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.

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.

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.

Potentiation of Anticancer Activity of G2/M Blockers by Mild Hyperthermia.

BACKGROUND/AIM: Hyperthermia (HT), combined with chemotherapy, has been used to treat various types of cancer. This study aimed to investigate the HT-sensitivity of malignant and non-malignant cells, and then evaluate the combination effect of docetaxel (DTX) and a newly synthesized chromone derivative (compound A) with HT. MATERIALS AND METHODS: The number of viable cells was determined using the MTT method. Cell cycle distribution was analyzed using a cell sorter, and DNA fragmentation pattern was detected using agarose gel electrophoresis. RESULTS: Among 12 cultured cells, oral squamous cell carcinoma (OSCC), especially Ca9-22 cells, and myelogenous leukemia cells showed higher sensitivity to HT than lung carcinoma and glioblastoma cell lines, while normal oral cells were the most resistant. Cytotoxicity of DTX on Ca9-22 cells was maximum at 41-42°C and 45~60 min exposure to HT. DXT, compound A, and HT induced G2/M arrest of Ca-22 cells. Mild HT enhanced the DTX- and compound A-induced subG1 arrest, in a synergistic fashion. CONCLUSION: The combination G2/M blockers and mild-HT can potentially be used for the treatment of OSCC.

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.

Prognostic factors and treatment outcomes of advanced maxillary gingival squamous cell carcinoma treated by intra-arterial infusion chemotherapy concurrent with radiotherapy.

BACKGROUND: The aim of this study was to evaluate the prognostic factors and treatment outcomes of advanced maxillary gingival squamous cell carcinoma (SCC) treated with intra-arterial infusion chemotherapy concurrent with radiotherapy. METHODS: A total of 46 patients were reviewed retrospectively in this study. The treatment schedule comprised intra-arterial chemotherapy (total, 60 mg/m2 docetaxel and 150 mg/m2 cisplatin) and three-dimensional computed tomography based, daily conventional radiotherapy (total, 60 Gy/30 fr) for 6 weeks. RESULTS: The median follow-up period was 40 months (range, 3-110 months). The 3-year overall survival and locoregional control rates for all patients were 64.3% and 84.3%, respectively. The OS rate of the patients with N0-1 was significantly higher than that of the patients with N ≥ 2 (P < .05). No grade 5 toxicities were observed. CONCLUSIONS: Intra-arterial infusion chemotherapy concurrent with radiotherapy was effective for advanced maxillary gingival SCC.

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.

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.

The iron chelating agent, deferoxamine detoxifies Fe(Salen)-induced cytotoxicity.

Iron-salen, i.e., µ-oxo-N,N'-bis(salicylidene)ethylenediamine iron (Fe(Salen)) was a recently identified as a new anti-cancer compound with intrinsic magnetic properties. Chelation therapy has been widely used in management of metallic poisoning, because an administration of agents that bind metals can prevent potential lethal effects of particular metal. In this study, we confirmed the therapeutic effect of deferoxamine mesylate (DFO) chelation against Fe(Salen) as part of the chelator antidote efficacy. DFO administration resulted in reduced cytotoxicity and ROS generation by Fe(Salen) in cancer cells. DFO (25 mg/kg) reduced the onset of Fe(Salen) (25 mg/kg)-induced acute liver and renal dysfunction. DFO (300 mg/kg) improves survival rate after systematic injection of a fatal dose of Fe(Salen) (200 mg/kg) in mice. DFO enables the use of higher Fe(Salen) doses to treat progressive states of cancer, and it also appears to decrease the acute side effects of Fe(Salen). This makes DFO a potential antidote candidate for Fe(Salen)-based cancer treatments, and this novel strategy could be widely used in minimally-invasive clinical settings.

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.

Simultaneous hyperthermia-chemotherapy with controlled drug delivery using single-drug nanoparticles.

We previously investigated the utility of µ-oxo N,N'- bis(salicylidene)ethylenediamine iron (Fe(Salen)) nanoparticles as a new anti-cancer agent for magnet-guided delivery with anti-cancer activity. Fe(Salen) nanoparticles should rapidly heat up in an alternating magnetic field (AMF), and we hypothesized that these single-drug nanoparticles would be effective for combined hyperthermia-chemotherapy. Conventional hyperthermic particles are usually made of iron oxide, and thus cannot exhibit anti-cancer activity in the absence of an AMF. We found that Fe(Salen) nanoparticles induced apoptosis in cultured cancer cells, and that AMF exposure enhanced the apoptotic effect. Therefore, we evaluated the combined three-fold strategy, i.e., chemotherapy with Fe(Salen) nanoparticles, magnetically guided delivery of the nanoparticles to the tumor, and AMF-induced heating of the nanoparticles to induce local hyperthermia, in a rabbit model of tongue cancer. Intravenous administration of Fe(Salen) nanoparticles per se inhibited tumor growth before the other two modalities were applied. This inhibition was enhanced when a magnet was used to accumulate Fe(Salen) nanoparticles at the tongue. When an AMF was further applied (magnet-guided chemotherapy plus hyperthermia), the tumor masses were dramatically reduced. These results indicate that our strategy of combined hyperthermia-chemotherapy using Fe(Salen) nanoparticles specifically delivered with magnetic guidance represents a powerful new approach for cancer treatment.