Effect of electroacupuncture on urodynamics and Raf/MEK/ERK signaling pathway in spinal cord tissue of rats with detrusor hyperreflexia after suprasacral spinal cord injury. / ç”µé’ˆå¯¹éª¶ä¸Šè„Šé«“æŸä¼¤åŽé€¼å°¿è‚Œåå°„äº¢è¿›åž‹å¤§é¼ å°¿æµåŠ¨åŠ›å­¦åŠè„Šé«“Raf/MEK/ERK信号通路的影响.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on urodynamics and Raf/MEK/ERK signaling pathway in spine cord tissue of rats after suprasacral spinal cord injury (SSCI), so as to explore its possible mechanism in improving bladder function in rats with detrusor hyperreflexia after SSCI. METHODS: Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at "Ciliao" (BL32), "Zhongji" (CV3), "Sanyinjiao" (SP6) and "Dazhui" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot. RESULTS: Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group. CONCLUSIONS: EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.

Unraveling the interplay between RAS/RAF/MEK/ERK signaling pathway and autophagy in cancer: From molecular mechanisms to targeted therapy.

RAS/RAF/MEK/ERK signaling pathway is one of the most important pathways of Mitogen-activated protein kinases (MAPK), which widely participate in regulating cell proliferation, differentiation, apoptosis and signaling transduction. Autophagy is an essential mechanism that maintains cellular homeostasis by degrading aged and damaged organelles. Recently, some studies revealed RAS/RAF/MEK/ERK signaling pathway is closely related to autophagy regulation and has a dual effect in tumor cells. However, the specific mechanism by which RAS/RAF/MEK/ERK signaling pathway participates in autophagy regulation is not fully understood. This article provides a comprehensive review of the research progress with regard to the RAS/RAF/MEK/ERK signaling pathway and autophagy, as well as their interplay in cancer therapy. The impact of small molecule inhibitors that target the RAS/RAF/MEK/ERK signaling pathway on autophagy is discussed in this study. The advantages and limitations of the clinical combination of these small molecule inhibitors with autophagy inhibitors are also explored. The findings from this study may provide additional perspectives for future cancer treatment strategies.

Quercetin-targeted AKT1 regulates the Raf/MEK/ERK signaling pathway to protect against doxorubicin-induced nephropathy in mice.

BACKGROUND: Doxorubicin is an anthracycline antitumor agent commonly used in clinical practice, which has some nephrotoxicity and is often used to establish mouse models of kidney injury for basic medical research. This study will investigate the protective effect of quercetin on renal function in doxorubicin-induced nephropathy mice. METHODS: C57BL/6 mice were divided into control, model, and quercetin low-, and high-dose groups. Serum and urine were collected to analyze markers of kidney function. H&E staining was used to detect pathological changes in renal tissues. Transmission electron microscopy was performed to observe the ultrastructural changes in renal tissues. Immunohistochemistry was performed to detect the changes of Ang II. RT-qPCR was performed to detect the changes of cytokines. ELISA was used to detect changes in serum inflammatory factors. Molecular docking was performed to verify the targeting relationship between quercetin and AKT1. Western blot was performed to detect Bax, Bcl-2, Cyt-c, AKT1, Raf, MEK, and ERK proteins. RESULTS: Quercetin could induce the recovery of kidney function in kidney-injured mice; H&E results showed that kidney tissue damage and tissue fibrosis were reduced in kidney-injured mice under quercetin. The mitochondrial swollen structure was destroyed by doxorubicin, while the mitochondrial structure was restored under quercetin. The levels of abnormal apoptotic proteins Bax and Bcl-2 were regulated to normal by quercetin. The high expression of Ang II caused by doxorubicin was down-regulated by quercetin. Abnormal inflammatory factors caused by doxorubicin were reversed by quercetin. Western blot experiments showed that quercetin regulated the protein levels of AKT1 and Raf/MEK/ERK and inhibited the detrimental effects of doxorubicin. CONCLUSION: Quercetin may mitigate doxorubicin-induced kidney injury in mice by regulating renal cell inflammatory factors and Raf/MEK/ERK signaling pathway through AKT1 to promote recovery of renal function.

