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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

ω3PUFAs restore doxorubicin cytotoxicity in chemoresistant colon cancer cells. 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). 5 μM doxorubicin (DOX) was added for 24 h, alone or during the last 24 h of incubation with fatty acids. Cycloheximide (4 μM for 24 h, CHX) was chosen as positive control of cytotoxicity in both chemosensitive and chemoresistant cells. (A) The intracellular accumulation of doxorubicin was measured fluorimetrically in duplicate. Data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005; versus CTRL HT29-dx: ° p < 0.005. (B) Cells were stained with Neutral Red solution and the absorbance of viable cells was measured in triplicate spectrophotometrically. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.002; versus DOX alone: ° p < 0.005. (C) Surface levels of calreticulin were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) The phagocytosis rate by DCs was evaluated in duplicate by flow cytometry. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.05; versus DOX alone: p < 0.005.
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Figure 6: ω3PUFAs restore doxorubicin cytotoxicity in chemoresistant colon cancer cells. 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). 5 μM doxorubicin (DOX) was added for 24 h, alone or during the last 24 h of incubation with fatty acids. Cycloheximide (4 μM for 24 h, CHX) was chosen as positive control of cytotoxicity in both chemosensitive and chemoresistant cells. (A) The intracellular accumulation of doxorubicin was measured fluorimetrically in duplicate. Data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005; versus CTRL HT29-dx: ° p < 0.005. (B) Cells were stained with Neutral Red solution and the absorbance of viable cells was measured in triplicate spectrophotometrically. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.002; versus DOX alone: ° p < 0.005. (C) Surface levels of calreticulin were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) The phagocytosis rate by DCs was evaluated in duplicate by flow cytometry. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.05; versus DOX alone: p < 0.005.

Mentions: The anthracycline doxorubicin, used to generate the resistant HT29-dx cell population[41], is a substrate of both Pgp and MRP1[27]. Although anthracyclines are not used in colon cancer therapy, we chose doxorubicin as a reliable tool to clarify whether ω3PUFAs chemosensitize resistant cells to anticancer drugs effluxed by Pgp and MRP1. Doxorubicin was indeed less accumulated in HT29-dx cells (Figure 6A) and did not reduce their viability (Figure 6B). In correlation with the reduction of Pgp and MRP1 at cell surface, DHA and EPA increased the intracellular doxorubicin retention (Figure 6A) and restored its cytotoxicity in HT29-dx cells (Figure 6B). Anthracyclines are amongst the few anticancer drugs able to kill cancer cells by exerting direct cytotoxicity on tumor cell and by inducing an immunogenic cell death. This type of cell death is characterized by the extracellular release of ATP and HMGB1, by the surface translocation of calreticulin, and by the subsequent phagocytosis of tumor cells by dendritic cells (DCs)[42,43]. While doxorubicin elicited all these events in HT29 cells, it was ineffective in HT29-dx cells (Additional file4A-B; Figure 6C-D; Table 6). EPA and DHA, which did not further enhance the effects of doxorubicin in chemosensitive cells, restored the doxorubicin-mediated release of extracellular ATP (Additional file4A) and HMGB1 (Additional file4B), the translocation of calreticulin on cell surface (Figure 6C; Table 6) and the DC-mediated phagocytosis of HT29-dx cells (Figure 6D), confirming that they fully restored the pro-immunogenic effects of doxorubicin. Of note, neither DHA nor EPA alone increased ATP and HMGB1 release, calreticulin translocation and cells phagocytosis (Additional file4A-B; Figure 6C-D; Table 6), suggesting that they were not immune-activating agents per se, but only enhancers of doxorubicin activity. Again, AA was completely ineffective in restoring doxorubicin cytotoxicity and pro-immunogenic effects in HT29-dx cells (Additional file4; Figure 6; Table 6).


