Autophagy In Nasopharyngeal Carcinoma Cells Research Articles

Trifolirhizin targets PTK6 to induce autophagy and exerts antitumor effects in nasopharyngeal carcinoma.

Trifolirhizin, a natural flavonoid glycoside, has been proved to exert antitumor activities in various human malignant tumors. PTK6 was identified as a direct target of trifolirhizin based on public database SuperPred (https://prediction.charite.de/). Overexpressed PTK6 in a variety of tumors is closely associated with the malignant development of tumors. Herein, this present research was formulated to elaborate the effects of trifolirhizin on the biological behaviors of nasopharyngeal carcinoma (NPC) cells and to probe into the intrinsic mechanisms. The current study firstly elucidated the tumor-inhibiting functions of trifolirhizin in NPC malignant progression from the perspective of targeting inhibition of PTK6. In this work, CCK-8 for cell viability, EdU staining for cell proliferation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for cell apoptosis and immunofluorescence staining for LC3 expression were performed. Besides, levels of proliferation-related, apoptosis-related and autophagy-related proteins were detected by western blot analysis. Moreover, molecular docking of trifolirhizin with PTK6 was conducted to seek the compound-protein binding potential. It was demonstrated that trifolirhizin treatment inhibited the proliferation and promoted the apoptosis of NPC cells as well as strengthened autophagy in NPC cells. Furthermore, it was verified that trifolirhizin targeted PTK6 and negatively regulated PTK6 expression. The suppressive effects of trifolirhizin on the malignant behaviors of NPC cells and the enhancing effect of trifolirhizin on autophagy in NPC cells were partly abolished upon upregulation of PTK6. To conclude, findings suggested that trifolirhizin may downregulate PTK6 expression to induce autophagy and exert the antitumor activities in NPC.

RNA-binding domain 2 of nucleolin is important for the autophagy induction of curcumol in nasopharyngeal carcinoma cells

Background & AimsExcessive autophagy induces cell death and is regarded as the treatment of cancer therapy. We have confirmed that the anti-cancer mechanism of curcumol is related to autophagy induction. As the main target protein of curcumol, RNA binding protein nucleolin (NCL) interacted with many tumor promoters accelerating tumor progression. However, the role of NCL in cancer autophagy and in curcumol's anti-tumor effects haven't elucidated. The purpose of the study is to identify the role of NCL in nasopharyngeal carcinoma autophagy and reveal the immanent mechanisms of NCL played in cell autophagy. Methods & ResultsIn the current study, we have found that NCL was markedly upregulated in nasopharyngeal carcinoma (NPC) cells. NCL overexpression effectively attenuated the level of autophagy in NPC cells, and NCL silence or curcumol treatment obviously aggravated the autophagy of NPC cells. Moreover, the attenuation of NCL by curcumol lead a significant suppression on PI3K/AKT/mTOR signaling pathway in NPC cells. Mechanistically, NCL was found to be directly interact with AKT and accelerate AKT phosphorylation, which caused the activation of the PI3K/AKT/mTOR pathway. Meanwhile, the RNA Binding Domain (RBD) 2 of NCL interacts with Akt, which was also influenced by curcumol. Notably, the RBDs of NCL delivered AKT expression was related with cell autophagy in the NPC. ConclusionThe results demonstrated that NCL regulated cell autophagy was related with interaction of NCL and Akt in NPC cells. The expression of NCL play an important role in autophagy induction and further found that was associated with its effect on NCL RNA-binding domain 2. This study may provide a new perspective on the target protein studies for natural medicines and confirm the effect of curcumol not only regulating the expression of its target protein, but also influencing the function domain of its target protein.

Parkin enhances sensitivity of paclitaxel to nasopharyngeal carcinoma by activating BNIP3/NIX-mediated mitochondrial autophagy.

