|Year : 2009 | Volume
| Issue : 9 | Page : 61-66
Association between the unfolded protein response, induced by 2-deoxyglucose, and hypersensitivity to cisplatin: A mechanistic study employing molecular genomics
Shobhan Gaddameedhi1, Satadal Chatterjee2
1 Department of Pharmaceutical Sciences, College of Pharmacy, Nursing and Allied Sciences, North Dakota State University, Fargo, ND - 58105, USA
2 Department of Biochemistry and Biophysics, University of North Carolin, School of Medicine, Chapel Hill, NC 27599, USA
|Date of Web Publication||21-Aug-2009|
Department of Pharmaceutical Sciences, College of Pharmacy, Nursing and Allied Sciences, North Dakota State University, Fargo, ND - 58105
Source of Support: None, Conflict of Interest: None
Background: The specific signaling that occurs between the endoplasmic reticulum (ER) and the nucleus in response to ER stress is known as the unfolded protein response (UPR). Specific induction of GRP78 (glucose-regulated protein of Mr 78 kDa) is an integral component of ER stress and the UPR. We first discovered that the up-regulation of GRP78 is associated with augmented sensitivity/apoptosis of cancer cells to clinically used alkylating/platinating agents.
Objectives: To decipher molecular mechanisms that associate induction of the UPR/GRP78 with augmented sensitivity/apoptosis to cisplatin.
Materials and Methods: A549 cells were exposed to 2-deoxyglucose (2dG) to induce the UPR/GRP78, followed by cisplatin treatment. We used human cDNA microarray containing 42,000 ESTs as well as pathway-specific macroarrays for apoptosis, cell cycle, and MAP kinase signaling pathways containing 100-280 genes and subsequently examined the pertinent transcript levels. The results obtained from these studies were confirmed by examining relevant protein levels and the enzymatic activity.
Results: We demonstrate that the induction of UPR/GRP78 alone causes a decrease in the transcript levels of DNA repair genes and DNA damage check point genes, and an increase in the transcript levels of apoptotic genes. Furthermore, we show that cisplatin treatment after the induction of UPR/GRP78 is facilitating the mitochondria-mediated apoptotic cascades through the initial activation of caspase-2 and down-regulation of genes involved in DNA repair.
Conclusions: Our study will shed new insight as to the increased understanding of the mechanisms of the UPR/GRP78 modulation of molecular and cellular responses to cisplatin that will allow strategies for transferring bench side results to the bed.
Keywords: Apoptosis, cisplatin sensitivity, glucose-regulated stress protein of molecular weight 78 kDa, unfolded protein response, 2-deoxyglucose
|How to cite this article:|
Gaddameedhi S, Chatterjee S. Association between the unfolded protein response, induced by 2-deoxyglucose, and hypersensitivity to cisplatin: A mechanistic study employing molecular genomics. J Can Res Ther 2009;5, Suppl S1:61-6
|How to cite this URL:|
Gaddameedhi S, Chatterjee S. Association between the unfolded protein response, induced by 2-deoxyglucose, and hypersensitivity to cisplatin: A mechanistic study employing molecular genomics. J Can Res Ther [serial online] 2009 [cited 2019 May 22];5:61-6. Available from: http://www.cancerjournal.net/text.asp?2009/5/9/61/55146
| > Introduction|| |
Endogenous or exogenous stress can adversely affect the process of protein folding in endoplasmic reticulum (ER) resulting in ER stress. The specific signaling that occurs between the ER and the nucleus in response to ER stress is known as the unfolded protein response (UPR). The mechanisms that govern or disrupt the balance between survival and apoptotic responses during ER stress are not well understood. Broadly speaking, during ER stress, ER chaperone protein GRP78 (glucose-regulated stress protein of molecular weight 78 kDa) is released from its sensors/transducers such as PERK (protein kinase-like ER kinase), IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), and ATF6 (activation of transcription factor 6), which become activated, triggering the UPR. Thus, apparently, specific induction/overexpression of GRP78 is an integral component of ER stress and the UPR. , We have previously shown that overexpression of GRP78 is associated with augmented sensitivity to alkylating/platinating agents in V79 Chinese hamster normal lung fibroblasts and a variety of human colon cancer cell lines irrespective of the mutations they are harboring. , However, the mechanisms of this association remained obscured.
