Preclinical toxicological assessment of amido-bridged nucleic acid-modified antisense oligonucleotides targeting synaptotagmin XIII for intra-abdominal treatment of peritoneal metastasis of gastric cancer.

BACKGROUND: Peritoneal metastasis of gastric cancer is closely associated with dismal prognosis. In previous preclinical proof-of-concept studies, an amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotide (ASO), designated ASO-4733 that targets the gene encoding synaptotagmin XIII (SYT13), inhibited cellular functions required for the formation of peritoneal metastasis of gastric cancer cells. ASO-4733 achieved therapeutic effects when intra-abdominally administered to mouse xenograft models. Here, we conducted an analysis of Syt13-deficient mice to determine the pharmacokinetics and toxicity of intra-abdominal administration of ASO-4733. METHODS: The effects of Syt13-deficiency in mice were determined. Good Laboratory Practice toxicity tests and the toxicokinetics of intra-abdominal administration of ASO-4733 were conducted in cynomolgus monkeys and rats. The pharmacokinetics of ASO-4733 administered intravenously or intra-abdominally to rats were investigated. RESULTS: Syt13-deficient mice exhibited normal reproduction, organ functions, and motor functions. Weekly intra-abdominal administration of ASO-4733 (125 mg/kg), corresponding to a 50-fold increase of the estimated clinical dose for 4 weeks, was well tolerated by cynomolgus monkeys. In rats, off-target toxicity (not attributable to hybridization) was observed after weekly intra-abdominal administration of ASO-4733. Blood concentrations of ASO-4733 were lower and rose more slowly after intra-abdominal administration compared with intravenous administration. CONCLUSIONS: The preclinical profile of intra-abdominal administration of ASO-4733 demonstrated its suitability for entry into clinical trials of patients with peritoneal metastasis of gastric cancer.

Biological characterization of adipose-derived regenerative cells used for the treatment of stress urinary incontinence.

OBJECTIVE: To assess the characteristics of adipose-derived regenerative cells, and provide supportive data explaining the mechanism of efficacy observed for the use of these cells in the treatment of stress urinary incontinence. METHODS: Adipose tissues were harvested by abdominal liposuction from healthy donors and patients with stress urinary incontinence. Adipose-derived regenerative cells were isolated from tissues using the Celution system, and assessed for their characteristics and ability to differentiate into smooth muscle cells. RESULTS: Adipose-derived regenerative cells isolated by the Celution system developed into fibroblastic colonies. Flow cytometric analysis of adipose-derived stem cell markers showed that adipose-derived regenerative cells were positive for CD34 and CD44, and negative for CD31. Immunofluorescence staining after differentiation showed that colony-forming cells were positive for alpha-smooth muscle actin, calponin and desmin, which are smooth muscle cell markers. A cytokine release assay showed that adherent cells secreted cytokines associated with angiogenesis, including vascular endothelial growth factor-A, angiopoietin-2 and placental growth factor. CONCLUSIONS: Adipose-derived regenerative cells collected by the Celution system might have clonogenic capacity and an angiogenetic function. These properties might contribute to the mechanisms through which regenerative cell therapy by periurethral injection of autologous adipose-derived regenerative cells ameliorates stress urinary incontinence.

Non-thermal plasma-activated lactate solution kills U251SP glioblastoma cells in an innate reductive manner with altered metabolism.

Ringer's lactate solution irradiated by non-thermal plasma, comprised of radicals, electrons, and ions, is defined as plasma-activated lactate (PAL). PAL exhibited antitumor effects in glioblastoma U251SP cells, which we termed PAL-specific regulated cell death. In contrast to the oxidative stress condition typical of cells incubated in plasma-activated medium (PAM), U251SP cells treated with Ringer's lactate solution or PAL exhibited changes in intracellular metabolites that were reductive in the redox state, as measured by the ratio of oxidative/reductive glutathione concentrations. In the metabolomic profiles of PAL-treated cells, the generation of acetyl-CoA increased for lipid metabolism from alanine and asparagine. PAL thus induces regulated death of U251SP glioblastoma cells in more innate microenvironments than PAM.