As a malignant head and neck cancer, nasopharyngeal carcinoma (NPC) has high morbidity. Parkin expression has been reported to be reduced in NPC tissues and its upregulation could enhance paclitaxel-resistant cell cycle arrest. This study was performed to explore the possible mechanism of Parkin related to B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3)/BNIP3-like (NIX)-mediated mitochondrial autophagy in NPC cells. Initially, after Parkin overexpression or silencing, cell viability and proliferation were evaluated by lactate dehydrogenase and colony formation assays. JC-1 staining was used to assess the mitochondrial membrane potential. In addition, the levels of cellular reactive oxygen species (ROS) and mitochondrial ROS were detected using DCFH-DA staining and mitochondrial ROS (MitoSOX) red staining. The expression of proteins was measured using Western blot. Results showed that Parkin overexpression inhibited, whereas Parkin knockdown promoted the proliferation of paclitaxel-treated NPC cells. Besides, Parkin overexpression induced, whereas Parkin knockdown inhibited mitochondrial apoptosis in paclitaxel-treated NPC cells, as evidenced by the changes of Cytochrome C (mitochondria), Cytochrome C (cytoplasm), BAK, and Bcl-2 expression. Moreover, the levels of ROS, mitochondrial membrane potential, and LC3II/LC3I in paclitaxel-treated C666-1 cells were hugely elevated by Parkin overexpression and were all declined by Parkin knockdown in CNE-3 cells. Furthermore, Parkin upregulation activated, whereas Parkin downregulation inactivated BNIP3/NIX signaling. Further, BNIP3 silencing or overexpression reversed the impacts of Parkin upregulation or downregulation on the proliferation and mitochondrial apoptosis of paclitaxel-treated NPC cells. Particularly, Mdivi-1 (mitophagy inhibitor) or rapamycin (an activator of autophagy) exerted the same effects on NPC cells as BNIP3 silencing or overexpression, respectively. Collectively, Parkin overexpression activated BNIP3/NIX-mediated mitochondrial autophagy to enhance sensitivity to paclitaxel in NPC.

LINC00324 suppresses apoptosis and autophagy in nasopharyngeal carcinoma through upregulation of PAD4 and activation of the PI3K/AKT signaling pathway.

Nasopharyngeal carcinoma (NPC) has high incidence in Southern China and is derived from the mucosal epithelium of the nasopharynx. Accumulating evidence has revealed that peptidyl arginine deiminase 4 (PAD4) exerts carcinogenic effect on certain cancers. We designed this study to probe the specific role that PAD4 plays in NPC and its molecular mechanism. PAD4 expression in NPC cells was detected by RT-qPCR analysis. MTT, colony formation, flow cytometry, TUNEL staining, and LC3-II punctuation experiments were done to probe into the biological functions of PAD4 on NPC cellular behaviors in vitro. Subsequently, the upstream regulatory mechanism of PAD4 was investigated by luciferase reporter, RNA pull-down, and RIP assays. The impact of PAD4 on NPC tumor growth in mice was assessed by in vivo xenograft tumor assay. PAD4 was upregulated in NPC cells. PAD4 knockdown suppressed proliferative ability and promoted apoptosis and autophagy in NPC cells. Additionally, PAD4 expression was negatively regulated by microRNA 3164 (miR-3164). LINC00324 positively upregulated PAD4 expression by interacting with miR-3164 and recruiting HuR protein. The LINC00324/miR-3164/PAD4 axis modulated the PI3K/AKT pathway in NPC cells. Moreover, PAD4 upregulation countervailed the influences of LINC00324 deficiency on NPC cell proliferation, apoptosis, and autophagy and on NPC tumor growth in mice. LINC00324 promoted NPC malignancy by upregulation of PAD4 to activate the PI3K/AKT pathway.

Tetrandrine sensitizes nasopharyngeal carcinoma cells to irradiation by inducing autophagy and inhibiting MEK/ERK pathway.

