In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAF^V600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.     

Pseudophosphatases as Regulators of MAPK Signaling

Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes, including the cell cycle, apoptosis, differentiation, protein biosynthesis, and oncogenesis; therefore, tight control of the activity of MAPK is critical. Kinases and phosphatases are well established as MAPK activators and inhibitors, respectively. Kinases phosphorylate MAPKs, initiating and controlling the amplitude of the activation. In contrast, MAPK phosphatases (MKPs) dephosphorylate MAPKs, downregulating and controlling the duration of the signal. In addition, within the past decade, pseudoenzymes of these two families, pseudokinases and pseudophosphatases, have emerged as bona fide signaling regulators. This review discusses the role of pseudophosphatases in MAPK signaling, highlighting the function of phosphoserine/threonine/tyrosine-interacting protein (STYX) and TAK1-binding protein (TAB 1) in regulating MAPKs. Finally, a new paradigm is considered for this well-studied cellular pathway, and signal transduction pathways in general.     

Apoptosis Pathways Triggered by a Potent Antiproliferative Hybrid Chalcone on Human Melanoma Cells

The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage and was prevented by a non-specific caspase inhibitor. Using SK-MEL-1 as a model, we found that the mechanism of cell death involves (i) the generation of reactive oxygen species, (ii) activation of the extrinsic and intrinsic apoptotic and mitogen-activated protein kinase pathways, (iii) upregulation of TRAIL, DR4 and DR5, (iv) downregulation of p21^Cip1/WAF1 and, inhibition of the NF-κB pathway.     

Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic β-Cells

Pancreatic β-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in β-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic β-cells.     

Blocking ERK-DAPK1 Axis Attenuates Glutamate Excitotoxicity in Epilepsy

Glutamate excitotoxicity induces neuronal cell death during epileptic seizures. Death-associated protein kinase 1 (DAPK1) expression is highly increased in the brains of epilepsy patients; however, the underlying mechanisms by which DAPK1 influences neuronal injury and its therapeutic effect on glutamate excitotoxicity have not been determined. We assessed multiple electroencephalograms and seizure grades and performed biochemical and cell death analyses with cellular and animal models. We applied small molecules and peptides and knocked out and mutated genes to evaluate the therapeutic efficacy of kainic acid (KA), an analog of glutamate-induced neuronal damage. KA administration increased DAPK1 activity by promoting its phosphorylation by activated extracellular signal-regulated kinase (ERK). DAPK1 activation increased seizure severity and neuronal cell death in mice. Selective ERK antagonist treatment, DAPK1 gene ablation, and uncoupling of DAPK1 and ERK peptides led to potent anti-seizure and anti-apoptotic effects in vitro and in vivo. Moreover, a DAPK1 phosphorylation-deficient mutant alleviated glutamate-induced neuronal apoptosis. These results provide novel insight into the pathogenesis of epilepsy and indicate that targeting DAPK1 may be a potential therapeutic strategy for treating epilepsy.     

Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK,SRC, and JNK Pathways

Bladder cancer is a common global cancer with a high percentage of metastases and high mortality rate. Thus, it is necessary to identify new biomarkers that can be helpful in diagnosis. Pyruvate dehydrogenase kinase 4 (PDK4) belongs to the PDK family and plays an important role in glucose utilization in living organisms. In the present study, we evaluated the role of PDK4 in bladder cancer and its related protein changes. First, we observed elevated PDK4 expression in high-grade bladder cancers. To screen for changes in PDK4-related proteins in bladder cancer, we performed a comparative proteomic analysis using PDK4 knockdown cells. In bladder cancer cell lines, PDK4 silencing resulted in a lower rate of cell migration and invasion. In addition, a PDK4 knockdown xenograft model showed reduced bladder cancer growth in nude mice. Based on our results, PDK4 plays a critical role in the metastasis and growth of bladder cancer cells through changes in ERK, SRC, and JNK.     

