Enhancing nasopharyngeal carcinoma cell radiosensitivity by suppressing AKT/mTOR via CENP-N knockdown
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ABSTRACT:
SUBMITTER: Wu L
PROVIDER: S-EPMC10631041 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor. VEGF was reported to promote the occurrence of autophagy, which enhanced the radioresistance of tumors. The purpose of this study was to investigate the influence of VEGF silencing on the radiosensitivity of nasopharyngeal carcinoma (NPC) cells and the underlying mechanisms. The radiosensitivity of NPC cells after VEGF silencing was detected by cell counting kit 8 (CCK-8) and clonogenic assay, while cell cycle and apoptosis were detected by flow cytometry. The processes of DNA damage, repair and autophagy were examined by immunofluorescence and western blotting. The interaction between VEGF and mTOR was confirmed by western blotting and co-immunoprecipitation studies. The effect of VEGF on radiosensitivity of NPC cells was investigated in vivo using a xenograft model. Furthermore, immunohistochemistry and TUNEL assays were used to verify the relationship between autophagy and radiosensitivity in NPC after VEGF depletion. Downregulation of VEGF significantly inhibited cell proliferation and induced apoptosis of NPC cells after radiotherapy in vitro and in vivo. In addition, VEGF knockdown not only decreased autophagy level, but also delayed the DNA damage repair in NPC cells after irradiation. Mechanistically, silencing VEGF suppressed autophagy through activation of the mTOR pathway. VEGF depletion increased radiosensitivity of NPC cells by suppressing autophagy via activation of the mTOR pathway.
| S-EPMC7531011 | biostudies-literature
Project description:BackgroundThe transferrin receptor (TfR) encoded by TFRC gene is the main cellular iron importer. TfR is highly expressed in many cancers and is expected to be a promising new target for cancer therapy; however, its role in nasopharyngeal carcinoma (NPC) remains unknown.MethodsThe TfR levels were investigated in NPC tissues and cell lines using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. Knockdown of TFRC using two siRNA to investigate the effects on intracellular iron level and biological functions, including proliferation by CKK-8 assay, colony formation, cell apoptosis and cell cycle by flow cytometry, migration and invasion, and tumor growth in vivo by nude mouse xenografts. RNA sequencing was performed to find possible mechanism after TFRC knockdown on NPC cells and further verified by western blotting.ResultsTfR was overexpressed in NPC cell lines and tissues. Knockdown of TFRC inhibited cell proliferation concomitant with increased apoptosis and cell cycle arrest, and it decreased intracellular iron, colony formation, migration, invasion, and epithelial-mesenchymal transition in HK1-EBV cells. Western blotting showed that TFRC knockdown suppressed the levels of the iron storage protein FTH1, anti-apoptotic marker BCL-xL, and epithelial-mesenchymal transition markers. We confirmed in vivo that TFRC knockdown also inhibited NPC tumor growth and decreased Ki67 expression in tumor tissues of nude mouse xenografts. RNA sequencing and western blotting revealed that TFRC silencing inhibited the PI3K/Akt/mTOR signaling pathway.ConclusionsThese results indicated that TfR was overexpressed in NPC, and TFRC knockdown inhibited NPC progression by suppressing the PI3K/Akt/mTOR signaling pathway. Thus, TfR may serve as a novel biomarker and therapeutic target for NPC.
| S-EPMC10466839 | biostudies-literature
Project description:To explore the relationship between autophagy and cell function, we investigated how PLAC8-mediated autophagy influences proliferation, apoptosis and epithelial-mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP-GFP-tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3-methyladenine or by knocking down Beclin-1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co-immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.
| S-EPMC7348153 | biostudies-literature
Project description:Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy. EOC control remains difficult, and EOC patients show poor prognosis regarding metastasis and chemotherapy resistance. The aim of this study was to estimate the effect of CXCR4 knockdown-mediated reduction of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) stemness and enhancement of chemotherapy sensitivity in EOC. Mechanisms contributing to these effects were also explored. Our data showed distinct contribution of CXCR4 overexpression by dependent PI3K/Akt/mTOR signaling pathway in EOC development. CXCR4 knockdown resulted in a reduction in CSCs and EMT formation and enhancement of chemotherapy sensitivity in tumor cells, which was further advanced by blocking CXCR4-PI3K/Akt/mTOR signaling. This study also documented the critical role of silencing CXCR4 in sensitizing ovarian CSCs to chemotherapy. Thus, targeting CXCR4 to suppress EOC progression, specifically in combination with paclitaxel (PTX) treatment, may have clinical application value.
