title: A novel MMP-2 inhibitor 3-azidowithaferin A (3-azidoWA) abrogates cancer cell invasion and angiogenesis by modulating extracellular Par-4.
abstract: BACKGROUND
  Withaferin A, which is a naturally derived steroidal lactone, has been found to prevent angiogenesis and metastasis in diverse tumor models. It has also been recognized by different groups for prominent anti-carcinogenic roles. However, in spite of these studies on withanolides, their detailed anti-metastatic mechanism of action remained unknown. The current study has poised to address the machinery involved in invasion regulation by stable derivative of Withaferin A, 3-azido Withaferin A (3-azidoWA) in human cervical HeLa and prostate PC-3 cells.
  
  
  RESULTS
  Sub-toxic concentration of 3-azidowithaferin A (3-azido WA) inhibited cancer cell motility and invasion in wound healing and Boyden chamber invasion by suppressing MMP-2 activity in gelatin zymography and its expression has proved to be a major obstacle in chemo-sensitivity. We have uncovered a novel mechanism of 3-azidoWA induced extracellular pro-apoptotic candidate tumor suppressor Par-4 protein stimulation in conditioned media and also noticed a concomitant marked reduction in pAkt and pERK signaling by immunoblot analysis. Furthermore, our zymography results suggest 3-azidoWA induced MMP-2 inhibition was mediated through secretory Par-4. The inhibition of apoptosis by 3-azidoWA could not restore MMP-2 gelatinase activity. In addition to this, our in vivo animal experiments data showed 3-azidoWA abrogated neovascularisation in dose dependent manner in mouse Matrigel plug assay.
  
  
  CONCLUSIONS
  For this report, we found that 3-azidoWA suppressed motility and invasion of HeLa and PC-3 cells in MMP-2 dependent manner. Our in vitro result strongly suggests that sub-toxic doses of 3-azidoWA enhanced the secretion of extracellular Par-4 that abolished secretory MMP-2 expression and activity. Depletion of secretory Par-4 restored MMP-2 expression and invasion capability of HeLa and PC-3 cells. Further, our findings implied that 3-azidoWA attenuated internal phospho-ERK and phospho-Akt expression in a dose dependent manner might play a key role in inhibition of mouse angiogenesis by 3-azidoWA.
authors.affiliation: Molecular Signal Transduction Laboratory, Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Jammu Tawi, India.
authors.forename: Bilal
authors.initials: B
authors.lastname: Rah
authors.forename: Hina
authors.initials: H
authors.lastname: Amin
authors.forename: Khalid
authors.initials: K
authors.lastname: Yousuf
authors.forename: Sheema
authors.initials: S
authors.lastname: Khan
authors.forename: Gayatri
authors.initials: G
authors.lastname: Jamwal
authors.forename: Debaraj
authors.initials: D
authors.lastname: Mukherjee
authors.forename: Anindya
authors.initials: A
authors.lastname: Goswami
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001386:Azides / Q000138:chemical synthesis / Q000494:pharmacology*; D001681:Biological Assay; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D003094:Collagen; D004338:Drug Combinations; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007797:Laminin; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009361:Neoplasm Invasiveness / Q000517:prevention & control*; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010766:Phosphorylation; D011509:Proteoglycans; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D054358:Withanolides / Q000138:chemical synthesis / Q000494:pharmacology*
chemical_list: C577558:3-azidowithaferin A; D000970:Antineoplastic Agents; D001386:Azides; D004338:Drug Combinations; D007797:Laminin; D061965:Matrix Metalloproteinase Inhibitors; D011509:Proteoglycans; D018179:Receptors, Thrombin; D054358:Withanolides; C059485:matrigel; D003094:Collagen; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020778:Matrix Metalloproteinase 2; C112908:protease-activated receptor 4