Orthogonal targeting of osteoclasts and myeloma cells for radionuclide stimulated dynamic therapy induces multidimensional cell death pathways.

Rationale: Multiple myeloma (MM) is a multifocal malignancy of bone marrow plasma cells, characterized by vicious cycles of remission and relapse that eventually culminate in death. The disease remains mostly incurable largely due to the complex interactions between the bone microenvironment (BME) and MM cells (MMC). In the "vicious cycle" of bone disease, abnormal activation of osteoclasts (OCs) by MMC causes severe osteolysis, promotes immune evasion, and stimulates the growth of MMC. Disrupting these cancer-stroma interactions would enhance treatment response. Methods: To disrupt this cycle, we orthogonally targeted nanomicelles (NM) loaded with non-therapeutic doses of a photosensitizer, titanocene (TC), to VLA-4 (α4ß1, CD49d/CD29) expressing MMC (MM1.S) and αvß3 (CD51/CD61) expressing OC. Concurrently, a non-lethal dose of a radiopharmaceutical, 18F-fluorodeoxyglucose ([18F]FDG) administered systemically interacted with TC (radionuclide stimulated therapy, RaST) to generate cytotoxic reactive oxygen species (ROS). The in vitro and in vivo effects of RaST were characterized in MM1.S cell line, as well as in xenograft and isograft MM animal models. Results: Our data revealed that RaST induced non-enzymatic hydroperoxidation of cellular lipids culminating in mitochondrial dysfunction, DNA fragmentation, and caspase-dependent apoptosis of MMC using VLA-4 avid TC-NMs. RaST upregulated the expression of BAX, Bcl-2, and p53, highlighting the induction of apoptosis via the BAK-independent pathway. The enhancement of multicopper oxidase enzyme F5 expression, which inhibits lipid hydroperoxidation and Fenton reaction, was not sufficient to overcome RaST-induced increase in the accumulation of irreversible function-perturbing α,ß-aldehydes that exerted significant and long-lasting damage to both DNA and proteins. In vivo, either VLA-4-TC-NM or αvß3-TC-NMs RaST induced a significant therapeutic effect on immunocompromised but not immunocompetent MM-bearing mouse models. Combined treatment with both VLA-4-TC-NM and αvß3-TC-NMs synergistically inhibited osteolysis, reduced tumor burden, and prevented rapid relapse in both in vivo models of MM. Conclusions: By targeting MM and bone cells simultaneously, combination RaST suppressed MM disease progression through a multi-prong action on the vicious cycle of bone cancer. Instead of using the standard multidrug approach, our work reveals a unique photophysical treatment paradigm that uses nontoxic doses of a single light-sensitive drug directed orthogonally to cancer and bone cells, followed by radionuclide-stimulated generation of ROS to inhibit tumor progression and minimize osteolysis in both immunocompetent murine and immunocompromised human MM models.

[Effect of jianpi-jiedu formula on tumor angiogenesis-relevant genes expression in colorectal cancer].

OBJECTIVE: To investigate the effect of the jianpi-jiedu formula (JPJD) on the expression of angiogenesis-relevant genes in colon cancer.
 Methods: Crude extract was obtained from JPJD by water extract method. The effect of JPJD crude extract on colon cancer cell proliferation capacity was determined by MTT assays. The IC50 value was calculated by GraphPad Prism5 software. Affymetrix gene expression profiling chip was used to detect significant differences in expressions of genes after JPJD intervention, and pathway enrichment analysis was performed to analyze the differentially expressed genes. Ingenuity Pathway Analysis software was applied to analyze differentially expressed genes relevant to tumor angiogenesis based on mammalian target of rapamycin (mTOR) signaling pathway and then the network diagram was built. Western blot was used to verify the protein levels of key genes related to tumor angiogenesis.
 Results: JPJD crud extract inhibited the proliferation capacity in colon cancer cells. The IC50 values in 24, 48, and 72 hours after treatment were 13.060, 9.646 and 8.448 mg/mL, respectively. The results of chip showed that 218 genes significantly upgraded, and 252 genes significantly downgraded after JPJD treatment. Most of the genes were related to the function of biosynthesis, metabolism, cell apoptosis, antigen extraction, angiogenesis and so on. There were 12 differentially expressed angiogenesis genes. IPA software analysis showed that the JPJD downregulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), VEGF, vascular endothelial growth factor A (VEGFA), integrin subunit alpha 1 (ITGA1), cathepsin B (CTSB), and cathepsin S (CTSS) genes, while upregulated expressions of GAB2 and plasminogen activator, urokinase receptor (PLAUR) genes in the colorectal cancer cell. Western blot results demonstrated that JPJD obviously downregulated expressions of phospho-mTOR (P-mTOR), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α (HIF-1α), and VEGF proteins, while obviously upregulated the level of phospho-P53 (P-P53) protein.
 Conclusion: JPJD may inhibit colorectal tumor angiogenesis through regulation of the mTOR-HIF-1α-VEGF signal pathway.

