METTL3/YTHDF1 m6A axis promotes tumorigenesis by enhancing DDR2 expression in ovarian cancer.

Ovarian cancer has the highest mortality among all gynecological malignancies. Therefore, it is urgent to determine the molecular mechanism of ovarian cancer progression. As the most prevalent modification of messenger RNA (mRNA), N6-Methyladenosine (m6A) modification is recognized as a key regulatory role in the progression of various tumors. However, the specific role of m6A and its related regulatory pathways in ovarian cancer (OV) remains unclear. In this study, we demonstrated that the METTL3/YTHDF1 m6A axis plays an important role in the progression of ovarian cancer. Depletion of METTL3/YTHDF1 impaired cancer proliferation and metastasis in vitro and in vivo. Mechanistically, The METTL3/YTHDF1 m6A axis directly binds to the mRNA of DDR2, thereby promoting the expression levels of the tumor promoter DDR2 and thus contributing to the progression of ovarian cancer. Collectively, our findings on the METTL3/YTHDF1/DDR2 m6A axis provide the insight into the underlying mechanism of ovarian carcinogenesis and highlight potential therapeutic targets for cancer treatment.

Rutaecarpine targets hERG channels and participates in regulating electrophysiological properties leading to ventricular arrhythmia.

Drug-mediated or medical condition-mediated disruption of hERG function accounts for the main cause of acquired long-QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG-HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (IhERG ) was measured by patch-clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the IhERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell-based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD90 and APD50 and the increase of DF, numbers of PSs, incidence of early after-depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the IhERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long-term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.

Thioridazine Induces Cardiotoxicity via Reactive Oxygen Species-Mediated hERG Channel Deficiency and L-Type Calcium Channel Activation.

Thioridazine (THIO) is a phenothiazine derivative that is mainly used for the treatment of psychotic disorders. However, cardiac arrhythmias especially QT interval prolongation associated with the application of this compound have received serious attention after its introduction into clinical practice, and the mechanisms underlying the cardiotoxicity induced by THIO have not been well defined. The present study was aimed at exploring the long-term effects of THIO on the hERG and L-type calcium channels, both of which are relevant to the development of QT prolongation. The hERG current (I hERG) and the calcium current (I Ca-L) were measured by patch clamp techniques. Protein levels were analyzed by Western blot, and channel-chaperone interactions were determined by coimmunoprecipitation. Reactive oxygen species (ROS) were determined by flow cytometry and laser scanning confocal microscopy. Our results demonstrated that THIO induced hERG channel deficiency but did not alter channel kinetics. THIO promoted ROS production and stimulated endoplasmic reticulum (ER) stress and the related proteins. The ROS scavenger N-acetyl cysteine (NAC) significantly attenuated hERG reduction induced by THIO and abolished the upregulation of ER stress marker proteins. Meanwhile, THIO increased the degradation of hERG channels via disrupting hERG-Hsp70 interactions. The disordered hERG proteins were degraded in proteasomes after ubiquitin modification. On the other hand, THIO increased I Ca-L density and intracellular Ca2+ ([Ca2+]i) in neonatal rat ventricular cardiomyocytes (NRVMs). The specific CaMKII inhibitor KN-93 attenuated the intracellular Ca2+ overload, indicating that ROS-mediated CaMKII activation promoted calcium channel activation induced by THIO. Optical mapping analysis demonstrated the slowing effects of THIO on cardiac repolarization in mouse hearts. THIO significantly prolonged APD50 and APD90 and increased the incidence of early afterdepolarizations (EADs). In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), THIO also resulted in APD prolongation. In conclusion, dysfunction of hERG channel proteins and activation of L-type calcium channels via ROS production might be the ionic mechanisms for QT prolongation induced by THIO.

Expression signature of miRNAs and the potential role of miR-195-5p in high-glucose-treated rat cardiomyocytes.

MicroRNAs are endogenous small noncoding RNAs that posttranscriptionally regulate the expression of target genes and play crucial roles in diverse physiopathologic processes. In the current study, we examined the microRNA (miRNA) expression profile of high-glucose-treated neonatal rat cardiomyocytes and the potential mechanisms. Differentially expressed miRNAs were analyzed by a miRNA microarray and validated by a quantitative real-time polymerase chain reaction in high-glucose-treated rat cardiomyocytes. Based on the results of our previous study and the bioinformatics prediction, we identified miR-195-5p/SGK1/Nedd4-2/hERG as the top-ranked signal pathway in diabetes cell model in vitro. In summary, our present study provides novel insights into the regulatory mechanism of miR-195-5p/SGK1/Nedd4-2/hERG in rat cardiomyocytes under high-glucose stress, which may provide a novel idea for the development of diagnostic and therapeutic strategies for diabetic cardiomyopathy in the future.

