Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes.

Background: Macrophages constitute the main part of infiltrating immune cells in Malignant pleural mesothelioma (MPM) and abnormally high ratios of M2 macrophages are present in both pleural effusion and tissue samples of MPM patients. Whether MPM cells affect formation of M2 macrophages is poorly understood. In this study, we focused on identification of MPM-cells-derived soluble factors with M2-promoting effects. Methods: Media of malignant pleural mesothelioma cells were collected and soluble factors affecting macrophages were analyzed by mass spectrometry. TGF-ß receptor inhibitor SB431542 was used as the entry point to explore the downstream mechanism of action by qRT-PCR, WB and immunofluorescence. Results: The serum-free culture media collected from the human MPM cells Meso1 and Meso2 significantly enhanced expression of the M2 signature molecules including IL-10, TGF-ß and CD206 in the human macrophages THP-1, while the culture medium of the human MPM cells H2452 did not show such M2-promoting effects. Analysis of proteins by mass spectrometry and ELISA suggested that Leucine rich α2 glycoprotein 1(LRG1) was a potential candidate. LRG1 time- and dose-dependently increased expression of the M2 signature molecules, confirming its M2-promoting effects. Furthermore, LRG1's M2-promoting effects were reduced by the TGF-ß receptor inhibitor SB431542, and LRG1 increased phosphorylation of Smad2, indicating that M2-promoting effects of LRG1 were via the TGF-ß receptor/Smad2 signaling pathway. Conclusions: Our results provide a potential M2-promoting new member, LRG1, which contributes to the immune escape of MPM via the TGF-ß receptor/Smad2 signaling pathway.

Posterior tibial nerve stimulation improves neurogenic bladder in rats with spinal cord injury through transient receptor potential/P2X signaling pathway.

BACKGROUND: To study the influences of posterior tibial nerve stimulation (PTNS) on neurogenic bladder and the expression of transient receptor potential (TRP) channels and P2X receptors in rats with spinal cord injury (SCI) and explore the possible mechanism. METHODS: SCI model was established by modified Allen's method and PTNS was performed. Urodynamic indexes and Haematoxylin and Eosine staining of bladder tissue were used to evaluate the therapeutic effect. The expression of TRP channels and P2X receptors in the bladder and dorsal root ganglia (DRG) was detected by real-time PCR and Western blot. RESULTS: The low compliance of bladder in treatment group was significantly improved compared with SCI group, and the infiltration of inflammatory cells in bladder tissue was significantly reduced. At the same time, the expression of TRP and P2X in bladder and DRG was partially restored after the treatment of PTNS. CONCLUSIONS: PTNS is an effective therapy for SCI-induced neurogenic bladder via the TRP/P2X signaling pathway.

Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study.

BACKGROUND: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure. METHODS: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice. RESULTS: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans. CONCLUSIONS: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled carbon nanotube administered by intra-tracheal instillation in the rat.

BACKGROUND: Multi-walled carbon nanotubes can be divided into two general subtypes: tangled and straight. MWCNT-N (60 nm in diameter) and MWCNT-7 (80-90 nm in diameter) are straight-type MWCNTs, and similarly to asbestos, both are carcinogenic to the lung and pleura when administered to rats via the airway. Injection of straight-type MWCNTs into the peritoneal cavity also induces the development of mesothelioma, however, injection of tangled-type MWCNTs into the peritoneal cavity does not induce carcinogenesis. To investigate these effects in the lung we conducted a 2-year comparative study of the potential carcinogenicities of a straight-type MWCNT, MWCNT-A (approximately 150 nm in diameter), and a tangled-type MWCNT, MWCNT-B (7.4 nm in diameter) after administration into the rat lung. Crocidolite asbestos was used as the reference material, and rats administered vehicle were used as the controls. Test materials were administered by intra-Tracheal Intra-Pulmonary Spraying (TIPS) once a week over a 7 week period (8 administrations from day 1 to day 50), followed by a 2-year observation period without further treatment. Rats were administered total doses of 0.5 or 1.0 mg MWCNT-A and MWCNT-B or 1.0 mg asbestos. RESULTS: There was no difference in survival between any of the groups. The rats administered MWCNT-A or asbestos did not have a significant increase in bronchiolo-alveolar hyperplasia or tumors in the lung. However, the rats administered MWCNT-B did have significantly elevated incidences of bronchiolo-alveolar hyperplasia and tumors in the lung: the incidence of bronchiolo-alveolar hyperplasia was 0/20, 6/20, and 9/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively, and the incidence of adenoma and adenocarcinoma combined was 1/19, 5/20, and 7/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively. Malignant pleural mesothelioma was not induced in any of the groups. CONCLUSIONS: The results of this initial study indicate that tangled-type MWCNT-B is carcinogenic to the rat lung when administered via the airway, and that straight-type MWCNT-A did not have higher carcinogenic potential in the rat lung than tangled-type MWCNT-B.

