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Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition.

Gelsomino G, Corsetto PA, Campia I, Montorfano G, Kopecka J, Castella B, Gazzano E, Ghigo D, Rizzo AM, Riganti C - Mol. Cancer (2013)

Bottom Line: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells.We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments.They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, University of Torino, via Santena 5/bis, 10126 Torino, Italy. dario.ghigo@unito.it.

ABSTRACT

Background: The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors.

Methods: We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells.

Results: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.

Conclusions: Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

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Related in: MedlinePlus

Effects of ω3PUFAs on cholesterol and ABC transporters in whole cell and DRMs. HT29 and HT29-dx cells were incubated for 48 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total lipids were extracted and the amount of cholesterol was measured in triplicate as reported under Methods. Data are presented as mean ± SD (n = 3). Versus respective CTRL: * p < 0.01; HT29-dx versus HT29 cells: ° p < 0.01. (B) DRMs were isolated as detergent-resistant membranes by separation on sucrose gradient; then the amount of cholesterol was measured as reported in Methods. Data are presented as mean ± SD (n = 4). Versus respective CTRL: * p < 0.05; HT29-dx versus HT29 cells: ° p < 0.01. (C) Surface levels of Pgp, MRP1 and BCRP were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) Western blot detection of Pgp, MRP1 and BCRP on DRM extracts. Flotillin expression was used as control of equal protein loading. The figure is representative of two experiments with similar results. The band density ratio between each protein and flotillin was expressed as arbitrary units. Versus CTRL HT29: * p < 0.05; versus CTRL HT29-dx: p < 0.005.
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Figure 5: Effects of ω3PUFAs on cholesterol and ABC transporters in whole cell and DRMs. HT29 and HT29-dx cells were incubated for 48 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total lipids were extracted and the amount of cholesterol was measured in triplicate as reported under Methods. Data are presented as mean ± SD (n = 3). Versus respective CTRL: * p < 0.01; HT29-dx versus HT29 cells: ° p < 0.01. (B) DRMs were isolated as detergent-resistant membranes by separation on sucrose gradient; then the amount of cholesterol was measured as reported in Methods. Data are presented as mean ± SD (n = 4). Versus respective CTRL: * p < 0.05; HT29-dx versus HT29 cells: ° p < 0.01. (C) Surface levels of Pgp, MRP1 and BCRP were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) Western blot detection of Pgp, MRP1 and BCRP on DRM extracts. Flotillin expression was used as control of equal protein loading. The figure is representative of two experiments with similar results. The band density ratio between each protein and flotillin was expressed as arbitrary units. Versus CTRL HT29: * p < 0.05; versus CTRL HT29-dx: p < 0.005.

Mentions: Consistently with the higher rate of cholesterol synthesis (Figure 1A), HT29-dx cells had twice more cholesterol in whole cell than HT29 cells (Figure 5A). DHA and EPA decreased cholesterol levels only in HT29-dx cells (Figure 5A). As expected, DRMs were characterized by a high cholesterol content in both cell lines (Figure 5B); again, only in HT29-dx cells DHA and EPA were able to significantly decrease cholesterol (Figure 5B).


Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition.

Gelsomino G, Corsetto PA, Campia I, Montorfano G, Kopecka J, Castella B, Gazzano E, Ghigo D, Rizzo AM, Riganti C - Mol. Cancer (2013)

Effects of ω3PUFAs on cholesterol and ABC transporters in whole cell and DRMs. HT29 and HT29-dx cells were incubated for 48 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total lipids were extracted and the amount of cholesterol was measured in triplicate as reported under Methods. Data are presented as mean ± SD (n = 3). Versus respective CTRL: * p < 0.01; HT29-dx versus HT29 cells: ° p < 0.01. (B) DRMs were isolated as detergent-resistant membranes by separation on sucrose gradient; then the amount of cholesterol was measured as reported in Methods. Data are presented as mean ± SD (n = 4). Versus respective CTRL: * p < 0.05; HT29-dx versus HT29 cells: ° p < 0.01. (C) Surface levels of Pgp, MRP1 and BCRP were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) Western blot detection of Pgp, MRP1 and BCRP on DRM extracts. Flotillin expression was used as control of equal protein loading. The figure is representative of two experiments with similar results. The band density ratio between each protein and flotillin was expressed as arbitrary units. Versus CTRL HT29: * p < 0.05; versus CTRL HT29-dx: p < 0.005.
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Figure 5: Effects of ω3PUFAs on cholesterol and ABC transporters in whole cell and DRMs. HT29 and HT29-dx cells were incubated for 48 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total lipids were extracted and the amount of cholesterol was measured in triplicate as reported under Methods. Data are presented as mean ± SD (n = 3). Versus respective CTRL: * p < 0.01; HT29-dx versus HT29 cells: ° p < 0.01. (B) DRMs were isolated as detergent-resistant membranes by separation on sucrose gradient; then the amount of cholesterol was measured as reported in Methods. Data are presented as mean ± SD (n = 4). Versus respective CTRL: * p < 0.05; HT29-dx versus HT29 cells: ° p < 0.01. (C) Surface levels of Pgp, MRP1 and BCRP were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) Western blot detection of Pgp, MRP1 and BCRP on DRM extracts. Flotillin expression was used as control of equal protein loading. The figure is representative of two experiments with similar results. The band density ratio between each protein and flotillin was expressed as arbitrary units. Versus CTRL HT29: * p < 0.05; versus CTRL HT29-dx: p < 0.005.
Mentions: Consistently with the higher rate of cholesterol synthesis (Figure 1A), HT29-dx cells had twice more cholesterol in whole cell than HT29 cells (Figure 5A). DHA and EPA decreased cholesterol levels only in HT29-dx cells (Figure 5A). As expected, DRMs were characterized by a high cholesterol content in both cell lines (Figure 5B); again, only in HT29-dx cells DHA and EPA were able to significantly decrease cholesterol (Figure 5B).

Bottom Line: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells.We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments.They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, University of Torino, via Santena 5/bis, 10126 Torino, Italy. dario.ghigo@unito.it.

ABSTRACT

Background: The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors.

Methods: We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells.

Results: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.

Conclusions: Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

Show MeSH
Related in: MedlinePlus