Indomethacin can induce cell death in rat gastric parietal cells through alteration of some apoptosis- and autophagy-associated molecules.

In clinical medicine, indomethacin (IND, a non-steroidal anti-inflammatory drug) is used variously in the treatment of severe osteoarthritis, rheumatoid arthritis, gouty arthritis or ankylosing spondylitis. A common complication found alongside the therapeutic characteristics is gastric mucosal damage. This complication is mediated through apoptosis and autophagy of the gastrointestinal mucosal epithelium. Apoptosis and autophagy are critical homeostatic pathways catalysed by caspases downstream of the gastrointestinal mucosal epithelial injury. Both act through molecular signalling pathways characterized by the initiation, mediation, execution and regulation of the cell regulatory cycle. In this study we hypothesized that dysregulated apoptosis and autophagy are associated with IND-induced gastric damage. We examined the spectra of in vivo experimental gastric ulcers in male Sprague-Dawley rats through gastric gavage of IND. Following an 18-hour fast, IND was administered to experimental rats. They were sacrificed at 3-, 6- and 12-hour intervals. Parietal cells (H+ , K+ -ATPase ß-subunit assay) and apoptosis (TUNEL assay) were determined. The expression of apoptosis-signalling caspase (caspases 3, 8, 9 and 12), DNA damage (anti-phospho-histone H2A.X) and autophagy (MAP-LC3, LAMP-1 and cathepsin B)-related molecules in gastric mucosal cells was examined. The administration of IND was associated with gastric mucosal erosions and ulcerations mainly involving the gastric parietal cells (PCs) of the isthmic and upper neck regions and a time-dependent gradual increase in the number of apoptotic PCs with the induction of both apoptotic (upregulation of caspases 3 and 8) cell death and autophagic (MAP-LC3-II, LAMP-1 and cathepsin B) cell death. Autophagy induced by fasting and IND 3 hours initially prompted the degradation of caspase 8. After 6 and 12 hours, damping down of autophagic activity occurred, resulting in the upregulation of active caspase 8 and its nuclear translocation. In conclusion we report that IND can induce time-dependent apoptotic and autophagic cell death of PCs. Our study provides the first indication of the interactions between these two homeostatic pathways in this context.

Interleukin-24 is a novel diagnostic biomarker for the severity of acute kidney injury.

There is a clinical need for sensitive acute kidney injury (AKI) biomarkers that enable early therapeutic interventions and prediction of disease prognosis. In this study, we monitored interleukin (IL)-24 expressed in kidneys with severe AKI that progresses to atrophic kidney in a mouse model of ischemia-reperfusion injury (IRI). Therefore, we evaluated IL-24 as a potential biomarker not only for early diagnosis of AKI, but also for predicting progression to chronic kidney disease (CKD). Serum IL-24 was detected earlier than the elevation of serum creatinine levels and urinary IL-24 was detected as early as neutrophil gelatinase associated lipocalin (NGAL) in severe AKI (60 min of IRI). In addition, serum and urine IL-24 levels tended to increase in relation to ischemia duration. In such kidneys, vascular smooth muscle cells expressed IL-24 in response to the injury in the renal tubular epithelial cell and its target was the renal tubular epithelial cell itself. IL-24 may play a pivotal role in the communication between tubular epithelial cells and vascular smooth muscle cells and, in conclusion, IL-24 can be used as a sensitive biomarker for AKI.

Ethanol-Induced Autophagy in Sertoli Cells Is Specifically Marked at Androgen-Dependent Stages of the Spermatogenic Cycle: Potential Mechanisms and Implications.

