Suppressive Effects of Plumbagin on Invasion and Migration of Breast Cancer Cells via the Inhibition of STAT3 Signaling and Down-regulation of Inflammatory Cytokine Expressions

Abstract

Objective: The aim of this study was to investigate the effects of plumbagin (PL), a naphthoquinone derived from the medicinal plant plumbago zeylanica, on the invasion and migration of human breast cancer cells. Methods: Human breast cancer MDA-MB-231SArfp cells were treated with different concentrations of plumbagin for 24 h. The effects of plumbagin on the migration and invasion were observed by a transwell method. The expressions of IL-1α, IL-1β, IL-6, IL-8, TGF-β, TNFα, MMP-2 and MMP-9 mRNA in MDA-MB-231SArfp cells were detected using Real-Time PCR. MDA-MB-231SArfp cells were treated with plumbagin at different concentrations for 45 minutes. The activation of STAT3 was detected by western blot. Following this analysis, STAT3 in MDA-MB-231SArfp cells was knocked out using specific siRNA. mRNA levels of IL-1α, TGF-β, MMP-2 and MMP-9 were then detected. Consequently, MDA-MB-231SArfp cells were injected intracardially into BALB/c nude mice to construct a breast cancer bone metastatic model. The mice were injected intraperitoneally with plumbagin. Non-invasive in vivo monitoring, X-ray imaging and histological staining were performed to investigate the effects of plumbagin on the invasion and migration of breast cancer cells in vivo. Results: The in vitro results showed that plumbagin could suppress the migration and invasion of breast cancer cells and down-regulate mRNA expressions of IL-1α, TGF-β, MMP-2 and MMP-9. Western blotting demonstrated that plumbagin inhibited the activation of STAT3 signaling in MDA-MB-231SArfp cells. The inactivation of STAT3 was found to have an inhibitory effect on the expressions of IL-1α, TGF-β, MMP-2 and MMP-9. In vivo studies showed that plumbagin inhibited the metastasis of breast cancer cells and decreased osteolytic bone metastases, as well as the secretion of MMP-2 and MMP-9 by tumor cells at metastatic lesions. Conclusions: Plumbagin can suppress the invasion and migration of breast cancer cells via the inhibition of STAT3 signaling and by downregulation of IL-1α, TGF-β, MMP-2 and MMP-9.

References

[1] Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol, 2006, 24: 2137-2150

[2] Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer, 2002, 2: 584-593

[3] Rose AA, Siegel PM. Breast cancer-derived factors facilitate osteolytic bone metastases. Bull Cancer, 2006, 93: 931-943

[4] Guise TA, Yin JJ, Taylor SD, Kumagai Y, Dallas M, Boyce BF, Yoneda T, Mundy GR. Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest, 1996, 98: 1544-1549

[5] Mundy GR. Mechanisms of bone metastases. Cancer, 1997, 80: 1546-1556

[6] Lipton A, Theriault RL, Hortobagyi GN, Simeone J, Knight RD, Mellars K, Reitsma DJ, Heffernan M, Seaman JJ. Pamidronate prevents skeletal complications and is effective palliative treatment in women with breast carcinoma and osteolytic bone metastases. Cancer, 2000, 88: 1082-1090

[7] Bamias A, Kastritis E, Bamia C, Moulopoulos LA, Melakopoulos I, Bozas G, Koutsoukou V, Gika D, Anagnostopoulos A, Papadimi-triou C, Terpos E, Dimopoulos MA. Osteonecrosis of the jaw in cancer after treatment with bisphosphonates: incidence and risk factors. J Clin Oncol, 2005, 23: 8580-8587

[8] Durie BG, Katz M, Crowley J. Osteonecrosis of the jaw and bisphosphonates. N Engl J Med, 2005, 353: 99-102

[9] Dimopoulos MA, Kastritis E, Anagnostopoulos A, Melakopoulos I, Gika D, Moulopoulos LA, Bamia C, Terpos E, Tsionos K, Bamias A. Osteonecrosis of the jaw in patients with multiple myeloma treated with bisphosphonates: evidence of increased risk after treatment with zoledronic acid. Haematologica, 2006, 91: 968-971

[10] Hoff AO, Toth BB, Altundag K, Johnson MM, Warneke CL, Hu M, Nooka A, Sayegh G, Guarneri V, Desrouleaux K, Cui J, Adamus A, Gagel RF, Hortobagyi GN. Frequency and risk factors associated with osteonecrosis of the jaw in cancer patients treated with intravenous bisphosphonates. J Bone Miner Res, 2008, 23: 826-836

[11] de Paiva SR, Figueiredo MR, Aragão TV, Kaplan MA. Antimicrobial activity in vitro of plumbagin isolated from Plumbago species. Mem Inst Oswaldo Cruz, 2003, 98: 959-961