Prevention of Tendon Adhesions by ERK2 Small Interfering RNAs

Tendon adhesions are one of the most concerning complications after surgical repair of flexor tendon injury. Extracellular signal-regulated kinase (ERK) 2 plays crucial roles in fibroblast proliferation and collagen expression which contributes to the formation of tendon adhesions after flexor tendon surgery. Using a chicken model, we have examined the effects of a small interfering RNA (siRNA) targeting ERK2 delivered by a lentiviral system on tendon adhesion formation with an adhesion scoring system, histological assessment, and biomechanical evaluation. It was found that ERK2 siRNA effectively suppressed the increase of fibroblasts and the formation of tendon adhesions (p < 0.05 compared with the control group). Moreover, no statistically significant reduction in breaking force was detected between the ERK2 siRNA group and the control group. These results show that the lentiviral-mediated siRNA system is effective in preventing tendon adhesion formation but not to tendon healing, and may be used for tendon repair after confirmation and improvement by future detailed studies.     

Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

MicroRNAs have been demonstrated as key regulators of gene expression in the etiology of a range of diseases including Alzheimer’s disease (AD). Recently, we identified miR-483-5p as the most upregulated miRNA amongst a panel of miRNAs in blood plasma specific to prodromal, early-stage Alzheimer’s disease patients. Here, we investigated the functional role of miR-483-5p in AD pathology. Using TargetScan and miRTarBase, we identified the microtubule-associated protein MAPT, often referred to as TAU, and the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), known to phosphorylate TAU, as predicted direct targets of miR-483-5p. Employing several functional assays, we found that miR-483-5p regulates ERK1 and ERK2 at both mRNA and protein levels, resulting in lower levels of phosphorylated forms of both kinases. Moreover, miR-483-5p-mediated repression of ERK1/2 resulted in reduced phosphorylation of TAU protein at epitopes associated with TAU neurofibrillary pathology in AD. These results indicate that upregulation of miR-483-5p can decrease phosphorylation of TAU via ERK pathway, representing a compensatory neuroprotective mechanism in AD pathology. This miR-483-5p/ERK1/TAU axis thus represents a novel target for intervention in AD.     

Interplay between Cartilage and Subchondral Bone Contributing to Pathogenesis of Osteoarthritis

Osteoarthritis (OA) is a common debilitating joint disorder, affecting large sections of the population with significant disability and impaired quality of life. During OA, functional units of joints comprising cartilage and subchondral bone undergo uncontrolled catabolic and anabolic remodeling processes to adapt to local biochemical and biological signals. Changes in cartilage and subchondral bone are not merely secondary manifestations of OA but are active components of the disease, contributing to its severity. Increased vascularization and formation of microcracks in joints during OA have suggested the facilitation of molecules from cartilage to bone and vice versa. Observations from recent studies support the view that both cartilage and subchondral bone can communicate with each other through regulation of signaling pathways for joint homeostasis under pathological conditions. In this review we have tried to summarize the current knowledge on the major signaling pathways that could control the cartilage-bone biochemical unit in joints and participate in intercellular communication between cartilage and subchondral bone during the process of OA. An understanding of molecular communication that regulates the functional behavior of chondrocytes and osteoblasts in both physiological and pathological conditions may lead to development of more effective strategies for treating OA patients.     

熊果酸对血管内皮细胞增殖的影响及其机制

目的研究熊果酸(Ursolic acid,UA)对人脐静脉血管内皮细胞(Human umbilical vein endothelial cells,HUVECs)增殖及ERK1、c-Jun、c-Myc、Cyclin D1基因mRNA和蛋白表达的影响,探讨熊果酸抑制血管生长的机制。方法体外培养HU-VECs,用不同浓度熊果酸(31.5、62.5、125、250、500μg/ml)处理不同时间(12、24、48 h),MTT法检测细胞增殖抑制率。用125μg/ml熊果酸和100μmol/L PD98059(ERK抑制剂)处理HUVECs 48 h;不同浓度熊果酸处理24 h;125μg/ml熊果酸处理不同时间,RT-PCR和Western blot分别检测HUVECs中ERK1、c-Jun、c-Myc、Cyclin D1基因mRNA的转录水平和蛋白的表达水平。结果不同浓度的熊果酸对HUVECs的增殖均有明显的抑制作用,且呈剂量和时间依赖性;经熊果酸和PD98059处理的HUVECs中ERK1、c-Jun、c-Myc、Cyclin D1基因mRNA和蛋白的表达水平均明显降低,且呈剂量和时间依赖性。结论熊果酸可通过抑制ERK信号转导通路而抑制血管内皮细胞增殖。     