In a recent study, we reported that acute ethanol exposure enhanced autophagy in Sertoli cells (SCs) of adult rats. However, further research is needed to clarify the specific spermatogenic stage exhibiting the highest autophagic response, the mechanisms behind such specificity, and the related relevance to sperm. This brief report provides results indicating that stages VII⁻VIII (androgen-dependent or spermiation stages) of the spermatogenic cycle exhibited more marked autophagic response in acute-ethanol treated rats (ETRs) than other stages based on suppression of androgen receptor (AR), analysis of microtubule-associated protein 1 light chain 3 (LC3) (an autophagosomal marker) immunostaining in SCs, double labeling of LC3 and lysosomal proteins and electron microscopy. Ultrastructural observations and TUNEL method revealed a notable presence of phagocytosed apoptotic germ cells and retained sperm in SCs of ETRs at these specific stages-a finding rarely observed in control testes. In addition, PTEN-induced putative kinase 1 ( PINK1) (a sensor of mitochondrial damage and mitophagy) and giant lipid droplets were found to have accumulated in SCs of ETRs at same stages. Our data show novel findings indicating that stages VII⁻VIII of the spermatogenic cycle exhibit high levels of autophagy, specifically under stress conditions, as expressed by the term autophagic stages. This stage-specific upregulation of autophagy in SCs may be related to AR suppression, mitochondrial damage, lipid accumulation, and phagocytosis of apoptotic cells. The phenomenon may be an essential part of ensuring the viability of SCs and supporting germ cells in toxic environments.

GABAB receptor regulates proliferation in the high-grade chondrosarcoma cell line OUMS-27 via apoptotic pathways.

BACKGROUND: High-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line. METHODS: We evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells. RESULTS: The GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways. CONCLUSIONS: The GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells.

Quantified kidney echogenicity in mice with renal ischemia reperfusion injury: evaluation as a noninvasive biomarker of acute kidney injury.

The purpose is to evaluate quantified kidney echogenicity as a biomarker for the early diagnosis of acute kidney injury (AKI) and predicting progression to chronic kidney disease (CKD) in a mouse model of ischemia-reperfusion injury (IRI). Two separate protocols of murine models of IRI were used: (1) 10, 30, and 40 min of bilateral ischemia duration and (2) 45 and 60 min of unilateral ischemia duration. Renal echogenicity was measured with ultrasound and compared with serum creatinine or urine neutrophil gelatinase-associated lipocalin (NGAL) at various timepoints after IRI. In mice subjected to 10, 30, and 40 min of bilateral ischemia, renal echogenicity increased about 2 h after IRI for all ischemia times, earlier than serum creatinine or urine NGAL. In those subjected to 45 and 60 min of unilateral ischemia, 60 min of unilateral ischemia, which represents atrophic changes 28 days after IRI, resulted in a sustained high level of echogenicity and was significantly different 24 h after IRI, while 45 min of unilateral ischemia resulted in trivial levels of histological damage 28 days after IRI. Renal echogenicity might have the potential to be a biomarker for the early diagnosis of AKI and the prognosis of CKD.

Positive feedback of DDX6/c-Myc/PTB1 regulated by miR-124 contributes to maintenance of the Warburg effect in colon cancer cells.

The human DEAD/H-box RNA helicase gene DDX6 is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein has been shown to cause malignant transformation. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, ribosome assembly, and more. However, details of the regulatory mechanism of DDX6 and functions of DDX6 in cancer cells are largely unknown. On the other hand, the Warburg effect is a well-known feature of cancer cells. Pyruvate kinase in muscle (PKM), which is a rate-limiting glycolytic enzyme, has 2 isoforms, PKM1 and PKM2. It has been frequently reported that PKM2 is a tumor-specific isoform and promotes the Warburg effect. However, the functions of the PKM1 gene have been hardly mentioned. Here, we showed that DDX6 was overexpressed in colorectal cancer specimens and regulated by microRNA (miR)-124 in colon cancer cells. Also, a DDX6/c-Myc/PTB1 positive feedback circuit regulated by miR-124 was shown to be established and to contribute to maintenance of the Warburg effect. Moreover, we showed that knockdown of DDX6 induced mainly apoptosis through an imbalance of PKM gene expression, especially causing down-regulation of PKM1 in colon cancer cells. These results suggest that miR-124 is a fine tuner of the Warburg effect and that DDX6 is one of the key molecules in Warburg effect-related miR-124 targeting various genes.

