Nutr Res Pract.  2022 Apr;16(2):161-172. 10.4162/nrp.2022.16.2.161.

β-carotene regulates cancer stemness in colon cancer in vivo and in vitro

• Lee KE
• Kwon M
• Kim YS
• Kim Y
• Chung MG
• Heo SC
• Kim Y

• KMID: 2527808
• DOI: http://doi.org/10.4162/nrp.2022.16.2.161

Abstract

BACKGROUND/OBJECTIVES
Colorectal cancer (CRC) is the third most common cancer worldwide and has a high recurrence rate, which is associated with cancer stem cells (CSCs). β-carotene (BC) possesses antioxidant activity and several anticancer mechanisms. However, no investigation has examined its effect on colon cancer stemness.
MATERIALS/METHODS
CD133 + CD44 + HCT116 and CD133⁺ CD44⁺ HT-29 cells were isolated and analyzed their self-renewal capacity by clonogenic and sphere formation assays. Expressions of several CSCs markers and Wnt/β-catenin signaling were examined. In addition, CD133⁺ CD44⁺ HCT116 cells were subcutaneously injected in xenograft mice and analyzed the effect of BC on tumor formation, tumor volume, and CSCs markers in tumors.
RESULTS
BC inhibited self-renewal capacity and CSC markers, including CD44, CD133, ALDH1A1, NOTCH1, Sox2, and β-catenin in vitro. The effects of BC on CSC markers were confirmed in primary cells isolated from human CRC tumors. BC supplementation decreased the number and size of tumors and delayed the tumor-onset time in xenograft mice injected with CD133⁺ CD44⁺ HCT116 cells. The inhibitory effect of BC on CSC markers and the Wnt/β-catenin signaling pathway in tumors was confirmed in vivo as well.
CONCLUSIONS
These results suggest that BC may be a potential therapeutic agent for colon cancer by targeting colon CSCs.

Keyword

Beta carotene; colorectal cancer; cancer stem cells

Figure

• 

  Fig. 1 BC suppressed self-renewal capacity of colon cancer stem cells by inhibiting clonogenicity and sphere formation. CD133+CD44+ HCT116 and CD133+CD44+ HT-29 cells were treated with BC (0, 20, and 40 μM) for 8–14 days. Clonogenic assay (A-C) and sphere formation assay (D-F) were performed. (A, D) Representative images (magnification, 100 ×) and number of colonies or spheres are shown in (B, E) CD133+CD44+ HCT116 and (C, F) CD133+CD44+ HT-29 cells.Ctrl, control group; BC, β-carotene.*,†,‡The labels on the bar graph indicate the values that significantly differ from each other (P < 0.05) by one-way analysis of variance for multiple comparisons.

• 

  Fig. 2 BC suppressed mRNA expression of colon CSC markers in human colon CSCs and primary cells from CRC patients' tissues. After BC treatment (0, 20, and 40 μM) for 6 or 8 days, mRNA levels of colon CSC markers, CD44, CD133, ALDH1A1, and NOTCH1, were evaluated in (A) CD133+CD44+ HCT116 and (B) CD133+CD44+ HT-29 cells. (C) mRNA levels of colon CSC markers, CD44, CD133, and ALDH1A1, were analyzed in human primary cells from CRC patients by real-time polymerase chain reaction. GAPDH was used as a loading control.CSC, cancer stem cell; Ctrl, control group; BC, β-carotene; CRC, colorectal cancer.*,†,‡The labels on the bar graph indicate the values that significantly differed from each other (P < 0.05) according to one-way analysis of variance for multiple comparisons.

• 

  Fig. 3 BC suppressed protein expression of CSC markers and the Wnt/β-catenin signaling pathway in human colon CSCs. After BC treatment (0, 20, and 40 μM) for 6 or 8 days, protein levels of CD44, CD133, Notch1, SOX2, and β-catenin in (A, B) CD133+CD44+ HCT116 and (C, D) CD133+CD44+ HT-29 cells were detected by western blot analysis. β-actin was used as a loading control.Ctrl, control group; BC, β-carotene; CSC, cancer stem cell.*,†The labels on the bar graph indicate the values that significantly differ from each other (P < 0.05) by one-way analysis of variance for multiple comparisons.

• 

  Fig. 4 BC supplementation suppressed tumor formation and expression of CSC markers in vivo. Balb/c nu/nu mice were pretreated with BC for 3 weeks and CD133+CD44+ HCT116 cells were injected subcutaneously for 7 weeks with BC feeding. (A) Tumor latency was measured. (B) mRNA expressions of CSC markers, CD44, CD133, ALDH1A1, and NOTCH1, were detected using real-time polymerase chain reaction. (C) Protein expressions of Notch1, SOX2, and β-catenin in tumors were detected by western blot. β-actin was used as a loading control.TC, mice were fed control diet (tumor control); BC, β-carotene; BC 6, tumor injection + BC at 6 mg/kg BW; BC 12, tumor injection + BC at 12 mg/kg BW.*,†The labels on the bar graph indicate the values that significantly differed from each other (P < 0.05) by one-way analysis of variance for multiple comparisons.

