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Coenzyme Q10 and lipid-related gene induction in HeLa cells
American Journal of Obstetrics and Gynecology (2004) 190, 1432e4
www.elsevier.com/locate/ajog
Coenzyme Q10 and lipid-related gene induction in HeLa cells Constantine Gorelick, MD,a,c Melissa Lopez-Jones, BA,c Gary L. Goldberg, MD,a,b,c Seymour L. Romney, MD,a,c Dineo Khabele, MDa,b,c Albert Einstein College of Medicine,a Albert Einstein Cancer Centerb and Montefiore Medical Center Bronx,c NY Received for publication September 10, 2003; accepted January 21, 2004
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Coenzyme Q10 Microarray Lipid response
Objective: Coenzyme Q10 is an antioxidant that may have a therapeutic role in cervical cancer. Study design: We investigated the cellular and molecular effects of 30 mmol/L Coenzyme Q10 in HeLa cells. Cell growth assays, fluorescence-activated cell sorting analyses, and Oil Red O staining were performed. Microarray experiments were performed in duplicate and analyzed on the basis of 2-fold changes in levels of gene expression. Results: Coenzyme Q10 inhibited cell growth and led to apoptosis. Microarray analysis showed that 264 sequences were altered over time, with enrichment in lipid-related genes. Enhanced lipid accumulation was confirmed with Oil Red O staining. Conclusion: A lipid response to Coenzyme Q10 may affect mechanisms of growth inhibition in HeLa cells. Ó 2004 Elsevier Inc. All rights reserved.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Coenzyme Q10 (CoQ10) is an antioxidant that is thought to reduce alpha-tocopherol, ascorbic acid, and beta-carotene sequentially.1 Decreased levels of antioxidants have been detected in women with cervical neoplasias.2,3 Antioxidants have been investigated as potential chemopreventive and chemotherapeutic agents.4 To study its potential therapeutic role, we treated HeLa cells with CoQ10.
Partially supported by P30-13330 from the National Cancer Institute. Reprint requests: Dineo Khabele, MD, Department of Obstetrics and Gynecology and Women’s Health, Division of Gynecologic Oncology, Montefiore Medical Center, 3332 Rochambeau Ave, Suite C, Bronx,NY10467. E-mail: dkhabele@montefiore.org 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.01.076
Methods CoQ10 was donated by Dr Raj Chopra of the Tishcon Corporation (Westbury, NY). All other reagents and media were purchased from Invitrogen Life Technologies (Carlsbad, Calif), and Sigma-Aldrich (St. Louis, Mo), and Amersham Biosciences (Piscataway, NJ). We maintained the established cervical cancer cell line (HeLa cells) in standard media. MTT (3-[4, 5-dimethylthiazol-2-yl] 2, 5-diophenyl tetrazolium bromide) Amersham assays in 8 replicates determined growth inhibition. Cells were treated with 30 mmol/L CoQ10 or with media at 24, 48, and 72 hours. The percent apoptosis was quantified by the sub-diploid cell fraction with the use of propridium iodide staining and fluorescence-activated cell sorting. Assays were
1433
Gorelick et al
Figure
Oil Red O staining in HeLa cells. Controls stained 0 to 1C, and CoQ10-treated cells stained 2C to 3C.
performed in triplicate. Lipid accumulation was detected with Oil Red O (Sigma) staining and scored 0 to 3C. Equal densities of CoQ10-treated and untreated cells were harvested at each time point for the microarrays. Total RNA was extracted, with Trizol (Invitrogen, Carlsbad, Calif) reagent. Complementary DNA (cDNA) that was synthesized from 50 mg of RNA was used as a probe and labeled with Cy3 or Cy5 fluorescent dyes. Samples were hybridized simultaneously to glass slides that contained 9216 human cDNA sequences. Relative gene expression levels were quantified, and sequences withO2-fold change were selected for further analysis, as previously described.5 RNA aliquots (5 mg) were reverse-transcribed into cDNA with SuperScript II RT (Invitrogen, Carlsbad, Calif). Quantitative polymerase chain reaction (QPCR) was performed with 10 ng cDNA, sequence specific primers, the SYBR Green core reagents kit (Applied Biosystems, Foster City, Calif), and a 7900HT real-time PCR instrument (Applied Biosystems, Foster City, Calif ). Samples were run in triplicate, and relative gene expression levels in CoQ10-treated and untreated cells were quantified, with probability values of !.05 calculated by the Student t tests.