Recombinant human soluble thrombomodulin improved lipopolysaccharide/d-galactosamine-induced acute liver failure in mice.

The effect of recombinant human soluble thrombomodulin (TM-α) on acute liver failure (ALF) is unclear, and we elucidated the effect of TM-α in lipopolysaccharide (LPS)/d-galactosamine (GalN)-induced ALF in mice. Placebo (saline) or TM-α (100 mg/kg) was administered 1 h after LPS/GalN administration. Survival rates were evaluated for 24 h after LPS/GalN administration. Plasma and liver samples were evaluated 1, 3, and 7 h after LPS/GalN administration. Survival rates were significantly higher in the TM-α-treated group than in the placebo group. A significant augmentation of plasma high-mobility group box 1 protein (HMGB1) was observed 7 h after LPS/GalN administration. In the TM-α-treated mice, plasma HMGB1 was significantly lower than in the placebo group. A significant augmentation of hepatic nuclear factor (NF)-κB p65 was observed in the placebo-treated group, whereas a significant reduction, relative to placebo, was observed in the TM-α-treated group. Hepatic expression of tumor necrosis factor (TNF)-α and myeloperoxidase were significantly increased in the placebo group, and were similarly significantly attenuated in the TM-α-treated group. TM-α treatment also produced a significant attenuation of liver neutrophil accumulation after LPS/GalN administration. Thus, TM-α may become a useful treatment strategy for reducing the symptoms of ALF via the attenuation of LPS/GalN-induced HMGB1 levels.

Chymase inhibition attenuates lipopolysaccharide/ d-galactosamine-induced acute liver failure in hamsters.

BACKGROUND/AIMS: Chymase inhibition has been shown to attenuate matrix metalloproteinase (MMP)-9 and tumor necrosis factor (TNF)-α, both of which are associated with the pathogenesis of acute liver failure (ALF). This study investigated the effects of the chymase inhibitor TY-51469 on lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced ALF in hamsters. METHODS: TY-51469 (10 or 30 mg/kg) or placebo was administered 1 h before the LPS (160 µg/kg)/GalN (400 mg/kg) injection. RESULTS: Hepatic chymase activity was significantly increased after the LPS/GalN injection, but the significant increase was dose-dependently and significantly attenuated by treatment with TY-51469. Significant increases in hepatic MMP-9 activity and TNF-α concentration were observed after the LPS/GalN injection, but these increases were also attenuated by treatment with TY-51469. Plasma aspartate aminotransferase and alanine aminotransferase activities were significantly increased after LPS/GalN injection in the placebo-treated group, but the increases were significantly attenuated in the TY-51469-treated group. The area of hepatic necrotic after LPS/GalN injection was significantly reduced by treatment with TY-51469. Treatment with TY-51469 resulted in significant reductions in the hepatic malondialdehyde concentration, mast cell numbers, and gene expressions of interleukin-1ß and myeloperoxidase. DISCUSSION: Chymase inhibition could be a useful strategy to attenuate LPS/GalN-induced ALF in hamsters.

Regulation of bcl-2 transcription by estrogen receptor-α and c-Jun in human endometrium.

The estrogen-estrogen receptor (ER) signaling pathway plays crucial physiologic roles in not only the control of reproduction, but also in the generation of cancer in the breast and uterus. While some ER target genes have been identified containing the estrogen-responsive element (ERE), others are activated eventually by ER via protein-protein interaction without binding to ERE. In a previous study, we identified that the proliferative phase-specific expression of the bcl-2 gene in glandular cells could be regulated by the binding of c-Jun to its motifs in the promoter. Results from our present study indicate that the menstrual cyclic expression of bcl-2 could be controlled by either direct binding of ERα to ERE in the c-Jun promoter or the interaction of ERα with c-Jun that binds to its motifs in the bcl-2 gene. Intriguingly, the transcriptionally active form of c-Jun phosphorylated at Ser63 was identified binding to its motifs in the bcl-2 gene in a menstrual cyclic non-specific manner. Our study revealed a novel mechanism that transcriptionally regulates the expression of bcl-2 in the normal human endometrium.

