Insights into the prediction of the liquid density of refrigerant systems by artificial intelligent approaches.

This study presents a novel model for accurately estimating the densities of 48 refrigerant systems, categorized into five groups: Hydrofluoroethers (HFEs), Hydrochlorofluorocarbons (HCFCs), Perfluoroalkylalkanes (PFAAs), Hydrofluorocarbons (HFCs), and Perfluoroalkanes (PFAs). Input variables, including pressure, temperature, molecular weight, and structural groups, were systematically considered. The study explores the efficacy of both the multilayer perceptron artificial neural network (MLP-ANN) and adaptive neuro-fuzzy inference system (ANFIS) methodologies in constructing a precise model. Utilizing a comprehensive dataset of 3825 liquid density measurements and outlier analysis, the models achieved R2 and MSE values of 0.975 & 0.5575 and 0.967 & 0.7337 for MLP-ANN and ANFIS, respectively, highlighting their remarkable predictive performance. In conclusion, the ANFIS model is proposed as an effective tool for estimating refrigerant system densities, particularly advantageous in scenarios where experimental measurements are resource-intensive or sophisticated analysis is required.

Targeted deep sequencing reveals APC mutations as predictors of overall survival in Chinese colorectal patients receiving adjuvant chemotherapy.

Objective: Targeted deep sequencing was used to characterize the mutational spectrum of APC in Chinese colorectal tumors in comparison to that in Caucasians from The Cancer Genome Atlas (TCGA) and to investigate whether APC mutations can predict overall survival in CRC patients receiving adjuvant chemotherapy.Methods: A total of 315 Chinese CRC patients including 241 stage II/III patients receiving fluorouracil-based adjuvant chemotherapy were included in this study. Next generation sequencing was carried out to detect somatic mutations on all APC exons. The associations between APC mutations and overall survival were determined by the Cox proportional hazards model.Results:APC was mutated in 221 of 315 colorectal tumors (70.2%). Chinese CRC had a much higher frequency of missense mutations (16.2% vs. 2.4%), but a lower frequency of nonsense (41.0% vs. 54.2%) and frameshift mutations (10.5% vs. 18.4%) than Caucasian CRC. Among stage II/III patients receiving fluorouracil-based adjuvant chemotherapy, APC mutations showed a significant association with worse survival (HR = 1.69; 95% CI, 1.10-2.62; p = .0179). Of the mutation types, frameshift mutations conferred the highest risk of death (HR = 2.88; 95% CI, 1.54-5.37; p =.0009). Among individual mutation sites, Arg232Ter, the most frequent mutation in Chinese CRC, exhibited the strongest negative impact on survival (HR = 2.65; 95% CI, 1.16-6.03; p =.0202).Conclusion:APC overall mutation was an independent predictor for overall survival of stage II/III CRC patients receiving fluorouracil-based chemotherapy.

Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1-mediated MAPK signaling.

BACKGROUND: Colorectal cancer (CRC) is one of the most malignant tumors with high incidence, yet its molecular mechanism is not fully understood, hindering the development of targeted therapy. Metabolic abnormalities are a hallmark of cancer. Targeting dysregulated metabolic features has become an important direction for modern anticancer therapy. In this study, we aimed to identify a new metabolic enzyme that promotes proliferation of CRC and to examine the related molecular mechanisms. METHODS: We performed RNA sequencing and tissue microarray analyses of human CRC samples to identify new genes involved in CRC. Squalene epoxidase (SQLE) was identified to be highly upregulated in CRC patients. The regulatory function of SQLE in CRC progression and the therapeutic effect of SQLE inhibitors were determined by measuring CRC cell viability, colony and organoid formation, intracellular cholesterol concentration and xenograft tumor growth. The molecular mechanism of SQLE function was explored by combining transcriptome and untargeted metabolomics analysis. Western blotting and real-time PCR were used to assess MAPK signaling activation by SQLE. RESULTS: SQLE-related control of cholesterol biosynthesis was highly upregulated in CRC patients and associated with poor prognosis. SQLE promoted CRC growth in vitro and in vivo. Inhibition of SQLE reduced the levels of calcitriol (active form of vitamin D3) and CYP24A1, followed by an increase in intracellular Ca2+ concentration. Subsequently, MAPK signaling was suppressed, resulting in the inhibition of CRC cell growth. Consistently, terbinafine, an SQLE inhibitor, suppressed CRC cell proliferation and organoid and xenograft tumor growth. CONCLUSIONS: Our findings demonstrate that SQLE promotes CRC through the accumulation of calcitriol and stimulation of CYP24A1-mediated MAPK signaling, highlighting SQLE as a potential therapeutic target for CRC treatment.