Effects of As2O3 and Resveratrol on the Proliferation and Apoptosis of Colon Cancer Cells and the hERG-mediated Potential Mechanisms.

BACKGROUND: As2O3 and resveratrol have been widely considered to be effective in anti-cancer therapies and the underlying mechanisms have been reported extensively. However, the combined treatment effect and potential target of As2O3 and resveratrol in the treatment of tumors remains elusive. The purpose of this study was to investigate the benefits and efficacy of As2O3 in combination with resveratrol in the treatment of colon cancer, as well as looking for new targets that could provide alternative explanation of the efficacy of drugs. METHODS: The proliferation of cancer cells was measured by the MTT and EdU staining assay, while the apoptosis of cancer cells was determined by the flow cytometry. Western blot and immunoprecipitation were performed to measure the expression levels of proteins and the interaction between hERG and integrin ß1, respectively. RESULTS: In this study, we found that both As2O3 and resveratrol can effectively inhibit cell proliferation and promote cell apoptosis in colon cancer, and the combined effect of the two drugs on colon cancer cells is more preeminent. The combination of As2O3 with resveratrol, on the one hand reduced the expression of hERG channels on the membrane, and on the other hand weaken the binding between hERG and integrin ß 1, which may be the main cause of downstream signaling pathways alterations, including the activation of the apoptotic pathway. CONCLUSION: Taken together, hERG, as a subunit of potassium ion channel on the cell membrane, is highly likely to be involved in the As2O3 and resveratrol induced intracellular signaling cascade disorder, and this novel signaling pathway that sustains the progression of colon cancer may be a promising therapeutic target for human colon cancer treatment in the future.

Intracellular Mechanism of Rosuvastatin-Induced Decrease in Mature hERG Protein Expression on Membrane.

The hERG potassium channel (IKr) encoded by human ether-a-go-go-related gene plays an important role in cardiac repolarization. Decreased IKr may lead to long QT syndrome, which subsequently causes torsade de pointes and sudden cardiac death. Previous studies have shown that statins inhibit IKr and are more potent in inhibiting hERG currents when combined with other drugs. Since chemical structure of rosuvastatin is similar to that of several IKr blockers (ibutilide and E-4031), the present study aimed to reveal the mechanism that underlies rosuvastatin-induced hERG current reduction and to evaluate the possibility of cardiac toxicity. The results showed that rosuvastatin reduced hERG currents by accelerating the inactivation and prolonged action potential duration (APD) in hiPSC-CMs. Meanwhile, it was observed that rosuvastatin reduced the expression of the mature hERG. Transcription factor Sp1 was involved in hERG protein downregulation induced by rosuvastatin, and the result was verified by Sp1 siRNA and Sp1 agonist epicatechin. These results indicated that rosuvastatin could potentially inhibit transcription and reduce hERG mRNA expression. The interaction between hERG and heat shock protein was evaluated to study the mechanism of trafficking inhibition through co-immunoprecipitation. We found that rosuvastatin reduces the interaction of heat shock protein 70 (Hsp70) with the hERG protein, thereby affecting the folding of the hERG channel. Additionally, rosuvastatin significantly activates ATF6, which plays a key role in the activation of the unfolded protein response (UPR) pathway. Increased expression of the molecular chaperone calnexin and calreticulin, which are activated by ATF6 to help channel folding, further confirmed UPR activation. Meanwhile, the degradation of the hERG channel was mediated by lysosomes and proteasomes. In conclusion, Rosuvastatin reduced the expression of hERG plasma membrane by two pathways, the first is to disrupt the transport of immature hERG channels to the membrane, and the second is to increase the degradation of mature hERG channels. In addition, Rosuvastatin potently blocked hERG current, delayed cardiac repolarization, and thereby prolonged APDs and QTc intervals. Therefore, caution should be taken when rosuvastatin is used in the treatment of hyperlipidemia, especially when combined with drugs that can prolong the QT interval.

Dual effects of arsenic trioxide on tumor cells and the potential underlying mechanisms.