IQGAP2 Inhibits Migration and Invasion of Gastric Cancer Cells via Elevating SHIP2 Phosphatase Activity.

Previous studies have shown reduced expression of Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) and its tumor-suppressive role in gastric cancer (GC). However, the precise role of SHIP2 in the migration and invasion of GC cells remains unclear. Here, an IQ motif containing the GTPase-activating protein 2 (IQGAP2) as a SHIP2 binding partner, was screened and identified by co-immunoprecipitation and mass spectrometry studies. While IQGAP2 ubiquitously expressed in GC cells, IQGAP2 and SHIP2 co-localized in the cytoplasm of GC cells, and this physical association was confirmed by the binding of IQGAP2 to PRD and SAM domains of SHIP2. The knockdown of either SHIP2 or IQGAP2 promoted cell migration and invasion by inhibiting SHIP2 phosphatase activity, activating Akt and subsequently increasing epithelial-mesenchymal transition (EMT). Furthermore, knockdown of IQGAP2 in SHIP2-overexpressing GC cells reversed the inhibition of cell migration and invasion by SHIP2 induction, which was associated with the suppression of elevated SHIP2 phosphatase activity. Moreover, the deletion of PRD and SAM domains of SHIP2 abrogated the interaction and restored cell migration and invasion. Collectively, these results indicate that IQGAP2 interacts with SHIP2, leading to the increment of SHIP2 phosphatase activity, and thereby inhibiting the migration and invasion of GC cells via the inactivation of Akt and reduction in EMT.

Pleural translocation and lesions by pulmonary exposed multi-walled carbon nanotubes.

Carbon nanotubes (CNTs) are recently developed tubular nanomaterials, with diameters ranging from a few nanometers to tens of nanometers, and the length reaching up to several micrometers. They can be either single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs). Due to their nano-scaled structure, CNTs have a unique set of mechanical, electrical, and chemical properties that make them useful in information technologies, optoelectronics, energy technologies, material sciences, medical technologies, and other fields. However, with the wide application and increasing production of CNTs, their potential risks have led to concerns regarding their impact on environment and health. The shape of some types of CNTs is similar to asbestos fibers, which suggests that these CNTs may cause characteristic pleural diseases similar to those found in asbestos-exposed humans, such as pleural plaques and malignant mesothelioma. Experimental data indicate that CNTs can induce lung and pleural lesions, inflammation, pleural fibrosis, lung tumors, and malignant mesothelioma upon inhalation in the experimental animals. In this review, we focus on the potential of MWCNTs to induce diseases similar to those by asbestos, molecular and cellular mechanisms associated with these diseases, and we discuss a method for evaluating the pleural toxicity of MWCNTs.

Serum levels of the chemokine CCL2 are elevated in malignant pleural mesothelioma patients.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a debilitating disease of the pleural cavity. It is primarily associated with previous inhalation of asbestos fibers. These fibers initiate an oxidant coupled inflammatory response. Repeated exposure to asbestos fibers results in a prolonged inflammatory response and cycles of tissue damage and repair. The inflammation-associated cycles of tissue damage and repair are intimately involved in the development of asbestos-associated cancers. Macrophages are a key component of asbestos-associated inflammation and play essential roles in the etiology of a variety of cancers. Macrophages are also a source of C-C motif chemokine ligand 2 (CCL2), and a variety of tumor-types express CCL2. High levels of CCL2 are present in the pleural effusions of mesothelioma patients, however, CCL2 has not been examined in the serum of mesothelioma patients. METHODS: The present study was carried out with 50 MPM patients and 356 subjects who were possibly exposed to asbestos but did not have disease symptoms and 41 healthy volunteers without a history of exposure to asbestos. The levels of CCL2 in the serum of the study participants was determined using ELISA. RESULTS: Levels of CCL2 were significantly elevated in the serum of patients with advanced MPM. CONCLUSIONS: Our findings are consistent with the premise that the CCL2/CCR2 axis and myeloid-derived cells play an important role in MPM and disease progression. Therapies are being developed that target CCL2/CCR2 and tumor resident myeloid cells, and clinical trials are being pursued that use these therapies as part of the treatment regimen. The results of trials with patients with a similar serum CCL2 pattern as MPM patients will have important implications for the treatment of MPM.

