Gopal C. Kundu

The Multifaceted Roles of Osteopontin in Cell Signaling, Tumor Progression and Angiogenesis

Osteopontin (OPN) is a chemokine like phosphorylated glycoprotein that plays important role in cancer progression. Extensive research from various laboratories has demonstrated the likely role of OPN in regulating the cell signaling that ultimately controls tumor growth and metastasis. Several earlier reports indicated that OPN is associated with various cancers; but its functional role in carcinogenesis is still not well defined. Besides the role of OPN in tumor biology, several studies have demonstrated the pathophysiological role of OPN in diverse biological events. This review will focus on recent advances in understanding the molecular mechanism by which OPN regulates a series of signaling cascades through activation of various kinases and transcription factors that ultimately control the expression of downstream effector genes, which contribute to tumor progression and angiogenesis in vitro and animal models. We will also provide evidences that suggest the enhanced expression of OPN is not only associated with several tumor types, but its level of expression is directly correlated to various stages of the clinical specimens of breast and prostate cancers. These studies may be useful for identifying novel OPN-based therapeutic approach for the treatment of cancer.

Activation of JAK2/STAT3 signaling by osteopontin promotes tumor growth in human breast cancer cells

Deregulation of signal transducer and activator of transcription (STAT)-3 signaling plays crucial role in oncogenesis of various cancers. However, the molecular mechanism by which osteopontin (OPN), a chemokine-like extracellular matrix-associated protein, regulates STAT3 activation that leads to tumor progression and inhibits apoptosis in breast cancer cells is not well understood. In this study, we for the first time report that OPN upregulates alphavbeta3 integrin-mediated Janus kinase 2 (JAK2) phosphorylation and STAT3 activation in breast cancer (MDA-MB-468 and MCF-7) cells. Pretreatment of cells with JAK2 inhibitor (AG 490) suppresses OPN-induced STAT3 phosphorylation, its nuclear localization and DNA binding indicating that JAK2 is involved in this process. Transfection of cells with wild-type (wt) STAT3 enhanced whereas mutant STAT3 (STAT3 Y705F) suppressed OPN-induced breast tumor cell migration. Treatment of cells with OPN followed by staurosporine (STS) showed that OPN protects the cells from STS-induced apoptosis. Moreover, transfection of cells with wt STAT3 upregulates whereas STAT3 Y705F downregulates Bcl2 and cyclin D1 expressions in response to OPN. Interestingly, STAT3-overexpressing cells when injected to non-obese diabetic/severe combined immunodeficiency mice followed by OPN treatment, the mice developed enhanced tumor growth as compared with STAT3 Y705F-injected mice or mice injected with OPN alone. The levels of Bcl2 and cyclin D1 in wt STAT3 tumors were significantly higher than controls. Clinical specimen analysis revealed that increased OPN and pSTAT3 expressions correlate with enhanced breast tumor progression. Thus, targeting OPN and its regulated STAT3 signaling could be a potent therapeutic approach and understanding these mechanisms may form the basis of new therapeutic regimen for the management of breast cancer.

Rapid efficient synthesis and characterization of silver, gold, and bimetallic nanoparticles from the medicinal plant Plumbago zeylanica and their application in biofilm control