Reference

1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68:394–424. PMID: 30207593.

2. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM. Cancer stem cells--perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res. 2006; 66:9339–9344. PMID: 16990346.

3. Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev Biol. 2007; 23:675–699. PMID: 17645413.

4. Cherciu I, Bărbălan A, Pirici D, Mărgăritescu C, Săftoiu A. Stem cells, colorectal cancer and cancer stem cell markers correlations. Curr Health Sci J. 2014; 40:153–161. PMID: 25729599.

5. Sikandar SS, Pate KT, Anderson S, Dizon D, Edwards RA, Waterman ML, Lipkin SM. NOTCH signaling is required for formation and self-renewal of tumor-initiating cells and for repression of secretory cell differentiation in colon cancer. Cancer Res. 2010; 70:1469–1478. PMID: 20145124.

6. Lundberg IV, Edin S, Eklöf V, Öberg Å, Palmqvist R, Wikberg ML. SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer. BMC Cancer. 2016; 16:471. PMID: 27411517.

7. Roy S, Majumdar AP. Signaling in colon cancer stem cells. J Mol Signal. 2012; 7:11. PMID: 22866952.

8. Brabletz S, Schmalhofer O, Brabletz T. Gastrointestinal stem cells in development and cancer. J Pathol. 2009; 217:307–317. PMID: 19031475.

9. Comstock GW, Alberg AJ, Huang HY, Wu K, Burke AE, Hoffman SC, Norkus EP, Gross M, Cutler RG, Morris JS, et al. The risk of developing lung cancer associated with antioxidants in the blood: ascorbic acids, carotenoids, alpha-tocopherol, selenium, and total peroxyl radical absorbing capacity. Am J Epidemiol. 2008; 168:831–840. PMID: 18820277.

10. Lin J, Cook NR, Albert C, Zaharris E, Gaziano JM, Van Denburgh M, Buring JE, Manson JE. Vitamins C and E and beta carotene supplementation and cancer risk: a randomized controlled trial. J Natl Cancer Inst. 2009; 101:14–23. PMID: 19116389.

11. Temple NJ, Basu TK. Protective effect of beta-carotene against colon tumors in mice. J Natl Cancer Inst. 1987; 78:1211–1214. PMID: 3473257.

12. Pham DN, Leclerc D, Lévesque N, Deng L, Rozen R. β,β-carotene 15,15′-monooxygenase and its substrate β-carotene modulate migration and invasion in colorectal carcinoma cells. Am J Clin Nutr. 2013; 98:413–422. PMID: 23803888.

13. Lim JY, Kim YS, Kim KM, Min SJ, Kim Y. B-carotene inhibits neuroblastoma tumorigenesis by regulating cell differentiation and cancer cell stemness. Biochem Biophys Res Commun. 2014; 450:1475–1480. PMID: 25019987.

14. Lee HA, Park S, Kim Y. Effect of β-carotene on cancer cell stemness and differentiation in SK-N-BE(2)C neuroblastoma cells. Oncol Rep. 2013; 30:1869–1877. PMID: 23900747.

15. Min SJ, Lim JY, Kim HR, Kim SJ, Kim Y. Sasa quelpaertensis leaf extract inhibits colon cancer by regulating cancer cell stemness in vitro and in vivo . Int J Mol Sci. 2015; 16:9976–9997. PMID: 25941936.

16. Kim Y, Lin Q, Zelterman D, Yun Z. Hypoxia-regulated delta-like 1 homologue enhances cancer cell stemness and tumorigenicity. Cancer Res. 2009; 69:9271–9280. PMID: 19934310.

17. Kim YS, Gong X, Rubin LP, Choi SW, Kim Y. β-Carotene 15,15′-oxygenase inhibits cancer cell stemness and metastasis by regulating differentiation-related miRNAs in human neuroblastoma. J Nutr Biochem. 2019; 69:31–43. PMID: 31048207.

18. Kim E, Shin JH, Seok PR, Kim MS, Yoo SH, Kim Y. Phyllodulcin, a natural functional sweetener, improves diabetic metabolic changes by regulating hepatic lipogenesis, inflammation, oxidative stress, fibrosis, and gluconeogenesis in db/db mice. J Funct Foods. 2018; 42:1–11.

