Molecular and Cellular Biochemistry

, Volume 436, Issue 1–2, pp 1–12 | Cite as

β-carotene at physiologically attainable concentration induces apoptosis and down-regulates cell survival and antioxidant markers in human breast cancer (MCF-7) cells

  • G. Sowmya Shree
  • K. Yogendra Prasad
  • H. S. Arpitha
  • U. R. Deepika
  • K. Nawneet Kumar
  • Priya Mondal
  • P. Ganesan


Although β-carotene is known for its anti-carcinogenic and antioxidant properties, a few recent epidemiological and experimental evidence show that at higher concentration it acts as pro-oxidant and induces cancer. Since the global burden of breast cancer exceeds all other types of cancer, and its incidence rates is also in increasing trend, the present study attempted to evaluate the anti-cancer molecular mechanism of β-carotene (at 1 µM concentration) isolated from Spinacia oleracea in human breast cancer (MCF-7) cells. The carotenoid was purified by open column chromatography and identified by LC–MS. The anti-proliferative effect of β-carotene at different concentrations was evaluated by WST-1 assay and the changes in cell morphology were examined by microscopic observation. The induction of apoptosis by β-carotene was observed by DAPI staining and colorimetric caspase-3 assay. The expression of cell survival, apoptotic, and antioxidant marker proteins was measured by western blot analysis. Purified β-carotene inhibited the viability of MCF-7 cells in a dose-dependent manner, which was well correlated with changes in cell morphology. Increased apoptotic cells were observed in β-carotene (1 µM)-treated cells. This apoptosis induction was associated with increased caspase-3 activity. The protein expression studies showed that β-carotene at 1 µM concentration effectively decreases the expression of the anti-apoptotic protein, Bcl-2 and PARP, and survival protein, NF-kB. It also inhibited the activation of intracellular growth signaling proteins, Akt and ERK1/2. The inhibition of Akt activation by β-carotene results in decreased phosphorylation of Bad. Further, it down-regulated antioxidant enzyme, SOD-2, and its transactivation factor (Nrf-2), and endoplasmic reticulum (ER) stress marker, XBP-1, at protein levels. These findings exhibit the key role of β-carotene even at a low physiological concentration in MCF-7 cells which further explains its predominant anti-cancer activity.


Beta-carotene Physiological concentration MCF-7 cells Apoptosis Antioxidant markers 



This study was supported by the Science and Engineering Research Board (SERB), Government of India under the Young Scientist Research grant (GAP-0460), and the 12th Five Year Plan Project of the Council for Scientific and Industrial Research (CSIR), New Delhi. The authors thank the Director, CSIR-CFTRI for the constant support to carry out this work.