Cigarette smoke extract induces select matrix metalloproteinases and integrin expression in periodontal ligament fibroblasts.

BACKGROUND: The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. METHODS: PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. RESULTS: Treatment with CSE-induced cell death at concentrations of ≥5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Vα3 and decreased collagen XIα1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins α1, α2, and α10 (collagen receptors) and α9 (a tenascin receptor). CONCLUSIONS: This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion.

Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors.

Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5'-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.

Effect of plasma exchange in accelerating natalizumab clearance and restoring leukocyte function.

BACKGROUND: Accelerating the clearance of therapeutic monoclonal antibodies (mAbs) from the body may be useful to address uncommon but serious complications from treatment, such as progressive multifocal leukoencephalopathy (PML). Treatment of PML requires immune reconstitution. Plasma exchange (PLEX) may accelerate mAb clearance, restoring the function of inhibited proteins and increasing the number or function of leukocytes entering the CNS. We evaluated the efficacy of PLEX in accelerating natalizumab (a therapy for multiple sclerosis [MS] and Crohn disease) clearance and alpha4-integrin desaturation. Restoration of leukocyte transmigratory capacity was evaluated using an in vitro blood-brain barrier (ivBBB). METHODS: Twelve patients with MS receiving natalizumab underwent three 1.5-volume PLEX sessions over 5 or 8 days. Natalizumab concentrations and alpha4-integrin saturation were assessed daily throughout PLEX and three times over the subsequent 2 weeks, comparing results with the same patients the previous month. Peripheral blood mononuclear cell (PBMC) migration (induced by the chemokine CCL2) across an ivBBB was assessed in a subset of six patients with and without PLEX. RESULTS: Serum natalizumab concentrations were reduced by a mean of 92% from baseline to 1 week after three PLEX sessions (p < 0.001). Although average alpha4-integrin saturation was not reduced after PLEX, it was reduced to less than 50% when natalizumab concentrations were below 1 mug/mL. PBMC transmigratory capacity increased 2.2-fold after PLEX (p < 0.006). CONCLUSIONS: Plasma exchange (PLEX) accelerated clearance of natalizumab, and at natalizumab concentrations below 1 mug/mL, desaturation of alpha4-integrin was observed. Also, CCL2-induced leukocyte transmigration across an in vitro blood-brain barrier was increased after PLEX. Therefore, PLEX may be effective in restoring immune effector function in natalizumab-treated patients.

Involvement of central immunity in uncomplicated diverticular disease.

OBJECTIVE: The pathogenesis of symptoms of uncomplicated diverticular disease (UDD) is unclear, but changes in gut microflora and physiologic inflammation may be implicated. The objective of the study was to investigate the distribution of gut homing lymphocytes in peripheral blood and intestinal mucosa of UDD patients, and the effects of luminal antibiotic treatment. MATERIAL AND METHODS: Ten UDD patients and 10 age- and gender-matched healthy subjects underwent peripheral blood sampling, and colonoscopy with biopsies taken from the transverse and sigmoid colon. Treatment consisted of a 2-month course of rifaximin 1.2 g/day for 15 days/month. Blood sample and mucosal biopsies were repeated in UDD patients at the end of treatment. Flow cytometry was performed using monoclonal antibodies (CD3, CD4, CD8, CD25, CD19, CD45, CD62L, CD103). RESULTS: In peripheral blood, both CD4+ and CD8+/CD103+ were significantly higher in patients at baseline than in controls (0.95% versus 0.36%, and 0.5% versus 0.09%, respectively). After treatment, peripheral CD4+/CD103+ decreased (0.27%), while CD8+/CD103+ did not change (0.35%); on the contrary, peripheral CD25+ increased, the CD4+ subpopulation showing significantly higher levels than those in controls. No difference was found between lymphocytes in the diverticular sigmoid mucosa of patients at baseline and those in controls, but there was a significant decrease in CD8+/CD62L+ after treatment. In the normal transverse colon, CD4+/CD62L+ of patient at baseline were significantly lower than in controls. After treatment, CD4+/CD103+ levels significantly increased, while CD8+/CD62L+ levels significantly decreased. CONCLUSIONS: Both central and mucosal immunity may be modified in UDD patients, with an increased recruitment of CD103+ lymphocytes. A 2-month course of rifaximin appears to reduce CD103+ levels, suggesting a decrease in mobilization of mucosal homing.

