Fibroblast-derived exosomal microRNA regulates NKX3-1 expression in androgen-sensitive, androgen receptor-dependent prostate cancer cells.

Androgen deprivation therapy (ADT) targeting androgen production and androgen receptor (AR) signaling is the primary antihormonal therapy in the treatment of advanced prostate cancer (PCa). However, no clinically established molecular biomarkers have been identified to predict the effectiveness of ADT before starting ADT. The tumor microenvironment of PCa contains fibroblasts that regulate PCa progression by producing multiple soluble factors. We have previously reported that AR-activating factor-secreted fibroblasts increase the responsiveness of androgen-sensitive, AR-dependent PCa cells to ADT. Thus, we hypothesized that fibroblast-derived soluble factors may affect cancer cell differentiation by regulating cancer-related gene expression in PCa cells and that the biochemical characteristics of fibroblasts may be used to predict the effectiveness of ADT. Here, we investigated the effects of normal fibroblasts (PrSC cells) and three PCa patient-derived fibroblast lines (pcPrF-M5, -M28, and -M31 cells) on the expression of cancer-related genes in androgen-sensitive, AR-dependent human PCa cells (LNCaP cells) and three sublines showing different androgen sensitivities and AR dependencies. The mRNA expression of the tumor suppressor gene NKX3-1 in LNCaP cells and E9 cells (which show low androgen sensitivity and AR dependency) was significantly increased by treatment with conditioned media from PrSC and pcPrF-M5 cells but not from pcPrF-M28 and pcPrF-M31 cells. Notably, no upregulation of NKX3-1 was observed in F10 cells (AR-V7-expressing, AR-independent cells with low androgen sensitivity) and AIDL cells (androgen-insensitive, AR-independent cells). Among 81 common fibroblast-derived exosomal microRNAs that showed 0.5-fold lower expression in pcPrF-M28 and pcPrF-M31 cells than in PrSC and pcPrF-M5 cells, miR-449c-3p and miR-3121-3p were found to target NKX3-1. In only LNCaP cells, the NKX3-1 mRNA expression was significantly increased by transfection of an miR-3121-3p mimic but not that of the miR-449c-3p mimic. Thus, fibroblast-derived exosomal miR-3121-3p may be involved in preventing the oncogenic dedifferentiation of PCa cells by targeting NKX3-1 in androgen-sensitive, AR-dependent PCa cells.

Heterogeneous induction of an invasive phenotype in prostate cancer cells by coculturing with patient-derived fibroblasts.

Prostate cancer (PCa) cells frequently invade the surrounding stroma, leading to heterogeneous formation of structural atypia. The surrounding stroma contains multiple functionally diverse populations of fibroblasts that trigger numerous changes in PCa cells including motility. Thus, we hypothesized that direct or indirect contact of PCa cells with fibroblasts determines an invasive phenotype in PCa cells. We investigated the effects of 10 different patient-derived fibroblast lines on the three-dimensional (3D) morphogenesis of PCa cells growing on a viscous substrate in vitro. When grown alone, all 10 patient-derived fibroblast lines clumped on the viscous substrate, whereas the human androgen-sensitive PCa cell line LNCaP did not. Cocultures of LNCaP cells with seven of the patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M7, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a thick fibroblast layer that resembled human prostate stromal structures. In contrast, cocultures of LNCaP cells with the remaining three fibroblast lines (NPF-M13, pcPrF-M10, and pcPrF-M26) did not form a thick fibroblast layer. Of the seven fibroblast lines that caused thick layer formation, four patient-derived fibroblast lines (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) induced an invasive phenotype in LNCaP cells with a cord-like infiltrating growth pattern, whereas the other three fibroblast lines (pcPrF-M7, pcPrF-M23, and pcPrF-M24) induced no or a very weak invasive phenotype. Using cell culture inserts, none of the four patient-derived fibroblast lines that induced an invasive phenotype (PrSC, pcPrF-M5, pcPrF-M28, and pcPrF-M31) affected CDH1 mRNA expression in LNCaP cells; yet, two patient-derived fibroblast lines (pcPrF-M5 and pcPrF-M28) increased CDH2 mRNA expression in LNCaP cells, whereas the other two fibroblast lines (PrSC and pcPrF-M31) did not. These results suggest that the existence of multiple functionally diverse populations of fibroblasts in PCa tissue may be responsible for the diversity in PCa cell invasion, leading to heterogeneous formation of structural atypia.

Autopsy findings for a case of acute gastric volvulus in a child.