Conflict of interest

The authors declare no conflict of interest


  1. 1.
    Parker RS (1996) Absorption, metabolism, and transport of carotenoids. FASEB J 10:542–551PubMedGoogle Scholar
  2. 2.
    van Bennekum A, Werder M, Thuahnai ST, Han C-H, Duong P, Williams DL, Wettstein P, Schulthess G, Phillips MC, Hauser H (2005) Class B scavenger receptor-mediated intestinal absorption of dietary β-carotene and cholesterol. Biochem 44:4517–4525CrossRefGoogle Scholar
  3. 3.
    Perera CO, Yen GM (2007) Functional properties of carotenoids in human health. Int J Food Prop 10:201–230CrossRefGoogle Scholar
  4. 4.
    Sporn MB, Suh N (2002) Chemoprevention: an essential approach to controlling cancer. Nature Rev. Cancer 2:537–543CrossRefGoogle Scholar
  5. 5.
    Tanaka T, Shnimizu M, Moriwaki H (2012) Cancer chemoprevention by carotenoids. Molecules 17:3202–3242CrossRefPubMedGoogle Scholar
  6. 6.
    Pashkow FJ, Watumull DG, Campbell CL (2008) Astaxanthin: a novel potential treatment for oxidative stress and inflammation in cardiovascular disease. Am J Cardiol 101(10, Supplement 1):S58–S68CrossRefGoogle Scholar
  7. 7.
    Eliassen AH, Hendrickson SJ, Brinton LA, Buring JE, Campos H, Dai Q, Dorgan JF, Franke AA, Gao Y, Goodman MT, Hallmans G, Helzlsouer KJ, Hoffman-Bolton J, Hultén K, Sesso HD, Sowell AL, Tamimi RM, Toniolo P, Wilkens LR, Winkvist A, Zeleniuch-Jacquotte A, Zheng W, Hankinson SE (2012) Circulating carotenoids and risk of breast cancer: pooled analysis of eight prospective studies. J Nat Cancer Inst 104:1905–1916CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Okuyama Y, Ozasa K, Oki K, Nishino H, Fujimoto S, Watanabe Y (2014) Inverse associations between serum concentrations of zeaxanthin and other carotenoids and colorectal neoplasm in Japanese. Int J Clin Oncol 19:87–97CrossRefPubMedGoogle Scholar
  9. 9.
    Niranjana R, Gayathri R, Nimish Mol S, Sugawara T, Hirata T, Miyashita K, Ganesan P (2015) Carotenoids modulate the hallmarks of cancer cells. J Func Foods 18:968–985CrossRefGoogle Scholar
  10. 10.
    La Vecchia C, Decarli A, Fasoli M, Parazzini F, Franceschi S, Gentile A, Negri E (1988) Dietary vitamin A and the risk of intraepithelial and invasive cervical neoplasia. Gynecol Oncol 30:187–195CrossRefPubMedGoogle Scholar
  11. 11.
    Nagao T, Ikeda N, Warnakulasuriya S, Fukano H, Yuasa H, Yano M, Miyasaki H, Ito Y (2000) Serum antioxidant micronutrients and the risk of oral leukoplakia among Japanese. Oral Oncol 36:466–470CrossRefPubMedGoogle Scholar
  12. 12.
    Kim J, Kim MK, Lee JK, Kim J-H, Son SK, Song E-S, Lee KB, Lee JP, Lee JM, Yun YM (2010) Intakes of vitamin A, C, and E, and beta-carotene are associated with risk of cervical cancer: a case-control study in Korea. Nutr Cancer 62:181–189CrossRefPubMedGoogle Scholar
  13. 13.
    Jung S, Wu K, Giovannucci E, Spiegelman D, Willett WC, Smith-Warner SA (2013) Carotenoid intake and risk of colorectal adenomas in a cohort of male health professionals. Cancer Causes Cont: CCC 24:705–717CrossRefGoogle Scholar
  14. 14.
    Cook NR, Le IM, Manson JE, Buring JE, Hennekens CH (2000) Effects of beta-carotene supplementation on cancer incidence by baseline characteristics in the Physicians’ Health Study (United States). Cancer Causes Cont: CCC 11:617–626CrossRefGoogle Scholar
  15. 15.
    Rohan TE, Jain M, Howe GR, Miller AB (2002) A cohort study of dietary carotenoids and lung cancer risk in women (Canada). Cancer Causes Cont: CCC 13:231–237CrossRefGoogle Scholar
  16. 16.
    Liu C, Wang XD, Bronson RT, Smith DE, Krinsky NI, Russell RM (2000) Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets. Carcinogenesis 21:2245–2253CrossRefPubMedGoogle Scholar