Results The growth assays demonstrated that 30 mmol/L of CoQ10 led to 40% cell growth inhibition by 72 hours.
Table I
Number of genes altered in expression over time
Time (hr)
N > 2.0 (range) Fold change
N!0.5 (range) Fold change
Total
24 48 72 Total
18 27 204 249
1 5 9 15
19 32 213 264
(2.13-6.40) (2.02-8.63) (2.04-9.22) (2.02-9.22)
(0.42) (0.31-0.44) (0.16-0.48) (0.16-0.48)
N, number of genes altered.
The percent apoptosis by fluorescence-activated cell sorting analysis was higher in the treated group: 9.4% apoptosis at 24 hours, 10.8% at 48 hours, and 17.7% at 72 hours versus !3.7% for untreated cells. A step-wise increase in gene induction over time was observed (Table I; database website: http://www.genlichtlab.com). Seven of 19 genes (37%) were related to lipids at 24 hours; 10 of 32 genes (31%) were related to lipids at 48 hours, and 10 of 213 genes (4.7%) were related to lipids at 72 hours, 3 of which were validated with QPCR (Table II). Enhanced staining with Oil Red O was indicative of lipid accumulation in CoQ10-treated cells (Figure).
Comment In this in vitro model of cervical cancer, 30 mmol/L of CoQ10 inhibited growth and induced apoptosis. There was a parallel induction of genes, with an overexpression of lipid-related sequences. CoQ10 has complex intracellular functions and intersects with lipid metabolic
1434 Table II
Gorelick et al Three unregulated lipid sequences validated with QPCR 24 Hours
Primer sequences Farnesyl diphosphatc farnesy transferase F-GCAATCCTGCTGTTTCTCTCTGA R-GCAAACTGCCTTTTCCACACA Isopenteayl-diphosphate delta isomerase F-TGCATCGAGCTTTTAGTGTCTTCT R-TCCGTAAAACAACCCTGGAAA Lanosterol synthase F-CAGCAAGTCAGTTCACAAAGTTTCA R-AGTGGAGTATTTTAGTCTAAAGGCTTTTCA Beta Actin F-GATGAGATTGGCATGGCTTT R-CACCTTCACCGTTCCAGTTT
Fold change
48 Hours
72 Hours
P value
Fold change
P value
6.77
.0008
13.84
.02
7.97
.04
7.48
.01
19.54
.04
20.07
.04
2E-06
10.96
.03
13.90
.02
22.90
Fold change
P value
F, Forward; R, Reverse.
pathways.1,6,7 Our data suggest that a CoQ10-induced lipid response may contribute to mechanisms of growth inhibition in HeLa cells.
Acknowledgments We thank Adolfo Alvino, BA, for his technical assistance and Leonard Augenlicht, PhD, and his laboratory members for their resources and advice. We also thank Dr. Geoff Childs and Aldo Massini: for printing and scanning at the C:NA microarrays, Ray Chopra, PhD, for the use of C:Q10.
References 1. Crane FL. Biochemical functions of coenzyme Q10. J Am Coll Nutr 2001;20:591-8.
2. Palan PR, Mikhail MS, Shaban DW, Romney SL. Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer. Eur J Cancer Prev 2003;12:321-6. 3. Palan PR, Mikhail MS, Goldberg GL, Basu J, Runowicz CD, Romney SL. Plasma levels of beta-carotene, lycopene, canthaxanthin, retinol, and alpha- and tau-tocopherol in cervical intraepithelial neoplasia and cancer. Clin Cancer Res 1996;2:181-5. 4. Seifried HE, McDonald SS, Anderson DE, Greenwald P, Milner JA. The antioxidant conundrum in cancer. Cancer Res 2003;63: 4295-8. 5. Mariadason JM, Corner GA, Augenlicht LH. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res 2000;60:4561-72. 6. Hargreaves IP. Ubiquinone: cholesterol’s reclusive cousin. Ann Clin Biochem 2003;40:207-18. 7. Ernster L, Dallner G. Biochemical, physiological and medical aspects of ubiquinone function. Biochim Biophys Acta 1995;1271: 195-204.