Angiopoietin-like 2 upregulation promotes human chondrocyte injury via NF-κB and p38/MAPK signaling pathway.

Several cellular and molecular processes participate in the pathologic changes of osteoarthritis (OA). However, the core molecular regulators of these processes are unclear, and no effective treatment for OA disease has been developed so far. ANGPTL2 is well known for its tissue remolding and pro-inflammation properties. However, the role of ANGPTL2 in osteoarthritis (OA) still remains unclear. To explore the expression level of ANGPTL2 in human OA cartilage and investigate the function of ANGPTL2 in human chondrocytes injury, qRT-PCR, western blot and immunohistochemistry were employed to investigate the expression of ANGPTL2 between human OA and normal cartilage samples. Next, human primary chondrocytes were treated with IL-1ß to mimic OA progress in vitro, and the expression of ANGPTL2 were tested by qRT-PCR and western blot. Furthermore, the effect of ANGPTL2 in the expression of pro-inflammation cytokines (IL-1ß, IL-6), proteolytic enzymes (MMP-1, MMP-13) and component of the cartilage matrix (COL2A1 and aggrecan) in human primary chondrocyte were explored by gain-of-function and loss-of-function methods. Finally, the nuclear factor kappa B (NF-κB) and p38/MAPK signaling pathways were also tested by western blot analysis. In this study, firstly, the expression level of ANGPTL2 was elevated both in human OA cartilage samples and IL-1ß stimulated human chondrocytes. Secondly, ANGPTL2 upregulation promotes extracellular matrix (ECM) degradation and inflammation mediator production in human chondrocytes. Finally, ANGPTL2 activated the NF-κB and p38/MAPK signaling pathways via integrin α5ß1. This study, for the first time, highlights that ANGPTL2 secreted by human chondrocytes plays a negative role in the pathogenesis of osteoarthritis, and it may be a potential therapeutic target in OA.

Persistent Pleural Lesions and Inflammation by Pulmonary Exposure of Multiwalled Carbon Nanotubes.

Translocation of multiwalled carbon nanotubes (MWCNTs) from the lung to the pleural cavity, deposition of the fibers in the pleural tissue, induction of pleural fibrosis, and mesothelial proliferation have been found in rodents administered MWCNTs by different pulmonary exposure methods. However, whether the translocation and deposition and the subsequent pleural inflammation are associated with the pleural lesions is unclear. In the present study, male F344 rats were given 250 µg of two types of MWCNTs, with crocidolite as a positive control, 2 times/week for 4 weeks by intratracheal spraying. At 24 h and at 3 months after the last spraying, the rats were sacrificed for histological examination of the lung and chest wall; pleural cavity lavage was also collected at sacrifice for observation of pleural inflammatory reactions. The results indicated that intratracheally sprayed MWCNTs, like crocidolite fibers, translocated into the pleural cavity, deposited in the pleura, and induced persistent infiltration of immune cells into the pleural cavity, persistent pleural fibrosis, and mesothelial proliferation. The number of MWCNT fibers detected in the pleural cavity lavage was parallel to the number of infiltrating immune cells, which were mainly composed of macrophages. Analysis of cytokines in the fluids of the pleural cavity lavages by suspension array indicated that levels of IL-2, IL-18, and IP-10 were significantly increased both at 24 h and at 3 months after the last spraying. In vitro proliferation assays revealed that a mixture of IL-2, IL-18, and IP-10, but not any of these cytokines alone, promoted cell proliferation of human fibroblasts and mesothelial cells. These results suggest that translocated and deposited MWCNTs induce subsequent pleural inflammation and that increased IL-2, IL-18, and IP-10 synergistically promote the development of pleural fibrosis and mesothelial proliferation.

