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Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Growth of Gefitinib-resistant Non-small Cell Lung Cancer Cells by Inducing Apoptosis.

Jeong CH - J Cancer Prev (2015)

Bottom Line: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs.Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Keimyung University, Daegu, Korea.

ABSTRACT

Background: Therapeutic approach by treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib or erlotinib to non-small cell lung cancer (NSCLC) patients has been limited due to emergence of acquired drug resistance. Our study was aimed to investigate whether the inhibition of ubiquitin-specific peptidase 8 (USP8) could be an alternative strategy capable of overcoming acquired resistance to EGFR-TKIs for treatment of NSCLCs.

Methods: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs. The immunoprecipitation and western blotting were conducted to check molecular interaction and signaling pathway followed by USP8 inhibition.

Results: Inhibition of USP8 induced overall degradation of oncogenic receptor tyrosine kinases including EGFR and Met, leading to a suppression of anchorage-dependent or independent cell growth of gefitinib-sensitive or resistant NSCLCs. Also, treatment with the USP8 inhibitor markedly induced apoptosis in HCC827GR cells. Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.

Conclusions: Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

No MeSH data available.


Related in: MedlinePlus

Ubiquitin-specific peptidase (USP8) inhibitor suppresses anchorage-independent growth of H1299 and H1650 cells by down-regulation of oncogenic receptor tyrosine kinases. (A) Chemical structure of USP8 inhibitor. (B) Effect of USP8 inhibitor on non-small cell lung cancers (NSCLCs) signaling. H1299 and H1650 cells were treated with the indicated concentrations of USP8 inhibitor for 24 hours to analyze molecular responsiveness. (C) Interaction of epidermal growth factor receptor (EGFR) and USP8 in NSCLCs. Cell lysates from H1299 and H1650 cells were subjected to immunoprecipitation (IP) and western blotting (WB) using the control immunoglobulin G (IgG), EGFR, and USP8 antibodies. (D, E) Colony formation of H1299 and H1650 cells after exposure to the increasing concentration of USP8 inhibitor or gefitinib for 7 days. Random areas were scanned (five areas per well, three wells per set) in colonies grown in soft agar. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01).
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f1-jcp-20-57: Ubiquitin-specific peptidase (USP8) inhibitor suppresses anchorage-independent growth of H1299 and H1650 cells by down-regulation of oncogenic receptor tyrosine kinases. (A) Chemical structure of USP8 inhibitor. (B) Effect of USP8 inhibitor on non-small cell lung cancers (NSCLCs) signaling. H1299 and H1650 cells were treated with the indicated concentrations of USP8 inhibitor for 24 hours to analyze molecular responsiveness. (C) Interaction of epidermal growth factor receptor (EGFR) and USP8 in NSCLCs. Cell lysates from H1299 and H1650 cells were subjected to immunoprecipitation (IP) and western blotting (WB) using the control immunoglobulin G (IgG), EGFR, and USP8 antibodies. (D, E) Colony formation of H1299 and H1650 cells after exposure to the increasing concentration of USP8 inhibitor or gefitinib for 7 days. Random areas were scanned (five areas per well, three wells per set) in colonies grown in soft agar. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01).

Mentions: To investigate that the targeting USP8 with its specific inhibitor might exhibit anti-cancer effect in NSCLCs, we first examined the effect of USP8 inhibitor on protein levels of RTKs including EGFR, Met and downstream signaling molecules in H1299 and H1650 cells. To do this, cells were treated with a recently synthesized USP8 inhibitor, 9-ehtyloxyimino9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile (Fig. 1A).25,26 Our data revealed that treatment with 0.25 to 0.5 μM USP8 inhibitor effectively downregulated the expression levels of EGFR, Met, Akt in gefitinib-sensitive H1650 cells, whereas did not affect in gefitinib-resistant H1299 cells (Fig. 1B). However, treatment with the higher concentration of USP8 inhibitor (5 to 10 μM) showed a robust downregulation of total levels of EGFR, Met, and Akt proteins in H1299 cells (data not shown), suggesting the differential potency of USP8 inhibitor in two cells. Because USP8 has been reported to regulate a number of cancer targets including EGFR,20–22 we first explored whether USP8 might interact with EGFR in NSCLCs, H1299 and H1650 cells. By conducting an Immunoprecipitation assay, we found an interaction between endogenous USP8 and EGFR in both cell lines, confirming that EGFR is a USP8’s client protein (Fig. 1C). Next, we investigate the anticancer effect of USP8 inhibitor in NSCLCs by performing the anchorage-independent colony formation assay. Expectedly, our data revealed that treatment with the USP8 inhibitor in H1299 cells resulted in no effect in colony number up to 1 μM, whereas significant decrease in colony number was observed at a concentration of 5 to 10 μM USP8 inhibitor (Fig. 1D). Also, treatment with the USP8 inhibitor in H1650 cells markedly decreased the soft-agar formation in a dose-dependent manner (Fig. 1E). Moreover, a more potent growth inhibitory effect was observed in USP8 inhibitor treated cells compared to gefitinib-treated cells, implying that USP8 inhibitor might have a potential therapeutic efficacy.


Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Growth of Gefitinib-resistant Non-small Cell Lung Cancer Cells by Inducing Apoptosis.