www.elsevier.com/locate/ajog
Coenzyme Q10 and lipid-related gene induction in HeLa cells Constantine Gorelick, MD,a,c Melissa Lopez-Jones, BA,c Gary L. Goldberg, MD,a,b,c Seymour L. Romney, MD,a,c Dineo Khabele, MDa,b,c Albert Einstein College of Medicine,a Albert Einstein Cancer Centerb and Montefiore Medical Center Bronx,c NY Received for publication September 10, 2003; accepted January 21, 2004
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Coenzyme Q10 Microarray Lipid response
Objective: Coenzyme Q10 is an antioxidant that may have a therapeutic role in cervical cancer. Study design: We investigated the cellular and molecular effects of 30 mmol/L Coenzyme Q10 in HeLa cells. Cell growth assays, fluorescence-activated cell sorting analyses, and Oil Red O staining were performed. Microarray experiments were performed in duplicate and analyzed on the basis of 2-fold changes in levels of gene expression. Results: Coenzyme Q10 inhibited cell growth and led to apoptosis. Microarray analysis showed that 264 sequences were altered over time, with enrichment in lipid-related genes. Enhanced lipid accumulation was confirmed with Oil Red O staining. Conclusion: A lipid response to Coenzyme Q10 may affect mechanisms of growth inhibition in HeLa cells. Ó 2004 Elsevier Inc. All rights reserved.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Coenzyme Q10 (CoQ10) is an antioxidant that is thought to reduce alpha-tocopherol, ascorbic acid, and beta-carotene sequentially.1 Decreased levels of antioxidants have been detected in women with cervical neoplasias.2,3 Antioxidants have been investigated as potential chemopreventive and chemotherapeutic agents.4 To study its potential therapeutic role, we treated HeLa cells with CoQ10.
Partially supported by P30-13330 from the National Cancer Institute. Reprint requests: Dineo Khabele, MD, Department of Obstetrics and Gynecology and Women’s Health, Division of Gynecologic Oncology, Montefiore Medical Center, 3332 Rochambeau Ave, Suite C, Bronx,NY10467. E-mail: dkhabele@montefiore.org 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.01.076
Methods CoQ10 was donated by Dr Raj Chopra of the Tishcon Corporation (Westbury, NY). All other reagents and media were purchased from Invitrogen Life Technologies (Carlsbad, Calif), and Sigma-Aldrich (St. Louis, Mo), and Amersham Biosciences (Piscataway, NJ). We maintained the established cervical cancer cell line (HeLa cells) in standard media. MTT (3-[4, 5-dimethylthiazol-2-yl] 2, 5-diophenyl tetrazolium bromide) Amersham assays in 8 replicates determined growth inhibition. Cells were treated with 30 mmol/L CoQ10 or with media at 24, 48, and 72 hours. The percent apoptosis was quantified by the sub-diploid cell fraction with the use of propridium iodide staining and fluorescence-activated cell sorting. Assays were
1433
Gorelick et al
Figure
Oil Red O staining in HeLa cells. Controls stained 0 to 1C, and CoQ10-treated cells stained 2C to 3C.
performed in triplicate. Lipid accumulation was detected with Oil Red O (Sigma) staining and scored 0 to 3C. Equal densities of CoQ10-treated and untreated cells were harvested at each time point for the microarrays. Total RNA was extracted, with Trizol (Invitrogen, Carlsbad, Calif) reagent. Complementary DNA (cDNA) that was synthesized from 50 mg of RNA was used as a probe and labeled with Cy3 or Cy5 fluorescent dyes. Samples were hybridized simultaneously to glass slides that contained 9216 human cDNA sequences. Relative gene expression levels were quantified, and sequences withO2-fold change were selected for further analysis, as previously described.5 RNA aliquots (5 mg) were reverse-transcribed into cDNA with SuperScript II RT (Invitrogen, Carlsbad, Calif). Quantitative polymerase chain reaction (QPCR) was performed with 10 ng cDNA, sequence specific primers, the SYBR Green core reagents kit (Applied Biosystems, Foster City, Calif), and a 7900HT real-time PCR instrument (Applied Biosystems, Foster City, Calif ). Samples were run in triplicate, and relative gene expression levels in CoQ10-treated and untreated cells were quantified, with probability values of !.05 calculated by the Student t tests.
