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IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer.

Liu YN, Chang TH, Tsai MF, Wu SG, Tsai TH, Chen HY, Yu SL, Yang JC, Shih JY - Oncotarget (2015)

Bottom Line: The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines.IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity.Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

No MeSH data available.


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Knockdown IL-8 increased gefitinib-induced apoptosisIL-8 mRNA A. and IL-8 secretion B. in PC9/gef-shCTL and PC9/gef-shIL8 cells were analyzed by RT-qPCR and ELISA, respectively. The bar graph represents the mean ± s.d. for n = 3 independent experiments (***p < 0.001). C. Cellular viability of PC9/gef-shCTL and PC9/gef-shIL8 cells was determined in the absence or presence of gefitinib for 72 hours by MTT assays. Herein, a two-fold serial dilution was used for the experiment resulting in concentration curves of gefitinib from 2.5 μM to 0.078 μM (*p < 0.05). D. Caspase-9 activity of these cells was analyzed by luminescent assay after treatment of gefitinib for 24 hours or 48 hours. Each condition was normalized to the corresponding vehicle-treated PC9/gef-shCTL group. The bar graph represents the mean ± s.d. for n = 3 independent experiments (*p < 0.05, **p < 0.01).
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Figure 3: Knockdown IL-8 increased gefitinib-induced apoptosisIL-8 mRNA A. and IL-8 secretion B. in PC9/gef-shCTL and PC9/gef-shIL8 cells were analyzed by RT-qPCR and ELISA, respectively. The bar graph represents the mean ± s.d. for n = 3 independent experiments (***p < 0.001). C. Cellular viability of PC9/gef-shCTL and PC9/gef-shIL8 cells was determined in the absence or presence of gefitinib for 72 hours by MTT assays. Herein, a two-fold serial dilution was used for the experiment resulting in concentration curves of gefitinib from 2.5 μM to 0.078 μM (*p < 0.05). D. Caspase-9 activity of these cells was analyzed by luminescent assay after treatment of gefitinib for 24 hours or 48 hours. Each condition was normalized to the corresponding vehicle-treated PC9/gef-shCTL group. The bar graph represents the mean ± s.d. for n = 3 independent experiments (*p < 0.05, **p < 0.01).

Mentions: To investigate whether knockdown of IL-8 could result in increasing gefitinib sensitivity, small hairpin RNA (shRNA) against IL-8 was used to knockdown IL-8 in PC9/gef, and we established two stable shIL8 cell lines with independent target sequences against IL-8 (PC9/gef-shIL8–1 and PC9/gef-shIL8–2) (Supplementary Table S4). We showed that both PC9/gef-shIL8 cell lines expressed lower levels of IL-8 than the control cells (PC9/gef-shCTL) (Fig. 3a–b). Both PC9/gef-shIL8 cell lines were more sensitive to the gefitinib treatment than PC9/gef-shCTL cells (Fig. 3c). Gefitinib-induced caspase-9 activity was significantly increased in PC9/gef-shIL8 cells compared with PC9/gef-shCTL cells (Fig. 3d). Moreover, we showed that knockdown of IL-8 with small interfering RNA (siIL-8) also resulted in recovery of gefitinib-induced apoptosis in PC9/gef or HCC827/gef cells (Supplementary Fig. S3). Collectively, these results indicate that IL-8 plays a crucial role in gefitinib resistance.


IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer.

Liu YN, Chang TH, Tsai MF, Wu SG, Tsai TH, Chen HY, Yu SL, Yang JC, Shih JY - Oncotarget (2015)

Knockdown IL-8 increased gefitinib-induced apoptosisIL-8 mRNA A. and IL-8 secretion B. in PC9/gef-shCTL and PC9/gef-shIL8 cells were analyzed by RT-qPCR and ELISA, respectively. The bar graph represents the mean ± s.d. for n = 3 independent experiments (***p < 0.001). C. Cellular viability of PC9/gef-shCTL and PC9/gef-shIL8 cells was determined in the absence or presence of gefitinib for 72 hours by MTT assays. Herein, a two-fold serial dilution was used for the experiment resulting in concentration curves of gefitinib from 2.5 μM to 0.078 μM (*p < 0.05). D. Caspase-9 activity of these cells was analyzed by luminescent assay after treatment of gefitinib for 24 hours or 48 hours. Each condition was normalized to the corresponding vehicle-treated PC9/gef-shCTL group. The bar graph represents the mean ± s.d. for n = 3 independent experiments (*p < 0.05, **p < 0.01).
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Related In: Results  -  Collection

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Figure 3: Knockdown IL-8 increased gefitinib-induced apoptosisIL-8 mRNA A. and IL-8 secretion B. in PC9/gef-shCTL and PC9/gef-shIL8 cells were analyzed by RT-qPCR and ELISA, respectively. The bar graph represents the mean ± s.d. for n = 3 independent experiments (***p < 0.001). C. Cellular viability of PC9/gef-shCTL and PC9/gef-shIL8 cells was determined in the absence or presence of gefitinib for 72 hours by MTT assays. Herein, a two-fold serial dilution was used for the experiment resulting in concentration curves of gefitinib from 2.5 μM to 0.078 μM (*p < 0.05). D. Caspase-9 activity of these cells was analyzed by luminescent assay after treatment of gefitinib for 24 hours or 48 hours. Each condition was normalized to the corresponding vehicle-treated PC9/gef-shCTL group. The bar graph represents the mean ± s.d. for n = 3 independent experiments (*p < 0.05, **p < 0.01).
Mentions: To investigate whether knockdown of IL-8 could result in increasing gefitinib sensitivity, small hairpin RNA (shRNA) against IL-8 was used to knockdown IL-8 in PC9/gef, and we established two stable shIL8 cell lines with independent target sequences against IL-8 (PC9/gef-shIL8–1 and PC9/gef-shIL8–2) (Supplementary Table S4). We showed that both PC9/gef-shIL8 cell lines expressed lower levels of IL-8 than the control cells (PC9/gef-shCTL) (Fig. 3a–b). Both PC9/gef-shIL8 cell lines were more sensitive to the gefitinib treatment than PC9/gef-shCTL cells (Fig. 3c). Gefitinib-induced caspase-9 activity was significantly increased in PC9/gef-shIL8 cells compared with PC9/gef-shCTL cells (Fig. 3d). Moreover, we showed that knockdown of IL-8 with small interfering RNA (siIL-8) also resulted in recovery of gefitinib-induced apoptosis in PC9/gef or HCC827/gef cells (Supplementary Fig. S3). Collectively, these results indicate that IL-8 plays a crucial role in gefitinib resistance.

Bottom Line: The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines.IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity.Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

No MeSH data available.


Related in: MedlinePlus