Alternative magnetic field exposure suppresses tumor growth via metabolic reprogramming.

Application of physical forces, ranging from ultrasound to electric fields, is recommended in various clinical practice guidelines, including those for treating cancers and bone fractures. However, the mechanistic details of such treatments are often inadequately understood, primarily due to the absence of comprehensive study models. In this study, we demonstrate that an alternating magnetic field (AMF) inherently possesses a direct anti-cancer effect by enhancing oxidative phosphorylation (OXPHOS) and thereby inducing metabolic reprogramming. We observed that the proliferation of human glioblastoma multiforme (GBM) cells (U87 and LN229) was inhibited upon exposure to AMF within a specific narrow frequency range, including around 227 kHz. In contrast, this exposure did not affect normal human astrocytes (NHA). Additionally, in mouse models implanted with human GBM cells in the brain, daily exposure to AMF for 30 min over 21 days significantly suppressed tumor growth and prolonged overall survival. This effect was associated with heightened reactive oxygen species (ROS) production and increased manganese superoxide dismutase (MnSOD) expression. The anti-cancer efficacy of AMF was diminished by either a mitochondrial complex IV inhibitor or a ROS scavenger. Along with these observations, there was a decrease in the extracellular acidification rate (ECAR) and an increase in the oxygen consumption rate (OCR). This suggests that AMF-induced metabolic reprogramming occurs in GBM cells but not in normal cells. Our results suggest that AMF exposure may offer a straightforward strategy to inhibit cancer cell growth by leveraging oxidative stress through metabolic reprogramming.

Cytotoxic effects of the cigarette smoke extract of heated tobacco products on human oral squamous cell carcinoma: the role of reactive oxygen species and CaMKK2.

BACKGROUND: The increasing prevalence of heated tobacco products (HTPs) has heightened concerns regarding their potential health risks. Previous studies have demonstrated the toxicity of cigarette smoke extract (CSE) from traditional tobacco's mainstream smoke, even after the removal of nicotine and tar. Our study aimed to investigate the cytotoxicity of CSE derived from HTPs and traditional tobacco, with a particular focus on the role of reactive oxygen species (ROS) and intracellular Ca2+. METHODS: A human oral squamous cell carcinoma (OSCC) cell line, HSC-3 was utilized. To prepare CSE, aerosols from HTPs (IQOS) and traditional tobacco products (1R6F reference cigarette) were collected into cell culture media. A cell viability assay, apoptosis assay, western blotting, and Fluo-4 assay were conducted. Changes in ROS levels were measured using electron spin resonance spectroscopy and the high-sensitivity 2',7'-dichlorofluorescein diacetate assay. We performed a knockdown of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) by shRNA lentivirus in OSCC cells. RESULTS: CSE from both HTPs and traditional tobacco exhibited cytotoxic effects in OSCC cells. Exposure to CSE from both sources led to an increase in intracellular Ca2+ concentration and induced p38 phosphorylation. Additionally, these extracts prompted cell apoptosis and heightened ROS levels. N-acetylcysteine (NAC) mitigated the cytotoxic effects and p38 phosphorylation. Furthermore, the knockdown of CaMKK2 in HSC-3 cells reduced cytotoxicity, ROS production, and p38 phosphorylation in response to CSE. CONCLUSION: Our findings suggest that the CSE from both HTPs and traditional tobacco induce cytotoxicity. This toxicity is mediated by ROS, which are regulated through Ca2+ signaling and CaMKK2 pathways.

EP4-induced mitochondrial localization and cell migration mediated by CALML6 in human oral squamous cell carcinoma.

Lymph node metastasis, primarily caused by the migration of oral squamous cell carcinoma (OSCC) cells, stands as a crucial prognostic marker. We have previously demonstrated that EP4, a subtype of the prostaglandin E2 (PGE2) receptor, orchestrates OSCC cell migration via Ca2+ signaling. The exact mechanisms by which EP4 influences cell migration through Ca2+ signaling, however, is unclear. Our study aims to clarify how EP4 controls OSCC cell migration through this pathway. We find that activating EP4 with an agonist (ONO-AE1-473) increased intracellular Ca2+ levels and the migration of human oral cancer cells (HSC-3), but not human gingival fibroblasts (HGnF). Further RNA sequencing linked EP4 to calmodulin-like protein 6 (CALML6), whose role remains undefined in OSCC. Through protein-protein interaction network analysis, a strong connection is identified between CALML6 and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), with EP4 activation also boosting mitochondrial function. Overexpressing EP4 in HSC-3 cells increases experimental lung metastasis in mice, whereas inhibiting CaMKK2 with STO-609 markedly lowers these metastases. This positions CaMKK2 as a potential new target for treating OSCC metastasis. Our findings highlight CALML6 as a pivotal regulator in EP4-driven mitochondrial respiration, affecting cell migration and metastasis via the CaMKK2 pathway.