RNF173 suppresses RAF/MEK/ERK signaling to regulate invasion and metastasis via GRB2 ubiquitination in Hepatocellular Carcinoma.

BACKGROUND: The role of the membrane-associated RING-CH (MARCH) family in carcinogenesis has been widely studied, but the member of this family, RNF173, has not yet been thoroughly explored in the context of hepatocellular carcinoma (HCC). METHODS: With the use of an HCC tissue microarray and IHC staining, we aim to determine the differential expression of RNF173 in HCC patients and its clinical significance. The biological role of RNF173 is investigated through in vitro and in vivo experiments. RNA sequencing, mass spectrometry, and immunoprecipitation are performed to uncover the underlying mechanism of RNF173's impact on the development of HCC. RESULTS: The mRNA and protein levels of RNF173 were significantly lower in HCC tissues than in normal tissues. HCC patients with low RNF173 expression had shorter overall survival and recurrence-free survival, and RNF173 was significantly correlated with tumor number, tumor capsule, tumor differentiation, and BCLC stage. In addition, in vitro and in vivo experiments showed that RNF173 downregulation exacerbated tumor progression, including migration, invasion, and proliferation. GRB2 is a key molecule in the RAF/MEK/ERK pathway. RNF173 inhibits the RAF/MEK/ERK signaling by ubiquitinating and degrading GRB2, thereby suppressing HCC cell proliferation, invasion and migration. Combining clinical samples, we found that HCC patients with high RNF173 and low GRB2 expression had the best prognosis. CONCLUSION: RNF173 inhibits the invasion and metastasis of HCC by ubiquitinating and degrading GRB2, thereby suppressing the RAF/MEK/ERK signaling pathway. RNF173 is an independent risk factor for the survival and recurrence of HCC patients. RNF173 may serve as a novel prognostic molecule and potential therapeutic target for HCC. Video Abstract Graphical abstract Model of RNF173 on RAF/MEK/ERK signaling. RNF173 knockdown resulted in impaired ubiquitination and degradation of GRB2, leading to the activation of the RAF/MEK/ERK signaling pathway and promotion of invasion and metastasis in HCC cells.

Preclinical evaluation of Mito-LND, a targeting mitochondrial metabolism inhibitor, for glioblastoma treatment.

BACKGROUND: Glioblastoma (GBM) is a brain tumor with the highest level of malignancy and the worst prognosis in the central nervous system. Mitochondrial metabolism plays a vital role in the occurrence and development of cancer, which provides critical substances to support tumor anabolism. Mito-LND is a novel small-molecule inhibitor that can selectively inhibit the energy metabolism of tumor cells. However, the therapeutic effect of Mito-LND on GBM remains unclear. METHODS: The present study evaluated the inhibitory effect of Mito-LND on the growth of GBM cells and elucidated its potential mechanism. RESULTS: The results showed that Mito-LND could inhibit the survival, proliferation and colony formation of GBM cells. Moreover, Mito-LND induced cell cycle arrest and apoptosis. Mechanistically, Mito-LND inhibited the activity of mitochondrial respiratory chain complex I and reduced mitochondrial membrane potential, thus promoting ROS generation. Importantly, Mito-LND could inhibit the malignant proliferation of GBM by blocking the Raf/MEK/ERK signaling pathway. In vivo experiments showed that Mito-LND inhibited the growth of GBM xenografts in mice and significantly prolonged the survival time of tumor-bearing mice. CONCLUSION: Taken together, the current findings support that targeting mitochondrial metabolism may be as a potential and promising strategy for GBM therapy, which will lay the theoretical foundation for further clinical trials on Mito-LND in the future.