Immunohistochemical analysis of O(6)-methylguanine-DNA methyltransferase in human melanoma in comparison with skin squamous cell carcinoma.

Alkylating agents, often used for chemotherapy in patients with melanoma, can produce O(6)-alkylguanine (O(6)AG) which is related to tumor cell killing after treatment with alkylating agents. O(6)AG is effectively eliminated by O(6)-methylguanine-DNA methyltransferase (O(6)MGMT) and its level is correlative to the resistance to alkylating agents. However, little is known about the relationship of O(6)MGMT to the characteristics of melanoma. This study investigated the expression of O(6)MGMT in 12 melanomas and compared it with that in 11 skin squamous cell cancers (SCCs) immunohistochemically to evaluate the O(6)MGMT activity in melanoma and its clinical significance. All of the SCC samples had high O(6)MGMT expression, while the expression of O(6)MGMT in melanoma was diverse and 4 out of 12 samples had no or extremely low O(6)MGMT activity. Out of 6 lesions obtained from metastasis, 4 had a high O(6)MGMT activity. Two out of 3 cases with a low O(6)MGMT activity in each primary lesion did not show any evidence of metastasis or local recurrence. The evaluation of O(6)MGMT activity in melanoma may, therefore, be useful to determine the characteristics of tumor in each melanoma case. In addition, the present study implies the possibility of selective cancer chemotherapy for melanoma in the near future.

Significance of matrix metalloproteinase-9 inhibition by imidapril for prevention of abdominal aortic aneurysms in angiotensin II type 1 receptor-knockout mice.

To clarify the matrix metalloproteinase (MMP)-9 inhibitory effect of an angiotensin-converting enzyme (ACE) inhibitor in vivo, we evaluated the effect of an ACE inhibitor against elastase-induced abdominal aortic aneurysm (AAA) progression in mice. Molecular models showed that imidapril bound directly to the mouse MMP-9 active center. An active form of imidapril, imidaprilat, dose-dependently inhibited MMP-9 activity in the extract from elastase-induced AAA in wild-type mice. Imidapril (10 mg/kg per day) was administered to wild-type or angiotensin II type 1 (AT1) receptor knockout mice. Blood pressure was significantly lower in AT1 receptor-knockout mice than in wild-type mice, but imidapril did not affect blood pressure in AT1 receptor-knockout mice. The aortic diameter was significantly expanded after elastase application, but the expansion was significantly lower in AT1 receptor-knockout mice than in wild-type mice. In AT1 receptor-knockout mice, the aortic expansion was further attenuated by imidapril. MMP-9 activity in aorta was significantly augmented after elastase application. The MMP-9 activity was significantly lower in AT1 receptor-knockout mice than in wild-type mice, and it was further attenuated by imidapril. In conclusion, MMP-9 inhibition by imidapril might contribute to the attenuation of AAA progression in AT1 receptor-knockout mice.

Chymase inhibitor ameliorates hepatic steatosis and fibrosis on established non-alcoholic steatohepatitis in hamsters fed a methionine- and choline-deficient diet.