The doses of plasmid backbone plays a major role in determining the HBV clearance in hydrodynamic injection mouse model.

BACKGROUND: Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, causing a major risk of liver disease and hepatocellular carcinoma (HCC). Many mouse models have been tried to establish HBV infection through injection with various HBV-containing plasmids. However, it is not well understood that different plasmids, all of which contain the similar HBV genome, even the same plasmids with different dose, results in opposite immune responses toward HBV. METHODS: In this study, we investigated the role of HBV-containing plasmid backbones and the HBcAg in determining the HBV persistence. C57BL/6 mice were injected hydrodynamically with 6 µg or 20 µg of WT pAAV/HBV1.2 plasmid, e/core-null pAAV/HBV1.2 plasmid, or none-HBV genome pAAV/control plasmid. Serum levels of HBV-related markers were measured by quantitative immunoradiometric assay (IRMA). Liver HBcAg expression was detected by immunohistochemical staining. The mRNA levels of cytokines and Th1-related immune factors were quantified by qRT-PCR. RESULTS: All mice injected with 6 µg of the pAAV/HBV1.2 plasmid shows HBsAg positive at week 6 after hydrodynamic injection (AHI) as previously investigated. However, the mice injected with 20 µg pAAV/HBV1.2 or 6µgpAAV/HBV1.2 plus 14µgpAAV/control plasmid results in HBV clearance within 4 weeks AHI, indicating the anti-HBV activity is induced by 20 µg plasmid DNA, but not by the inserted viral genome. This anti-HBV activity is independent of HBcAg and Toll like receptor (TLR) signaling pathway, since the lack of HBcAg in pAAV/HBV1.2 plasmid or stimulation with TLRs agonists does not influence the kinetics of serum HBsAg in mice. The mRNA levels of t-bet and cxcr3 were dramatically up-regulated in the liver of the mice injected with 20 µg plasmid DNA. CONCLUSION: Our studies demonstrate that plasmid backbones are responsible for modulating immune responses to determine HBV persistence or clearance in our HBV mouse model by hydrodynamic injection of HBV-containing plasmid, and Th1 cells play key roles on HBV clearance.

Decreased Sp1 Expression Mediates Downregulation of SHIP2 in Gastric Cancer Cells.

Past studies have shown that the Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) is commonly downregulated in gastric cancer, which contributes to elevated activation of PI3K/Akt signaling, proliferation and tumorigenesis of gastric cancer cells. However, the mechanisms underlying the reduced expression of SHIP2 in gastric cancer remain unclear. While gene copy number variation analysis and exon sequencing indicated the absence of genomic alterations of SHIP2, bisulfite genomic sequencing (BGS) showed promoter hypomethylation of SHIP2 in gastric cancer cells. Analysis of transcriptional activity of SHIP2 promoter revealed Specificity protein 1 (Sp1) was responsible for the regulation of SHIP2 expression in gastric cancer cells. Furthermore, Sp1 expression, but not Sp3, was frequently downregulated in gastric cancer compared with normal gastric mucosa, which was associated with a paralleled reduction in SHIP2 levels in gastric cancer. Moreover, overexpression of Sp1 inhibited cell proliferation, induced apoptosis, suppressed cell motility and invasion in gastric cancer cells in vitro, which was, at least in part, due to transcriptional activation of SHIP2 mediated by Sp1, thereby inactivating Akt. Collectively, these results indicate that decreased expression of transcription factor Sp1 contributes to suppression of SHIP2 in gastric cancer cells.

Chemokine (C-C motif) ligand 3 detection in the serum of persons exposed to asbestos: A patient-based study.