Targeting RAS-RAF-MEK-ERK signaling pathway in human cancer: Current status in clinical trials.

Molecular target inhibitors have been regularly approved by Food and Drug Administration (FDA) for tumor treatment, and most of them intervene in tumor cell proliferation and metabolism. The RAS-RAF-MEK-ERK pathway is a conserved signaling pathway that plays vital roles in cell proliferation, survival, and differentiation. The aberrant activation of the RAS-RAF-MEK-ERK signaling pathway induces tumors. About 33% of tumors harbor RAS mutations, while 8% of tumors are driven by RAF mutations. Great efforts have been dedicated to targeting the signaling pathway for cancer treatment in the past decades. In this review, we summarized the development of inhibitors targeting the RAS-RAF-MEK-ERK pathway with an emphasis on those used in clinical treatment. Moreover, we discussed the potential combinations of inhibitors that target the RAS-RAF-MEK-ERK signaling pathway and other signaling pathways. The inhibitors targeting the RAS-RAF-MEK-ERK pathway have essentially modified the therapeutic strategy against various cancers and deserve more attention in the current cancer research and treatment.

Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

Enterovirus 71 (EV71) is the major pathogen responsible for hand, foot, and mouth disease (HFMD) outbreaks; to date, there is no specific anti-EV71 agent. HSP90 is a crucial host factor for the viral life cycle and an ideal therapeutic target for limiting viral proliferation. However, the specific role of HSP90 in EV71-related signaling pathways and anti-EV71 agents targeting HSP90 remains unclear. This study aimed to verify the role of HSP90 in signaling pathways involved in EV71 replication and investigate the antiviral effects of a small molecule of VER-50589, a potent HSP90 inhibitor, against EV71 both in vitro and in vivo. Viral plaque assay, western blotting, and qPCR results showed that VER-50589 diminished the plaque formation induced by EV71 and inhibited EV71 mRNA and protein synthesis. A single daily dose of VER-50589 treatment significantly improved the survival rate of EV71-infected mice (p < 0.005). Interestingly, VER-50589 also exhibits activities against a series of human enteroviruses, including Coxsackievirus B3 (CVB3), Coxsackievirus B4-5 (CVB4-5), Coxsackievirus B4-7 (CVB4-7), and Echovirus 11 (Echo11). EV71 infection activated the AKT and ERK signaling pathways, and phosphorylation of AKT and RAF/MEK/ERK was weakened by VER-50589 administration. Thus, VER-50589 exhibits robust antiviral activity by inhibiting HSP90 and mediating the AKT and RAF/MEK/ERK signaling pathways. Considering that there are no effective antivirals or vaccines for the prevention and cure of HFMD in a clinical setting, the development of an anti-EV71 agent would be a straightforward and feasible therapeutic approach.

PLX8394, a RAF inhibitor, inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling.

Enterovirus 71 (EV71) poses a serious threat to human health, with scattered outbreaks worldwide. There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection. Therefore, it is urgent and of significance to develop anti-EV71 drugs. Here, we found that PLX8394, a RAF inhibitor, possesses high antiviral activity against EV71 in vitro, being superior to the traditional clinical drug ribavirin. Moreover, PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses. Notably, in a suckling mouse model, PLX8394 provided a 70% protection rate for EV71-infected mice, reduced the viral load in liver and heart tissues, and relieved the inflammatory response. A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway. Thus, PLX8394 lays a foundation for the development of new drugs against EV71.

NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP.