Our present study clearly demonstrates that A549 human lung cancer cells exhibit increased sensitivity/apoptosis to cisplatin following induction of the UPR by 2dG (2-deoxyglucose). The overall objective of this study is to determine the biological consequences of the induction of UPR and to understand the molecular mechanisms that associate this induction with augmented sensitivity/apoptosis to cisplatin in A549 cells. The identification of specific components related to the underlying mechanisms could then potentially serve as targets for cancer therapy. Our study is driven by the following hypotheses:
It should be noted that, we are not suggesting that these two pathways are mutually exclusive. Rather, they can be complementary to each other.
- Since GRP78 is a strong Ca 2+ binding protein, its induction will lead to an altered mobilization of Ca 2+ from the ER which has the potential to modify the signal transduction pathways and/or proteins associated with apoptosis and cell-cycle regulation. Treatment with cisplatin under those conditions will lead to hyperactivation of those pathways resulting in increased sensitivity/apoptosis.
- During the UPR, GRP78 translocates into the nucleus that is a residence of many DNA repair enzymes. Since GRP78 is a chaperone, it will downregulate the activity of DNA repair enzymes through direct/indirect interaction with these enzymes. Consequently, treatment with cisplatin will lead to attenuation in repair of DNA adducts, strand breaks, and cross-links causing apoptosis/hypersensitivity.
| > Materials and Methods|| |
Cell culture maintenance and 2dG treatment
Experiments were performed following the schematic protocol outlined below [Figure 1].
A 549 human lung cancer cells were cultured at 37°C with 5% CO 2 in the RPMI-1640 medium (Mediatech, Inc., Herndon, VA, USA) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin (Mediatech), and 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Inc., Lawrenceville, GA, USA). 2dG was obtained from Sigma-Aldrich Co., St. Louis, MO, USA. It was dissolved in distilled water to prepare a stock solution of 2 M, and aliquots were kept frozen at 20°C for future use. It should be noted that the concentration and exposure time of 2dG is not toxic to the cells as evidenced by clonogenic survival assays (data not shown); however, under the conditions of the experiments, 2dG induces the UPR as evidenced by GRP78 overexpression [Figure 2]a. The cells were allowed to grow for an additional 12 h in a normal medium after 2dG treatment to avoid the potential artifacts that might have resulted from 2dG treatment. For example, 2dG depletes the ATP pool, and these levels become normal after 12 h of the treatment (data not shown); however, GRP78 levels remain elevated [Figure 2]a thus allowing us to specifically associate the UPR induction with the results obtained.
cDNA microarray for global gene expression analysis
Human cDNA microarrays containing ~42,000 genes from Stanford Functional Genomics Facility were used to identify changes in gene transcript profiles after the UPR induction. Total RNA was isolated employing QIAGEN RNeasy mini kit (QIAGEN, Valencia, CA, USA), followed by cDNA labeling using Invitrogen Superscript TM Direct cDNA Labeling System (Invitrogen Corporation, Carlsbad, CA, USA). DNA immobilization, prehybridization, and hybridization were performed as recommended by CORNING Life Sciences. Images were scanned by Affymetrix 428 scanner (Affymetrix, Inc., Santa Clara, CA, USA), followed by image processing using GenePix pro 6.0 software (Molecular Devices Corporation, Sunnyvale, CA, USA). Microarray normalization and analysis were done using the GeneSpring 7.2 software package (Agilent Technologies, Inc., Palo Alto, CA, USA). The average values of two biological replicates were taken after data normalization and analysis. Results were expressed as a list of the genes whose transcript levels were up-regulated (positive values) and down-regulated (negative values) after the UPR induction, and the numerical values indicate the fold change of transcript levels compared to normal control (NC).
Macroarrays for apoptosis, cell-cycle regulation, and MAP kinase signaling pathways
Human cell-cycle, apoptosis, and MAP kinase signaling pathways were monitored using macroarrays (SuperArray Bioscience Corporation, Frederick, MD, USA) containing 100-280 genes following all the protocols recommended by the manufacturer. The average values of two biological replicates were taken after normalization using GE Array Expression Analysis Suite Software. Results were expressed as a list of the genes whose transcript levels were changed in response to cisplatin as well as the UPR induction followed by cisplatin treatment, and the numerical values indicate the fold changes in transcript levels compared to normal control (NC).
