.; Wei, Z.; Wong, G.W.; Zhang, J. PI3K/Akt signaling requires spatial compartmentalization in plasma membrane microdomains. Proc. Natl. Acad. Sci. USA 2011, 108, 14509–14514. [Google Scholar] [CrossRef]
- Payapilly, A.; Malliri, A. Compartmentalisation of RAC1 signalling. Curr. Opin. Cell Biol. 2018, 54, 50–56. [Google Scholar] [CrossRef] [PubMed]
- Cantley, L.C. The phosphoinositide 3-kinase pathway. Science 2002, 296, 1655–1657. [Google Scholar] [CrossRef]
- Maehama, T.; Dixon, J.E. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 1998, 273, 13375–13378. [Google Scholar] [CrossRef]
- Stambolic, V.; Suzuki, A.; de la Pompa, J.L.; Brothers, G.M.; Mirtsos, C.; Sasaki, T.; Ruland, J.; Penninger, J.M.; Siderovski, D.P.; Mak, T.W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998, 95, 29–39. [Google Scholar] [CrossRef]
- Cheung, L.W.; Walkiewicz, K.W.; Besong, T.M.; Guo, H.; Hawke, D.H.; Arold, S.T.; Mills, G.B. Regulation of the PI3K pathway through a p85alpha monomer-homodimer equilibrium. eLife 2015, 4, e06866. [Google Scholar] [CrossRef]
- Raftopoulou, M.; Etienne-Manneville, S.; Self, A.; Nicholls, S.; Hall, A. Regulation of cell migration by the C2 domain of the tumor suppressor PTEN. Science 2004, 303, 1179–1181. [Google Scholar] [CrossRef]
- Megha; London, E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): Implications for lipid raft structure and function. J. Biol. Chem. 2004, 279, 9997–10004. [Google Scholar] [CrossRef] [PubMed]
- Lee, A.S. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 2005, 35, 373–381. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Y.; Tseng, C.C.; Tsai, Y.L.; Fu, X.; Schiff, R.; Lee, A.S. Cancer cells resistant to therapy promote cell surface relocalization of GRP78 which complexes with PI3K and enhances PI(3,4,5)P3 production. PLoS ONE 2013, 8, e80071. [Google Scholar] [CrossRef] [PubMed]
- Arap, M.A.; Lahdenranta, J.; Mintz, P.J.; Hajitou, A.; Sarkis, A.S.; Arap, W.; Pasqualini, R. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands. Cancer Cell 2004, 6, 275–284. [Google Scholar] [CrossRef] [PubMed]
- Kim, Y.; Lillo, A.M.; Steiniger, S.C.; Liu, Y.; Ballatore, C.; Anichini, A.; Mortarini, R.; Kaufmann, G.F.; Zhou, B.; Felding-Habermann, B.; et al. Targeting heat shock proteins on cancer cells: Selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry 2006, 45, 9434–9444. [Google Scholar] [CrossRef]
- Liu, Y.; Steiniger, S.C.; Kim, Y.; Kaufmann, G.F.; Felding-Habermann, B.; Janda, K.D. Mechanistic studies of a peptidic GRP78 ligand for cancer cell-specific drug delivery. Mol. Pharm. 2007, 4, 435–447. [Google Scholar] [CrossRef] [PubMed]
- Han, J.; Kaufman, R.J. The role of ER stress in lipid metabolism and lipotoxicity. J. Lipid Res. 2016, 57, 1329–1338. [Google Scholar] [CrossRef]
- Cook, K.L.; Soto-Pantoja, D.R.; Clarke, P.A.; Cruz, M.I.; Zwart, A.; Warri, A.; Hilakivi-Clarke, L.; Roberts, D.D.; Clarke, R. Endoplasmic Reticulum Stress Protein GRP78 Modulates Lipid Metabolism to Control Drug Sensitivity and Antitumor Immunity in Breast Cancer. Cancer Res. 2016, 76, 5657–5670. [Google Scholar] [CrossRef]