BACKGROUND: NeiyiKangfu tablets (NYKF) are widely used clinically for the treatment of endometriosis (EMS), whose mechanism of action has been extensively studied. Researchers have found that NYKF may control the development of ectopic lesions by inhibiting angiogenesis and inflammatory cytokine secretion. Nevertheless, NYKF's mechanism of action remains unclear. METHODS: In the present study, the function of NYKF in the progression of EMS and the associated underlying mechanism was investigated by in vivo and in vitro experiments. EMS model mice were treated with NYKF and the pro-inflammatory factors and apoptosis of ectopic endometrium as well as RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling activation were assessed. In addition, human endometriosis-derived immortalized entopic stromal (hEM15A) cells transfected with or without RAF kinase inhibitor protein (RKIP)-small-interfering RNA (siRNA) were also treated with NYKF and the proliferation, migration, apoptosis, and RAF/MEK/ERK signaling activation were measured by Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, and western blot, respectively. RESULTS: Results showed that NYKF increased the expression of RKIP, inhibited RAF/MEK/ERK signaling activation, and induced apoptosis while inhibiting proliferation and migration both in EMS mice and hEM15A cells. RKIP knockdown could inhibit the effect of NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and the proliferation and migration of hEM15A cells. CONCLUSIONS: In conclusion, these results suggest that NYKF treatment promotes apoptosis and inhibits proliferation and migration in EMS by inhibiting the RAF/MEK/ERK signaling pathway by targeting RKIP.

Astragaloside trigger autophagy: Implication a potential therapeutic strategy for pulmonary fibrosis.

Pulmonary fibrosis is an abnormal wound-healing response to repeated alveolar injury, characterized by continuous inflammation and abnormal collagen deposition. Its treatment is problematic. Astragaloside (AST) is an active component of Astragalus membranaceus with anti-inflammatory and anti-tumor properties. Although the underlying mechanisms are unknown, AST is also used to treat fibrotic diseases. This study aimed to investigate the mechanisms of action of AST in pulmonary fibrosis treatment. We found that AST significantly improved restrictive ventilatory impairment, compliance, total lung capacity, and functional residual capacity. In mice with pulmonary fibrosis, extracellular matrix deposition in the pulmonary parenchyma and intemperate inflammation were reversed. This therapeutic effect can be attributed to autophagy, activating the genes for autophagy flux and autophagic vacuoles. Impaired autophagy increased susceptibility to pulmonary fibrosis by exacerbating collagen deposition in vitro and in vivo. Using a combination of molecular docking and network pharmacology, the Ras/Raf/MEK/ERK signaling pathway was identified as a possible candidate for the pharmacologic target of AST. Functional dephosphorylation of MEK and ERK inhibited the Ras/Raf/MEK/ERK signaling pathway, which converges at the rapamycin switch to initiate autophagy. Inhibitors of Ras and MEK regulated autophagy. These findings suggest that AST might treat pulmonary fibrosis by modulating the Ras/Raf/MEK/ERK signaling pathway mediated by depression.

Long non­coding RNA BANCR promotes proliferation, invasion and migration in esophageal squamous cell carcinoma cells via the Raf/MEK/ERK signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a major histological type of esophageal cancer, identified as a leading cause of tumor­associated death worldwide. In addition, long non­coding RNA (lncRNA) BRAF­activated non­coding RNA (BANCR) expression is increased in the plasma of patients with ESCC, which can be reversed by tumor resection. Thus, the aim of the present study was to investigate the underlying mechanism of BANCR in ESCC progression. The relative mRNA expression of BANCR was determined via reverse transcription­quantitative PCR. The cell behaviors of Eca­109 cells were detected using Cell Counting Kit­8, colony formation, wound healing and Transwell chamber assays. Finally, the expression levels of proteins involved in the Raf/MEK/ERK signaling pathway and cell metastasis were analyzed with western blotting. The results revealed that lncRNA BANCR was highly expressed in ESCC cells compared with in normal esophageal cells. BANCR overexpression enhanced proliferation, migration and invasion of ESCC cells, and BANCR silencing exerted opposite effects. Moreover, BANCR overexpression induced activation of the Raf/MEK/ERK signaling pathway in ESCC cells. Notably, U0126, a specific MEK inhibitor, decreased MEK and ERK expression, and blocked the promotive effects of BANCR overexpression on the proliferation, migration and invasion of ESCC cells. Overall, lncRNA BANCR facilitated the proliferation, migration and invasion of ESCC cells via the Raf/MEK/ERK signaling pathway. Thus, lncRNA BANCR may be a promising target for inhibiting ESCC growth and metastasis.

