Set-up errors of the neck are underestimated using the overall registration frame of head and neck in IMRT for NPC.

BACKGROUND: There is no standardized registration frame of cone beam CT (CBCT) in intensity modulated radiotherapy (IMRT) for nasopharyngeal carcinoma (NPC). The overall registration frame that covers the whole head and neck is the most commonly used CBCT registration frame for NPC patients in IMRT. OBJECTIVE: To compare the set-up errors using different registration frames of CBCT for NPC to assess the set-up errors for different region of the commonly used clinical overall registration frame. METHODS: 294 CBCT images of 59 NPC patients were collected. Four registration frames were used for matching. The set-up errors were obtained using an automatic matching algorithm and then compared. The expansion margin from the clinical target volume (CTV) to the planned target volume (PTV) in the four groups was also calculated. RESULTS: The average range of the isocenter translation and rotation errors of four registration frames are 0.89∼2.41 mm and 0.49∼1.53°, respectively, which results in a significant difference in the set-up errors (p < 0.05). The set-up errors obtained from the overall frame are smaller than those obtained from the head, upper neck, and lower neck frames. The margin ranges of the overall, head, upper neck, and lower neck frames in three translation directions are 1.49∼2.39 mm, 1.92∼2.45 mm, 1.86∼3.54 mm and 3.02∼4.78 mm, respectively. The expansion margins calculated from the overall frame are not enough, especially for the lower neck. CONCLUSION: Set-up errors of the neck are underestimated by the overall registration frame. Thus, it is important to improve the position immobilization of the neck, especially the lower neck. The margin of the target volume of the head and neck region should be expanded separately if circumstances permit.

The development and validation of a m6A-lncRNAs based prognostic model for overall survival in lung squamous cell carcinoma.

Background: No biomarkers have been identified for the prognosis of lung squamous cell carcinoma (LUSC). Risk models based on m6A-lncRNAs help to predict survival in some cancers. However, very few studies have reported m6A-lncRNA risk models in LUSC. We aimed to construct a prognostic model based on m6A-lncRNAs in LUSC. Methods: The clinical and RNA-sequencing information of 504 LUSC patients were downloaded from The Cancer Genome Atlas (TCGA) database. Prognostic m6A-lncRNAs were identified by a Pearson correlation analysis and univariate Cox regression analysis. The ConsensusClusterPlus algorithm was used to cluster the prognostic m6A-lncRNAs. The overall survival (OS) and clinicopathological characteristics of the 2 clusters were compared. A gene set enrichment analysis (GSEA) analysis was performed to analyze the genes enriched in the 2 clusters. A least absolute shrinkage and selection operator (LASSO) Cox regression analysis was used to construct the risk-score model. Two hundred and forty eight patients were randomly chosen from TCGA-LUSC cohort for the training set. The receiver operating characteristic (ROC) curve analysis was used to assess the predictive ability of the model. The clinical characteristics and OS in the high- and low-risk groups were compared. The independent prognostic value of the model was tested by Cox regression analyses. Results: Thirteen m6A-lncRNAs were identified as prognostic lncRNAs and classified into cluster A and cluster B. The OS of patients in cluster A was better than that of patients in cluster B (P<0.001). Patients in cluster B had higher expressions of immune checkpoints. Immune score, stromal score, and ESTIMATE score were higher in cluster B (P<0.001). Seven of the 13 lncRNAs were used to construct the risk-score model. Patients in the high-risk group had a worse OS. ROC curves showed a under the curve (AUC) of 0.639 in the training set and 0.624 in the validation set. A high risk was associated with cluster B, a high immune score, and stage III-IV disease. Patients in the high-risk group had increased expressions of immune checkpoints. The Cox regression analyses showed that the risk-score model had independent prognostic value for OS. The risk-score model retained its prognostic value in different subgroups. Conclusions: The m6A-lncRNA risk-score model is an independent prognostic factor for OS in LUSC patients. However, the risk-score model need to be further tested clinically.

Prognostic value and potential mechanism of long non-coding RNA Lnc-SMIM20-1 in acute myeloid leukemia.