Background Nanoparticles (NPs) have gained significance in medical fields due to their high surface-area-to-volume ratio. In this study, we synthesized NPs from a medicinally important plant – Plumbago zeylanica. Materials and methods Aqueous root extract of P. zeylanica (PZRE) was analyzed for the presence of flavonoids, sugars, and organic acids using high-performance thin-layer chromatography (HPTLC), gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), and biochemical methods. The silver NPs (AgNPs), gold NPs (AuNPs), and bimetallic NPs (AgAuNPs) were synthesized from root extract and characterized using ultraviolet-visible spectra, X-ray diffraction (XRD), energy-dispersive spectrometry (EDS), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The effects of these NPs on Acinetobacter baumannii, Staphylococcus aureus, and Escherichia coli biofilms were studied using quantitative biofilm inhibition and disruption assays, as well as using fluorescence, scanning electron microscopy, and atomic force microscopy. Results PZRE showed the presence of phenolics, such as plumbagin, and flavonoids, in addition to citric acid, sucrose, glucose, fructose, and starch, using HPTLC, GC-TOF-MS, and quantitative analysis. Bioreduction of silver nitrate (AgNO3) and chloroauric acid (HAuCl4) were confirmed at absorbances of 440 nm (AgNPs), 570 nm (AuNPs), and 540 nm (AgAuNPs), respectively. The maximum rate of synthesis at 50°C was achieved with 5 mM AgNO3 within 4.5 hours for AgNPs; and with 0.7 mM HAuCl4 within 5 hours for AuNPs. The synthesis of AgAuNPs, which completed within 90 minutes with 0.7 mM AgNO3 and HAuCl4, was found to be the fastest. Fourier-transform infrared spectroscopy confirmed bioreduction, while EDS and XRD patterns confirmed purity and the crystalline nature of the NPs, respectively. TEM micrographs and DLS showed about 60 nm monodispersed Ag nanospheres, 20–30 nm Au nanospheres adhering to form Au nanotriangles, and about 90 nm hexagonal blunt-ended AgAuNPs. These NPs also showed antimicrobial and antibiofilm activity against E. coli, A. baumannii, S. aureus, and a mixed culture of A. baumannii and S. aureus. AgNPs inhibited biofilm in the range of 96%–99% and AgAuNPs from 93% to 98% in single-culture biofilms. AuNPs also showed biofilm inhibition, with the highest of 98% in S. aureus. AgNPs also showed good biofilm disruption, with the highest of 88% in A. baumannii. Conclusion This is the first report on rapid and efficient synthesis of AgNPs, AuNPs and AgAuNPs from P. zeylanica and their effect on quantitative inhibition and disruption of bacterial biofilms.

Osteopontin as a therapeutic target for cancer

Introduction: Cancer is a complex pathological disorder, established as a result of accumulation of genetic and epigenetic changes, which lead to adverse alterations in the cellular phenotype. Tumor progression involves intricate signaling mediated through crosstalk between various growth factors, cytokines and chemokines. Osteopontin (OPN), a chemokine-like protein, is involved in promotion of neoplastic cancer into higher grade malignancies by regulating various facets of tumor progression such as cell proliferation, angiogenesis and metastasis. Areas covered: Tumors as well as stroma-derived OPN play key roles in various signaling pathways involved in tumor growth, angiogenesis and metastasis. OPN derived from tumor-activated macrophages modulates the tumor microenvironment and thereby regulate melanoma growth and angiogenesis. OPN also regulates hypoxia-inducible factor-1α-dependent VEGF expression leading to breast tumor growth and angiogenesis in response to hypoxia. Thus, a clear understanding of the molecular mechanism underlying OPN-mediated regulation will shed light on exciting avenues for further investigation of targeted therapies. Silencing of OPN using RNAi technology, blocking OPN activity using specific antibodies and small-molecule inhibitors might provide novel strategies, which would aid in developing effective therapeutics for the treatment of various types of cancer. Expert opinion: This review focuses on new possibilities to exploit OPN as a tumor and stroma-derived therapeutic target to combat cancer.

Osteopontin selectively regulates p70S6K/mTOR phosphorylation leading to NF-κB dependent AP-1-mediated ICAM-1 expression in breast cancer cells

