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Down regulation of Wnt signaling mitigates hypoxia-induced chemoresistance in human osteosarcoma cells.

Scholten DJ, Timmer CM, Peacock JD, Pelle DW, Williams BO, Steensma MR - PLoS ONE (2014)

Bottom Line: We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells.Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05).Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01).

View Article: PubMed Central - PubMed

Affiliation: Michigan State University College of Human Medicine, Grand Rapids, Michigan, United States of America; Van Andel Research Institute, Grand Rapids, Michigan, United States of America.

ABSTRACT
Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

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Hypoxia results in chemoresistance of human OS cells to doxorubicin.A, Dose-response curves for the 143B and MNNG/HOS (mHOS) cell lines treated with increasing concentrations of doxorubicin under normoxic and hypoxic conditions (72 hour, 0.5% O2). Luminescence (viability) was determined as a percent of untreated control (0 µM doxorubicin). B, Average half maximal inhibitory concentration (IC50) values were obtained from the dose-response curves and compared between normoxic and hypoxic conditions for the cell lines MNNG/HOS (mHOS), 143B, and a patient-derived OS cell line, 206-2. Asterisks indicate statistical significance (**p<0.01, ***p<0.001).
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pone-0111431-g004: Hypoxia results in chemoresistance of human OS cells to doxorubicin.A, Dose-response curves for the 143B and MNNG/HOS (mHOS) cell lines treated with increasing concentrations of doxorubicin under normoxic and hypoxic conditions (72 hour, 0.5% O2). Luminescence (viability) was determined as a percent of untreated control (0 µM doxorubicin). B, Average half maximal inhibitory concentration (IC50) values were obtained from the dose-response curves and compared between normoxic and hypoxic conditions for the cell lines MNNG/HOS (mHOS), 143B, and a patient-derived OS cell line, 206-2. Asterisks indicate statistical significance (**p<0.01, ***p<0.001).

Mentions: Hypoxia has been shown to promote resistance to cytotoxic drugs in other human OS cell lines [12]. Thus we asked whether a more broad panel of OS cell lines, and particularly a patient-derived cell line isolate, show resistance under hypoxia to doxorubicin treatment, which is part of the mainstay chemotherapy regimen for OS patients [28]. We cultured both MNNG/HOS cells and 143B cells under normoxic and hypoxic conditions for 72 hours in the presence of increasing concentrations of doxorubicin. In both cell lines we saw a dramatic right shift in the dose–response curve under hypoxic conditions, indicating that these cells were more resistant to doxorubicin-mediated growth inhibition (Figure 4A). We calculated the average half maximal inhibitory concentration (IC50) between normoxic and hypoxic conditions for each cell line and found that hypoxia resulted in a statistically significant increase in the doxorubicin IC50 (MNNG/HOS, 5.9-fold increase; 143B, 13.6-fold increase, Figure 4B, p<0.01). We tested a patient-derived OS cell line (206-2) and found that it too was significantly more resistant to doxorubicin treatment under hypoxic conditions (7.5-fold increase, Figure 4B, p<0.01). We also examined the response to doxorubicin treatment under hypoxia and normoxia in MNNG/HOS cells in the context of either HIF-1 or HIF-2 knockdown. We were not able to determine any statistically significant difference between the non-targeting shRNA and the HIFα shRNA cell lines (Figure S3).


Down regulation of Wnt signaling mitigates hypoxia-induced chemoresistance in human osteosarcoma cells.

Scholten DJ, Timmer CM, Peacock JD, Pelle DW, Williams BO, Steensma MR - PLoS ONE (2014)

Hypoxia results in chemoresistance of human OS cells to doxorubicin.A, Dose-response curves for the 143B and MNNG/HOS (mHOS) cell lines treated with increasing concentrations of doxorubicin under normoxic and hypoxic conditions (72 hour, 0.5% O2). Luminescence (viability) was determined as a percent of untreated control (0 µM doxorubicin). B, Average half maximal inhibitory concentration (IC50) values were obtained from the dose-response curves and compared between normoxic and hypoxic conditions for the cell lines MNNG/HOS (mHOS), 143B, and a patient-derived OS cell line, 206-2. Asterisks indicate statistical significance (**p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4210185&req=5

pone-0111431-g004: Hypoxia results in chemoresistance of human OS cells to doxorubicin.A, Dose-response curves for the 143B and MNNG/HOS (mHOS) cell lines treated with increasing concentrations of doxorubicin under normoxic and hypoxic conditions (72 hour, 0.5% O2). Luminescence (viability) was determined as a percent of untreated control (0 µM doxorubicin). B, Average half maximal inhibitory concentration (IC50) values were obtained from the dose-response curves and compared between normoxic and hypoxic conditions for the cell lines MNNG/HOS (mHOS), 143B, and a patient-derived OS cell line, 206-2. Asterisks indicate statistical significance (**p<0.01, ***p<0.001).
Mentions: Hypoxia has been shown to promote resistance to cytotoxic drugs in other human OS cell lines [12]. Thus we asked whether a more broad panel of OS cell lines, and particularly a patient-derived cell line isolate, show resistance under hypoxia to doxorubicin treatment, which is part of the mainstay chemotherapy regimen for OS patients [28]. We cultured both MNNG/HOS cells and 143B cells under normoxic and hypoxic conditions for 72 hours in the presence of increasing concentrations of doxorubicin. In both cell lines we saw a dramatic right shift in the dose–response curve under hypoxic conditions, indicating that these cells were more resistant to doxorubicin-mediated growth inhibition (Figure 4A). We calculated the average half maximal inhibitory concentration (IC50) between normoxic and hypoxic conditions for each cell line and found that hypoxia resulted in a statistically significant increase in the doxorubicin IC50 (MNNG/HOS, 5.9-fold increase; 143B, 13.6-fold increase, Figure 4B, p<0.01). We tested a patient-derived OS cell line (206-2) and found that it too was significantly more resistant to doxorubicin treatment under hypoxic conditions (7.5-fold increase, Figure 4B, p<0.01). We also examined the response to doxorubicin treatment under hypoxia and normoxia in MNNG/HOS cells in the context of either HIF-1 or HIF-2 knockdown. We were not able to determine any statistically significant difference between the non-targeting shRNA and the HIFα shRNA cell lines (Figure S3).

Bottom Line: We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells.Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05).Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01).

View Article: PubMed Central - PubMed

Affiliation: Michigan State University College of Human Medicine, Grand Rapids, Michigan, United States of America; Van Andel Research Institute, Grand Rapids, Michigan, United States of America.

ABSTRACT
Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6-13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.

Show MeSH
Related in: MedlinePlus