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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

Autophagy was induced by nedaplatin in HNE1/DDP cells.(A) Nedaplatin induced formation of autophagosomes. HNE1/DDP cells were either untreated or treated with 6.0 μg/ml of nedaplatin for 48 h. A magnified view of the electron photomicrograph shows a characteristic autophagosome. (B) HNE1/DDP cells were treated with 6.0 μg /ml of nedaplatin for 24 h or with 500 nM of rapamycin for 4 h, and then the cells were stained with Cyto-ID Green autophagy dye and analyzed by confocal microscopy. (C) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12, 24, and 48 h. (D) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with nedaplatin for 48 h at 0, 1.5, 3.0 and 6.0 μg/ml.
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pone.0135236.g002: Autophagy was induced by nedaplatin in HNE1/DDP cells.(A) Nedaplatin induced formation of autophagosomes. HNE1/DDP cells were either untreated or treated with 6.0 μg/ml of nedaplatin for 48 h. A magnified view of the electron photomicrograph shows a characteristic autophagosome. (B) HNE1/DDP cells were treated with 6.0 μg /ml of nedaplatin for 24 h or with 500 nM of rapamycin for 4 h, and then the cells were stained with Cyto-ID Green autophagy dye and analyzed by confocal microscopy. (C) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12, 24, and 48 h. (D) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with nedaplatin for 48 h at 0, 1.5, 3.0 and 6.0 μg/ml.

Mentions: Autophagy occurs at low basal levels in all cells and is rapidly up-regulated when cell faces stress. The ability of autophagy to promote cell survival during metabolic stress may promote resistance to cytotoxic therapy [22]. To assess whether nedaplatin induced autophagy in NPC cells, we initially examined the accumulation of autophagosomes by transmission electron microscopy (TEM). As shown in Fig 2A, nedaplatin-treated HNE1/DDP cells showed numerous cytosolic autophagic vacuoles with clearly double-layered membrane structures compared with untreated cells. Since autophagosome was recognized as characteristic double membrane vacuolar structures containing cytoplasmic contents [23], our results indicated that nedaplatin might induce autophagosome accumulation in HNE1/DDP cells. To confirm the TEM result, nedaplatin-treated HNE1/DDP cells were incubated with autophagic vacuoles specific marker, Cyto-ID, for 30 min and fixed for confocal fluorescence microscopy analysis. As shown in Fig 2B, treatment of HNE1/DDP cells either with nedaplatin or autophagy inducer, rapamycin, resulted in a substantial increase in autophagosomes in the cells. Similar results were discovered in nedaplatin-treated HNE1 and CNE2/DDP cells (S2E and S2F Fig).


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)

Autophagy was induced by nedaplatin in HNE1/DDP cells.(A) Nedaplatin induced formation of autophagosomes. HNE1/DDP cells were either untreated or treated with 6.0 μg/ml of nedaplatin for 48 h. A magnified view of the electron photomicrograph shows a characteristic autophagosome. (B) HNE1/DDP cells were treated with 6.0 μg /ml of nedaplatin for 24 h or with 500 nM of rapamycin for 4 h, and then the cells were stained with Cyto-ID Green autophagy dye and analyzed by confocal microscopy. (C) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12, 24, and 48 h. (D) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with nedaplatin for 48 h at 0, 1.5, 3.0 and 6.0 μg/ml.
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Related In: Results  -  Collection

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pone.0135236.g002: Autophagy was induced by nedaplatin in HNE1/DDP cells.(A) Nedaplatin induced formation of autophagosomes. HNE1/DDP cells were either untreated or treated with 6.0 μg/ml of nedaplatin for 48 h. A magnified view of the electron photomicrograph shows a characteristic autophagosome. (B) HNE1/DDP cells were treated with 6.0 μg /ml of nedaplatin for 24 h or with 500 nM of rapamycin for 4 h, and then the cells were stained with Cyto-ID Green autophagy dye and analyzed by confocal microscopy. (C) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12, 24, and 48 h. (D) Immunoblot analysis of LC3-I/II levels. HNE1/DDP cells were treated with nedaplatin for 48 h at 0, 1.5, 3.0 and 6.0 μg/ml.
Mentions: Autophagy occurs at low basal levels in all cells and is rapidly up-regulated when cell faces stress. The ability of autophagy to promote cell survival during metabolic stress may promote resistance to cytotoxic therapy [22]. To assess whether nedaplatin induced autophagy in NPC cells, we initially examined the accumulation of autophagosomes by transmission electron microscopy (TEM). As shown in Fig 2A, nedaplatin-treated HNE1/DDP cells showed numerous cytosolic autophagic vacuoles with clearly double-layered membrane structures compared with untreated cells. Since autophagosome was recognized as characteristic double membrane vacuolar structures containing cytoplasmic contents [23], our results indicated that nedaplatin might induce autophagosome accumulation in HNE1/DDP cells. To confirm the TEM result, nedaplatin-treated HNE1/DDP cells were incubated with autophagic vacuoles specific marker, Cyto-ID, for 30 min and fixed for confocal fluorescence microscopy analysis. As shown in Fig 2B, treatment of HNE1/DDP cells either with nedaplatin or autophagy inducer, rapamycin, resulted in a substantial increase in autophagosomes in the cells. Similar results were discovered in nedaplatin-treated HNE1 and CNE2/DDP cells (S2E and S2F Fig).

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