Cell Lines HONE1 Research Articles

Study on the sensitizing effect of SM-1 combined with irradiation on head and neck squamous cell carcinoma

Purpose Head and neck squamous cell carcinoma (HNSCC) is globally prevalent with high recurrence, low survival rate, and poor quality of life for patients. Derived from PAC-1, SM-1 can activate procaspase-3 and induce apoptosis in cancer cells to exert anti-tumor effects. However, the inhibitory effect of SM-1 on HNSCC after combination with radiation are unclear. This study aims to investigate the radiosensitizing effect of SM-1 on HNSCC in vitro and in vivo. Methods MTT method was used to detect the effect of SM-1 on the viability of HNSCC cell lines (HONE1, HSC-2, and CAL27). The effects of SM-1 combined with radiation on the survival index of HONE1, HSC-2, and CAL27 cell lines were determined by colony formation assay. Flow cytometry was used to investigate the effects of SM-1 and radiation combination on cell apoptosis and cell cycle, and western blot experiments were performed to detect the expression of apoptosis and cell cycle-related proteins. Finally, a xenograft tumor model of CAL27 was established to evaluate the anti-tumor effect of SM-1 combined with radiation in vivo. Results In vitro, SM-1 effectively inhibited the activity of HNSCC cell lines HONE1, HSC-2, and CAL27 cells, and synergistically showed anti-proliferation activity during combined irradiation. Meanwhile, anti-tumor effect of SM-1 on HNSCC was higher than that of Debio1143, and the radiosensitivity of cells was greatly increased. Flow cytometry and western blot analysis showed that SM-1 induced G2/M phase arrest of head and neck squamous cell carcinoma cells via inhibiting the expression of CyclinB1 and CDC2. Moreover, SM-1 activated caspase-3 activity and up-regulated the cleaved form of PARP1 to induce cell apoptosis. In vivo, SM-1 combined irradiation showed a good anti-tumor effect. Conclusion SM-1 enhances HNSCC cell radiation sensitivity in vitro and in vivo, supporting its potential as a radiosensitizer for clinical trials in combination with radiotherapy.

Effects of biological clock gene BMAL1 and hypoxia-inducible factor HIF-1α on proliferation, migration and radiotherapy sensitivity of nasopharyngeal carcinoma cells HONE1

Objective To understand the effects of clock gene BMAL1 and HIF-1α(Hypoxia inducible factor-1α) on proliferation, migration and sensitivity to radiotherapy of nasopharyngeal carcinoma cells HONE1.At the same time, whether the biological clock gene BMAL1 can affect the expression of HIF-1α protein was investigated.It will lay the foundation for further study on the correlation between clock gene BMAL1 and HIF pathway.Methods BMAL1 gene overexpression and interference lentivirus and HIF-1α gene interference lentivirus were constructed respectively, and were transfected into nasopharyngeal carcinoma cells HONE1. Western blot was used to verify the establishment of overexpressed and knockdown BMAL1 cell lines and HIF-1α gene knockdown cell line, and to investigate the expression of HIF-1α protein in overexpressed and knockdown BMAL1 cell lines.CCK-8 cell proliferation test and scratch test were used to analyze the proliferation and migration ability of cells.Cell apoptosis after radiotherapy was analyzed by flow cytometry.The effects of BMAL1 and HIF-1α on the sensitivity of HONE1 radiotherapy in nasopharyngeal carcinoma cells after X-ray irradiation at different doses (0Gy, 2Gy, 4Gy, 6Gy) were detected by clone formation assay.Results The overexpression of BMAL1 gene and lentivirus interference were constructed to effectively up regulate and down regulate the expression of BMAL1 protein in nasopharyngeal carcinoma cells HONE1.Meanwhile, HIF-1α gene interference lentivirus was constructed to effectively down-regulate the expression of HIF-1α protein in nasopharyngeal carcinoma cell line HONE1, and successfully screen out stable nasopharyngeal carcinoma cell lines.Western blot results showed that overexpression of BMAL1 gene could inhibit the expression of HIF-1α protein in HONE1 of nasopharyngeal carcinoma cells, while knockdown of BMAL1 gene promoted the expression of HIF-1α protein in HONE1 of nasopharyngeal carcinoma cells(P < 0.05).CCK-8 cell proliferation and scratch test showed that overexpression of BMAL1 gene or knockdown of HIF-1α gene could inhibit the proliferation and migration of HONE1 cells (P < 0.05).Flow cytometry results showed that after 8Gy irradiation for 72 h, the apoptosis rate of BMALl gene overexpression group was higher than that of the overexpression control group, similarly, the apoptosis rate of HIF-1α gene knockdown group was higher than that of the knockdown control group (P < 0.05).After X-ray irradiation at different doses (0Gy, 2Gy, 4Gy, 6Gy), clon-formation experiment showed that the clon-formation rate and cell survival fraction of BMALl overexpression group or HIF-1α knockdown group were lower than those of negative control group (P < 0.05).Sigmaplot analysis showed that the D0, Dq and SF2 of the BMAL1 overexpression group or HIF-1α knockdown group were lower than those of the negative control group, and the radiosensitization ratios were 1.381 and 1.063, respectively.ConclusionOverexpression of BMAL1 gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1, increase apoptosis after radiotherapy and improve radiosensitivity.Knock down HIF-1α Gene can inhibit the proliferation and migration of nasopharyngeal carcinoma cell line HONE1, increase apoptosis after radiotherapy and improve radiosensitivity.In nasopharyngeal carcinoma cells HONE1, overexpression of BMAL1 gene can inhibit the expression of HIF-1α protein while knockdown of BMAL1 gene can promote the expression of HIF-1α protein.

