Hyaluronic acid-based hydrogels as 3D matrices for in vitro tumor engineering

Recreating the tumor microenvironment in a bilayer, hyaluronic acid hydrogel construct for the growth of prostate cancer spheroids

Heparin-Binding Epidermal Growth Factor–Like Growth Factor as a Critical Mediator of Tissue Repair and Regeneration

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Cancer of the ovary is the leading cause of death for women with gynecologic malignancies. Over 90% of ovarian cancers derive from malignant transformation of the ovarian surface epithelium (OSE), from which cells disseminate into the peritoneal cavity, invade locally or spread via lymphatics, resulting in ascites formation and abdominal distension. Whereas early-stage ovarian cancer is curable in >90% of patients, it is asymptomatic, and most patients are diagnosed with advanced disease that carries a poor prognosis. Current tools for early detection of ovarian cancer are limited: neither ovarian palpation, transvaginal ultrasonography nor serum CA125 levels have sufficient sensitivity or specificity for general screening. Among candidate biomarkers for ovarian cancer are MUC1, claudin-3, or lysophosphatidic acid (LPA), which may be elevated in malignant ascites or even serum of ovarian cancer patients. Experimentally, LPA has been shown to stimulate mitogenic signaling cascades (Fig. 1), which may be mediated, at least in part, through metalloprotease-induced cleavage of EGF-like growth factors, notably heparin-binding epidermal growth factor–like growth factor (HB-EGF), that activate the epidermal growth factor receptor (EGFR) and downstream signaling pathways, a phenomenon termed transactivation (ref. 1; Fig. 2). The pathophysiologic role of LPA or EGF-like ligands and their receptors in ovarian carcinogenesis is poorly understood. In this issue of Clinical Cancer Research, Tanaka et al. have extended their previous studies on the expression and clinical significance of molecules involved in signaling through the LPA and EGFR axes (2–4) in ovarian cancer. Together, their findings suggest a pathophysiologic role for LPA-induced a disintegrin and metalloprotease-17 (ADAM-17/TACE)–mediated HB-EGF cleavage and EGFR transactivation in ovarian cancer, identifying HB-EGF as a prognostic marker and promising therapeutic target (5). This commentary will discuss the biology of both LPA and EGFR-associated signaling in ovarian physiology and cancer from the perspective of EGFR transactivation.LPA has emerged as an important intercellular signaling molecule implicated not only in physiologic processes such as brain development or angiogenesis but also in the pathophysiology of cancer promotion (6, 7). Being generated through hydrolysis of lysophosphatidyl choline by lysophospholipase D/autotaxin or via hydrolysis of phosphatidic acid by phospholipase A2 or A1, LPA is present and bioactive in numerous extracellular fluids, including serum, ascites or malignant effusions. Binding of LPA to one of at least four different heptahelical transmembrane G protein–coupled receptors (GPCR; LPA1/endothelial differentiation gene (Edg)2, LPA2/Edg4, LPA3/Edg7, and LPA4/GPR23/P2Y9) results in activation of at least three distinct G protein subfamilies (Gq, Gi, G12/13) and initiation of multiple signaling pathways, including Ras/Raf/mitogen-activated protein kinase, phosphoinositide-3-kinase/Akt, phospholipase C/protein kinase C, or RhoA small GTPase signaling (Fig. 1). Subsequent activation of cell surface metalloproteases such as members of the ADAM family may induce cleavage of EGF-like ligand precursors and autocrine or paracrine stimulation of the human EGFR (HER/erbB) family of transmembrane tyrosine kinases (Fig. 2). This transactivation process seems to involve multiple signaling pathways, including mitogen-activated protein kinase (p38 and p44/42), protein kinase C or c-Src, and may be amplified through additional mechanisms, such as LPA-induced production of interleukin-8. Moreover, there seems to exist a signal amplification loop through activation of protein kinase C, which up-regulates LPA production. Thus, LPA induces cancer cell proliferation, survival, drug resistance, invasion, opening of intercellular tight junctions and gap junction closure, cell migration or metastasis (Fig. 1). Membrane-bound lipid ectophosphatases of the LPP family rapidly inactivate LPA by converting it to monoacylglycerol. LPA signaling may further be attenuated through receptor desensitization and ligand-induced receptor internalization, although the fate of internalized LPA receptors—degradation versus recycling—remains to be elucidated (7). Recently, LPA