Poly(d,l-lactide-co-glycolide) Surface-Anchored Biotin-Loaded Irinotecan Nanoparticles for Active Targeting of Colon Cancer.

A poly(d,l-lactide-co-glycolide) (PLGA) copolymer was synthesized using the ring-opening polymerization of d,l-lactide and glycolide monomers in the presence of zinc proline complex in bulk through the green route and was well characterized using attenuated total reflectance-Fourier transform infrared, 1H and 13C nuclear magnetic resonance, gel permeation chromatography, differential scanning calorimetry, X-ray diffraction, matrix-assisted laser desorption/ionization time-of-flight, etc. Furthermore, PLGA-conjugated biotin (PLGA-B) was synthesized using the synthesized PLGA and was employed to fabricate nanoparticles for irinotecan (Ir) delivery. These nanoparticles (PLGA-NP-Ir and PLGA-B-NP-Ir) were tested for physicochemical and biological characteristics. PLGA-B-NP-Ir exhibited a stronger cellular uptake and anticancer activity as compared to PLGA-NP-Ir in CT-26 cancer cells (log p < 0.05). The accumulation and retention of fluorescence-labeled nanoparticles were observed to be better in CT-26-inoculated solid tumors in Balb/c mice. The PLGA-B-NP-Ir-treated group inhibited tumor growth significantly more (log p < 0.001) than the untreated control, PLGA-NP-Ir, and Ir-treated groups. Furthermore, no body weight loss, hematological, and blood biochemical tests demonstrated the nanocarriers' nontoxic nature. This work presents the use of safe PLGA and the demonstration of a proof-of-concept of biotin surface attached PLGA nanoparticle-mediated active targeted Ir administration to combat colon cancer. To treat colon cancer, PLGA-B-NP-Ir performed better due to specific active tumor targeting and greater cellular uptake due to biotin.

Tumor-associated macrophage derived IL-6 enriches cancer stem cell population and promotes breast tumor progression via Stat-3 pathway.

BACKGROUND: Cancer stem cells (CSCs) play crucial role in tumor progression, drug resistance and relapse in various cancers. CSC niche is comprised of various stromal cell types including Tumor-associated macrophages (TAMs). Extrinsic ques derived from these cells help in maintenance of CSC phenotype. TAMs have versatile roles in tumor progression however their function in enrichment of CSC is poorly explored. METHODS: Mouse macrophages (RAW264.7) cells were activated by interaction with conditioned media (CM) of murine breast cancer cells (4T1) into TAMs and the effect of activated macrophage (TAM) derived factors was examined on enrichment of cancer stem cells (CSCs) and tumor growth using in vitro and in vivo models. RESULTS: In this study, we report that macrophages upon interaction with breast cancer cells activate tumor promoting function and exhibit differential expression of various proteins as shown by secretome analysis using proteomics studies. Based on secretome data, we found that Interleukin-6 (IL-6) is one of the up-regulated genes expressed in activated macrophages. Further, we confirm that TAMs produce high levels of IL-6 and breast cancer cell derived factors induce IL-6 production in activated macrophages via p38-MAPK pathway. Furthermore, we demonstrate that tumor activated macrophages induce enrichment of CSCs and expression of CSC specific transcription factors such as Sox-2, Oct-3/4 and Nanog in breast cancer cells. We further prove that TAM derived IL-6 plays a key role in TAM mediated CSC enrichment through activation of Signal transducer and activator of transcription 3 (STAT-3) signaling. TAM derived IL-6 influences breast cancer cell migration and angiogenesis. Moreover, our in vivo findings indicated that TAM derived IL-6 induces CSC population and resulting tumor growth in breast cancer. CONCLUSION: These finding provide evidence that TAM derived IL-6 plays a major role in CSC enrichment and tumor progression in breast cancer and IL-6 and its regulated signalling network may act as potential therapeutic target for management of breast cancer.

Tumor-derived osteopontin drives the resident fibroblast to myofibroblast differentiation through Twist1 to promote breast cancer progression.