Preparation of monoclonal antibodies against Epstein-Barr virus glycoprotein 350.

Epstein-Barr virus (EBV) is the first identified human oncogenic herpesvirus infecting over 90% of the adults worldwide. However, the safe and effective prophylactic vaccine has not been licensed. The major glycoprotein 350 (gp350) on the EBV envelope is the main target for neutralizing antibodies, and gp350 (aa15-320) was used for the development of monoclonal antibodies in present study. The purified recombinant gp35015-320aa with an estimated molecular weight of 50kDa was used to immunize six-week-old BALB/c mice, and the hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) were obtained. The ability of developed mAbs for capturing and neutralizing EBV was evaluated, and mAb 4E1 presented better performance to block the infection of EBV in cell line Hone-1. The mAb 4E1 recognized the epitope. Its sequence of variable region genes (VH and VL) presented a unique identity which hadn't been reported. The developed mAbs might benefit the antiviral therapy and immunologic diagnosis for EBV infection.

Curcuma xanthorrhiza Rhizome Extract Induces Apoptosis in HONE-1 Nasopharyngeal Cancer Cells Through Bid

BACKGROUND: Curcuma xanthorrhiza rhizomes have been demonstrated to have anticancer properties toward various types of cancer cells. The effect of C. xanthorrhiza rhizome extract (CXRE) on nasopharyngeal cancer (NPC) cells, including HONE-1 cell line has not been elucidated yet. Therefore, the effect of CXRE on the apoptosis of HONE-1 cells and its possible underlying mechanism are necessary to be explored.METHODS: C. xanthorrhiza rhizomes were minced, dried, extracted with distilled ethanol, filtered, and evaporated to produce CXRE. HONE-1 cells were seeded, starved, and treated with dimethyl sulfoxide (DMSO), Doxorubicin, or various concentrations of CXRE. Treated HONE-1 cells were stained with 4',6'-diamidino-2-phenylindole (DAPI) and the number of viable cells was counted. HONE-1 cells were also collected, lysed, and further processed for immunoblotting analysis to measure Bid activity.RESULTS: The number of viable HONE-1 cells decreased in concentration- and time-dependent manner. The number of viable cells in 50 and 250 μg/mL CXRE-treated groups were significantly lower compared with that in the DMSO-treated group after 24 h. At 48 h incubation period, the number of viable cells in 10, 50 and 250 μg/mL CXRE-treated groups were significantly lower compared with that in the DMSO-treated group. The number of viable cells in 250 μg/mL CXRE-treatment group was not significantly different compared with that in the Doxorubicin-treated group after 48 h. Bid expression levels in CXRE-treated groups were lower compared with that in the DMSO-treated group.CONCLUSION: CXRE could induce apoptosis via Bid activation, hence reducing the viability of HONE-1 cells.KEYWORDS: Curcuma xanthorrhiza, nasopharyngeal cancer, HONE-1 cells, apoptosis, Bid

Saucerneol attenuates nasopharyngeal carcinoma cells proliferation and metastasis through selectively targeting Grp94

