Original Article

Decreased Inhibition of Proliferation and Induction of Apoptosis in Breast Cancer Cell Lines (T47D and MCF7) from Treatment with Conditioned Medium Derived from Hypoxia-Treated Wharton’s Jelly MSCs Compared with Normoxia-Treated MSCs

Abstract

Background: Mesenchymal stem cells (MSCs) are an appealing source of adult stem cells for cell therapy due to the high rate of proliferation, self-renewal capability, and applicable therapy. Wharton’s jelly (WJ), the main component of the umbilical cord extracellular matrix, comprises multipotent stem cells with a high proliferation rate and self-renewal capability and has anti-cancer properties. MSCs have been reported to secrete a variety of cytokines that have a cytotoxic effect in various cancers. Oxygen tension affects MSCs proliferation, cytokines level but no in surface markers expression, MSCs’ differentiation.

We explored the cytotoxic effect and inducing apoptosis of Wharton’s jelly derived mesenchymal stem cells (WJMSCs) secretions from normoxic WJMSCs (WJMSCs-norCM) (CM: conditioned medium) and hypoxic WJMSCs (WJMSCs-hypoCM) in breast cancer cell lines (T47D and MCF7).

Materials and Methods: Cytotoxic activity was determined using the MTS assay. RT-PCR was performed to measure the expression of apoptosis-inducing genes, specifically P53, BAX, and CASP9, and the antiapoptotic gene BCL-2.

Results: WJMSCs-norCM and WJMSCs-hypoCM were potent inhibitors of the proliferation in both cell lines. WJMSCs-norCM had more anticancer activity in T47D and MCF7. The IC50 value of WJMSCs-norCM on MCF7 was 42.34%, and on T47D was 42.36%. WJMSCs-norCM significantly induced the gene expression of apoptotic P53, BAX, and CASP9 and insignificantly decreased the antiapoptotic gene BCL-2 in both MCF7 and T47D cells. WJMSCs-CM has anticancer activity by inducing P53, BAX, and CASP9 apoptotic genes.

Conclusion: WJMSCs-norCM has more anticancer activity than WJMSCs-hypoCM.