has also been suggested as an intracellular messenger. Although glycerolipid synthesis at the endoplasmatic reticulum or the outer mitochondrial membrane continuously involves LPA production, the kinetics of further acylation to phosphatidic acid are high, and LPA accumulation seems to be negligible at these sites. More interestingly, LPA has been shown to bind the lipid-inducible transcription factor PPARγ and induce vascular remodeling, suggesting that LPA actions may in part be independent of classical LPA receptors (Fig. 2). However, it remains unclear how the charged phospholipid LPA may traverse the plasma membrane and translocate into the nucleus in vivo, or conversely if PPARγ is activated in an indirect manner.EGFR signaling is involved in the regulation of a myriad of biological and pathophysiologic processes including proliferation, differentiation, apoptosis, angiogenesis, or metastasis and is a convergence point for diverse signaling pathways (8). Overactivity of the receptor tyrosine kinase is considered a hallmark of malignancy and has been associated with poor prognosis for cancer patients. Ligands of the EGF superfamily are synthesized as transmembrane precursors, from which mature growth factors are released through metalloprotease-mediated cleavage of the ectodomain (9). These ligands bind to and activate one or more of the four human EGFRs designated ErbB-1 (EGFR), ErbB-2 (HER-2/neu), ErbB-3, and ErbB-4, resulting in receptor homo- and heterodimerization, a necessary prelude to receptor phosphorylation and activation of three major intracellular pathways: Ras/Raf/mitogen-activated protein kinase, phosphoinositide-3-kinase/Akt, and phospholipase C/protein kinase C. Additionally, multiple other signaling molecules are stimulated; these include c-Src, the STAT family of transcription factors as well as Rho, Rac and Cdc42 small GTPases (ref. 10; Fig. 2).The EGF-like ligand HB-EGF is expressed in a wide variety of cells and has been implicated in numerous physiologic or pathologic processes, including malignant tumor growth, where its expression has been correlated with survival in various cancers of epithelial origin. Although membrane-bound ligands can activate EGFR in adjacent cells and induce proliferation in a process called juxtacrine stimulation, metalloprotease-induced shedding seems to be critical for the majority of EGFR-related processes, including GPCR-mediated EGFR transactivation (11). Recent results confirm distinct roles for soluble versus membrane-associated HB-EGF. Whereas the membrane-anchored proform promotes cell interactions and decreases migration, soluble HB-EGF induces opposite effects, promoting a transformed phenotype in terms of proliferative rates, colony-forming ability, activation of the cyclin D1 promoter or induction of matrix metalloproteases (MMP). Likewise, LPA-induced ectodomain shedding of HB-EGF has been shown to promote tumor formation of ovarian cancer cells in nude mice (5). The regulated proteolytic cleavage of proHB-EGF yields at least two fragments: the amino-terminal soluble ligand for EGFR as well as the carboxyl-terminal cell-associated remnant, HB-EGF-c. The latter has recently been shown to translocate to the nucleus, where it interacts with the promyelocytic leukemia zinc finger (PLZF) transcriptional repressor, resulting in CRM1-dependent nuclear export and functional loss of PLZF. Thus, HB-EGF shedding results in dual intracellular signaling that is in part independent of EGFR activation (Fig. 2).Proteolytic processing of EGFR ligands and their receptors is a key regulatory switch in EGFR signaling (12). Enzymes implicated in HB-EGF shedding seem to be cell/tissue type-, localization- and/or stimulus-specific; they include MMP-3/stromelysin-1, MMP-7/matrilysin, and the ADAM family members ADAM-9/meltrin-γ, ADAM-10/kuzbanian, ADAM-12/meltrin-α, and ADAM-17/TACE. ADAMs may be activated through removal of their pro-domain by a furin-type pro-protein convertase or through autocatalysis in the trans-Golgi network; additionally, proteins interacting with the cytoplasmic tails of ADAMs may influence their activity. To date, bona fide enzymes implicated in LPA-induced HB-EGF cleavage are ADAM-10 and ADAM-17 (13, 14). Importantly, tissue inhibitors of metalloproteinases can counteract the catalytic activity of various ADAMs, and the balance between the level of active metalloproteases and tissue inhibitors of metalloproteinases may determine the net metalloprotease activity in a cellular microenvironment (15).Metalloprotease-dependent release of EGF-like ligands may transactivate the EGFR in response to multiple stimuli, including