Ononin promotes radiosensitivity in lung cancer by inhibiting HIF-1α/VEGF pathway.

BACKGROUND: In our previous study, we provided evidence that Astragalus mongholicus Bunge(AM) and its extracts possess a protective capability against radiation-induced damage, potentially mediated through the reduction of reactive oxygen species (ROS) and nitric oxide (NO). However, we were pleasantly surprised to discover during our experimentation that AM not only offers protection against radiation damage but also exhibits a radiation sensitization effect. This effect may be attributed to a specific small molecule present in AM known as ononin. Currently, radiation sensitizers are predominantly found in nitrazole drugs and nanomaterials, with no existing reports on the radiation sensitization properties of ononin, nor its underlying mechanism. PURPOSE: This study aims to investigate the sensitization effect of the small molecule ononin derived from AM on lung cancer radiotherapy, elucidating its specific molecular mechanism of action. Additionally, the safety profile of combining astragalus small molecule ononin with radiation therapy will be evaluated. METHODS: The effective concentration of ononin was determined through cell survival experiments, and the impact of ononin combined with varying doses of radiation on lung cancer cells was observed using CCK-8 and cell cloning experiments. The apoptotic effect of ononin combined with radiation on lung cancer cells was assessed using Hochester staining, flow cytometry, and WB assay. Additionally, WB and immunofluorescence analysis were conducted to investigate the influence of ononin on HIF-1α/VEGF pathway. Furthermore, Molecular Dynamics Simulation was employed to validate the targeted binding ability of ononin and HIF-1α. A lung cancer cell line was established to investigate the effects of knockdown and overexpression of HIF-1α. Subsequently, the experiment was repeated using tumor bearing nude mice and C57BL/6 mouse models in an in vivo study. Tumor volume was measured using a vernier caliper, while HE, immunohistochemistry, and immunofluorescence techniques were employed to observe the effects of ononin combined with radiation on tumor morphology, proliferation, and apoptosis. Additionally, Immunofluorescence was employed to examine the impact of ononin on HIF-1α/VEGF pathway in vivo, and its effect on liver function in mice was assessed through biochemistry analysis. RESULTS: At a concentration of 25 µM, ononin did not affect the proliferation of lung epithelial cells but inhibited the survival of lung cancer cells. In vitro experiments demonstrated that the combination of ononin and radiation could effectively inhibit the growth of lung cancer cells, induce apoptosis, and suppress the excessive activation of the Hypoxia inducible factor 1 alpha/Vascular endothelial growth factor pathway. In vivo experiments showed that the combination of ononin and radiation reduced the size and proliferation of lung cancer tumors, promoted cancer cell apoptosis, mitigated abnormal activation of the Hypoxia inducible factor 1 alpha pathway, and protected against liver function damage. CONCLUSION: This study provides evidence that the combination of AM and its small molecule ononin can enhance the sensitivity of lung cancer to radiation. Additionally, it has been observed that this combination can specifically target HIF-1α and exert its effects. Notably, ononin exhibits the unique ability to protect liver function from damage while simultaneously enhancing the tumor-killing effects of radiation, thereby demonstrating a synergistic and detoxifying role in tumor radiotherapy. These findings contribute to the establishment of a solid basis for the development of novel radiation sensitizers derived from traditional Chinese medicine.

A comprehensive analysis of partial peristalsis recovery after peroral endoscopic myotomy in patients with achalasia.