1. Jemal A, Bary F, Center MM, et al. Global cancer statistics. CA Cancer J Clin. 2011; 61(2): 69–90.
2. Caplan AI. Why are MSCs therapeutic? new data:new insight. J Pathol. 2009; 217(2):318-24.
3. Hartmann I, Hollweck T, Haffner S, et al. Umbilical cord tissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypic and functional properties. J Immunol Methods. 2010; 363(1):80–9.
4. Batsali AK, Kastrinaki MC, Papadaki HA, et al. Mesenchymal stem cells derived from Wharton's Jelly of the umbilical cord: biological properties and emerging clinical applications. Curr Stem Cell Res Ther. 2013;8(2):144-55.
5. Fong CY, Chak LL, Biswas A, et al. Human Wharton’s jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells. Stem Cell Rev. 2011;7(1):1–16.
6. Can A, Karahuseyinoglu S. Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells. 2007;25(11):2886-95.
7. Troyer DL, Weiss ML. Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells. 2008;26(3):591-9.
8. Bongso A, Fong C-F, Gauthaman K. Taking Stem Cells to the Clinic: Major Challenges. J Cell Biochem. 2008;105(6):1352–60.
9. Widowati W, Wijaya L, Bachtiar I, et al. Effect of oxygen tension on proliferation and characteristics of wharton’s jelly-derivied mesenchymal stem cells. BGM.2014;6(1):43-8.
10. Ganta C, Chiyo D, Ayuzawa R, et al. Rat umbilical cord stem cells completely abolish rat mammary carcinomas with no evidence of metastasis or recurrence 100 days post-tumor cell inoculation. Cancer Res. 2009;69(5):1815–20.
11. Ayuzawa R, Doi C, Rachakatla RS, et al. Naive human umbilical cord matrix derived stem cells significantly attenuate growth of human breast cancer cells in vitro and in vivo. Cancer Lett. 2009; 280(1):31-7.
12. Yang C, Lei D, Ouyang W, et al. Conditioned media from human adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells efficiently induced the apoptosis and differentiation in human glioma cell lines in vitro. Biomed Res Int . 2014 ;2014: 109389
13. Ezashi T, Das P, Roberts RM. Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci USA. 2005;102(13):4783-8.
14. Gassmann M, Fandrey J, Bichet S, et al. Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells. Proc Natl Acad Sci USA. 1996; 93(7):2867-72.
15. Lee MJ, Kim JY, Suk K. Identification of the hypoxiainducible factor 1 alpha-responsive HGTD-P gene as a mediator in the mitochondrial apoptotic pathway. Mol Cell Biol. 2004; 24(9):3918-3927.
16. Hao P, Liang Z, Piao H, et al. Conditioned medium of human adipose-derived mesenchymal stem cells mediates protection in neurons following glutamate excitotoxicity by regulating energy metabolism and GAP-43 expression. Metab Brain Dis. 2014;29(1):193–205.
17. Widowati W, Wijaya L, Murti H, et al. Conditioned medium from normoxia (WJMSCs-norCM) and hypoxia-treated WJMSCs (WJMSCs-hypoCM) in inhibiting cancer cell proliferation. BGM .2015; 7(1):8-17.
18. Kim ES, Jeon HB, Lim H, et al. Conditioned media from human umbilical cord blood-derived mesenchymal stem cells inhibits melanogenesis by promoting proteasomal degradation of MITF. Plos One. 2015;10(5):e0128078.
19. Antonius AA, Widowati W, Wijaya L, et al. Human platelet lysate enhances the proliferation of Wharton’s jelly-derived mesenchymal stem cells. BGM. 2015;7(3):87-97.
20. Nekanti U, Dastidar S, Venugopal P, et al. Increased proliferation and analysis of differential gene expression in human Wharton’s jelly-derived mesenchymal stromal cells under hypoxia. Int J Biol Sci. 2010;6(5):499-512.
21. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells.Cytotherapy. 2006;8(4):315-7.
22. Zheng L, Zhang D, Chen X, et al. Antitumor activities of human placenta-derived mesenchymal stem cells expressing endostatin on ovarian cancer. Plos One. 2012;7(7):e39119.
23. Jun EK, Zhang Q, Yoon BS, et al. Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-β/SMAD2 and PI3K/Akt pathways. Int J Mol Sci. 2014;15(1):605-28.
24. Nakahara M, Okumura N, Kay EP, et al. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium. Plos One. 2013;8(7):e69009.
25. Widowati W, Mozef T, Risdian C, et al. Anticancer and free radical scavenging potency of Catharanthus roseus, Dendrophthoe petandra, Piper betle and Curcuma mangga extracts in breast cancer cell lines. Oxid Antioxid Med Sci. 2013;2(2):137-42.