Regenerative treatment for male stress urinary incontinence by periurethral injection of adipose-derived regenerative cells: Outcome of the ADRESU study.

OBJECTIVES: To report the outcome of the ADRESU study, a multicenter, single-arm, investigator-initiated clinical trial to confirm the efficacy and safety of regenerative treatment for male patients with stress urinary incontinence. METHODS: The participants were male patients with mild-to-moderate stress urinary incontinence persisting for >1 year after prostatectomy. Autologous adipose-derived regenerative cells were isolated using the Celution system from adipose tissue obtained by liposuction. Adipose-derived regenerative cells and mixture of adipose-derived regenerative cells with adipose tissue were transurethrally injected into the rhabdosphincter and submucosal space of the urethra, respectively. The primary end-point was the proportion of patients with improvement of the urine leakage volume at 52 weeks (or last visit within 52 weeks). Improvement of leakage volume was defined as a decrease from baseline >50% by the 24-h pad test. A total of 10 secondary end-points were set. RESULTS: A total of 45 patients satisfying the eligibility criteria were enrolled. The primary end-point was met; the proportion of patients with improvement in leakage volume at 52 weeks was 37.2% (95% confidence interval 23.0-53.3%). No serious adverse events with causal relationships to the adipose-derived regenerative cells were encountered. There was a progressive improvement in secondary end-points. In the King's Health Questionnaire, improvement of quality of life scores showed greater improvement in responders, as compared with non-responders. CONCLUSIONS: Findings from the ADRESU study suggest the efficacy and safety of regenerative treatment for male patients with mild-to-moderate stress urinary incontinence.

Development of novel diagnostic system for pancreatic cancer, including early stages, measuring mRNA of whole blood cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most life-threating disease among all digestive system malignancies. We developed a blood mRNA PDAC screening system using real-time detection PCR to detect the expression of 56 genes, to discriminate PDAC from noncancer subjects. We undertook a clinical study to assess the performance of the developed system. We collected whole blood RNA from 53 PDAC patients, 102 noncancer subjects, 22 patients with chronic pancreatitis, and 23 patients with intraductal papillary mucinous neoplasms in a per protocol analysis. The sensitivity of the system for PDAC diagnosis was 73.6% (95% confidence interval, 59.7%-84.7%). The specificity for noncancer volunteers, chronic pancreatitis, and patients with intraductal papillary mucinous neoplasms was 64.7% (54.6%-73.9%), 63.6% (40.7%-82.8%), and 47.8% (26.8%-69.4%), respectively. Importantly, the sensitivity of this system for both stage I and stage II PDAC was 78.6% (57.1%-100%), suggesting that detection of PDAC by the system is not dependent on the stage of PDAC. These results indicated that the screening system, relying on assessment of changes in mRNA expression in blood cells, is a viable alternative screening strategy for PDAC.

Non-thermal plasma-activated medium modified metabolomic profiles in the glycolysis of U251SP glioblastoma.

Non-equilibrium atmospheric pressure plasma (NEAPP) is a mixture of radicals, electrons, anions, cations and light at near body temperature. Plasma-activated medium (PAM) is realized using NEAPP provided by engineered devices and irradiated to a cell culture medium for a period of 600 s. Glioblastoma cells U251SP cultivated in PAM previously indicated that antitumor effects induced PAM-specific apoptotic cell-death. Metabolomic profiles of a hundred intracellular metabolites were analyzed using capillary electrophoresis mass spectrometry. The metabolomic profiles of the PAM-treated U251SP cells were changed significantly with inhibition of the glycolysis pathway and with enhancement of the pentose phosphate pathway.

Design of a Randomized Controlled Clinical Study of tissue-engineered osteogenic materials using bone marrow-derived mesenchymal cells for Maxillomandibular bone defects in Japan: the TEOM study protocol.