Gonadotropin-releasing hormone agonists suppress melanoma cell motility and invasiveness through the inhibition of alpha3 integrin and MMP-2 expression and activity.

Cutaneous melanoma represents the leading cause of skin cancer deaths. The prognosis of highly aggressive, metastatic melanoma is still very poor, due to the resistance of the disseminated tumor to existing therapies. The clarification of the molecular mechanisms regulating melanoma growth and progression might help identify novel molecular targets for the development of new therapeutic interventions. We previously showed that gonadotropin-releasing hormone (GnRH) receptors are expressed in melanoma cells; activation of these receptors by means of GnRH agonists significantly reduces cell proliferation. In the current study, we first showed that GnRH agonists significantly reduced the metastatic behavior of melanoma cells in terms of both cell motility (haptotactic assay using laminin as the chemoattractant) and invasiveness (cell invasion assay evaluating the capacity of the cells to invade a reconstituted extracellular matrix barrier). On the basis of this observation, we then investigated the molecular mechanisms underlying the antimetastatic activity of GnRH agonists. We found that, in melanoma cells, a) the activity of the alpha3 integrin subunit is crucial for the migratory behavior of the cells; b) GnRH agonists significantly reduced alpha3 integrin expression (Western blotting and immunofluorescence studies); c) GnRH agonists significantly reduced MMP-2 expression (comparative RT-PCR) and activity (zymographic analysis performed on cell culture media). These data indicate that GnRH agonists, in addition to the previously reported antiproliferative effect, elicit a strong inhibitory activity on the migratory/invasive behavior of melanoma cells expressing GnRH receptors. These compounds reduce the metastatic potential of melanoma cells by interfering with the expression/activity of cell adhesion molecules (alpha3 integrin) and matrix metalloproteinase (MMP-2).

Integrin expression on monocytes and lymphocytes in unstable angina short term effects of atorvastatin.

Inflammatory reactions in coronary plaques play an important role in the pathogenesis of acute atherothrombotic events. The most powerful class of lipid-lowering drugs available-statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors)--have additional actions, unrelated to cholesterol reduction, including anti-inflammatory and immunomodulatory properties. This study sought to determine if atorvastatin affects monocyte and lymphocyte activation in patients with unstable angina and mild primary hypercholesterolemia. Following a 4-weeks hypolipemiant-free baseline period, 22 patients-12 with unstable angina (UA) and 10 patients with stable coronary heart disease (SCHD) - were treated with Atorvastatin 20 mg/day. Lipopolysaccharide (LPS)-receptor (CD14) and HLA-DR expression on monocytes and beta-integrins (CD11b, 11c, 49d) on monocytes and lymphocytes were measured by flow cytometry before and after treatment with atorvastatin for 8 weeks. Monocyte CD11b, 11c and CD14 expression and T lymphocytes CD11b expression were significantly (p < 0.001) higher in UA patients before treatment when compared with that in SCHD patients. In patients with UA, they decreased markedly with atorvastatin treatment. The reduction in expression of adhesion molecule on monocytes and lymphocytes and the concentrations of CRP and sICAM-1 may crucially contribute to the clinical benefit of atorvastatin in coronary artery disease, independent of cholesterol lowering effects.

Mixture of sugar and povidone--iodine stimulates wound healing by activating keratinocytes and fibroblast functions.