Cell lysate preparation, gel electrophoresis, and western blotting
The procedure was described by us previously. , Briefly, after the treatments, cells were trypsinized, washed with HBSS, and collected by centrifugation. Cell pellets were lysed with a lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with complete mini protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and PMSF (Sigma-Aldrich). Lysate was sonicated to shear the DNA, and the protein concentration was determined using a Bio-Rad protein assay kit. Protein samples were separated by SDS-PAGE, transferred to Immobilin-P membrane (Millipore, Bedford, MA, USA), blocked, incubated with primary antibodies followed by secondary antibodies and finally with chemiluminescence detection reagents. Blots were exposed to Hyperfilm and protein band intensities quantified by densitometry. Actin was used as an internal standard. The primary antibodies and sources are as follows: Caspase-2, phospho bcl-2, actin, antirabbit IgG, and antimouse IgG (Cell Signaling Technology); GRP78 (SPA-827, Stressgen Biotechnology,Victoria, British Columbia, Canada); cytochrome c (Santa Cruz Biotechnology, Santa Cruz,
CA, USA); topo IIa (611327, BD Biosciences, Pharmingen, San Jose, CA, USA). Two to four biological replicates were performed for each experiment.
Caspase-3 and caspase-9 fluorometric-based assays were performed as recommended by the manufacturer (BioVision, Inc., Mountain View, CA, USA).
Cytosol, nucleus, and mitochondrial isolation and measurement of cytochrome c oxidase levels
Mitochondrial isolation was done using the BioVision mitochondrial/cytosol fractionation kit. The intact mitochondria were used to measure cytochrome c oxidase levels as suggested by Sigma-Aldrich. The results are derived from the average of two biological replicates.
Detection of internucleosomal DNA fragmentation
DNA fragmentation was detected by a slightly modified protocol described by us previously.  Briefly, cells were harvested after the treatment, trypsinized, and washed with HBSS. Cells (about 2×10 6 ) were fixed in 70% ethanol at −20°C for 24 h and centrifuged at 800 g for 5min to remove the ethanol thoroughly. The cell pellet was resuspended in a 40-µl phosphate-citrate buffer (PCB) and incubated at room temperature for 30min and then centrifuged at 14,000 rpm for 10min at 4°C to eliminate intact high-molecular-weight DNA. The supernatant containing low-molecular-weight DNA was treated with 3 µl 0.25% nonide NP-40, 3 µl RNase (1 mg/ml) for 30min at 37°C followed by 3 µl proteinase K (1 mg/ml) for 30min at 37°C, and then electrophoresed in a 2% agarose gel for 3 h at 4 V/cm. DNA was visualized by ethidium bromide under UV light. Cells treated with 1 µM staurosprine, a kinase inhibitor, for 12 h followed by 12 h in a normal medium to induce apoptosis served as a positive control for monitoring apoptosis.
| > Results|| |
Since the up-regulation of GRP78 is the hallmark of the UPR induction, we used overexpression of GRP78 as an indicator of the UPR. [Figure 2]a demonstrates that 2dG treatment results in a significant elevation in GRP78 levels in 2dG and 2dG + CIS compared to NC and CIS. We have previously shown in V79 Chinese hamster cells that the up-regulation of GRP78 causes a down-regulation of DNA topoisomerase IIa (topo IIa), a nuclear enzyme involved in DNA replication and repair. , For A549 cells, a down-regulation of topo IIa was also observed in 2dG and 2dG + CIS. The decrease in topo IIa is much more pronounced in 2dG +CIS as evidenced from [Figure 2]b. This fact prompted us to postulate that GRP78 enters nucleus following up-regulation and interacts with topo IIa. We verified this concept under the condition of 2dG and subsequently determining the levels of GRP78 in cytosolic, nuclear, and membrane fractions. As illustrated in [Figure 2]c, GRP78 translocates into the nucleus following activation by 2dG. [Figure 3] depicts augmented apoptosis in 2dG + CIS compared to NC, 2dG, and CIS as carried out by the DNA fragmentation assay. In fact, no detectable apoptosis was observed under the latter conditions. Thus, the induction of the UPR is clearly associated with increased sensitivity/apoptosis to cisplatin in A549 cells also. Clearly, this finding is true irrespective of the cell lines studied by us so far. Topo IIa is involved in DNA repair and its deficiency stemming from the UPR induction can potentially compromise the repair of DNA damage inflicted by cisplatin thus rendering the cells to become hypersensitive to it. However, we became more interested in exploring other pathways contributing to the augmented sensitivity to cisplatin after the UPR induction.