BACKGROUND: Maxillomandibular bone defects arise from maxillofacial injury or tumor/cyst removal. While the standard therapy for bone regeneration is transplantation with autologous bone or artificial bone, these therapies are still unsatisfactory. Autologous bone harvesting is invasive and occasionally absorbed at the implanted site. The artificial bone takes a long time to ossify and it often gets infected. Therefore, we have focused on regenerative therapy consisting of autologous bone marrow-derived mesenchymal cells (BM-MSCs), which decreases the burden on patients. Based on our previous research in patients with maxillomandibular bone defects or alveolar bone atrophy using a mixture of BM-MSCs, platelet-rich plasma (PRP), thrombin, and calcium, we confirmed the efficacy and acceptable safety profile of this treatment. In this investigator-initiated clinical study (the TEOM study), we intended to add ß-tricalcium phosphate (ß-TCP) owing to large defect with patients. The TEOM study aimed to evaluate the efficacy and safety of bone regeneration using mixtures of BM-MSCs in patients with bone defects resulting from maxillofacial injury, and tumor/cyst removal in the maxillomandibular region. METHODS: The TEOM study is an open-label, single-center, randomized controlled study involving a total of 83 segments by the Fédération Dentaire Internationale numbering system in maxillomandibular bone defects that comprise over 1/3 of the maxillomandibular area with a remaining bone height of ≤10 mm. The primary endpoint is rate of procedure sites with successful bone regeneration defined as a computed tomography (CT) value of more than 400 and a bone height of more than 10 mm. Our specific hypothesis is that the number of required regions was calculated assuming that the rate of procedure sites with successful bone regeneration is similar and the non-inferiority margin is 15.0%. DISCUSSION: The TEOM study is the first randomized controlled study of regenerative treatment using BM-MSCs for large maxillomandibular bone defects. We will evaluate the efficacy and safety in this study to provide an exploratory basis for the necessity of BM-MSCs for these patients. TRIAL REGISTRATION: This trial was registered at the University Hospital Medical information Network Clinical Trials Registry (UMIN-CTR Unique ID: UMIN000020398; Registration Date: Jan 15, 2016; URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000016543 ).

Non-thermal plasma specifically kills oral squamous cell carcinoma cells in a catalytic Fe(II)-dependent manner.

Oral cancer accounts for ~2% of all cancers worldwide, and therapeutic intervention is closely associated with quality of life. Here, we evaluated the effects of non-thermal plasma on oral squamous cell carcinoma cells with special reference to catalytic Fe(II). Non-thermal plasma exerted a specific killing effect on oral squamous cell carcinoma cells in comparison to fibroblasts. Furthermore, the effect was dependent on the amounts of catalytic Fe(II), present especially in lysosomes. After non-thermal plasma application, lipid peroxidation occurred and peroxides and mitochondrial superoxide were generated. Cancer cell death by non-thermal plasma was promoted dose-dependently by prior application of ferric ammonium citrate and prevented by desferrioxamine, suggesting the association of ferroptosis. Potential involvement of apoptosis was also observed with positive terminal deoxynucleaotidyl transferase-mediated dUTP nick end labeling and annexin V results. Non-thermal plasma exposure significantly suppressed the migratory, invasive and colony-forming abilities of squamous cell carcinoma cells. The oral cavity is easily observable; therefore, non-thermal plasma can be directly applied to the oral cavity to kill oral squamous cell carcinoma without damaging fibroblasts. In conclusion, non-thermal plasma treatment is a potential therapeutic option for oral cancer.

Molecular mechanisms of non-thermal plasma-induced effects in cancer cells.