cytokines, osmotic stressors, phorbol 12-myristate 13-acetate or GPCR activation (1). Besides LPA, numerous other GPCR agonists have been implicated in this process (including angiotensin-II or insulin-like growth factor I), and a role for multiple metalloproteases (including ADAM-9, -10, -12, -15, -17, or MMP-9) and EGF-like ligands (predominantly HB-EGF but also transforming growth factor-α or amphiregulin) has been suggested (16). Multiple signaling pathways seem to control ligand shedding in response to diverse stimuli, including p38 and p44/42 mitogen-activated protein kinase, protein kinase C or c-Src. A major signaling route of LPA is Gi-mediated activation of Ras and the downstream Raf/mitogen-activated protein kinase cascade in a tyrosine kinase-dependent fashion (Fig. 1). The precise role of receptor tyrosine kinases in LPA receptor-mediated Ras activation, however, is controversial with recent evidence suggesting that these receptor types may independently control distinct signaling cascades leading to Ras activation (17). Indeed, EGFR downstream pathways overlap with prototypical LPA signal transduction, and cross-talk between these cascades may occur at various levels, which may or may not involve ligand-induced receptor transactivation (Fig. 2).LPA and LPA receptors. In the normal ovary, LPA is present and active in follicular fluid, suggesting its local synthesis and physiologic function. LPA had first been related to carcinomatous growth in the mid-1990s, when excess concentrations were found in ascites of ovarian cancer patients. Although the mechanism of LPA production in ovarian cancer has not been fully identified, both lysophospholipase D/autotaxin and phospholipase A2 seem to play a role. Autotaxin and its major substrate LPC are widely produced in the human body and are abundantly present in plasma. Both are also generated in excess by a variety of human cancers and have been found in the supernatant of transformed cells, including ovary-derived cells, most likely as part of secreted microvesicles. Thus, not surprisingly, mammalian serum contains high levels of bioactive LPA. Several studies have reported 10-fold increases of serum-LPA in ovarian cancer patients compared with healthy subjects or other cancer conditions, suggesting its use as a biomarker for ovarian cancer screening, early detection, or prognosis prediction. Discrepancies between these and opposite findings that fail to establish a correlation between LPA plasma levels and carcinomatous growth may be attributable in part to differences in sample handling and/or the method of detection employed. Whether accumulation of LPA in the tumor microenvironment constitutes the major mechanism through which autotaxin might promote tumor growth and angiogenesis or whether autotaxin might exert LPA-independent effects in vivo remains to be determined. LPA alone exerts similar trophic effects as total ascites fluid from ovarian cancer patients when added to tumor cells, inducing proliferation, survival, metastasis, or cisplatinum resistance. Conversely, expression of a lipid phosphatase that hydrolyzes LPA may reduce survival, growth and tumorigenesis of ovarian cancer cells (18). Interestingly, whereas normal OSE cells only express significant amounts of the LPA receptors LPA1/Edg2 and LPA4/GPR23, LPA2/Edg4 and LPA3/Edg7 are abnormally expressed in ovarian cancer cells, suggesting differential functions for these receptors—alone or in combination—in ovarian physiology and pathology. Recently, LPA has been shown to induce ectodomain shedding of HB-EGF essential for tumor formation of ovarian cancer cells in nude mice (5), underscoring the role of this lysophospholipid in ovarian carcinogenesis.Metalloproteases. ADAMs and MMPs are widely expressed in the human body and have been found up-regulated or decreased in various cancers; both metalloprotease families may be induced by LPA. Dynamic equilibrium between metalloproteases and their inhibitors (tissue inhibitors of metalloproteinases) plays an important role in ovarian physiology (follicular growth and ovulation), and ovarian cancer cells may exploit that proteolytic potential to promote degradation of extracellular matrix components and release membrane-anchored growth factors, their receptors or adhesion molecules. Metalloproteases may thus play a role in both, initiation or termination of mitogenic signaling (19). Interestingly, metalloproteinases may be up-regulated by expression of their substrates or related enzymes (e.g., overexpression of HB-EGF or MMP-7 may induce MMP-3; ref. 20). The concomitant up-regulation of ADAM-17 and its substrate HB-EGF in ovarian