  17. 17.
    Palozza P, Serini S, Torsello A, Boninsegna A, Covacci V, Maggiano N, Ranelletti FO, Wolf FI, Calviello G (2002) Regulation of cell cycle progression and apoptosis by beta-carotene in undifferentiated and differentiated HL-60 leukemia cells: possible involvement of a redox mechanism. Int J Cancer 97:593–600CrossRefPubMedGoogle Scholar
  18. 18.
    Yeh S-L, Wang H-M, Chen P-Y, Wu T-C (2009) Interactions of β-carotene and flavonoids on the secretion of pro-inflammatory mediators in an in vitro system. Chem-Biol Inter 179:386–393CrossRefGoogle Scholar
  19. 19.
    Bouayed J, Bohn T (2010) Exogenous antioxidants-double-edged swords in cellular redox state: health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid Med Cell Longev 3:228–237CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Eroglu A, Hruszkewycz DP, Curley RW, Harrison EH (2010) The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα. Arch Biochem Biophys 504:11–16CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Eroglu A, Hruszkewycz DP, dela Sena C, Narayanasamy S, Riedl KM, Kopec RE, Schwartz SJ, Schwartz SJ, Jr. Curley RW, Harrison EH (2012) Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors. J Biol Chem 287:15886–15895CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Prince MR, Frisoli JK (1993) Beta-carotene accumulation in serum and skin. Am J Clin Nutr 57:175–181PubMedGoogle Scholar
  23. 23.
    Hu P, Reuben DB, Crimmins EM, Harris TB, Huang M-H, Seeman TE (2004) The effects of serum beta-carotene concentration and burden of inflammation on all-cause mortality risk in high-functioning older persons: macArthur studies of successful aging. J Gerontol Series A, Biol Sci Med Sci 59:849–854CrossRefGoogle Scholar
  24. 24.
    Lakshminarayana R, Raju M, Krishnakantha TP, Baskaran V (2005) Determination of major carotenoids in a few Indian leafy vegetables by high-performance liquid chromatography. J Agric Food Chem 53:2838–2842CrossRefPubMedGoogle Scholar
  25. 25.
    Ganesan P, Matsubara K, Ohkubo T, Tanaka Y, Noda K, Sugawara T, Hirata T (2010) Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile. Phytomed 17:1140–1144CrossRefGoogle Scholar
  26. 26.
    Harada-Shiba M, Kinoshita M, Kamido H, Shimokado K (1998) Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms. J Biol Chem 273:9681–9687CrossRefPubMedGoogle Scholar
  27. 27.
    Johnson J, Maher P, Hannekan A (2009) The flavonoid, eriodictyol, induces long-term protection in ARPE-19 cells through its effects on Nrf2 activation and phase 2 gene expression. Invest Ophthalmol Vis Sci 50:2398–2406CrossRefPubMedGoogle Scholar
  28. 28.
    Sangeetha RK, Baskaran V (2010) Retinol-deficient rats can convert a pharmacological dose of astaxanthin to retinol: antioxidant potential of astaxanthin, lutein, and β-carotene. Can J Physiol Pharmacol 88:977–985CrossRefPubMedGoogle Scholar
  29. 29.
    Mamatha BS, Baskaran V (2011) Effect of micellar lipids, dietary fiber and β-carotene on lutein bioavailability in aged rats with lutein deficiency. Nutrition 27:960–966CrossRefPubMedGoogle Scholar
  30. 30.
    Roy SK, Srivastava RK, Shankar S (2010) Inhibition of PI3 K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer. J Mol Signal 5:10CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Will M, Qin ACR, Toy W, Yao Z, Rodrik-Outmezguine V, Schneider C, Huang X, Monian P, Jiang X, de Stanchina E, Baselga J, Liu N, Chandarlapaty S, Rosen N (2014) Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS-ERK signaling. Cancer Disc 4:334–347CrossRefGoogle Scholar
  32. 32.
