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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

Suppression of ROS production inhibited nedaplatin-induced cell death.(A) HNE1/DDP cells were incubated with varied concentrations of nedaplatin for 12 h. Then, the samples were prepared as described in Materials and Methods. All data are expressed as means ± SD from five independent experiments. *p< 0.05. (B) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin in the presence or absence of Baf A1 for 6 h. Then, the cells were treated as described in A. (C) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12 h in the absence or presence of NAC (10 mM). The cells were treated as described in A. *p<0.05. (D) The LC3 I/II levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h in the absence or presence of NAC (10 mM). The cell viability was determined as described in Materials and Methods. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only.
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pone.0135236.g004: Suppression of ROS production inhibited nedaplatin-induced cell death.(A) HNE1/DDP cells were incubated with varied concentrations of nedaplatin for 12 h. Then, the samples were prepared as described in Materials and Methods. All data are expressed as means ± SD from five independent experiments. *p< 0.05. (B) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin in the presence or absence of Baf A1 for 6 h. Then, the cells were treated as described in A. (C) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12 h in the absence or presence of NAC (10 mM). The cells were treated as described in A. *p<0.05. (D) The LC3 I/II levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h in the absence or presence of NAC (10 mM). The cell viability was determined as described in Materials and Methods. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only.

Mentions: Previous report has demonstrated that ROS played an important role in cisplatin-induced cell death [25]. To explore whether nedaplatin treatment induced ROS generation in HNE1/DDP cells, we applied DCFH-DA, a well-established compound to detect and quantify intracellular generated ROS. Our results showed that treatment of nedaplatin for 12 h did not lead to obvious change of ROS level in HNE1/DDP cells (Fig 4A). The lack of ROS production in response to nedaplatin stimulation may be caused by up-regulation of autophagy in HNE1/DDP cells, since there was evidence to demonstrate that ROS accumulation was suppressed by autophagy [26]. To test this idea, HNE1/DDP cells were treated with autophagy inhibitor, BafA1, and the level of ROS production in response to nedaplatin stimulation was measured. As shown in Fig 4B, in the presence of autophagy inhibitor, BafA1, nedaplatin resulted in 2.78-fold ROS production in HNE1/DDP cells over nedaplatin alone treated cells. To further verify that ROS contributed to nedaplatin-induced cell killing effect, HNE1/DDP cells were pretreated with an antioxidant, NAC. In the presence of NAC, ROS level in HNE1/DDP cells was significantly reduced (Fig 4C). Previous studies showed that autophagy was induced under oxidative stress [27,28]. Consistently, we found that pretreatment with NAC remarkably reduced LC3-II conversion (Fig 4D), indicating that NAC could block nedaplatin-induced autophagy. Further, the combination of nedaplatin with NAC could rescue HNE1/DDP cells from nedaplatin-induced cell death (Fig 4E). These results indicated that accumulation of ROS played a critical role in the resensitization of HNE1/DDP cells to nedaplatin-induced cell death under conditions of suppressed autophagy.


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)

Suppression of ROS production inhibited nedaplatin-induced cell death.(A) HNE1/DDP cells were incubated with varied concentrations of nedaplatin for 12 h. Then, the samples were prepared as described in Materials and Methods. All data are expressed as means ± SD from five independent experiments. *p< 0.05. (B) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin in the presence or absence of Baf A1 for 6 h. Then, the cells were treated as described in A. (C) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12 h in the absence or presence of NAC (10 mM). The cells were treated as described in A. *p<0.05. (D) The LC3 I/II levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h in the absence or presence of NAC (10 mM). The cell viability was determined as described in Materials and Methods. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4543554&req=5

pone.0135236.g004: Suppression of ROS production inhibited nedaplatin-induced cell death.(A) HNE1/DDP cells were incubated with varied concentrations of nedaplatin for 12 h. Then, the samples were prepared as described in Materials and Methods. All data are expressed as means ± SD from five independent experiments. *p< 0.05. (B) HNE1/DDP cells were incubated with 6.0 μg/ml nedaplatin in the presence or absence of Baf A1 for 6 h. Then, the cells were treated as described in A. (C) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 12 h in the absence or presence of NAC (10 mM). The cells were treated as described in A. *p<0.05. (D) The LC3 I/II levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h in the absence or presence of NAC (10 mM). The cell viability was determined as described in Materials and Methods. Data are mean ± SD from five independent experiments. ***p<0.001 compared to nedaplatin only.
Mentions: Previous report has demonstrated that ROS played an important role in cisplatin-induced cell death [25]. To explore whether nedaplatin treatment induced ROS generation in HNE1/DDP cells, we applied DCFH-DA, a well-established compound to detect and quantify intracellular generated ROS. Our results showed that treatment of nedaplatin for 12 h did not lead to obvious change of ROS level in HNE1/DDP cells (Fig 4A). The lack of ROS production in response to nedaplatin stimulation may be caused by up-regulation of autophagy in HNE1/DDP cells, since there was evidence to demonstrate that ROS accumulation was suppressed by autophagy [26]. To test this idea, HNE1/DDP cells were treated with autophagy inhibitor, BafA1, and the level of ROS production in response to nedaplatin stimulation was measured. As shown in Fig 4B, in the presence of autophagy inhibitor, BafA1, nedaplatin resulted in 2.78-fold ROS production in HNE1/DDP cells over nedaplatin alone treated cells. To further verify that ROS contributed to nedaplatin-induced cell killing effect, HNE1/DDP cells were pretreated with an antioxidant, NAC. In the presence of NAC, ROS level in HNE1/DDP cells was significantly reduced (Fig 4C). Previous studies showed that autophagy was induced under oxidative stress [27,28]. Consistently, we found that pretreatment with NAC remarkably reduced LC3-II conversion (Fig 4D), indicating that NAC could block nedaplatin-induced autophagy. Further, the combination of nedaplatin with NAC could rescue HNE1/DDP cells from nedaplatin-induced cell death (Fig 4E). These results indicated that accumulation of ROS played a critical role in the resensitization of HNE1/DDP cells to nedaplatin-induced cell death under conditions of suppressed autophagy.

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