OBJECTIVES: Acute myeloid leukemia (AML) is a common hematologic malignancy with high heterogeneity and poor prognosis. Although long non-coding RNAs (lncRNAs) have been used as biomarkers for tumors, the clinical relevance of numerous lncRNAs in AML remains to be investigated. RESEARCH DESIGN AND METHODS: Differentially expressed lncRNAs between AML and normal peripheral blood samples were identified using DESeq2. Pan-cancer analysis was performed by GEPIA tool. Kaplan-Meier survival curve was applied for prognosis analysis. KEGG pathway analysis and GSEA were used for functional enrichment. The ceRNA network was constructed by GDCRNAtools. RESULTS: Lnc-SMIM20-1 was most highly expressed in AML and up-regulated in the TCGA-AML cohort compared to normal tissues. Patients with high expression of Lnc-SMIM20-1 had poor overall prognosis both in the TCGA adult AML cohort and the TARGET pediatric AML cohort, no matter whether they were treated with chemotherapy or allo-HSCT. Lnc-SMIM20-1 might participate in cancer-associated signaling pathways and immune-related signaling pathways by interacting with four microRNAs and 20 mRNAs. CONCLUSION: Lnc-SMIM20-1 was up-regulated in AML acting as a stable poor prognostic factor. The prognostic impact of Lnc-SMIM20-1 cannot be overcome by allo-HSCT. Our findings provide insight into the clinical relevance of Lnc-SMIM20-1 in AML; aiming to progress the development of novel therapeutics.

Long non-coding RNA LOC644135 is a potential prognostic indicator in cytogenetically normal acute myeloid leukemia.

OBJECTIVES: Acute myeloid leukemia (AML) is a hematological malignancy with highly clinical heterogeneity resulting in poor outcomes. We aim to identify novel prognostic lncRNA in AML expecting to provide new clues for therapy in AML. METHODS: Three cohorts were enrolled in this study. Differentially expressed lncRNAs between TCGA-AML cohort and GTEx cohort was identified by DESeq2. The relationship between expression level of LOC644135 and prognosis in AML was analyzed by multiple methods. RESULTS: Pan-cancer analysis indicated that LOC644135 was most highly expressed in AML across 33 types of cancer. Patients with high expression of LOC644135 had poor overall prognosis in both TCGA-AML cohort and the TARGET-AML cohort. Especially, high expression of LOC644135 indicated inferior overall survival and event-free survival in CN-AML patients in the TCGA-AML cohort. Besides, CN-AML patients had higher expression of LOC644135 than normal samples. Multivariable analysis suggested that LOC644135 was an independent prognostic factor in AML. GSEA analysis showed that LOC644135 was associated with some immune-related pathways. Besides, high expression of LOC644135 was associated with less infiltration of CD8+ T cell. CONCLUSION: Our findings indicated that LOC644135 was an independent prognostic factor in AML and provided a new idea in the development of therapy in AML.

Prognostic and Therapeutic Values of Autophagy-related Genes in Triple-negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with a poor prognosis. Therefore, it is imperative to develop new prognostic or therapeutic biomarkers for TNBC. OBJECTIVES: To explore the prognostic and therapeutic values of autophagy-related genes (ARGs) in TNBC. METHODS: Overall, 157 TNBC patients' data were obtained from The Cancer Genome Atlas database, and the ARGs were acquired from the Human Autophagy Database. Differentially expressed ARGs (DEGs) between tumor and normal tissues were identified, and the prognostic ARGs were developed using R software. Kaplan-Meier survival curves and receiver operating characteristic (ROC) curves were both used to evaluate the accuracy of the signature. Patents about prognostic ARGs were reviewed through Worldwide Espacenet® and Patentscope®. RESULTS: We obtained 28 DEGs and two prognostic ARGs (EIF4EBP1 and PARP1). The Kaplan- Meier survival curves showed that the survival rate of patients with low 2-ARG signature risk score was significantly higher than that of patients with high-risk score (P =0.003). ROC at 5 years indicated that the signature had good prognostic accuracy (AUC =0.929). The signature was independent of T, N, M, and TNM stages (P <0.05). The patent review suggested that many mTOR inhibitors alone or in combination with another anticancer agent have been provided for the treatment of many cancers and shown promising results. No drug patents about PARP1 overexpression were disclosed. CONCLUSION: We developed a 2-ARG signature (EIF4EBP1 and PARP1), which was an independent prognostic biomarker for TNBC. As EIF4EBP1 was upregulated in TNBC, mTOR inhibitors which blocked the mTOR/4EBP1/eIF4E pathway, may be a promising therapeutic strategy for TNBC.