| S-EPMC9217704 | biostudies-literature
Project description:Tripartite motif-containing 29 (TRIM29) has been reported to be dysregulated in human cancers. Up-regulation of TRIM29 was first observed in NPC cell lines by a genome-wide transcriptome analysis in our previous study. However, its expression biological function and clinical significance in nasopharyngeal carcinoma (NPC) remain unclear. In this study, TRIM29 expression was validated by qRT-PCR and immunohistochemistry in 69 NPC samples. Notably, TRIM29 protein expression was significantly and positively correlated with the tumor size, clinical stage and metastasis. TRIM29 was identified as the direct target of miR-335-5p and miR-15b-5p, both of which were down-regulated and negatively associated with TRIM29 expression in NPC cell lines and clinical samples. Ectopic TRIM29 expression promoted proliferation, epithelial-mesenchymal transition (EMT), migration and invasion in NPC cells, while its depletion inhibited cell invasion and EMT phenotype. Mechanistically, TRIM29 overexpression reduced PTEN expression and increase phosphorylated protein level of AKT, p70S6K and 4E-BP1. Correspondingly, AKT inhibitor and Rapamycin blocked the effect of TRIM29 on cell invasion. In conclusion, our results suggest that miR-335-5p and miR-15b-5p down-regulation results in TRIM29 over-expression, which induces proliferation, EMT and metastasis of NPC through the PTEN/AKT/mTOR signaling pathway.
| S-EPMC4924667 | biostudies-literature
Project description:Radiotherapy is a conventional and effective treatment method for nasopharyngeal carcinoma (NPC), although it can fail, mainly because radioresistance results in residual or recurrent tumors. However, the mechanisms and predictive markers of NPC radioresistance are still obscure. In this study, we identified Annexin A6 (ANXA6) as a candidate radioresistance marker by using Tandem Mass Tag quantitative proteomic analysis of NPC cells and gene chip analysis of NPC clinical samples with different radiosensitivities. It was observed that a high expression level of ANXA6 was positively correlated with radioresistance of NPC and that inhibition of ANXA6 by siRNA increased the radiosensitivity. The incidence of autophagy was enhanced in the established radioresistant NPC cells in comparison with their parent cells, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, ANXA6 siRNA (siANXA6) suppressed cellular autophagy by activating the PI3K/AKT/mTOR pathway, ultimately leading to radiosensitization. The combination of siANXA6 and CAL101 (an inhibitor of PI3K, p-AKT, and mTOR, concurrently) significantly reversed the above siANAX6-reduced autophagy. Suppression of PI3K/AKT/mTOR by CAL101 also increased the expression of ANXA6 in a negative feedback process. In conclusion, this study revealed for the first time that ANXA6 could promote autophagy by inhibiting the PI3K/AKT/mTOR pathway and that it thus contributes to radioresistance of NPC. The significance of this is that ANXA6 could be applied as a new predictive biomarker of NPC prognosis after radiotherapy.
| S-EPMC7176914 | biostudies-literature
Project description:Increasing evidence reveals that the Rho GTPase-activating protein is a crucial negative regulator of Rho family GTPase involved in tumorigenesis. The Rho GTPase-activating protein 25 (ARHGAP25) has been shown to specifically inactivate the Rho family GTPase Rac1, which plays an important role in pancreatic adenocarcinoma (PAAD) progression. Therefore, here we aimed to clarify the expression and functional role of ARHGAP25 in PAAD. The ARHGAP25 expression was lower in PAAD tissues than that in normal pancreatic tissues based on bioinformatics analysis and immunohistochemistry staining. Overexpression of ARHGAP25 inhibited cell growth of AsPC-1 human pancreatic cancer cells in vitro, while opposite results were observed in BxPC-3 human pancreatic cancer cells with ARHGAP25 knockdown. Consistently, in vivo tumorigenicity assays also confirmed that ARHGAP25 overexpression suppressed tumor growth. Mechanically, overexpression of ARHGAP25 inactivated AKT/mTOR signaling pathway by regulating Rac1/PAK1 signaling, which was in line with the results from the Gene set enrichment analysis on The Cancer Genome Atlas dataset. Furthermore, we found that ARHGAP25 reduced HIF-1α-mediated glycolysis in PAAD cells. Treatment with PF-04691502, a dual PI3K/mTOR inhibitor, hampered the increased cell growth and glycolysis due to ARHGAP25 knockdown in PAAD cells. Altogether, these results conclude that ARHGAP25 acts as a tumor suppressor by inhibiting the AKT/mTOR signaling pathway, which might provide a therapeutic target for PAAD.