Physiological functions of calcium signaling via Orai1 in cancer.

Intracellular calcium (Ca2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca2+ entry (SOCE) is a major mechanism by which Ca2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca2+ entry (SOCE) is also a receptor-regulated Ca2+ entry pathway that maintains Ca2+ homeostasis by sensing reduced Ca2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors located in the ER membrane. The, IP3 receptors in the ER membrane trigger a rapid and transient release of Ca2+ from the ER store. The resulting depletion of ER Ca2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca2+ channel subunits (also known as CRACM1) on the PM, leading to Ca2+ influx from the extracellular space to increase intracellular Ca2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca2+ release-activated Ca2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial-mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings.

Store-operated calcium entry via ORAI1 regulates doxorubicin-induced apoptosis and prevents cardiotoxicity in cardiac fibroblasts.

Despite exhibiting cardiotoxicity, doxorubicin (DOX) is widely used for cancer treatments. Cardiac fibroblasts (CFs) are important in the pathogenesis of heart failure. This necessitates the study of the effect of DOX on CFs. The impairment of calcium (Ca2+) homeostasis is a common mechanism of heart failure. Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2⁺ entry pathway that maintains calcium balance by sensing reduced calcium stores in the endoplasmic reticulum. ORAI1, a calcium channel protein and the most important component of SOCE, is highly expressed in human cardiac fibroblasts (HCFs). It is upregulated in CFs from failing ventricles. However, whether ORAI1 in HCFs is increased and/or plays a role in DOX-induced cardiotoxicity remains unknown. In this study, we aimed to elucidate the relationship between ORAI1/SOCE and DOX-induced heart failure. Induction of apoptosis by DOX was characterized in HCFs. Apoptosis and cell cycle analyses were performed by fluorescence-activated cell sorting (FACS). Reactive oxygen species (ROS) production was measured using fluorescence. YM-58483 was used as an ORAI1/SOCE inhibitor. ORAI1-knockdown cells were established by RNA interference. In vivo experiments were performed by intraperitoneally injecting YM-58483 and DOX into mice. We first demonstrated that DOX significantly increased the protein expression level of p53 in HCFs by western blotting. FACS analysis revealed that DOX increased early apoptosis and induced cell cycle arrest in the G2 phase in fibroblasts. DOX also increased ROS production. DOX significantly increased the expression level of ORAI1 in CFs. Both YM-58483 and ORAI1 gene knockdown attenuated DOX-induced apoptosis. Similarly, YM-58483 attenuated cell cycle arrest in the G2 phase, and ORAI1 knockdown attenuated DOX-induced ROS production in HCFs. In the animal experiment, YM-58483 attenuated DOX-induced apoptosis. In HCFs, ORAI1/SOCE regulates p53 expression and plays an important role in DOX-induced cardiotoxicity. ORAI1 may serve as a new target for preventing DOX-induced heart failure.

Multilayered Human Skeletal Muscle Myoblast Sheets Promote the Healing Process After Colonic Anastomosis in Rats.