We investigated the global changes in gene transcript levels, after the UPR induction by 2dG treatment, employing human cDNA microarrays. Overall, we observed a significant up-regulation of 75 genes and down-regulation of 460 genes. Nonetheless, we presented only those genes pertinent to the scope of this manuscript and the results are shown in [Table 1] which is self-explanatory. We also used pathway-specific macroarrays to monitor transcript levels of the genes associated with apoptosis, cell cycle, and MAP kinase signaling pathways after CIS and 2dG + CIS. The results, pertinent to this manuscript, are depicted in [Table 2]. Although gene transcript levels are very good indicators of genes of interest, they do not necessarily represent gene expression levels. Thus, subsequently, we chose some of the relevant genes from micro- and macroarray experiments. We contemplated that a further exploration of expression of those genes might provide insight as to the mechanisms responsible for hypersensitivity to cisplatin after the UPR induction. Active caspase-2 results from the cleavage of procaspase-2. Caspase-2 activity is required for the translocation of the proapoptotic protein Bax to mitochondria. Consequently, mitochondrial membrane permeability is increased; cytochrome c is released into cytosol that triggers a series of events eventually causing apoptosis. In contrast, antiapoptotic protein bcl-2 prevents such action.  Thus, broadly speaking, the decision of cells to undergo apoptosis hangs between the balance of the activity of Bax and bcl-2. [Figure 4]a illustrates that the UPR followed by cisplatin (2dG + CIS) activates procaspase-2 to a larger extent compared to the UPR alone (2dG) or CIS which consequently releases significantly higher amount of cytochrome c into cytosol [Figure 4]b. Bcl-2 is activated upon phosphorylation. Cytochrome c release into cytosol is not inhibited by bcl-2 since phosphorylated bcl-2 is practically absent under the UPR induction (2dG) and UPR followed by cisplatin treatment (2dG + CIS) as evidenced from [Figure 4]c.
The released cytochrome c binds to a cytosolic scaffold protein called "apoptotic protease activating factor (Apaf-1)" to form a ternary complex with procaspase-9. Consequently, procaspase-9 is converted to active caspase-9; active caspase-9 then turns on downstream effector caspases such as caspase-3 and caspase-7 resulting in apoptosis. , These facts prompted us to investigate the activity levels of caspase-3 and caspase-9. It should be noted that caspase assays were performed at different time intervals immediately after the (1) growth of cells in a normal medium for 48 h, (2) growth of cells in a 2dG-containing medium for 48 h, (3) growth of cells in a normal medium for 48 h followed by a 2-h cisplatin treatment, and (4) growth of cells in a 2dG-containing medium for 48 h followed by a 2-h cisplatin treatment. It was necessary to design the experiments in this fashion since the activation of caspases precedes apoptosis and we observed apoptosis at 12 h after the growth of cells in a 2dG-containing medium for 48 h followed by a 2-h cisplatin treatment. [Figure 5] shows that the increased activity of caspase-3 and caspase-9 reaches peak at 4 h under 2dG, CIS, and 2dG + CIS conditions, and the highest activity was observed under the 2dG + CIS condition.
| > Discussion|| |
In this study, we have clearly shown that, under the conditions of the experiments, cisplatin fails to cause any apoptosis. In contrast, the UPR followed by cisplatin treatment exhibits significant augmentation in apoptosis. Microarray results show the transcript levels of many of the genes involved in signaling, apoptosis, and tumor suppression, such as calreticulin, JNK2, caspase-3, programmed cell death 5, death-associated protein, and caveolin 2, are increased after the UPR induction. In contrast, transcript levels of DNA repair proteins, such as, DNA polymerase b, XRCC4 and TP53BP1 are concomitantly decreased. The UPR followed by cisplatin treatment exhibits elevated transcript levels of mostly apoptotic genes, for example, caspase-2, caspase-7, bcl2L11 and Apaf-1.
We have verified the validity of the transcript levels, by examining relevant protein/enzymatic levels of various genes involved in apoptosis, signaling, and DNA repair mechanism. We have shown under the 2DG + CIS condition that caspase-2 is activated to a significant extent compared to 2dG causing translocation of the apoptotic protein Bax into the mitochondria (data not shown and to be published elsewhere), while blocking the antiapoptotic effects of bcl-2 as evidenced from the absence of phospho-bcl-2 under the 2dG + CIS condition. Subsequently, we have demonstrated, as a consequence, that activated caspase-2 significantly increases the levels of cytochrome c in cytosol by increasing the permeability of the mitochondrial membrane as evidenced from cytochrome c oxidase assay (data not shown and to be published elsewhere) under the 2dG + CIS condition. Furthermore, we have shown that caspase-3 and caspase-9 attain their highest activity at 4 h, as alluded to at the end of the "Results" section, which is downstream of the cytochrome c release. Based on these observations, we propose a model to decipher the mechanisms of association of the UPR and hypersensitivity to cisplatin [Figure 6].