BackgroundNasopharyngeal carcinoma (NPC) is highly prevalent in southern China. The remote metastasis of advanced NPC requires chemotherapeutic treatments to reduce the mortality. Our previous work revealed that saucerneol (SN) showed cytotoxicity against several nasopharyngeal carcinoma (NPC) cells. This work aims to investigate the effect of SN in NPC growth and exploring the mechanism of action. Study designApplying in vivo study, in vitro study and in silico study to indicate the mechanism of SN in inhibiting NPC growth. MethodsSaucerneol (SN) toxicity was measured with MTT assay. NPC proliferation was measured with EdU and colony formation assays, cell cycle was detected with flow cytometry. NPC migration and invasion were measured with scratch assay and matrigel transwell method. Further, human NPC xenograft tumor models were established in nude mice to evaluate the therapeutic efficacy of SN in vivo. Toxicological analysis was performed on H&E staining and IHC. Quantitative real-time PCR and Western blot analyses were used to evaluate the expression levels of key molecules in PI3K/AKT/mTOR, MAPK, NF-κB, and HIF-1α signal pathways. Target predicting was conducted using computational method, and target identification was carried out by ATPase assay and TSA. ResultsSN, a potent NPC inhibitor that was previously isolated from Saururus chinensis in our lab, is proven to inhibit the proliferation and metastasis of HONE1 cell lines and inhibit the growth of human NPC xenografts in nude mice. Moreover, we further articulate the molecular mechanism of action for SN and, reveal that SN promotes the expression of cell cycle-dependent kinase inhibitory protein p21 Waf1/Cip1 through targeting Grp94 and then inhibiting PI3K/AKT signaling pathway as well as up-regulating p53 to disrupt the progression of HONE1 cells. ConclusionSN significantly inhibits NPC cells proliferation and metastasis in vitro and in vivo via selectively inhibit Grp94 and then blocking PI3K/AKT/mTOR/HIF-1α signaling pathway. This study firstly provides a novel selective Grp94 inhibitor as a NPC candidate.

MiRNA profiling of human nasopharyngeal carcinoma cell lines HONE1 and CNE2 after X-ray therapy.

Radiotherapy is the main treatment for nasopharyngeal carcinoma. The radioresistance mechanism of cells is related to miRNAs. To investigate the miRNA profiling of HONE1 and CNE2 after X-ray therapy. The HONE1 and CNE2 cells were treated with X-ray at 4 Gy, 8 Gy, 16 Gy, and 20 Gy doses. The cell lines CNE2 with the best therapy effects and HONE1 with the worst therapy effects were screened out. Apoptosis and cell viability were detected with flow cytometry and Cell Counting Kit-8 (CCK-8). High-throughput sequencing was performed. A miRNA library was constructed. The miRNA annotation expression distribution, family prediction and target gene interaction, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted. The 24-hour 20 Gy dose X-rays were selected as the optimal therapy conditions. The CNE2_C, CNE2_M, HONE1_C and HONE1_M miRNAs accounted for 26.5%, 31.7%, 21.3%, and 22.9% of the Cleandata reads count, respectively, and the contents of rRNAs accounted for 24.9%, 14.7%, 25.1%, and 25.1% of the Cleandata reads count, respectively. The miRNAs with differential expression between the HONE1 and CNE2 cell lines including hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-125a-5p, hsa-miR-26a-5p, hsa-let-7f-5p, hsa-miR-20a-5p, and hsa-miR-24a-3p. There were also differentially expressed miRNAs in HONE1_C vs. HONE1_M, such as hsa-miR-21-5p and hsa-let-7i-5p. The differentially expressed miRNA in CNE2_C vs. CNE2_M was hsa-miR-148b-3p. The Gene Ontology analysis showed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were mainly enriched in biological process such as negative and positive regulation of transcription from RNA polymerase II promoter, cellular component such as cytosol and molecular function such as protein binding factor. The KEGG pathway analysis revealed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were enriched in the cancer-related pathways, such as pathways in cancer, MAPK signaling pathway and Wnt signaling pathway. Twelve miRNAs and 9 genes which contribute to X-ray radiation resistance were identified. Among those with differential expression between the HONE1 and CNE2 cell lines, which played a regulatory role in multiple pathways, were hsa-miR-20a-5p, hsa-let-7a-5p, hsa-let-7f5p, hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-148b-3p, and hsa-miR-200c-3p. The corresponding genes were MAPK1, SOS1, TGFBR1, TGFBR2, TP53, CASP3, CCNE2, PTEN, and CDK2.

