|Year : 2014 | Volume
| Issue : 3 | Page : 507-513
Evaluation of β-catenin expression in muscle-invasive urothelial bladder carcinoma
Fatma Ahmed Elserafy1, Tarek Mohamed Abdel Elbaky1, Eid Abdel-Rasoul Elsherif1, Noha Mohamed Nor-Eldin-Elkady2, Marwa Mohamed Dawood2, Mohamed Sayed El Gharabawy1
1 Department of Urology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||12-Dec-2013|
|Date of Acceptance||19-Jan-2014|
|Date of Web Publication||26-Nov-2014|
MSc Mohamed Sayed El Gharabawy
Urology Department, Faculty of Medicine, Menoufia University, Menoufia
Source of Support: None, Conflict of Interest: None
This study aimed at evaluation of the expression of β-catenin in muscle-invasive urothelial bladder carcinoma and its correlation with other clinicopathological parameters of prognostic importance.
β-Catenin is a protein that is encoded by the CTNNB1 gene that can function as an oncogene. Activation of the Wnt/β-catenin signaling pathway by aberrant accumulation of stabilized β-catenin contributes to the progression of several human cancers. Therefore, inhibition of the Wnt pathway may have a major therapeutic potential.
Patients and methods
This study was conducted as a retrospective study at Urology and Pathology Departments, Faculty of Medicine, Menoufia University, during the period between May 2011 and May 2013. A total of 40 patients were included and divided into two groups. The first one included 10 patients with chronic nonspecific cystitis as a control group and the second one included 30 patients with muscle-invasive urothelial bladder carcinoma who underwent radical cystectomy. The pathological blocks of both groups were investigated for the expression of β-catenin using immunohistochemical staining.
β-Catenin expression was noticed in 100% (30 cases) of muscle-invasive bladder carcinoma and in 70% (seven cases) of nonmalignant cases. In the malignant group, 26 (87%) cases showed nucleocytoplasmic expression and four (13%) cases showed membranous expression only. There was a significant correlation between nucleocytoplasmic localization of β-catenin and some pathological parameters such as tumor grade, stage, and mitosis. Furthermore, the study showed statistically significant correlation between intensity of β-catenin expression and tumor grade, stage, lymph node metastasis, vascular and perineural invasion, mitosis, and microvessel density.
Nucleocytoplasmic expression of β-catenin and high intensity of its expression was associated with poor prognostic parameters.
Keywords: bladder, β-catenin, carcinoma, invasive, urothelial
|How to cite this article:|
Elserafy FA, Abdel Elbaky TM, Elsherif EA, Nor-Eldin-Elkady NM, Dawood MM, El Gharabawy MS. Evaluation of β-catenin expression in muscle-invasive urothelial bladder carcinoma. Menoufia Med J 2014;27:507-13
|How to cite this URL:|
Elserafy FA, Abdel Elbaky TM, Elsherif EA, Nor-Eldin-Elkady NM, Dawood MM, El Gharabawy MS. Evaluation of β-catenin expression in muscle-invasive urothelial bladder carcinoma. Menoufia Med J [serial online] 2014 [cited 2020 Mar 30];27:507-13. Available from: http://www.mmj.eg.net/text.asp?2014/27/3/507/145491
| Introduction|| |
Bladder cancer is the fourth most common cancer occurring in the USA, the second most prevalent cancer in men, and the 10th most prevalent cancer in women, with incidence rate of 24.5 per 100 000 populations and mortality rate of 4.2 per 100 000 populations per year  . Because of its high recurrence rate, the actual prevalence of active bladder cancer is estimated to be about 10 times the number of new cases  . Among Egyptian men, bladder cancer is still the most frequent malignant tumor representing 16.2% of male cancers. Among Egyptian women, its frequency is 4%. For both sexes together, the frequency of bladder cancer is 10.1%  .