Colorectal anastomotic leakage is one of the most feared and fatal complications of colorectal surgery. To date, no external coating material that can prevent anastomotic leakage has been developed. As myoblasts possess anti-inflammatory capacity and improve wound healing, we developed a multilayered human skeletal muscle myoblast (HSMM) sheet by periodic exposure to supraphysiological hydrostatic pressure during repeated cell seeding. We assessed whether the application of an HSMM sheet can promote the healing process after colonic anastomosis. Partial colectomy and insufficient suturing were employed to create a high-risk colo-colonic anastomosis model in 60 nude rats. Rats were divided into a control group (n = 30) and an HSMM sheet group (n = 30). Macroscopic findings, anastomotic bursting pressure, and histology at the colonic anastomotic site were evaluated on postoperative day (POD) 3, 5, 7, 14, and 28. The application of an HSMM sheet significantly suppressed abscess formation at the anastomotic site compared to the control group on POD3 and 5. The anastomotic bursting pressure in the HSMM sheet group was higher than that in the control group on POD3 and 5. Inflammatory cell infiltration in the HSMM sheet group was significantly suppressed compared to that in the control group throughout the time course. Collagen deposition in the HSMM sheet group on POD3 was significantly abundant compared to that in the control group. Regeneration of the mucosa at the colonic anastomotic site was promoted in the HSMM sheet group compared to that in the control group on POD14 and 28. Immunohistochemical analysis demonstrated that surviving cells in the HSMM sheet gradually decreased with postoperative time and none were detected on POD14. These results suggest that the application of a multilayered HSMM sheet may prevent postoperative colonic anastomotic leakage.

Regulators of Epithelial Sodium Channels in Aldosterone-Sensitive Distal Nephrons (ASDN): Critical Roles of Nedd4L/Nedd4-2 and Salt-Sensitive Hypertension.

Ubiquitination is a representative, reversible biological process of the post-translational modification of various proteins with multiple catalytic reaction sequences, including ubiquitin itself, in addition to E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligase, deubiquitinating enzymes, and proteasome degradation. The ubiquitin-proteasome system is known to play a pivotal role in various molecular life phenomena, including the cell cycle, protein quality, and cell surface expressions of ion-transporters. As such, the failure of this system can lead to cancer, neurodegenerative diseases, cardiovascular diseases, and hypertension. This review article discusses Nedd4-2/NEDD4L, an E3-ubiquitin ligase involved in salt-sensitive hypertension, drawing from detailed genetic dissection analysis and the development of genetically engineered mice model. Based on our analyses, targeting therapeutic regulations of ubiquitination in the fields of cardio-vascular medicine might be a promising strategy in future. Although the clinical applications of this strategy are limited, compared to those of kinase systems, many compounds with a high pharmacological activity were identified at the basic research level. Therefore, future development could be expected.

Excessive EP4 Signaling in Smooth Muscle Cells Induces Abdominal Aortic Aneurysm by Amplifying Inflammation.

OBJECTIVE: Excessive prostaglandin E2 production is a hallmark of abdominal aortic aneurysm (AAA). Enhanced expression of prostaglandin E2 receptor EP4 (prostaglandin E receptor 4) in vascular smooth muscle cells (VSMCs) has been demonstrated in human AAAs. Although moderate expression of EP4 contributes to vascular homeostasis, the roles of excessive EP4 in vascular pathology remain uncertain. We aimed to investigate whether EP4 overexpression in VSMCs exacerbates AAAs. Approach and Results: We constructed mice with EP4 overexpressed selectively in VSMCs under an SM22α promoter (EP4-Tg). Most EP4-Tg mice died within 2 weeks of Ang II (angiotensin II) infusion due to AAA, while nontransgenic mice given Ang II displayed no overt phenotype. EP4-Tg developed much larger AAAs than nontransgenic mice after periaortic CaCl2 application. In contrast, EP4fl/+;SM22-Cre;ApoE-/- and EP4fl/+;SM22-Cre mice, which are EP4 heterozygous knockout in VSMCs, rarely exhibited AAA after Ang II or CaCl2 treatment, respectively. In Ang II-infused EP4-Tg aorta, Ly6Chi inflammatory monocyte/macrophage infiltration and MMP-9 (matrix metalloprotease-9) activation were enhanced. An unbiased analysis revealed that EP4 stimulation positively regulated the genes binding cytokine receptors in VSMCs, in which IL (interleukin)-6 was the most strongly upregulated. In VSMCs of EP4-Tg and human AAAs, EP4 stimulation caused marked IL-6 production via TAK1 (transforming growth factor-ß-activated kinase 1), NF-κB (nuclear factor-kappa B), JNK (c-Jun N-terminal kinase), and p38. Inhibition of IL-6 prevented Ang II-induced AAA formation in EP4-Tg. In addition, EP4 stimulation decreased elastin/collagen cross-linking protein LOX (lysyl oxidase) in both human and mouse VSMCs. CONCLUSIONS: Dysregulated EP4 overexpression in VSMCs promotes inflammatory monocyte/macrophage infiltration and attenuates elastin/collagen fiber formation, leading to AAA exacerbation.