Salidroside suppresses the activation of nasopharyngeal carcinoma cells via targeting miR-4262/GRP78 axis

ABSTRACT To study the effect of Salidroside on nasopharyngeal carcinoma (NPC) cells and its mechanism. NPC cells were cultured, MTT was used to detect the effect of Salidroside on cell proliferation, apoptosis detected by flow cytometry assay, Western blot was used to detect the related protein expression. MiR-4262 and GRP78 used qRT-PCR for evaluation. Mimics/mimic NC and miR-4262 inhibitor/inhibitor NC were transfected into CNE2 and HONE1 cell lines, and cell viability was detected by MTT. Caspase-3, −8 and −9 activities were detected by caspase colorimetric assay kit. Targetscan predicted that downstream target of miR-4262. Relative luciferase activity was detected by luciferase assay. The effect of Salidroside on the growth of transplanted tumor in nude mice was observed. After Salidroside treatment, cell proliferation decreased and apoptosis increased, Bax protein expression increased and Bcl-2 decreased; miR-4262 expression level in nasopharyngeal carcinoma tissues was lower than that in adjacent tissues. GRP78 was the target of miR-4262 and downregulate the expression of miR-4262 in NPC cells can increase the expression of GRP78, and the expression of GRP78 decreased after upregulating the expression of miR-4262. Salidroside could inhibit the growth of NPC xenografts in nude mice. The level of Bax was increased and Bcl-2 was decreased in Salidroside group. Salidroside can significantly inhibit the proliferation and promote the apoptosis of NPC cells via regulating miR-4262/GRP78 signal axis.

Effect of miR-25 on Proliferation of Nasopharyngeal Carcinoma Cells through Wnt/β-Catenin Signaling Pathway.

Objective To investigate the biological role and potential mechanism of miR-25 in nasopharyngeal carcinoma. Methods The expression of miR-25 in nasopharyngeal carcinoma cell lines was detected by qRT-PCR. The effect of inhibition of miR-25 expression on the proliferative activity of nasopharyngeal carcinoma cell line HONE-1 was examined by CCK-8 method. Flow cytometry was used to detect the effect of miR-25 expression inhibition on the apoptosis rate of nasopharyngeal carcinoma cell line HONE-1. The miRNA target gene prediction site TargetScan predicts the target protein action site of miR-124 and verifies whether miR-25 interacts with the target by luciferase activity assay, qPCR, and Western experiments. The miR-25 inhibitor and target egg gene expression plasmids were cotransfected into HONE-1 cells for rescue experiments to investigate whether miR-25 inhibits proliferation of nasopharyngeal carcinoma cells by target genes. At the same time, qRT-PCR was used to detect the mRNA expression levels of Wnt/β-catenin pathway key proteins TCF4, c-Myc, and Cyclin D1 in different transfected cells. Results miR-25 expression was upregulated in nasopharyngeal carcinoma cell lines. Functional studies showed that inhibition of miR-25 expression significantly inhibited the proliferation of nasopharyngeal carcinoma cell line HONE-1 (p < 0.05). Inhibition of miR-25 expression by flow cytometry significantly promoted apoptosis (p < 0.05). Detection of dual luciferase activity indicated that DKK3 is a direct target site for miR-25. Western blots showed that inhibition of miR-25 significantly upregulated DKK3 mRNA and protein levels. Supplementation with DKK3 significantly attenuated the inhibitory effect of miR-25 on the proliferation of nasopharyngeal carcinoma cell line HONE-1 (p < 0.05). qRT-PCR found that mRNA levels of TCF4, c-Myc, and Cyclin D1 were significantly upregulated in miR-25-transfected cells compared to control transfection. QRT PCR showed that the mRNA and protein levels of Tcf4, c-myc, and Cyclin D1 were significantly upregulated in miR-25 overexpression-transfected cells. Conclusion Inhibition of miR-25 expression promotes DKK3 gene expression, and inactivation of Wnt/β-catenin signaling pathway inhibits proliferation and promotes apoptosis of nasopharyngeal carcinoma cells.

LncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway.