Plasma is the fourth state of matter with higher energy than gas; non-thermal plasma (NTP) is currently available. As NTP is useful in sterilization, promoting wound healing and cancer treatments, the molecular mechanisms of plasma-induced effects in living cells and microorganisms are of significant interest in plasma medicine with medical-engineering collaboration. Molecular mechanisms of plasma-induced effects in cancer cells will be described in this minireview. Both direct and indirect methods to treat cancer cells with NTP have been developed. NTP interacts directly with not only cancer cells but also the liquids surrounding cancer cells and the immune cells that target them. Reactive oxygen and nitrogen species play key roles in NTP-induced effects; however, other mechanisms have been suggested. The complex interactions between NTP, cells and liquids have been extensively studied. In the future, details regarding NTP-induced effects on gene regulatory networks, signaling networks, and metabolic networks will be elucidated.

Effect of Plasma-Activated Lactated Ringer's Solution on Pancreatic Cancer Cells In Vitro and In Vivo.

BACKGROUND: The medical applications of nonequilibrium atmospheric pressure plasma in cancer therapy have attracted attention. We previously reported on the antitumor effect of plasma-activated medium. However, this approach requires plasma-activated liquids that are administrable to the human body. In this study, we produced plasma-activated lactated Ringer's solution (PAL) and evaluated its antitumor effect and mechanism. Furthermore, we evaluated the effect of the intraperitoneal administration of PAL using a peritoneal dissemination mouse tumor model. METHODS: The antitumor effect of PAL on pancreatic cancer cell lines was evaluated using proliferation and apoptosis assays. In addition, cellular reactive oxygen species (ROS) generation was examined. The role of ROS was assessed using a proliferation assay with N-acetyl cysteine (NAC). An adhesion assay was performed to evaluate the effect of PAL on cell adhesion. Finally, pancreatic cancer cells stably expressing luciferase (AsPC-1/CMV-Luc) were injected intraperitoneally into mice, followed by intraperitoneal injection of PAL. Peritoneal dissemination was monitored using in vivo bioluminescent imaging. RESULTS: The antitumor effect of PAL was shown in all cell lines in vitro. The TUNEL assay showed that PAL induced apoptosis. ROS uptake was observed in PAL-treated cells, and the antitumor effect was inhibited by NAC. Cell adhesion also was suppressed by PAL. The intraperitoneal administration of PAL suppressed the formation of peritoneal nodules in vivo. CONCLUSIONS: Our study demonstrated the antitumor effects of PAL in vitro and in vivo. Intraperitoneal administration of PAL may be a novel therapeutic option for peritoneal metastases.

Long-Term Clinical Outcomes Survey of Bone Marrow-Derived Cell Therapy in Critical Limb Ischemia in Japan.

BACKGROUND: The Therapeutic Angiogenesis by Cell Transplantation (TACT) trial demonstrated the efficacy and safety of autologous bone marrow-derived mononuclear cells (BM-MNCs) in patients with critical limb ischemia (CLI). The present study aimed to assess the long-term clinical outcomes of therapeutic angiogenesis using autologous BM-MNC implantation under advanced medical treatment in Japan.Methods and Results:The study was retrospective, observational, and non-controlled. We assessed no-option CLI patients who had BM-MNC implantation performed in 10 institutes. Overall survival (OS), major amputation-free (MAF), and amputation-free survival (AFS) rates were primary endpoints of this study. The median follow-up duration was 31.7 months. The 10-year OS rate was 46.6% in patients with arteriosclerosis obliterans (ASO) (n=168), 90.5% in patients with thromboangiitis obliterans (TAO) (n=108), and 67.6% in patients with collagen disease-associated vasculitis (CDV) (n=69). The 10-year MAF rate was 70.1%, 87.9%, and 90.9%, respectively. The 10-year AFS rate was 37.8%, 80.9%, and 61.2%, respectively. Major adverse cardiovascular events occurred in 6.0% of patients with ASO, 1.9% of patients with TAO, and no patients with CDV. CONCLUSIONS: Therapeutic angiogenesis using autologous BM-MNC implantation may be feasible and safe in patients with no-option CLI, particularly those with CLI caused by TAO or CDV.

