ABSTRACT
Objective: To investigate the changes of autophagy after spinal cord injury (SCI) in rats and its relationship with multisite phosphorylation of B-cell lymphoma-2 (Bcl-2) protein. Methods: Forty male Sprague-Dawley rats aged 8 weeks were used to prepare SCI models by modified Allen method, and the SCI model were prepared successfully in 36 rats. The 36 SCI models were randomly divided into SCI group, autophagy inhibitor group, and autophagy promoter group, with 12 rats in each group. Another 12 rats were selected as sham operation group with only laminectomy and no spinal cord injury. At the end of modeling, the autophagy inhibitor group and the autophagy promoter group were intrathecally injected with 20 μL of 600 nmol/L 3-methyladenine and 25 nmol/L rapamycin, respectively, once a day for 4 weeks. The sham operation group and the SCI group were injected with only 20 μL of normal saline at the same time point. The motor function of rat in each group was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and 1, 2, 4 weeks after modeling. The rats in each group were sacrificed at 24 hours after the last injection and the spinal cord tissues were taken. ELISA assay was used to detect the levels of inflammatory factors in spinal cord tissues, including myeloperoxidase (MPO), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β); the morphological changes of spinal cord were observed by HE staining; the autophagy of mitochondria in spinal cord tissues was observed by transmission electron microscopy; the expressions of Beclin1 and microtubule-associated protein light chain 3 (LC3) were detected by immunofluorescence staining; neuronal apoptosis in spinal cord tissues were observed by TUNEL staining; LC3/TUNEL positive cells were calculated by immunofluorescence double staining; the expressions of Bcl-2 associated X protein (Bax), Bcl-2, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) were detected by Western blot. Results: Compared with sham operation group, BBB score of SCI group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; peripheral space of nerve cells enlarged, cells swelled, vacuoles appeared, and autophagic bodies appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins, and apoptotic rate of neurons significantly increased; the LC3/TUNEL positive cells significantly increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased; all showing significant differences ( P<0.05). Compared with SCI group, BBB score in autophagy inhibitor group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; a few autophagic vesicles appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins decreased and the apoptotic rate of neurons increased significantly; the LC3 positive cells decreased and the TUNEL positive cells increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased. The results of autophagy promoter group were opposite to those of autophagy inhibitor group; all showing significant differences between groups ( P<0.05). Conclusion: Induction of autophagy after SCI in rats can reduce neuronal apoptosis and protect spinal cord function, which may be related to the inhibition of Bcl-2 protein multisite phosphorylation.
ABSTRACT
PURPOSE: B-cell lymphoma 2 (BCL2) is an antiapoptosis protein and an important clinical breast cancer prognostic marker. As the role of BCL2 is dependent on the estrogen receptor (ER) status, this effect might differ according to molecular subtypes. The aim of this study was to evaluate the relationship between the prognostic outcomes and BCL2 expression among the molecular subtypes. METHODS: We retrieved the data of 1,356 patients who were newly diagnosed with malignant breast cancer between November 2006 and November 2011. Immunohistochemistry was used to measure ER, progesterone receptor, human epidermal growth factor receptor 2 (HER2), Ki-67, and BCL2 expression. We classified breast cancer into five molecular subtypes based on the 13th St. Gallen International Expert Consensus, including luminal A, luminal B (HER2-negative), luminal B (HER2-positive), HER2-overexpression, and triple-negative subtypes. We analyzed the clinicopathological features and assessed the correlation between BCL2 expression and clinical outcomes, such as relapse-free survival (RFS) and disease-specific survival (DSS) according to the five molecular subtypes. RESULTS: A total of 605 cases of breast cancer (53.8%) showed BCL2 expression. BCL2-positive expression was associated with young age (<50 years, p=0.036), lower histological grade (p<0.001), low Ki-67 level (<14%, p<0.001), hormone receptor positivity (p<0.001), HER2 negativity (p<0.001), luminal breast cancer (p<0.001), and low recurrence rate (p=0.016). BCL2-positive expression was also associated with favorable 5-year RFS (p=0.008, 91.4%) and DSS (p=0.036, 95.6%) in all the patients. BCL2-positive expression in luminal A breast cancer resulted in significantly favorable 5-year RFS and DSS (p=0.023 and p=0.041, respectively). However, BCL2 expression was not associated with the prognosis in the other subtypes. CONCLUSION: The prognostic role of BCL2 expression in breast cancer is subtype-specific. BCL2 expression differs according to the molecular subtype and is a good prognostic marker for only luminal A breast cancer.