Approximately 20-30% of urothelial bladder carcinoma (UBC) cases initially present with muscle-invasive tumors (pT2-4) , and its treatment requires a radical cystectomy or chemotherapy with radiation protocol. Radical cystectomy has many quality of life implications. In addition, the absolute survival benefit of neoadjuvant or adjuvant chemotherapy is debatable, and toxicity can be significant. Despite the current treatments, distant metastases eventually may develop in as many as 50% of patients with muscle-invasive tumor. Treatment options for metastatic bladder cancers are extremely limited, with a 5-year survival rate of 6% and a median survival time of 12-20 months. Therefore, it is generally believed that there is an urgent need to expand the current paradigm of therapy by integrating novel targeted therapies for muscle-invasive bladder cancer  .
The role of Wnt signaling pathway is increasingly recognized in many different tissues, and new data indicate cross-talk with other key cancer pathways regulating angiogenesis, proliferation, invasion, and metastasis  . Thus, aberrant activation of the Wnt pathway contributes to the progression of several major human cancers, and inhibition of Wnt effects has major therapeutic potential  .
β-Catenin is a subunit of the cadherin protein complex; it also acts as an intracellular signal transducer in the Wnt signaling pathway  .
β-Catenin plays a dual role by its function in cell adhesion, by transmitting the contact inhibition signal, and also in the regulation of gene transcription in normal cells in a variety of biological processes including development and differentiation, the regulation of embryonic and adult stem cells, and also in the course of cancer development ,, .
| Patients and methods|| |
This retrospective study was conducted during the period between May 2011 and May 2013 by collaboration between Urology and Histopathology Departments, Faculty of Medicine, Menoufia University.
Forty patients were included in this study. Ten patients with chronic nonspecific cystitis, as a control group, and 30 patients with muscle-invasive UBC who underwent radical cystectomy. The pathological blocks of both groups were investigated for the expression of β-catenin.
Urinary bladder specimens were obtained by different procedures such as cold cup biopsy and transuretheral resection of bladder tumor for the nonmalignant group and by radical cystectomy for the malignant group. All specimens were fixed in 10% neutral-buffered formalin.
Samples were subjected to routine tissue processing that ended with paraffin-embedded blocks ready for sectioning. Four-micrometer-thick tissue sections were cut and stained with hematoxylin and eosin and examined under light microscope.
Examination of hematoxylin and eosin-stained slides
Malignant samples were evaluated for tumor grade according to the 2004 WHO criteria where urothelial carcinoma is graded into low and high grades. Tumor staging was performed according to the TNM staging system and stage grouping  . Stage II was considered as an early stage, whereas stages III and IV were lumped together to represent advanced stages. Other pathological parameters such as vascular invasion, perineural invasion, apoptotic index, mitotic index, microvessel density (MVD), presence of bilharziasis, necrosis, and adjacent bladder tissue were also evaluated.
Four-micrometer-thick sections were cut from the paraffin-embedded blocks with subsequent steps of deparaffinization and rehydration in xylene and a graded series of alcohol, respectively. Antigen was retrieved by boiling in 10 mmol/l citrate buffer (pH 6.0) for 10-20 min and then cooled at room temperature. The slides were incubated overnight at room temperature with β-catenin (purified rabbit polyclonal antibody against C-terminus of β-catenin in PBS/1% BSA buffer and sodium azide, Cat. #RB-9035-P0; Thermo Fisher Scientific, UK). One to two drops of biotinylated goat-polyvalent secondary antibody were applied and incubated for 10 min at room temperature at humidity chamber (antipolyvalent, HRP/DAB+(PTU), Cat. #TP-015-HD; Labvision Corp., Thermoscientific, USA). The reaction was visualized by an appropriate substrate/chromogen (diaminobenzidine tetrahydrochloride) DAB reagent with Mayer's hematoxylin as a counterstain (Cat. #94583; BioGenex). Breast cancer tissue was used as a positive control. The negative controls obtained by substitution of primary antibodies with blocking buffer were included in the staining procedure.