Intraperitoneal Administration of Plasma-Activated Medium: Proposal of a Novel Treatment Option for Peritoneal Metastasis From Gastric Cancer.

BACKGROUND: The administration of fluid irradiated with non-equilibrium atmospheric pressure plasma (NEAPP) has attracted much interest as a novel therapeutic method for cancer. The authors previously reported on the efficacy of plasma-activated medium (PAM) for treating cancer cell lines through the induction of apoptosis. In this study, the therapeutic effect of PAM was evaluated in vivo using a peritoneal metastasis mouse model. METHODS: Two gastric cancer cell lines were used in proliferation assays performed to optimize the production of PAM by changing the distance between the plasma source and the medium surface and by altering the volume of irradiated medium. Wound-healing and adhesion assays were conducted to determine the effect of PAM therapy on cell migration and adhesion capacity in vitro. Finally, a mouse model established by the intraperitoneal injection of enhanced green fluorescent protein-tagged gastric cancer cells was used to explore the efficacy of PAM administered intraperitoneally in inhibiting peritoneal metastasis formation. RESULTS: Shorter distances between the plasma source and the medium surface and smaller volumes of treated medium increased the anti-tumor effect of PAM. The PAM treatment attenuated gastric cancer cell migration and adhesion in vitro. The intraperitoneal administration of PAM decreased the formation of peritoneal metastatic nodules by 60% in the mouse model, and no adverse events were observed. CONCLUSIONS: Plasma-activated liquids may represent a novel therapeutic method for the treatment of peritoneal metastases in gastric cancer.

Design of a single-arm clinical trial of regenerative therapy by periurethral injection of adipose-derived regenerative cells for male stress urinary incontinence in Japan: the ADRESU study protocol.

BACKGROUND: Male stress urinary incontinence is a prevalent condition after radical prostatectomy. While the standard recommendation for the management of urine leakage is pelvic floor muscle training, its efficacy is still unsatisfactory. Therefore, we have focused on regenerative therapy, which consists of administering a periurethral injection of autologous regenerative cells from adipose tissue, separated using the Celution® system. Based on an interim data analysis of our exploratory study, we confirmed the efficacy and acceptable safety profile of this treatment. Accordingly, we began discussions with Japanese regulatory authorities regarding the development of this therapy in Japan. The Ministry of Health, Labour and Welfare suggested that we implement a clinical trial of a new medical device based on the Pharmaceutical Affaires Act in Japan. Next, we discussed the design of this investigator-initiated clinical trial (the ADRESU study) aimed at evaluating the efficacy and safety of this therapy, in a consultation meeting with the Pharmaceuticals and Medical Device Agency. METHODS: The ADRESU study is an open-label, multi-center, single-arm study involving a total of 45 male stress urinary incontinence patients with mild-to-moderate urine leakage persisting more than 1 year after prostatectomy, in spite of behavioral and pharmacological therapies. The primary endpoint is the rate of patients at 52 weeks with improvement of urine leakage volume defined as a reduction from baseline greater than 50% by 24-h pad test. Our specific hypothesis is that the primary endpoint result will be higher than a pre-specified threshold of 10%. DISCUSSION: The ADRESU study is the first clinical trial of regenerative treatment for stress urinary incontinence by adipose-derived regenerative cells using the Celution® system based on the Japanese Pharmaceutical Affaires Act. We will evaluate the efficacy and safety in this trial to provide an adequate basis for marketing approval with the final objective of making this novel therapy widely available for Japanese patients. TRIAL REGISTRATION: This trial was registered at the University Hospital Medical information Network Clinical Trial Registry (UMIN-CTR Unique ID: UMIN000017901 ; Registered July 1, 2015) and at ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT02529865 ; Registered August 18, 2015).

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells.

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by l-ascorbate. l-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM l-ascorbate. However, brief pre-treatment with a pharmacological dose (250-750 µM) of l-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of l-ascorbate was completely abolished by a prolonged 4 h pre-incubation with l-ascorbate (500 µM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2',7'-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and l-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.