Reactive fibrosis precedes doxorubicin-induced heart failure through sterile inflammation.

AIMS: Doxorubicin (DOX)-induced heart failure has a poor prognosis, and effective treatments have not been established. Because DOX shows cumulative cardiotoxicity, we hypothesized that minimal cardiac remodelling occurred at the initial stage in activating cardiac fibroblasts. Our aim was to investigate the initial pathophysiology of DOX-exposed cardiac fibroblasts and propose prophylaxis. METHODS AND RESULTS: An animal study was performed using a lower dose of DOX (4 mg/kg/week for 3 weeks, i.p.) than a toxic cumulative dose. Histological analysis was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, picrosirius red staining, and immunohistochemical staining. The mechanism was analysed in vitro with a low dose of DOX, which did not induce cell apoptosis. Microarray analysis was performed. Differentially expressed genes were confirmed by enrichment analysis. Mitochondrial damage was assessed by mitochondrial membrane potential. The production of inflammatory cytokines and fibrosis markers was assessed by western blot, quantitative polymerase chain reaction, and ELISA. A phosphokinase antibody array was performed to detect related signalling pathways. Low-dose DOX did not induced cell death, and fibrosis was localized to the perivascular area in mice. Microarray analysis suggested that DOX induced genes associated with the innate immune system and inflammatory reactions, resulting in cardiac remodelling. DOX induced mitochondrial damage and increased the expression of interleukin-1. DOX also promoted the expression of fibrotic markers, such as alpha smooth muscle actin and galectin-3. These responses were induced through stress-activated protein kinase/c-Jun NH2-terminal kinase signalling. A peroxisome proliferator-activated receptor (PPARγ) agonist attenuated the expression of fibrotic markers through suppressing stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, this molecule also suppressed DOX-induced early fibrotic responses in vivo. CONCLUSIONS: Low-dose DOX provoked reactive fibrosis through sterile inflammation evoked by the damaged mitochondria.

Prostaglandin E2 receptor EP4 regulates cell migration through Orai1.

The EP4 prostanoid receptors are one of four receptor subtypes for prostaglandin E2 (PGE2 ). Therefore, EP4 may play an important role in cancer progression. However, little information is available regarding their function per se, including migration and the cellular signaling pathway of EP4 in oral cancer. First, we found that mRNA and protein expression of EP4 was abundantly expressed in human-derived tongue squamous cell carcinoma cell lines HSC-3 and OSC-19. The EP4 agonist (ONO-AE1-437) significantly promoted cell migration in HSC-3 cells. In contrast, knockdown of EP4 reduced cell migration. Furthermore, we confirmed that knockdown of EP4 suppressed metastasis of oral cancer cells in the lungs of mice in vivo. Therefore, we focused on the mechanism of migration/metastasis in EP4 signaling. Interestingly, EP4 agonist significantly induced intracellular Ca2+ elevation not in only oral cancer cells but also in other cells, including normal cells. Furthermore, we found that EP4 activated PI3K and induced Ca2+ influx through Orai1 without activation of store depletion and stromal interaction molecule 1 (STIM1). Immunoprecipitation showed that EP4 formed complexes with Orai1 and TRPC1, but not with STIM. Moreover, the EP4 agonist ONO-AE1-437 phosphorylated ERK and activated MMP-2 and MMP-9. Knockdown of Orai1 negated EP4 agonist-induced ERK phosphorylation. Taken together, our data suggested that EP4 activated PI3K and then induced Ca2+ influx from the extracellular space through Orai1, resulting in ERK phosphorylation and promoting cell migration. Migration is regulated by EP4/PI3K/Orai1 signaling in oral cancer.

Doxorubicin induces trans-differentiation and MMP1 expression in cardiac fibroblasts via cell death-independent pathways.