OBJECTIVE: To determine whether peroral endoscopic myotomy (POEM) improves esophageal peristalsis and to investigate the association between recovery of esophageal peristalsis after POEM and clinical features of the patients. METHODS: In this single-center retrospective study, data were collected from medical records of the patients with achalasia who underwent POEM between January 2014 and May 2016. Demographics data, high-resolution esophageal manometry parameters, Eckardt score, and gastroesophageal reflux disease questionnaire (GERD-Q) score were collected. Weak and fragmented contraction was defined as partial recovery of esophageal peristalsis based on the Chicago classification version 3.0. Logistic regression analysis was used to identify variables associated with the partial recovery of peristalsis after POEM. RESULTS: A total of 103 patients were enrolled. Esophageal contractile activity was observed in the distal two-thirds of the esophagus in 24 patients. The Eckardt score, integrated relaxation pressure, and lower esophageal sphincter (LES) resting pressure were significantly decreased after POEM. Multivariate analysis revealed that preprocedural LES resting pressure (P = 0.013) and preprocedural Eckardt score (P = 0.002) were related to the partial recovery of peristalsis after POEM. Symptoms of gastroesophageal reflux and reflux esophagitis after POEM were less frequent in those with partial recovery of peristalsis (both P < 0.05). CONCLUSIONS: Normalization of esophagogastric junction relaxation pressure achieved by POEM is associated with the partial recovery of esophageal peristalsis in patients with achalasia. Preprocedural LES resting pressure and the Eckardt score are predictive of the recovery of esophageal peristalsis.

A Bayesian network meta-analysis of the primary definitive therapies for locoregionally advanced nasopharyngeal carcinoma: IC+CCRT, CCRT+AC, and CCRT alone.

BACKGROUND: The major aim of this Bayesian network analysis was to determine the optimal treatment strategy for locoregionally advanced nasopharyngeal carcinoma (LANPC). METHOD: We systematically searched databases and extracted data from randomized clinical trials involving LANPC patients randomly assigned to receive induction chemotherapy followed by concurrent chemoradiotherapy (IC+CCRT), CCRT followed by adjuvant chemotherapy (CCRT+AC), or CCRT. RESULTS: In the network analysis, IC+CCRT was significantly better than CCRT alone for 5-year FFS (odds ratio [OR]: 1.63, 95% credible interval [CrI] 1.16-2.29), DMFS (OR: 1.56, 95% CrI 1.08-2.22), and LFRS (OR: 1.62, 95% CrI 1.02-2.59), but not OS (OR: 1.35, 95% CrI 0.92-2.00). Rank probabilities showed that IC+CCRT was ranked the best followed by CCRT+AC and CCRT for all 5-year outcomes. Although compared to IC+CCRT and CCRT, CCRT+AC did not significantly improve survival but had the highest 5-year survival rates. CONCLUSIONS: IC+CCRT could be recommended as a front-preferred primary definitive therapy for patients with LANPC.

The expression profile of lung long non-coding RNAs and mRNAs in a mouse model of smoke inhalation injury.

To study the potential expression of lung long non-coding RNAs (lncRNAs) and mRNAs during smoke inhalation injury (SII), using a SII mouse model that we created in our previous work. Microarray was used to investigate the lncRNAs and mRNAs profiles. A bioinformatics analysis was performed. Changes in the top 10 down-regulated and 10 up-regulated lncRNAs were validated using Quantitative Reverse Transcription-PCR (RT-qPCR). The acute lung injury (ALI) mouse model was successfully induced by smoke inhalation, as confirmed by the aberrantly modified cell numbers of red blood cells and neutrophils counts, increased levels of TNF-α, IL-1ß, Bax, caspase-7, caspase-3, and decreased Bcl-2 content in lung tissues. When compared to the control mice, 577 lncRNAs and 517 mRNAs were found to be aberrantly expressed in the SII mice. According to the Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, the altered mRNAs were enriched in acute-phase response, oxidoreductase activity, oxidation-reduction process, glutathione metabolism, the wnt signaling pathway, and ferroptosis. A lncRNA-related competitive endogenous RNA (ceRNA) network, including 383 lncRNAs, 318 MicroRNAs (miRNAs), and 421 mRNAs specific to SII, was established. The changes in NONMMUT026843.2, NONMMUT065071.2, ENSMUST00000235858.1, NONMMUT131395.1, NONMMUT122516.1, NONMMUT057916.2, and NONMMUT013388.2 in the lung matched the microarray results. Our findings help to provide a more comprehensive understanding of the pathogenesis of SII as well as new insights into potential therapeutic targets.

Evaluation of Minimal Change Lesions Using Linked Color Imaging in Patients With Nonerosive Reflux Esophagitis.