Exposure to asbestos results in serious risk of developing lung and mesothelial diseases. Currently, there are no biomarkers that can be used to diagnose asbestos exposure. The purpose of the present study was to determine whether the levels or detection rate of chemokine (C-C motif) ligand 3 (CCL3) in the serum are elevated in persons exposed to asbestos. The primary study group consisted of 76 healthy subjects not exposed to asbestos and 172 healthy subjects possibly exposed to asbestos. The secondary study group consisted of 535 subjects possibly exposed to asbestos and diagnosed with pleural plaque (412), benign hydrothorax (10), asbestosis (86), lung cancer (17), and malignant mesothelioma (10). All study subjects who were possibly exposed to asbestos had a certificate of asbestos exposure issued by the Japanese Ministry of Health, Labour and Welfare. For the primary study group, levels of serum CCL3 did not differ between the two groups. However, the detection rate of CCL3 in the serum of healthy subjects possibly exposed to asbestos (30.2%) was significantly higher (P < 0.001) than for the control group (6.6%). The pleural plaque, benign hydrothorax, asbestosis, and lung cancer groups had serum CCL3 levels and detection rates similar to that of healthy subjects possibly exposed to asbestos. The CCL3 chemokine was detected in the serum of 9 of the 10 patients diagnosed with malignant mesothelioma. Three of the patients with malignant mesothelioma had exceptionally high CCL3 levels. Malignant mesothelioma cells from four biopsy cases and an autopsy case were positive for CCL3, possibly identifying the source of the CCL3 in the three malignant mesothelioma patients with exceptionally high serum CCL3 levels. In conclusion, a significantly higher percentage of healthy persons possibly exposed to asbestos had detectable levels of serum CCL3 compared to healthy unexposed control subjects.

Size- and shape-dependent pleural translocation, deposition, fibrogenesis, and mesothelial proliferation by multiwalled carbon nanotubes.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure similar to asbestos, raising concern that MWCNT exposure may lead to asbestos-like diseases. Previously we showed that MWCNT translocated from the lung alveoli into the pleural cavity and caused mesothelial proliferation and fibrosis in the visceral pleura. Multiwalled carbon nanotubes were not found in the parietal pleura, the initial site of development of asbestos-caused pleural diseases in humans, probably due to the short exposure period of the study. In the present study, we extended the exposure period to 24 weeks to determine whether the size and shape of MWCNT impact on deposition and lesion development in the pleura and lung. Two different MWCNTs were chosen for this study: a larger sized needle-like MWCNT (MWCNT-L; l = 8 µm, d = 150 nm), and a smaller sized MWCNT (MWCNT-S; l = 3 µm, d = 15 nm), which forms cotton candy-like aggregates. Both MWCNT-L and MWCNT-S suspensions were administered to the rat lung once every 2 weeks for 24 weeks by transtracheal intrapulmonary spraying. It was found that MWCNT-L, but not MWCNT-S, translocated into the pleural cavity, deposited in the parietal pleura, and induced fibrosis and patchy parietal mesothelial proliferation lesions. In addition, MWCNT-L induced stronger inflammatory reactions including increased inflammatory cell number and cytokine/chemokine levels in the pleural cavity lavage than MWCNT-S. In contrast, MWCNT-S induced stronger inflammation and higher 8-hydroxydeoxyguanosine level in the lung tissue than MWCNT-L. These results suggest that MWCNT-L has higher risk of causing asbestos-like pleural lesions relevant to mesothelioma development.

Inhibition of intestinal polyp growth by oral ingestion of bovine lactoferrin and immune cells in the large intestine.

Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.

Nanosized zinc oxide particles do not promote DHPN-induced lung carcinogenesis but cause reversible epithelial hyperplasia of terminal bronchioles.

Zinc oxide (ZnO) is known to induce lung toxicity, including terminal bronchiolar epithelial hyperplasia, which gives rise to concerns that nanosized ZnO (nZnO) might lead to lung carcinogenesis. We studied the tumor promoting activity of nZnO by an initiation-promotion protocol using human c-Ha-ras proto-oncogene transgenic rats (Hras128 rats). The rats were given 0.2 % N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water for 2 weeks and then treated with 0.5 ml of 250 or 500 µg/ml nZnO suspension by intra-pulmonary spraying once every 2 weeks for a total of 7 times. Treatment with nZnO particles did not promote DHPN-induced lung carcinogenesis. However, nZnO dose-dependently caused epithelial hyperplasia of terminal bronchioles (EHTB) and fibrosis-associated interstitial pneumonitis (FAIP) that were independent of DHPN treatment. Tracing the fate of EHTB lesions in wild-type rats indicated that the hyperplastic lesions almost completely disappeared within 12 weeks after the last nZnO treatment. Since nZnO particles were not found in the lung and ZnCl2 solution induced similar lung lesions and gene expression profiles, the observed lesions were most likely caused by dissolved Zn(2+). In summary, nZnO did not promote carcinogenesis in the lung and induced EHTB and FAIP lesions that regressed rapidly, probably due to clearance of surplus Zn(2+) from the lung.