Although doxorubicin (DOX)-induced cardiomyopathy causes lethal heart failure (HF), no early detection or effective treatment methods are available. The principal mechanisms of cardiotoxicity are considered to involve oxidative stress and apoptosis of cardiomyocytes. However, the effect of DOX on cardiac fibroblasts at non-lethal concentrations remains unknown. The aim of this study was to investigate the direct effect of doxorubicin on the activation of cardiac fibroblasts independent of cell death pathways. We first found that DOX induced α-SMA expression (marker of trans-differentiation) at a low concentration range, which did not inhibit cell viability. DOX also increased MMP1, IL-6, TGF-ß and collagen expression in human cardiac fibroblasts (HCFs). In addition, DOX promoted Akt and Smad phosphorylation. A Smad inhibitor prevented DOX-induced α-SMA and IL-6 protein expression. An PI3K inhibitor also prevented MMP1 mRNA expression in HCFs. These findings suggest that DOX directly induces fibrotic changes in HCFs via cell death-independent pathways. Furthermore, we confirmed that these responses are organ- and species-specific for HCFs based on experiments using different types of human and murine fibroblast cell lines. These results suggest potentially new mechanisms of DOX-induced cardiotoxicity from the viewpoint of fibrotic changes in cardiac fibroblasts.

Translationally controlled tumor protein (TCTP) plays a pivotal role in cardiomyocyte survival through a Bnip3-dependent mechanism.

Prevention of cardiomyocyte death is an important therapeutic strategy for heart failure. In this study, we focused on translationally controlled tumor protein (TCTP), a highly conserved protein that is expressed ubiquitously in mammalian tissues, including heart. TCTP plays pivotal roles in survival of certain cell types, but its function in cardiomyocytes has not been examined. We aimed to clarify the role of TCTP in cardiomyocyte survival and the underlying mechanism. Here, we demonstrated that downregulation of TCTP with siRNA induced cell death of cardiomyocytes with apoptotic and autophagic features, accompanied with mitochondrial permeability transition pore (mPTP) opening. TCTP loss did not induce cell death of cardiac fibroblasts. Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) was found to mediate the TCTP-loss-induced cardiomyocyte death. In exploring the clinical significance of the TCTP expression in the heart, we found that DOX treatment markedly downregulated the protein expression of TCTP in cultured cardiomyocytes and in mouse heart tissue. Exogenous rescue of TCTP expression attenuated DOX-induced cardiomyocyte death. In mice, cardiomyocyte-specific overexpression of TCTP resulted in decreased susceptibility to DOX-induced cardiac dysfunction, accompanied with attenuated induction of Bnip3. Dihydroartemisinin, a pharmacological TCTP inhibitor, induced development of heart failure and cardiomyocyte death in control mice, but not in mice with cardiomyocyte-specific TCTP overexpression. Our findings revealed TCTP has a pivotal role in cardiomyocyte survival, at least in part through a Bnip3-dependent mechanism. TCTP could be considered as a candidate therapeutic target to prevent DOX-induced heart failure.

Epac1 deficiency inhibits basic fibroblast growth factor-mediated vascular smooth muscle cell migration.

Vascular smooth muscle cell (VSMC) migration and the subsequent intimal thickening play roles in vascular restenosis. We previously reported that an exchange protein activated by cAMP 1 (Epac1) promotes platelet-derived growth factor (PDGF)-induced VSMC migration and intimal thickening. Because basic fibroblast growth factor (bFGF) also plays a pivotal role in restenosis, we examined whether Epac1 was involved in bFGF-mediated VSMC migration. bFGF-induced lamellipodia formation and migration were significantly decreased in VSMCs obtained from Epac1-/- mice compared to those in Epac1+/+-VSMCs. The bFGF-induced phosphorylation of Akt and glycogen synthase kinase 3ß (GSK3ß), which play a role in bFGF-induced cell migration, was attenuated in Epac1-/--VSMCs. Intimal thickening induced by the insertion of a large wire was attenuated in Epac1-/- mice, and was accompanied by the decreased phosphorylation of GSK3ß. These data suggest that Epac1 deficiency attenuates bFGF-induced VSMC migration, possibly via Akt/GSK3ß pathways.

Usefulness of Exchanged Protein Directly Activated by cAMP (Epac)1-Inhibiting Therapy for Prevention of Atrial and Ventricular Arrhythmias in Mice.

