溶瘤腺病毒结合阿特珠单抗通过AMPK靶点对甲状腺癌动物模型的治疗作用

Oncolytic Adenovirus Combined with Atezolizumab Exerts Therapeutic Effects on a Thyroid Cancer Animal Model via the AMPK Pathway

关键词:  溶瘤腺病毒  阿特珠单抗  AMPK  甲状腺癌

Key words:  Oncolytic adenovirus  Atezolizumab  AMPK  Thyroid cancer

摘要:

目的 探究溶瘤腺病毒(rAd.Nulli)联合阿特珠单抗对甲状腺癌细胞的增殖和凋亡的影响,并在裸鼠移植瘤模型中评估其抗肿瘤作用及AMPK信号通路的潜在机制。方法? 采用CCK-8检测rAd.Nulli、阿特珠单抗及其联合治疗对TPC-1细胞增殖的影响,流式细胞术检测细胞凋亡情况。建立TPC-1裸鼠甲状腺癌移植瘤模型,随机分为移植瘤对照组、rAd.Null组、阿特珠单抗组及联合组(每组6只),测量移植瘤体积和瘤重,并计算抑瘤率。HE染色观察肿瘤组织的病理变化,Western blot检测肿瘤组织中AMPK蛋白的表达水平,Co-IP探究rAd.Nulli与AMPK蛋白相互间作用。结果 rAd.Nulli组、阿特珠单抗组及联合组TPC-1细胞的存活率显著低于对照组(P<0.05),联合组的增殖抑制作用显著高于单独用药组(P<0.05)。流式结果显示,联合组的凋亡率显著高于rAd.Nulli组、阿特珠单抗组(P<0.05)。体内实验结果表明rAd.Null组、阿特珠单抗组及联合组的瘤体积、瘤重显著低于对照组(P<0.05),联合组对TCP-1肿瘤模型的抑瘤率显著高于rAd.Null组、阿特珠单抗组(P<0.05)。对照组肿瘤组织坏死程度较轻,细胞排列密集、聚积成巢rAd.Nulli组、阿特珠单抗组及联合组肿瘤组织呈现坏死区域,表现为胞质溶解和核固缩,联合组坏死区域显著高于单独用药组(P<0.05)。Western blot结果显示,rAd.Nulli、阿特珠单抗可上调p-AMPK表达,联合组p-AMPK表达量显著高于rAd.Nulli组、阿特珠单抗组(P<0.05),Co-IP实验表明,rAd.Nulli与AMPK在TPC-1细胞中特异性结合。结论 溶瘤腺病毒结合阿特珠单抗可通过上调AMPK信号通路,抑制甲状腺癌细胞增殖并促进凋亡,从而增强抗肿瘤效果,为溶瘤病毒免疫联合疗法在甲状腺癌治疗中的应用提供了依据。

Abstract:

Objective To investigate? the effects of oncolytic adenovirus (rAd.Nulli) in combination with atezolizumab on thyroid cancer cell proliferation and apoptosis, as well as its antitumor efficacy and the potential involvement of the AMPK signaling pathway in a nude mouse xenograft model. Methods The CCK-8 assay was used to evaluate the impact of rAd.Nulli, atezolizumab, and their combination on TPC-1 cell proliferation, while apoptosis was assessed by flow cytometry. A TPC-1 nude mouse xenograft model was established and randomly assigned to four groups: control, rAd.Null, atezolizumab, and combination therapy (n=6 per group). Tumor volume and weight were measured, and tumor inhibition rates were calculated. Histopathological changes were examined using hematoxylin and eosin (HE) staining. AMPK protein expression levels in tumor tissues were analyzed via Western blot, and co-immunoprecipitation (Co-IP) was performed to investigate the interaction between rAd.Nulli and AMPK. Results Cell viability in the rAd.Nulli, atezolizumab, and combination treatment groups was significantly lower than in the control group (P<0.05), with the combination group demonstrating a stronger inhibitory effect than monotherapy (P<0.05). Flow cytometry revealed that apoptosis rates were significantly higher in the combination group than in the rAd.Nulli and atezolizumab groups (P<0.05). In vivo, tumor volume and weight in the treatment groups were significantly reduced compared to the control (P<0.05), with the combination therapy exhibiting the highest tumor inhibition rate (P<0.05). Histological analysis showed mild necrosis in the control group, whereas treatment groups exhibited necrotic regions with cytoplasmic dissolution and nuclear pyknosis, with the most extensive necrosis observed in the combination group (P<0.05). Western blot analysis indicated that both rAd.Nulli and atezolizumab increased p-AMPK expression, with the combination group showing the highest expression levels (P<0.05). Co-IP confirmed a specific interaction between rAd.Nulli and AMPK in TPC-1 cells. Conclusion Oncolytic adenovirus combined with atezolizumab enhances antitumor efficacy in thyroid cancer by upregulating the AMPK signaling pathway, inhibiting tumor cell proliferation, and promoting apoptosis. These findings provide a rationale for the potential application of oncolytic virus-based immunotherapy in thyroid cancer treatment.