BACKGROUND AND AIMS: High prevalence of minimal change lesion (MCL) in nonerosive reflux esophagitis (NERD) patients is commonly recognized by many endoscopists. However, it is difficult to detect MCL with conventional white-light imaging (WLI) endoscopy. Linked color imaging (LCI), a novel image-enhanced endoscopy technology with strong, unique color enhancement, is used for easy recognition of early gastric cancer and detection of Helicobacter pylori infection. The aim of the study was to compare the efficacy of LCI and WLI endoscopy in evaluating MCL in patients with NER. MATERIALS AND METHODS: Forty-one patients with NERD and 38 subjects with nongastroesophageal reflux disease (non-GERD) were recruited in this study between August 2017 and July 2018. During upper gastrointestinal endoscopy, the distal 5 cm of the esophageal mucosal morphology at the squamocolumnar junction was visualized using WLI followed by LCI. MCL was defined as areas of erythema, blurring of the Z-line, friability, decreased vascularity, white turbid discoloration, and edema or accentuation of the mucosal folds. Three experienced endoscopists evaluated the color patterns for MCL on WLI images and on WLI combined with LCI images in both groups. A biopsy was taken 2 cm above the esophagogastric junction. Histologic slides were scored by a pathologist in a blinded manner. RESULTS: The proportion of MCL was higher in the patients with NERD (70.7%, 29/41) than in patients with non-GERD (39.5%, 15/38) using WLI combined with LCI. In 12 patients with NERD, both WLI and LCI showed normal mucosa. The MCL detection rate was significantly higher when using WLI combined with LCI than when using WLI (70.7% vs. 51.2%, P=0.039) in patients with NERD. The histopathologic score of MCL (+) was significantly higher than that of MCL (-) patients in both the NERD group (4.59±0.32 vs. 2.36±0.34, P<0.01) and the non-GERD group (3.47±0.50 vs. 2.00±0.28, P<0.01). The intraobserver reproducibility levels and interobserver agreement were better with LCI than with WLI alone. CONCLUSIONS: Frequency of MCL was higher in patients with NERD than in those with non-GERD. MCL can be identified by using WLI combined with LCI in patients with NERD. By enhancing endoscopic images, LCI is more sensitive in detecting MCL compared with WLI.

Prognostic value of pretreatment prognostic nutritional index and lactated dehydrogenase in locally advanced nasopharyngeal carcinoma patients.

BACKGROUND: To evaluate the prognostic value of pretreatment prognostic nutritional index (PNI), lactated dehydrogenase (LDH) and their combination (PNI-LDH) in patients with locally advanced NPC receiving induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CCRT). METHODS: A total of 213 patients diagnosed with locally advanced (III-IVA) NPC between January 2013 and December 2017 were retrospectively reviewed. The optimal PNI and LDH cutoff values were determined by the quartiles. The association between PNI and LDH and the clinicopathological characteristics of the patients was examined. Survival curves were analyzed using the Kaplan-Meier method and compared by the log-rank test between the different PNI and LDH subgroups. Univariate and multivariate analyses were performed by the Cox proportional hazards regression model to evaluate the prognostic impact on overall survival (OS), progression-free survival (PFS), locoregional recurrence free survival (LRFS) and distant metastasis-free survival (DMFS). Furthermore, the prognostic values of the PNI, LDH, and PNI-LDH were evaluated by comparing the AUC area. RESULTS: The optimal cut-off values of PNI and LDH were 52 and 177, respectively. Multivariate analyses revealed that patients with a higher PNI had inferior OS (P=0.027), PFS (P=0.040), LRFS (P=0.015) and DMFS (P=0.040), and patients with a higher LDH level had poorer OS (P=0.040), PFS (P=0.001), LRFS (P=0.001) and DMFS (P=0.001). Furthermore, EBV DNA positive, stage IVA were independent prognostic factors for survival outcomes in the multivariate analyses. Moreover, we further demonstrated that low PNI-high LDH in locally advanced NPC patients was significantly related to poor OS (P=0.012), PFS (P=0.001), LRFS (P=0.001) and DMFS (P=0.001). The AUC of the PNI, LDH and PNI-LDH were 0.653 (P=0.021), 0.647 (P=0.028) and 0.751 (P=0.001), respectively, indicating that PNI-LDH is superior to either score alone. CONCLUSIONS: Pretreatment PNI and LDH could be considered as valuable predictors for survival outcomes in locally advanced NPC patients. The combination of them, superior to either score alone, can be used as a supplement to the traditional TNM staging system.

