Abstract：
BACKGROUND: The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy. Here, we studied the efficacy of the genotoxic agent etoposide (Eto) and irradiation in inducing senescence of Panc02 pancreatic cancer cells, and the capability of the Bcl-2 inhibitor navitoclax (ABT-263; Nav) to trigger senolysis.
METHODS: Panc02 cells were treated with Eto or irradiated with 5-20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav.
RESULTS: Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive. Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable.
CONCLUSIONS: In vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.
METHODS: Panc02 cells were treated with Eto or irradiated with 5-20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav.
RESULTS: Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive. Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable.
CONCLUSIONS: In vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.
摘要：
背景：衰老触发剂与抗衰老药物的组合被认为是一种有希望的癌症治疗新方法。这里,我们研究了基因毒性剂依托泊苷（Eto）和辐射在诱导Panc02胰腺癌细胞衰老中的功效，以及Bcl-2抑制剂navitoclax（ABT-263；Nav）触发衰老的能力。
方法:Panc02细胞在暴露于Nav之前用Eto处理或用5-20Gy照射。细胞存活,扩散,通过锥虫蓝染色评估衰老，DNA合成的定量，和染色的衰老相关的β-半乳糖苷酶（SA-β-Gal）-阳性细胞，分别。通过实时聚合酶链反应测定mRNA水平，并通过免疫印迹分析蛋白质表达。Panc02细胞也在小鼠中生长为胰腺肿瘤，随后用Eto和Nav治疗。
结果：Eto和辐射对Panc02细胞具有抗增殖作用，该作用被Nav显着或逐步增强。在体内,Eto和Nav在一起,但不是只有埃托，显著降低了增殖细胞的比例。衰老标志物γH2AX的表达和T细胞的肿瘤浸润不受治疗的影响。体外，几乎所有暴露于Eto的细胞和20Gy照射的细胞中的很大一部分均为SA-β-Gal阳性。Nav的应用降低了辐射后SA-β-Gal阳性细胞的百分比，但在用Eto预处理后没有降低。为了应对衰老的触发，培养的Panc02细胞显示γH2AX和自噬标志物LC3B-II的蛋白质水平增加，和更高的Cdkn1amRNA水平，Mdm2和PAI-1，而Nav的影响是可变的。
结论：体外和体内，衰老触发剂与Nav的组合比单独触发剂更有效地抑制肿瘤细胞生长。我们的数据也为Nav的体外衰老作用提供了一些证据。
方法:Panc02细胞在暴露于Nav之前用Eto处理或用5-20Gy照射。细胞存活,扩散,通过锥虫蓝染色评估衰老，DNA合成的定量，和染色的衰老相关的β-半乳糖苷酶（SA-β-Gal）-阳性细胞，分别。通过实时聚合酶链反应测定mRNA水平，并通过免疫印迹分析蛋白质表达。Panc02细胞也在小鼠中生长为胰腺肿瘤，随后用Eto和Nav治疗。
结果：Eto和辐射对Panc02细胞具有抗增殖作用，该作用被Nav显着或逐步增强。在体内,Eto和Nav在一起,但不是只有埃托，显著降低了增殖细胞的比例。衰老标志物γH2AX的表达和T细胞的肿瘤浸润不受治疗的影响。体外，几乎所有暴露于Eto的细胞和20Gy照射的细胞中的很大一部分均为SA-β-Gal阳性。Nav的应用降低了辐射后SA-β-Gal阳性细胞的百分比，但在用Eto预处理后没有降低。为了应对衰老的触发，培养的Panc02细胞显示γH2AX和自噬标志物LC3B-II的蛋白质水平增加，和更高的Cdkn1amRNA水平，Mdm2和PAI-1，而Nav的影响是可变的。
结论：体外和体内，衰老触发剂与Nav的组合比单独触发剂更有效地抑制肿瘤细胞生长。我们的数据也为Nav的体外衰老作用提供了一些证据。