CUL5-ASB6 Complex Promotes p62/SQSTM1 Ubiquitination and Degradation to Regulate Cell Proliferation and Autophagy.

p62/SQSTM1 (sequestosome-1) is a key protein involved in multiple cellular bioprocesses including autophagy, nutrient sensing, cell growth, cell death, and survival. Therefore, it is implicated in human diseases such as obesity and cancer. Here, we show that the CUL5-ASB6 complex is a ubiquitin E3 ligase complex mediating p62 ubiquitination and degradation. Depletion of CUL5 or ASB6 induced p62 accumulation, and overexpression of ASB6 promoted ubiquitination and degradation of p62. Functionally, ASB6 overexpression can inhibit the proliferation of MEF and hepatocellular carcinoma cells by reducing p62 protein level, and impair the occurrence of autophagy. Overall, our study identified a new molecular mechanism regulating p62 stability, which may provide additional insights for understanding the delicate control of p62 and cell proliferation-autophagy control in physiological and pathological settings.

Anti-aging effects exerted by Tetramethylpyrazine enhances self-renewal and neuronal differentiation of rat bMSCs by suppressing NF-kB signaling.

In order to improve the therapeutic effects of mesenchymal stem cell (MSC)-based therapies for a number of intractable neurological disorders, a more favorable strategy to regulate the outcome of bone marrow MSCs (bMSCs) was examined in the present study. In view of the wide range of neurotrophic and neuroprotective effects, Tetramethylpyrazine (TMP), a biologically active alkaloid isolated from the herbal medicine Ligusticum wallichii, was used. It was revealed that treatment with 30-50 mg/l TMP for 4 days significantly increased cell viability, alleviated senescence by suppressing NF-κB signaling, and promoted bMSC proliferation by regulating the cell cycle. In addition, 40-50 mg/l TMP treatment may facilitate the neuronal differentiation of bMSCs, verified in the present study by presentation of neuronal morphology and expression of neuronal markers: microtubule-associated protein 2 (MAP-2) and neuron-specific enolase (NSE). The quantitative real-time polymerase chain reaction (qRT-PCR) revealed that TMP treatment may promote the expression of neurogenin 1 (Ngn1), neuronal differentiation 1 (NeuroD) and mammalian achaete-scute homolog 1 (Mash1). In conclusion, 4 days of 40-50 mg/l TMP treatment may significantly delay bMSC senescence by suppressing NF-κB signaling, and enhancing the self-renewal ability of bMSCs, and their potential for neuronal differentiation.

Prognostic significance of lymphovascular invasion in colorectal cancer and its association with genomic alterations.

BACKGROUND: Lymphovascular invasion (LVI) is suggested to be an early and important step in tumor progression toward metastasis, but its prognostic value and genetic mechanisms in colorectal cancer (CRC) have not been well investigated. AIM: To investigate the prognostic value of LVI in CRC and identify the associated genomic alterations. METHODS: We performed a retrospective analysis of 1219 CRC patients and evaluated the prognostic value of LVI for overall survival by the Kaplan-Meier method and multivariate Cox regression analysis. We also performed an array-based comparative genomic hybridization analysis of 47 fresh CRC samples to examine the genomic alterations associated with LVI. A decision tree model was applied to identify special DNA copy number alterations (DCNAs) for differentiating between CRCs with and without LVI. Functional enrichment and protein-protein interaction network analyses were conducted to explore the potential molecular mechanisms of LVI. RESULTS: LVI was detected in 150 (12.3%) of 1219 CRCs, and the presence was positively associated with higher histological grade and advanced tumor stage (both P < 0.001). Compared with the non-LVI group, the LVI group showed a 1.77-fold (95% confidence interval: 1.40-2.25, P < 0.001) increased risk of death and a significantly lower 5-year overall survival rate (P < 0.001). Based on the comparative genomic hybridization data, 184 DCNAs (105 gains and 79 losses) were identified to be significantly related to LVI (P < 0.05), and the majority were located at 22q, 17q, 10q, and 6q. We further constructed a decision tree classifier including seven special DCNAs, which could distinguish CRCs with LVI from those without it at an accuracy of 95.7%. Functional enrichment and protein-protein interaction network analyses revealed that the genomic alterations related to LVI were correlated with inflammation, epithelial-mesenchymal transition, angiogenesis, and matrix remodeling. CONCLUSION: LVI is an independent predictor for survival in CRC, and its development may correlate with inflammation, epithelial-mesenchymal transition, angiogenesis, and matrix remodeling.

