Holmium (III)-doped multifunctional nanotheranostic agent for ultra-high-field magnetic resonance imaging-guided chemo-photothermal tumor therapy.

Ultra-high-field (UHF) MRI has shown great advantages over low-field magnetic resonance imaging (MRI). Despite being the most commonly used MRI contrast agents, gadolinium chelates perform poorly in high magnetic fields, which significantly weakens their T1 intensity. In comparison, the rare element Holmium (Ho)-based nanoparticles (NPs) have demonstrated great potential as T2-weighted MRI contrast agents in UHF MRI due to their extremely short electron relaxation times (∼ 10-13s). In this study, a multifunctional nanotherapeutic probe was designed for UHF MRI-guided chemotherapy and photothermal therapy. The Ho (III)-doped mesoporous polydopamine (Ho-MPDA, HM) nanosphere was loaded with the chemotherapeutic drug mitoxantrone (MTO) and then coated with 4T1 cell membranes to enhance active targeting delivery to breast cancer. The prepared nanotherapeutic probe MTO@HMM@4T1 (HMM@T) exhibited good biocompatibility, high drug-loading capability and great potential as Ho (III)-based UHF MRI contrast agents. Moreover, the biodegradation of HMM@T in response to the intratumor pH and glutathione (GSH) promotes MTO release. Near-infrared (NIR) light irradiation of HM induced photothermal therapy and further enhanced drug release. Consequently, HMM@T effectively acted as an MRI-guided tumor-targeting chemo-photothermal therapy against 4T1 breast cancer. STATEMENT OF SIGNIFICANCE: Ultra-high-field (UHF) MRI has shown great advantages over low-field magnetic resonance imaging (MRI). Although gadolinium chelates are the most commonly used MRI contrast agents in clinical practice, they exhibit a significantly decreased T1 relaxivity at UHF. Holmium exhibits outstanding UHF magnetic resonance capabilities in comparison with gadolinium chelates currently used in clinic. Herein, a theranostic nanodrug (HMM@T) was designed for UHF MRI-guided chemo-photothermal therapy. The nanodrug possessed remarkable UHF T2 MRI properties (r2 = 152.13 mM-1s-1) and high drug loading capability of 18.4 %. The biodegradation of HMM@T NPs under triple stimulations of pH, GSH, and NIR led to an efficient release of MTO in tumor microenvironment. Our results revealed the potential of a novel UHF MRI-guided multifunctional nanosystem in cancer treatment.

Carbon Monoxide Impairs CD11b+Ly-6Chi Monocyte Migration from the Blood to Inflamed Pancreas via Inhibition of the CCL2/CCR2 Axis.

Acute pancreatitis (AP) is a sterile inflammation, in which inflammatory monocytes (CD11b+Ly-6Chi) are recruited into the inflamed tissue at the onset of disease. Monocyte infiltration and activation at the site of inflammation are critical to the pathogenesis of AP. Our previous studies have shown a protective role for CO in AP, which is partially mediated by inhibition of macrophage activation via TLR4 signaling. In the current study, to gain a better understanding of CO's therapeutic effect, we further investigated whether CO could affect inflammatory monocyte trafficking during AP. In a mouse model of AP, we found that treatment with CO-releasing molecule-2 (CORM-2) impaired recruitment of inflammatory monocytes, but not that of neutrophils, from peripheral blood to inflamed pancreas. During the early stage of AP, a single dose of CORM-2 decreased pancreatic CCL2 and soluble ICAM-1 expression. In addition, using in vivo and in vitro experiments, we found that CORM-2 had the ability to inhibit CD11b+Ly-6Chi monocyte migration via blockade of CCR2 endocytosis. Notably, we showed that CORM-2 inhibited CCR2 endocytosis of inflammatory monocytes (CD14hiCD16-) from AP patients. Taken together, our results highlighted CO's effect on inflammatory monocyte trafficking, shedding additional light on its therapeutic potential in AP.

Dopamine D2 receptor signalling controls inflammation in acute pancreatitis via a PP2A-dependent Akt/NF-κB signalling pathway.