The human ether-a-go-go related gene (hERG) encodes the rapid delayed rectifier K+ channel. hERG not only serves an important role in heart muscle and cardiomyocyte excitability by regulating action potential repolarization, but also represents a selective advantage for cancer cell proliferation. Arsenic trioxide is a traditional Chinese medicine, which has been previously identified as an efficient tumor suppressor, particularly in the treatment of acute pro-myelocytic leukemia. However, studies have also reported that long-term exposure to arsenicals may lead to the formation of malignant tumors. In the present study, the effect of low-dose arsenic trioxide on the proliferation and apoptosis of tumor cells was investigated, as were the potential underlying mechanisms of this effect. The data demonstrated that low-dose arsenic trioxide (0.1 µM) enhanced the viability and apoptosis of tumor cells expressing hERG channels following long-term incubation. However, in tumor cells lacking hERG channels, low-dose arsenic trioxide had no effect. Therefore, we hypothesized that this hormesis effect of low-dose arsenic trioxide on tumor cells may be associated with the hERG channel. Furthermore, low dose arsenic trioxide promoted the hERG-channel current by changing the kinetics of channel gating and prolonging the open-channel stage. Simultaneously, high-dose As2O3 (1 or 10 µM) significantly reduced the expression of hERG in tumor cells compared with the control group, which resulted in reduced proliferation rate and promotion of apoptotic rate. The results of the present study demonstrate that the dual effects of arsenic trioxide on hERG channels vary according to concentration, resulting in the dual effects on tumor cells. This provides a theoretical basis for the potential clinical application of arsenic trioxide, suggesting that hERG channels are an important target in preventing and treating tumorigenesis during arsenicosis.

Photothermal therapeutic application of gold nanorods-porphyrin-trastuzumab complexes in HER2-positive breast cancer.

Gold nanorods are effective photothermal agents in diagnosis and treatment of cancer due to their specific near-infrared laser absorption. However, tumor photothermal therapy by nanorods alone is lack of targeting. Here, we described a novel nanocomplex made up of gold nanorods, porphyrin, and trastuzumab, called TGNs and investigated the TGN-mediated photothermal therapy as a potential alternative treatment of targeting HER2-positive breast cancers. By conjugating trastuzumab and porphyrin to the surface of gold nanorods, we have increased the targeting specificity and amplified the detecting effectiveness at the same time. TGN-mediated photothermal ablation by near-infrared laser led to a selective destruction of HER2-positive cancer cells and significantly inhibited tumor growth in mouse models bearing HER2 over-expressed breast cancer xenograft with less toxicity. Moreover, TGNs provided better therapeutic efficacy in comparison with the conventional molecule targeted therapy. Our current data suggest a highly promising future of TGNs for its therapeutic application in trastuzumab-resistant breast cancers.

miR-493 inhibits proliferation and invasion in pancreatic cancer cells and inversely regulated hERG1 expression.

The human ether-a-go-go-related potassium channel 1 (hERG1) is a component of the voltage-gated Kv11.1 potassium channel, which has been recently indicated to have a crucial role in the tumorigenesis of multiple tumors, including pancreatic carcinoma. Pancreatic carcinoma is one of the most malignant human cancer types, which has an extremely poor prognosis. The present study demonstrated that the expression levels of hERG1 were markedly elevated in pancreatic cancer tissues and pancreatic cancer cell lines, and that the abnormal hERG1 expression was significantly associated with the proliferation and invasion ability of pancreatic cancer. Furthermore, hERG1 was identified to be a direct target of miR-493, which is generally reduced in pancreatic cancer tissues and cell lines. These findings provide a novel insight into the regulatory mechanism of miR-493/hERG1 in pancreatic cancer cell proliferation and invasion, which may aid the development of novel diagnostic and therapeutic strategies for pancreatic cancer in the future.

The prognostic and predictive value of the lymphocyte to monocyte ratio in luminal-type breast cancer patients treated with CEF chemotherapy.

Several reports have suggested that peripheral blood-based parameters are associated with host immunity response, which is an essential component of the pathogenesis and progression of cancer. The purpose of the present study was to identify the prognostic significance of various peripheral blood-based biomarkers and to determine the optimal cut-off value suitable for luminal breast cancer patients. We found that lymphocyte-to-monocyte ratio (LMR) was significant prognostic predictors. And the patients with a CEF regimen and LMR ratio ≥ 5.2 gained a good prognosis. This study suggested that the LMR could be regarded as an independent prognostic factor in luminal breast cancer patients. The elevated LMR level also had enhanced 5-fluorouracil sensitivity in luminal breast cancer patients.