26. Widowati W, Wijaya L, Wargasetia TL, et al. Antioxidant, anticancer, and apoptosis-inducing effects of Piper extracts in HeLa cells. J Exp Integr Med. 2013;3(3):225-30.
27. Afifah E, Mozef T, Sandra F, et al. Induction of matrix metalloproteinases in chondrocytes by Interleukin IL-1β as an osteoarthritis model. J Math Fund Sci. 2019; 51(2):103-11.
28. Widowati W, Jasaputra DK, Onggowidjaja D, et al. Effects of conditioned medium of co-culture IL-2 induced NK cells and human Wharton’s jelly mesenchymal stem cells (hWJMSCs) on apoptotic gene expression in a breast cancer cell line (MCF-7). J Math Fund Sci. 2019; 51(3):205-24.
29. Kusuma HSW, Widowati W, Gunanegara RF, et al. Effect of conditioned medium from IGF1-induced human Wharton’s jelly mesenchymal stem cells (IGF1-hWJMSCs-CM) on osteoarthritis. Avicenna J Med Biotechnol. 2020;12(3):172-8.
30. Simon MC, Keith B. The role of oxygen availability in embryonic development and stem cell function. Nat Rev Mol Cell Biol. 2008; 9(4):285-96.
31. Raheja LF, Genetos DC, Yellowley CE. The effect of oxygen tension on the long-term osteogenic differentiation and MMP/TIMP expression of human mesenchymal stem cells. Cell Tissues Organs. 2010;191(3):175-84.
32. Kim JW, Tchernyshyov I, Semenza GL, et al. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 2006;3(3):177-85.
33. Kalaszczynska I, Ferdyn K. Wharton's jelly derived mesenchymal stem cells: future of regenerative medicine? recent findings and clinical significance. Biomed Res Int. 2015;2015:430847.
34. Albersen M, Fandel T, Lin G, et al. Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury. J Sex Med. 2010;7(10):3331-40.
35. Widowati W, Widyastuti H, Murti H, et al. Interleukins and VEGF secretome of human wharton’s jelly mesenchymal stem cells-conditioned medium (hwjmscs-CM) in different passages and oxygen tensions. Biosci Res. 2017;14(4):776-87.
36. Qiao L, Xu Z, Zhao Z, et al. Supression of tumorigenesis by human mesenchymal stem cells in a hepatoma model. Cell Res. 2008;18(4):500-7.
37. Fong C, Gauthaman K, Cheyyatraivendran S, et al. Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo. J Cell Biochem. 2012;113(2):658-68.
38. Li L, Tian H, Chen Z, et al. Inhibition of lung cancer cell proliferation mediated by human mesenchymal stem cells. Acta Biochem Biophys Sin(Shanghai). 2011;43(2):143-8.
39. Chao KC, Yang HT, Chen MW. Human umbilical cord mesenchymal stem cells suppress breast cancer tumourigenesis through direct cell-cell contact and internalization. J Cell Mol Med. 2012;16(8):1803–15.
40. Gauthaman K, Yee F, Cheyyatraivendran S, et al. Human umbilical cord Wharton’s jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro. J Cell Biochem. 2012;113(6):2027-39.
41. Madrigal M, Rao KS, Riodan NH. A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med. 2014;12:260.
42. Matsuzuka T, Rachakatla RS, Doi C, et al. Human umbilical cord matrix-derived stem cells expressing interferon-beta gene significantly attenuate bronchioloalveolar carcinoma xenograft in SCID mice. Lung Cancer. 2010;70(1):28-36.
43. Lin H, Fong C, Biswas A, et al. Human Wharton's jelly stem cells, its conditioned medium and cell-free lysate inhibit the growth of human lymphoma cells. Stem Cell Rev Rep. 2014;10(4):573-86.
44. Fong C, Biswas A, Subramanian A, et al. Human keloid cell characterization and inhibitio of growth with human Wharton's jelly stem cell extracts. J Cell Biochem. 2014;115(5):826-38.
45. Nguyen DP, Li J, Tewari AK. Inflammation and prostate cancer: the role of interleukin 6 (IL-6). BJU Int. 2014;113(6):986–92.
46. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins BCL2 and A1 in vascular endothelial cells. J Biol Chem. 1998;273(21):13313–6.
47. Sa-nguanraksa D, O-charoenrat P. The Role of Vascular Endothelial Growth Factor A Polymorphisms in Breast Cancer. Int J Mol Sci. 2012;13(11):14845-64.
48. Tsai CC, Yew TL, Yang DC, et al. Benefits of hypoxic culture on bone marrow multipotent stromal cells. Am J Blood Res. 2012;2(3):148-59.