Nomogram for Predicting Risk of Intestinal Complications After Colorectal Cancer Surgery.

BACKGROUND Intestinal complications are a major cause of morbidity after colorectal cancer surgery. This study aimed to develop an effective nomogram for predicting risk of intestinal complications following colorectal cancer surgery. MATERIAL AND METHODS We retrospectively analyzed 1876 patients who underwent colorectal cancer surgery at Yangpu and Zhuji hospitals from January 2013 to October 2018. Intestinal complications were defined as intestinal obstruction, leakage or bleeding, or peritonitis within 30 days after surgery. A logistic regression model was used to identify the risk factors associated with postoperative intestinal complications, and a nomogram for intestinal complications was established. The predictive accuracy of the nomogram was assessed using area under the receiver operating characteristic curve (AUC) and calibration plot. RESULTS A total of 164 patients (8.7%) developed intestinal complications after colorectal cancer surgery; 35 (21.3%) of whom died in the postoperative period. Multivariate logistic analysis showed that male gender, history of abdominal surgery, preoperative intestinal obstruction/perforation, metastatic cancer, and lower level of hemoglobin and prognostic nutrition index were independent risk factors (P<0.05 for all). A nomogram was then constructed, and it displayed good accuracy in predicting postoperative intestinal complications with an AUC of 0.76. The calibration plot also showed an excellent agreement between the predicted and observed probabilities. CONCLUSIONS We constructed a nomogram based on clinical variables, which could provide individual prediction of postoperative intestinal complications with good accuracy.

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis, biological activity, and molecular docking studies.

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC50 values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC50 values of 2.0 and 0.9 µM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100 µg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.

Correction: Development and validation of nomograms for predicting survival in patients with non-metastatic colorectal cancer.

[This corrects the article DOI: 10.18632/oncotarget.16167.].

PIK3CA and TP53 mutations predict overall survival of stage II/III colorectal cancer patients.

AIM: To investigate the predictive value of PIK3CA and TP53 mutation status in colorectal cancer (CRC) patients treated with 5-fluorouracil-based chemotherapy. METHODS: In this study, a total of 315 patients with histologically proven CRC were enrolled from Yangpu Hospital affiliated to Shanghai Tongji University between 2007 and 2011. Of these patients, 241 with stage II/III CRC received 5-fluorouracil-based adjuvant chemotherapy. Formalin-fixed paraffin-embedded lesion samples of the patients with curatively resected CRC were collected. Next-generation sequencing was performed to identify somatic gene mutations. The correlation of PIK3CA and TP53 mutation status with overall survival (OS) was analyzed using a Cox proportional hazard model and the Kaplan-Meier method. RESULTS: Among the 241 patients with stage II/III in this cohort, the PIK3CA and/or TP53 mutation was detected in 177 patients, among which 54 patients had PIK3CA and TP53 double mutations. The PIK3CA or TP53 mutation was not significantly correlated with OS in univariate and multivariate analyses. Compared with patients without PIK3CA and TP53 mutations, those with double PIK3CA-TP53 mutations showed a significantly worse survival (univariate HR = 2.21; 95%CI: 1.15-4.24; multivariate HR = 2.02; 95%CI: 1.04-3.91). The PIK3CA mutation located in the kinase domain showed a trend toward a shorter OS compared with wild-type tumors (multivariate HR = 1.56; 95%CI: 1.00-2.44; P = 0.052). The Kaplan-Meier curve showed that patients harboring the PIK3CA mutation located in the kinase domain had a worse clinical outcome than those with wild-type status (Log-rank P = 0.041). CONCLUSION: Double mutation of PIK3CA and TP53 is correlated with a shorter OS in stage II/III CRC patients treated with 5-fluorouracil-based therapy.