Interpretation of immunostaining
Assessment of immunohistochemical sections was performed using a semiquantitative visual approach. Unintentional bias was prevented by coding patient's slides. The entire slide was scanned for immunostaining evaluation. Scoring was carried out using an Olympus CH2 light microscope (Olympus, Tokyo, Japan) with wide angle (field size of 0.274 mm 2 and field diameter of 0.59 mm) at ×400 magnification. β-Catenin expression was evaluated in the non-neoplastic and malignant group. The cases were labeled positive when any number of cells displayed brown staining. Thereafter, the localization was classified into membranous, cytoplasmic, and/or nuclear. The score of β-catenin staining was assessed and classified into low and high score according to Xi-Qing et al.  (0: ≤5%; 1: 5-25%; 2: 25-75%; 3: ≥75%). The score 0 was defined as low and 1, 2, 3 as high.
| Results|| |
This study included 40 patients with a mean age ± SD of 62.63 ± 4.88 years, with 28 (70%) male patients and 12 (30%) female patients.
They were classified into two groups. The first control group 'chronic nonspecific cystitis' included 10 cases, five (50%) were men and five (50%) were women with a mean age ± SD of 58.3 ± 3.37 years. None of these cases showed specific infections, dysplasia, or metaplasia. Seven (70%) patients of the control group showed positive β-catenin expression. All β-catenin-positive cases showed membranous localization. On examining the seven positive cases, three of them were of a low β-catenin score, whereas four of them were of a high score.
The second malignant group included 30 cases that showed male predominance [23 cases (77%)]. The patients' age ranged between 51 and 70 years with a mean age ± SD of 64.07 ± 4.46 years. The second group showed positive expression of β-catenin in all 30 (100%) samples. In all, 26 (87%) cases of them showed nucleocytoplasmic expression and four (13%) cases showed membranous expression only. Eight (26.67%) cases were of low tumor grade and 22 (73.33%) were of high grade. Eleven (36.37%) cases belonged to tumor stage II (early stage) and 19 (63.33%) were of advanced stages (III and IV). Lymph node positivity was encountered in eight (26.67%) cases. Vascular invasion was seen in 11 (36.37%) cases, whereas 13 (43.33%) cases showed perineural invasion. Associated bilharziasis was seen in eight (26.67%) cases. Thirteen (43.33%) cases showed tumor necrosis. The apoptotic index ranged between 5 and 20 with a mean ± SD of 13.77 ± 4.04 and median of 15, whereas mitotic index ranged between 10 and 25 with a mean ± SD of 17.33 ± 4.13 and a median of 18. MVD ranged between 13 and 25 with a mean ± SD of 19.43 ± 2.80 and a median of 20 ([Table 1]).
|Table 1 Clinicopathological data of the muscle-invasive urothelial bladder carcinoma group |
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When comparing the malignant muscle-invasive UBC group and nonmalignant group, there was a statistical significant difference regarding β-catenin positivity (P = 0.01) and nucleocytoplasmic localization (P = 0.01), where positive expression and nucleocytoplasmic localization were seen in the malignant cases than in the control ones.
Correlation of β-catenin nucleocytoplasmic localization with age and sex showed no statistical significant difference (P = 0.238 and 1.0, respectively). Correlation with histopathological data revealed a statistically significant relationship between β-catenin nucleocytoplasmic localization and high tumor grade (P = 0.003), advanced stage (P = 0.012), and mitotic index (P = 0.005), but there was no statistical significance with respect to the lymph node metastasis (P = 0.55), vascular invasion (P = 0.268), perineural invasion (P = 0.113), associated bilharziasis (P = 0.55), tumor necrosis (P = 0.113), apoptosis (P = 0.065), and MVD (P = 0.139) ([Table 2]).
|Table 2 Correlation between localization of b -catenin-positive urothelial bladder carcinoma cases and the various histopathological parameters |
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Regarding β-catenin score and its correlation with age, 11 samples showed low score with a median of 63 years, whereas 19 cases showed high score with a median of 66 years. This difference was not statistically significant (P = 0.729). In addition, the correlation between sex and β-catenin score showed no statistically significant difference (P = 0.515).
