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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

HNE1/DDP cells were resistant to nedaplatin-induced cell death.(A&B) HNE1 cells and HNE1/DDP cells were treated with the indicated concentrations of nedaplatin (A) or cispaltin (B) for 48 h. Cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. *p<0.05, **p<0.01 and ***p<0.0001 compared to HNE1 cells. (C) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with the indicated concentrations of nedaplatin for 48 h. (D) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with 6.0 μg/ml nedaplatin as indicated time.
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pone.0135236.g001: HNE1/DDP cells were resistant to nedaplatin-induced cell death.(A&B) HNE1 cells and HNE1/DDP cells were treated with the indicated concentrations of nedaplatin (A) or cispaltin (B) for 48 h. Cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. *p<0.05, **p<0.01 and ***p<0.0001 compared to HNE1 cells. (C) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with the indicated concentrations of nedaplatin for 48 h. (D) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with 6.0 μg/ml nedaplatin as indicated time.

Mentions: To assess whether cisplatin-resistant NPC cells would resist to nedaplatin-induced cell death, both cisplatin-sensitive nasopharyngeal cancer cells and cisplatin-resistant NPC cells were treated with nedaplatin at increasing doses for 48 h. In a parallel set of experiment, the cells (HNE1, CNE2, HNE1/DDP and CNE2/DDP) were treated with cisplatin for comparison. As shown in Fig 1A and 1B and S1 Fig, both nedaplatin and cisplatin induced dose-dependent cell death of HNE1 and CNE2 cells with IC50 (2.75±0.37 μg/ml for nedaplatin vs. 2.83±0.25 μg/ml for cisplatin, 1.49±0.20 μg/ml for nedaplatin v.s. 1.24±0.15 μg/ml for cisplatin, respectively). In contrast, the cisplatin-resistant HNE1/DDP and CNE2/DDP cells were less sensitive to nedaplatin-induced cell death with the IC50 of 6.74±0.58 μg/ml and 7.86±0.73 μg/ml, which was similar to its IC50 for cisplatin (6.02±0.72 μg/ml and 6.21±0.58 μg/ml, respectively). These results were in line with previous observation, indicating that nedaplatin was cross-resistant with cisplatin [19]. Since previous study showed that nedaplatin induced tumor cell death through induction of apoptosis [20], we reasoned that the resistance to nedaplatin was most likely due to a lack of apoptosis [21]. To test our hypothesis, we treated HNE1 and HNE1/DDP cells with nedaplatin and analyzed the level of cleaved form of caspase 3, a typical marker for apoptotic cell death. As shown in Fig 1C, nedaplatin treatment significantly promoted the cleavage of caspase 3 in HNE1 cells, while no obvious cleavage caspase 3 could be detected in nedaplatin-treated HNE1/DDP cells. Moreover, time-course experiment showed that nedaplatin-induced cleavage of caspase 3 in HNE1/DDP cells was delayed by 24 hours compared to HNE1 cells. (Fig 1D). These results indicated that cisplatin-resistant NPC cells (HNE1/DDP and CNE2/DDP cells) were resistant to nedaplatin.


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)

HNE1/DDP cells were resistant to nedaplatin-induced cell death.(A&B) HNE1 cells and HNE1/DDP cells were treated with the indicated concentrations of nedaplatin (A) or cispaltin (B) for 48 h. Cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. *p<0.05, **p<0.01 and ***p<0.0001 compared to HNE1 cells. (C) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with the indicated concentrations of nedaplatin for 48 h. (D) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with 6.0 μg/ml nedaplatin as indicated time.
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pone.0135236.g001: HNE1/DDP cells were resistant to nedaplatin-induced cell death.(A&B) HNE1 cells and HNE1/DDP cells were treated with the indicated concentrations of nedaplatin (A) or cispaltin (B) for 48 h. Cell viability was determined by MTT assay at the wavelength of 570 nm. Data are mean ± SD from five independent experiments. *p<0.05, **p<0.01 and ***p<0.0001 compared to HNE1 cells. (C) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with the indicated concentrations of nedaplatin for 48 h. (D) Western blot analysis for the expression of cleaved caspase 3 protein in HNE1 and HNE1/DDP cells treated with 6.0 μg/ml nedaplatin as indicated time.
Mentions: To assess whether cisplatin-resistant NPC cells would resist to nedaplatin-induced cell death, both cisplatin-sensitive nasopharyngeal cancer cells and cisplatin-resistant NPC cells were treated with nedaplatin at increasing doses for 48 h. In a parallel set of experiment, the cells (HNE1, CNE2, HNE1/DDP and CNE2/DDP) were treated with cisplatin for comparison. As shown in Fig 1A and 1B and S1 Fig, both nedaplatin and cisplatin induced dose-dependent cell death of HNE1 and CNE2 cells with IC50 (2.75±0.37 μg/ml for nedaplatin vs. 2.83±0.25 μg/ml for cisplatin, 1.49±0.20 μg/ml for nedaplatin v.s. 1.24±0.15 μg/ml for cisplatin, respectively). In contrast, the cisplatin-resistant HNE1/DDP and CNE2/DDP cells were less sensitive to nedaplatin-induced cell death with the IC50 of 6.74±0.58 μg/ml and 7.86±0.73 μg/ml, which was similar to its IC50 for cisplatin (6.02±0.72 μg/ml and 6.21±0.58 μg/ml, respectively). These results were in line with previous observation, indicating that nedaplatin was cross-resistant with cisplatin [19]. Since previous study showed that nedaplatin induced tumor cell death through induction of apoptosis [20], we reasoned that the resistance to nedaplatin was most likely due to a lack of apoptosis [21]. To test our hypothesis, we treated HNE1 and HNE1/DDP cells with nedaplatin and analyzed the level of cleaved form of caspase 3, a typical marker for apoptotic cell death. As shown in Fig 1C, nedaplatin treatment significantly promoted the cleavage of caspase 3 in HNE1 cells, while no obvious cleavage caspase 3 could be detected in nedaplatin-treated HNE1/DDP cells. Moreover, time-course experiment showed that nedaplatin-induced cleavage of caspase 3 in HNE1/DDP cells was delayed by 24 hours compared to HNE1 cells. (Fig 1D). These results indicated that cisplatin-resistant NPC cells (HNE1/DDP and CNE2/DDP cells) were resistant to nedaplatin.

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