Prognostic efficacy of the combination of the pretreatment systemic Immune-Inflammation Index and Epstein-Barr virus DNA status in locally advanced Nasopharyngeal Carcinoma Patients.

Background: The systemic immune-inflammation index (SII) and Epstein-Barr virus DNA (EBV DNA) levels has been used as a prognostic marker for nasopharyngeal carcinoma (NPC) patients, but there is no in-depth study in locally advanced NPC patients and no research on the predictive value of their combination. Our study aimed to evaluate the prognostic efficacy of the pretreatment SII, EBV DNA levels and their combination in locally advanced NPC patients receiving induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CCRT). Materials and methods: 319 patients diagnosed with locally advanced NPC receiving IC followed by CCRT were retrospectively reviewed (213 in the training cohort and 106 in the validation cohort). The cut-off value for the SII was determined using receiver operating characteristic (ROC) curve. Correlations between characteristics of patients were assessed using the Pearson correlation coefficient. Survival curves for the SII, EBV DNA levels and their combination were analyzed using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses were performed by the Cox proportional hazards regression model to evaluate the prognostic impact on overall survival (OS) and progression-free survival (PFS). A prognostic nomogram was generated and its prediction ability was measured by the concordance index (C-index). Results: The optimal cutoff point for the SII was 402.10. A higher SII and EBV DNA positivity were demonstrated to be related to poorer survival outcomes (P < 0.05). Multivariate analyses showed that a higher SII, EBV DNA positivity and their combination were powerful independent risk factors for OS and PFS (P < 0.05). The SII - EBV DNA had the largest area under the curve (AUC) compared to either score alone. The incorporation of the SII - EBV DNA into established nomogram achieved higher C-index in the prediction of OS and PFS, indicating its superior for predicting survival. All results were found in the training cohort and confirmed in the validation cohort. Conclusions: The pretreatment SII and EBV DNA levels are promising factors for predicting survival in locally advanced NPC patients. The combination of them, which was superior to either score alone, was a complement to the conventional TNM staging system.

Effects of Apatinib on the "Stemness" of Non-Small-Cell Lung Cancer Cells In Vivo and Its Related Mechanisms.

Objective: To investigate the effects of Apatinib on the "stemness" of lung cancer cells in vivo and to explore its related mechanisms. Methods: A xenograft model of lung cancer cells A549 was established in nude mice and randomized into a control group (n = 4) and an Apatinib group (n = 4). Tumor tissues were harvested after 2 weeks, and mRNA was extracted to detect changes in stemness-related genes (CD133, EPCAM, CD13, CD90, ALDH1, CD44, CD45, SOX2, NANOG, and OCT4) and Wnt/ß-catenin, Hedgehog, and Hippo signal pathways. Results: Compared with the control group, the volume and weight of nude mice treated with Apatinib were different and had statistical significance. Apatinib inhibited the expressions of ABCG2, CD24, ICAM-1, OCT4, and SOX2 and upregulated the expressions of CD44, CD13, and FOXD3. Apatinib treatment also inhibited the Wnt/ß-catenin, Hedgehog, and Hippo signaling pathways. Conclusion: Apatinib suppressed the growth of non-small-cell lung cancer cells by repressing the stemness of lung cancer through the inhibition of the Hedgehog, Hippo, and Wnt signaling pathways.

R-spondin2 Suppresses the Progression of Hepatocellular Carcinoma via MAPK Signaling Pathway.