The topical application of a mixture of sugar and povidone--iodine (PI) has been reported to accelerate the healing of cutaneous wounds and ulcers by promoting re-epithelialization and granulation tissue formation as well as having an anti-microbial effect. To clarify the mechanisms accounting for the efficacy of a 70% sugar and 3% PI paste (U-PASTAtrade mark) (SP), various keratinocytes and fibroblasts functions, including proliferation, collagen synthesis, integrin expression, and cytokine and proteinase secretions in the presence of SP were investigated. Cultured human keratinocytes and fibroblasts were treated with various concentrations of SP, SU and PI. The secretion of urokinase-type plasminogen activator (u-PA), transforming growth factor (TGF)-alpha and interleukin-1alpha from keratinocytes, was detected by ELISA. Collagen synthesis of fibroblasts was examined by means of detecting proline uptake. Furthermore, integrin expressions of these cells were analyzed using a flow cytometer. SP and PI increased intra-cellular u-PA of keratinocytes and stimulated the secretion of u-PA and TGF-alpha. Sugar accelerated the extra-cellular u-PA level only. Both SP and sugar increased the collagen synthesis of fibroblasts. SP and PI also remarkably induced the expressions of extra-cellular matrix receptor integrins, alpha1, alpha2, alpha3, alpha4, alpha5 and beta1, on the surface of keratinocytes and fibroblasts. SP, the mixture of sugar and PI, is likely to act on wounds not only as an antibiotic agent, but also as a modulator for keratinocytes and fibroblasts.

Expression and function of C/EBP homology protein (GADD153) in podocytes.

Podocytes are crucial for the permeability of the glomerular filtration barrier. In glomerular disease, however, reactive oxygen species (ROS) may be involved in podocyte injury and subsequent proteinuria. Here, we describe ROS-dependent gene induction in differentiated podocytes stimulated with H(2)O(2) or xanthine/xanthine-oxidase. Superoxide anions and H(2)O(2) increased mRNA and protein expression of GAS5 (growth arrest-specific protein 5) and CHOP (C/EBP homology protein). Cultured podocytes overexpressing CHOP showed increased generation of superoxide anions compared to controls. In addition, the expression of alpha(3)/beta(1) integrins, crucial for cell-matrix interaction of podocytes, was down-regulated, leading to increased cell-matrix adhesion and cell displacement. The altered cell-matrix adhesion was antagonized by the ROS scavenger 1,3-dimethyl-2-thiourea, and the increase in cell displacement could be mimicked by stimulating untransfected podocytes with puromycin, an inductor of ROS. We next performed immunohistochemical staining of human kidney tissue (normal, membranous nephropathy, focal segmental glomerulosclerosis, and minimal change nephropathy) as well as sections from rats with puromycin nephrosis, a model of minimal change nephropathy. CHOP was weakly expressed in podocytes of control kidneys but up-regulated in most proteinuric human kidneys and in rat puromycin nephrosis. Our data suggest that CHOP-via increased ROS generation-regulates cell-matrix adhesion of podocytes in glomerular disease.

Involvement of beta(1) integrin in betaAP-induced apoptosis in human neuroblastoma cells.

Integrin-mediated cell adhesion is required for cell survival and differentiation. Recently, integrins have been proposed as a target for beta-amyloid peptide (betaAP) neurotoxicity. We report here that treatment with betaAP (1-42) or with the active betaAP fragment (25-35) induced a great deal of apoptosis in SK-N-BE and SH-SY5Y cell lines. In the presence of either collagen I degrees, fibronectin, or laminin, betaAP toxicity was severely reduced. This protective effect seems to be mediated by integrins, because preincubation of neuroblastoma cells with antibodies directed against beta(1) and alpha(1) integrin subunits greatly enhanced betaAP-induced apoptosis. In addition, treatment with betaAP induced a strong reduction of beta(1) and alpha(1) integrin subunits expressed in plasma membrane, which occurred 3 h after treatment, before the appearance of the apoptotic morphology. The rapid downregulation of the alpha(1)beta(1) integrin was almost completely recovered 15-24 h after betaAP treatment and was not prevented by cycloheximide. In conclusion, our data indicate a relationship between betaAP neurotoxicity and modulation of alpha(1)beta(1) integrin expression, and support the hypothesis that aberrant integrin function may play a significant role in betaAP-mediated neurotoxicity.

Heparin modulates integrin-mediated cellular adhesion: specificity of interactions with alpha and beta integrin subunits.

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.