A Novel MMP-2 Inhibitor 3-azidowithaferin A (3- azidoWA) Abrogates Cancer Cell Invasion and Angiogenesis by Modulating Extracellular Par-4 Bilal Rah 1 , Hina Amin1, Khalid Yousuf2, Sheema Khan 1 , Gayatri Jamwal1, Debaraj Mukherjee2, Anindya Goswami1* 1 Molecular Signal Transduction Laboratory, Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Jammu Tawi, India, 2 Nautral Product Chemistry, Indian Institute of Integrative Medicine (CSIR), Jammu Tawi, India 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. Methods and Principal Findings: 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. Conclusion/Significance: 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. Citation: Rah B, Amin H, Yousuf K, Khan S, Jamwal G, et al. (2012) A Novel MMP-2 Inhibitor 3-azidowithaferin A (3-azidoWA) Abrogates Cancer Cell Invasion and Angiogenesis by Modulating Extracellular Par-4. PLoS ONE 7(9): e44039. doi:10.1371/journal.pone.0044039 Editor: Daotai Nie, Southern Illinois University School of Medicine, United States of America Received May 23, 2012; Accepted August 1, 2012; Published September 4, 2012 Copyright: 2012 Rah et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: agoswami@iiim.ac.in Introduction Extracellular secretory pathways are considered to play pivotal role in human physiology. Body’s vital hormones and growth factors are secreted and they control the development and differentiation of organs in normal physiological condition. Likewise, systemic (extracellular) proteins attribute major in vivo function during tissue growth and apoptosis [1]. Prostate apoptotic response 4 (Par-4) is ubiquitously expressed and evolutionary conserved pro-apoptotic protein whose expression was mainly correlated with the cells that undergo apoptosis due to exogenous insults [2]. Apart from its intracellular function, the new perspective of extracellular secretion in different cancer cells has augmented the therapeutic potential of Par-4 [3]. Recently, Burikhanov et al. have shown that mammalian cells in general caused secretion of Par-4. However, the apoptotic induction by extracellular Par-4 occurring via cell- surface GRP-78 was found to promote cell invasion and tumorigenesis [3]. The stabilization of pro-angiogenic GRP-78 by Par-4 has been designated an anti- invasive role of extracellular Par-4. Metastasis is a multi-step process involving cell migration and pericellular proteolysis of ECM that mediates cancer cells protrusion [4]. Matrix metalloproteinases (MMP’s) are responsible for the degradation of environmental barriers, such as the extracellular matrix and basement membrane [5,6]. Amongst the MMP family members, MMP-2 and -9 are generally considered to be the malignancy of various tumors as well as poor prognosis of many cancers [6]. Thus, MMP’s are capable of cleaving type IV basement membrane collagen (MMP-2 and -9) and add value for drug development. Compelling preclinical studies from diverse laboratories have provided overwhelming support for direct relationship between MMP-2 over expression PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44039