Abstract：
BACKGROUND: Chemobrain is widespread in breast cancer patients receiving chemotherapy. However, the exact mechanism, especially the associated signalling pathway, is not currently clear. This study was to evaluate the behavioural changes in breast cancer mice after chemotherapy and to further explore the role of Wnt3a/glycogen synthase kinase (GSK3β)/β-catenin signalling in chemobrain.
METHODS: MMTV-PyMT(+) breast cancer mice were injected intraperitoneally with doxorubicin (4 mg/kg) once a week for three weeks to establish a chemobrain model. The Morris water maze (MWM) and novel object recognition (NOR) tests were performed to assess the learning and memory ability. Electron microscopy was used to observe the structural changes in the hippocampal CA1 region. The brain tissue of breast cancer mice after chemotherapy was taken out for mRNA-seq detection. Then, the expression levels and phosphorylation of key proteins in the Wnt3a/GSK3 β/β-catenin signalling pathway were evaluated through Western blotting (WB) and immunofluorescence.
RESULTS: Doxorubicin-induced spatial and short-term memory impairment was observed in breast cancer mice, and obvious neuronal damage could be seen in the hippocampal CA1 region. Immunofluorescence staining for GSK3β was increased. Wnt signalling pathway is highly enriched from mRNA-seq analysis, with GSK3β genes at important nodes. The relative protein levels of p-PI3K, p-AKT, p-GSK3 β, Wnt3a and TCF-1 were decreased significantly, while the p-β-catenin level was increased. After injection of the GSK3β inhibitor sb216763 (1 ng/0.5 µl/side), hippocampal neuronal injury was alleviated to some extent, and the changes in the expression of proteins upstream and downstream of this signalling pathway were reversed.
CONCLUSIONS: Wnt3a/GSK3 β/β-catenin signalling is likely involved in doxorubicin-induced memory impairment. This result provides basic evidence for the further study of chemobrain in breast cancer.
METHODS: MMTV-PyMT(+) breast cancer mice were injected intraperitoneally with doxorubicin (4 mg/kg) once a week for three weeks to establish a chemobrain model. The Morris water maze (MWM) and novel object recognition (NOR) tests were performed to assess the learning and memory ability. Electron microscopy was used to observe the structural changes in the hippocampal CA1 region. The brain tissue of breast cancer mice after chemotherapy was taken out for mRNA-seq detection. Then, the expression levels and phosphorylation of key proteins in the Wnt3a/GSK3 β/β-catenin signalling pathway were evaluated through Western blotting (WB) and immunofluorescence.
RESULTS: Doxorubicin-induced spatial and short-term memory impairment was observed in breast cancer mice, and obvious neuronal damage could be seen in the hippocampal CA1 region. Immunofluorescence staining for GSK3β was increased. Wnt signalling pathway is highly enriched from mRNA-seq analysis, with GSK3β genes at important nodes. The relative protein levels of p-PI3K, p-AKT, p-GSK3 β, Wnt3a and TCF-1 were decreased significantly, while the p-β-catenin level was increased. After injection of the GSK3β inhibitor sb216763 (1 ng/0.5 µl/side), hippocampal neuronal injury was alleviated to some extent, and the changes in the expression of proteins upstream and downstream of this signalling pathway were reversed.
CONCLUSIONS: Wnt3a/GSK3 β/β-catenin signalling is likely involved in doxorubicin-induced memory impairment. This result provides basic evidence for the further study of chemobrain in breast cancer.
摘要：
背景：化疗药物广泛存在于接受化疗的乳腺癌患者中。然而,确切的机制，尤其是相关的信号通路，目前还不清楚。本研究旨在评估化疗后乳腺癌小鼠的行为变化，并进一步探讨Wnt3a/糖原合成酶激酶（GSK3β）/β-catenin信号在化疗中的作用。
方法：MMTV-PyMT(+)乳腺癌小鼠腹腔注射阿霉素（4mg/kg），每周1次，连续3周，建立化疗药物模型。进行了Morris水迷宫（MWM）和新型物体识别（NOR）测试以评估学习和记忆能力。电镜观察海马CA1区的结构变化。将化疗后的乳腺癌小鼠脑组织取出进行mRNA-seq检测。然后，通过蛋白质印迹(WB)和免疫荧光检测Wnt3a/GSK3β/β-catenin信号通路中关键蛋白的表达水平和磷酸化水平。
结果：在乳腺癌小鼠中观察到阿霉素诱导的空间和短期记忆障碍，海马CA1区可见明显的神经元损伤。GSK3β的免疫荧光染色增加。Wnt信号通路从mRNA-seq分析高度富集，在重要节点有GSK3β基因。p-PI3K的相对蛋白水平,p-AKT,p-GSK3β,Wnt3a和TCF-1显著降低,而P-β-catenin水平升高。注射GSK3β抑制剂sb216763（1ng/0.5μl/侧）后，海马神经元损伤得到一定程度的缓解,并且该信号通路上游和下游蛋白质表达的变化被逆转。
结论：Wnt3a/GSK3β/β-连环蛋白信号可能与多柔比星诱导的记忆障碍有关。这一结果为乳腺癌化疗的进一步研究提供了基础依据。
方法：MMTV-PyMT(+)乳腺癌小鼠腹腔注射阿霉素（4mg/kg），每周1次，连续3周，建立化疗药物模型。进行了Morris水迷宫（MWM）和新型物体识别（NOR）测试以评估学习和记忆能力。电镜观察海马CA1区的结构变化。将化疗后的乳腺癌小鼠脑组织取出进行mRNA-seq检测。然后，通过蛋白质印迹(WB)和免疫荧光检测Wnt3a/GSK3β/β-catenin信号通路中关键蛋白的表达水平和磷酸化水平。
结果：在乳腺癌小鼠中观察到阿霉素诱导的空间和短期记忆障碍，海马CA1区可见明显的神经元损伤。GSK3β的免疫荧光染色增加。Wnt信号通路从mRNA-seq分析高度富集，在重要节点有GSK3β基因。p-PI3K的相对蛋白水平,p-AKT,p-GSK3β,Wnt3a和TCF-1显著降低,而P-β-catenin水平升高。注射GSK3β抑制剂sb216763（1ng/0.5μl/侧）后，海马神经元损伤得到一定程度的缓解,并且该信号通路上游和下游蛋白质表达的变化被逆转。
结论：Wnt3a/GSK3β/β-连环蛋白信号可能与多柔比星诱导的记忆障碍有关。这一结果为乳腺癌化疗的进一步研究提供了基础依据。