Dihydrotanshinone I inhibits gallbladder cancer growth by targeting the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation.

BACKGROUND: Gallbladder cancer (GBC) poses a significant risk to human health. Its development is influenced by numerous factors, particularly the homeostasis of reactive oxygen species (ROS) within cells. This homeostasis is crucial for tumor cell survival, and abnormal regulation of ROS is associated with the occurrence and progression of many cancers. Dihydrotanshinone I (DHT I), a biologically effective ingredient isolated from Salvia miltiorrhiza, has exhibited cytotoxic properties against various tumor cells by inducing apoptosis. However, the precise molecular mechanisms by which dht I exerts its cytotoxic effects remain unclear. PURPOSE: To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms. METHODS: The proliferation of GBC cells, NOZ and SGC-996, was assessed using various assays, including CCK-8 assay, colony formation assay and EdU staining. We also examined cell apoptosis, cell cycle progression, ROS levels, and alterations in mitochondrial membrane potential to delve into the intricate molecular mechanism. Quantitative PCR (qPCR), immunofluorescence staining, and Western blotting were performed to evaluate target gene expression at both the mRNA and protein levels. The correlation between nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1) were examined using co-immunoprecipitation. Finally, the in vivo effect of dht I was investigated using a xenograft model of gallbladder cancer in mice. RESULTS: Our research findings indicated that dht I exerted cytotoxic effects on GBC cells, including inhibiting proliferation, disrupting mitochondrial membrane potential, inducing oxidative stress and apoptosis. Our in vivo studies substantiated the inhibition of dht I on tumor growth in xenograft nude mice. Mechanistically, dht I primarily targeted Nrf2 by promoting Keap1 mediated Nrf2 degradation and inhibiting protein kinase C (PKC) induced Nrf2 phosphorylation. This leads to the suppression of Nrf2 nuclear translocation and reduction of its target gene expression. Moreover, Nrf2 overexpression effectively counteracted the anti-tumor effects of dht I, while Nrf2 knockdown significantly enhanced the inhibitory effect of dht I on GBC. Meanwhile, PKC inhibitors and nuclear import inhibitors increased the sensitivity of GBC cells to dht I treatment. Conversely, Nrf2 activators, proteasome inhibitors, antioxidants and PKC activators all antagonized dht I induced apoptosis and ROS generation in NOZ and SGC-996 cells. CONCLUSION: Our findings indicated that dht I inhibited the growth of GBC cells by regulating the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation. These insights provide a strong rationale for further investigation of dht I as a potential therapeutic agent for GBC treatment.

Demethylase FTO enhances the PI3K/Akt signaling to promote gastric cancer malignancy.

Enhanced expression of fat mass and obesity-associated protein (FTO) has been reported in gastric cancer (GC). Bioinformatical studies indicate that FTO expression is correlated with the patients' overall survival (OS). How FTO exerts its promotion effects on GC development and affects OS remains largely unknown. In this study, the prognostic relevance of FTO expression in human GC tissues and the molecular mechanisms underlying FTO's promotion roles were investigated. Kaplan-Meier survival curve analysis revealed that the patients with high FTO levels had shorter OS compared to those with low FTO expression (p < 0.0001). Univariate and multivariate COX regression analyses showed that the patients' OS was affected by FTO status (p < 0.0001, p = 0.001, respectively). FTO knockdown in HGC27 cells by shRNAs reduced cell proliferation, colony formation, migration and invasion, while FTO overexpression in AGS cells had reverse effects. FTO knockdown in HGC27 cells also suppressed the tumor growth in a mouse xenograft model. High-throughput transcriptome sequencing indicated that FTO enhanced the PI3K/Akt signaling, which was confirmed in vitro. In summary, our research revealed that FTO is a potent prognostic biomarker of GC. FTO enhances the PI3K/Akt signaling and thus, promotes GC development.