Mechanisms underlying probucol-induced hERG-channel deficiency.

The hERG gene encodes the pore-forming α-subunit of the rapidly activating delayed rectifier potassium channel (I Kr), which is important for cardiac repolarization. Reduction of I hERG due to genetic mutations or drug interferences causes long QT syndrome, leading to life-threatening cardiac arrhythmias (torsades de pointes) or sudden death. Probucol is a cholesterol-lowering drug that could reduce hERG current by decreasing plasma membrane hERG protein expression and eventually cause long QT syndrome. Here, we investigated the mechanisms of probucol effects on I hERG and hERG-channel expression. Our data demonstrated that probucol reduces SGK1 expression, known as SGK isoform, in a concentration-dependent manner, resulting in downregulation of phosphorylated E3 ubiquitin ligase Nedd4-2 expression, but not the total level of Nedd4-2. As a result, the hERG protein reduces, due to the enhanced ubiquitination level. On the contrary, carbachol could enhance the phosphorylation level of Nedd4-2 as an alternative to SGK1, and thus rescue the ubiquitin-mediated degradation of hERG channels caused by probucol. These discoveries provide a novel mechanism of probucol-induced hERG-channel deficiency, and imply that carbachol or its analog may serve as potential therapeutic compounds for the handling of probucol cardiotoxicity.

Up-regulation of miR-21 and miR-23a Contributes to As2 O3 -induced hERG Channel Deficiency.

Arsenic trioxide (As2O3) is used to treat acute pro-myelocytic leukaemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the hERG current via disturbing its trafficking to cellular membrane. Consistent with these findings, in this study, we reported that As2O3 inhibited hERG channel at both protein and mRNA levels and damaged hERG current but did not affect channel kinetics. Further, we demonstrated that As2O3 up-regulated miR-21 and miR-23a expression in hERG-HEK293 cells and neonatal cardiomyocytes. In addition, knock-down of miR-21 by its specific antisense molecules AMO-21 was able to rescue Sp1 and hERG inhibition caused by As2O3. Consistently, phosphorylation of NF-κB, the upstream regulatory factor of miR-21, was significantly up-regulated by As2O3 . This finding revealed that regulation of the NF-κB-miR-21-Sp1 signalling pathway is a novel mechanism for As2O3-induced hERG inhibition. Meanwhile, the expression of Hsp90 and hERG was rescued by transfection with AMO-23a. And the hERG channel inhibition induced by As2O3 was rescued after being transfected with AMO-23a, which may be a molecular mechanism for the role of As2O3 in hERG trafficking deficiency. In brief, our study revealed that miR-21 and miR-23a are involved in As2O3-induced hERG deficiency at transcriptional and transportational levels. This discovery may provide a novel mechanism of As2O3-induced hERG channel deficiency, and these miRNAs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

The rescuable function and mechanism of resveratrol on As2O3-induced hERG K⁺ channel deficiency.

Arsenic trioxide (As2O3) is used to treat acute promyelocytic leukemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the human ether-a-go-go-related gene (hERG) current via disturbing its trafficking to cellular membrane. This study aimed to investigate whether the As2O3-insulted hERG channel can be rescued by resveratrol, a recognized cardioprotective agent. The whole-cell patch clamp technique was used to record the hERG current and action potential duration. Co-immunoprecipitation and Western blot assay were applied to determine the function of hERG-Hsp70/Hsp90 chaperone complexes and the expression alteration of protein-folding-related proteins, respectively. Compared with treatment of As2O3 alone, co-treatment with resveratrol successfully restored the current and surface expression of hERG and obviously shortened action potential duration in guinea pig ventricular myocytes. Further experiments demonstrate that resveratrol relieved As2O3-caused endoplasmic reticulum (ER) stress by restoring the function of hERG-Hsp70/Hsp90 chaperone complexes and downregulating the protein expression of ER chaperone proteins (calnexin and calreticulin) and activating transcription factor 6. In conclusion, resveratrol was able to rescue the trafficking deficiency and relieve the ER stress (ERS). Our findings suggest that resveratrol has a potential effect to alleviate the adverse effect of As2O3 on cardiotoxicity.