Correlation of the invasive potential of glioblastoma and expression of caveola-forming proteins caveolin-1 and CAVIN1.

INTRODUCTION: Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15 months after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. Caveolae are plasma membrane subdomains that participate in numerous biological functions. Caveolin-1 and Caveolae Associated Protein 1 (CAVIN1), formerly termed Polymerase I and Transcript Release Factor, are both necessary for caveola formation. We hypothesized that high expression of caveola-forming proteins in GBM promotes invasiveness via modulation of the production of matrix-degrading enzymes. METHODS: The mRNA expression of caveola-forming proteins and matrix proteases in GBM samples, and survival after stratifying patients according to caveolin-1 or CAVIN1 expression, were analyzed from TCGA and REMBRANDT databases. The proteolytic profile of cell lines expressing or devoid of caveola-forming proteins was investigated using zymography and real-time qPCR. Invasion through basement membrane-like protein was investigated in vitro. RESULTS: Expression of both caveolin-1 and CAVIN1 was increased in GBM compared to normal samples and correlated with expression of urokinase plasminogen activator (uPA) and gelatinases. High expression of caveola-forming proteins was associated with shorter survival time. GBM cell lines capable of forming caveolae expressed more uPA and matrix metalloproteinase-2 (MMP-2) and/or -9 (MMP-9) and were more invasive than GBM cells devoid of caveola-forming proteins. Experimental manipulation of caveolin-1 or CAVIN1 expression in GBM cells recapitulated some, but not all of these features. Caveolae modulate GBM cell invasion in part via matrix protease expression.

Bone marrow sinusoidal endothelium as a facilitator/regulator of cell egress from the bone marrow.

Despite more attention to cell migration from circulation into the bone marrow (BM), particularly homing of haematopoietic stem/progenitor cells, the process and mechanisms of cell mobilisation from the BM into the circulation remain largely underexplored. The process of cell mobilisation or transcellular cell migration from BM into the circulation (cell egress) is a crucial biological process in mammals as it is important to maintain homeostasis of various physiological functions including, but not limited to, the immune system, erythropoiesis, platelet release, and stem cell migration. The BM microvascular system composes of a monolayer of specialized endothelial cells, called sinusoidal endothelial cells (SECs). While it is very well evident that the process of cell egress occurs exclusively through BM SECs, there is a lack of systematic analyses addressing the extent of contribution of BM SECs to the process of cell egress from the BM. Therefore, this review aims to address the potential ability of BM SECs in regulating and/or facilitating the process of cell egress from BM. In this review, we address, firstly, the unique ultra-/structural and molecular features of BM SECs and discuss the possible biological interactions between BM SECs and various egressing cells in physiological conditions. Secondly, we propose the potential role of BM SECs in egress of leukemic cells from BM into the circulation. Finally, we discuss the potential role of BM SECs in homing of haematopoietic stem cells. Collectively, the current review suggests that the BM SECs may not be merely a neutral gatekeeper for cell intravasation and extravasation, but rather is a dynamic trafficking surveillance system, thereby the process of BM cell egress/mobilisation can be regulated.

Retracted: Icariin attenuates methotrexate chemotherapy-induced bone marrow microvascular damage and bone loss in rats.

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX-induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX-treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin-treated rats than those of MTX alone-treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis-related genes but suppressed the induction of adipogenesis-related genes in bones of MTX-treated rats. In addition, icariin treatment attenuated MTX-induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX-treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide.

Opioids and matrix metalloproteinases: the influence of morphine on MMP-9 production and cancer progression.

Opioids are widely administered to alleviate pain, including chronic pain in advanced cancer patients. Among opioids, morphine is one of the most clinically effective drugs for the palliative management of severe pain. In the last few decades, there has been a debate around the possible influence of opioids such as morphine on tumour growth and metastasis. Whilst several in vitro and in vivo studies suggest the possible modulatory effects of morphine on tumour cells, little is known about the impact of this analgesic drug on other mediators such as matrix metalloproteinases (MMPs) that play a key role in the control of cancer cell invasion and metastasis. MMP-9 has been considered as one of the principal mediators in regulation of not only the initial steps of cancer but during the invasion and spreading of cancer cells to distant organs. Herein, current studies regarding the direct and indirect effects of morphine on regulation of MMP-9 production are discussed. In addition, drawing from previous in vivo and in vitro studies on morphine action in regulating MMP-9 production, the potential roles of several underlying factors are summarised, including nuclear factor kappa-B and intracellular molecules such as nitric oxide.

Critical limb ischemia: Current and novel therapeutic strategies.