Tumor-stroma interactions are important determinants for the disease course in cancer. While stromal influence has been known to often play a tumor-promoting role, incomplete mechanistic insight into this phenomenon has prevented its therapeutic targeting. Stromal fibroblasts can be activated by tumor cells to differentiate into cancer-associated fibroblasts (CAFs), that exhibit the traits of myofibroblasts, and in turn, they increase cancer aggressiveness. Here, we report the crosstalk between the cancer cells and stromal fibroblasts that leads to tumor progression. The process is initiated by secretion of a chemokine like protein, osteopontin (OPN) from the cancer cells that differentiates the fibroblasts to myofibroblasts. Tumor-derived OPN achieves this transition by engaging CD44 and αvß3 integrins on the fibroblast surface, which mediates signaling via Akt and ERK to induce Twist1-dependent gene expression. The OPN-driven CAFs then secrete CXCL12, which in turn triggers epithelial to mesenchymal transition (EMT) in the tumor cells. OPN, produced by the cancer cells, and CXCL12, secreted by activated fibroblasts, are necessary and sufficient to perpetuate the crosstalk. Knocking out OPN in carcinogen-induced mammary tumors or knocking down OPN in cancer cells and fibroblast co-implanted xenografts abrogates myofibroblast differentiation, Twist1, and CXCL12 expression. OPN expression is correlated with CAF-specific gene signature as shown by breast tumor tissue microarray consisting of 100 patient specimens. Bioinformatics analyses have confirmed that the expression of OPN is significantly correlated with the expression of myofibroblast-specific markers as demonstrated in human breast carcinoma dataset of 2509 patients. Our findings describe OPN and CXCL12 act as compelling targets to curb the tumor-promoting features of the stromal components and further suggested that OPN-regulated CXCL12 network might act as potential therapeutic target for the management of CAF-mediated breast cancer progression.

RGD functionalized chitosan nanoparticle mediated targeted delivery of raloxifene selectively suppresses angiogenesis and tumor growth in breast cancer.

Acidic pH is a crucial intrinsic property of the microenvironment of most solid tumors. Hence, the use of pH sensitive tumor targeting nanoparticles is an attractive approach to enhance the therapeutic efficacy of anti-cancer agents in solid tumors. Chitosan nanoparticles (CHNPs) have been widely explored in the area of cancer drug delivery; nevertheless their true potential as a pH responsive targeted drug delivery vehicle in cancer therapy has not been deciphered yet as most of the research is limited to pH dependent stability and drug release. In the present study, we investigate the direct effect of pH in synergy with RGD peptide based targeting on the therapeutic efficacy of chitosan nanoparticles (RGD-CHNPs) in breast cancer. Furthermore, for the first time we performed a comprehensive study showing the anti-tumor, anti-migratory and anti-angiogenic effect of raloxifene (Rlx) loaded CHNPs in breast cancer. We prepared stable formulations of raloxifene encapsulated CHNPs and RGD-CHNPs by the nontoxic ionic gelation method. pH dependent studies revealed that NPs possess higher stability and zeta potential along with enhanced cellular uptake at acidic pH (as present in solid tumors) compared to physiological pH. Furthermore, RGD conjugation enhanced the in vitro cellular uptake of CHNPs in αvß3 integrin expressing breast cancer cells and induced higher cellular apoptosis in breast cancer cells which was further augmented by lower pH. Moreover, Rlx-RGD-CHNPs significantly inhibited breast cancer cell migration and angiogenesis. In vivo studies showed that Cy5.5 conjugated RGD-CHNPs can distinctly visualize tumors and Rlx-RGD-CHNPs significantly inhibit breast tumor growth without causing any toxic effect to normal tissue as confirmed by hematology and blood biochemical studies. Therefore, RGD-CHNPs could potentially enhance the therapeutic efficacy of chemotherapeutic drugs due to the synergistic effect of pH responsiveness and tumor specific targeting in breast cancer.

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: 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.

Status of research on matrix metalloproteinases (MMPs) in India.

INTRODUCTION: MMPs are extracellular matrix (ECM)-degrading enzymes that play a crucial role in embryogenesis, tissue remodeling, inflammation and angiogenesis. MMP-2 and -9 (also called type 2 and type 4 collagenase, or gelatinase A and B) are the key molecules that control invasion, tumor growth and metastasis. Tissue inhibitor of matrix metalloproteinase (TIMP) -2 and -1 are specific inhibitors of MMP-2 and MMP-9 respectively, and play a crucial role in regulation of MMP-2 and -9 activation, during pathophysiological processes. MMPs can specifically degrade native gelatin, collagens, fibronectin, ectactin and elastin. MMP-2 and -9 are overexpressed in almost all types of cancers, and so act as important therapeutic targets. AREAS COVERED: The status of MMP research in India from 1998 to 2010. In this review, the authors cover the role of matrix metalloproteinase inhibitors in cancer therapy. EXPERT OPINION: As compared with other parts of the world, Indian scientists have not generated a significant number of specific MMP inhibitors for the treatment of cancer, or other diseases. MMPs and membrane type (MT)-MMPs are potentially important therapeutic targets in many diseases, including cancers, therefore, designing specific inhibitors from natural products or through synthetic routes, is crucial.