Acute gastric volvulus resulting in abdominal compartment syndrome was determined to be the cause of death in a 4-year-old girl who presented with abdominal distension. At about 1AM on the day of her death, she was brought to our emergency medical center. Physical examination and plain abdominal X-ray revealed pronounced gastric dilatation. A decompression procedure was performed, followed by observation. She went into cardiopulmonary arrest around 1PM on the same day and died. Postmortem investigation, including an autopsy and computed tomography (CT), was performed to determine the cause of death. The findings included that the stomach was severely distended. Evidence was seen of mucosal hemorrhage in the gastric mucosa on the greater curvature side, which was thinned in places but without perforation. No necrosis of the gastric mucosa was observed; reversible changes were evident on histopathological examination. The postmortem CT images suggested that the pyloric region was positioned cranioventrally to the cardiac region. None of the findings indicated sudden blockage, and the cause of death was determined to be acute gastric volvulus resulting in abdominal compartment syndrome. The abnormal placement of the organs was difficult to determine based on physical examination alone; postmortem CT and careful examination were helpful in conducting the autopsy in this case.

Acute brainstem compression by intratumoral hemorrhages in an intracranial hypoglossal schwannoma.

A 77-year-old female in the hospital was found tachycardic and hypothermic by a nurse, and the patient's respiration subsequently ceased. Forensic autopsy revealed an intracranial cystic tumor that would have compressed the brainstem. On microscopic examination, the tumor was diagnosed as an Antoni A schwannoma growth, and recent multiple intratumoral hemorrhages in the intracranial schwannoma were observed, suggesting the sudden enlargement of the intracranial schwannoma due to intratumoral hemorrhaging. Accordingly, we diagnosed the cause of death as brainstem compression induced by the intratumoral hemorrhaging in the intracranial schwannoma. Meanwhile, a rhinopharyngeal tumor was also detected by the autopsy, which was compatible with an antemortem diagnosis of a dumbbell-shaped hypoglossal schwannoma.

Molecular-biological analysis of acute lung injury (ALI) induced by heat exposure and/or intravenous administration of oleic acid.

The aim of this study was to molecular-biologically investigate the interaction between heat exposure and pulmonary fat embolization in regards to the development of acute lung injury (ALI). Ten-week-old Wistar male rats were divided into four groups: (1) oleic acid injected into caudal vein after heat exposure, (2) oleic acid injected without heat exposure, (3) soybean oil injected after heat exposure, and (4) soybean oil injected without heat exposure, and then mRNA expression of eight inflammatory mediators related to ALI/acute respiratory distress syndrome (ARDS) and heat shock protein 70 (Hsp70) in lung was determined 1h after the injection. mRNA expression of interleukin 1 beta (Il1b), tumor necrosis factor alpha (Tnfa), vascular endothelial growth factor A (Vegfa), transforming growth factor beta 1 (Tgfb1) and Hsp70 was significantly increased by heat exposure, while that of Il1b, interleukin 6 (Il6), Tnfa, macrophage inflammatory protein 2 (Mip2) and granulocyte macrophage-colony stimulating factor (Gm-csf) was significantly elevated by the injection of oleic acid. Moreover, the expressions of inflammatory cytokines and chemokines in lung almost paralleled their mRNA expressions. In particular, IL-1ß expression was synergistically elevated by heat exposure followed by injection of oleic acid. Additionally, IL-6 expression tended to increase under the same conditions as well. It is likely that heat exposure itself injures lung tissue within a short time, and that more than two conditions which induce ALI/ARDS interact with each other synergistically, exacerbating the development of ALI/ARDS.

The blood concentration and organ distribution of haloperidol at therapeutic and toxic doses in severe fatty liver disease.

The aim of this study was to investigate the difference between the pharmacokinetics of haloperidol in normal rats and in rats with fatty liver disease. A therapeutic dosage (0.1 mg/kg) and a toxic dose (15 mg/kg) of haloperidol were administrated to normal 9-week-old male rats or those with severe fatty liver disease, and the blood concentration of haloperidol was determined 15 min, 1, 2, and 3 h following haloperidol administration. The concentration of haloperidol in the organs was determined 1, 2, and 3 h after the haloperidol administration. Additionally, the volume of the portal vein blood flow was measured 3 h after haloperidol administration. When given at the therapeutic dosage, the concentrations of haloperidol in both the blood and organs of the rats with fatty liver disease were significantly higher than those in the normal rats. However, when given at the toxic level, the blood and organ haloperidol concentrations 1 h after administration tended to be lower in the rats with fatty liver disease than those in the normal rats; these lower haloperidol levels returned to be the levels in the normal rats 3 h after the administration of haloperidol. The volume of the portal vein blood flow significantly increased following the toxic haloperidol dose as compared with the volume pre-administration and following the therapeutic haloperidol dose in the normal rats. However, the volume did not change after the toxic or the therapeutic dose of haloperidol compared with pre-administration in rats with severe fatty liver disease, although it was significantly higher than in the normal rats. The pathway for haloperidol metabolism might have been saturated before the administration of haloperidol in rats with fatty liver disease; thus, it is possible that the blood concentration of haloperidol tends to be much higher in individuals with severe fatty liver disease than in those with normal livers in an inverse proportion to the dosage of haloperidol.

