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Inhibition of Autophagy Potentiated the Antitumor Effect of Nedaplatin in Cisplatin-Resistant Nasopharyngeal Carcinoma Cells.

Liu Z, Liu J, Li L, Nie D, Tao Q, Wu J, Fan J, Lin C, Zhao S, Ju D - PLoS ONE (2015)

Bottom Line: Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity.While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells.Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, China.

ABSTRACT
Nedaplatin, a cisplatin analog, was developed to reduce the toxicity of cisplatin, whereas it can be cross-resistant with cisplatin in some circumstances. This study aimed to investigate the role of autophagy in nedaplatin induced cell death in cisplatin-resistant nasopharyngeal carcinoma cells. Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity. Nedaplatin treatment resulted in autophagosome accumulation and increased expression of LC3-II, indicating the induction of autophagy by nedaplatin in HNE1/DDP and CNE2/DDP cells. Inhibition of autophagy by Bafilomycin A1 (Baf A1) and 3-Methyladenine (3-MA) remarkably enhanced the antitumor efficacy of nedaplatin in HNE1/DDP and CNE2/DDP cells, suggesting that the resistance to nedaplatin-induced cell death was caused by enhanced autophagy in nedaplatin-resistant NPC cells. Additionally, Baf A1 enhanced reactive oxygen species (ROS) generation and apoptosis induced by nedaplatin in HNE1/DDP cells. Mechanistically, nedaplatin treatment caused activation of ERK1/2 and suppression of Akt/mTOR signaling pathways. While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells. Furthermore, suppression of ROS could inhibit nedaplatin-induced ERK activation in HNE1/DDP cells, indicating that ROS and ERK were involved in nedaplatin-induced autophagy. Together, these findings suggested that autophagy played a cytoprotective role in nedaplatin-induced cytotoxicity of HNE1/DDP and CNE2/DDP cells. Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.

No MeSH data available.


Related in: MedlinePlus

Inhibition of autophagy enhanced nedaplatin-induced apoptosis and suppression of cell growth in HNE1/DDP cells.(A) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin for 48 h, in the presence or absence of Baf A1 (5 nM) for 48 h, and the levels of LC3-I/II were detected by western blot. (B)HNE1/DDP cells were untreated or treated with nedaplatin at indicated concentrations in the absence or presence of Baf A1 (5 nM) for 48h. The cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only. (C) HNE1/DDP cells were treated with 6.0 μg/ml with or without Baf A1 (5 nM) for 48 h and then analyzed by FACS after PI and FITC-annexin V staining. (D) HNE1/DDP cells were incubated with or without 6.0 μg/ml of nedaplatin in the presence or absence of the autophagy inhibitors Baf A1 for 48 h. The whole protein was extracted, and PARP, cleaved PARP and cleaved caspase 3 were analyzed by Western blot.
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pone.0135236.g003: Inhibition of autophagy enhanced nedaplatin-induced apoptosis and suppression of cell growth in HNE1/DDP cells.(A) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin for 48 h, in the presence or absence of Baf A1 (5 nM) for 48 h, and the levels of LC3-I/II were detected by western blot. (B)HNE1/DDP cells were untreated or treated with nedaplatin at indicated concentrations in the absence or presence of Baf A1 (5 nM) for 48h. The cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only. (C) HNE1/DDP cells were treated with 6.0 μg/ml with or without Baf A1 (5 nM) for 48 h and then analyzed by FACS after PI and FITC-annexin V staining. (D) HNE1/DDP cells were incubated with or without 6.0 μg/ml of nedaplatin in the presence or absence of the autophagy inhibitors Baf A1 for 48 h. The whole protein was extracted, and PARP, cleaved PARP and cleaved caspase 3 were analyzed by Western blot.

Mentions: Given that nedaplatin-treated HNE1/DDP and CNE2/DDP cells exhibited significant autophagic activity, we further tested whether enhanced autophagy was associated with the cell death resistance in response to nedaplatin. Baf A1 inhibits autophagy by blocking the fusion of autophagosome and lysosome and leads to significant elevation of LC3-II expression. Western blot analysis revealed that the addition of Baf A1 to nedaplatin significantly enhanced LC3-II expression in HNE1/DDP cells when compared to the cells treated with nedaplatin alone (Fig 3A), while treatment with Baf A1 alone did not affect the growth of HN1/DDP cells (data not shown). Interestingly, inhibition of autophagy by Baf A1 substantially potentiated nedaplatin-induced cell death (Fig 3B). Similar results were obtained in CNE2/DDP cells (S3D and S3E Fig). To understand the role of autophagy in nedaplatin-induced apoptosis, apoptotic rate of HN1/DDP cells was determined after they were co-incubated with nedaplatin and Baf A1. Compared with the cells treated with nedaplatin alone, HNE1/DDP cells treated with both nedaplatin and Baf A1 exhibited significant increases in apoptotic rate (Fig 3C) as well as the protein levels of cleaved caspase 3 and cleaved PARP (Fig 3D). Moreover, another autophagy inhibitor 3-MA was used to confirm these results, which could block autophagy initiation and lead to the decrease in LC3-II level. As illustrated in S3A–S3C Fig, inhibition of autophagy using 3-MA significantly enhanced nedaplatin-induced cell death and caspase 3 cleavage in HNE1/DDP cells.


