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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 HMGCoAR transcription, phosphorylation and ubiquitination in colon cancer cells. HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total RNA was extracted, reverse-transcribed and subjected to qRT-PCR for HMGCoAR gene. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005. (B) Cells were subjected to ultracentrifugation to isolate the microsomal fraction. Left panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-phosphoserine (pSer) antibody, anti-ubiquitin antibody or anti-HMGCoAR antibody, to detect the amount of the immunoprecipitated (IP) enzyme. No Ab: extracts in the absence of the anti-HMGCoAR antibody, a condition used as internal control. Right panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-ubiquitin antibody. As a control of proteasome involvement in HMGCoAR degradation, the proteasome inhibitor MG-132 (10 μM, MG) was added for 16 h, alone or during the last 16 h of the incubation with DHA. The figures are representative of three experiments with similar results. The 95 kDa band corresponding to native HMGCoAR protein is indicated by the arrow.
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Figure 3: Effects of ω3PUFAs on HMGCoAR transcription, phosphorylation and ubiquitination in colon cancer cells. HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total RNA was extracted, reverse-transcribed and subjected to qRT-PCR for HMGCoAR gene. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005. (B) Cells were subjected to ultracentrifugation to isolate the microsomal fraction. Left panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-phosphoserine (pSer) antibody, anti-ubiquitin antibody or anti-HMGCoAR antibody, to detect the amount of the immunoprecipitated (IP) enzyme. No Ab: extracts in the absence of the anti-HMGCoAR antibody, a condition used as internal control. Right panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-ubiquitin antibody. As a control of proteasome involvement in HMGCoAR degradation, the proteasome inhibitor MG-132 (10 μM, MG) was added for 16 h, alone or during the last 16 h of the incubation with DHA. The figures are representative of three experiments with similar results. The 95 kDa band corresponding to native HMGCoAR protein is indicated by the arrow.

Mentions: HT29-dx cells had higher levels of HMGCoAR (Figure 3A) and HMGCoAS (Additional file2A) mRNA than HT29 cells, and higher nuclear levels of the transcription factor SREBP-2 (Additional file2B). Neither ω6PUFA nor ω3PUFAs changed the levels of HMGCoAR (Figure 3A) and HMGCoAS (Additional file2A) mRNA, and the amount of nuclear SREBP-2 (Additional file2B). These results suggest that DHA and EPA did not decrease HMGCoAR expression by down-regulating gene transcription. SREBP-1 was unmodified in each experimental condition and did not differ between HT29 and HT29-dx cells (Additional file2B).


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 HMGCoAR transcription, phosphorylation and ubiquitination in colon cancer cells. HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total RNA was extracted, reverse-transcribed and subjected to qRT-PCR for HMGCoAR gene. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005. (B) Cells were subjected to ultracentrifugation to isolate the microsomal fraction. Left panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-phosphoserine (pSer) antibody, anti-ubiquitin antibody or anti-HMGCoAR antibody, to detect the amount of the immunoprecipitated (IP) enzyme. No Ab: extracts in the absence of the anti-HMGCoAR antibody, a condition used as internal control. Right panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-ubiquitin antibody. As a control of proteasome involvement in HMGCoAR degradation, the proteasome inhibitor MG-132 (10 μM, MG) was added for 16 h, alone or during the last 16 h of the incubation with DHA. The figures are representative of three experiments with similar results. The 95 kDa band corresponding to native HMGCoAR protein is indicated by the arrow.
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Figure 3: Effects of ω3PUFAs on HMGCoAR transcription, phosphorylation and ubiquitination in colon cancer cells. HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total RNA was extracted, reverse-transcribed and subjected to qRT-PCR for HMGCoAR gene. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005. (B) Cells were subjected to ultracentrifugation to isolate the microsomal fraction. Left panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-phosphoserine (pSer) antibody, anti-ubiquitin antibody or anti-HMGCoAR antibody, to detect the amount of the immunoprecipitated (IP) enzyme. No Ab: extracts in the absence of the anti-HMGCoAR antibody, a condition used as internal control. Right panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-ubiquitin antibody. As a control of proteasome involvement in HMGCoAR degradation, the proteasome inhibitor MG-132 (10 μM, MG) was added for 16 h, alone or during the last 16 h of the incubation with DHA. The figures are representative of three experiments with similar results. The 95 kDa band corresponding to native HMGCoAR protein is indicated by the arrow.
Mentions: HT29-dx cells had higher levels of HMGCoAR (Figure 3A) and HMGCoAS (Additional file2A) mRNA than HT29 cells, and higher nuclear levels of the transcription factor SREBP-2 (Additional file2B). Neither ω6PUFA nor ω3PUFAs changed the levels of HMGCoAR (Figure 3A) and HMGCoAS (Additional file2A) mRNA, and the amount of nuclear SREBP-2 (Additional file2B). These results suggest that DHA and EPA did not decrease HMGCoAR expression by down-regulating gene transcription. SREBP-1 was unmodified in each experimental condition and did not differ between HT29 and HT29-dx cells (Additional file2B).

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