Neutrophil Extracellular Traps in Colorectal Cancer Progression and Metastasis

Neutrophils form sticky web-like structures known as neutrophil extracellular traps (NETs) as part of innate immune response. NETs are decondensed extracellular chromatin filaments comprising nuclear and cytoplasmic proteins. NETs have been implicated in many gastrointestinal diseases including colorectal cancer (CRC). However, the regulatory mechanisms of NET formation and potential pharmacological inhibitors in the context of CRC have not been thoroughly discussed. In this review, we intend to highlight roles of NETs in CRC progression and metastasis as well as the potential of targeting NETs during colon cancer therapy.     

CTI-2 Inhibits Metastasis and Epithelial-Mesenchymal Transition of Breast Cancer Cells by Modulating MAPK Signaling Pathway

Although some breast cancer patients die due to tumor metastasis rather than from the primary tumor, the molecular mechanism of metastasis remains unclear. Therefore, it is necessary to inhibit breast cancer metastasis during cancer treatment. In this case, after designing and synthesizing CTI-2, we found that CTI-2 treatment significantly reduced breast cancer cell metastasis in vivo and in vitro. Notably, with the treatment of CTI-2 in breast cancer cells, the expression level of E-cadherin increased, while the expression level of N-cadherin and vimentin decreased. In addition, after CTI-2 treatment, those outflow levels for p-ERK, p-p38, and p-JNK diminished, while no significant changes in the expression levels of ERK, JNK, or p38 were observed. Our conclusion suggested that CTI-2 inhibits the epithelial-mesenchymal transition (EMT) of breast carcinoma cells by inhibiting the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, thereby inhibiting the metastasis of breast tumor cells. Therefore, we believe that CTI-2 is another candidate for breast tumor medication.     

Picrasidine I Triggers Heme Oxygenase-1-Induced Apoptosis in Nasopharyngeal Carcinoma Cells via ERK and Akt Signaling Pathways

Nasopharyngeal carcinoma (NPC) has a higher incidence in Taiwan than worldwide. Although it is a radiosensitive malignancy, cancer recurrence is still high in the advanced stages because of its ability to induce lymph node metastasis. Picrasidine I from Picrasma quassioides has been reported as a potential drug for targeting multiple signaling pathways. The present study aimed to explore the role of picrasidine I in the apoptosis of NPC cells. Our results show that picrasidine I induced cytotoxic effects in NPC cells and caused cell cycle arrest in the sub-G1, S, and G2/M phases. Western blot analysis further demonstrated that the modulation of apoptosis through the extrinsic and intrinsic pathways was involved in picrasidine I-induced cell death. Downregulation of the ERK1/2 and Akt signaling pathways was also found in picrasidine I-induced apoptosis. Additionally, the apoptosis array showed that picrasidine I significantly increased heme oxygenase-1 (HO-1) expression, which could act as a critical molecule in picrasidine I-induced apoptosis in NPC cells. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets also revealed that the HMOX1 mRNA level (HO-1) is lower in patients with head and neck squamous carcinoma (HNSCC) and NPC than in patients without cancer. Our study indicated that picrasidine I exerts anticancer effects in NPC by modulating HO-1 via the ERK and Akt signaling pathways.     