The R-spondin family plays important roles in embryonic development, including in humans. However, information on the relationship between R-spondin2 and hepatocellular carcinoma (HCC) is lacking. This study aimed was to explore the mechanisms of R-spondin2 action in the progression of HCC. By analyzing R-spondin2 expression levels in HCC tissues by IHC and database, we identified that HCC tissues had lower expression levels of R-spondin2, correlated with a poor prognosis. We also established R-spondin2-overexpressing and knockdown cell lines and measured their viabilities and invasion abilities in vitro and their oncogenic capacity in vivo. Human mRNA microarray analysis was performed to screen for mRNAs that were differentially expressed between R-spondin2-overexpressing and control HCC cells. Microarray and Western blot analyses showed significant changes in the MAPK signaling pathway after transfection. Furthermore, in vivo experiments indicated that R-spondin2 knockdown increased the tumorigenicity of HCC cells after subcutaneous implantation in mice. Altogether, our results indicate that the R-spondin2, which might be a novel tumor suppressor gene, were responsible for inhibiting the proliferation and invasion of HCC via the MAPK signaling pathway. IMPLICATIONS: R-spondin2 gene might be a novel tumor suppressor gene providing new clues to clarify the biological behavior of HCC and thus reduce patient mortality and prolong survival.

KCNN4 promotes invasion and metastasis through the MAPK/ERK pathway in hepatocellular carcinoma.

Hepatocellular Carcinoma (HCC) is one of the most common malignancies in the world, and is well-known for its bad prognosis. Potassium calcium-activated channel subfamily N member 4 (KCNN4) is a type of intermediate conductance calcium-activated potassium channel, and increasing evidence suggests that KCNN4 contributes to the regulation of invasion and metastasis in a number of cancers. However, its clinical significance and biological function remain unclear in the HCC disease process. In this study, the expression levels of KCNN4 in 86 HCC samples were compared with corresponding paracancerous tissues. sh-RNA was used to reduce the expression of KCNN4 in Hep3B HCC cells in vitro; this was confirmed by Real time-PCR and western blotting. Wound healing, transwell assays and high content analysis were performed to investigate the tumor-promoting characteristics of KCNN4 in Hep3B HCC cells. As results, KCNN4 expression was significantly associated with preoperative serum alpha-fetoprotein level (p=0.038) and TNM stage (p=0.039). Additionally, patients with high KCNN4 amplification in HCC tissue exhibited shorter disease-free survival, whereas there was no statistical significance between KCNN4 amplification and overall survival. Wound healing and transwell assays showed that knockdown of KCNN4 expression could reduce migration and invasion abilities of HCC cells. High content analysis result showed that down-regulated KCNN4 could inhibit the ability of HCC cell proliferation. The mitogen-activated protein kinase (MAPK) pathway is active in cell proliferation, differentiation, migration, senescence, and apoptosis. Matrix metallopeptidase 9 and extracellular signal regulated kinase 1/2 (ERK1/2) were important biomarkers of MAPK/ERK pathway, knockdown of KCNN4 reduced the expression of MMP9 and ERK1/2. These findings showed that KCNN4 promotes HCC invasion and metastasis through the MAPK/ERK pathway.

A meta-analysis of cisplatin-based concurrent chemoradiotherapy with or without cetuximab for locoregionally advanced nasopharyngeal carcinoma.

BACKGROUND: It is unclear whether cetuximab (CTX) plus cisplatin-based concurrent chemoradiotherapy (CCRT) delivers equivalent or improved results over standard CCRT in locoregionally advanced nasopharyngeal carcinoma (NPC). METHODS: The strategy involved searching the PubMed, Embase, Cochrane Library, and Web of Science. Pooled hazard ratios (HRs) for overall survival (OS), distant metastasis-free survival (DMFS), locoregional relapse-free survival (LRFS), and disease-free survival (DFS), and pooled risk ratios for adverse events were meta-analyzed. RESULTS: In all, 1744 patients in 5 clinical trials were included in the analysis. Compared with CCRT group, CTX plus CCRT significantly improved DFS (HR = 0.59, 95% confidence interval [CI]: 0.41-0.86, P = .006) and distant metastasis failure-free survival (HR = 0.54, 95% CI: 0.38-0.76, P = .0004), rather than OS (HR = 0.70, 95% CI: 0.44-1.09, P = .12) and local-regional failure-free survival (HR = 0.82, 95% CI: 0.54-1.22, P = .33). CONCLUSIONS: CTX plus CCRT might achieve higher DFS and DMFS with no significant difference in OS and LRFS. CTX plus CCRT group was associated with more grade 3-4 skin rash, mucositis and dermatitis. Large randomized trials were urgent to fully explore the usefulness of this treatment in the locally advanced NPC patients.