- Choy, K.W.; Murugan, D.; Mustafa, M.R. Natural products targeting ER stress pathway for the treatment of cardiovascular diseases. Pharmacol. Res. 2018, 132, 119–129. [Google Scholar] [CrossRef]
- Suyama, K.; Watanabe, M.; Sakabe, K.; Otomo, A.; Okada, Y.; Terayama, H.; Imai, T.; Mochida, J. GRP78 suppresses lipid peroxidation and promotes cellular antioxidant levels in glial cells following hydrogen peroxide exposure. PLoS ONE 2014, 9, e86951. [Google Scholar] [CrossRef]
- Dauer, P.; Sharma, N.S.; Gupta, V.K.; Durden, B.; Hadad, R.; Banerjee, S.; Dudeja, V.; Saluja, A.; Banerjee, S. ER stress sensor, glucose regulatory protein 78 (GRP78) regulates redox status in pancreatic cancer thereby maintaining “stemness”. Cell Death Dis. 2019, 10, 132. [Google Scholar] [CrossRef] [PubMed]
- Lee, A.S. GRP78 induction in cancer: Therapeutic and prognostic implications. Cancer Res. 2007, 67, 3496–3499. [Google Scholar] [CrossRef]
- Reddy, M.V.; Shen, Y.C.; Yang, J.S.; Hwang, T.L.; Bastow, K.F.; Qian, K.; Lee, K.H.; Wu, T.S. New bichalcone analogs as NF-kappaB inhibitors and as cytotoxic agents inducing Fas/CD95-dependent apoptosis. Bioorganic Med. Chem. 2011, 19, 1895–1906. [Google Scholar] [CrossRef] [PubMed][Green Version]
- Sahni, S.; Hickok, J.R.; Thomas, D.D. Nitric oxide reduces oxidative stress in cancer cells by forming dinitrosyliron complexes. Nitric Oxid. Biol. Chem. 2018, 76, 37–44. [Google Scholar] [CrossRef]
- Caneba, C.A.; Yang, L.; Baddour, J.; Curtis, R.; Win, J.; Hartig, S.; Marini, J.; Nagrath, D. Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells. Cell Death Dis. 2014, 5, e1302. [Google Scholar] [CrossRef] [PubMed]
- Lin, C.T.; Wong, C.I.; Chan, W.Y.; Tzung, K.W.; Ho, J.K.; Hsu, M.M.; Chuang, S.M. Establishment and characterization of two nasopharyngeal carcinoma cell lines. Lab. Investig. J. Tech. Methods Pathol. 1990, 62, 713–724. [Google Scholar]
- Lin, M.L.; Lu, Y.C.; Chung, J.G.; Li, Y.C.; Wang, S.G.; NG, S.H.; Wu, C.Y.; Su, H.L.; Chen, S.S. Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway. Cancer Lett. 2010, 291, 46–58. [Google Scholar] [CrossRef]
- Lin, M.L.; Lu, Y.C.; Chen, H.Y.; Lee, C.C.; Chung, J.G.; Chen, S.S. Suppressing the formation of lipid raft-associated Rac1/PI3K/Akt signaling complexes by curcumin inhibits SDF-1alpha-induced invasion of human esophageal carcinoma cells. Mol. Carcinog. 2014, 53, 360–379. [Google Scholar] [CrossRef]
- Zong, W.X.; Li, C.; Hatzivassiliou, G.; Lindsten, T.; Yu, Q.C.; Yuan, J.; Thompson, C.B. Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J. Cell Biol. 2003, 162, 59–69. [Google Scholar] [CrossRef]
- Taha, M.S.; Nouri, K.; Milroy, L.G.; Moll, J.M.; Herrmann, C.; Brunsveld, L.; Piekorz, R.P.; Ahmadian, M.R. Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP) with nucleolin. PLoS ONE 2014, 9, e91465. [Google Scholar] [CrossRef]
- Wu, C.W.; Wang, S.G.; Lin, M.L.; Chen, S.S. Downregulation of miR-144 by triptolide enhanced p85alpha-PTEN complex formation causing S phase arrest of human nasopharyngeal carcinoma cells. Eur. J. Pharmacol. 2019, 855, 137–148. [Google Scholar] [CrossRef] [PubMed]