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

Tubulin polymerization promoting protein family member 3 (TPPP3) overexpression inhibits cell proliferation and invasion in nasopharyngeal carcinoma

ABSTRACT The function of tubulin polymerization promoting protein family member 3 (TPPP3) in tumor cells is complicated, and the role of TPPP3 in nasopharyngeal carcinoma (NPC) remains unclear. This study aims to explore the expression of TPPP3 in NPC and its effect on NPC cells. The expression of TPPP3 in NPC tissues and other cancers were analyzed by using the Oncomine and Gene Expression Omnibus (GEO) databases. The mRNA and protein of TPPP3 were detected in NPC tissues by quantitative real-time PCR and immunohistochemistry. Furthermore, TPPP3 was overexpressed in 5–8 F and HONE1 cell lines by lentivirus transfection, and functional analysis of TPPP3 in NPC was evaluated through in vitro experiments. The expression of TPPP3 was significantly down-regulated in NPC tissues and cells. Overexpression of TPPP3 significantly inhibited proliferation of 5–8 F and HONE1 cells in vitro. In addition, overexpression of TPPP3 significantly attenuated the invasion ability of 5–8 F, HONE1 cells in vitro, but have no significant effect on migration ability. Furthermore, TPPP3 overexpression diminished the expression of MMP-2 and MMP-9 mRNA. By analyzing dataset GSE12452, it was interesting that TPPP3 high expression group mainly functioned in B cell receptor signaling pathway, cell cycle and DNA replication. In conclusion, our results suggest that TPPP3 may be considered as an antioncogene, which plays an important role in the occurrence and progression of NPC. Abbreviations: TPPP3: tubulin polymerization promoting protein family member 3; NPC: nasopharyngeal carcinoma; GEO: Gene Expression Omnibus; qRT-PCR: quantitative real-time PCR; GFP: green fluorescence protein; MOI, transfected multiplicity of infection; CCK-8: cell counting kit-8; OD: optical density; GSEA: gene set enrichment analysis; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; MMP-2: matrix metalloproteinase-2; MMP-9: matrix metalloproteinase-9.

Implication of hsa_circ_0028007 in reinforcing migration, invasion, and chemo-tolerance of nasopharyngeal carcinoma cells.

BackgroundGiven the reliability of circRNAs in symbolizing cancer progression, this investigation was designed to expound the involvement of hsa_circ_0028007 in regulating chemosensitivity of nasopharyngeal carcinoma (NPC) cells.MethodsAltogether, 241 pairs of NPC tissues and para‐cancerous normal tissues were collected to identify NPC‐symbolic circRNAs, which have been screened by circRNA microarray in advance. Expressions of the circRNAs were determined by means of real‐time polymerase chain reaction (PCR). Besides, human NPC cell lines (ie, CNE2 and HONE1) were transfected by si‐hsa_circ_0028007 and si‐NC. Scratch assay, transwell assay, and MTT assay were performed to assess migration, invasion, and paclitaxel/cisplatin‐resistance of NPC cell lines.ResultsHsa_circ_0028007 expression was abnormally heightened within NPC tissues in comparison with matched non‐tumor tissues (P < .05). Over‐expressed hsa_circ_0028007 was strongly associated with advanced (III‐IV) tumor stage, aggressive infiltration, and metastatic lymph nodes of NPC patients (P < .05). Regarding in vitro experiments, hsa_circ_0028007 expression was elevated in CNE2 and HONE1 cell lines as compared with HENE cell line (P < .05). Silencing of hsa_circ_0028007 not merely sensitized CNE2 and HONE1 cells against paclitaxel and cisplatin (P < .05), but also significantly repressed migration and invasion of the cell lines (P < .05).ConclusionHsa_circ_0028007 was involved in facilitating progression and chemo‐resistance of NPC, which might offer an alternative for NPC treatment.

Two new lignans from the aerial parts of Saururus chinensis with cytotoxicity toward nasopharyngeal carcinoma

Two new lignans (1 and 12), together with 15 known compounds (2–11 and 13–17), were isolated from the aerial parts of Saururus chinensis Baill. Their structures were determined through extensive spectroscopic analyses. All the isolates were evaluated for their cytotoxicity against four human nasopharyngeal carcinoma cells (HONE1, CNE1, CNE2, and SUNE1). Compound 13 showed the most potent cytotoxicity toward HONE1, SUNE1, CNE2, and CNE1 cells with IC50 values of 0.76, 5.42, 5.86 and 6.28 μM, respectively. Further studies revealed that compound 13 suppressed cell growth by arresting the cell cycle at the S phase and induced cell apoptosis in the HONE1 cell line.