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)

ω3PUFAs restore doxorubicin cytotoxicity in chemoresistant colon cancer cells. 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). 5 μM doxorubicin (DOX) was added for 24 h, alone or during the last 24 h of incubation with fatty acids. Cycloheximide (4 μM for 24 h, CHX) was chosen as positive control of cytotoxicity in both chemosensitive and chemoresistant cells. (A) The intracellular accumulation of doxorubicin was measured fluorimetrically in duplicate. Data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005; versus CTRL HT29-dx: ° p < 0.005. (B) Cells were stained with Neutral Red solution and the absorbance of viable cells was measured in triplicate spectrophotometrically. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.002; versus DOX alone: ° p < 0.005. (C) Surface levels of calreticulin were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) The phagocytosis rate by DCs was evaluated in duplicate by flow cytometry. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.05; versus DOX alone: p < 0.005.
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Figure 6: ω3PUFAs restore doxorubicin cytotoxicity in chemoresistant colon cancer cells. 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). 5 μM doxorubicin (DOX) was added for 24 h, alone or during the last 24 h of incubation with fatty acids. Cycloheximide (4 μM for 24 h, CHX) was chosen as positive control of cytotoxicity in both chemosensitive and chemoresistant cells. (A) The intracellular accumulation of doxorubicin was measured fluorimetrically in duplicate. Data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005; versus CTRL HT29-dx: ° p < 0.005. (B) Cells were stained with Neutral Red solution and the absorbance of viable cells was measured in triplicate spectrophotometrically. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.002; versus DOX alone: ° p < 0.005. (C) Surface levels of calreticulin were measured in non-permeabilized cells by flow cytometry. The figures shown here are representative of three similar experiments, performed in triplicate. (D) The phagocytosis rate by DCs was evaluated in duplicate by flow cytometry. Data are presented as means ± SD (n = 4). Versus respective CTRL: * p < 0.05; versus DOX alone: p < 0.005.
Mentions: The anthracycline doxorubicin, used to generate the resistant HT29-dx cell population[41], is a substrate of both Pgp and MRP1[27]. Although anthracyclines are not used in colon cancer therapy, we chose doxorubicin as a reliable tool to clarify whether ω3PUFAs chemosensitize resistant cells to anticancer drugs effluxed by Pgp and MRP1. Doxorubicin was indeed less accumulated in HT29-dx cells (Figure 6A) and did not reduce their viability (Figure 6B). In correlation with the reduction of Pgp and MRP1 at cell surface, DHA and EPA increased the intracellular doxorubicin retention (Figure 6A) and restored its cytotoxicity in HT29-dx cells (Figure 6B). Anthracyclines are amongst the few anticancer drugs able to kill cancer cells by exerting direct cytotoxicity on tumor cell and by inducing an immunogenic cell death. This type of cell death is characterized by the extracellular release of ATP and HMGB1, by the surface translocation of calreticulin, and by the subsequent phagocytosis of tumor cells by dendritic cells (DCs)[42,43]. While doxorubicin elicited all these events in HT29 cells, it was ineffective in HT29-dx cells (Additional file4A-B; Figure 6C-D; Table 6). EPA and DHA, which did not further enhance the effects of doxorubicin in chemosensitive cells, restored the doxorubicin-mediated release of extracellular ATP (Additional file4A) and HMGB1 (Additional file4B), the translocation of calreticulin on cell surface (Figure 6C; Table 6) and the DC-mediated phagocytosis of HT29-dx cells (Figure 6D), confirming that they fully restored the pro-immunogenic effects of doxorubicin. Of note, neither DHA nor EPA alone increased ATP and HMGB1 release, calreticulin translocation and cells phagocytosis (Additional file4A-B; Figure 6C-D; Table 6), suggesting that they were not immune-activating agents per se, but only enhancers of doxorubicin activity. Again, AA was completely ineffective in restoring doxorubicin cytotoxicity and pro-immunogenic effects in HT29-dx cells (Additional file4; Figure 6; Table 6).

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