AIM: Chymase plays a role in the augmentation of angiotensin II formation, which is involved in liver fibrosis. The therapeutic effects of a chymase inhibitor, TY-51469, on established hepatic steatosis and fibrosis were investigated in a model of developed non-alcoholic steatohepatitis. METHODS: Hamsters were fed a normal diet or methionine- and choline-deficient (MCD) diet for 12 weeks. Then, treatment with TY-51469 (1 mg/kg per day) or placebo was initiated, and the treatment was continued concurrently with the MCD diet for an additional 12 weeks. RESULTS: At 12 weeks after initiating the MCD diet, marked hepatic steatosis and fibrosis were observed in MCD diet-fed hamsters. Malondialdehyde and gene expression levels of collagen I, collagen III, α-smooth muscle actin (α-SMA) and Rac-1 in liver extracts were also increased in the MCD-diet-fed hamsters at 12 weeks. At 24 weeks, hepatic steatosis and fibrosis were more prominent in the placebo-treated hamsters that were fed the MCD-diet for 24 weeks versus 12 weeks. Hamsters treated with TY-51469 for 12 weeks after being on a 12-week MCD diet had significant ameliorations in both hepatic steatosis and fibrosis, and there were no significant differences compared to normal diet-fed hamsters. There were significant augmentations in angiotensin II and malondialdehyde, and gene expressions of collagen I, collagen III, α-SMA and Rac-1 in the placebo-treated hamsters at 24 weeks; however, these levels were reduced to normal levels in the TY-51469-treated hamsters. CONCLUSION: TY-51469 not only prevented the progression of hepatic steatosis and fibrosis, but also ameliorated hepatic steatosis and fibrosis.

Alterations in cell cycle and induction of apoptotic cell death in breast cancer cells treated with α-mangostin extracted from mangosteen pericarp.

The development of molecularly targeted drugs has greatly advanced cancer therapy, despite these drugs being associated with some serious problems. Recently, increasing attention has been paid to the anticancer effects of natural products. α-Mangostin, a xanthone isolated from the pericarp of mangosteen fruit, has been shown to induce apoptosis in various cancer cell lines and to exhibit antitumor activity in a mouse mammary cancer model. In this study, we investigated the influence of α-mangostin on apoptosis and cell cycle in the human breast cancer cell line MDA-MB231 (carrying a p53 mutation, and HER2, ER, and PgR negative) in order to elucidate its anticancer mechanisms. In α-mangostin-treated cells, induction of mitochondria-mediated apoptosis was observed. On cell-cycle analysis, G1-phase arrest, increased p21(cip1) expression and decreases in cyclins, cdc(s), CDKs and PCNA were observed. In conclusion, α-mangostin may be useful as a therapeutic agent for breast cancer carrying a p53 mutation and having HER2- and hormone receptor-negative subtypes.

Outside fibroblasts play a key role in the development of inner neointima after the implantation of polytetrafluoroethylene grafts.

The neointima formation inside of polytetrafluoroethylene (PTFE) grafts may be associated with the migration of outside fibroblasts to the luminal surfaces. This study aimed to verify whether blockade of fibroblast migration can prevent neointima formation by testing two types of prosthetic vessels, the porous PTFE graft and the impermeable Grasil graft, respectively. After implantation of the PTFE graft in dogs, a time-dependent migration of outside fibroblasts to the luminal side occurred. Compared with the PTFE grafts, the total neointima formation in the Grasil grafts was significantly less. Although the neointima formation at the arterial or venous anastomotic regions did not significantly differ between the two grafts, the neointima at the middle region of the PTFE grafts was significantly evident than the Grasil grafts. The components of the renin­angiotensin system (RAS), such as angiotensin II and its receptor AT1, as well as the forming enzymes of the RAS (angiotensin-converting enzyme and chymase), were all detectable in the grafts' surrounding tissues. Neointima formation at the middle region of the prosthetic vessels could be suppressed almost completely by the blockade of outside fibroblast migration, indicating that outside fibroblasts play a key role in the formation of neointima in this region.

Assessment of On-Board and Laboratory Gas Measurement Systems for Future Heavy-Duty Emissions Regulations.