The Prognostic Value of a Tumor Microenvironment-Based Immune Cell Infiltration Score Model in Colon Cancer.

The current study aimed to construct a prognostic predictive model based on tumor microenvironment. CIBERSORT and ESTIMATE algorithms were used to reveal the immune cell infiltration (ICI) landscape of colon cancer. Patients were classified into three clusters by ConsensusClusterPlus algorithm. ICI scores of each patient were determined by principal component analysis. Patients were divided into high and low ICI score groups. Survival, gene expression, and somatic mutation of the two groups were compared. We found that patients with no lymph node invasion, no metastasis, T1-2 disease, and stage I-II had higher ICI scores. Calcium signaling pathway, leukocyte transendothelial migration pathway, MAPK signaling pathway, TGF ß pathway, and Wnt signaling pathway were enriched in the high ICI score group. Immune-checkpoint and immune-activity associated genes were decreased in high ICI score patients. Patients in the high ICI score group had better survival. Prognostic value of ICI score was independent of tumor mutational burden (TMB). The ICI score model constructed in the current study may serve as an independent prognostic biomarker in colon cancer.

Upregulation of HSPA1A/HSPA1B/HSPA7 and Downregulation of HSPA9 Were Related to Poor Survival in Colon Cancer.

The human HSP70 family is a type of heat shock protein (HSP), consisting of 13 members encoded by the HSPA genes. HSPs play important roles in regulating cellular responses and functions during carcinogenesis, but their relationship with colon cancer is unclear. In our study, we found that the expressions of HSPA1B, HSPA4, HSPA5, HSPA6, HSPA8, HSPA9, HSPA13, and HSPA14 were significantly increased, while those of HSPA1A, HSPA2, HSPA7, and HSPA12B were significantly decreased in colon cancer tissues. The expression of HSPA gene family members was associated with some clinicopathological characteristics, including age, gender, TNM stage, pathological stage, and CEA level. Furthermore, the Kaplan-Meier method and Cox regression analysis showed that high HSPA1A, HSPA1B, and HSPA7 expressions were related to unfavorable survival, and high HSPA9 was associated with favorable survival. The relationships between HSPA1A and HSPA9 expression and survival were validated in the GEO dataset, and the HSPA1A and HSPA9 protein expression differences between colon cancer tissues and normal tissues were validated in the UALCAN database. Methylation of HSPA1A and HSPA9 was also analyzed, and it was found that the methylation of the HSPA1A promoter was significantly increased, and the methylation of the HSPA9 promoter was significantly decreased in colon cancer tissues. Increasing the methylation level of the HSPA1A gene and decreasing the methylation level of HSPA9 were related to favorable prognosis. The expression difference of HSPA1A/HSPA1B/HSPA7/HSPA9 was verified in colon cancer cell lines and colonic epithelial cells. Gene ontology analysis was used to screen signal pathways related to HSPA1A-, HSPA1B-, HSPA7-, and HSPA9- high phenotype. In summary, the increased expressions of HSPA1A1, HSPA1B, and HSPA7 were associated with poor prognosis, while that of HSPA9 was related to favorable prognosis for colon cancer patients.

Chemoradiotherapy and Increased Prescription Dose in Esophageal Squamous Cell Cancer: A Retrospective Study.

