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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

Akt/mTOR and ERK signaling pathways were involved in nedaplatin-induced autophagy in HNE1/DDP cells.(A) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (B) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (C) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2 (Thr202/Tyr204) by western blot. (D) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2(Thr202/Tyr204) by western blot. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h with or without the pretreatment of U0126 (20 μM) for 2h. Protein extracts were examined by Western blot using LC3 I/II and phospho-Erk1/2 (Thr202/Tyr204) antibodies. (F) The phospho-Erk 1/2 (Thr202/Tyr204) levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h.
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pone.0135236.g005: Akt/mTOR and ERK signaling pathways were involved in nedaplatin-induced autophagy in HNE1/DDP cells.(A) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (B) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (C) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2 (Thr202/Tyr204) by western blot. (D) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2(Thr202/Tyr204) by western blot. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h with or without the pretreatment of U0126 (20 μM) for 2h. Protein extracts were examined by Western blot using LC3 I/II and phospho-Erk1/2 (Thr202/Tyr204) antibodies. (F) The phospho-Erk 1/2 (Thr202/Tyr204) levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h.

Mentions: Our result demonstrated that inhibition of mTOR by rapamycin induced autophagy in HNE1/DDP cells (Fig 2B), suggesting that PI3K/Akt/mTOR may be involved in nedaplatin-induced autophagy in HNE1/DDP cells. To test this speculation, HNE1/DDP cells were treated with nedaplatin, and the phosphorylation of key components of PI3K/Akt/mTOR pathway including Akt, mTOR, p70S6K and, 4E-BP1 were examined by western blotting analysis. As shown in Fig 5A and 5B, nedaplatin treatment caused a time- and does-dependent inhibition of phosphorylation of AKT, mTOR, p70S6K and 4E-BP1, which was consistent with previous reports showing that autophagy was suppressed by activated PI3K/Akt/mTOR signaling pathway [29,30]. Additionally, Sustained ERK activity that occured in tumors has been shown previously to have prosurvival effect by contributing to autophagy [31]. To test whether ERK was involved in nedaplatin-induced autophagy, HNE1/DDP cells were treated with nedaplatin, and phosphorylation of ERK was examined by Western blot analysis. As illustrated in Fig 5C and 5D, nedaplatin induced a time- and does-dependent increase in phospho-ERK1/2 in HNE1/DDP cells. Phosphorylation of ERK1/2 occured 12 hours after nedaplatin treatment and sustained until 48 hours after the treatment. Inhibition of ERK1/2 phosphorylation by MEK1/2 inhibitor, U0126, blocked nedaplatin-induced increase in LC3-II level (Fig 5E), indicating that nedaplatin-induced autophagy was mediated by MEK/ERK1/2 signaling pathway.


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)

Akt/mTOR and ERK signaling pathways were involved in nedaplatin-induced autophagy in HNE1/DDP cells.(A) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (B) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (C) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2 (Thr202/Tyr204) by western blot. (D) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2(Thr202/Tyr204) by western blot. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h with or without the pretreatment of U0126 (20 μM) for 2h. Protein extracts were examined by Western blot using LC3 I/II and phospho-Erk1/2 (Thr202/Tyr204) antibodies. (F) The phospho-Erk 1/2 (Thr202/Tyr204) levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h.
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pone.0135236.g005: Akt/mTOR and ERK signaling pathways were involved in nedaplatin-induced autophagy in HNE1/DDP cells.(A) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (B) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Levels of pAkt, pmTOR, pP70S6K, and p4E-BP1 were detected by western blot. (C) HNE1/DDP cells were treated with different concentrations of nedaplatin for 48 h. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2 (Thr202/Tyr204) by western blot. (D) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin as indicated times. Protein extracts were analyzed using Erk1/2 and phospho-Erk1/2(Thr202/Tyr204) by western blot. (E) HNE1/DDP cells were treated with 6.0 μg/ml nedaplatin for 48 h with or without the pretreatment of U0126 (20 μM) for 2h. Protein extracts were examined by Western blot using LC3 I/II and phospho-Erk1/2 (Thr202/Tyr204) antibodies. (F) The phospho-Erk 1/2 (Thr202/Tyr204) levels were examined by western blot after the nedaplatin treatment with or without of NAC (10 mM) for 48 h.
Mentions: Our result demonstrated that inhibition of mTOR by rapamycin induced autophagy in HNE1/DDP cells (Fig 2B), suggesting that PI3K/Akt/mTOR may be involved in nedaplatin-induced autophagy in HNE1/DDP cells. To test this speculation, HNE1/DDP cells were treated with nedaplatin, and the phosphorylation of key components of PI3K/Akt/mTOR pathway including Akt, mTOR, p70S6K and, 4E-BP1 were examined by western blotting analysis. As shown in Fig 5A and 5B, nedaplatin treatment caused a time- and does-dependent inhibition of phosphorylation of AKT, mTOR, p70S6K and 4E-BP1, which was consistent with previous reports showing that autophagy was suppressed by activated PI3K/Akt/mTOR signaling pathway [29,30]. Additionally, Sustained ERK activity that occured in tumors has been shown previously to have prosurvival effect by contributing to autophagy [31]. To test whether ERK was involved in nedaplatin-induced autophagy, HNE1/DDP cells were treated with nedaplatin, and phosphorylation of ERK was examined by Western blot analysis. As illustrated in Fig 5C and 5D, nedaplatin induced a time- and does-dependent increase in phospho-ERK1/2 in HNE1/DDP cells. Phosphorylation of ERK1/2 occured 12 hours after nedaplatin treatment and sustained until 48 hours after the treatment. Inhibition of ERK1/2 phosphorylation by MEK1/2 inhibitor, U0126, blocked nedaplatin-induced increase in LC3-II level (Fig 5E), indicating that nedaplatin-induced autophagy was mediated by MEK/ERK1/2 signaling pathway.

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