Cell survival of glioblastoma grown in medium containing hydrogen peroxide and/or nitrite, or in plasma-activated medium.

Non-equilibrium atmospheric pressure plasmas generate a high electron density (on the order of 10(16) electrons per cm(-3)) using Ar gas. Culture medium in air at room temperature was plasma-irradiated for several hundred seconds. Tens of micromolar hydrogen peroxide (H2O2) and millimolar levels of nitrous ion (NO2(-)) were detected in the plasma-irradiated culture medium (plasma activated medium; PAM) and selectively induced the apoptotic death of glioblastoma tumor cells, but did not kill normal mammary epithelial cells. A similar antitumor effect was induced by spiking the medium with comparable concentrations of H2O2 and NO2(-). The PAM remained still a somewhat difference that it should also be assessed for understanding other latent mechanisms.

Effectiveness of plasma treatment on gastric cancer cells.

BACKGROUND: Treatment of peritoneal carcinomatosis arising from gastric cancer remains a considerable challenge. In recent years, the anticancer effect of nonequilibrium atmospheric pressure plasma (NEAPP) has been reported in several cancer cell lines. Use of NEAPP may develop into a new class of anticancer therapy that augments surgery, chemotherapy, and radiotherapy. METHOD: Gastric cancer cells were assessed for changes in cell morphology and rate of proliferation after treatment with NEAPP-exposed medium (PAM). To explore the functional mechanism, caspase 3/7, annexin V, and uptake of reactive oxygen species (ROS) were evaluated, along with the effect of the ROS scavenger N-acetylcysteine (NAC). RESULTS: PAM treatment for 24 h affected cell morphology, suggestive of induction of apoptosis. PAM cytotoxicity was influenced by the time of exposure to PAM, the type of cell line, and the number of cells seeded. Cells treated with PAM for 2 h demonstrated activated caspase 3/7 and an increased proportion of annexin V-positive cells compared with untreated cells. Additionally, ROS uptake was observed in PAM-treated cells, whereas NAC reduced the cytotoxicity induced by PAM presumably through reduction of ROS uptake. Furthermore, CD44 variant 9, which reportedly leads to glutathione synthesis and suppresses stress signaling of ROS, was overexpressed in PAM-resistant cells. CONCLUSIONS: PAM treatment induced apoptosis of gastric cancer cells through generation and uptake of ROS. Local administration of PAM could develop into an option to treat peritoneal carcinomatosis.

Direct exposure of non-equilibrium atmospheric pressure plasma confers simultaneous oxidative and ultraviolet modifications in biomolecules.

Thermal plasmas and lasers are used in medicine to cut and ablate tissues and for coagulation. Non-equilibrium atmospheric pressure plasma (NEAPP) is a recently developed, non-thermal technique with possible biomedical applications. Although NEAPP reportedly generates reactive oxygen/nitrogen species, electrons, positive ions, and ultraviolet radiation, little research has been done into the use of this technique for conventional free radical biology. Recently, we developed a NEAPP device with high electron density. Electron spin resonance spin-trapping revealed (•)OH as a major product. To obtain evidence of NEAPP-induced oxidative modifications in biomolecules and standardize them, we evaluated lipid peroxidation and DNA modifications in various in vitro and ex vivo experiments. Conjugated dienes increased after exposure to linoleic and α-linolenic acids. An increase in 2-thiobarbituric acid-reactive substances was also observed after exposure to phosphatidylcholine, liposomes or liver homogenate. Direct exposure to rat liver in saline produced immunohistochemical evidence of 4-hydroxy-2-nonenal- and acrolein-modified proteins. Exposure to plasmid DNA induced dose-dependent single/double strand breaks and increased the amounts of 8-hydroxy-2'-deoxyguanosine and cyclobutane pyrimidine dimers. These results indicate that oxidative biomolecular damage by NEAPP is dose-dependent and thus can be controlled in a site-specific manner. Simultaneous oxidative and UV-specific DNA damage may be useful in cancer treatment.