To analyze the outcomes and adverse events of patients with esophageal squamous cell carcinoma (ESCC) treated with definitive chemoradiation with modified radiotherapy volume and increased radiation dose. This was a retrospective analysis of patients with ESCC treated with definitive chemoradiotherapy at the Sun Yat-sen University Cancer Center (02/2015 to 02/2017). The dose to the planning gross tumor volume (PGTV) and planning clinical tumor volume (PTV1) was 66-68 Gy (2.0-2.2 Gy/fraction). The dose to the planning regional lymph node drainage area volume (PTV2) was 46 Gy (2.0 Gy/fraction). Treatment response, adverse events, progression-free survival (PFS), overall survival (OS), and locoregional failure-free survival (LRFFS) were analyzed. Twenty-six patients were included. The median follow-up was 31 (range, 4.3-51.3) months. Sixteen (61.5%) patients had a complete response, and four (15.4%) achieved a partial response. The objective response rate was 76.9%, and the disease control rate was 80.8%. The median PFS and OS were not achieved. The 4-year PFS was 63.9%, and the 4-year OS was 71.0%. Grade 1-2 and 3-4 radiation-related esophagitis was observed in 15 (57.7%) and one (4.5%) patients, respectively. Grade 1-2 and 3-4 radiation-related pneumonitis was observed in 12 (46.2%) and one (4.5%) patients, respectively. No patients developed radiation-related heart or skin damage. The modified target volume definition and increased dose of definitive radiotherapy combined with chemotherapy in patients with ESCC had low toxicity and might improve survival, but additional trials are necessary to prove the superiority of this strategy.

Erastin decreases radioresistance of NSCLC cells partially by inducing GPX4-mediated ferroptosis.

The aim of the present study was to examine whether erastin influences radioresistance in non-small cell lung cancer (NSCLC) cells and produce a preliminary investigation into its mechanism of action. The radioresistant subtype of NSCLC cells, A549-R and H460-R, were induced by high-dose hypofractionated irradiation. Erastin was used to treat the radioresistant cells and radiosensitivity was examined by colony formation assays. Cell death was determined after the cells were treated with erastin, irradiation (IR) or erastin together with IR. The expression of glutathione peroxidase 4 (GPX4) expression in the parental cells and radioresistance cells was detected by western blotting. GPX4 expression in the radioresistance cells was subsequently inhibited, radiosensitivity and cell death was measured, and erastin enhanced radiosensitivity in A549-R and H460-R cells. Erastin and IR exhibited a combined effect on killing cells, as co-treatment with erastin and IR demonstrated a higher effect on killing cells compared with erastin or IR alone. GPX4 expression was inhibited by erastin in the radioresistant cells. Inhibiting GPX4 expression also radiosensitized NSCLC cells to radiation in the radioresistant cell lines. Erastin-induced and GPX4-inhibition-induced cell death could partially be rescued by deferoxamine, but not Z-VAD-FMK and olaparib, which indicated that erastin and GPX4-inhibition induced ferroptosis in the radioresistant cells. Erastin decreased radioresistance of NSCLC cells partially by inducing GPX4-mediated ferroptosis.

Quinalizarin enhances radiosensitivity of nasopharyngeal carcinoma cells partially by suppressing SHP-1 expression.

The purpose of this study was to investigate the influence of quinalizarin on the radiosensitivity of nasopharyngeal carcinoma (NPC) cells and the relevant underlying mechanisms. Human NPC cell lines CNE-1, CNE-2 and 5-8F were treated with quinalizarin and then irradiated with different X-rays doses. Cell viability, survival, DNA double-strand breaks (DSB), apoptosis, cell cycle distribution, expression of SHP-1 and other related proteins were detected with MTT assay, colony formation assay, immunofluorescent assay, flow cytometry and western blot analysis, respectively. We also examined how the effects of quinalizarin were affected by SHP-1-overexpression by lentivirus transfection. Quinalizarin at 25 µM enhanced radiosensitivity of NPC cells. This increased radiosensitivity was due to inhibition of cell viability, which delayed DSB repair as seen by significantly increased γ-H2AX foci, promoting apoptosis by 34% in CNE-1 and 9% in CNE-2 cells compared to controls and changing cell cycle distribution in CNE-1, but not CNE-2 cells. Quinalizarin treatment obviously decreased SHP-1 protein expression. Overexpressing SHP-1 partially reversed the radiosensitive effect of quinalizarin. Quinalizarin inhibited binding of p65 and the promoter of SHP-1, and decreased the activities of SHP-1 promoter and SHP-1. Quinalizarin enhanced radiosensitivity of NPC cells partially by suppressing SHP-1 expression.

MicroRNA-378g enhanced radiosensitivity of NPC cells partially by targeting protein tyrosine phosphatase SHP-1.