BACKGROUND: It has been suggested that protein directly activated by cAMP (Epac), one of the downstream signaling molecules of ß-adrenergic receptor (ß-AR), may be an effective target for the treatment of arrhythmia. However, there have been no reports on the anti-arrhythmic effects or cardiac side-effects of Epac1 inhibitors in vivo. Methods and Results: In this study, the roles of Epac1 in the development of atrial and ventricular arrhythmias are examined. In addition, we examined the usefulness of CE3F4, an Epac1-selective inhibitor, in the treatment of the arrhythmias in mice. In Epac1 knockout (Epac1-KO) mice, the duration of atrial fibrillation (AF) was shorter than in wild-type mice. In calsequestrin2 knockout mice, Epac1 deficiency resulted in a reduction of ventricular arrhythmia. In both atrial and ventricular myocytes, sarcoplasmic reticulum (SR) Ca2+ leak, a major trigger of arrhythmias, and spontaneous SR Ca2+ release (SCR) were attenuated in Epac1-KO mice. Consistently, CE3F4 treatment significantly prevented AF and ventricular arrhythmia in mice. In addition, the SR Ca2+ leak and SCR were significantly inhibited by CE3F4 treatment in both atrial and ventricular myocytes. Importantly, cardiac function was not significantly affected by a dosage of CE3F4 sufficient to exert anti-arrhythmic effects. CONCLUSIONS: These findings indicated that Epac1 is involved in the development of atrial and ventricular arrhythmias. CE3F4, an Epac1-selective inhibitor, prevented atrial and ventricular arrhythmias in mice.

Simultaneous hyperthermia-chemotherapy effect by arterial injection of Fe(Salen) for femur tumor.

We previously identified a novel nanomagnetic particle, N,N'-bis(salicylidene)ethylenediamine iron [Fe(Salen)]. Fe(Salen) not only shows antitumor effects but also magnetic properties. We found that Fe(Salen) can be used for magnet-guided drug delivery and visualization of accumulated drug by magnetic resonance imaging (MRI) because of its magnetism. In addition, Fe(Salen) can generate heat by itself when exposed to an alternating current magnetic field (AMF), resulting in a hyperthermia effect. Herein, we partly elucidated the antitumor mechanism of Fe(Salen) and carried out an i.v. repeated dose toxicity study to decide the therapeutic amount. Furthermore, we evaluated the antitumor effect of selective intra-arterial injection or i.v. injection of Fe(Salen) by catheter and the hyperthermia effect of Fe(Salen) when exposed to AMF in vivo. We used a rabbit model grafted with VX2 cells (rabbit squamous cell carcinoma) on the right leg. Intra-arterial injection of Fe(Salen) showed a greater antitumor effect than did i.v. injection. The combination of Fe(Salen) intra-arterial injection and AMF exposure showed a greater antitumor effect than did either Fe(Salen) or methotrexate (MTX) without AMF exposure, suggesting that AMF exposure greatly enhanced the antitumor effect of Fe(Salen) by arterial injection by catheter. This is the first report that the effectiveness of Fe(Salen) was evaluated in the point of administration route; that is, selective intra-arterial injection by catheter. Taken together, these results indicate a new administration route; that is, selective arterial injection of Fe(Salen) by catheter, and the development of a new strategy of simultaneous hyperthermia-chemotherapy in the future.

Alternating magnetic field enhances cytotoxicity of Compound C.

We previously reported the efficacy of anti-cancer therapy with hyperthermia using an alternating magnetic field (AMF) and a magnetic compound. In the course of the study, unexpectedly, we found that an AMF enhances the cytotoxicity of Compound C, an activated protein kinase (AMPK) inhibitor, although this compound is not magnetic. Therefore, we examined the cellular mechanism of AMF-induced cytotoxicity of Compound C in cultured human glioblastoma (GB) cells. An AMF (280 kHz, 250 Arms) for 30 minutes significantly enhanced the cytotoxicity of Compound C and promoted apoptosis towards several human GB cell lines in vitro. The AMF also increased Compound C-induced cell-cycle arrest of GB cells at the G2 phase and, thus, inhibited cell proliferation. The AMF increased Compound C-induced reactive oxygen species production. Furthermore, the AMF decreased ERK phosphorylation in the presence of Compound C and suppressed the protective autophagy induced by this compound. The application of an AMF in cancer chemotherapy may be a simple and promising method, which might reduce the doses of drugs used in future cancer treatment and, therefore, the associated side effects.

Hydrostatic pressure suppresses fibrotic changes via Akt/GSK-3 signaling in human cardiac fibroblasts.