Elaborate cooperation of poly(rC)-binding proteins 1/2 and glutathione in ferroptosis induced by plasma-activated Ringer's lactate.

Reactive species are involved in various aspects of neoplastic diseases, including carcinogenesis, cancer-specific metabolism and therapeutics. Non-thermal plasma (NTP) can directly provide reactive species, by integrating atmospheric and interjacent molecules as substrates, to represent a handy strategy to load oxidative stress in situ. NTP causes apoptosis and/or ferroptosis specifically in cancer cells of various types. Plasma-activated Ringer's lactate (PAL) is another modality at the preclinical stage as cancer therapeutics, based on more stable reactive species. PAL specifically kills malignant mesothelioma (MM) cells, employing lysosomal ·NO as a switch from autophagy to ferroptosis. However, the entire molecular mechanisms have not been elucidated yet. Here we studied cytosolic iron regulations in MM and other cancer cells in response to PAL exposure. We discovered that cells with higher catalytic Fe(II) are more susceptible to PAL-induced ferroptosis. PAL caused a cytosolic catalytic Fe(II)-associated pathology through iron chaperones, poly (rC)-binding proteins (PCBP)1/2, inducing a disturbance in glutathione-regulated iron homeostasis. PCBP1/NCOA4-mediated ferritinophagy started at a later phase, further increasing cytosolic catalytic Fe(II), ending in ferroptosis. In contrast, PCBP2 after PAL exposure contributed to iron loading to mitochondria, leading to mitochondrial dysfunction. Therapeutic effect of PAL was successfully applied to an orthotopic MM xenograft model in mice. In conclusion, PAL can selectively sensitize MM cells to ferroptosis by remodeling cytoplasmic iron homeostasis, where glutathione and PCBPs play distinct roles, resulting in lethal ferritinophagy and mitochondrial dysfunction. Our findings indicate the clinical application of PAL as a ferroptosis-inducer and the potential of PCBPs as novel targets in cancer therapeutics.

U-shaped link of health checkup data and need for care using a time-dependent cox regression model with a restricted cubic spline.

We explored risk indicators likely to result in older adults needing certified long-term care in Japan and ascertained whether this relationship forms a U-shaped link. We analyzed a community-based cohort of residents in Kitanagoya City, Aichi Prefecture, Japan. Participants were 3718 individuals aged 65 years and above who underwent health examinations between April 1, 2011 and March 31, 2012. For continuous clinical variables, we applied a time-dependent Cox regression model. Two types of models were applied-a linear and nonlinear model with restricted cubic splines-to assess the U-shaped association. Statistical significance (set at 0.05) for the nonlinearity was tested by comparing the spline and linear models. Among the participants, 701 were certified as needing Level 1 care or higher during a follow-up. Among the continuous clinical variables, the nonlinear model for body mass index, systolic blood pressure, high-density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transpeptidase revealed significant U-shaped associations as compared with the linear model in which the outcome was a certification of the need for nursing care. These results provide an important insight into the usefulness of nonlinear models for predicting the risk of such certification.

Plasma activated Ringer's lactate solution.

Low-temperature plasma (LTP) has been widely used in life science. Plasma-activated solutions were defined as solutions irradiated with LTP, and water, medium, and Ringer's solutions have been irradiated with LTP to produce plasma-activated solutions. They contain chemical compounds produced by reactions among LTP, air, and solutions. Reactive oxygen and nitrogen species (RONS) are major components in plasma-activated solutions and recent studies revealed that plasma-activated organic compounds are produced in plasma-activated Ringer's lactate solution (PAL). Many in vitro and in vivo studies demonstrated that PAL exhibits anti-tumor effects on cancers, and biochemical analyses revealed intracellular molecular mechanisms of cancer cell death by PAL.