Critical limb ischemia (CLI) is the advanced stage of peripheral artery disease spectrum and is defined by limb pain or impending limb loss because of compromised blood flow to the affected extremity. Current conventional therapies for CLI include amputation, bypass surgery, endovascular therapy, and pharmacological approaches. Although these conventional therapeutic strategies still remain as the mainstay of treatments for CLI, novel and promising therapeutic approaches such as proangiogenic gene/protein therapies and stem cell-based therapies have emerged to overcome, at least partially, the limitations and disadvantages of current conventional therapeutic approaches. Such novel CLI treatment options may become even more effective when other complementary approaches such as utilizing proper bioscaffolds are used to increase the survival and engraftment of delivered genes and stem cells. Therefore, herein, we address the benefits and disadvantages of current therapeutic strategies for CLI treatment and summarize the novel and promising therapeutic approaches for CLI treatment. Our analyses also suggest that these novel CLI therapeutic strategies show considerable advantages to be used when current conventional methods have failed for CLI treatment.

Adipose-derived stem cells for wound healing.

Wound healing is a complex but a fine-tuned biological process in which human skin has the ability to regenerate itself following damage. However, in particular conditions such as deep burn or diabetes the process of wound healing is compromised. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose-derived stem cells (ASCs) seem to possess the least limitations for clinical applications, and literature showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. In the present review, advantages and disadvantages of various stem cells which can be used for promoting wound healing are discussed. In addition, potential mechanisms of action by which ASCs may accelerate wound healing are summarised. Finally, clinical studies applying ASCs for wound healing and the associated limitations are reviewed.

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

Bone marrow sinusoidal endothelium: damage and potential regeneration following cancer radiotherapy or chemotherapy.

It is very well known that bone marrow (BM) microvasculature may possess a crucial role in the maintenance of homeostasis of BM due to mutual interactions between BM microvascular system and other physiological functions including haematopoiesis and osteogenesis. Chemotherapy and radiotherapy are known as main approaches for cancer treatment and also are known as the main cause of damage to the BM microvascular system. However, despite the importance of BM microvasculature in orchestrating various biological functions, less attention has been drawn to address the underlying mechanisms for the damage and to explore cellular and molecular mechanisms by which the recovery/regeneration of chemotherapy- and/or radiotherapy-induced BM microvascular system damage can occur. Therefore, in this review we firstly discuss the ultra-/structure and biological characteristics of BM microvascular system (sinusoids). Secondly, potential contribution of BM sinusoids is discussed in pathophysiological circumstances (bone remodelling, haematopoiesis, cancer bone metastasis, and haematological cancers). Thirdly, we address previous preclinical and clinical studies regarding chemotherapy- and irradiation-induced BM microvasculature damage. Finally, potential cellular and molecular mechanisms are discussed for the recovery/regeneration of damaged BM microvascular system, including the potential roles of endothelial progenitor cells, haematopoietic stem/progenitor cells, and stimulation of VEGF/VEGFR and Ang-1/Tie-2 signalling pathways.

Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

Opioids modulate the tumor microenvironment with potential functional consequences for tumor growth and metastasis. We evaluated the effects of morphine administration on the circulating proteolytic profile of tumor-free mice. Serum from morphine-treated (1 or 10 mg/kg, i.p. every 12 h) or saline-treated mice was collected at different time points and tested ex vivo in endothelial, lymphatic endothelial, and breast cancer cell migration assays. Serum from mice that were treated with 10 mg/kg morphine for 3 d displayed reduced chemotactic potential for endothelial and breast cancer cells, and elicited reduced cancer cell invasion through reconstituted basement membrane compared with serum from saline controls. This was associated with decreased circulating matrix metalloproteinase 9 (MMP-9) and increased circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-3/4 as assessed by zymography and reverse zymography. By using quantitative RT-PCR, we confirmed morphine-induced alterations in MMP-9 and TIMP expression and identified organs, including the liver and spleen, in which these changes originated. Pharmacologic inhibition of MMP-9 abrogated the difference in chemotactic attraction between serum from saline-treated and morphine-treated mice, which indicated that reduced proteolytic ability mediated the decreased migration toward serum from morphine-treated mice. This novel mechanism may enable morphine administration to promote an environment that is less conducive to tumor growth, invasion, and metastasis.-Xie, N., Khabbazi, S., Nassar, Z. D., Gregory, K., Vithanage, T., Anand-Apte, B., Cabot, P. J., Sturgess, D., Shaw, P. N., Parat, M.-O. Morphine alters the circulating proteolytic profile in mice: functional consequences on cellular migration and invasion.

Effect of the Biphenyl Neolignan Honokiol on Aß42-Induced Toxicity in Caenorhabditis elegans, Aß42 Fibrillation, Cholinesterase Activity, DPPH Radicals, and Iron(II) Chelation.