Regulatory effect of annexin A5 on glioma cell invasion andmigration and its mechanisms / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. 【Methods】 The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5 (si-Annexin A5) in human glioma cell line (U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. 【Results】 Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively (P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased, and the tumor grade and positive rate were significantly positively correlated (r=1.000, P=0.000). The cell viability of U251 cells in the si-Annexin A5 group after 48 h and 72 h of culture was significantly reduced by 29.46% and 40.43%, respectively, compared with that in the control group (P<0.05). Compared with the control group, in the si-Annexin A5 group the colony formation rate was reduced by 68.58%, while the apoptosis rate was increased by 24.41 times (P<0.05); the cell migration rate and invasion rate were reduced by 65.35% and 68.80% (P<0.05). The protein expression of p-Raf, p-MEK1/2, p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%, 70.37%, 60.26% and 54.95%, respectively, and that of E-Cadherin was increased by 3.58 times (P<0.05). 【Conclusion】 Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.

BRAF Mutation in Colorectal Cancers: From Prognostic Marker to Targetable Mutation.

The Raf murine sarcoma viral oncogene homolog B (BRAF) mutation is detected in 8-12% of metastatic colorectal cancers (mCRCs) and is strongly correlated with poor prognosis. The recent success of the BEACON CRC study and the development of targeted therapy have led to the determination of BRAF-mutated mCRCs as an independent category. For nearly two decades, a growing body of evidence has established the significance of the BRAF mutation in the development of CRC. Herein, we overview both basic and clinical data relevant to BRAF-mutated CRC, mainly focusing on the development of treatment strategies. This review is organized into eight sections, including clinicopathological features, molecular features, prognosis, the predictive value of anti-epidermal growth factor receptor (EGFR) therapy, resistant mechanisms for BRAF-targeting treatment, the heterogeneity of the BRAF mutation, future perspectives, and conclusions. A characterization of the canonical mitogen-activated protein kinase (MAPK) pathway is essential for controlling this malignancy, and the optimal combination of multiple interventions for treatments remains a point of debate.

Long non-coding RNA SNHG16 regulates cell behaviors through miR-542-3p/HNF4α axis via RAS/RAF/MEK/ERK signaling pathway in pediatric neuroblastoma cells.

Neuroblastoma (NB) is an extracranial solid tumor in children with complex mechanism. Increasing reports indicated that long non-coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) account for the pathogenesis of NB. Nevertheless, the precise functions of SNHG16 needed to be further exposed in NB progression. Our data revealed that SNHG16 and hepatocyte nuclear factor 4 α (HNF4α) were up-regulated, but miR-542-3p was down-regulated in NB. Knockdown of SNHG16 or HNF4α could impede cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Interestingly, the role of SNHG16 detetion in cell behaviors was rescued by HNF4α overexpression in NB cells. Mechanically, SNHG16 modulated the progression of tumor growth via miR-542-3p/HNF4α axis in NB. Also, SNHG16 knockdown inactivated rat sarcoma/effector of RAS/mitogen-activated extracellular signal-regulated kinase/extracellular regulated protein kinases (RAS/RAF/MEK/ERK) signaling pathway through HNF4α. Therefore, SNHG16/miR-542-3p/HNF4α axis modified NB progression via RAS/RAF/MEK/ERK signaling pathway, might highlight a novel therapeutic approach for NB.

miR-145-5p restrained cell growth, invasion, migration and tumorigenesis via modulating RHBDD1 in colorectal cancer via the EGFR-associated signaling pathway.