In Vivo Study on the Effects of Xiaoaiping on the Stemness of Hepatocellular Carcinoma Cells.

AIMS: The aim of this study was to examine the effects of Xiaoaiping on the stemness of hepatocellular carcinoma (HCC) cells in vivo and to investigate the underlying molecular mechanism. METHODS: A subcutaneous xenograft nude mouse model was established using Hep3B-derived HCC cells. The mice were randomly assigned to the 100 mg/kg Xiaoaiping or 100 µL/20 g normal saline (control) groups (n =3/sex/group) for daily intragastric administration for 14 days. The tumor size was closely monitored during the dosing phase. After the treatment period, the tumor tissues were weighed and harvested for mRNA and protein isolation. qPCR and Western blotting were used to evaluate the expression of cancer stemness markers (epithelial cell adhesion molecule [EpCAM], cluster of differentiation [CD13], CD90, aldehyde dehydrogenase 1 [ALDH1], CD44, and CD45), totipotency factors (sex determining region Y-box 2 [Sox2], Nanog, and octamer-binding transcription factor 4 [Oct4]), and genes involved in the Notch, Wnt/ß-catenin, Hedgehog, and Hippo signaling pathways. KEY FINDINGS: The tumor size and weight were significantly reduced in the nude mice treated with 100 mg/kg Xiaoaiping when compared with the controls. The Xiaoaiping effects on the stemness markers and totipotency factors included decreased expression of EpCAM, CD24, CD47, Sox2, Oct4, and sal-like protein 4 (SALL4), as well as increased expression of CD13 and ALDH1. In addition, Xiaoaiping inhibited the Hippo, Wnt, and Hedgehog signaling pathways. CONCLUSION: Xiaoaiping significantly inhibited the growth of HCC xenograft in nude mice. These antitumor effects may be mediated by modulating the expression of multiple stemness markers and totipotency factors and inhibition of the Hippo, Wnt, and Hedgehog signaling pathways.

High Expression of ANXA2 Pseudogene ANXA2P2 Promotes an Aggressive Phenotype in Hepatocellular Carcinoma.

OBJECTIVE: Accumulating evidence suggests that pseudogenes play potential roles in the regulation of their cognate wild-type genes, oncogenes, and tumor suppressor genes. ANXA2P2 (annexin A2 pseudogene 2) is one of three pseudogenes of annexin A2 that have recently been shown to be aberrantly transcribed in hepatocellular carcinoma (HCC) cells. However, its clinical meaning and biological function in HCC have remained unclear. Therefore, the present study was aimed at exploring the prognostic value of a high expression of ANXA2P2 in HCC tissue and at identifying whether it can affect the efficacy of targeted drugs (sorafenib, regorafenib, and lenvatinib). METHODS: We obtained ANXA2P2 mRNA expression levels from The Cancer Genome Atlas (TCGA) RNA sequence database. The expression levels of ANXA2P2 in 49 pairs of intratumoral and peritumoral liver tissues were examined by RT-PCR. Wound healing and transwell assays were performed to confirm the tumor-promoting properties of ANXA2P2 in HCC cells. CCK8 assay was conducted to identify whether ANXA2P2 can affect the growth of HCC cells when administered with targeted drugs (sorafenib, regorafenib, and lenvatinib). RESULTS: The expression of ANXA2P2 in HCC tissues was significantly higher than that in adjacent cancerous tissues from TCGA database and validation group. Additionally, patients with high ANXA2P2 expression in HCC tissue had a shorter overall survival, whereas no statistically significant correlation was found between ANXA2P2 expression and disease-free survival (p = 0.08) as well as other clinical parameters, such as age, gender, histological grade, T classification, stage, albumin level, alpha-fetoprotein, and vascular invasion (p = 0.7323, 0.8807, 0.5762, 0.8515, 0.7113, 0.242, 1.0000, and 0.7685, respectively). Furthermore, in vitro experiments showed that knockdown of ANXA2P2 inhibited migration and invasion of HCC cells but did not have an influence on the HCC cell proliferation when treated with targeted drugs (sorafenib, regorafenib, and lenvatinib). CONCLUSION: Our study confirmed elevated ANXA2P2 expression levels in HCC tissue compared with adjacent noncancerous tissue and a worse prognosis of patients with high ANXA2P2 levels in the HCC tissue. The newly found properties of promoting migration and invasion of ANXA2P2 in HCC help to explain this phenomenon. ANXA2P2 could be a novel and suitable predicative biomarker for the risk assessment of recurrence or metastasis of HCC patients but may not be effective to predict the efficacy of targeted drugs.