Prevalence and Risk Factors of Chronic Constipation Among Women Aged 50 Years and Older in Shanghai, China.

BACKGROUND Chronic constipation (CC) is a major public health problem worldwide, especially in elderly women. This study aimed to investigate the prevalence and risk factors of CC among women aged 50 years and older in Shanghai, China. MATERIAL AND METHODS A cross-sectional survey was conducted on 1950 women aged 50 years and older, randomly sampled in Yangpu District of Shanghai from April to October 2015. Information on demographic characteristics, lifestyle habits, medical history, and defecation situation was collected through in-person interviews. CC was defined according to Rome III criteria. The data were analyzed by chi-square test and multiple logistic regression analysis. RESULTS The response rate to the survey was 80.4%. Of the 1568 participants, 77 were diagnosed with CC, with a prevalence of 4.9%. Moreover, the prevalence increased with advancing age. Multiple logistic analyses showed that body mass index (BMI) ≥25.0 kg/m², non-manual occupation, premenopausal period, no delivery history, poor sleep quality, meat-based diet, and less physical exercise were significant risk factors for CC in the population of women aged 50 years and older. CONCLUSIONS CC was a common health problem among women aged 50 years and older in Shanghai, and the prevalence was positively associated with BMI ≥25.0 kg/m², non-manual occupation, premenopausal period, no delivery history, poor sleep quality, meat-based diet, and less physical exercise. Further studies are needed to identify the risk factors and potential interventions for CC.

Development and validation of nomograms for predicting survival in patients with non-metastatic colorectal cancer.

BACKGROUND: This study aimed to develop nomograms for predicting survival in patients with non-metastatic colorectal cancer (CRC). RESULTS: On multivariate analyses of the derivation set, the nomograms for OS and CSS shared common significant prognostic factors: age, first-degree relative cancer history, differentiation grade, vessels/nerves invasion, TNM stage, CEA, CA19-9 and PNI. The nomograms displayed good accuracy in predicting OS and CSS, with C-indexes of 0.75 and 0.76, respectively. The calibration plots also showed an excellent agreement between the predicted and observed survival probabilities. Furthermore, the predictive accuracy of the nomograms was confirmed in the validation set, with C-indexes of 0.79 and 0.83 for OS and CSS, respectively. MATERIALS AND METHODS: On the basis of data from 822 patients with resected non-metastatic CRC, nomograms for predicting overall survival (OS) and cancer-specific survival (CSS) were established using Cox regression model. The predictive performance of the nomograms was assessed by concordance index (C-index) and calibration plot. An independent external cohort of 171 patients was used to validate the nomograms. CONCLUSIONS: We developed and validated two nomograms for patients with non-metastatic CRC, which could provide individual prediction of OS and CSS with high accuracy.

Prognostic Value of the Combination of Preoperative Hemoglobin, Lymphocyte, Albumin, and Neutrophil in Patients with Locally Advanced Colorectal Cancer.

BACKGROUND Systemic inflammatory response and nutritional status are important to the prognosis of patients with colorectal cancer (CRC). This study aimed to investigate the prognostic value of the combination of preoperative hemoglobin, lymphocyte, albumin, and neutrophil (HLAN) in patients with locally advanced CRC (LACRC). MATERIAL AND METHODS We performed a retrospective analysis in 536 LACRC patients undergoing radical surgery. The value of HLAN was defined as follow: HLAN=Hemoglobin (g/L)×Lymphocyte (/L)×Albumin (g/L)/Neutrophil (/L)/100. The X-tile program was used to determine the optimal cut-point of HLAN, and the prognostic value of HLAN for overall survival (OS) was evaluated with the Cox proportional hazard model. RESULTS The cut-point of HLAN was set at 19.5. Compared with the high-HLAN group, the low-HLAN group had a 1.50-fold (95% confidence interval 1.09-2.05) increased risk of death and a significantly lower OS rate (P<0.001). Furthermore, the risk stratification model based on HLAN (AUC=0.72) displayed better accuracy in OS prediction than the TNM system (AUC=0.61). CONCLUSIONS HLAN is a valuable prognostic marker for patients with LACRC.