| S-EPMC8120455 | biostudies-literature
Project description:Tenacissoside H (TEH), which has anti-inflammatory and anti-tumor effects, is a major active ingredient extracted from the stem of Marsdenia tenacissima. However, the effect of TEH on hepatocellular carcinoma (HCC) as well as the underlying mechanisms are still indistinct. Presently, HCC cells (including Huh-7 and HepG2) were dealt with different concentrations of TEH. The proliferation and apoptosis of HCC cells were determined via Cell Counting Kit-8 (CCK8) assay and flow cytometry. In addition, Western blot was conducted to evaluate the expressions of autophagy-and apoptosis-related proteins. Tissue immunofluorescence was carried out to evaluate LC3B expression in the tumor tissues. The data showed that TEH suppressed the growth of HCC cells in a concentration-dependent manner. Besides, TEH enhanced radiosensitivity and promoted the apoptosis of HCC cells. Moreover, the mRNA and protein levels of autophagy-related genes (LC3-II/LC2-I, ATG5, Beclin-1) were significantly promoted by TEH. Mechanistically, TEH attenuated the activation of PI3K/Akt/mTOR signaling pathway. However, inhibition of PI3 K pathway abolished the anti-tumor effects of TEH in HCC cells. Collectively, this study suggested that TEH increases the radiosensitivity of HCC cells via inducing autophagy and apoptosis through downregulating PI3K/Akt/mTOR signaling pathway.
| S-EPMC8127767 | biostudies-literature
Project description:BackgroundAfatinib, a second-generation tyrosine kinase inhibitor (TKI), exerts its radiosensitive effects in nasopharyngeal carcinoma (NPC). However, the detailed mechanism of afatinib-mediated sensitivity to radiation is still obscure in NPC.MethodsQuantitative phosphorylated proteomics and bioinformatics analysis were performed to illustrate the global phosphoprotein changes. The activity of the CD44-Stat3 axis and Epithelial-Mesenchymal Transition (EMT)-linked markers were evaluated by Western blotting. Wound healing and transwell assays were used to determine the levels of cell migration upon afatinib combined IR treatment. Cell proliferation was tested by CCK-8 assay. A pharmacological agonist by IL-6 was applied to activate Stat3. The xenograft mouse model was treated with afatinib, radiation or a combination of afatinib and radiation to detect the radiosensitivity of afatinib in vivo.ResultsIn the present study, we discovered that afatinib triggered global protein phosphorylation alterations in NPC cells. Further, bioinformatics analysis indicated that afatinib inhibited the CD44-Stat3 signaling and subsequent EMT process. Moreover, functional assays demonstrated that afatinib combined radiation treatment remarkably impeded cell viability, migration, EMT process and CD44-Stat3 activity in vitro and in vivo. In addition, pharmacological stimulation of Stat3 rescued radiosensitivity and biological functions induced by afatinib in NPC cells. This suggested that afatinib reversed the EMT process by blocking the activity of the CD44-Stat3 axis.ConclusionCollectively, this work identifies the molecular mechanism of afatinib as a radiation sensitizer, thus providing a potentially useful combination treatment and drug target for NPC radiosensitization. Our findings describe a new function of afatinib in radiosensitivity and cancer treatment.
| S-EPMC9864417 | biostudies-literature
Project description: Not available
| S-EPMC10777026 | biostudies-literature