Mechanical stresses play important roles in the process of constructing and modifying heart structure. It has been well established that stretch force acting on cardiac fibroblasts induces fibrosis. However, the effects of compressive force, that is, hydrostatic pressure (HP), have not been well elucidated. We thus evaluated the effects of HP using a pressure-loading apparatus in human cardiac fibroblasts (HCFs) in vitro. In this study, high HP (200 mmHg) resulted in significant phosphorylation of Akt in HCFs. HP then greatly inhibited glycogen synthase kinase 3 (GSK-3)α, which acts downstream of the PI3K/Akt pathway. Similarly, HP suppressed mRNA transcription of inflammatory cytokine-6, collagen I and III, and matrix metalloproteinase 1, compared with an atmospheric pressure condition. Furthermore, HP inhibited collagen matrix production in a three-dimensional HCF culture. Taken together, high HP suppressed the differentiation of fibroblasts into the myofibroblast phenotype. HP under certain conditions suppressed cardiac fibrosis via Akt/GSK-3 signaling in HCFs. These results might help to elucidate the pathology of some types of heart disease.

Treatment of oral cancer using magnetized paclitaxel.

N,N'-Bis(salicylidene)ethylenediamine iron (Fe(Salen)) is an anti-cancer agent with intrinsic magnetic property. Here, we covalently linked Fe(Salen) to paclitaxel (PTX), a widely used anti-cancer drug, to obtain a magnetized paclitaxel conjugate (M-PTX), which exhibited magnetic characteristics for magnet-guided drug delivery and MRI visualization. M-PTX increased apoptosis and G2/M arrest of cultured human oral cancer cell lines in the same manner as PTX. Furthermore, marked contrast intensity was obtained in magnetic resonance imaging (MRI) of M-PTX. In a mouse oral cancer model, a permanent magnet placed on the body surface adjacent to the tumor resulted in distinct accumulation of M-PTX, and the anti-cancer effect was greater than that of M-PTX without the magnet. We believe that this strategy may improve future cancer chemotherapy by providing conventional anti-cancer drugs with novel functionalities such as magnet-guided drug delivery or MRI-based visualization/quantitation of drug distribution.

Acute Hyperthermia Inhibits TGF-ß1-induced Cardiac Fibroblast Activation via Suppression of Akt Signaling.

Transforming growth factor-ß1 (TGF-ß1) induces phenotypic changes in fibroblasts to become myofibroblasts with increased production of extracellular matrix (ECM) components and cytokines. It is also known that excessive activation of myofibroblasts accelerates cardiac fibrosis, remodeling, and thus cardiac dysfunction. However, no effective therapy has been established to prevent this process although recent clinical studies have demonstrated the effectiveness of hyperthermia in cardiac dysfunction. The aim of this study was to examine the molecular mechanism of hyperthermia on TGF-ß1-mediated phenotypic changes in cardiac fibroblasts. TGF-ß1 increased the expression of IL-6, α-smooth muscle actin (α-SMA), and collagen in human cardiac fibroblasts (HCFs). Hyperthermia (42 °C) significantly prevented these changes, i.e., increases in IL-6, α-SMA, and collagen, as induced by TGF-ß1 in a time-dependent manner. Immunoblotting showed that hyperthermia decreased Akt/S6K signaling, but did not affect Smad2 and Smad3 signaling. Pharmacological inhibition of Akt signaling mimicked these effects of hyperthermia. Furthermore, hyperthermia treatment prevented cardiac fibrosis in Ang II infusion mice model. Putting together, our findings suggest that hyperthermia directly inhibits TGF-ß-mediated activation of HCFs via suppressing Akt/S6K signaling.

Epac activation inhibits IL-6-induced cardiac myocyte dysfunction.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic and thus catecholamine signaling is activated thereafter to compensate for cardiac dysfunction. The mechanism of such compensation by catecholamine signaling has been traditionally understood to be cyclic AMP-dependent protein kinase (PKA)-mediated enforcement of cardiac contractility. We hypothesized that the exchange protein activated by cAMP (Epac), a newly identified target of cAMP signaling that functions independently of PKA, also plays a key role in this mechanism. In cultured cardiac myocytes, activation of Epac attenuated the inhibitory effect of interleukin-6 on the increase of intracellular Ca2+ concentration and contractility in response to isoproterenol, most likely through inhibition of the Jak-STAT pathway via SOCS3, with subsequent changes in inducible nitric oxide synthase expression. These findings suggest a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac and its downstream pathway may be a novel target for treating cardiac dysfunction in endotoxemia.