cancer observed by Nakano's group in this issue of Clinical Cancer Research (2) is intriguing and warrants further validation at a cell-biological level.EGF-like ligands and their receptors. Numerous tumors overexpress the EGFR, its heterodimeric partner ErbB-2 and/or its ligands, including up to 75% of ovarian cancers, and accumulating evidence underscores the importance of EGF-like ligands for malignant transformation of ovarian epithelial cells. The prognostic impact of EGFR expression in ovarian cancer, however, remains controversial. As for HB-EGF, previous studies have reported this ligand in the stroma but not in the surface epithelium of the normal ovary (21). The fact that Tanaka et al. did not detect HB-EGF in the normal ovary might be attributable to differences in antibody sensitivity and/or specificity. Nakano's group had previously reported increased HB-EGF concentrations in the peritoneal fluid from ovarian cancer patients (4). They now relate these findings to abnormal expression of HB-EGF in malignant OSE cells (mRNA/fluorescence in situ hybridization) that express ADAM-17 (2) and in the surrounding interstitial tissue (protein/immunohistochemistry), indicating that HB-EGF produced from malignant OSE cells is constitutively secreted into the microenvironment. Interestingly, the tetraspanin CD9 has previously been detected on OSE cells only, and association of proHB-EGF with CD9 has been shown to increase HB-EGF's juxtacrine activity and cytoprotective capacity in epithelial cell models. Thus, aberrant expression of HB-EGF in OSE cells might entail interactions with different cell surface proteins than those present in stromal cells, resulting in distinct biological functions. Increasing evidence suggests the organization of metalloproteases, ligand growth factors and/or their receptors into preformed complexes within membrane microdomains and shed membrane vesicles (22). Examples include the interaction of CD9 with HB-EGF and ADAM-10 in complexes that mediate EGFR transactivation by GPCRs (14) or the association of CD44 with HB-EGF, its sheddase MMP-7 and its receptor erbB4 on the surface of tumor cell lines (23). A detailed study of the colocalization of HB-EGF and distinct ADAM proteins, especially ADAM-10, in ovarian cancer tissue is therefore warranted to judge the physiologic relevance of the presumed interaction. Furthermore, important insights into the (patho-) physiology of both GPCR and EGFR signaling axes may be gained through studies of their polarized distribution and function in normal epithelia and during loss of polarity characteristic of malignant transformation.Drugability. The link between LPA and ovarian carcinogenesis has been mostly correlative, such as elevated levels of LPA in serum or ascites fluid and in vitro studies with exogenous LPA. The study by Tanaka et al. in this issue of Clinical Cancer Research complements their previous observations, now suggesting a functional role for LPA-induced, ADAM-17-mediated HB-EGF cleavage in ovarian cancer. This first laudable dispatch raises several questions: what is the role of other membrane-anchored or soluble metalloproteases (in particular ADAM-10, ADAM-15, or MMPs) in ovarian cancer? Will cell-permeable metalloprotease inhibitors be required to block metalloprotease action in preformed intracellular complexes with their substrates? Is loss of polarity and aberrant access of LPA to its receptors of importance? Will specific inhibitors of LPA and/or erbB receptors and/or other components of the GPCR/EGFR signaling axes (e.g., metalloproteases or HB-EGF) stand the test of clinical application? Thus far, metalloprotease- or erbB receptor-targeted agents have exhibited only limited activity in ovarian cancer patients. Now, in the light of functional redundancy and incomplete overlap of LPA and EGFR signaling, the additional exploration of LPA receptors as novel drug targets seems promising. What about other signaling events? Abundant crosstalk between diverse mitogenic pathways such as EGFR, insulin-like growth factor receptor or estrogen receptor signaling has been reported, and these interactions will have to be considered in our efforts to combat the disease. The stage is set for more mechanistic studies into the complex pathophysiology of ovarian cancer, which, hopefully, will advance to effective treatment strategies that are urgently needed in the clinics.We apologize to all colleagues, whose original work could not be cited due to strict space limitations. Interested readers are referred to the references indicated in the mentioned reviews. We thank Jeffrey L. Franklin for critical review of the manuscript.

