Increased Bax/Bcl-2 Ratio Up-regulates Caspase-3 and Increases Apoptosis in the Thymus of Patients with Myasthenia Gravis

Abstract

Background: In this study the possible relation of Bax (an apoptosis promoter) to Bcl-2 (an apoptosis inhibitor) ratio with the apoptosis co-ordination enzyme, caspase-3, in the thymus of patients with myasthenia gravis (MG) was investigated in correlation with long-term clinical prognosis. Patients and Methods: The study included 46 patients (17M/29F, mean age 36.60±16.09 yr) with MG, who underwent thymectomy for treatment. The clinical staging (Osserman classification) included: stage I-5, IIA-21, IIB-17, III-3. The pathology of the thymus showed: hyperplasia-26, atrophy-8, thymoma B1 and B2 type-9, thymoma B3 type (well differentiated thymic carcinoma) -3. The patients were evaluated 39-166 (mean 91.87±38.38) months after thymectomy. At the end of the follow-up period, the patients were classified as follows: group A: complete stable remission, group B: pharmacological remission+minimal manifestations+improvement+deterioration. Paraffin sections of thymic tissue were subjected to: a) immunohistochemistry (bax, bcl-2, caspase-3 protein); b) in situ hybridization (bax, bcl-2 mRNA); and c) TUNEL-stain (apoptotic cells). Bax to bcl-2 mRNA and protein ratio was determined for each sample by dividing the %bax (+) cells by the % bcl-2 (+) cells. Results: Follow-up data were available for 39/46 patients: 13/39 patients belonged to group A and 26/39 to group B. The Bax/Bcl-2 mRNA and protein ratios were increased towards advanced disease stages (+370% for mRNA and +391% for protein, from MG stage I to stage III). These ratios were correlated with caspase-3 expression (r=0.782 and 0.583, p<0.01) and apoptosis (r=0.591 and 0.358 p<0.01 and p<0.05). All the 13 cases in group A had a Bax/Bcl-2 ratio<1 (mean±SD: 0.58±0.04 for mRNA and 0.62±0.03 for protein), whereas all the 26 cases of group B had a ratio>1(1.47±0.07 for mRNA and 1.52±0.18 for protein). The Kaplan-Meier survival curve showed higher, free of disease, survival in group A (p=0.0082). Cox regression analysis revealed that the Bax/Bcl-2 ratio was an independent prognostic factor, however the p-value was marginally significant (95% CI:1.078-44.073, p=0.041). Conclusion: This study has demonstrated that in patients with MG who underwent thymectomy: a) the Bax/Bcl-2 ratio may up-regulate caspase-3 expression and modulate apoptosis associated with progress of the disease; b) the Bax/Bcl-2 ratio<1 was associated with complete stable remission after thymectomy; and c) Bax/Bcl-2 ratio was an independent predictive marker for therapeutic response after thymectomy.