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 1-5, 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.