Molecularly targeted anti-cancer drugs inhibit the invasion and metastasis of hepatocellular carcinoma by regulating the expression of MMP and TIMP gene families.

To investigate the effect of multi-kinase kinase inhibitors (sorafenib; regorafenib; lenvatinib) on the invasion and metastasis of human hepatocellular carcinoma (HCC) cells, and the outcome of this effect on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), yet unclarified. Cells were subjected to four different treatments: blank control group, sorafenib (10 µmol/L) treatment group, regorafenib (20 mmol/L) treatment group, and lenvatinib (4 µmol/L) treatment group. Anti-invasion and anti-metastasis effects were tested using the wound-healing assay and transwell invasion assay. Real-time PCR and Western blot analyses were used to determine the impact of sorafenib, regorafenib, and lenvatinib on the gene expression of MMPs and TIMPs in the two HCC lines (Hep3B and SMMC-7721). Results from the wound-healing and transwell invasion assays showed the three tested anti-cancer drugs to have a significant inhibitory effect on the metastasis and invasion of HCC cells. Real-time PCR and western blot analyses revealed that sorafenib down-regulated the expressions of MMP-7,10,16 and up-regulated those of TIMP-1,3,4, regorafenib down-regulated the expression of MMP-1 and up-regulated TIMP-3 gene expression, and lenvatinib down-regulated the expressions of MMP-1,2,7,9,10,16 and up-regulated those of TIMP-1,3,4. However, these three targeted anti-cancer drugs seem to have no significant regulatory effect on the expressions of other MMPs and TIMPs family genes. In conclusion, sorafenib, regorafenib, and lenvatinib inhibit the invasion and metastasis of HCC cells by regulating MMPs/TIMPs expression levels.

Finite element simulation of lower limb injuries to the driver in minibus frontal collisions.

PURPOSE: This study aims to explore the biomechanical mechanism of lower limb injuries to the driver by establishing a finite element (FE) simulation model of collisions. METHODS: First a minibus FE model was integrated with a seat belt system. Then it was used to rebuild two collisions together with the total human model for safety (THUMS) provided by Toyota Motor Corporation: a rear-end collision between a minibus and a truck and a head-on collision of a minibus to a rigid wall. The impact velocities of both collisions were set at 56 km/h. The vehicle dynamic response, vehicle deceleration, and dashboard intrusion in the two collisions were compared. RESULTS: In the minibus rear-end truck collision, the peak values of the von Mises equivalent stress at the tibia and the femur were 133 MPa and 126 MPa respectively; while in the minibus head-on rigid wall collision, the data were 139 MPa and 99 MPa. Compared with the minibus head-on rigid wall collision, the vehicle deceleration was smaller and the dashboard intrusion was larger in the minibus rear-end truck collision. CONCLUSION: The results illustrate that a longer dashboard incursion distance corresponds to a higher von Mises equivalent stress at the femur. The simulation results are consistent with the driver's autopsy report on lower limbs injuries. These findings verify that FE simulation method is reliable and useful to analyze the mechanisms of lower limb injuries to the driver in minibus frontal collisions.