[12] Checker R, Sharma D, Sandur SK, Khanam S, Poduval TB. Anti-inflammatory effects of plumbagin are mediated by inhibition of NF-kappaB activation in lymphocytes. Int Immunopharmacol, 2009, 9: 949-958

[13] Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther, 2006, 5: 3209-3221

[14] Aziz MH, Dreckschmidt NE, Verma AK. Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res, 2008, 68: 9024-9032

[15] Sand JM, Hafeez BB, Jamal MS, Witkowsky O, Siebers EM, Fischer J, Verma AK. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), isolated from Plumbago zeylanica, inhibits ultraviolet radiation-induced development of squamous cell carcinomas. Carcinogenesis, 2012, 33: 184-190

[16] Sung B, Oyajobi B, Aggarwal BB. Plumbagin inhibits osteoclastogenesis and reduces human breast cancer-induced osteolytic bone metastasis in mice through suppression of RANKL signaling. Mol Cancer Ther, 2012, 11: 350-359

[17] Manu KA, Shanmugam MK, Rajendran P, Li F, Ramachandran L, Hay HS, Kannaiyan R, Swamy SN, Vali S, Kapoor S, Ramesh B, Bist P, Koay ES, Lim LH, Ahn KS, Kumar AP, Sethi G. Plumbagin inhibits invasion and migration of breast and gastric cancer cells by downregulating the expression of chemokine receptor CXCR4. Mol Cancer, 2011, 10: 107

[18] Li Z, Xiao J, Wu X, Li W, Yang Z, Xie J, Xu L, Cai X, Lin Z, Guo W, Luo J, Liu M. Plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment. Curr Mol Med, 2012, 12: 967-981

[19] Yoneda T, Williams PJ, Hiraga T, Niewolna M, Nishimura R. A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res, 2001, 16: 1486-1495

[20] Price JT, Quinn JM, Sims NA, Vieusseux J, Waldeck K, Docherty SE, Myers D, Nakamura A, Waltham MC, Gillespie MT, Thompson EW. The heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastases of a human breast cancer cell line. Cancer Res, 2005, 21: 4929-4938

[21] Tu B, Du L, Fan QM, Tang Z, Tang TT. STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastases of osteosarcoma. Cancer Lett, 2012, 325: 80-88

[22] Apte RN, Dotan S, Elkabets M, White MR, Reich E, Carmi Y, Song X, Dvozkin T, Krelin Y, Voronov E. The involvement of IL-1 in tumorigenesis, tumor invasiveness, metastasis and tumor-host interactions. Cancer Metastasis Rev, 2006, 25: 387-408

[23] Guo S, Gonzalez-Perez RR. Notch, IL-1 and leptin crosstalk outcome (NILCO) is critical for leptin-induced proliferation, migration and VEGF/VEGFR-2 expression in breast cancer. PloS one, 2011, 6: e21467

[24] Baselga J, Rothenberg ML, Tabernero J, Seoane J, Daly T, Cleverly A, Berry B, Rhoades SK, Ray CA, Fill J. TGF-β signalling-related markers in cancer patients with bone metastases. Biomarkers, 2008, 13: 217-236

[25] Wright LE, Frye JB, Lukefahr AL, Timmermann BN, Mohammad KS, Guise TA, Funk JL. Curcuminoids block TGF-beta signaling in human breast cancer cells and limit osteolysis in a murine model of breast cancer bone metastasis. J Nat Prod, 2013, 76: 316-321

[26] Fagan-Solis KD, Schneider SS, Pentecost BT, Bentley BA, Otis CN, Gierthy JF, Arcaro KF. The RhoA pathway mediates MMP-2 and MMP-9-independent invasive behavior in a triple-negative breast cancer cell line. J Cell Biochem, 2013, 114: 1385-1394

[27] Zhao S, Ma W, Zhang M, Tang D, Shi Q, Xu S, Zhang X, Liu Y, Song Y, Liu L, Zhang Q. High expression of CD147 and MMP-9 is correlated with poor prognosis of triple-negative breast cancer (TNBC) patients. Med Oncol, 2013, 30: 335

[28] Germain D, Frank DA. Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res, 2007, 13: 5665-5669

[29] Fukuda A, Wang SC, Morris JP IVth, Folias AE, Liou A, Kim GE, Akira S, Boucher KM, Firpo MA, Mulvihill SJ, Hebrok M. Stat3 and MMP7 contribute to pancreatic ductal adenocarcinoma initiation and progression. Cancer Cell, 2011, 19: 441-455

[30] Darnell JE. Validating Stat3 in cancer therapy. Nat Med, 2005, 11: 595-596

[31] Ahmad A, Banerjee S, Wang Z, Kong D, Sarkar FH. Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2. J Cell Biochem, 2008, 105: 1461-1471