PURPOSE: To investigate the influence of microRNA-378g (miR-378g) on radiosensitivity and metastasis of nasopharyngeal carcinoma cells and study how miR-378g regulated Src homology region 2 domain-containing phosphatase-1 (SHP-1) expression. MATERIALS AND METHODS: Polymerase chain reaction (PCR) was used to detect the expression level of miR-378g and SHP-1 mRNA in different nasopharyngeal carcinoma (NPC) cell lines. MiR-378g mimics were transfected into NPC cells and radiosensitivity was determined by colony formation assay. Cell apoptotic rate was determined by flow cytometry analysis. Cell invasion was examined by transwell assay. SHP-1 transcriptional activity was examined by luciferase assay. SHP-1 expression level was determined by Western blot. Lentivirus containing SHP-1 gene and miR-378g mimics were co-transfected into NPC cells and radiosensitivity and metastasis were detected by colony formation assay and transwell assay again. RESULTS: Expression of miR-378g and SHP-1 mRNA was negatively correlated in NPC cell lines. MiR-378g mimics enhanced radiosensitivity, promoted apoptosis and decreased invasion in NPC cells. SHP-1 expression was inhibited by miR-378g mimics. Luciferase reporter assay showed that miR-378g directly targeted SHP-1 by binding to 3' untranslated region (3'UTR) of SHP-1 mRNA. Overexpression of SHP-1 partially inversed the effect of miR-378g mimics on radiosensitivity, but had no effect on cell invasion. CONCLUSION: MiR-378g enhanced radiosensitivity partially by targeting SHP-1 in NPC cells. Cell invasion was also partially inhibited by miR-378g, but the effect was not mediated by SHP-1.

Increased SHP-1 expression results in radioresistance, inhibition of cellular senescence, and cell cycle redistribution in nasopharyngeal carcinoma cells.

BACKGROUND: Radioresistance is the main limit to the efficacy of radiotherapy in nasopharyngeal carcinoma (NPC). SHP-1 is involved in cancer progression, but its role in radioresistance and senescence of NPC is not well understood. This study aimed to assess the role of SHP-1 in the radioresistance and senescence of NPC cells. METHODS: SHP-1 was knocked-down and overexpressed in CNE-1 and CNE-2 cells using lentiviruses. Cells were irradiated to observe their radiosensitivity by colony forming assay. BrdU incorporation assay and flow cytometry were used to monitor cell cycle. A ß-galactosidase assay was used to assess senescence. Western blot was used to assess SHP-1, p21, p53, pRb, Rb, H3K9Me3, HP1γ, CDK4, cyclin D1, cyclin E, and p16 protein expressions. RESULTS: Compared with CNE-1-scramble shRNA cells, SHP-1 downregulation resulted in increased senescence (+107%, P < 0.001), increased radiosensitivity, higher proportion of cells in G0/G1 (+33%, P < 0.001), decreased expressions of CDK4 (-44%, P < 0.001), cyclin D1 (-41%, P = 0.001), cyclin E (-97%, P < 0.001), Rb (-79%, P < 0.001), and pRb (-76%, P = 0.001), and increased expression of p16 (+120%, P = 0.02). Furthermore, SHP-1 overexpression resulted in radioresistance, inhibition of cellular senescence, and cell cycle arrest in the S phase. Levels of p53 and p21 were unchanged in both cell lines (all P > 0.05). CONCLUSION: SHP-1 has a critical role in radioresistance, cell cycle progression, and senescence of NPC cells. Down-regulating SHP-1 may be a promising therapeutic approach for treating patients with NPC.

MicroRNA-4649-3p inhibits cell proliferation by targeting protein tyrosine phosphatase SHP-1 in nasopharyngeal carcinoma cells.