19. Puck TT, Marcus PI. Action of X-rays on mammalian cells. J Exp Med. 1956; 103:653–666. PMID: 13319584.

20. Kim D, Kim Y, Kim Y. Effects of β-carotene on expression of selected microRNAs, histone acetylation, and DNA methylation in colon cancer stem cells. J Cancer Prev. 2019; 24:224–232. PMID: 31950022.

21. O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007; 445:106–110. PMID: 17122772.

22. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007; 445:111–115. PMID: 17122771.

23. Ong CW, Kim LG, Kong HH, Low LY, Iacopetta B, Soong R, Salto-Tellez M. CD133 expression predicts for non-response to chemotherapy in colorectal cancer. Mod Pathol. 2010; 23:450–457. PMID: 20081809.

24. Horst D, Scheel SK, Liebmann S, Neumann J, Maatz S, Kirchner T, Jung A. The cancer stem cell marker CD133 has high prognostic impact but unknown functional relevance for the metastasis of human colon cancer. J Pathol. 2009; 219:427–434. PMID: 19621338.

25. Afify A, Purnell P, Nguyen L. Role of CD44s and CD44v6 on human breast cancer cell adhesion, migration, and invasion. Exp Mol Pathol. 2009; 86:95–100. PMID: 19167378.

26. Ju SY, Chiou SH, Su Y. Maintenance of the stemness in CD44⁺ HCT-15 and HCT-116 human colon cancer cells requires miR-203 suppression. Stem Cell Res (Amst). 2014; 12:86–100.

27. Haraguchi N, Ohkuma M, Sakashita H, Matsuzaki S, Tanaka F, Mimori K, Kamohara Y, Inoue H, Mori M. CD133⁺CD44⁺ population efficiently enriches colon cancer initiating cells. Ann Surg Oncol. 2008; 15:2927–2933. PMID: 18663533.

28. Tomita H, Tanaka K, Tanaka T, Hara A. Aldehyde dehydrogenase 1A1 in stem cells and cancer. Oncotarget. 2016; 7:11018–11032. PMID: 26783961.

29. Kanwar SS, Yu Y, Nautiyal J, Patel BB, Majumdar AP. The Wnt/beta-catenin pathway regulates growth and maintenance of colonospheres. Mol Cancer. 2010; 9:212. PMID: 20691072.

30. Li Y, Zhang T, Korkaya H, Liu S, Lee HF, Newman B, Yu Y, Clouthier SG, Schwartz SJ, Wicha MS, et al. Sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells. Clin Cancer Res. 2010; 16:2580–2590. PMID: 20388854.

31. Ryu MJ, Cho M, Song JY, Yun YS, Choi IW, Kim DE, Park BS, Oh S. Natural derivatives of curcumin attenuate the Wnt/beta-catenin pathway through down-regulation of the transcriptional coactivator p300. Biochem Biophys Res Commun. 2008; 377:1304–1308. PMID: 19000900.

32. van Staveren WC, Solís DY, Hébrant A, Detours V, Dumont JE, Maenhaut C. Human cancer cell lines: experimental models for cancer cells in situ? For cancer stem cells? Biochim Biophys Acta. 2009; 1795:92–103. PMID: 19167460.

33. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008; 22:659–661. PMID: 17942826.

34. Mech-Nowak A, Swiderski A, Kruczek M, Luczak I, Kostecka-Gugała A. Content of carotenoids in roots of seventeen cultivars of Daucus carota L. Acta Biochim Pol. 2012; 59:139–141. PMID: 22428130.

35. Liu C, Wang XD, Bronson RT, Smith DE, Krinsky NI, Russell RM. Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets. Carcinogenesis. 2000; 21:2245–2253. PMID: 11133814.

36. Yang CM, Yen YT, Huang CS, Hu ML. Growth inhibitory efficacy of lycopene and β-carotene against androgen-independent prostate tumor cells xenografted in nude mice. Mol Nutr Food Res. 2011; 55:606–612. PMID: 21462328.

37. Männistö S, Yaun SS, Hunter DJ, Spiegelman D, Adami HO, Albanes D, van den Brandt PA, Buring JE, Cerhan JR, Colditz GA, et al. Dietary carotenoids and risk of colorectal cancer in a pooled analysis of 11 cohort studies. Am J Epidemiol. 2007; 165:246–255. PMID: 17158857.

38. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL Jr, Valanis B, Williams JH Jr, et al. Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial. J Natl Cancer Inst. 1996; 88:1550–1559. PMID: 8901853.

39. Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med. 1994; 330:1029–1035. PMID: 8127329.

40. Yan M, Liu Q. Differentiation therapy: a promising strategy for cancer treatment. Chin J Cancer. 2016; 35:3. PMID: 26739838.