On analyzing the correlation between β-catenin score and various histopathological characters, the study showed statistically significant correlation between high β-catenin score and high tumor grade (P = 0.001), lymph node metastasis (P = 0.014), advanced stage (P = 0.001), vascular invasion (P = 0.02), perinural invasion (P = 0.001), high mitotic index (P = 0.001), and high MVD (P = 0.003). However, the study showed no statistically significant correlation between β-catenin score and apoptotic index (P = 0.07), associated bilharziasis (P = 0.199), and tumor necrosis (P = 0.259) ([Table 3]).
|Table 3 Correlation between score of b -catenin-positive urothelial bladder carcinoma cases and the various histopathologic data |
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| Discussion|| |
In this study, we evaluated β-catenin expression, localization, and score in patients with muscle-invasive UBC and correlated these findings with different prognostic factors.
β-Catenin controls cellular adhesion through its binding with cadherin at the cellular membrane; in addition, it acts as a transcriptional activator in the nucleus where it interacts with lymphoid enhancer factor (LEF1) and T-cell factor (TCF) transcription factors and regulates transcription of target genes that are responsible for cell proliferation and differentiation. Thus, subcellular translocation of β-catenin from cell membrane to the nucleus determines its functions  .
Osterheld et al.  found abnormal β-catenin expression, consisting of the loss of membranous staining and the appearance of the nuclear staining in 43 (61%) cases of adenocarcinoma of the esophagus. Urakami et al.  also found that nuclear β-catenin accumulation was significantly higher in bladder tumor than in normal bladder mucosa. These results were in concordance with our results, as we found nucleocytoplasmic expression in 26 (86.7%) cases of 30 invasive UBC specimens and only four (13.3%) cases showed membranous expression ([Figure 1]). In the nonmalignant group, all the seven (70%) specimens that showed positive β-catenin expression were localized to the membrane ([Figure 2]). When comparing the localization of β-catenin in the nonmalignant and the UBC groups, there was a statistical significant difference between them (P = 0.001).
|Figure 1: Nucleocytoplasmic expression of β-catenin in invasive high-grade urothelial carcinoma (immunostaining, ×200).|
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|Figure 2: Membranous β-catenin expression in urothelial covering in a case of chronic cystitis (immunostaining, ×200).|
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It has been hypothesized that the loss of normal expression or function of any component of the β-catenin-cadherin complex may contribute to the malignant phenotype  . A study conducted by Xu et al.  had shown that cytoplasmic accumulation and, subsequently, nuclear β-catenin can activate β-catenin/Tcf-4 downstream target genes, such as c-Myc, cyclin D1, c-jun/fra-1, uPAR, PPARd, matrix metalloproteinase 7 (MMP-7), and WISP.
In our study, there was a statistically significant correlation between nucleocytoplasmic localization of β-catenin and high tumor grade (P = 0.003), high tumor stage (P = 0.012), and high mitotic index (P = 0.005).
Cytoplasmic and nuclear β-catenin expression has been reported to be associated with a poorer prognosis in patients with cancers of breast, liver, and colon ,, .
In addition, in breast cancer, nuclear and cytoplasmic accumulation of β-catenin was enriched in basal-like breast cancers. In contrast, membrane-associated β-catenin was observed in all breast cancer subtypes, and its expression decreased with tumor progression  .
Another study by Xi-Qing et al.  found that expression of either cytoplasmic or nuclear β-catenin was strongly associated with poor prognosis and was an independent prognostic factor for overall survival in non-small-cell lung cancer.
In contrast to our results is the work by Bilim et al.  who worked on RCCs and 81 primary TCCs cell lines. They failed to find overexpression of β-catenin with cytoplasmic and/or nuclear immunoreactivity. Among these 81 primary TCCs, only 24.7% lost β-catenin expression and they correlated it only with tumor stage III and multiplicity of tumors but was not correlated with either tumor grade or lymph nodes.