Radioresistance was the main reason for local recurrence and metastasis of nasopharyngeal carcinoma. Tetrandrine is reported as an antitumor drug via inducing cell cycle arrest and apoptosis. In this study, the radiosensitization effects of maximum noncytotoxic doses of tetrandrine in nasopharyngeal carcinoma were analyzed both in vitro and in vivo, using MTT assay, western blot, TUNEL, and HE staining. It was found that the maximum dose of tetrandrine inhibited the phosphorylation of ERK and MEK induced by irradiation, and significantly enhanced irradiation‐induced cell growth inhibition in nasopharyngeal carcinoma cells CNE1, CNE2, and C666‐1. The ERK activator and overexpression of ERK reversed the radiosensitization effect of tetrandrine. About 50 mg/kg of tetrandrine which was used as the maximum noncytotoxic dose of tetrandrine in vivo, enhanced the radiosensitivity of the xenograft tumor and increased the apoptosis rate of the xenograft tumor cells caused by irradiation, while did not raise the side effect of the treatment. Moreover, tetrandrine increased autophagy in nasopharyngeal carcinoma cells. These results suggested that the maximum noncytotoxic dose of tetrandrine sensitized nasopharyngeal carcinoma to irradiation by inhibiting MEK/ERK pathway and inducing autophagy.

Autophagy inhibition impairs the epithelial-mesenchymal transition and enhances cisplatin sensitivity in nasopharyngeal carcinoma.

Drug resistance restricts the efficacy of cisplatin in the treatment of nasopharyngeal carcinoma (NPC). Increasing evidence indicates that autophagy and the epithelial-mesenchymal transition (EMT) participate in cancer progression and drug sensitivity. The aim of the present study was to investigate the function of autophagy and EMT in cisplatin treatment, and to reveal the underlying impact of autophagy on the EMT process in NPC. Transmission electron microscopy assays and western blot analyses confirmed that cisplatin activates autophagy in NPC cells. Alterations in cell morphology and biomolecular markers confirmed that cisplatin induces the EMT phenotype in NPC cells. Cell viability assays showed that the combination of the autophagy inhibitor chloroquine (CQ) increased the cytotoxicity of cisplatin in NPC cells and that the EMT inducer transforming growth factor β1 promoted the resistance to cisplatin in NPC cells. Moreover, autophagy inhibition by CQ and microtubule-associated protein 1 light chain 3B-knockdown reversed the EMT phenotype in NPC cells. In conclusion, autophagy and the EMT process promote cisplatin resistance in NPC cells, while the inhibition of autophagy impairs the EMT process.

MicroRNA-138-5p controls sensitivity of nasopharyngeal carcinoma to radiation by targeting EIF4EBP1.

Radiation therapy is the standard treatment for primary nasopharyngeal carcinoma (NPC). MicroRNA regulates cancer responsiveness to radiation therapy by controlling the genes involved in radiation responses. Recent studies suggested that downregulation of microRNA-138-5p was clinically significant in NPC. Here, we evaluated the effect of miR-138-5p on radiosensitivity of NPC cells and explored the underlying mechanisms by identifying its target gene that impacted sensitivity to radiation. Our results revealed that overexpression of miR-138-5p reduced the ability to form colonies, inhibited proliferation, and enhanced radiation-induced DNA damage and autophagy in NPC cells upon radiation treatment. By integrating predicted targets with the transcripts downregulated by miR-138-5p, EIF4EBP1 was identified to be a target gene of miR-138-5p. Results from luciferase reporter assay demonstrated that miR-138-5p downregulated the expression of EIF4EBP1 by binding to the 3'-UTR. Silence of EIF4EBP1 enhanced radiosensitivity of NPC cells as evidenced by reduced ability to form colonies after radiation exposure. In summary, our results indicated that miR-138-5p enhanced radiosensitivity of NPC cells by targeting EIF4EBP1. Further studies are warranted to investigate the potential use of miR-138-5p in the clinical management and treatment prediction of NPC patients.

Autophagy inhibitor 3-methyladenine enhances the sensitivity of nasopharyngeal carcinoma cells to chemotherapy and radiotherapy