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.

Nuclear factor inducing kinase: a key regulator in osteopontin- induced MAPK/IkappaB kinase dependent NF-kappaB-mediated promatrix metalloproteinase-9 activation.

Osteopontin (OPN) is a secreted, non-collagenous, sialic-acid rich, glycosylated adhesive phospho- protein. Several highly metastatic transformed cells synthesized a higher level of OPN compared with non-tumorigenic cells. We have recently reported that OPN induces nuclear factor-kappaB (NF-kappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IKK signaling pathways. However, the molecular mechanism(s) by which OPN regulates pro-matrix metalloproteinase-9 (pro-MMP-9) activation and involvement of upstream kinases in regulation of these processes that ultimately control cell motility and tumor growth in murine melanoma cells are not well defined. Here we report that OPN induces alphavbeta3 integrin-mediated phosphorylation and activation of nuclear factor inducing kinase (NIK) and enhances the interaction between phosphorylated NIK and IkappaBalpha kinase alpha/beta (IKKalpha/beta) in B16F10 cells. Moreover, NIK is involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induces NIK-dependent NF-kappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore, OPN enhances NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, and cell motility. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. Taken together, NIK acts as crucial regulator in OPN-induced MAPK/IKK-mediated NF-kappaB-dependent uPA secretion and MMP-9 activation thereby controlling melanoma cell motility and chemoinvasion.

Osteopontin: role in cell signaling and cancer progression.

Cell migration and degradation of the extracellular matrix (ECM) are crucial steps in tumor progression. Several matrix-degrading proteases, including matrix metalloproteases, are highly regulated by growth factors, cytokines and ECM proteins. Osteopontin (OPN), a chemokine-like, calcified ECM-associated protein, plays a crucial role in determining the metastatic potential of various cancers. Since its first identification in bone, the multifaceted roles of OPN have been an area of intense investigation. Extensive research has elucidated the pivotal role of OPN in regulating the cell signaling that controls tumor progression and metastasis. This review focuses on recent advances in understanding the functional role of the OPN-induced signaling pathway in the regulation of cell migration and tumor progression and the implications for identifying novel targets for cancer therapy.

Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression.

Cancer progression depends on an accumulation of metastasis supporting cell signaling molecules that target signal transduction pathways and ultimately gene expression. Osteopontin (OPN) is one such chemokine like metastasis gene which plays a key signaling event in regulating the oncogenic potential of various cancers by controlling cell motility, invasiveness and tumor growth. We have reported that OPN stimulates tumor growth and nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkappaBalpha/IKK (IkappaBalpha kinase) signaling pathway in melanoma cells. Urokinase type plasminogen activator (uPA), a widely acting serine protease degrades the ECM components and plays a pivotal role in cancer progression. However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and uPA secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt/PKB (protein kinase B) in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with dominant negative mutant of p85 domain of PI 3'-kinase (Deltap85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECM-invasion. Furthermore, cells transfected with Deltap85 or super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkappaB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN induced PI 3'-kinase dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells.

JNK1 differentially regulates osteopontin-induced nuclear factor-inducing kinase/MEKK1-dependent activating protein-1-mediated promatrix metalloproteinase-9 activation.