Results The growth assays demonstrated that 30 mmol/L of CoQ10 led to 40% cell growth inhibition by 72 hours.
Table I
Number of genes altered in expression over time
Time (hr)
N > 2.0 (range) Fold change
N!0.5 (range) Fold change
Total
24 48 72 Total
18 27 204 249
1 5 9 15
19 32 213 264
(2.13-6.40) (2.02-8.63) (2.04-9.22) (2.02-9.22)
(0.42) (0.31-0.44) (0.16-0.48) (0.16-0.48)
N, number of genes altered.
The percent apoptosis by fluorescence-activated cell sorting analysis was higher in the treated group: 9.4% apoptosis at 24 hours, 10.8% at 48 hours, and 17.7% at 72 hours versus !3.7% for untreated cells. A step-wise increase in gene induction over time was observed (Table I; database website: http://www.genlichtlab.com). Seven of 19 genes (37%) were related to lipids at 24 hours; 10 of 32 genes (31%) were related to lipids at 48 hours, and 10 of 213 genes (4.7%) were related to lipids at 72 hours, 3 of which were validated with QPCR (Table II). Enhanced staining with Oil Red O was indicative of lipid accumulation in CoQ10-treated cells (Figure).
Comment In this in vitro model of cervical cancer, 30 mmol/L of CoQ10 inhibited growth and induced apoptosis. There was a parallel induction of genes, with an overexpression of lipid-related sequences. CoQ10 has complex intracellular functions and intersects with lipid metabolic
1434 Table II
Gorelick et al Three unregulated lipid sequences validated with QPCR 24 Hours
Primer sequences Farnesyl diphosphatc farnesy transferase F-GCAATCCTGCTGTTTCTCTCTGA R-GCAAACTGCCTTTTCCACACA Isopenteayl-diphosphate delta isomerase F-TGCATCGAGCTTTTAGTGTCTTCT R-TCCGTAAAACAACCCTGGAAA Lanosterol synthase F-CAGCAAGTCAGTTCACAAAGTTTCA R-AGTGGAGTATTTTAGTCTAAAGGCTTTTCA Beta Actin F-GATGAGATTGGCATGGCTTT R-CACCTTCACCGTTCCAGTTT
Fold change
48 Hours
72 Hours
P value
Fold change
P value
6.77
.0008
13.84
.02
7.97
.04
7.48
.01
19.54
.04
20.07
.04
2E-06
10.96
.03
13.90
.02
22.90
Fold change
P value
F, Forward; R, Reverse.
pathways.1,6,7 Our data suggest that a CoQ10-induced lipid response may contribute to mechanisms of growth inhibition in HeLa cells.
Acknowledgments We thank Adolfo Alvino, BA, for his technical assistance and Leonard Augenlicht, PhD, and his laboratory members for their resources and advice. We also thank Dr. Geoff Childs and Aldo Massini: for printing and scanning at the C:NA microarrays, Ray Chopra, PhD, for the use of C:Q10.
References 1. Crane FL. Biochemical functions of coenzyme Q10. J Am Coll Nutr 2001;20:591-8.
2. Palan PR, Mikhail MS, Shaban DW, Romney SL. Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer. Eur J Cancer Prev 2003;12:321-6. 3. Palan PR, Mikhail MS, Goldberg GL, Basu J, Runowicz CD, Romney SL. Plasma levels of beta-carotene, lycopene, canthaxanthin, retinol, and alpha- and tau-tocopherol in cervical intraepithelial neoplasia and cancer. Clin Cancer Res 1996;2:181-5. 4. Seifried HE, McDonald SS, Anderson DE, Greenwald P, Milner JA. The antioxidant conundrum in cancer. Cancer Res 2003;63: 4295-8. 5. Mariadason JM, Corner GA, Augenlicht LH. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res 2000;60:4561-72. 6. Hargreaves IP. Ubiquinone: cholesterol’s reclusive cousin. Ann Clin Biochem 2003;40:207-18. 7. Ernster L, Dallner G. Biochemical, physiological and medical aspects of ubiquinone function. Biochim Biophys Acta 1995;1271: 195-204.