Effects of PI3K inhibitor NVP-BKM120 on acquired resistance to gefitinib of human lung adenocarcinoma H1975 cells.

The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 µmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 µmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 µmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 µmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 µmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 µmol/L)-treated group or between DMSO-treated control group and gefitinib (1 µmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 µmol/L), and NVP-BKM120 (1 µmol/L) or NVP-BKM120 (1 µmol/L) plus gefitinib (1 µmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 µmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.

Involvement of interstitial cells of Cajal in experimental severe acute pancreatitis in rats.

AIM: To observe the changes in interstitial cells of Cajal (ICC) in rats with experimental severe acute pancreatitis (SAP). METHODS: A total of twenty-four SD rats were randomly divided into two groups (n = 12), namely the sham (S) group and the SAP group; the SAP rat model was established by retrograde injection of 5% sodium taurocholate (1.0 mL/kg) into the pancreatic duct. Twenty-four hours later intestinal motility was assessed by testing small intestinal propulsion rate, and then the rats were sacrificed. The pancreas and jejunum were resected and underwent routine pathologic examination. Immunohistochemical staining was used to detect c-kit-positive cells in the jejunum. Expression of c-kit mRNA was detected by real-time polymerase chain reaction, and the expression of c-kit protein was evaluated by Western blotting. Ultrastructure of ICC was evaluated by transmission electron microscopy. RESULTS: There was bleeding, necrosis and a large amount of inflammatory cell infiltration in pancreatic tissue in the SAP group, while in jejunal tissue we observed a markedly denuded mucosal layer, loss of villous tissue and a slightly dilated muscular layer. The small intestinal propulsion rate was 68.66% ± 2.66% in the S group and 41.55% ± 3.85% in the SAP group. Compared with the S group, the rate of the SAP group decreased sharply. The density of c-kit-positive cells in the SAP group was significantly lower than in the S group; the respective mean densities were 88.47 ± 10.49 in the S group and 56.11 ± 7.09 in the SAP group. The levels of c-kit protein and mRNA were 0.36 ± 0.04 and 1.29 ± 0.91 in the SAP group, respectively, which were significantly lower than those in the S group (0.53 ± 0.06, 0.64 ± 0.33, respectively). In the SAP group, ICC profiles showed the same change tendency, such as vacuolation of mitochondria, irregular vacuoles and loosened desmosome-like junctions. CONCLUSION: Decreased c-kit-positive cells and ultrastructural changes in ICC resulting from blockade of the c-kit signaling pathway are involved in the intestinal dysmotility associated with SAP.

Antidepressant effects of a plant-derived flavonoid baicalein involving extracellular signal-regulated kinases cascade.

Depression and related mood disorders are among the world's greatest public health problems. Previous studies have demonstrated that baicalein (Bai), one plant-derived active flavonoid, exhibits neuroprotection against ischemic brain injury and stimulates the levels of phosphorylation of extracellular signal-regulated kinase (pERK) and brain-derived neurotrophic factor (BDNF) expression in vivo. In this study, the antidepressant-like effects of baicalein was investigated using acute and chronic animal models of depression. The results showed that acute application of Bai at doses of 1, 2 and 4 mg/kg by intraperitoneal injection (i.p.) significantly reduced the immobility time in the forced swimming test (FST) and tail suspending test (TST) of mice. In addition, the chronic application of Bai by i.p. for 21 d also reduced the immobility time and improved locomotor activity in chronic unpredictable mild stress (CMS) model rats. Furthermore, it was shown that Bai reversed the reduction of extracellular ERKs phosphorylation and the level of BDNF expression in the hippocampus of CMS model rats. These results suggest that Bai produce an antidepressant-like effect and this effect is at least partly mediated by hippocampal ERK-mediated neurotrophic action.