MiR-122 exerts anti-proliferative and apoptotic effects on nasopharyngeal carcinoma cells via the PI3K/AKT signaling pathway

To investigate the effects of microRNA-122 (miR-122) on the proliferation and apoptosis of nasopharyngeal carcinoma (NPC) HONE-1 cells, and its correlation with the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Human NPC cell line (HONE-1) was transfected with miR-122 inhibitor (anti-miR-122 group), negative controls (vector control group) via lipofectamines, and HONE-1 cell lines undergoing no transfection were selected (non-transfection group). The expression of miR-122, cell proliferation, apoptosis, and expressions of PI3K/AKT pathway and downstream target proteins in the three groups were determined using fluorescence quantitative polymerase chain reaction (qPCR), cell counting kit-8 (CCK8), immunofluorescence (IF) and Western blotting, respectively. The expression of miR-122 in the anti-miR-122 group was significantly lower than corresponding expressions in the non-transfection and vector control groups after 48h of transfection (p &lt;0.05). The proliferation of cells in the anti-miR-122 group was significantly reduced with time after transfection (p &lt;0.05). After 48h of transfection, the extent of apoptosis in the anti-miR-122 group (47.11 ± 1.95%) was significantly higher than that in normal control (7.37 ± 0.82%) and vector control group (8.54 ± 0.96%; p &lt;0.05). There were no significant differences in the expressions of PI3K, AKT, mTOR protein, and the downstream signal proteins (p70S6K and 4E-BP1) in the three groups (p &gt;0.05). However, the expressions of phosphorylated forms of these proteins were significantly lower in the anti-miR-122 group than in the non-transfection and vector control groups (p &lt;0.05). IF results revealed that there were no significant differences in the fluorescence intensity value of PI3K and Akt among the three groups of patients (p&gt;0.05). Inhibition of the expression of miR-122 in NPC suppresses the proliferation, and promotes their apoptosis through the PI3K/AKT signal transduction pathway.

Nuclear Klf4 accumulation is associated with cetuximab drug-resistance and predicts poor prognosis of nasopharyngeal carcinoma

BackgroundThe functions of the protein expressed in the nucleus and cytoplasm were different or opposite. The previous study found that oncogene Klf4 played a role of tumor suppressor in the nasopharyngeal cytoplasm. Cetuximab targeted epidermal growth factor receptor (EGFR) for the treatment of nasopharyngeal carcinoma.MethodsA cohort of 231 cases of advanced nasopharyngeal carcinoma (7th AJCC III–IVa) samples was assessed by immunohistochemistry (IHC), of which, 63 cases were treated with basic treatment without cetuximab, the basic treatment include chemotherapy and radiotherapy, the regent of the chemotherapy include cisplatin and fluorouracil and 168 cases were treated with cetuximab in addition to the basic treatment. The expression of the KLF4 protein was detected in nucleus and cytoplasm, c-Met protein and nuclear EGFR protein (nEGFR) by IHC, and H-Ras and PI3K mutations by an arms-PCR method in vivo. KLF4 was found to specifically express in the cytoplasm by deleting the NES, while H-Ras and PI3K genes were mutated in the nasopharyngeal carcinoma 5–8F and HONE1cell line. The cetuximab resistance in differentially mutated 5–8F and HONE1 cells was analyzed.ResultsThe expression of Klf4 in the nucleus was associated with prognosis in 168 patients with cetuximab-treated nasopharyngeal carcinoma, which was found by retrospective analysis. The KLF4 expression in the nucleus was not significantly correlated with the prognosis in 63 nasopharyngeal carcinoma patients treated with basic treatment (P = 0.261). The expression of Klf4 in the nucleus was correlated with mutations of H-Ras and PI3K in 168 cases of nasopharyngeal carcinoma with cetuximab treatment. In vitro experiments showed that Klf4 was specifically expressed in the nucleus of 5–8F and HONE1 cells as assessed by deleting nuclear export signal, which led to cetuximab resistance. H-Ras and PI3K mutations in 5–8F and HONE1 cells also led to the expression of Klf4 in the nucleus and resistance to cetuximab. In HONE1 cells, Klf4 was specifically localized in the cytoplasm by deleting the NES, and the H-Ras and PI3K mutations did not result in an increased expression of Klf4 in the nucleus and cetuximab resistance.ConclusionThe prognosis of nasopharyngeal carcinoma was not significantly improved by cetuximab treatment when the Klf4 was highly expressed in the nucleus of nasopharyngeal carcinoma tissues. The expression of Klf4 in the nucleus can be used as a biomarker for predicting the effects of cetuximab treatment in nasopharyngeal carcinoma, which might be attributed to the H-RAS and PI3K mutations, leading to cetuximab resistance.