Tumor-activated neutrophils promote metastasis in breast cancer via the G-CSF-RLN2-MMP-9 axis.

The immune component of the tumor microenvironment is essential for the regulation of cancer progression. In breast cancer (BC), a patient's tumor mass is frequently infiltrated by neutrophils (tumor-associated neutrophils, TANs). Our study addressed the role of TANs and their mechanism of action in BC. Using quantitative IHC, ROC, and Cox analysis, we demonstrated that a high density of TANs infiltrating the tumor parenchyma was predictive of poor prognosis and of decreased progression-free survival of patients with BC, who underwent surgical tumor removal without previous neoadjuvant chemotherapy, in 3 different cohorts: training, validation, and independent cohorts. Conditioned medium from human BC cell lines prolonged the lifespan of healthy donor neutrophils ex vivo. Neutrophils activated by the supernatants of BC lines demonstrated an increased ability to stimulate proliferation, migration, and invasive activity of BC cells. Cytokines involved in this process were identified using antibody arrays. The relationship between these cytokines and the density of TANs was validated by ELISA and IHC in fresh BC surgical samples. It was determined that tumor-derived G-CSF significantly extended the lifespan and increased the metastasis-promoting activities of neutrophils via the PI3K-AKT and NF-κB pathways. Simultaneously, TAN-derived RLN2 promoted the migratory abilities of MCF7 cells via PI3K-AKT-MMP-9. Analysis of tumor tissues from 20 patients with BC identified a positive correlation between the density of TANs and the activation of the G-CSF-RLN2-MMP-9 axis. Finally, our data demonstrated that TANs in human BC have detrimental effects, supporting malignant cell invasion and migration.

Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats.

Multi-walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 µg/mL suspensions of multi-walled carbon nanotubes or crocidolite five times over a 9-day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi-walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi-walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10-fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammation-induced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi-walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi-walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development.

Agonists Specific for κ-Opioid Receptor Induces Apoptosis of HCC Cells Through Enhanced Endoplasmic Reticulum Stress.

Cancer pain is an important factor affecting life quality of patients especially in the advanced stage and relieving pain is one of fundamental strategies for cancer treatment. Opioids such as morphine are the most widely used in clinics. However, they have been reported to be associated with the occurrence and development of several types of cancer. Thus, search for an opioid that has analgesic effect and can retard cancer progress simultaneously is critical for cancer management. In this study, we first examined the expression of µ and κ (MOR and KOR) in cell lines and tumor tissues of hepatocellular carcinoma (HCC), a malignant tumor with high mortality, and then compared the effects of opioid receptors-specific agonists on malignant phenotypes of HCC cells in vitro and tumor growth in an HCC xenograft mouse model. KOR and MOR were found to be highly expressed in HCC cell lines and HCC tissues. The KOR-specific agonist U50488h, oxycodone (agonist for both KOR and MOR) and the MOR-specific agonist morphine inhibited HCC cell proliferation, while only U50488h and oxycodone suppressed colony formation and migration of HCC cells. U50488h and oxycodone, but not morphine, induced HCC apoptosis. Further detection of PERK, GRP78 and CHOP revealed that PERK signaling was upregulated by treatment with U50488h, while treatment with the PERK inhibitor GSK2656157 partially reversed the promotion of apoptosis and inhibition of cell proliferation by U50488h, indicating that endoplasmic reticulum stress is associated with its suppressing effect on HCC malignant phenotypes. Similar to the in vitro results, HCC growth was significantly reduced by administration of U50488h and oxycodone, but not by morphine, in the HCC xenograft mouse model. PERK and caspase-3 in the HCC tissues were up-regulated by U50488h treatment as detected by immunohistochemistry and western blotting. Taken together, our results revealed that activation of KOR by U50488h inhibited malignant phenotypes of HCC both in vitro and in vivo, while activation of MOR by morphine did not have such effect. Because of their dual roles in the relief of pain and in the suppression of malignant phenotypes, opioids such as U50488h that act on KOR should be considered as the first choice for HCC management.