The present study aimed to investigate the influence of microRNA-4649-3p on nasopharyngeal carcinoma (NPC) cell proliferation and how it regulated SHP-1 expression. The online software TargetScan was used to predict the microRNAs targeting SHP-1 and identified that miR-4649-3p was one of the possible miRNAs targeting SHP-1. Subsequently, quantitative polymerase chain reaction (PCR) was used to detect the expression level of miR-4649-3p and SHP-1 mRNA in different NPC cell lines. The miR-4649-3p mimics and inhibitors were transfected into NPC cells and cell proliferation was examined by the MTT assay. The SHP-1 expression level was determined by PCR and western blot analysis. Lentivirus containing the SHP-1 gene and miR-4649-3p mimics was co-transfected into the NPC cells and cell proliferation was detected by the MTT assay. The expression level of miR-4649-3p and SHP-1 mRNA was negatively correlated in the NPC cell lines. miR-4649-3p mimics suppressed NPC cell proliferation whereas miR-4649-3p inhibitors promoted NPC cell proliferation. The SHP-1 expression level was suppressed when transfected with miR-4649-3p mimics in NPC cells. The miR-4649-3p inhibitors increased SHP-1 expression. The luciferase reporter assay showed that miR-4649-3p directly targeted SHP-1 by binding to the 3'-untranslated region of SHP-1 mRNA. Overexpression of SHP-1 inversed the inhibited effect of miR-4649-3p mimics on cell proliferation. In conclusion, miR-4649-3p inhibits cell proliferation by targeting SHP-1 in NPC cells.

MicroRNA-18a enhances the radiosensitivity of cervical cancer cells by promoting radiation-induced apoptosis.

Evidence has demonstrated that microRNAs (miRNAs) are important in the regulation of cellular radiosensitivity of various types of human cancer. The aim of this study was to examine the role of miR-18a in regulating the radiosensitivity of cervical cancer, in order to understand the underlying mechanism and to assess the potential of miR-18a as a biomarker for predicting radiosensitivity. The expression of miR-18a was investigated in 48 cervical cancer patients. The results revealed that miR-18a expression was significantly higher in radiosensitive patients than in radioresistant patients by RT-qPCR (P<0.05). Transient transfection experiments showed that miR-18a was upregulated by the miR-18a mimic and downregulated by the miR-18a inhibitor in the SiHa and HeLa cells. Without irradiation treatment, a similar growth was observed in the cells with or without transfection of miR-18a. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Hoechst staining assays showed that miR-18a had no effect on the proliferation and apoptosis of cervical cancer cells after transfection. However, the upregulation of miR-18a suppressed the level of ataxia-telangiectasia mutated and attenuated DNA double-strand break repair after irradiation, which re-sensitized the cervical cancer cells to radiotherapy by promoting apoptosis. Taken together, these results demonstrated that miR-18a is a potential molecule predictor of radiosensitivity in cervical cancer patients and played an important role in the response to radiotherapy.

SHP-1 overexpression increases the radioresistance of NPC cells by enhancing DSB repair, increasing S phase arrest and decreasing cell apoptosis.

The present study aimed to investigate the influence of SHP-1 on the radioresistance of the nasopharyngeal carcinoma (NPC) cell line CNE-2 and the relevant underlying mechanisms. The human NPC cell line CNE-2 was transfected with a lentivirus that contained the SHP-1 gene or a nonsense sequence (referred to as LP-H1802Lv201 and LP-NegLv201 cells, respectively). Cells were irradiated with different ionizing radiation (IR) doses. Cell survival, DNA double-strand breaks (DSBs), apoptosis, cell cycle distribution, and the expression of related proteins were assessed using colony formation assay, immunofluorescent assays (IFAs), flow cytometry (FCM) and western blot analyses, respectively. Compared with the control (CNE-2 cells) and LP-NegLv201 cells, LP-H1802Lv201 cells were more resistant to IR. IFAs showed that IR caused less histone H2AX phosphorylation (γH2AX) and RAD51 foci in the LP-H1802Lv201 cells. Compared with the control and LP-NegLv201 cells, LP-H1802Lv201 cells showed increased S phase arrest. After IR, the apoptotic rate of the LP-H1802Lv201 cells was lower in contrast to the control and LP-NegLv201 cells. Western blot analyses showed that IR increased the phosphorylation of ataxia telangiectasia mutated (ATM) kinase, checkpoint kinase 2 (CHK2), ataxia telangiectasia and Rad3-related (ATR) protein, checkpoint kinase 1 (CHK1) and p53. In LP-H1802Lv201 cells, the phosphorylation levels of ATM and CHK2 were significantly increased while the p53 phosphorylation level was decreased compared to these levels in the control and LP-NegLv201 cells. Phosphorylation of ATR and CHK1 did not show significant differences in the three cell groups. Overexpression of SHP-1 in the CNE-2 cells led to radioresistance and the radioresistance was related to enhanced DNA DSB repair, increased S phase arrest and decreased cell apoptosis.

