The mechanism of epithelial-mesenchymal transition (EMT) consists of the cellular phenotypic transition from epithelial to mesenchymal status. The cells exhibiting EMT exist in cancer stem cell (CSC) population, which is involved in drug resistance. CSCs demonstrating EMT feature remain after cancer treatment, which leads to drug resistance, recurrence, metastasis and malignancy of cancer. In this context, the recent advance of nanotechnology in the medical application has ascended the possibility to target CSCs using nanomedicines. In this review article, we focused on the mechanism of CSCs and EMT, especially into the signaling pathways in EMT, regulation of EMT and CSCs by microRNAs and nanomedicine-based approaches to target CSCs.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH₂-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Gα_12/13-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Gα_12/13, but not by an RGS domain specific for Gα_q. Constitutively active Gα₁₂- or Gα₁₃-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Gα₁₃, which was completely inhibited by the Gα_12/13-specific RGS domain. Furthermore, the Gα_12/13-specific but not the Gα_q-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Gα_12/13-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Gα_12/13-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.

Sustained elevation of [Ca(2+)](i) has been implicated in many cellular events. We previously reported that alpha subunits of G(12) family G proteins (Galpha(12/13)) participate in sustained Ca(2+) influx required for the activation of nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor, in rat neonatal cardiac fibroblasts. Here, we demonstrate that Galpha(12/13)-mediated up-regulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca(2+) influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active Galpha(12) or Galpha(13) increased the expression of TRPC6 proteins and basal Ca(2+) influx activity. The treatment with ET-1 increased TRPC6 protein levels through Galpha(12/13), reactive oxygen species, and c-Jun N-terminal kinase (JNK)-dependent pathways. NFAT is activated by sustained increase in [Ca(2+)](i) through up-regulated TRPC6. A Galpha(12/13)-inhibitory polypeptide derived from the regulator of the G-protein signaling domain of p115-Rho guanine nucleotide exchange factor and a JNK inhibitor, SP600125, suppressed the ET-1-induced increase in expression of marker proteins of myofibroblast formation through a Galpha(12/13)-reactive oxygen species-JNK pathway. The ET-1-induced myofibroblast formation was suppressed by overexpression of TRPC6 and CA NFAT, whereas it was enhanced by TRPC6 small interfering RNAs and cyclosporine A. These results suggest two opposite roles of Galpha(12/13) in cardiac fibroblasts. First, Galpha(12/13) mediate ET-1-induced myofibroblast formation. Second, Galpha(12/13) mediate TRPC6 up-regulation and NFAT activation that negatively regulates ET-1-induced myofibroblast formation. Furthermore, TRPC6 mediates hypertrophic responses in cardiac myocytes but suppresses fibrotic responses in cardiac fibroblasts. Thus, TRPC6 mediates opposite responses in cardiac myocytes and fibroblasts.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Local hyperthermia has been widely used as physical therapy for a number of diseases such as inflammatory osteoarticular disorders, tendinitis, and muscle injury. Local hyperthermia is clinically applied to improve blood and lymphatic flow to decrease swelling of tissues (e.g., skeletal muscle). As for muscle repair following injury, the mechanisms underlying the beneficial effects of hyperthermia-induced muscle repair are unknown. In this study, we investigated the direct effects of continuous heat stress on the differentiation of cultured mammalian myoblasts. Compared with control cultures grown at 37°C, incubation at 39°C (continuous mild heat stress; CMHS) enhanced myotube diameter, whereas myotubes were poorly formed at 41°C by primary human skeletal muscle culture cells, human skeletal muscle myoblasts (HSMMs), and C2C12 mouse myoblasts. In HSMMs and C2C12 cells exposed to CMHS, mRNA and protein levels of myosin heavy chain (MyHC) type I were increased compared with the control cultures. The mRNA level of MyHC IIx was unaltered in HSMMs and decreased in C2C12 cells, compared with cells that were not exposed to heat stress. These results indicated a fast-to-slow fiber-type shift in myoblasts. We also examined upstream signals that might be responsible for the fast-to-slow shift of fiber types. CMHS enhanced the mRNA and protein levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α in HSMMS and C2C12 cells but not the activities of MAPKs (ERK1/2 and p38 MAPK) in HSMMs and C2C12 cells. These data suggest that CMHS induces a fast-to-slow fiber-type shift of mammalian myoblasts through PGC-1α.

In neonatal cardiomyocytes, activation of the G _q -coupled α ₁ -adrenergic receptor (α ₁ AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH ₂ -terminal kinase (JNK). Here, we show that JNK activation is essential for α ₁ AR-induced hypertrophy, in that α ₁ AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate α ₁ AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. α ₁ AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Gα _q , Gα ₁₂ , and Gα ₁₃ . JNK activation was also inhibited by the Gα _q/11 - or Gα _12/13 -specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein–coupled receptor kinase 2, a polypeptide that sequesters Gβγ. α ₁ AR-induced hypertrophic responses were inhibited by Gα _q/11 - and Gα _12/13 -specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein–coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Gα ₁₂ and Gα ₁₃ but not by Gα _q . Our findings suggest that α ₁ AR-induced hypertrophic responses are mediated in part by a Gα _12/13 -Rho-JNK pathway, in part by a G _q/11 -JNK pathway that is Rho independent, and in part by a Gβγ pathway that is JNK independent.