Nanoconstructs, such as liposomes, polymeric micelles, gold nanoparticles, carbon nanomaterials and nanocrystalline quantum dots, have been widely investigated for diagnostic, bioimaging and therapeutic applications [1]. Nanoconstructs have been also extensively explored in the field of dermatology. For example, liposomes and polymeric nanoparticles loaded with drugs were applied as topical administration agents for the treatment of skin diseases such as psoriasis, dermatitis and skin cancer [2,3]. Titanium dioxide and zinc oxide (ZnO) nanoparticles have been used as sun-screen formulation for the protection of skin from UV by the scattering, absorption and reflection of UV [4]. Silver nanoparticles are commercially used as wound and burn dressing agents with antibacterial effects [5]. Quantum dots and ZnO nanoparticles were utilized as bioimaging agents for the diagnosis of skin diseases [6,7]. Moreover, gold nanoconstructs and carbon nanomaterials have been actively investigated as promising agents for the photothermal ablation therapy of skin cancers due to their high light-to-heat conversion capability [8,9]. For further applications of nanoconstructs in dermatology, we need efficient transdermal delivery carriers of the nanoconstructs. The transdermal delivery has several advantages over other administration routes, such as oral delivery and needle based injection. The advantages include noninvasive treatment, self-administration, improved patient compliance and avoidance of hepatic first-pass metabolism or digestion system [10]. Despite these benefits, the low skin permeability of nanoconstructs such as polymers, proteins, hydrophilic drugs and nanoparticles limited their wide applications to the transdermal delivery. To facilitate the transdermal delivery of nanoconstructs, additional treatments have been adopted using penetration enhancers, iontophoresis, ultrasound and microneedles [11]. However, these methods require physical perturbations to the skin tissue, causing skin damage in some cases [12]. A noninvasive molecular carrier for transdermal delivery would have compelling advantages. The understanding for the characteristics of skin layers can be a good starting point for the development of transdermal delivery carriers of nanoconstructs. Stratum corneum (SC), the outermost skin layer, is the main barrier composed of densely packed dead cells forming hydrophobic surfaces. For the penetration of hydrophobic SC, hydrophobic molecules have clear benefits over hydrophilic molecules. The hydrophobic molecules can infiltrate into densely packed lipid layers in SC. However, nanoconstructs are usually formulated to have hydrophilic surfaces enhancing the physiological stability in the biological conditions. This contradicting requirement of hydrophobicity and hydrophilicity for transdermal delivery should be reconciled to enhance the physiological stability and the skin permeability by lipid disruption in SC. Recently, hyaluronic acid (HA) has been investigated as a promising transdermal delivery carrier. HA is a naturally occurring linear polysaccharide composed of repeating units of d-glucuronic acid and N-acetylEnhancing the transdermal penetration of nanoconstructs: could hyaluronic acid be the key?

Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-Binding Epidermal Growth Factor–Like Growth Factor/Diphtheria Toxin Receptor Expression by Acute Myeloid Leukemia Cells

Atorvastatin (ATO) and other statins demonstrate anti-inflammatory, anti-proliferative, and pro-apoptotic activities that suggest possible utility in cancer chemoprevention. Bisphosphonates such as zoledronic acid (ZOL) are used clinically to manage bone metastases from prostate and other cancers, and also demonstrate anti-apoptotic and growth inhibitory effects on cancer cells. Should these agents demonstrate significant chemopreventive activity in preclinical cancer models, their desirable clinical safety profiles would make them high priority candidates for clinical evaluation as chemopreventive agents. The present studies were performed to characterize the effects of ATO and ZOL on the in vitro proliferation kinetics of parental LNCaP and PC3 human prostate cancer cells, and in sub-clones of LNCaP and PC3 cells that differ phenotypically from the parental cell lines. When administered as single agents, ATO and ZOL both inhibited the proliferation of parental LNCaP cells and LNCaP cell sub-clones in a dose-dependent (1 - 20 µM) and time-dependent manner. Administration of ATO and ZOL in combination at concentrations at which each agent was minimally active as a single agent resulted in synergistic antiproliferative activity in both LNCaP cells and LNCaP sub-clones. For example, at 5 µM, ATO and ZOL each inhibited LNCaP cell proliferation by 5 to 10%; combined administration of ATO + ZOL at 5 µM inhibited LNCaP cell proliferation by nearly 50%. PC3 cells and sub-clones were also sensitive to statin administration: exposure of parental PC3 cells to ATO (5 µM) for 6 days induced significant cell death. ZOL inhibited proliferation in both PC3 cells and PC3 sub-clone cells in a dose and time-dependent manner; enhanced antiproliferative activity was seen in cells exposed to ATO + ZOL in combination. Because the autophagosomal marker, LC3-II, is reported to be induced by ATO in PC3 cells; LC3-II expression was evaluated in parental LNCaP cells and in PC3 cells by immunoblotting. ATO consistently induced LC3-II expression in PC3 cells, but had minimal effect on LC3-II expression in LNCaP cells. ZOL did not induce LC3-II expression in either cell line. Exposure to ATO + ZOL in combination induced LC3-II expression in PC3 cells only. These data demonstrate synergistic antiproliferative effects of ATO + ZOL in both LNCaP prostate cancer cells and PC3 prostate cancer cells, as well as in sub-clones derived from both prostate cancer cell lines. Enhanced chemotherapeutic and/or chemopreventive efficacy may be achieved in prostate cancer cells by administration of drug combinations that act through different molecular mechanisms. (N01-CN-43303 from the NCI, DHHS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 613. doi:1538-7445.AM2012-613

BLADDER EPITHELIAL CELLS FROM PATIENTS WITH INTERSTITIAL CYSTITIS PRODUCE AN INHIBITOR OF HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH FACTOR PRODUCTION

Neuroblastoma is the most common cancer in infancy. Current therapies are only modestly effective; patients with high-risk disease have less than a 50% chance of survival. High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low Schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble proteins, including heparan sulfate proteoglycans (HSPGs), which act to promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent pro-differentiating factor in neuroblastoma. Using microarray analysis, HBEGF mRNA expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from high-stage disease having decreased stroma and HBEGF. Soluble HBEGF increased neuroblast differentiation and decreased proliferation, while HBEGF knockdown decreased neuroblast differentiation. In patient samples, HBEGF expression correlates with SOX10, a neural crest differentiation marker, and the cell cycle inhibitor, CDKN1A. Alternatively, HBEGF negatively correlates with ASCL1, a primitive neuroectodermal marker, and the cell cycle promoter CCND1. HSPGs, including TβRIII, GPC1, GPC3, and SDC3 further promote the differentiating effects of HBEGF treatment by forming a complex with the epidermal growth factor receptor (EGFR) via glycosaminoglycan modifications, leading to activation of the Erk1/2 pathway and upregulation of the inhibitor of DNA binding transcription factor, whose expression correlates with HBEGF in patient samples. Inhibition of EGFR with erlotinib, lapatinib, and gefitinib diminish HBEGF-induced differentiation. These data identify a novel member of the pro-differentiating secretome and support the use of heparan sulfate mimetics in neuroblastoma, while cautioning against the use of EGFR inhibitors for neuroblastoma treatment. Citation Format: Angela L. Gaviglio, Gerard C. Blobe. Heparin-binding epidermal growth factor-like growth factor is a pro-differentiating factor in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1182.