BackgroundBreast cancer is one of the most frequently diagnosed cancer and accounts for over 400,000 deaths each year worldwide. It causes premature death in women, despite progress in early detection, treatment, and advances in understanding the molecular basis of the disease. Therefore, it is important to understand the in depth mechanism of tumor progression and develop new strategies for the treatment of breast cancer. Thus, this study is aimed at gaining an insight into the molecular mechanism by which osteopontin (OPN), a member of SIBLING (Small Integrin Binding LIgand N-linked Glycoprotein) family of protein regulates tumor progression through activation of various transcription factors and expression of their downstream effector gene(s) in breast cancer.ResultsIn this study, we report that purified native OPN induces ICAM-1 expression in breast cancer cells. The data revealed that OPN induces NF-κB activation and NF-κB dependent ICAM-1 expression. We also observed that OPN-induced NF-κB further controls AP-1 transactivation, suggesting that there is cross talk between NF-κB and AP-1 which is unidirectional towards AP-1 that in turn regulates ICAM-1 expression in these cells. We also delineated the role of mTOR and p70S6 kinase in OPN-induced ICAM-1 expression. The study suggests that inhibition of mTOR by rapamycin augments whereas overexpression of mTOR/p70S6 kinase inhibits OPN-induced ICAM-1 expression. Moreover, overexpression of mTOR inhibits OPN-induced NF-κB and AP-1-DNA binding and transcriptional activity. However, rapamycin further enhanced these OPN-induced effects. We also report that OPN induces p70S6 kinase phosphorylation at Thr-421/Ser-424, but not at Thr-389 or Ser-371 and mTOR phosphorylation at Ser-2448. Overexpression of mTOR has no effect in regulation of OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424. Inhibition of mTOR by rapamycin attenuates Ser-371 phosphorylation but does not have any effect on Thr-389 and Thr-421/Ser-424 phosphorylation of p70S6 kinase. However, OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424 is being controlled by MEK/ERK pathway.ConclusionThese results suggest that blocking of OPN-induced ICAM-1 expression through mTOR/p70S6 kinase signaling pathway may be an important therapeutic strategy for the treatment of breast cancer.

The crucial role of cyclooxygenase-2 in osteopontin-induced protein kinase C α/c-Src/IκB Kinase α/β-dependent prostate tumor progression and angiogenesis

The regulation of tumor progression towards its malignancy needs the interplay among several cytokines, growth factors, and enzymes, which are controlled in the tumor microenvironment. Here, we report that osteopontin, a small integrin-binding ligand N-linked glycoprotein family of calcified extracellular matrix–associated protein, regulates prostate tumor growth by regulating the expression of cyclooxygenase-2 (COX-2). We have shown that osteopontin stimulates the activation of protein kinase C α/nuclear factor–inducing kinase/nuclear factor-κB–dependent signaling cascades that induces COX-2 expression, which in turn regulates the prostaglandin E 2 production, matrix metalloproteinase-2 activation, and tumor progression and angiogenesis. We have revealed that suppression of osteopontin-induced COX-2 expression by the nonsteroidal anti-inflammatory drug celecoxib or blocking the EP2 receptor by its blocking antibody resulted in significant inhibition of cell motility and tumor growth and angiogenesis. The data also showed that osteopontin-induced mice PC-3 xenograft exhibits higher tumor load, increased tumor cell infiltration, nuclear polymorphism, and neovascularization. Interestingly, use of celecoxib or anti-EP2 blocking antibody drastically suppressed osteopontin-induced tumor growth that further indicated that suppression of COX-2 or its metabolites could significantly inhibit osteopontin-induced tumor growth. Human clinical prostate cancer specimen analysis also supports our in vitro and animal model studies. Our findings suggest that blockage of osteopontin and/or COX-2 is a promising therapeutic approach for the inhibition of prostate tumor progression and angiogenesis. (Cancer Res 2006; 66(13): 6638-48)

Osteopontin: a potentially important therapeutic target in cancer.

Introduction: Cancer is an extremely complex disease and most cancer treatments are limited to chemotherapy, radiation and surgery. The progression of tumours towards malignancy requires the interaction of various cytokines, growth factors, transcription factors and effector molecules. Osteopontin is a cytokine-like, calcium-binding, extracelular-matrix- associated member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family of proteins. It plays an important role in determining the oncogenic potential of various cancers. The role of osteopontin in various pathophysiological conditions suggests that the alteration in post-translational modification result in different functional forms that might change its normal physiological functions. Areas covered: Osteopontin -based anticancer therapy, which may provide a new insight for the effective management of cancer. Expert opinion: A better understanding of the signalling mechanism by which osteopontin promotes tumourigenesis may be useful in crafting novel osteopontin -based anticancer therapy. The role of osteopontin in promoting cancer progression is the subject of in depth investigation and thus targeting osteopontin might be a suitable therapeutic approach for the treatment of cancer.