miR-145-5p has been reported to be downregulated and described functioning as a tumor suppressive gene in colorectal cancer (CRC), yet its detailed regulatory function and mechanism in malignant progression of the disease have not been thoroughly understood. In our study, miR-145-5p and rhomboid domain containing 1 (RHBDD1) in CRC tissues and cells were examined by qRT-PCR and western blot. MTT, colony formation, wound healing, Transwell invasion, and flow cytometry assays were performed to evaluate the malignant phenotypes of CRC cells. Xenograft tumor, qRT-PCR, and western blot assays were applied to validate the roles and mechanism of miR-145-5p in CRC in vivo. The interaction between miR-145-5p and RHBDD1 was investigated by luciferase reporter assay and western blot. The changes of the EGFR/Raf/MEK/ERK pathway were detected by western blot. We found miR-145-5p was lowly expressed and low miR-145-5p predicted poor prognosis in CRC, while RHBDD1 was greatly enhanced in CRC cells and tissues. RHBDD1 silencing resulted in inhibiting cell proliferative, invasive, and migratory potentials as well as elevating apoptotic ones in CRC cells. miR-145-5p was inversely related with RHBDD1 expression in CRC tissues. miR-145-5p was found to directly bind to RHBDD1 and restrained its expression in CRC cells. miR-145-5p overexpression repressed CRC cell proliferation, invasion, migration and induced apoptosis, and these effects were reversed by RHBDD1 upregulation. Moreover, in CRC xenograft tumor, its growth was impeded by miR-145-5p via suppressing RHBDD1. Furthermore, miR-145-5p inhibited the expression of EGFR, p-MEK1/2 and p-ERK1/2, in vitro and in vivo by targeting RHBDD1. In conclusion, our study revealed that miR-145-5p overexpression inhibited tumorigenesis in CRC by downregulating RHBDD1 via suppressing the EGFR-associated signaling pathway (EGFR/Raf/MEK/ERK cascades).

Diwu Yanggan capsule inhibits the occurrence and development of liver cancer in the Solt-Farber rat model by regulating the Ras/Raf/Mek/Erk signaling pathway.

This study sought to determine the effect and explore the mechanism of the Chinese medicinal compound preparation Diwu Yanggan (DWYG) capsule on the occurrence and development of liver cancer using the Solt-Farber rat model. Sprague-Dawley rats were randomly distributed into a normal group, sham group, DWYG group, sorafenib group, and model group. The DWYG group and sorafenib group were given DWYG capsule and sorafenib tablet, respectively, with induction of the model. Hematoxylin-eosin (HE) staining was used to detect liver pathological changes. The content of nuclear DNA in the liver was detected by Feulgen staining, and the expression of PCNA was detected by immunohistochemical staining. Molecular biology methods were used to detect the expression of liver regeneration-related factors and Ras/Raf/Mek/Erk signaling pathway-related proteins and mRNAs. HE staining showed that compared with those in the model group, the liver pathological changes in the DWYG group were significantly reduced (P < 0.05). The nuclear DNA content in the liver based on Feulgen staining and the expression of PCNA in the DWYG group was lower than that in the model group (P < 0.05). The expression of regeneration-related factors and Ras/Raf/Mek/Erk signaling pathway-related proteins and mRNAs was significantly lower in the DWYG group than in the model group (P < 0.05). In conclusion, DWYG capsules to some degree inhibit the occurrence and development of liver cancer in the Solt-Farber rat model, and the effect is not inferior to that of sorafenib. DWYG capsules likely delay the occurrence and development of liver cancer and improve the liver regeneration microenvironment by regulating the Ras/Raf/Mek/Erk signaling pathway and regeneration-related factors.