    Zong WX, Edelstein LC, Chen C, Bash J, Gélinas C (1999) The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Develop 13:382–387CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Catz SD, Johnson JL (2001) Transcriptional regulation of bcl-2 by nuclear factor kappa B and its significance in prostate cancer. Oncogene 20:7342–7351CrossRefPubMedGoogle Scholar
  34. 34.
    Schneider BL, Kulesz-Martin M (2004) Destructive cycles: the role of genomic instability and adaptation in carcinogenesis. Carcinogenesis 25:2033–2044CrossRefPubMedGoogle Scholar
  35. 35.
    Chen X, Iliopoulos D, Zhang Q, Tang Q, Greenblatt MB, Hatziapostolou M, Lim E, Tam WL, Ni M, Chen Y, Mai J, Shen H, Hu DZ, Adoro S, Hu B, Song M, Tan C, Landis MD, Ferrari M, Shin SJ, Brown M, Chang JC, Liu XS, Glimcher LH (2014) XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway. Nature 508:103–107CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Epstein JH (1977) Effects of beta-carotene on ultraviolet induced cancer formation in the hairless mouse skin. Photochem Photobiol 25:211–213CrossRefPubMedGoogle Scholar
  37. 37.
    Peto R, Doll R, Buckley JD, Sporn MB (1981) Can dietary beta-carotene materially reduce human cancer rates? Nature 290:201–208CrossRefPubMedGoogle Scholar
  38. 38.
    van Poppel G, Goldbohm RA (1995) Epidemiologic evidence for beta-carotene and cancer prevention. Am J Clin Nutr 62(6 Suppl):1393S–1402SPubMedGoogle Scholar
  39. 39.
    Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S (1996) Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. New Eng J Med 334:1150–1155CrossRefPubMedGoogle Scholar
  40. 40.
    Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, Belanger C, LaMotte F, Gaziano JM, Ridker PM, Willett W, Peto R (1996) Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. New Eng J Med 334:1145–1149CrossRefPubMedGoogle Scholar
  41. 41.
    Levin G, Mokady S (1994) Antioxidant activity of 9-cis compared to all-trans beta-carotene in vitro. Free Rad Biol Med 17:77–82CrossRefPubMedGoogle Scholar
  42. 42.
    Ben-Amotz A, Levy Y (1996) Bioavailability of a natural isomer mixture compared with synthetic all-trans beta-carotene in human serum. Am J Clin Nutr 63:729–734PubMedGoogle Scholar
  43. 43.
    Schwartz J, Shklar G (1992) The selective cytotoxic effect of carotenoids and α-tocopherol on human cancer cell lines in vitro. J Oral Maxillofac Surg 50:367–373CrossRefPubMedGoogle Scholar
  44. 44.
    Kotake-Nara E, Asai A, Nagao A (2005) Neoxanthin and fucoxanthin induce apoptosis in PC-3 human prostate cancer cells. Cancer Lett 220:75–84CrossRefPubMedGoogle Scholar
  45. 45.
    Palozza P, Torelli C, Boninsegna A, Simone R, Catalano A, Mele MC, Picci N (2009) Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells. Cancer Lett 283:108–117CrossRefPubMedGoogle Scholar
  46. 46.
    Ganesan P, Noda K, Manabe Y, Ohkubo T, Tanaka Y, Maoka T, Sugawara T, Hirata T (2011) Siphonaxanthin, a marine carotenoid from green algae, effectively induces apoptosis in human leukemia (HL-60) cells. Biochim Biophy Acta (BBA) 1810:497–503CrossRefGoogle Scholar
  47. 47.
    Pasquet V, Morisset P, Ihammouine S, Chepied A, Aumailley L, Berard J-B, Serive B, Kaas R, Lanneluc I, Thiery V, Lafferriere M, Piot J-M, Patrice T, Cadoret J-P, Picot L (2011) Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts. Mar Drugs 9:819–831CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Gloria NF, Soares N, Brand C, Oliveira FL, Borojevic R, Teodoro AJ (2014) Lycopene and beta-carotene induce cell-cycle arrest and apoptosis in human breast cancer cell lines. Anticancer Res 34:1377–1386PubMedGoogle Scholar
  49. 49.