Preoperative combined hemoglobin, albumin, lymphocyte and platelet levels predict survival in patients with locally advanced colorectal cancer.

More than 50% of patients with colorectal cancer (CRC) are initially diagnosed with locally advanced CRC (LACRC), and half of those patients develop recurrence or metastasis after resection. Here, we investigated whether the novel index HALP, which is a combination of preoperative hemoglobin, albumin, lymphocyte and platelet levels, correlates with survival in LACRC patients. A total of 820 patients with LACRC from two independent hospitals were included in our study. The correlations between HALP and overall and cancer-specific survival were calculated using training and validation sets. Lower HALP values correlated with an increased risk of death and cancer-related death in both sets. Moreover, the risk score based on HALP allowed stratification of patients into distinct prognostic groups with greater accuracy than previously proposed indexes. These results suggest that HALP may be useful as a clinical prognostic factor for patients with LACRC.

[Association between RIPK4 relative copy number and prognosis of colorectal cancer patient after oxaliplatin-based chemotherapy].

OBJECTIVE: To investigate the association between receptor-interacting kinase protein 4 (RIPK4) relative copy number (RCN) and prognosis of stage III( colorectal cancer (CRC) patients treated with oxaliplatin-based chemotherapy. METHODS: RIPK4 RCN was determined by real-time PCR and then dichotomized into high RIPK4 RCN group(n=35) and low RIPK4 RCN group (n=104) using the third quartile as the cut-off point. Overall survival (OS) and recurrence-free survival (RFS) were compared between high and low RIPK4 RCN groups. The subgroup prognostic analysis was also conducted based on tumor site. RESULTS: The median follow-up period was 49 months (ranged 4 to 98 months). Patients with high RIPK4 RCN had poorer OS than those with low RIPK4 RCN, which reached marginal significance(median OS, 43.0 months vs. 53.5 months, P=0.074). Meanwhile there was no significant difference of RFS between two groups (P=0.352). In colon cancer subgroup, high RIPK4 RCN was significantly associated with poor OS (median OS, 31.5 months vs. 56.6 months, P=0.015) but not with RFS (P=0.135). In rectal cancer subgroup, RIPK4 RCN was not associated with both OS and RFS (P=0.981, P=0.738). Multivariate analysis revealed that high RIPK4 RCN was an independent prognostic factor of OS in stage III( CRC patients treated with oxaliplatin-based chemotherapy (HR=2.903, 95% CI: 1.275 to 6.610). CONCLUSION: RIPK4 RCN is significantly associated with OS in stage III( colon cancer patients receiving oxaliplatin-based chemotherapy and may be a novel biomarker that can predict the efficacy of oxaliplatin in colon cancer patients.

PPP1R12A Copy Number Is Associated with Clinical Outcomes of Stage III CRC Receiving Oxaliplatin-Based Chemotherapy.

AIM: To investigate the correlation between PPP1R12A gene copy number and clinical outcomes of oxaliplatin-based regimen in stage III colorectal cancer (CRC). METHODS: A total of 139 paraffin-embedded tissue samples of stage III CRC patients who received oxaliplatin-based treatment after radical surgery were recruited. Genomic DNA was extracted and purified from paraffin-embedded sections. Quantitative PCR methods were used to detect the relative copy number (RCN) of PPP1R12A. RESULTS: Statistical analysis demonstrated that low PPP1R12A RCN was associated with poor RFS (HR = 2.186, 95% CI: 1.293-3.696; P = 0.003) and OS (HR = 2.782, 95% CI: 1.531-5.052; P < 0.001). Additionally, when patients were stratified according to subgroups of stage III and tumor location, poor RFS and OS were also observed in the low PPP1R12A RCN group with significance (RFS: IIIB HR = 2.870, P < 0.001; colon HR = 1.910, P = 0.037; OS: IIIB HR = 3.527, P < 0.001; IIIC HR = 2.662, P = 0.049; rectum HR = 4.229, P = 0.002). CONCLUSION: Our findings suggest the copy number of PPP1R12A can independently predict recurrence and overall survival of stage III colorectal cancer patients receiving oxaliplatin-based chemotherapy.