An integrative pharmacological-based study on network mechanism of Xinkeshu Tablets intervention in atherosclerosis / 中草药

Objective: To explore the mechanism of the intervention of Xinkeshu Tablets (XKST) on atherosclerosis (AS) and provide reference for the secondary development and clinical application of XKST. Methods: The integrated pharmacology platform was used to predict the key targets and pathways of the intervention of XKST on AS and its molecular mechanism was also explored. Results: In the integrative analysis of heterogeneous network of “TCM-component-target-pathway”, 80 relevant effective ingredients were found, including B4GALT4, B4GALT2, PRKCD, GCK, GNB1, and other key targets; Endocrine system, thyroid hormone signaling pathway, nervous system, estrogen signaling pathway, and chemokine signaling pathway were key pathways related with its anti-atherosclerosis. Conclusion: According to the analysis and prediction of the enrichment information, the effect of XKST on common regulating PI3K/Akt/eNOS and Raf/MEK/ERK signaling pathway and protecting vascular endothelial cells is first prompted, thus achieving the intervention in AS.

Vitamin C at high concentrations induces cytotoxicity in malignant melanoma but promotes tumor growth at low concentrations.

Vitamin C has been used in complementary and alternative medicine for cancers regardless of its ineffectiveness in clinical trials and the paradoxical effects antioxidants have on cancer. Vitamin C was found to induce cytotoxicity against cancers. However, the mechanisms of action have not been fully elucidated, and the effects of vitamin C on human malignant melanoma have not been examined. This study revealed that vitamin C at millimolar concentrations significantly reduced the cell viability as well as invasiveness, and induced apoptosis in human malignant melanoma cells. Vitamin C displayed stronger cytotoxicity against the Vemurafenib-resistance cell line A2058 compared with SK-MEL-28. In contrast, vitamin C at micromolar concentrations promoted cell growth, migration and cell cycle progression, and protected against mitochondrial stress. Vemurafenib paradoxically activated the RAS-RAF-MEK-ERK signaling pathway in the Vemurafenib-resistant A2058, however, vitamin C abolished the activations. Vitamin C displayed synergistic cytotoxicity with Vemurafenib against the Vemurafenib-resistant A2058. In vivo assay suggested that lower dosage (equivalent to 0.5 g/70 kg) of vitamin C administered orally increased the melanoma growth. Therefore, vitamin C may exert pro- or anti-melanoma effect depending on concentration. The combination of vitamin C at high dosage and Vemurafenib is promising in overcoming the action of drug resistance.

SOX9 was involved in TKIs resistance in renal cell carcinoma via Raf/MEK/ERK signaling pathway.

Renal cell carcinoma (RCC) is common genitourinary malignancy in human, 30-40% of patients with RCC would be diagnosed with metastatic RCC (mRCC). Even in the era of targeted therapy, patients with mRCC would inevitably progress due to drug resistance. Herein, exploration of the mechanisms of resistance is noteworthy to study. In the present study, we firstly reported the expression profile of SOX9 in renal carcinoma cells and tissues, and found that its expression was significantly associated with Fuhrman grading. Dual luciferase analysis confirmed that Raf/MEK/ERK pathway could directly be regulated by SOX9, and sequential experiments demonstrated that, renal carcinoma cells could sensitize to Sorafenib/Sunitinib through Raf/MEK/ERK signaling pathway inhibition regulated by SOX9 down-regulation. In a small cases with mRCC treated with Sorafenib/Sunitinib (n=38), comparative analysis showed that patients with SOX9 (-) had much better therapeutic response to TKIs than those with SOX9 (+) (PD: 9.1% vs. 56.2%, P=0.002, DCR: 90.9% vs. 43.8%, P=0.002). Based on these findings, we concluded that, SOX9 was firstly described to be highly expressed in renal cell carcinoma, and its expression was involved in TKIs drug resistance through activation of Raf/MEK/ERK pathway. In vitro, patients with SOX9 (-) was related to better response to TKIs treatment than those with SOX9 (+). SOX9 could be expected to be a promising biomarker predicting TKIs response and even expected to be another novel target in the treatment of mRCC.

Apoptosis in glioma-bearing rats after neural stem cell transplantation.

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of glioblastoma treatment by neural stem cell transplantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in suspension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly increased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.