    Cui Y, Lu Z, Bai L, Shi Z, Zhao W, Zhao B (2007) β-carotene induces apoptosis and up-regulates peroxisome proliferator-activated receptor γ expression and reactive oxygen species production in MCF-7 cancer cells. Eur J Cancer 43:2590–2601CrossRefPubMedGoogle Scholar
  50. 50.
    Kohno M, Pouyssegur J (2006) Targeting the ERK signaling pathway in cancer therapy. Ann Med 38:200–211CrossRefPubMedGoogle Scholar
  51. 51.
    Liu P, Cheng H, Roberts TM, Zhao JJ (2009) Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev. Drug Disc 8:627–644CrossRefGoogle Scholar
  52. 52.
    Yu K, Toral-Barza L, Shi C, Zhang W-G, Zask A (2008) Response and determinants of cancer cell susceptibility to PI3K inhibitors: combined targeting of PI3K and Mek1 as an effective anticancer strategy. Cancer Biol Ther 7:307–315PubMedGoogle Scholar
  53. 53.
    Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241CrossRefPubMedGoogle Scholar
  54. 54.
    Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-xl and Bcl-2, displaces Bax and promotes cell death. Cell 80:285–291CrossRefPubMedGoogle Scholar
  55. 55.
    Staudt LM (2010) Oncogenic activation of NF-kappaB. Cold Spring Harbor Perspect Biol 2:a000109CrossRefGoogle Scholar
  56. 56.
    DiDonato JA, Mercurio F, Karin M (2012) NF-κB and the link between inflammation and cancer. Immunol Rev 246:379–400CrossRefPubMedGoogle Scholar
  57. 57.
    Nagendraprabhu P, Sudhandiran G (2011) Astaxanthin inhibits tumor invasion by decreasing extracellular matrix production and induces apoptosis in experimental rat colon carcinogenesis by modulating the expressions of ERK-2, NFkB and COX-2. Invest New Drugs 29:207–224CrossRefPubMedGoogle Scholar
  58. 58.
    Kavitha K, Kowshik J, Kishore TKK, Baba AB, Nagini S (2013) Astaxanthin inhibits NF-κB and Wnt/β-catenin signaling pathways via inactivation of Erk/MAPK and PI3 K/Akt to induce intrinsic apoptosis in a hamster model of oral cancer. Biochim Biophy Acta (BBA) 1830:4433–4444CrossRefGoogle Scholar
  59. 59.
    Li J, Dai W, Xia Y, Chen K, Li S, Liu T, Zhang R, Wang J, Lu W, Zhou Y, Yin Q, Abudumijiti H, Chen R, Zheng Y, Wang F, Lu J, Zhou Y, Guo C (2015) Astaxanthin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells via inhibition of Nf-Κb P65 and Wnt/Β-Catenin in vitro. Mar Drugs 13:6064–6081CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Martínez-Sánchez G, Giuliani A (2007) Cellular redox status regulates hypoxia inducible factor-1 activity. Role in tumour development. J Exp Clin Cancer Res: CR 26:39–50PubMedGoogle Scholar
  61. 61.
    Palozza P, Serini S, Di Nicuolo F, Piccioni E, Calviello G (2003) Prooxidant effects of β-carotene in cultured cells. Mol Aspects Med 24:353–362CrossRefPubMedGoogle Scholar
  62. 62.
    Chen EI, Hewel J, Krueger JS, Tiraby C, Weber MR, Kralli A, Becker K, Yates JR, Felding-Habermann B (2007) Adaptation of energy metabolism in breast cancer brain metastases. Cancer Res 67:1472–1486CrossRefPubMedGoogle Scholar
  63. 63.
    Sullivan R, Paré GC, Frederiksen LJ, Semenza GL, Graham CH (2008) Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity. Mol Cancer Ther 7:1961–1973CrossRefPubMedGoogle Scholar
  64. 64.
    Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature Rev. Drug Disc 8:579–591CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • G. Sowmya Shree
    • 1
  • K. Yogendra Prasad
    • 1
  • H. S. Arpitha
    • 1
  • U. R. Deepika
    • 1
  • K. Nawneet Kumar
    • 2
  • Priya Mondal
    • 1
  • P. Ganesan
    • 1
  1. 1.Department of Molecular NutritionCSIR-Central Food Technological Research Institute (CFTRI)MysoreIndia
  2. 2.Department of BiochemistryCSIR-Central Food Technological Research Institute (CFTRI)MysoreIndia

Personalised recommendations