TRIM24 siRNA induced cell apoptosis and reduced cell viability in human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a common cancer occurring primarily in East Asia and Africa. The high rate of recurrence and metastasis of NPC continuously endangers the health of patients. The present study aimed to identify the underlying mechanisms involved in the progression of NPC and provide experimental basis to develop a novel and efficient agent against NPC. The present study measured the expression level of tripartite motif containing24 (TRIM24) in tumor tissues from NPC patients using reverse transcription quantitative polymerase chain reaction. Subsequently, Cell Counting kit‑8 and flow cytometry were used to detect the cell proliferation and apoptosis of NPC cell lines HONE1 and CNE1 cells where the TRIM24 gene was knocked‑down with small interfering RNA (siRNA). Further, caspase kits and western blot analysis were used to detect the expression of apoptosis and angiogenesis‑associated proteins. The present study detected a higher expression level of TRIM24 in tumor tissues and NPC cell lines and lower cell viability and higher apoptotic rate were observed when TRIM24 was silenced. Meanwhile, upregulated caspase‑3 and caspase‑9 indicated induced cell apoptosis in HONE1 and CNE1 cells following the treatment with TRIM24 siRNA. Additionally, the downregulated expression level of vascular endothelial growth factor (VEGF) and VEGF receptor 2 suggested inhibited angiogenesis of NPC cells. Additionally, the reduced levels of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) indicated a blocked JAK2/STAT3 signaling pathway. However, there was no direct evidence that inactivation of the JAK2/STAT3 signaling pathway was involved in regulation of siTRIM24, these results suggested that TRIM24 has an important role in the growth of NPC. Additionally, silenced TRIM24 may lead to inhibited cell proliferation and induced cell apoptosis in NPC cells. The limitation of this study was that HONE1, CNE1 and CNE2 cells may have been contaminated with other cells. Further experiments with validated NPC cells may be needed.

Pan-CDK inhibition augments cisplatin lethality in nasopharyngeal carcinoma cell lines and xenograft models

In addition to their canonical roles in regulating cell cycle transition and transcription, cyclin-dependent kinases (CDKs) have been shown to coordinate DNA damage response pathways, suggesting a rational pairing of CDK inhibitors with genotoxic chemotherapeutic agents in the treatment of human malignancies. Here, we report that roniciclib (BAY1000394), a potent pan-CDK inhibitor, displays promising anti-neoplastic activity as a single agent and potentiates cisplatin lethality in preclinical nasopharyngeal carcinoma (NPC) models. Proliferation of the NPC cell lines HONE-1, CNE-2, C666-1, and HK-1 was effectively curbed by roniciclib treatment, with IC50 values between 11 and 38 nmol/L. These anticancer effects were mediated by pleiotropic mechanisms consistent with successful blockade of cell cycle CDKs 1, 2, 3, and 4 and transcriptional CDKs 7 and 9, ultimately resulting in arrest at G1/S and G2/M, downregulation of the transcriptional apparatus, and repression of anti-apoptotic proteins. Considerably enhanced tumor cell apoptosis was achieved following combined treatment with 10 nmol/L roniciclib and 2.0 μmol/L cisplatin; this combination therapy achieved a response over 250% greater than either drug alone. Although roniciclib chemosensitized NPC cells to cisplatin, it did not sensitize untransformed (NP69) cells. The administration of 0.5 mg/kg roniciclib to BALB/c xenograft mice was well tolerated and effectively restrained tumor growth comparable to treatment with 6 mg/kg cisplatin, whereas combining these two agents produced far greater tumor suppression than either of the monotherapies. In summary, these data demonstrate that roniciclib has strong anti-NPC activity and synergizes with cisplatin chemotherapy at clinically relevant doses, thus justifying further evaluation of this combinatorial approach in clinical settings.

Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.

BackgroundCytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.MethodsPolymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF).ResultsIn CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.ConclusionCYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis, thereby suppressing tumor formation.

