Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell'Emilia, Italy.
Department of Veterinary Sciences, University of Turin, Grugliasco, Italy.
BMC Vet Res. 2021 Jan 18;17(1):30. doi: 10.1186/s12917-020-02709-5.
Doxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50 DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC).
Both cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50 DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis.
DOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.
多柔比星（DOX）广泛应用于人类和兽医肿瘤学领域，尽管肿瘤细胞中多药耐药（MDR）的出现常常导致化疗失败。更好地了解规避化疗疗效的细胞机制至关重要。本研究的目的是研究两种犬乳腺肿瘤细胞系 CIPp（来源于原发性肿瘤）和 CIPm（来源于淋巴结转移）在暴露于 EC50 DOX 后 12、24 和 48 小时时对 DOX 的反应。我们评估了 DOX 的摄取和亚细胞分布，以及 P-糖蛋白（P-gp）和乳腺癌耐药蛋白（BCRP）的表达和功能，这两种蛋白是重要的 MDR 介质。为了更好地理解这一现象，我们还通过免疫细胞化学（ICC）评估了 DOX 对细胞周期和 Ki67 细胞增殖指数以及 p53 和端粒酶逆转录酶（TERT）表达的影响。
两种细胞系在治疗 3 小时内均能将 DOX 摄取到细胞核内，而在 48 小时时，所有存活细胞的细胞内部分（通过荧光显微镜评估）均无 DOX 存在。来源于转移瘤的 CIPm 能够更有效地排出 P-gp 底物。通过 ICC 和 qRT-PCR，在两种细胞系中均观察到在 EC50 DOX 治疗 48 小时时 P-gp 和 BCRP 的总体增加，并与 ICC 中 p53 和 TERT 表达细胞的比例显著增加相关。在两种细胞系中，细胞增殖分数在 48 小时时均降低，细胞周期分析显示 CIPp 的 S 期发生 DOX 诱导的阻滞，而 CIPm 则出现细胞死亡增加而无阻滞。因此，两种细胞系均由对 DOX 敏感的细胞组成，这些细胞经历凋亡/坏死。
DOX 给药导致细胞群体中相互关联的变化，包括对细胞周期的重大影响，特别是 CIPp 的 S 期阻滞，以及选择具有 P-gp 和 BCRP 表达、TERT 激活、p53 积累和增殖分数降低特征的多药耐药表型的肿瘤细胞亚群。这些信息对于理解肿瘤细胞群体中耐药性发生的动态和机制非常重要。
