Nimbolide inhibits invasion and migration, and down-regulates uPAR chemokine gene expression, in two breast cancer cell lines.
OBJECTIVE
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women, worldwide. Urokinase type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis of breast cancer. Nimbolide is a potent cytotoxic limnoid isolated from Azadirachta indica. Our previous studies have shown that nimbolide elicits pleiotropic effects on breast cancer cells; however, its roles in invasion and migration have not previously been fully elucidated.


METHODS
Protein expression of pEGFR, VEGFR, NFκB, IKKα, IKKβ, MMP-2, MMP-9 and TIMP-2 were analysed by western blotting. We also analysed expressions of uPA, uPAR genes and chemokines by real-time PCR. Breast cancer cell invasion was assessed by transwell invasion assay and cell migration analysed by scratch wound healing assay.


RESULTS
Our results showed that reduced protein expression of pEGFR, VEGFR, NFκB, IKKα, β, MMP-2, MMP-9 and TIMP-2 was higher in nimbolide-treated breast cancer cells. mRNA expression of uPA, uPAR, chemokines and their receptors were also significantly reduced in response to nimbolide treatment. Nimbolide inhibited breast cancer cell migration and invasion as shown in transwell invasion and wound healing assays.


CONCLUSIONS
These results clearly proved inhibitory effects of nimbolide on tumour cell invasion and migration by down-regulating proteins critically involved in regulation of cell invasion and metastasis, suggesting a possible therapeutic role of nimbolide for breast cancer.
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, 600113, India.
P
P
Elumalai
A
A
Brindha Mercy
R
R
Arunkamar
G
G
Sharmila
F A
FA
Bhat
S
S
Balakrishnan
P
P
Raja Singh
J
J
Arunakaran
D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D005260:Female; D006801:Humans; D036701:Limonins / Q000633:toxicity*; D061986:MCF-7 Cells; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism
D018925:Chemokines; D036701:Limonins; D012333:RNA, Messenger; D055293:Receptors, Urokinase Plasminogen Activator; D019716:Tissue Inhibitor of Metalloproteinase-2; C042198:nimbolide; D014568:Urokinase-Type Plasminogen Activator; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9