Prostate cancer is the most common solid malignancy in men, with 32,000 deaths annually. Piperine, a major alkaloid constituent of black pepper, has previously been reported to have anti-cancer activity in variety of cancer cell lines. The effect of piperine against prostate cancer is not currently known. Therefore, in this study, we investigated the anti-tumor mechanisms of piperine on androgen dependent and androgen independent prostate cancer cells. Here, we show that piperine inhibited the proliferation of LNCaP, PC-3, 22RV1 and DU-145 prostate cancer cells in a dose dependent manner. Furthermore, Annexin-V staining demonstrated that piperine treatment induced apoptosis in hormone dependent prostate cancer cells (LNCaP). Using global caspase activation assay, we show that piperine-induced apoptosis resulted in caspase activation in LNCaP and PC-3 cells. Further studies revealed that piperine treatment resulted in the activation of caspase-3 and cleavage of PARP-1 proteins in LNCaP, PC-3 and DU-145 prostate cancer cells. Piperine treatment also disrupted androgen receptor (AR) expression in LNCaP prostate cancer cells. Our evaluations further show that there is a significant reduction of Prostate Specific Antigen (PSA) levels following piperine treatment in LNCaP cells. NF-kB and STAT-3 transcription factors have previously been shown to play a role in angiogenesis and invasion of prostate cancer cells. Interestingly, treatment of LNCaP, PC-3 and DU-145 prostate cancer cells with piperine resulted in reduced expression of phosphorylated STAT-3 and Nuclear factor-κB (NF-kB) transcription factors. These results correlated with the results of Boyden chamber assay, wherein piperine treatment reduced the cell migration of LNCaP and PC-3 cells. Finally, we show that piperine treatment significantly reduced the androgen dependent and androgen independent tumor growth in nude mice model xenotransplanted with prostate cancer cells. Taken together, these results support further investigation of piperine as a potential therapeutic agent in the treatment of prostate cancer.

ABSTRACTBackgroundNecrotizing enterocolitis (NEC) is associated with increased neurodevelopmental impairment. Gut-brain interactions through the brainstem may be central to NEC-related microglia-driven neuroinflammation. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has intestinal protective properties and is a potential therapy for NEC. The aim of this study is to test the hypothesis that HB-EGF in pregnant rats reduces both NEC incidence and proinflammatory changes in the brainstem microglia of newborn rats.MethodsWe compared four experimental groups, HB-EGF+/NEC–, HB-EGF–/NEC–, HB-EGF+/NEC+ and HB-EGF–/NEC+, depending on whether HB-EGF was given prenatally, and whether the newborn rats underwent the NEC induction protocol. We stained brainstem microglia and performed fractal analyses to provide objective measures of morphological changes.ResultsNEC incidence was lower in the HB-EGF+/NEC+ group (n=64, p<0.005) than in the HB-EGF–/NEC+ group. Brainstem microglia of breastfed rats had a larger cell area, perimeter, roughness, and less circularity compared with smaller, denser, compact cells in the NEC+ pups (p<0.0001, n=320 cells). HB-EGF+/NEC+ and HB-EGF–/NEC+ pups had similar-appearing microglia.ConclusionsPrenatal HB-EGF treatment reduces NEC incidence in neonatal rats. NEC-related proinflammatory changes are seen in microglial cells present in crucial centers controlling the gut-brain pathway. HB-EGF has a growth-promoting effect on healthy microglia in the offspring but does not avert microglial activation in the brainstem of newborn rats with NEC.

Hormonal therapy of prostate cancer, by inhibiting androgen production and/or androgen function, is the treatment of choice for advanced prostate cancer. Although most patients respond initially, the effect is only temporary, and the tumor cells will resume proliferation in an androgen-deprived environment. The mechanism for androgen-independent proliferation of cancer cells is unclear. Hormonal therapy induces neuroendocrine differentiation of prostate cancer cells, which is hypothesized to contribute to tumor recurrence by a paracrine mechanism. We studied signal transduction pathways of neuroendocrine differentiation in LNCaP cells after androgen withdrawal, and we showed that both the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway and ERK are activated, but only the former is required for neuroendocrine differentiation. A constitutively active AKT promotes neuroendocrine differentiation and a dominant negative AKT inhibits it. Activation of AKT by IGF-1 leads to neuroendocrine differentiation, and neuroendocrine differentiation induced by epinephrine requires AKT activation. We also show that the AKT pathway is likely responsible for neuroendocrine differentiation in DU145, an androgen-independent prostate cancer cell line. Therefore, our study demonstrated a novel function of the AKT pathway in prostate cancer progression and identified potential targets that may be explored for the treatment of androgen-independent cancer.

The chemotherapeutic agent VP16 increases the stability of HB-EGF mRNA by a mechanism involving the 3′-UTR