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.

[Doxorubicin directly induced fibrotic change of cardiac fibroblasts].

Doxorubicin (DOX)-induced cardiomyopathy has a poor prognosis. No early detection or effective treatment methods are available in clinical. The mechanisms of cardiotoxicity were considered as oxidative stress and apoptosis in cardiomyocytes. However, the effect of DOX on cardiac fibroblasts remains to be developed. We investigated the direct effect of DOX on the function of human cardiac fibroblasts (HCFs) independently of cell death pathway. Animal study showed that lower dose of DOX (4 mg/kg/week for 3 weeks, i.p.) than a toxic cumulate dose, induced perivascular fibrosis without cell death in hear of mice. DOX increased the protein expression of α-SMA (a marker of trans-differentiation) in HCFs culture cells, indicating that DOX promoted the trans-differentiation of HCFs into myofibroblast. DOX also increased the mRNA and protein expression of matrix metalloproteinase (MMP)-1 in less than 0.1 µM which did not induce cell apoptosis of HCFs cells via PI3K/Akt pathway in HCFs. DOX increased Interleukin-6 (IL-6) via transforming growth factor (TGF)-ß/Smad pathway. In addition, DOX induced the mitochondrial damage and increased the expression of Interleukin-1 (IL-1) via stress-activated protein kinases (SAPK)/ c-Jun NH-2termial kinase (JNK). A peroxisome proliferator-activated receptor gamma (PPARγ) agonist, pioglitazone hydrochloride attenuated the expression of fibrotic marker such as α-SMA and galectin-3 and collagen1 via SAPK/JNK signaling. Pioglitazone also suppressed DOX-induced early fibrotic response in vivo. In conclusion, these findings suggested that low dose DOX induced reactive fibrotic change of cardiac fibroblasts via cell death-independent pathway. There may be potentially new mechanisms of DOX induced cardiotoxicity in clinical usage.

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.

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.

Hyperthermia and chemotherapy using Fe(Salen) nanoparticles might impact glioblastoma treatment.

We previously reported that µ-oxo N,N'-bis(salicylidene)ethylenediamine iron [Fe(Salen)], a magnetic organic compound, has direct anti-tumor activity, and generates heat in an alternating magnetic field (AMF). We showed that Fe(Salen) nanoparticles are useful for combined hyperthermia-chemotherapy of tongue cancer. Here, we have examined the effect of Fe(Salen) on human glioblastoma (GB). Fe(Salen) showed in vitro anti-tumor activity towards several human GB cell lines. It inhibited cell proliferation, and its apoptosis-inducing activity was greater than that of clinically used drugs. Fe(Salen) also showed in vivo anti-tumor activity in the mouse brain. We evaluated the drug distribution and systemic side effects of intracerebrally injected Fe(Salen) nanoparticles in rats. Further, to examine whether hyperthermia, which was induced by exposing Fe(Salen) nanoparticles to AMF, enhanced the intrinsic anti-tumor effect of Fe(Salen), we used a mouse model grafted with U251 cells on the left leg. Fe(Salen), BCNU, or normal saline was injected into the tumor in the presence or absence of AMF exposure. The combination of Fe(Salen) injection and AMF exposure showed a greater anti-tumor effect than did either Fe(Salen) or BCNU alone. Our results indicate that hyperthermia and chemotherapy with single-drug nanoparticles could be done for GB treatment.

Transient receptor potential cation 3 channel regulates melanoma proliferation and migration.

Melanoma has an extremely poor prognosis due to its rapidly progressive and highly metastatic nature. Several therapeutic drugs have recently become available, but are effective only against melanoma with specific BRAF gene mutation. Thus, there is a need to identify other target molecules. We show here that Transient receptor potential, canonical 3 (TRPC3) is widely expressed in human melanoma. We found that pharmacological inhibition of TRPC3 with a pyrazole compound, Pyr3, decreased melanoma cell proliferation and migration. Similar inhibition was observed when the TRPC3 gene was silenced with short-hairpin RNA (shRNA). Pyr3 induced dephosphorylation of signal transducer and activator of transcription (STAT) 5 and Akt. Administration of Pyr3 (0.05 mg/kg) to mice implanted with human melanoma cells (C8161) significantly inhibited tumor growth. Our findings indicate that TRPC3 plays an important role in melanoma growth, and may be a novel target for treating melanoma in patients.