The UPR followed by cisplatin activates caspase-2. In turn, activated caspase-2 allows the mitochondrial release of cytochrome c into cytosol through the translocation of Bax into mitochondria. Antiapoptotic protein bcl-2 cannot prevent the mitochondrial release of cytochrome c since its active form is absent under the experimental conditions. Furthermore, it can be inactivated by overexpression of proapoptotic protein bcl2L11. Released cytochrome c binds to Apaf-1 resulting in the sequential activation of caspase-9 and caspase-3 which causes hypersensitivity/increased apoptosis.
We have shown GRP78 enters nucleus following the UPR activation. Subsequently, we pulled down GRP78-binding proteins employing the co-immunoprecipitation assay. GRP78-protein complexes were then separated by SDS-PAGE; protein bands were excised and subjected to tryptic digestion followed by mass spectrometry analysis by LC/MS/MS. Analysis of protein bands revealed many DNA repair proteins including topo IIa (data not shown and to be published elsewhere). Thus, we also propose that the UPR activation causes GRP78 to enter nucleus which then binds to various DNA repair and replication proteins forming complexes. These complexes are subsequently degraded by yet some unknown pathways causing deficiencies of those proteins leading to the impairment of repair of DNA damages inflicted by cisplatin and other alkylating agents. Clearly, these series of events can also promote hypersensitivity/augmented apoptosis. However, the questions remain as to whether the two proposed pathways leading to apoptosis are complimentary. For clarity, in this manuscript, we presented only the pathway stemming from the molecular genomics study.
A significant number of questions still remain to be answered. (1) How does the UPR induction affect so many networks of apparently unrelated proteins? (2) How does GRP78 translocate into nucleus despite lacking a nuclear localization signal? (3) What are the mechanisms of forming complexes with DNA repair/replication proteins? (4) What are the pathways contributing to the degradation of such complexes?This study has enormous clinical implications. For example, many solid tumors are hypoxic and resistant to radiation. However, these cells overexpress GRP78 and thus can be potential candidates for low-dose alkylating/platinating agent therapy. Most of the tumors harbor mutations in various genes such as p53, mismatch repair system which render them to become refractory to currently available alkylating/platinating agents. In these cases, the combination therapy of 2dG and low doses of alkylating/platinating agents should be very effective because these approaches efficiently induce apoptosis irrespective of the various mutations in the cancer cells.
| > Acknowledgements|| |
This study was supported, in part, by grants RO1CA65920 from the NCI/NIH, 00A1 from the American Institute for Cancer Research, EPSCoR, and BRIN from NDSU to SC.
| > References|| |
|1.||Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: Friend or foe? Nat Rev Cancer 2004;4:966-77. |
|2.||Li J, Lee AS. Stress induction of GRP78/BiP and its role in cancer. Curr Mol Med 2006;6:45-54. |
|3.||Chatterjee S, Hirota H, Belfi CA, Berger SJ, Berger NA. Hypersensitivity to DNA cross-linking agents associated with up-regulation of glucose- regulated stress protein GRP78. Cancer Res 1997;57:5112-16. |
|4.||Belfi CA, Chatterjee S, Gosky DM, Berger SJ, Berger NA. Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation. Biochem Biophys Res Commun 1999;257:361-8. |
|5.||Chatterjee S, Cheng MF, Berger RB, Berger SJ, Berger NA. Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide. Cancer Res 1995;55:868-73. |
|6.||Chatterjee S, Cheng MF, Berger SJ, Berger NA. Induction of M(r) 78,000 glucose-regulated stress protein in poly(adenosine diphosphate- ribose) polymerase- and nicotinamide adenine dinucleotide-deficient V79 cell lines and its relation to resistance to the topoisomerase II inhibitor etoposide. Cancer Res 1994;54:4405-11. |
|7.||Hashimoto H, Chatterjee S, Berger NA. Inhibition of etoposide (VP-16)- induced DNA recombination and mutant frequency by Bcl-2 protein overexpression. Cancer Res 1995;55:4029-35. |
|8.||Gosky D, Chatterjee S. Down-regulation of topoisomerase II alpha is caused by up-regulation of GRP78. Biochem Biophys Res Commun 2003;300:327-32. |
|9.||Hirota H, Gosky D, Berger NA, Chatterjee S. Interference with topoisomerase IIalpha potentiates melphalan cytotoxicity. Int J Oncol 2002;20:311-8. |
|10.||Lassus P, Opitz-Araya X, Lazebnik Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 2002;297:1352-4. |
|11.||Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, et al . bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993;74:597-608. |
|12.||Jemmerson R, LaPlante B, Treeful A. Release of intact, monomeric cytochrome c from apoptotic and necrotic cells. Cell Death Differ 2002;9:538-48. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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