To explore the effects of 3-methyladenine (3-MA, an autophagy inhibitor) on sensitivities of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy and the underlying mechanisms. Cell proliferation was examined by MTT and colony formation assay, while cell apoptosis was evaluated by annexin V/PI double staining and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. Mitochondrial membrane potential was measured by commercial kit (JC-1). The expression of endoplasmic reticulum stress (ERS)-related protein, glucose-regulated protein 78 (GRP78) and autophagy-related protein beclin1, microtubule-associated protein 1 light chain 3 (LC3) were examined by Western blot. Cisplatin (DDP), ionizing radiation (IR) or tunicamycin (TM) treatment obviously inhibited the proliferation of HONE-1 cells in a concentration-dependent and time-dependent manner. Compared with control group, pretreatment with 1 mmol/L of 3-MA significantly reduced cell viability and enhanced the apoptosis in the DDP (6.00 μmol/L), 4.00 Gy IR or TM (1.00μmol/L) groups. There was no significant difference in the apoptosis between the DDP (5.8%) and 4Gy IR (6.7%) groups. Compared with the control group, protein levels of GRP78, beclin1 and lipid-conjugated membrane-bound form (LC3-II) were significantly increased after the treatment of DDP, 4.00 Gy IR or TM, which were inhibited by pretreatment of 3-MA. 3-MA can sensitize HONE-1 cells to chemotherapy and radiotherapy, which is related to prevention of endoplasmic reticulum stress-induced autophagy in nasopharyngeal carcinoma cells.

Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo

AimProtein kinase B (AKT) signaling frequently is deregulated in human cancers and plays an important role in nasopharyngeal carcinoma (NPC). This preclinical study investigated the effect of MK-2206, a potent allosteric AKT inhibitor, on human NPC cells in vitro and in vivo.MethodsThe effect of MK-2206 on the growth and proliferation of CNE-1, CNE-2, HONE-1, and SUNE-1 cells was assessed by Cell Counting Kit 8 and colony formation assay. Flow cytometry was performed to analyze cell cycle and apoptosis. The effects of MK-2206 on the AKT pathway were analyzed by Western blotting. Autophagy induction was evaluated via electron microscopy and Western blot. To test the effects of MK-2206 in vivo, CNE-2 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with MK-2206 or placebo. Tumors were harvested for immunohistochemical analysis.ResultsIn vitro, MK-2206 inhibited the four NPC cell line growths and reduced the sizes of the colonies in a dose-dependent manner. At 72 and 96 hours, the half maximal inhibitory concentration (IC50) values of MK-2206 in CNE-1, CNE-2, and HONE-1 cell lines were 3–5 μM, whereas in SUNE-1, IC50 was less than 1 μM, and MK-2206 induced cell cycle arrest at the G1 phase. However, our study found no evidence of apoptosis. MK-2206 induced autophagy in NPC cells, as evidenced by electron microscopy and Western blot, and inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of downstream phosphorylation through the PRAS40 and S6 pathways seems to be the main mechanism for the MK-2206-induced growth inhibition.ConclusionOur preclinical study suggests that MK-2206’s antiproliferative effect may be useful for NPC treatment; however, strategies for reinforcing this effect are needed to maximize clinical benefit.

Low-intensity ultrasound-induced cellular destruction and autophagy of nasopharyngeal carcinoma cells

Ultrasound therapy, as a non-invasive modality, has been attracting extensive attention in the management of malignant tumors. The present study aimed to investigate low-intensity ultrasound-induced cellular destruction and autophagy in nasopharyngeal carcinoma cells in vitro. Nasopharyngeal carcinoma CNE2 cells were subjected to ultrasound exposure, as tumor model cells, at an intensity of 1.35 W/cm(2). Cytotoxicity was investigated 24 h after ultrasound treatment. Nuclear damage was observed using nuclear staining with Hoechst 33258. Mitochondrial dysfunction was measured using confocal laser scanning microscopy with rhodamine123 staining. Mitochondrial morphology and autophagy were observed using transmission electron microscopy (TEM). Low-intensity ultrasound significantly killed CNE2 cells proportional to the ultrasonic treatment time. Upon nuclear staining, nuclear condensation and typical apoptotic bodies were noted in the CNE2 cells exposed to ultrasound wave for 12 sec. A collapse in mitochondrial membrane potential was noted in the treated cells. Upon TEM, swollen mitochondria, more vacuoles and autophagy were noted after ultrasound treatment. Our findings demonstrate that low-intensity ultrasound significantly damages CNE2 cells and emphasize that autophagy may be an important event in ultrasound-induced cell death.