Inhibition of Autophagy Potentiated the Antitumor Effect of Nedaplatin in Cisplatin-Resistant Nasopharyngeal Carcinoma Cells.

Liu Z, Liu J, Li L, Nie D, Tao Q, Wu J, Fan J, Lin C, Zhao S, Ju D - PLoS ONE (2015)

Inhibition of autophagy enhanced nedaplatin-induced apoptosis and suppression of cell growth in HNE1/DDP cells.(A) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin for 48 h, in the presence or absence of Baf A1 (5 nM) for 48 h, and the levels of LC3-I/II were detected by western blot. (B)HNE1/DDP cells were untreated or treated with nedaplatin at indicated concentrations in the absence or presence of Baf A1 (5 nM) for 48h. The cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only. (C) HNE1/DDP cells were treated with 6.0 μg/ml with or without Baf A1 (5 nM) for 48 h and then analyzed by FACS after PI and FITC-annexin V staining. (D) HNE1/DDP cells were incubated with or without 6.0 μg/ml of nedaplatin in the presence or absence of the autophagy inhibitors Baf A1 for 48 h. The whole protein was extracted, and PARP, cleaved PARP and cleaved caspase 3 were analyzed by Western blot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4543554&req=5

pone.0135236.g003: Inhibition of autophagy enhanced nedaplatin-induced apoptosis and suppression of cell growth in HNE1/DDP cells.(A) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin for 48 h, in the presence or absence of Baf A1 (5 nM) for 48 h, and the levels of LC3-I/II were detected by western blot. (B)HNE1/DDP cells were untreated or treated with nedaplatin at indicated concentrations in the absence or presence of Baf A1 (5 nM) for 48h. The cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only. (C) HNE1/DDP cells were treated with 6.0 μg/ml with or without Baf A1 (5 nM) for 48 h and then analyzed by FACS after PI and FITC-annexin V staining. (D) HNE1/DDP cells were incubated with or without 6.0 μg/ml of nedaplatin in the presence or absence of the autophagy inhibitors Baf A1 for 48 h. The whole protein was extracted, and PARP, cleaved PARP and cleaved caspase 3 were analyzed by Western blot.
Mentions: Given that nedaplatin-treated HNE1/DDP and CNE2/DDP cells exhibited significant autophagic activity, we further tested whether enhanced autophagy was associated with the cell death resistance in response to nedaplatin. Baf A1 inhibits autophagy by blocking the fusion of autophagosome and lysosome and leads to significant elevation of LC3-II expression. Western blot analysis revealed that the addition of Baf A1 to nedaplatin significantly enhanced LC3-II expression in HNE1/DDP cells when compared to the cells treated with nedaplatin alone (Fig 3A), while treatment with Baf A1 alone did not affect the growth of HN1/DDP cells (data not shown). Interestingly, inhibition of autophagy by Baf A1 substantially potentiated nedaplatin-induced cell death (Fig 3B). Similar results were obtained in CNE2/DDP cells (S3D and S3E Fig). To understand the role of autophagy in nedaplatin-induced apoptosis, apoptotic rate of HN1/DDP cells was determined after they were co-incubated with nedaplatin and Baf A1. Compared with the cells treated with nedaplatin alone, HNE1/DDP cells treated with both nedaplatin and Baf A1 exhibited significant increases in apoptotic rate (Fig 3C) as well as the protein levels of cleaved caspase 3 and cleaved PARP (Fig 3D). Moreover, another autophagy inhibitor 3-MA was used to confirm these results, which could block autophagy initiation and lead to the decrease in LC3-II level. As illustrated in S3A–S3C Fig, inhibition of autophagy using 3-MA significantly enhanced nedaplatin-induced cell death and caspase 3 cleavage in HNE1/DDP cells.

Bottom Line: Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity.While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells.Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, China.

ABSTRACT
Nedaplatin, a cisplatin analog, was developed to reduce the toxicity of cisplatin, whereas it can be cross-resistant with cisplatin in some circumstances. This study aimed to investigate the role of autophagy in nedaplatin induced cell death in cisplatin-resistant nasopharyngeal carcinoma cells. Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity. Nedaplatin treatment resulted in autophagosome accumulation and increased expression of LC3-II, indicating the induction of autophagy by nedaplatin in HNE1/DDP and CNE2/DDP cells. Inhibition of autophagy by Bafilomycin A1 (Baf A1) and 3-Methyladenine (3-MA) remarkably enhanced the antitumor efficacy of nedaplatin in HNE1/DDP and CNE2/DDP cells, suggesting that the resistance to nedaplatin-induced cell death was caused by enhanced autophagy in nedaplatin-resistant NPC cells. Additionally, Baf A1 enhanced reactive oxygen species (ROS) generation and apoptosis induced by nedaplatin in HNE1/DDP cells. Mechanistically, nedaplatin treatment caused activation of ERK1/2 and suppression of Akt/mTOR signaling pathways. While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells. Furthermore, suppression of ROS could inhibit nedaplatin-induced ERK activation in HNE1/DDP cells, indicating that ROS and ERK were involved in nedaplatin-induced autophagy. Together, these findings suggested that autophagy played a cytoprotective role in nedaplatin-induced cytotoxicity of HNE1/DDP and CNE2/DDP cells. Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.

No MeSH data available.


Related in: MedlinePlus