Critical Roles of Circular RNA in Tumor Metastasis via Acting as a Sponge of miRNA/isomiR

Circular RNAs (circRNAs), a class of new endogenous non-coding RNAs (ncRNAs), are closely related to the carcinogenic process and play a critical role in tumor metastasis. CircRNAs can lay the foundation for tumor metastasis via promoting tumor angiogenesis, make tumor cells gain the ability of migration and invasion by regulating epithelial-mesenchymal transition (EMT), interact with immune cells, cytokines, chemokines, and other non-cellular components in the tumor microenvironment, damage the normal immune function or escape the immunosuppressive network, and further promote cell survival and metastasis. Herein, based on the characteristics and biological functions of circRNA, we elaborated on the effect of circRNA via circRNA-associated competing endogenous RNA (ceRNA) network by acting as miRNA/isomiR sponges on tumor angiogenesis, cancer cell migration and invasion, and interaction with the tumor microenvironment (TME), then explored the potential interactions across different RNAs, and finally discussed the potential clinical value and application as a promising biomarker. These results provide a theoretical basis for the further application of metastasis-related circRNAs in cancer treatment. In summary, we briefly summarize the diverse roles of a circRNA-associated ceRNA network in cancer metastasis and the potential clinical application, especially the interaction of circRNA and miRNA/isomiR, which may complicate the RNA regulatory network and which will contribute to a novel insight into circRNA in the future.     

乌司他丁和泰索帝对人乳腺癌细胞MDA-MB-231中uPA、uPAR、ERK表达的影响

目的探讨乌司他丁(Ulinastatin,UTI)和泰索帝(Taxotere,TXT)对体外培养的人乳腺癌细胞MDA-MB-231中u-PA、uPAR、ERK表达的影响。方法将MDA-MB-231(ER-)细胞分为4组:UTI组(UTI 800 U/ml)、TXT组(TXT 3.7μg/ml)、UTI+TXT组(UTI 800 U/ml+TXT 3.7μg/ml)、对照组(等量生理盐水)。给药后24 h,分别采用荧光定量RT-PCR检测各组细胞中uPA、uPAR、ERK基因mRNA的水平,Western blot法检测各组细胞中uPA、uPAR、p-ERK1/2蛋白的表达水平。结果 UTI组和UTI+TXT组MDA-MB-231(ER-)细胞中uPA和uPAR基因mRNA的水平均明显低于对照组(P<0.05),而TXT组中二者的表达水平均明显高于对照组(P<0.01),各组间ERK基因mRNA的水平差异无统计学意义(P>0.05);UTI组和UTI+TXT组中uPA、uPAR和p-ERK1/2蛋白的表达水平均明显低于对照组(P<0.01),而TXT组中3种蛋白的表达水平均明显高于对照组(P<0.05)。结论UTI可抑制MDA-MB-231细胞中uPA、uPAR、p-ERK的表达,而TXT可上调三者的表达。     

Cell Metabolomics Reveals the Potential Mechanism of Aloe Emodin and Emodin Inhibiting Breast Cancer Metastasis

Metastasis is one of the main obstacles for the treatment and prognosis of breast cancer. In this study, the effects and possible mechanisms of aloe emodin (AE) and emodin (EMD) for inhibiting breast cancer metastasis were investigated via cell metabolomics. First, a co-culture model of MCF-7 and HUVEC cells was established and compared with a traditional single culture of MCF-7 cells. The results showed that HUVEC cells could promote the development of cancer cells to a malignant phenotype. Moreover, AE and EMD could inhibit adhesion, invasion, and angiogenesis and induce anoikis of MCF-7 cells in co-culture model. Then, the potential mechanisms behind AE and EMD inhibition of MCF-7 cell metastasis were explored using a metabolomics method based on UPLC-Q-TOF/MS multivariate statistical analysis. Consequently, 27 and 13 biomarkers were identified in AE and EMD groups, respectively, including polyamine metabolism, methionine cycle, TCA cycle, glutathione metabolism, purine metabolism, and aspartate synthesis. The typical metabolites were quantitatively analyzed, and the results showed that the inhibitory effect of AE was significantly better than EMD. All results confirmed that AE and EMD could inhibit metastasis of breast cancer cells through different pathways. Our study provides an overall view of the underlying mechanisms of AE and EMD against breast cancer metastasis.