BACKGROUND AND PURPOSE: Dopamine has multiple anti-inflammatory effects, but its role and molecular mechanism in acute pancreatitis (AP) are unclear. We investigated the role of dopamine signalling in the inflammatory response in AP. EXPERIMENTAL APPROACH: Changes in pancreatic dopaminergic system and effects of dopamine, antagonists and agonists of D1 and D2 dopamine receptors were analysed in wild-type and pancreas-specific Drd2-/- mice with AP (induced by caerulein and LPS or L-arginine) and pancreatic acinar cells with or without cholecystokinin (CCK) stimulation. The severity of pancreatitis was assessed by measuring serum amylase and lipase and histological assessments. The NF-κB signalling pathway was evaluated, and macrophage and neutrophil migration assessed by Transwell assay. KEY RESULTS: Pancreatic dopamine synthetase and metabolic enzyme levels were increased, whereas D1 and D2 receptors were decreased in AP. Dopamine reduced inflammation in CCK-stimulated pancreatic acinar cells by inhibiting the NF-κB pathway. Moreover, the protective effects of dopamine were blocked by a D2 antagonist, but not a D1 antagonist. A D2 agonist reduced pancreatic damage and levels of p-IκBα, p-NF-κBp65, TNFα, IL-1ß and IL-6 in AP. Pancreas-specific Drd2-/- aggravated AP. Also, the D2 agonist activated PP2A and inhibited the phosphorylation of Akt, IKK, IκBα and NF-κB and production of inflammatory cytokines and chemokines. Furthermore, it inhibited the migration of macrophages and neutrophils by reducing the expression of CCL2 and CXCL2. A PP2A inhibitor attenuated these protective effects of the D2 agonist. CONCLUSIONS AND IMPLICATIONS: D2 receptors control pancreatic inflammation in AP by inhibiting NF-κB activation via a PP2A-dependent Akt signalling pathway.

Roles of endotoxin-related signaling molecules in the progression of acute necrotizing pancreatitis in mice.

OBJECTIVE: To study the potential roles of lipopolysaccharide (LPS) signaling molecules, LPS-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4) in mice with acute necrotizing pancreatitis (ANP). METHODS: The ANP model was made by 7 intraperitoneal injections of cerulein (50 mug/kg) at hourly intervals, challenged by LPS administration of a dose of 5 mg/kg intraperitoneally 5 hours after the first injection of cerulein. Fifty-nine Balb/C mice were divided into 4 groups: group A, ANP control group, n = 18, received physiological saline; group B, anti-LBP group, n = 18, received 200 mug anti-LBP antibody; group C, anti-CD14 group, n = 18, received 20 microg anti-CD14 antibody; group D, anti-TLR4 group, n = 5, received 20 mug anti-TLR4 antibody. All treatments were given at 15 minutes before LPS injection. Serum amylase and lactic dehydrogenase (LDH) were measured. Histologic alteration of the pancreas was evaluated. Expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and intercellular adhesion molecular (ICAM-1) mRNA were determined using reverse transcription polymerase chain reaction (RT-PCR). Myeloperoxidase (MPO) activity in pancreas was also analyzed. Nuclear factor-kappaB (NF-kappaB) p65 subunit was measured by immunohistochemistry and Western blotting. RESULTS: Pretreatment of animals with anti-CD14 antibody resulted in a significant decrease in serum amylase and LDH levels, reduction of the severity of pancreatic injury, down-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, decrease in pancreatic MPO activity, and decrease in NF-kappaB expression compared with ANP control mice. In contrast, mice pretreated with anti-LBP antibody or anti-TLR4 antibody resulted in increasing of serum amylase and LDH levels, aggravation of the severity of necrosis and inflammation in pancreas, up-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, increasing of pancreatic MPO activity, and up-regulation of NF-kappaB expression compared with ANP control mice. CONCLUSIONS: (1) LBP per se possesses a protective effect on ANP. It could facilitate clearance of endotoxin. (2) CD14 plays a crucial intermediate role in the progression of ANP. (3) TLR4, the essential transducer of LPS responses, may act independently of LPS. The antibody against TLR4 may mimic endotoxin and induce activation of downstream cytokines. For implication, recombinant LBP, anti-CD14 antibody, or silent TLR4 might alleviate the progression of ANP.

Effects of angiotensin II receptor antagonist, Losartan on the apoptosis, proliferation and migration of the human pancreatic stellate cells.

