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PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells.

Qi L, Toyoda H, Xu DQ, Zhou Y, Sakurai N, Amano K, Kihira K, Hori H, Azuma E, Komada Y - Cancer Cell Int. (2015)

Bottom Line: GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0-G1 ratio in LAN-1-MK than that in LAN-1 as well.PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan ; Department of Pediatrics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai, 200092 China.

ABSTRACT

Purpose: AKT plays a pivotal role in the signal transduction of cancer cells. MK2206, an AKT inhibitor, has been shown to be an effective anti-cancer drug to a variety of cancer cell lines. However, some cancer cells acquire resistance to MK2206 and new strategies to suppress these cell lines remain to be developed.

Experimental design: Acquired MK-2206-resistant neuroblastoma (NB) cell sublines were induced by stepwise escalation of MK-2206 exposure (4-12 weeks). MTT assay was used to validate cell proliferation. Flow cytometry was performed for cell cycle analysis. Western blot assay was used for cell signaling study.

Results: MK2206 (5-10 µmol) significantly suppressed cell growth of MK2206 non-resistant NB cells (LAN-1, KP-N-SIFA, NB-19 and SK-N-DZ), but is less efficient in inhibiting that of resistant sublines, even after 2-week MK2206-free incubation. MK2206 acted in mTOR-S6K dependent and independent methods. MK-2206 resistant sublines (LAN-1-MK, KP-N-SIFA-MK, and SK-N-DZ-MK) showed lower IC50 of GSK2334470 (PDK1 inhibitor). The cell growth of all sublines was prohibited by AZD8805 (mTOR inhibitor), with IC50 of AZD8805 3-10 times lower than MK2206 non-resistant cells. The signaling profiles of these resistant sublines were characterized by elevated PDK1-mTOR-S6K activity, accompanying by low phosphorylation of AKT compared with non-resistant counterparts. GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0-G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.

Conclusion: NB cells can acquire MK2206 resistance after exposure for 4-12 weeks. Resistant cells feature reliance on PDK1-mTOR-S6K pathway and are more sensitive to PDK1 and mTOR inhibitors than the non-resistant counterparts. Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.

No MeSH data available.


Related in: MedlinePlus

Effect of AZD8055 (AZD), mTOR inhibitor, in MK-2206-resistant sublines compared with non-resistant cells. a Indicated cell lines were treated with AZD at indicated concentrations, with/without MK-2206 (5 μM) in RPMI1640 + 10 % FBS. Cell growth was evaluated as cell numbers at 72 h, and it was repeated three times. Data are expressed as the mean (±SD). b The effect of AZD on cell cycle phase distribution in LAN-1 and LAN-MK. Indicated cells were treated with AZD (50 nM) with/without MK-2206 (5 μM) in RPMI1640 with 10 % FBS for 12 h followed by analysis of cell cycle phase distribution, as introduced in methods and material. Cells were stained with PI for 30 min followed by FACScan flow cytometer. c IC50 of AZD of indicated cell lines
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Fig5: Effect of AZD8055 (AZD), mTOR inhibitor, in MK-2206-resistant sublines compared with non-resistant cells. a Indicated cell lines were treated with AZD at indicated concentrations, with/without MK-2206 (5 μM) in RPMI1640 + 10 % FBS. Cell growth was evaluated as cell numbers at 72 h, and it was repeated three times. Data are expressed as the mean (±SD). b The effect of AZD on cell cycle phase distribution in LAN-1 and LAN-MK. Indicated cells were treated with AZD (50 nM) with/without MK-2206 (5 μM) in RPMI1640 with 10 % FBS for 12 h followed by analysis of cell cycle phase distribution, as introduced in methods and material. Cells were stained with PI for 30 min followed by FACScan flow cytometer. c IC50 of AZD of indicated cell lines

Mentions: To study inhibitory effect of MK-2206 on AKT pathway, cells were culture in RPMI + 10 % FBS with/without MK-2206, after 1 h serum starvation. P-AKT was detected by an anti-p-AKT antibody. AKT phosphorylation was suppressed by 5 μM of MK-2206 in LAN-1, KP-N-SIFA, NB-19, and SK-N-DZ cells (Fig. 5a–d). Stepwise escalation of MK-2206 exposure reduced p-AKT and total AKT levels in LAN-1-MK, NB-19-MK and SK-N-DZ-MK, comparing with the non-resistant opponents, after 1.5 and 12 h incubation without MK-2206 (Fig. 5a, c, d). KP-N-SIFA-MK had low levels of p-AKT but high levels of total AKT than its non-resistant counterpart (Fig. 5b).


PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells.

Qi L, Toyoda H, Xu DQ, Zhou Y, Sakurai N, Amano K, Kihira K, Hori H, Azuma E, Komada Y - Cancer Cell Int. (2015)

Effect of AZD8055 (AZD), mTOR inhibitor, in MK-2206-resistant sublines compared with non-resistant cells. a Indicated cell lines were treated with AZD at indicated concentrations, with/without MK-2206 (5 μM) in RPMI1640 + 10 % FBS. Cell growth was evaluated as cell numbers at 72 h, and it was repeated three times. Data are expressed as the mean (±SD). b The effect of AZD on cell cycle phase distribution in LAN-1 and LAN-MK. Indicated cells were treated with AZD (50 nM) with/without MK-2206 (5 μM) in RPMI1640 with 10 % FBS for 12 h followed by analysis of cell cycle phase distribution, as introduced in methods and material. Cells were stained with PI for 30 min followed by FACScan flow cytometer. c IC50 of AZD of indicated cell lines
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Fig5: Effect of AZD8055 (AZD), mTOR inhibitor, in MK-2206-resistant sublines compared with non-resistant cells. a Indicated cell lines were treated with AZD at indicated concentrations, with/without MK-2206 (5 μM) in RPMI1640 + 10 % FBS. Cell growth was evaluated as cell numbers at 72 h, and it was repeated three times. Data are expressed as the mean (±SD). b The effect of AZD on cell cycle phase distribution in LAN-1 and LAN-MK. Indicated cells were treated with AZD (50 nM) with/without MK-2206 (5 μM) in RPMI1640 with 10 % FBS for 12 h followed by analysis of cell cycle phase distribution, as introduced in methods and material. Cells were stained with PI for 30 min followed by FACScan flow cytometer. c IC50 of AZD of indicated cell lines
Mentions: To study inhibitory effect of MK-2206 on AKT pathway, cells were culture in RPMI + 10 % FBS with/without MK-2206, after 1 h serum starvation. P-AKT was detected by an anti-p-AKT antibody. AKT phosphorylation was suppressed by 5 μM of MK-2206 in LAN-1, KP-N-SIFA, NB-19, and SK-N-DZ cells (Fig. 5a–d). Stepwise escalation of MK-2206 exposure reduced p-AKT and total AKT levels in LAN-1-MK, NB-19-MK and SK-N-DZ-MK, comparing with the non-resistant opponents, after 1.5 and 12 h incubation without MK-2206 (Fig. 5a, c, d). KP-N-SIFA-MK had low levels of p-AKT but high levels of total AKT than its non-resistant counterpart (Fig. 5b).

Bottom Line: GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0-G1 ratio in LAN-1-MK than that in LAN-1 as well.PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan ; Department of Pediatrics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai, 200092 China.

ABSTRACT

Purpose: AKT plays a pivotal role in the signal transduction of cancer cells. MK2206, an AKT inhibitor, has been shown to be an effective anti-cancer drug to a variety of cancer cell lines. However, some cancer cells acquire resistance to MK2206 and new strategies to suppress these cell lines remain to be developed.

Experimental design: Acquired MK-2206-resistant neuroblastoma (NB) cell sublines were induced by stepwise escalation of MK-2206 exposure (4-12 weeks). MTT assay was used to validate cell proliferation. Flow cytometry was performed for cell cycle analysis. Western blot assay was used for cell signaling study.

Results: MK2206 (5-10 µmol) significantly suppressed cell growth of MK2206 non-resistant NB cells (LAN-1, KP-N-SIFA, NB-19 and SK-N-DZ), but is less efficient in inhibiting that of resistant sublines, even after 2-week MK2206-free incubation. MK2206 acted in mTOR-S6K dependent and independent methods. MK-2206 resistant sublines (LAN-1-MK, KP-N-SIFA-MK, and SK-N-DZ-MK) showed lower IC50 of GSK2334470 (PDK1 inhibitor). The cell growth of all sublines was prohibited by AZD8805 (mTOR inhibitor), with IC50 of AZD8805 3-10 times lower than MK2206 non-resistant cells. The signaling profiles of these resistant sublines were characterized by elevated PDK1-mTOR-S6K activity, accompanying by low phosphorylation of AKT compared with non-resistant counterparts. GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0-G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.

Conclusion: NB cells can acquire MK2206 resistance after exposure for 4-12 weeks. Resistant cells feature reliance on PDK1-mTOR-S6K pathway and are more sensitive to PDK1 and mTOR inhibitors than the non-resistant counterparts. Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.

No MeSH data available.


Related in: MedlinePlus