High-throughput RNAi screening of human kinases identifies predictors of clinical outcome in colorectal cancer patients treated with oxaliplatin.

The purpose of this study is to identify protein kinase genes that modulate oxaliplatin cytotoxicity in vitro and evaluate the roles of these genes in predicting clinical outcomes in CRC patients receiving oxaliplatin-based adjuvant chemotherapy. A high-throughput RNAi screening targeting 626 human kinase genes was performed to identify kinase genes whose inhibition potentiates oxaliplatin sensitivity in CRC cells. The associations between copy numbers of the candidate genes and recurrence-free survival and overall survival were analyzed in 142 stage III CRC patients receiving first-line oxaliplatin-based adjuvant chemotherapy who were enrolled from two independent hospitals. HT-RNAi screening identified 40 kinase genes whose inhibition potentiated oxaliplatin cytotoxicity in DLD1 cells. The relative copy number (RCN) of MAP4K1 and CDKL4 were associated with increased risks of both recurrence and death. Moreover, significant genes-based risk score and the ratios of RCN of different genes can further categorize patients into subgroups with distinctly differing outcomes. The estimated AUC for the prediction models including clinical variables plus kinase biomarkers was 0.77 for the recurrence and 0.82 for the survival models. The copy numbers of MAP4K1 and CDKL4 can predict clinical outcomes in CRC patients treated with oxaliplatin-based chemotherapy.

PIK3C2G copy number is associated with clinical outcomes of colorectal cancer patients treated with oxaliplatin.

PURPOSE: To investigate whether the copy number of PIK3C2G is associated with clinical outcomes for stage III colorectal cancer (CRC) patients treated with oxaliplatin-based chemotherapy. METHODS: A total of 142 CRC patients who received first-line oxaliplatin-based chemotherapy after curative surgery in Ruijin Hospital and The Tenth People's Hospital were recruited in this study. Patients were enrolled between June 2006 and December 2011, with follow-up to January 2014. Quantitative real-time PCR method was used to detect the copy number of PIK3C2G. Cox proportional hazards model and Kaplan-Meier curves were used to analyze the association between PIK3C2G copy number and clinical outcome. RESULTS: In patients with stage III disease, low copy number of PIK3C2G was associated with increased risk of both recurrence (HR, 2.44, 95% CI, 1.33-4.47, P=0.004) and death (HR, 2.89, 95% CI, 1.49-5.60, P=0.002). Multivariate analysis also indicated that low PIK3C2G copy number was a significant and independent predictor of OS and RFS of stage III CRC. CONCLUSIONS: PIK3C2G is capable of predicting the recurrence and overall survival of stage III CRC patients receiving oxaliplatin-based therapy.

Targeting lung cancer stem-like cells with TRAIL gene armed oncolytic adenovirus.

Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors-defined serum-free medium. A549 sphere cells displayed CSC properties, including chemo-resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55-EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55-TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55-TRAIL. In the A549 sphere cells xenograft models, ZD55-TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs.

GFP stable transfection facilitated the characterization of lung cancer stem cells.

Cancer stem cells (CSCs) are a subset of cancer cells that play key roles in metastasis and cancer relapse. The elimination of CSCs is very important during cancer therapy. To develop drugs that target CSCs, the isolation and identification of putative CSCs are required. Some of the characteristics of CSCs are assessed by cell survival assays. In such experiments, the density of the cells seeded on the plates may affect the experimental results, leading to potentially inaccurate conclusions. In this study, a new assay to facilitate the characterization of CSCs has been developed by stable transfection of GFP, using the A549 lung cancer cell line as a model. A putative CSC line, A549 sphere cells, was obtained by culturing A549 cells in ultra-low dishes in serum-free medium. To ensure that the putative CSCs were grown under the same conditions as the A549-GFP cells and were not affected by the number of cells seeded, A549 sphere cells were mixed with GFP stably transfected A549 (A549-GFP) cells. The mixture was subjected to flow cytometry assay and inverted fluorescence microscopy to detect changes in the proportion of GFP-positive cells after treatment. A549 sphere cells had a slower proliferation rate and an improved chemoresistance. They also showed differentiation ability. This work suggests that mixing GFP stably transfected cancer cells with putative CSCs may facilitate the identification of CSCs, making it convenient for studies of targeted CSCs.