AIM: To investigate the effects of AT1 (Type 1 angiotensin II receptor) antagonist (Losartan) on the apoptosis, proliferation and migration of the human pancreatic stellate cells (hPSCs). METHODS: hPSCs were isolated from pancreatic sample of patients with pancreatic carcinoma using radioimmunoassay (RIA) technique to detect the concentration of AngII in culture media and cell homogenate. Immunocytochemistry (ICC) and in situ hybridization (ISH) methods were utilized to test AT1 expression in hPSCs. Effects of Losartan on hPSCs proliferation, apoptosis and migration were investigated using BrdU incorporation, TUNEL, flow cytometry (FCM), and phase-contrast microscope separately when cells treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type I collagen in hPSCs. RESULTS: There exists AT1 expression in hPSCs, while no AngII was detected in culture media and cell homogenate. Losartan induces cell apoptosis in a dose- and time-dependent manner (apparently at 10(-5) mol/L), no pro-proliferative effect was observed in the same condition. Corresponding dosage of Losartan can also alleviate the motion capability and type I collagen content of hPSCs compared with AngII treatment and non-treatment control groups. CONCLUSION: These findings suggest that paracrine not autocrine functions of AngII may have effects on hPSCs, which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion and partly by inducing apoptosis.

[The role of lipopolysacchride-binding protein in the pathogenesis of animal model of acute necrotizing pancreatitis].

OBJECTIVE: To explore the pathogenic role of lipopolysacchride-binding protein (LBP) in the pathogenesis of acute necrotizing pancreatitis (ANP) by applying anti-LBP antibody to the animal model of ANP in mice. METHODS: Sixty BALB/c mice were randomly divided into four groups, including ANP group (n = 18), ANP treated with anti-LBP antibody group (n = 18), anti-LBP antibody group (n = 18) and normal control (n = 6). ANP model was induced by seven times administration of cerulein (50 micro g/kg.body weight), challenged by lipopolysaccharide (LPS) (5 mg/kg) intravenous injection. Treatment with anti-LBP antibody was started 15 minutes before LPS injection in ANP treated with anti-LBP antibody group. Anti-LBP antibody group only received intravenous injection of anti-LBP antibody, normal saline was administrated intraperitoneally instead of cerulein and LPS. At 9 h, 12 h and 24 h after the first injection of cerulein (or saline), the serum levels of amylase and lactate dehydrogenase (LDH) were measured. The severity of pancreatitis was evaluated by histological scoring system. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA expressions were studied by semi-quantitative RT-PCR. The activation of nuclear factor-kappaB (NF-kappaB) in the pancreas was investigated by the methods of immunohistochemistry and Western blot. The activity of PMN myeloperoxidase (MPO) was determined by zymohistochemistry. RESULTS: Compared with the ANP group, a marked elevation of serum amylase was observed 9 h and 12 h after cerulein administration and a marked elevation of serum LDH was observed 24 h after cerulein administration in the ANP treated with anti-LBP antibody group. Histologically, treatment with anti-LBP group increased the severity of pancreatic injury including edema at 9 h and 12 h after, and inflammatory cell infiltration and necrosis 24 h after. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA levels were increased. The activity of MPO was increased significantly 12 h and 24 h after in the anti-LBP antibody group. Immunohistochemistry and Western blotting showed up-regulation of NF-kappaB. However, there was no significant difference in serum parameters and pathologic scoring results between LBP antibody group and normal control. CONCLUSION: LBP plays a protective role in the pathogenesis of ANP, and this action may be mediated by inhibiting of NF-kappaB activation and down-regulation of proinflammatory mediators.

[Inhibition of angiogenesis in pancreatic carcinoma by cyclooxygenase-2 antisense oligodeoxynucleotides].