[Preliminary mechanism of paclitaxel enhanced radiation sensitivity for nasopharyngeal carcinoma cells].

OBJECTIVE: To study the radiation-sensitizing function and preliminary mechanism of paclitaxel in radiation-resistant nasopharyngeal carcinoma cells. METHOD: X-ray dose fractionated irradiation technology to build radiation-resistant subline of nasopharyngeal carcinoma; CNE-2S1 was treated with paclitaxel alone or combined with radiation therapy, while control group treated with radiation therapy; cell colony formation assay was used to observe sensitizing effect of paclitaxel on radiotherapy; flow cytometry analysis was used to analyze cell cycle distribution and apoptosis ratio of different treatment groups; immunoblotting was used to analyze SHP-1 expression levels of different treatment groups. RESULT: Nasopharyngeal carcinoma cells resistant to radiation was successfully established; cell colony formation assay showed that paclitaxel has obvious sensitizing effect on radiotherapy; FACS results showed that: CNE-2S1 treated by paclitaxel were arrested in G2M phase; paclitaxel and radiotherapy treatments significantly improved the CNE-2S1 apoptosis ratio; Western blot results showed that paclitaxel and combined radiotherapy can reduce the CNE-2S1 cells SHP-1 expression levels. CONCLUSION: Paclitaxel enhanced radiation therapy for nasopharyngeal carcinoma cells resistant to radiation, and SHP-1 may be involved in this progress.

[Interleukin-17 contributes to the macrophage secretion of interleukin-27 in a murine model of viral myocarditis].

OBJECTIVE: Interleukin-27 (IL-27) has been reported to reduce the levels of interleukin-17 (IL-17) and alleviate the severity of experimental autoimmune myocarditis. IL-17, an important tissue-protective cytokine in viral myocarditis (VMC), has been reported to increase synovial expression of IL-27 in rheumatoid arthritis. However, the influence of IL-17 on IL-27 expression in murine model of VMC remains unknown. METHODS: Wild-type (WT) and IL-17A-deficient (IL-17A(-/-)) mice on the BALB/c background were intraperitoneally (i.p) injected with coxsackievirus B3 (CVB3) for establishing VMC models. Cardiac tissue was obtained on day 7 after CVB3 injection. Myocardial histopathologic changes were observed by hematoxylin-eosin (HE) stained myocardial sections.Expression of IL-27 in heart and serum was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. Furthermore, splenic lymphocytes and peritoneal macrophages were purified 1 week after injection from WT mice.Isolated lymphocytes were cultured in the presence of different concentrations (0 and 25 ng/ml) of recombinant IL-17 (rIL-17) for 24 h. Macrophages were cultured with different concentrations of rIL-17 (0 and 10 ng/ml) for 48 h.IL-27 mRNA expression of cultured cells was assayed by RT-PCR, and their protein level in the culture supernatant was measured by ELISA. RESULTS: Compared with WT mice, significantly less cardiac inflammation was evidenced in the heart of IL-17A-/- mice (0.9 ± 0.3 vs.1.9 ± 0.5) , relative cardiac IL-27 p28 mRNA expressions (1.11 ± 0.24 vs.3.1 ± 0.8) and serum IL-27 protein[(72 ± 18) pg/ml vs.(95 ± 25) pg/ml] were also significantly lower in IL-17A-/- mice (all P < 0.05).In the culture lymphocytes, the relative mRNA (1.02 ± 0.13 vs.1.32 ± 0.21) and protein [(49 ± 9) pg/ml vs.(52 ± 11) pg/ml]expressions of IL-27 p28 and were similar post treatment with 0 and 25 ng/ml rIL-17 (all P > 0.05). Compared with 0 ng/ml rIL-17 culture with macrophages, higher relative mRNA (8.5 ± 3.1 vs.2.2 ± 0.7) and protein [(368 ± 95) pg/ml vs.(150 ± 38) pg/ml] expressions of IL-27 p28 were detected in 10 ng/ml rIL-17 group (all P < 0.05). CONCLUSION: Our data indicates that cytokine IL-17 may contribute to the secretion of IL-27 in VMC mice.Furthermore, macrophages but not lymphocytes may be the important IL-27-producing immune cells and major target cells for IL-17. Thus,IL-27 and IL-17 might be actively involved in the pathogenesis of VMC.