- Lin, M.L.; Chen, S.S.; Ng, S.H. CHM-1 Suppresses Formation of Cell Surface-associated GRP78-p85alpha Complexes, Inhibiting PI3K-AKT Signaling and Inducing Apoptosis of Human Nasopharyngeal Carcinoma Cells. Anticancer Res. 2015, 35, 5359–5368. [Google Scholar] [PubMed]
- Lin, M.L.; Lu, Y.C.; Su, H.L.; Lin, H.T.; Lee, C.C.; Kang, S.E.; Lai, T.C.; Chung, J.G.; Chen, S.S. Destabilization of CARP mRNAs by aloe-emodin contributes to caspase-8-mediated p53-independent apoptosis of human carcinoma cells. J. Cell. Biochem. 2011, 112, 1176–1191. [Google Scholar] [CrossRef] [PubMed]
- Dobrowsky, R.T.; Kolesnick, R.N. Analysis of sphingomyelin and ceramide levels and the enzymes regulating their metabolism in response to cell stress. Methods Cell Biol. 2001, 66, 135–165. [Google Scholar]
- Lu, H.L.; Chen, S.S.; Hsu, W.T.; Lu, Y.C.; Lee, C.C.; Wu, T.S.; Lin, M.L. Suppression of phospho-p85alpha-GTP-Rac1 lipid raft interaction by bichalcone analog attenuates cancer cell invasion. Mol. Carcinog. 2016, 55, 2106–2120. [Google Scholar] [CrossRef]
- Wheelock, M.J.; Johnson, K.R. Cadherins as modulators of cellular phenotype. Annu. Rev. Cell Dev. Biol. 2003, 19, 207–235. [Google Scholar] [CrossRef]
- Goswami, R.; Singh, D.; Phillips, G.; Kilkus, J.; Dawson, G. Ceramide regulation of the tumor suppressor phosphatase PTEN in rafts isolated from neurotumor cell lines. J. Neurosci. Res. 2005, 81, 541–550. [Google Scholar] [CrossRef]
- Oninla, V.O.; Breiden, B.; Babalola, J.O.; Sandhoff, K. Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2. J. Lipid Res. 2014, 55, 2606–2619. [Google Scholar] [CrossRef]
- Bai, A.; Kokkotou, E.; Zheng, Y.; Robson, S.C. Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses. Cell Death Dis. 2015, 6, e1828. [Google Scholar] [CrossRef]
- Nakanishi, C.; Toi, M. Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs. Nat. Rev. Cancer 2005, 5, 297–309. [Google Scholar] [CrossRef]
- Perona, R.; Montaner, S.; Saniger, L.; Sanchez-Perez, I.; Bravo, R.; Lacal, J.C. Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins. Genes Dev. 1997, 11, 463–475. [Google Scholar] [CrossRef] [PubMed]
- Brown, D.A.; London, E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 2000, 275, 17221–17224. [Google Scholar] [CrossRef] [PubMed]
- Murai, T. The role of lipid rafts in cancer cell adhesion and migration. Int. J. Cell Biol. 2012, 2012, 763283. [Google Scholar] [CrossRef] [PubMed]
- Bernardes, N.; Fialho, A.M. Perturbing the Dynamics and Organization of Cell Membrane Components: A New Paradigm for Cancer-Targeted Therapies. Int. J. Mol. Sci. 2018, 19, 3871. [Google Scholar] [CrossRef]
- Lee, A.S. Glucose-regulated proteins in cancer: Molecular mechanisms and therapeutic potential. Nat. Rev. Cancer 2014, 14, 263–276. [Google Scholar] [CrossRef]
- Sozen, E.; Ozer, N.K. Impact of high cholesterol and endoplasmic reticulum stress on metabolic diseases: An updated mini-review. Redox Biol. 2017, 12, 456–461. [Google Scholar] [CrossRef]
- Bellezza, I.; Scarpelli, P.; Pizzo, S.V.; Grottelli, S.; Costanzi, E.; Minelli, A. ROS-independent Nrf2 activation in prostate cancer. Oncotarget 2017, 8, 67506–67518. [Google Scholar] [CrossRef]