The current study showed that high β-catenin score is consistent with higher grade (P = 0.001) and advanced stage (P = 0.001), lymph node positivity (P = 0.014), vascular (P = 0, 02) and perineural invasion (P = 0.001), MVD (P = 0.003), and high mitotic index (P = 0.001). These results are concordant with the results of Deng et al.  who found that low β-catenin expression score was correlated with longer survival time. In addition, patients with β-catenin accumulation had shorter disease-free interval, whereas in multivariate analysis they had shorter disease-specific survival  .
In contrast to our results is the work by Fanelli et al.  who investigated the relationship between β-catenin score and several pathological and molecular factors in breast cancer and failed to find a statistical significant correlation except with lymph node metastasis. This may be due to the different stratification of cases in both studies, as most of their cases were of low grade and stage  .
The Wnt/β-catenin pathway has been shown to determine the proliferation/differentiation balance through its regulation of G1-S transition in several cellular systems (e.g. stem, progenitor, and colorectal cancer cells) , . The G1-S transition in cell cycle is driven mainly by cyclin-dependent kinase (CDK) 2 that is controlled by abundance of CDK inhibitors: p21/WAF1 (p21) and p27/Kip1  . The regulation of G1-S transition is physiologically required for cell fate determination, a cell undergoing apoptosis, proliferation, or differentiation. However, during oncogenic transformation, G1-S transition is deregulated by enhanced oncogenic growth signaling and/or by loss of tumor suppressors, which then leads to overgrowth of transformed cells. The activation of the Wnt/β-catenin pathway by Wnts elicits specific target genes (e.g. c-myc and cyclin D1) for cell cycle regulation and growth  .
Accumulated β-catenin leads to elevated level of the transcription factor TCF/LEF, which leads to upregulation of Wnt/β-catenin target gene MMP-7, redistribution of E-cadherin, rearrangement of actin filaments, and the elevation of active Rho family members, Cdc42 and Rac. These results suggest that aberrant accumulation of β-catenin can induce TCF/LEF-mediated transcriptional activity, upregulate MMP-7, and induce epithelial and mesenchymal transition, which can enhance the invasion and cellular migration  .
The MVD showed a statistical significant association with nucleocytoplasmic pattern and high score of β-catenin expression, which is consistent with the study by Hashimoto et al.  who confirmed the cross-talk between β-catenin and some growth factors that are important for angiogenesis such as FGF-1 and VEGF. Those two growth factors can regulate β-catenin expression by Wnt-independent pathways. VEGF is a major factor responsible for angiogenesis and FGF-1 is responsible for maturation of vessels  .
| Conclusion|| |
Nucleocytoplasmic localization and high score of β-catenin expression are associated with higher tumor grade, advanced stage, and other poor prognostic parameters in UBC.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
|1.||Pashos C, Botteman M, Laskin B, et al. Bladder cancer: epidemiology, diagnosis, and management. Cancer Pract 2002; 10 :311-322. |
|2.|| Stein J, Lieskovsky G, Cote R, et al. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1054 patients. J Clin Oncol 2001; 19 :666-675. |
|3.|| Hussein K. Systemic management of bladder cancer in egypt: revisited. J Egypt Nat Cancer Inst 2005; 17 :127-131. |
|4.|| Bostrom P, van Rhijn Bas WG, et al. Staging and staging errors in bladder cancer. Eur Urol Suppl 2010; 9 :2-9. |
|5.|| VnRhijn BWG, Burger M, Lotan Y, et al. Recurrence and progression of disease in non-muscle-invasive bladder cancer: from epidemiology to treatment strategy. Eur Urol 2009; 56 :430-442. |
|6.|| Snyder C, Harlan L, Knopf K, et al. Patterns of care for the treatment of bladder cancer. J Urol 2003; 169 :1697-1701. |