Semaphorin 3A Suppresses Tumor Growth and Metastasis in Mice Melanoma Model

Background Recent understanding on cancer therapy indicated that targeting metastatic signature or angiogenic switch could be a promising and rational approach to combat cancer. Advancement in cancer research has demonstrated the potential role of various tumor suppressor proteins in inhibition of cancer progression. Current studies have shown that axonal sprouting inhibitor, semaphorin 3A (Sema 3A) acts as a potent suppressor of tumor angiogenesis in various cancer models. However, the function of Sema 3A in regulation of melanoma progression is not well studied, and yet to be the subject of intense investigation. Methodology/Principal Findings In this study, using multiple in vitro and in vivo approaches we have demonstrated that Sema 3A acts as a potent tumor suppressor in vitro and in vivo mice (C57BL/6) models. Mouse melanoma (B16F10) cells overexpressed with Sema 3A resulted in significant inhibition of cell motility, invasiveness and proliferation as well as suppression of in vivo tumor growth, angiogenesis and metastasis in mice models. Moreover, we have observed that Sema 3A overexpressed melanoma clone showed increased sensitivity towards curcumin and Dacarbazine, anti-cancer agents. Conclusions Our results demonstrate, at least in part, the functional approach underlying Sema 3A mediated inhibition of tumorigenesis and angiogenesis and a clear understanding of such a process may facilitate the development of novel therapeutic strategy for the treatment of cancer.

Osteopontin: an emerging therapeutic target for anticancer therapy

Distant migration of malignant cells or metastasis is considered one of the hallmarks of tumour progression and makes cancer a most deadly disease. The elevated expression of osteopontin (OPN), a metastasis-associated small integrin-binding ligand N-linked glycoprotein family member has been observed in several cancers and, thus, this protein is considered as a potent prognostic marker during tumour progression. OPN regulates a series of signalling cascades and augments the expression of several oncogenic molecules. Therefore, understanding the molecular mechanism and the signalling pathways by which OPN promotes tumorigenesis may be helpful in designing a novel anticancer therapy. At present, the role of OPN in regulating cancer progression is the subject of intense investigation and targeting OPN might be an appropriate therapeutic strategy for the treatment of cancer. This review is focused on OPN-based anticancer therapy, which may provide a new dimension for the successful treatment of cancer.

Hyaluronan-binding protein 1 (HABP1/p32/gC1qR) induces melanoma cell migration and tumor growth by NF-kappa B dependent MMP-2 activation through integrin αvβ3 interaction

Cell migration is the hallmark of cancer regulating anchorage independent growth and invasiveness of tumor cells. Hyaluronan (HA), an ECM polysaccharide is shown to regulate this process. In the present report, we demonstrated, supplementation of purified recombinant hyaluronan binding protein 1(HABP1/p32/gC1qR) from human fibroblast cDNA enhanced migration potential of highly invasive melanoma (B16F10) cells. Exogenous HABP1 adhered to the cell surface transiently and was shown to interact and colocalize with α(v)β(3) integrin, a regulatory molecule of cell migration. In HABP1 treated cells, the phosphorylation of nuclear factor inducing kinase (NIK) and IκBα was observed, followed by nuclear translocation of p65 subunit of NFκB, along with its DNA-binding and transactivation, resulting in upregulation of MT1-MMP expression and finally MMP-2 activation. To substantiate our findings, prior to HABP1 treatment, the expression of NIK was reduced by small interfering RNA mediated knockdown and confirmed the inhibition of nuclear translocation of p65 subunit of NFκB and upregulation of MT1-MMP expression. In addition, the use of curcumin, an anti-cancer drug, or GRGDSP, the blocking peptide along with exogenous HABP1, inhibited such NFκB-dependent pathway, confirming that HABP1-induced cell migration is α(v)β(3) integrin-mediated and downstream signaling by NFκB. Finally, we translated the in vitro data in mice model and observed enhanced tumor growth with higher MT1-MMP expression and MMP-2 activation in the tumors upon injection of HABP1 treated melanoma cells. The treatment of curcumin, the anticancer drug along with HABP1, inhibited the migration, expression of MT1-MMP and activation of MMP-2 and finally tumor growth supports the involvement of HABP1 in tumor formation.