Jeong CH - J Cancer Prev (2015)

Ubiquitin-specific peptidase (USP8) inhibitor suppresses anchorage-independent growth of H1299 and H1650 cells by down-regulation of oncogenic receptor tyrosine kinases. (A) Chemical structure of USP8 inhibitor. (B) Effect of USP8 inhibitor on non-small cell lung cancers (NSCLCs) signaling. H1299 and H1650 cells were treated with the indicated concentrations of USP8 inhibitor for 24 hours to analyze molecular responsiveness. (C) Interaction of epidermal growth factor receptor (EGFR) and USP8 in NSCLCs. Cell lysates from H1299 and H1650 cells were subjected to immunoprecipitation (IP) and western blotting (WB) using the control immunoglobulin G (IgG), EGFR, and USP8 antibodies. (D, E) Colony formation of H1299 and H1650 cells after exposure to the increasing concentration of USP8 inhibitor or gefitinib for 7 days. Random areas were scanned (five areas per well, three wells per set) in colonies grown in soft agar. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4384715&req=5

f1-jcp-20-57: Ubiquitin-specific peptidase (USP8) inhibitor suppresses anchorage-independent growth of H1299 and H1650 cells by down-regulation of oncogenic receptor tyrosine kinases. (A) Chemical structure of USP8 inhibitor. (B) Effect of USP8 inhibitor on non-small cell lung cancers (NSCLCs) signaling. H1299 and H1650 cells were treated with the indicated concentrations of USP8 inhibitor for 24 hours to analyze molecular responsiveness. (C) Interaction of epidermal growth factor receptor (EGFR) and USP8 in NSCLCs. Cell lysates from H1299 and H1650 cells were subjected to immunoprecipitation (IP) and western blotting (WB) using the control immunoglobulin G (IgG), EGFR, and USP8 antibodies. (D, E) Colony formation of H1299 and H1650 cells after exposure to the increasing concentration of USP8 inhibitor or gefitinib for 7 days. Random areas were scanned (five areas per well, three wells per set) in colonies grown in soft agar. Error bars represent the mean ± SD. Statistical significance was determined by the Student’s t-test (*P < 0.01).
Mentions: To investigate that the targeting USP8 with its specific inhibitor might exhibit anti-cancer effect in NSCLCs, we first examined the effect of USP8 inhibitor on protein levels of RTKs including EGFR, Met and downstream signaling molecules in H1299 and H1650 cells. To do this, cells were treated with a recently synthesized USP8 inhibitor, 9-ehtyloxyimino9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile (Fig. 1A).25,26 Our data revealed that treatment with 0.25 to 0.5 μM USP8 inhibitor effectively downregulated the expression levels of EGFR, Met, Akt in gefitinib-sensitive H1650 cells, whereas did not affect in gefitinib-resistant H1299 cells (Fig. 1B). However, treatment with the higher concentration of USP8 inhibitor (5 to 10 μM) showed a robust downregulation of total levels of EGFR, Met, and Akt proteins in H1299 cells (data not shown), suggesting the differential potency of USP8 inhibitor in two cells. Because USP8 has been reported to regulate a number of cancer targets including EGFR,20–22 we first explored whether USP8 might interact with EGFR in NSCLCs, H1299 and H1650 cells. By conducting an Immunoprecipitation assay, we found an interaction between endogenous USP8 and EGFR in both cell lines, confirming that EGFR is a USP8’s client protein (Fig. 1C). Next, we investigate the anticancer effect of USP8 inhibitor in NSCLCs by performing the anchorage-independent colony formation assay. Expectedly, our data revealed that treatment with the USP8 inhibitor in H1299 cells resulted in no effect in colony number up to 1 μM, whereas significant decrease in colony number was observed at a concentration of 5 to 10 μM USP8 inhibitor (Fig. 1D). Also, treatment with the USP8 inhibitor in H1650 cells markedly decreased the soft-agar formation in a dose-dependent manner (Fig. 1E). Moreover, a more potent growth inhibitory effect was observed in USP8 inhibitor treated cells compared to gefitinib-treated cells, implying that USP8 inhibitor might have a potential therapeutic efficacy.

Bottom Line: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs.Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Keimyung University, Daegu, Korea.

ABSTRACT

Background: Therapeutic approach by treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib or erlotinib to non-small cell lung cancer (NSCLC) patients has been limited due to emergence of acquired drug resistance. Our study was aimed to investigate whether the inhibition of ubiquitin-specific peptidase 8 (USP8) could be an alternative strategy capable of overcoming acquired resistance to EGFR-TKIs for treatment of NSCLCs.

Methods: The anticancer effect of USP8 inhibitor was determined by testing anchorage-dependent or independent growth of gefitinib-sensitive or resistant NSCLCs. The immunoprecipitation and western blotting were conducted to check molecular interaction and signaling pathway followed by USP8 inhibition.

Results: Inhibition of USP8 induced overall degradation of oncogenic receptor tyrosine kinases including EGFR and Met, leading to a suppression of anchorage-dependent or independent cell growth of gefitinib-sensitive or resistant NSCLCs. Also, treatment with the USP8 inhibitor markedly induced apoptosis in HCC827GR cells. Notably, treatment with the USP8 inhibitor was more effective in suppressing cell growth and inducing apoptosis in gefitinib-resistant HCC827GR cells than that of gefitinib-sensitive HCC827 cells.

Conclusions: Inhibition of USP8 could be an effective strategy for overcoming gefitinib resistance in NSCLCs.

No MeSH data available.


Related in: MedlinePlus