The biphenyl neolignan honokiol is a neuroprotectant which has been proposed as a treatment for central nervous system disorders such as Alzheimer's disease (AD). The death of cholinergic neurons in AD is attributed to multiple factors, including accumulation and fibrillation of amyloid beta peptide (Aß) within the brain; metal ion toxicity; and oxidative stress. In this study, we used a transgenic Caenorhabditis elegans model expressing full length Aß42 as a convenient in vivo system for examining the effect of honokiol against Aß-induced toxicity. Furthermore, honokiol was evaluated for its ability to inhibit Aß42 oligomerization and fibrillation; inhibit acetylcholinesterase and butyrylcholinesterase; scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals; and chelate iron(II). Honokiol displayed activity similar to that of resveratrol and (-)-epigallocatechin gallate (EGCG) in delaying Aß42-induced paralysis in C. elegans, and it exhibited moderate-to-weak ability to inhibit Aß42 on-pathway aggregation, inhibit cholinesterases, scavenge DPPH radicals, and chelate iron(II). Moreover, honokiol was found to be chemically stable relative to EGCG, which was highly unstable. Together with its good drug-likeness and brain availability, these results suggest that honokiol may be amenable to drug development and that the synthesis of honokiol analogues to optimize these properties should be considered.

The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro.

Macrophages are abundant in the tumor microenvironment where they adopt a pro-tumor phenotype following alternative polarization induced by paracrine factors from cancer and stromal cells. In contrast, classically activated macrophages have tumoricidal activities, such that the polarization of tumor-associated macrophages has become a novel therapeutic target. Toll-like receptor 4 engagement promotes classical activation of macrophages, and recent literature suggests TLR4 agonism to prevent metastasis and promote survival in experimental metastasis models. A growing number of studies indicate that TLR4 can respond to opioids, including the opioid receptor-inactive morphine metabolite morphine-3-glucuronide (M3G). We measured the activation of TLR4 in a reporter cell line exogenously expressing TLR4 and TLR4 co-receptors, and confirmed that M3G weakly but significantly activates TLR4. We hypothesized that M3G would promote the expression of classical activation signature genes in macrophages in vitro. We exposed mouse and human macrophage cell lines to M3G or the TLR4 activator lipopolysaccharide (LPS), alone or in combination with interferon gamma (IFN-γ). The classical macrophage activation markers tested were iNOS, CD86, IL-6, or TNF-α in RAW 264.7 cells and IL-6, IL-12, IL-23, TNF-α, CXCL10, and CXCL11 in THP1 cells. Our results show that despite exhibiting TLR4-activation ability, M3G does not elicit the expression of classical activation markers in LPS-responsive macrophages.

Morphine decreases the pro-angiogenic interaction between breast cancer cells and macrophages in vitro.

Interactions between the various cell types that constitute a solid tumour are essential to the biology of the tumour. We evaluated the effect of morphine on the proangiogenic interaction taking place between macrophages and breast cancer cells in vitro. The conditioned medium (CM) from breast cancer cells co-cultured with macrophages elicited endothelial cell proliferation and tube formation. This effect was inhibited if the co-culture occurred in the presence of morphine. The CM from breast cancer cells or macrophages grown individually, whether or not prepared in the presence of morphine, was ineffective in stimulating EC proliferation or tube formation. Using a mouse antibody array, we identified several angiogenesis-regulating factors differentially expressed in the CM of co-cultured cells prepared in the presence or absence of morphine, amongst which interleukin (IL)-6, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A. VEGF was induced in both cell types by the co-culture and this was prevented by morphine in a non-naloxone reversible fashion. The effect of CM from co-cultured cells on endothelial tube formation, but not proliferation, was prevented by anti-VEGF neutralizing antibody. Our results indicate that morphine prevents, in part via modulating VEGF-A expression, the pro-angiogenic interaction between macrophages and breast cancer cells.

Morphine Modulates Interleukin-4- or Breast Cancer Cell-induced Pro-metastatic Activation of Macrophages.

Interactions between cancer cells and stromal cells in the tumour microenvironment play a key role in the control of invasiveness, metastasis and angiogenesis. Macrophages display a range of activation states in specific pathological contexts and alternatively activated (M2) macrophages can promote tumour aggressiveness. Opioids are able to modulate tumour growth and metastasis. We tested whether morphine modulates the activation of macrophages induced by (i) interleukin-4 (IL-4), the prototypical M2 polarization-inducing cytokine, or (ii) coculture with breast cancer cells. We showed that IL-4 causes increased MMP-9 production and expression of the alternative activation markers arginase-1 and MRC-1. Morphine prevented IL-4-induced increase in MMP-9 in a naloxone- and methylnaltrexone-reversible fashion. Morphine also prevented IL-4-elicited alternative activation of RAW264.7 macrophages. Expression of MMP-9 and arginase-1 were increased when RAW264.7 were subjected to paracrine activation by 4T1 cells, and this effect was prevented by morphine via an opioid receptor-mediated mechanism. Morphine further decreased 4T1 breast cancer cell invasion elicited by co-culture with RAW264.7. Reduction of MMP-9 expression and alternative activation of macrophages by morphine was confirmed using mouse bone marrow-derived macrophages. Taken together, our results indicate that morphine may modulate tumour aggressiveness by regulating macrophage protease production and M2 polarization within the tumour microenvironment.