Road transport contributes significantly to air pollution in cities. Regulations across the globe continuously reduce the limits that vehicles need to respect during their lifetimes. Furthermore, more pollutants are being subject to control with new regulations and, most important, testing tends to be done under real-world conditions on the road. In this study, various portable systems were compared with laboratory-grade equipment with a wide range of emissions, focusing on the lower end, where the measurement uncertainty of the instruments is crucial for the determination of emission limits. The engines were diesel- and compressed natural gas (CNG)-fueled. The results were promising, with relatively small differences between portable emissions measurement systems (PEMSs), portable Fourier transform infrared (FTIR) and quantum cascade laser infrared (QCL-IR) spectrometers, and the respective laboratory-grade analyzers based on chemiluminescence detection (CLD), non-dispersive infrared (NDIR), and FTIR principles. The results also highlighted the need for strict technical regulations regarding accuracy and drift for low emission limits in future.

α-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation.

BACKGROUND: The mangosteen fruit has a long history of medicinal use in Chinese and Ayurvedic medicine. Recently, the compound α-mangostin, which is isolated from the pericarp of the fruit, was shown to induce cell death in various types of cancer cells in in vitro studies. This led us to investigate the antitumor growth and antimetastatic activities of α-mangostin in an immunocompetent xenograft model of mouse metastatic mammary cancer having a p53 mutation that induces a metastatic spectrum similar to that seen in human breast cancers. METHODS: Mammary tumors, induced by inoculation of BALB/c mice syngeneic with metastatic BJMC3879luc2 cells, were subsequently treated with α-mangostin at 0, 10 and 20 mg/kg/day using mini-osmotic pumps and histopathologically examined. To investigate the mechanisms of antitumor ability by α-mangostin, in vitro studies were also conducted. RESULTS: Not only were in vivo survival rates significantly higher in the 20 mg/kg/day α-mangostin group versus controls, but both tumor volume and the multiplicity of lymph node metastases were significantly suppressed. Apoptotic levels were significantly increased in the mammary tumors of mice receiving 20 mg/kg/day and were associated with increased expression of active caspase-3 and -9. Other significant effects noted at this dose level were decreased microvessel density and lower numbers of dilated lymphatic vessels containing intraluminal tumor cells in mammary carcinoma tissues. In vitro, α-mangostin induced mitochondria-mediated apoptosis and G1-phase arrest and S-phase suppression in the cell cycle. Since activation by Akt phosphorylation plays a central role in a variety of oncogenic processes, including cell proliferation, anti-apoptotic cell death, angiogenesis and metastasis, we also investigated alterations in Akt phosphorylation induced by α-mangostin treatment both in vitro and in vivo. Quantitative analysis and immunohistochemistry showed that α-mangostin significantly decreased the levels of phospho-Akt-threonine 308 (Thr308), but not serine 473 (Ser473), in both mammary carcinoma cell cultures and mammary carcinoma tissues in vivo. CONCLUSIONS: Since lymph node involvement is the most important prognostic factor in breast cancer patients, the antimetastatic activity of α-mangostin as detected in mammary cancers carrying a p53 mutation in the present study may have specific clinical applications. In addition, α-mangostin may have chemopreventive benefits and/or prove useful as an adjuvant therapy, or as a complementary alternative medicine in the treatment of breast cancer.

The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model.

BACKGROUND: Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. A new splicing variant, endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2) that we recently identified is an endogenous selective inhibitor of lymphangiogenesis. To evaluate the antimetastatic potential of esVEGFR-2, gene therapy with vector expressing esVEGFR-2 (pesVEGFR-2) or endostatin (pEndo) as a positive control was conducted on murine metastatic mammary cancer. METHODS: Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of pesVEGFR-2, pEndo or pVec as control, once a week for six weeks. In vivo gene electrotransfer was performed on the tumors after each injection. RESULTS: Deaths from metastasis were much lower in the pesVEGFR-2 and pEndo groups than in those of the pVec. Tumor volume was significantly lower in the pesVEGFR-2 and the pEndo groups throughout the study. Multiplicity of lymph node and lung metastatic nodules was significantly suppressed in the pesVEGFR-2 and pEndo groups. Moreover, the total number of overall metastasis including the other organs was also decreased in these groups. However, pesVEGFR-2 was not able to decrease the number of lungs, ovaries, kidneys and adrenals with metastasis as counted by unilateral or bilateral metastasis. The number of CD34+/Lyve-1⁻ blood microvessels was significantly decreased in the pEndo group, while the number of CD34⁻/Lyve-1+ lymphatic vessels was significantly decreased in the pesVEGFR-2 and pEndo groups. In addition, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed in the pesVEGFR-2 and pEndo groups. Levels of apoptosis were significantly increased in the pEndo group, whereas the rates of cell proliferation were significantly decreased in the pesVEGFR-2 and pEndo groups. CONCLUSIONS: Our data demonstrate that esVEGFR-2 can inhibit mainly lymph node metastasis. The antimetastatic activity of esVEGFR-2 may be of high clinical significance in the treatment of metastatic breast cancer because lymph node involvement is a most important prognostic factor in cancer patients.