OBJECTIVE: To investigate the effect of cyclooxygenase-2 antisense oligodeoxynucleotides (COX-2 AS-ODNs) on the angiogenesis in pancreatic carcinoma and to evaluate the intermediary effect of prostaglandin 2 in this process. METHODS: Specific targeting COX-2 AS-ODNs were designed and synthesized, and transfected into the PC3 human pancreatic carcinoma cells cultured in vitro. Fluorescence microscopy was used to observe the PC3 cells 0.12. 24, 40, and 72 hours after the transfection. the second cultured PC3 cells were divided into 5 groups: control group, Lipo group (transfected with Lipofectin only), C1 group (transfected with 1 micro g COX-2 AS-ODN + Lipo/well), C2 group (transfected with 2 micro g COX-2 AS-ODN + Lipo/well), and C3 group (transfected with 3 micro g COX-2 AS-ODN + Lipo/well). RT-PCR was used to observe the expression of COX-2 mRNA in the PC3 cells. The third batch of PC3 cells were transfected with 3 micro g COX-2 AS-ODN + Lipo/well, and the expression of COX-2 mRNA was observed 0, 12, 24, 48, and 72 hours later by RT-PCR. 3 micro g COX-2 AS-ODN + Lipo/bottle and 9 micro g COX-2 AS-ODN + Lipo/bottle were added into the cultured PC3 cells and Western blotting was used to observe the expression of COX-2 protein 24 hours later. 24 chicken eggs were inoculated with PC3 cells into the chorio-allantoic membrane and then divided equally into 5 groups; control group, Lipo group, COX-2 AS-ODN + Lipo group, and COX-2 AS-ODN + Lipo + PGE2 group. Leica microscopy was used to observe the angiogenesis in the transplanted carcinoma. RESULTS: RT-PCR showed that the downregulation of expression of COX-2 mRNA in the PC3 cells with the increase of the COX-2 AS-ODN concentration, peaking at the concentration of 0.2 micro mol/L. The effect of COX-2 AS-ODN was strongest by the 12th hour after transfection and then began to decrease and basically disappeared 48 hours after. Western blotting showed that COX-2 AS-ODN, especially that of the concentration of the expression of 9 micro g/bottle, inhibited the expression of COX-2 AS-ODN. The angiogenesis of the transplanted carcinoma in the eggs was significantly inhibited in the Lipo + COX-2 AS-ODN group, the density of newly generated vessels in the Lipo + COX-2 AS-ODN + PGE2 group was between those of the other 2 groups. CONCLUSION: COX-2 AS-ODN significantly inhibits the angiogenesis in the pancreatic carcinoma. Endogenous COX-2 AS-ODN may play an important role in such a process and PGE2 may play an intermediate role therein.

[Regulatory effects of peroxisome proliferator-activated receptor gamma on the growth of pancreatic carcinoma].

OBJECTIVE: To examine the effects of peroxisome proliferator-activated receptor (PPAR)gamma activation on the growth of human pancreatic carcinoma both in vitro and in vivo. METHODS: The expression of PPARgamma and RXRalpha were examined by RT-PCR. SW1990 pancreatic cancer cells were treated with 9-cis-RA, ligand of PPARgamma, 15d-PGJ(2), and both. Antiproliferative effect was evaluated with cell viability by using MTT assay. Pancreatic cancer xenograft tumor model was established in nude mice by inoculating SW1990 cells subcutaneously and rosiglitazone, a PPARgamma activator, was administered via water drinking in experimental group. The nude mice were sacrificed after 75 days, the volume and weight of the xenograft tumor were measured. Expression of PCNA was observed by immunohistochemical staining. RESULTS: RT-PCR showed that PPARgamma and RXRalpha mRNA were expressed in SW1990 cell line. MTT assay demonstrated that 15d-PGJ(2), 9-cis-RA and the combination of both had a potent inhibitory effect on the growth of SW1990 cells with a dose-dependent manner. SW1990 cells were suppressed to more than 50% of the control at the concentration of 10 micro mol/L 15d-PGJ(2), 20 micro mol/L 9-cis-RA and 5 micro mol/L 15d-PGJ(2) plus 10 micro mol/L 9-cis-RA, respectively. 9-cis-RA had a synergic action with 15d- PGJ(2) on the growth inhibition of pancreatic carcinoma. In vivo studies, rosiglitazone suppressed the growth of pancreatic carcinoma in a statistically significant manner (P < 0.05). The average tumor volume and tumor weight in the experimental group were less than those in the control group, the growth inhibition rate of rosiglitazone was 80.7%. PCNA was present in both groups, but immunohistochemistry showed a down-regulation trend of PCNA in the experimental group as compared with the control group. CONCLUSIONS: Activation of PPARgamma exerts a negative regulatory effect on the growth of pancreatic carcinoma both in vitro and in vivo. These results suggest that PPARgamma might be a novel therapeutic target for the pancreatic carcinoma. Activation of RXRalpha has a synergic action with PPARgamma agonist on the growth inhibition of pancreatic carcinoma.