|7.|| Kastritis E, Murray S, Kyriakou F, et al. Somatic mutations of adenomatous polyposis coli gene and nuclear β-catenin accumulation have prognostic significance in invasive urothelial carcinomas: evidence for Wnt pathway implication. Int J Cancer 2009; 124 :103-108. |
|8.|| Tang Y, Simoneau AR, Liao W, et al. WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells. Mol Cancer Ther 2009; 8 :458-468. |
|9.|| MacDonald T, Tamai K, He X. Wnt/2-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009; 17 :9-26. |
|10.||Logan Y, Nusse R. The Wnt signaling pathway in development and disease. Ann Rev Cell Dev Biol 2004; 20 :781-810. |
|11.||Clevers H. Wnt/β-catenin signaling in development and disease. Cell 2006; 127: 469-480. |
|12.||Polakis P. The many ways of Wnt in cancer. Curr Opin Genet Dev 2007; 17: 45-51. |
|13.||Eble J, Sauter G, Epstein J, et al., editos. World Health Organization classification of tumours. Pathology and genetics of tumors of the urinary system and male genital organs. Lyon: IARC Press; 2004. |
|14.||Xi-Qing L, Xing-Long Y, et al. Nuclear β-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer. J Transl Med 2013; 11 :114. |
|15.||Osterheld M, Bian Y, Bosman F, et al. β-catenin expression and its association with prognostic factors in adenocarcinoma developed in Barrett esophagus. Am J Clin Pathol 2002; 117 : 451-456. |
|16.||Urakami S, Shiina H, Enokida H, et al. Epigenetic inactivation of Wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/β-catenin signaling pathway. Clin Cancer Res 2006; 2 :383-391. |
|17.||Xu L, Corcoran R, Welsh J, et al. WISP-1 is a Wnt-1- and β-catenin-responsive oncogene. Gene Dev 2000; 14 :585-595. |
|18.||López-Knowles E, Zardawi S, McNeil C, et al. Cytoplasmic localization of β-catenin is a marker of poor outcome in breast cancer patients. Cancer Epidemiol Biomarker Prev 2010; 19 :301-309. |
|19.||Yu B, Yang X, Xu Y, et al. Elevated expression of DKK1 is associated with cytoplasmic/nuclear β-catenin accumulation and poor prognosis in hepatocellular carcinomas. J Hepatol 2009; 50 :948-957. |
|20.||Baldus S, Mönig S, Huxel S, et al. MUC1 and nuclear β-catenin are coexpressed at the invasion front of colorectal carcinomas and are both correlated with tumor prognosis. Clin Cancer Res 2004; 10 :2790-2796. |
|21.||Khramtsov A, Khramtsova G, Tretiakova M, et al. Wnt/β-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. Am J Pathol 2010; 176 :2911-2920. |
|22.||Bilim V, Kawasaki T, Katagiri A, et al. Altered expression of β-catenin in renal cell cancer and transitional cell cancer with the absence of β-catenin gene mutations. Clin Cancer Res 2000; 6 :460-466. |
|23.||Deng Z, Niu G, Cai L, Wei R, Zhao X. The prognostic significance of CD44V6, CDH11, and β-catenin expression in patients with osteosarcoma. Biomed Res Int 2013:496193. |
|24.||Björklund P, Lindberg D, Åkerström G, et al. Stabilizing mutation of CTNNB1/β-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients. Mol Cancer 2008; 7 :53. |
|25.||Fanelli M, Montt-Guevara M, Diblasi A, et al. P-cadherin and β-catenin are useful prognostic markers in breast cancer patients; β-catenin interacts with heat shock protein Hsp27. Cell Stress Chaperones 2008; 13 :207-220. |
|26.||Van de Wetering M, Sancho E, Verweij C, et al. The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 2002; 111 :241-250. |
|27.||Jho E, Zhang T, Domon C, et al. Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol 2002; 22 :1172-1183. |
|28.||Pfeuty B, David-Pfeuty T, Kaneko K. Underlying principles of cell fate determination during G1 phase of the mammalian cell cycle. Cell Cycle 2008; 7 :3246-3257. |
|29.||Iwai S, Yonekawa A, Harada C, et al. Involvement of the Wnt-β-catenin pathway in invasion and migration of oral squamous carcinoma cells. Int J Oncol 2010; 37 :1095-1103. |
|30.||Hashimoto M, Sagara Y, Langford D, et al. Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3 beta pathway. Implications for neuroprotection. J Biol Chem 2002; 277 :32985-32991. |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]