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INTRODUCTION

Abemaciclib, a CDK4/6 inhibitor, is well-established for treating hormone receptor-positive and HER2-negative (HR/HER2) human breast cancer by inducing G1 cell cycle arrest. However, its antitumor effects in other cancers, including canine melanoma, remain largely unexplored. Canine melanoma often harbors CDK4/6 copy number gains and cell cycle dysregulation, suggesting it may be a suitable target for abemaciclib.

METHODS

Five canine melanoma cell lines (CMeC1, KMeC, LMeC, UCDK9M4, and UCDK9M5) were used to evaluate the antitumor effects of abemaciclib. Cell viability and migration were assessed using Cell Counting Kit-8 and wound healing assays, respectively. Cell cycle distribution was analyzed by flow cytometry, and the expression of cell cycle-related genes and proteins was examined using RT-PCR and western blotting. The origin of cytoplasmic vacuoles was investigated using FITC-dextran uptake and V-ATPase inhibitor assays. Impaired autophagic flux was assessed by immunofluorescence detection of p62 accumulation. Synergistic effects with fenbendazole were evaluated using the Highest Single Agent (HSA) synergy scoring method, and efficacy was assessed in a xenograft model.

RESULTS

Abemaciclib induced G1 cell cycle arrest and altered the expression of cell cycle-related genes and proteins. Autophagy, but not apoptosis, was activated. Vacuolization was observed and suggested to originate from lysosomes, as evidenced by FITC-dextran uptake and V-ATPase inhibitor co-treatment. p62 accumulation indicated impaired autophagy flux. Co-treatment with fenbendazole enhanced cytotoxicity and showed synergistic effects within specific dose ranges. , abemaciclib alone or in combination with fenbendazole significantly suppressed tumor growth.

DISCUSSION

These findings demonstrate that abemaciclib exerts potent antitumor effects in canine melanoma by inducing cell cycle arrest and disrupting lysosomal function. Its synergistic interaction with fenbendazole suggests a potential combinatorial therapeutic approach for canine melanoma.

INTRODUCTION

METHODS

RESULTS

DISCUSSION

引言

阿贝西利是一种细胞周期蛋白依赖性激酶4/6（CDK4/6）抑制剂，通过诱导G1期细胞周期停滞，在治疗激素受体阳性和人表皮生长因子受体2阴性（HR/HER2）的人类乳腺癌方面已得到充分证实。然而，其在包括犬黑色素瘤在内的其他癌症中的抗肿瘤作用在很大程度上仍未得到探索。犬黑色素瘤常存在CDK4/6拷贝数增加和细胞周期失调的情况，这表明它可能是阿贝西利的一个合适靶点。

方法

使用五种犬黑色素瘤细胞系（CMeC1、KMeC、LMeC、UCDK9M4和UCDK9M5）来评估阿贝西利的抗肿瘤作用。分别使用细胞计数试剂盒-8和伤口愈合试验评估细胞活力和迁移能力。通过流式细胞术分析细胞周期分布，并使用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测细胞周期相关基因和蛋白质的表达。使用异硫氰酸荧光素（FITC）-葡聚糖摄取试验和V-ATP酶抑制剂试验研究细胞质空泡的来源。通过免疫荧光检测p62积累来评估自噬流受损情况。使用最高单药（HSA）协同评分方法评估与芬苯达唑的协同作用，并在异种移植模型中评估疗效。

结果

阿贝西利诱导G1期细胞周期停滞，并改变细胞周期相关基因和蛋白质的表达。激活了自噬，但未激活凋亡。观察到空泡化现象，FITC-葡聚糖摄取试验和V-ATP酶抑制剂联合处理证明空泡化可能起源于溶酶体。p62积累表明自噬流受损。与芬苯达唑联合处理增强了细胞毒性，并在特定剂量范围内显示出协同作用。单独使用阿贝西利或与芬苯达唑联合使用均能显著抑制肿瘤生长。