- Li, Y.C.; Park, M.J.; Ye, S.K.; Kim, C.W.; Kim, Y.N. Elevated levels of cholesterol-rich lipid rafts in cancer cells are correlated with apoptosis sensitivity induced by cholesterol-depleting agents. Am. J. Pathol. 2006, 168, 1107–1118. [Google Scholar] [CrossRef]
- Yue, S.; Li, J.; Lee, S.Y.; Lee, H.J.; Shao, T.; Song, B.; Cheng, L.; Masterson, T.A.; Liu, X.; Ratliff, T.L.; et al. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. Cell Metab. 2014, 19, 393–406. [Google Scholar] [CrossRef]
- Grassme, H.; Jendrossek, V.; Bock, J.; Riehle, A.; Gulbins, E. Ceramide-rich membrane rafts mediate CD40 clustering. J. Immunol. 2002, 168, 298–307. [Google Scholar] [CrossRef]
- Boslem, E.; Weir, J.M.; MacIntosh, G.; Sue, N.; Cantley, J.; Meikle, P.J.; Biden, T.J. Alteration of endoplasmic reticulum lipid rafts contributes to lipotoxicity in pancreatic beta-cells. J. Biol. Chem. 2013, 288, 26569–26582. [Google Scholar] [CrossRef] [PubMed]
- Chagpar, R.B.; Links, P.H.; Pastor, M.C.; Furber, L.A.; Hawrysh, A.D.; Chamberlain, M.D.; Anderson, D.H. Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA 2010, 107, 5471–5476. [Google Scholar] [CrossRef] [PubMed]
- Otsu, M.; Hiles, I.; Gout, I.; Fry, M.J.; Ruiz-Larrea, F.; Panayotou, G.; Thompson, A.; Dhand, R.; Hsuan, J.; Totty, N.; et al. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell 1991, 65, 91–104. [Google Scholar] [CrossRef]
- Shepherd, P.R.; Withers, D.J.; Siddle, K. Phosphoinositide 3-kinase: The key switch mechanism in insulin signalling. Biochem. J. 1998, 333, 471–490. [Google Scholar] [CrossRef]
- Thorpe, L.M.; Spangle, J.M.; Ohlson, C.E.; Cheng, H.; Roberts, T.M.; Cantley, L.C.; Zhao, J.J. PI3K-p110alpha mediates the oncogenic activity induced by loss of the novel tumor suppressor PI3K-p85alpha. Proc. Natl. Acad. Sci. USA 2017, 114, 7095–7100. [Google Scholar] [CrossRef]
- Ueki, K.; Fruman, D.A.; Brachmann, S.M.; Tseng, Y.H.; Cantley, L.C.; Kahn, C.R. Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol. Cell. Biol. 2002, 22, 965–977. [Google Scholar] [CrossRef]
- Zhang, L.Y.; Ho-Fun Lee, V.; Wong, A.M.; Kwong, D.L.; Zhu, Y.H.; Dong, S.S.; Kong, K.L.; Chen, J.; Tsao, S.W.; Guan, X.Y.; et al. MicroRNA-144 promotes cell proliferation, migration and invasion in nasopharyngeal carcinoma through repression of PTEN. Carcinogenesis 2013, 34, 454–463. [Google Scholar] [CrossRef]
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Lee, T.-L.; Wang, S.-G.; Chan, W.-L.; Lee, C.-H.; Wu, T.-S.; Lin, M.-L.; Chen, S.-S. Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells. Cells 2020, 9, 371. https://doi.org/10.3390/cells9020371
Lee T-L, Wang S-G, Chan W-L, Lee C-H, Wu T-S, Lin M-L, Chen S-S. Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells. Cells. 2020; 9(2):371. https://doi.org/10.3390/cells9020371
Chicago/Turabian StyleLee, Tsung-Lin, Shyang-Guang Wang, Wen-Ling Chan, Ching-Hsiao Lee, Tian-Shung Wu, Meng-Liang Lin, and Shih-Shun Chen. 2020. "Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells" Cells 9, no. 2: 371. https://doi.org/10.3390/cells9020371
APA StyleLee, T.-L., Wang, S.-G., Chan, W.-L., Lee, C.-H., Wu, T.-S., Lin, M.-L., & Chen, S.-S. (2020). Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells. Cells, 9(2), 371. https://doi.org/10.3390/cells9020371