We have recently demonstrated that nuclear factor-inducing kinase (NIK) plays a crucial role in osteopontin (OPN)-induced mitogen-activated protein kinase/I kappa B alpha kinase-dependent nuclear factor kappa B (NF kappa B)-mediated promatrix metalloproteinase-9 activation (Rangaswami, H., Bulbule, A., and Kundu, G. C. (2004) J. Biol. Chem. 279, 38921-38935). However, the molecular mechanism(s) by which OPN regulates NIK/MEKK1-dependent activating protein-1 (AP-1)-mediated promatrix metalloproteinase-9 activation and whether JNK1 plays any role in regulating both these pathways that control the cell motility are not well defined. Here we report that OPN induces alpha v beta3 integrin-mediated MEKK1 phosphorylation and MEKK1-dependent JNK1 phosphorylation and activation. Overexpression of NIK enhances OPN-induced c-Jun expression, whereas overexpressed NIK had no role in OPN-induced JNK1 phosphorylation and activation. Sustained activation of JNK1 by overexpression of wild type but not kinase negative MEKK1 resulted in suppression of ERK1/2 activation. But this did not affect the OPN-induced NIK-dependent ERK1/2 activation. OPN stimulated both NIK and MEKK1-dependent c-Jun expression, leading to AP-1 activation, whereas NIK-dependent AP-1 activation is independent of JNK1. OPN also enhanced JNK1-dependent/independent AP-1-mediated urokinase type plasminogen activator (uPA) secretion, uPA-dependent promatrix metalloproteinase-9 (MMP-9) activation, cell motility, and invasion. OPN stimulates tumor growth, and the levels of c-Jun, AP-1, urokinase type plasminogen activator, and MMP-9 were higher in OPN-induced tumor compared with control. To our knowledge this is first report that OPN induces NIK/MEKK1-mediated JNK1-dependent/independent AP-1-mediated pro-MMP-9 activation and regulates the negative crosstalk between NIK/ERK1/2 and MEKK1/JNK1 pathways that ultimately controls the cell motility, invasiveness, and tumor growth.

Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation.

We have recently demonstrated that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IkappaBalpha kinase (IKK) signaling pathways. However, the molecular mechanism(s) by which OPN regulates promatrix metalloproteinase-9 (pro-MMP-9) activation, MMP-9-dependent cell motility, and tumor growth and the involvement of upstream kinases in regulation of these processes in murine melanoma cells are not well defined. Here we report that OPN induced alpha(v)beta(3) integrin-mediated phosphorylation and activation of nuclear factor-inducing kinase (NIK) and enhanced the interaction between phosphorylated NIK and IKKalpha/beta in B16F10 cells. Moreover, NIK was involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induced NIK-dependent NFkappaB activation through ERK/IKKalpha/beta-mediated pathways. Furthermore OPN enhanced NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, cell motility, and tumor growth. Wild type NIK, IKKalpha/beta, and ERK1/2 enhanced and kinase-negative NIK (mut NIK), dominant negative IKKalpha/beta (dn IKKalpha/beta), and dn ERK1/2 suppressed the OPN-induced NFkappaB activation, uPA secretion, pro-MMP-9 activation, cell motility, and chemoinvasion. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. The level of active MMP-9 in the OPN-induced tumor was higher compared with control. To our knowledge, this is the first report that NIK plays a crucial role in OPN-induced NFkappaB activation, uPA secretion, and pro-MMP-9 activation through MAPK/IKKalpha/beta-mediated pathways, and all of these ultimately control the cell motility, invasiveness, and tumor growth.

Matrix metalloproteinase-2: mechanism and regulation of NF-kappaB-mediated activation and its role in cell motility and ECM-invasion.

Matrix metalloproteinases belong to a family of enzymes that degrade the extracellular matrix (ECM) components and play an important role in tissue repair, tumor invasion, and metastasis. ECM proteins, cytokines, and certain other factors regulate MMP activity. OPN, an ECM protein, has been found to be overexpressed in various cancers, and it has been shown to correlate with the metastatic potential. Although such reports indicate that OPN plays an important role in the ability of tumor cells to survive and metastasize to secondary sites, the mechanism by which OPN regulates these processes is yet to be understood. In this study we report that native purified human OPN can induce cell migration and ECM invasion. Increased invasiveness and migration correlates with enhanced expression and activation of MMP-2. Our study provides evidence showing that OPN increases gelatinolytic activity by inducing MT1-MMP expression via activation of the NF-kappaB pathway. Suppression of MMP-2 by ASMMP-2 reduces the OPN-induced cell migration and ECM invasion. Curcumin blocks OPN-induced MT1-MMP expression and pro-MMP-2 activation. Curcumin, a known anti-inflammatory and anticarcinogenic compound, suppresses OPN-induced cell migration, invasion and induces apoptotic morphology in OPN-treated cells. The mechanism by which curcumin suppresses the OPN-induced effects has also been delineated. Curcumin inhibits MT1-MMP gene expression by blocking signals leading to IKK activation. This in turn inhibits IkappaBalpha phosphorylation and NF-kappaB activation.