Abstract 3098: The effects of a pan-cyclin dependent kinase (CDK) inhibitor and its combination with cisplatin in nasopharyngeal carcinoma (NPC)

Abstract Undifferentiated NPC is a unique epithelial malignancy endemic to South-east Asia and North Africa. Cell cycle dysregulation, mediated by mechanisms such as over-expression of cyclin D1 and down-regulation of p16INK4A is frequently found in NPC. Our previous work suggest that NPC cells are sensitive to the effects of cell cycle inhibition. BAY1000394 (BAY) is a potent pan-CDK inhibitor exhibiting broad anti-proliferation activity across a number of different cancer types. In this study, the anti-tumor effects of BAY alone and in combination with cisplatin on NPC cell lines were examined in vitro (MTS assay) and in a xenograft model. Cell cycle effects and apoptosis were examined via flowcytometry. Protein levels of relevant cell cycle regulators and mediators of apoptosis were assessed via Western Blot. Time-dependent inhibition of NPC cell lines HONE-1 and HK-1 by BAY was evaluated and demonstrated that growth inhibition of 50% of the cell population occurred by 48hrs of treatment. Mean IC50 of 30nM was seen. BAY inhibits phosphorylation of cellular CDK target proteins such as retinoblastoma (Ser807/811) (Rb), nucleophosmin (Thr199) (NPM) and RNA polymerase II (Ser2) (p-RNA POL II). Dephosphorylation of RNA POL II inhibits transcription of gene products resulting in rapid decline of MCL-1. Direct caspase-3 activation was observed with the concomitant appearance of active caspase-3; inducing PARP cleavage and inhibition of survivin. BAY was able to induce cell cycle arrest at multiple checkpoints thus enhancing apoptosis. Annexin V and PI staining after 96hrs of BAY exposure showed apoptosis of &amp;gt;80% of BAY treated cell. In vivo dosing of BAY1000394 at 0.5mg/kg and 1mg/kg showed significant growth inhibition as compared to control (p-value &amp;lt;0.05). Combination studies of BAY with cisplatin displayed synergistic cell kill. Cell cycle analysis of HONE-1 cells treated at 10nM and 20nM BAY with doses of cisplatin ranging from 0.5μM to 2μM showed marked increase in cell death as compared to control and NP69 epithelial cells. Protein expression of activated caspase-3, PARP cleavage and MCL-1 inhibition were observed in dual drug treatment groups. No change in expressions these proteins were seen in treated NP69 cells. In vivo assays of both drugs also demonstrated growth inhibition of tumor bearing mice at BAY 0.5mg/kg and 1.0mg/kg with cisplatin 6mg/kg with good tolerance. Tumor protein expression of p-RNA POL II, NPM and MCL-1 expression were suppressed, suggesting inhibition of CDK9/cyclin T1. Our data suggest that BAY inhibits NPC tumor cell proliferation in a time- and dose- dependent manner through inhibition of cell-cycle progression and induction of apoptosis. Combination studies with cisplatin suggest synergistic anti-tumor effect. This preliminary study strongly suggests that BAY and its combination with cisplatin has therapeutic potential in the treatment of NPC. Citation Format: Pei Li Lim, John SW Low, Gerhard Siemeister, Boon Cher Goh, Wen-son Hsieh. The effects of a pan-cyclin dependent kinase (CDK) inhibitor and its combination with cisplatin in nasopharyngeal carcinoma (NPC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3098. doi:10.1158/1538-7445.AM2015-3098

Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor

PurposePast studies have reported that S-allylcysteine (SAC) inhibits the migration and invasion of cancer cells through the restoration of E-cadherin, the reduction of matrix metalloproteinase (MMP) and Slug protein expression, and inhibition of the production of reactive oxygen species (ROS). Furthermore, evidence is emerging that shows that ROS induced by radiation could increase Met activation. Following on these reports of SAC and Met, we investigated whether SAC could suppress Met activation.Materials and MethodsWound healing, invasion, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), soft agar colony forming, western blotting, and gelatin zymography assays were performed in the human nasopharyngeal cancer cell lines HNE1 and HONE1 treated with SAC (0, 10, 20, or 40 mM) and hepatocyte growth factor (HGF).ResultsThis study showed that SAC could suppress the migration and invasion of HNE1 and HONE1 cell lines by inhibiting p-Met. An increase of migration and invasion induced by HGF and its decrease in a dose dependent manner by SAC in wound healing and invasion assays was observed. The reduction of p-Met by SAC was positively correlated with p-focal adhesion kinase (p-FAK) and p-extracellular related kinase (p-ERK in both cell lines). SAC reduced Slug, MMP2, and MMP9 involved in migration and invasion with the inhibition of Met-FAK signaling.ConclusionThese results suggest that SAC inhibited not only Met activation but also the downstream FAK, Slug, and MMP expression. Finally, SAC may be a potent anticancer compound for nasopharyngeal cancer treated with radiotherapy.

Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer&amp;nbsp;in&amp;nbsp;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.