[Effect of IL-17A on levels of antiheart autoantibodies in mice with viral myocarditis].

OBJECTIVE: To explore the effect of interleukin-17A (IL-17A) on the serum level of antiheart autoantibodies in mice with viral myocarditis. METHODS: Male wild-type (WT) and IL-17A-deficient (IL-17A(-/-)) BALB/c mice were intraperitoneally injected with Coxsackie virus B3 (CVB3) for establishing VMC models (VMC-WT group and VMC-IL-17A(-/-) group). Meanwhile, a control group (WT group) of WT mice were established by i.p. administration of phosphate buffered saline (PBS). Paraffin sections of cardiac tissues were made 14 days after CVB3 injection. Myocardial histopathologic changes were evaluated by HE staining. The levels of anti-adenine nucleotide translocator (ANT) autoantibody, anti-ß-myosin heavy chain (ß-MHC) autoantibody and anti-cardiac L-type calcium channel (CACH2) autoantibody in sera were measured by ELISA. RESULTS: Compared with WT group, the levels of anti-ANT-autoantibody and anti-ß-MHC-autoantibody significantly increased in VMC-WT group (P<0.01, P<0.05), while the concentration of anti-CACH2-autoantibody showed no significant difference between WT and VMC-WT groups (P>0.05). Compared with VMC-WT group, the level of anti-ANT-autoantibody was reduced in VMC-IL-17A(-/-) group (P<0.05), while the levels of anti-ß-MHC-autoantibody and anti-CACH2-autoantibody showed no significant difference between them (P>0.05). CONCLUSION: IL-17A contributed to the secretion of anti-ANT-autoantibody of VMC mice, but had no effect on the secretion of anti-ß-MHC-autoantibody and anti-CACH2-autoantibody in VMC mice.

1-Phenyl-4-benzoyl-1H-1,2,3-triazoles as orally bioavailable transcriptional function suppressors of estrogen-related receptor α.

Estrogen-related receptor α is a potential candidate target for therapeutic treatment of breast cancer. We describe the discovery and structure-activity relationship study of a series of 1-phenyl-4-benzoyl-1H-1,2,3-triazoles as novel suppressors of ERRα transcriptional functions. The most promising compound, 2-aminophenyl-(1-(3-isopropylphenyl)-1H-1,2,3-triazol-4-yl)methanone (14n), potently suppressed the transcriptional functions of ERRα with IC50 = 0.021 µM in a cell-based reporter gene assay and also decreased both the mRNA levels and the protein levels of ERRα and the downstream targets. This compound inhibited the proliferation and migration of breast cancer cells with high level of ERRα. Preliminary pharmacokinetic studies suggested that it possessed a good pharmacokinetic profile with an oral bioavailability of 71.8%. The compounds may serve as novel small molecule probes for further validation of ERRα as a molecular target for anticancer drug development.

Identification of GZD824 as an orally bioavailable inhibitor that targets phosphorylated and nonphosphorylated breakpoint cluster region-Abelson (Bcr-Abl) kinase and overcomes clinically acquired mutation-induced resistance against imatinib.

Bcr-Abl(T315I) mutation-induced imatinib resistance remains a major challenge for clinical management of chronic myelogenous leukemia (CML). Herein, we report GZD824 (10a) as a novel orally bioavailable inhibitor against a broad spectrum of Bcr-Abl mutants including T315I. It tightly bound to Bcr-Abl(WT) and Bcr-Abl(T315I) with K(d) values of 0.32 and 0.71 nM, respectively, and strongly inhibited the kinase functions with nanomolar IC(50) values. The compound potently suppressed proliferation of Bcr-Abl-positive K562 and Ku812 human CML cells with IC(50) values of 0.2 and 0.13 nM, respectively. It also displayed good oral bioavailability (48.7%), a reasonable half-life (10.6 h), and promising in vivo antitumor efficacy. It induced tumor regression in mouse xenograft tumor models driven by Bcr-Abl(WT) or the mutants and significantly improved the survival of mice bearing an allograft leukemia model with Ba/F3 cells harboring Bcr-Abl(T315I). GZD824 represents a promising lead candidate for development of Bcr-Abl inhibitors to overcome acquired imatinib resistance.