Molecular biological analysis of cardiac effect of high temperature in rats.

The aim of this study was to investigate direct effects of heat exposure on the heart molecular-biologically and pathohistologically, using rats exposed to high temperatures. The mRNA expression of natriuretic peptide type A (Nppa), natriuretic peptide type B (Nppb), actin alpha 1 skeletal muscle (Acta1), myosin heavy polypeptide 6 cardiac muscle alpha (Myh6) and myosin heavy polypeptide 7 cardiac muscle alpha (Myh7) was determined in the hearts of the rats. Whereas the expression of Nppa and Nppb rapidly increased immediately after the heat exposure, the expression of Acta1 was gradually reduced, which indicated cardiac overload. Moreover, the expression of Myh6 and Myh7 in the heart increased 4h after the heat exposure, which suggested the involvement of a compensatory mechanism. Immunohistochemical staining with anti-fibronectin antibody showed that positive cardiomyocytes could be detected sparsely 4h after the heat exposure, and they could be clearly observed 8h after the heat exposure. Our results showed that hyperthermia causes myocardial damage shortly after the exposure to heat and that the ventricle was more vulnerable to hyperthermia-induced damage than the atrium. Cardiac dysfunction may be induced not only by hypercytokinemia but also by the direct effect of heat exposure at the early period of heat stroke, which may be one of the mechanisms by which heat causes death. Elucidating the mechanism of death from heat stroke could lead to not only diagnostic improvement but also the prevention of death from heat stroke.

A subacute epidural haematoma extending over the occipital region and posterior cranial fossa due to a laceration in the transverse sinus.

A 6-year-old male was found dead on his stomach with massive reddish vomiting from his mouth and nose. Postmortem cranial CT revealed an epidural haematoma in the left occipital region, but the cause and origin of the haematoma were unclear. An autopsy revealed that the epidural haematoma expanded over the left temporal region and the left side of the occipital region and posterior cranial fossa, and its origin was a laceration in the left transverse sinus induced by diastases in the left lambdoidal and occipitomastoid sutures. A pathohistological examination revealed that one portion of the haematoma was an early-stage hemorrhage, while the other portion extended approximately 1 week after the hemorrhage. Moreover, approximately 1 week elapsed after the laceration of the transverse sinus. Thus, we believe that the primary haematoma was induced by the laceration in the transverse sinus approximately 1 week before death, but the haematoma ceased to enlarge due to hemostasis. However, later, the size of the haematoma rapidly increased again due to rebleeding from the laceration, which led to intracranial hypertension. Consequently, we diagnosed the direct cause of death as choking due to vomit aspiration that resulted from intracranial hypertension induced by a subacute epidural haematoma.

Molecular-biological analysis of the effect of methamphetamine on the heart in restrained mice.

In order to investigate the interaction in the heart between the administration of methamphetamine (MAP) and restraint of the body following it, we administrated MAP intraperitoneally to mice and restrained them, and then determined the level of mRNA expression of 22 genes in the heart using quantitative RT-PCR method. The mRNA expressions of Nfkbiz, Nr4a1 and Dusp1 changed significantly after the administration of MAP, suggesting the induction of an inflammatory condition such as damage to the myocardium. Moreover, the serum concentrations of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1 beta and IL-6 were significantly increased by the administration of MAP. On the other hand, the mRNA expressions of Rgs2 and Rasd1 were changed by both the administration of MAP and body restraint without interaction, which indicated that these insults affected the circulatory system additively or synergistically. From these results, it is likely that the administration of MAP, followed by body restraint, might cause acute myocardial damage due to the direct myocardial toxic effect of MAP, myocardial hypoxia and/or severe hypertension, which is one of the mechanisms for sudden death in MAP abusers who were restrained due to their excited state.