Raloxifene inhibits tumor growth and lymph node metastasis in a xenograft model of metastatic mammary cancer.

BACKGROUND: The effects of raloxifene, a novel selective estrogen receptor modulator, were studied in a mouse metastatic mammary cancer model expressing cytoplasmic ERα. METHODS: Mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879luc2 cells, were subsequently treated with raloxifene at 0, 18 and 27 mg/kg/day using mini-osmotic pumps. RESULTS: In vitro study demonstrated that the ERα in BJMC3879luc2 cells was smaller (between 50 and 64 kDa) than the normal-sized ERα (66 kDa) and showed cytoplasmic localization. A statistically significant but weak estradiol response was observed in this cell line. When BJMC3879luc2 tumors were implanted into mice, the ERα mRNA levels were significantly higher in females than in males. In vitro studies showed that raloxifene induced mitochondria-mediated apoptosis and cell-cycle arrest in the G1-phase and a decrease in the cell population in the S-phase. In animal experiments, tumor volumes were significantly suppressed in the raloxifene-treated groups. The multiplicity of lymph node metastasis was significantly decreased in the 27 mg/kg group. Levels of apoptosis were significantly increased in the raloxifene-treated groups, whereas the levels of DNA synthesis were significantly decreased in these groups. No differences in microvessel density in tumors were observed between the control and raloxifene-treated groups. The numbers of dilated lymphatic vessels containing intraluminal tumor cells were significantly reduced in mammary tumors in the raloxifene-treated groups. The levels of ERα mRNA in mammary tumors tended to be decreased in the raloxifene-treated groups. CONCLUSION: These results suggest that the antimetastatic activity of raloxifene in mammary cancer expressing cytoplasmic ERα may be a crucial finding with clinical applications and that raloxifene may be useful as an adjuvant therapy and for the chemoprevention of breast cancer development.

Riluzole induces apoptotic cell death in human prostate cancer cells via endoplasmic reticulum stress.

BACKGROUND: Ion channel modulators have been previously associated with cell proliferation and cell death in human cancer cell lines. MATERIALS AND METHODS: The effects of riluzole, an ion channel modulator, on cell proliferation, apoptosis and the apoptotic pathway in the LNCaP and C4-2 prostate cancer cell lines were investigated. RESULTS: Riluzole inhibited DNA synthesis and increased apoptotic cells in both cell lines. The activities of caspase-3, -8 and -9 were significantly increased, and caspase inhibitors for caspase-3, -8 and -9 significantly rescued the cell viability of both carcinoma cell lines treated with riluzole. However, a change in mitochondrial membrane potential, release of cytochrome c and cleavage of Bid were not observed in the riluzole-treated cells. Riluzole significantly induced elevation of caspase-4 activity, fluorescence indicating cytosolic calcium, and morphological changes in endoplasmic reticulum (ER) as observed by transmission electron microscopy. CONCLUSION: Riluzole induces inhibition of DNA synthesis and apoptotic cell death via ER stress in both the LNCaP and C4-2 prostate cancer cell lines.