49. Pawitan JA. Prospect of stem cell conditioned medium in regenerative medicine. Biomed Res Int. 2014;2014:965849.
50. Wu S, Ju G, Du T, et al. Microvesicles derived from human umbilical cord Wharton's jelly mesenchymal stem cells attenuate badder tumor cell growth in vitro and in vivo. Plos One. 2013;8(4):e61366.
51. Shah K. Mesenchymal stem cells engineered for cancer therapy. Adv Drug Deliv Rev. 2012;64(8):739–48.
52. Crisostomo PR, Wang Y, Markel TA, et al. Human mesenchymal stem cells stimulated by TNF-α, LPS, or hypoxia produce growth factors by an NFκB- but not JNK-dependent mechanism. Am J Physiol Cell Physiol. 2008;294(3):C675–82.
53. Lee EY, Xia Y, Kim WS, et al. Hypoxia-enhanced wound-healing function of adipose-derived stem cells: Increase in stem cell proliferation and up-regulation of VEGF and bFGF. Wound Repair Regen. 2009;17(4):540–7.
54. Jun EK, Zhang Q, Yoon BS, et al. Hypoxic Conditioned Medium from Human Amniotic Fluid-Derived Mesenchymal Stem Cells Accelerates Skin Wound Healing through TGF-β/SMAD2 and PI3K/Akt Pathways. Int J Mol Sci. 2014;15(1):605-28.
55. Chen L, Xu Y, Zhao J, et al. Conditioned Medium from Hypoxic Bone Marrow-Derived Mesenchymal Stem Cells Enhances Wound Healing in Mice. Plos One. 2014;9(4):e96161.
56. Madrigal M, Rao KS, Riodran NH. A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med. 2014;12:260.
57. Pantschenko AG, Pushkar I, Anderson KH, et al. The interleukin-1 family of cytokines and receptors in human breast cancer: implications for tumor progression. Int J Oncol. 2003;23(2):269-84.
58. Miller LJ, Kurtzman SH, Anderson K, et al. Interleukin-1 family expression in human breast cancer: interleukin-1 receptor antagonist. Cancer Invest. 2000;18(4):293-302.
59. Lewis AM, Varghese S, Xu H, et al. Interleukin-1 and cancer progression: the emerging role of interleukin-1 receptor antagonist as a novel therapeutic agent in cancer treatment. J Transl Med. 2006;4:48.
60. Apte RN, Krelin Y, Song X, et al. Effects of micro-environment- and malignant cell-derived interleukin-1 in carcinogenesis, tumour invasiveness and tumour-host interactions. Eur J Cancer. 2006;42(6):751-9.
61. Waugh DJJ, Wilson C. The Interleukin-8 Pathway in Cancer. Clin Cancer Res. 2008;14(21):6735-41.
62. Singh JK, Simoes BM, Howell SJ, et al. Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells. Breast Cancer Res. 2013;15(4):210.
63. Pang X, Li K, Wei L, et al. IL-8 inhibits the apoptosis of MCF-7 human breast cancer cells by up-regulating BCL2 and down-regulating caspase-3. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015; 31(3):307-11.
64. Aggarwal I. Targeting the Interleukin-8 Signaling Pathway in Colorectal Cancer: A Mini-review. J Young Investigator. 2013;25(8):108-13.
65. Becker C, Fantini MC, Wirtz S, et al. IL-6 signaling promotes tumor growth in colorectal cancer. Cell Cycle. 2005;4(2):217-20.
66. Lebrun J-J. The Dual Role of TGF in Human Cancer: From Tumor Suppression to Cancer Metastasis. ISRN Mol Biol. 2012;2012:381428.
67. Lederle W, Depner S, Schnur S, et al. IL-6 promotes malignant growth of skin SCCs by regulating a network of autocrine and paracrine cytokines. Int J Cancer. 2011;128(12):2803–14.
68. Yadav A, Kumar B, Datta J, et al. Epithelial–Mesenchymal Transition via the JAK-STAT3-SNAIL Signaling Pathway. Mol Cancer Res. 2011;9(12);1658–67.
69. Heinrich PC, Behrmann I, Haan S, et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J. 2003;374(Pt1):1–20.
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IssueVol 15 No 2 (2021) QRcode
SectionOriginal Article(s)
Published2021-04-11
DOI https://doi.org/10.18502/ijhoscr.v15i2.6038
Keywords
Apoptosis; Breast cancer; Conditioned medium; Hypoxia; Wharton’s jelly-derived mesenchymal stem cells

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1.
Widowati W, Murti H, Widyastuti H, Laksmitawati D, Rizal R, Kusuma H, Sumitro S, Widodo M, Bachtiar I. Decreased Inhibition of Proliferation and Induction of Apoptosis in Breast Cancer Cell Lines (T47D and MCF7) from Treatment with Conditioned Medium Derived from Hypoxia-Treated Wharton’s Jelly MSCs Compared with Normoxia-Treated MSCs. Int J Hematol Oncol Stem Cell Res. 15(2):77-89.