The utility of sputum supernatant as an alternative liquid biopsy specimen for next-generation sequencing-based somatic variation profiling.

Background: Comprehensive genomic profiling has become standard clinical practice in the management of advanced lung cancer. In addition to tissue and plasma, other body fluids are also being actively explored as alternative sources of tumor DNA. This study investigated the utility of induced sputum obtained from patients with non-small-cell lung cancer (NSCLC) for somatic variation profiling. Methods: Our study included 41 treatment-naïve patients diagnosed with locally advanced to advanced NSCLC between October 2018 and June 2019. Capture-based targeted sequencing was performed on matched tumor, plasma, and induced sputum samples of 41 patients using a 168-gene panel. We analyzed the somatic variations detected from each sample type and the concordance of variations detected between matched samples. The concordance rate was defined as the proportion of the total number of variations detected from one sample type relative to the reference sample type. Results: Comparative analysis on the somatic variation detection using matched tumor samples as a reference revealed detection rates of 76.9% for plasma, 72.4% for sputum-supernatant, and 65.7% for sputum-sediment samples. Plasma, sputum-supernatant, and sputum-sediment achieved positive predictive values of 73.3%, 80.4%, and 55.6% and sensitivities of 50.0%, 36.9%, 31.3%, respectively, relative to tumor samples for 168 genes. Sputum-supernatants had significantly higher concordance rates relative to matched tumor samples (69.2% vs. 37.8%; P=0.031) and maximum allelic fraction (P<0.001) than their matched sputum-sediments. Sputum-supernatants had comparable detection rates (71.4% vs. 67.9%; P=1.00) but with significantly higher maximum allelic fraction than their matched plasma samples (P=0.003). Furthermore, sputum-supernatant from smokers had a significantly higher maximum allelic fraction than sputum-supernatant from non-smokers (P=0.021). Conclusions: Our study demonstrated that supernatant fraction from induced sputum is a better sampling source than its sediment and performs comparably to plasma samples. Induced sputum from NSCLC patients could serve as an alternative media for next-generation sequencing (NGS)-based somatic variation profiling.

Anlotinib plus platinum-etoposide as a first-line treatment for extensive-stage small cell lung cancer: A single-arm trial.

BACKGROUND: Anlotinib as a third-line or beyond therapy for extensive-stage small-cell lung cancer (ES-SCLC) was studied. This single-arm phase II trial was to investigate the value of anlotinib plus platinum-etoposide as first-line treatment in ES SCLC. METHODS: The primary endpoint was progression-free survival (PFS) and objective response rate (ORR). The secondary endpoints included overall survival (OS), disease control rate (DCR), time to progression (TTP), duration of remission (DoR), and safety. The subgroups of preset liver metastasis and brain metastasis were analyzed. RESULTS: In 35 ES-SCLC patients, the median PFS, ORR, DCR, and OS were 8.02 months [95% confidence interval (CI): 6.90-9.66], 85.71% (95% CI: 69.74-95.19), 94.29% (95% CI: 80.84-99.30), and 15.87 months (95% CI: 10.38-18.89), respectively. The median PFS in the liver metastasis and brain metastasis subgroups was 7.33 months (95% CI: 4.76-9.69) and 7.34 months (95% CI: 5.68-9.20), respectively. The most common AEs with grade 3-4 were hand-foot syndrome (17%), granulocytosis (17%), stomatitis (14%), hypertriglyceridemia (11%), hypercholesterolemia (11%), as well as nausea and vomiting (11%), and no grade 5 AEs were recorded. CONCLUSIONS: Anlotinib combined with platinum-etoposide provided an effective and safe therapy for patients with ES-SCLC.

Effect of EGCG on bronchial epithelial cell premalignant lesions induced by cigarette smoke and on its CYP1A1 expression.

Epigallocatechin­3­gallate (EGCG) has been demonstrated to exhibit anticancer effects; however, the mechanisms behind these are not yet clear. The objective of the present study was to assess the effect of EGCG on smoking­induced, precancerous, bronchial epithelial cell lesions and determine a potential protective mechanism. Human bronchial epithelial (HBE) cells were treated with cigarette smoke extract (CSE). Benzopyrene­DNA adducts were detected by immunofluorescence cytochemistry. Changes to microRNA (miRNA) expression levels were detected via microarray. The effects of EGCG on smoke­induced benzopyrene­DNA adduct formation and the subsequent change in miRNA expression were analyzed. Subsequently, the protective effect of EGCG on smoke inhalation­induced precancerous lesions was investigated. The expression levels of miRNA target genes were also analyzed. After CSE treatment, benzopyrene­DNA adducts appeared in HBE cells, along with a resultant change in miRNA expression. EGCG inhibited the effects of CSE exposure; benzopyrene­DNA adduct formation was reduced and miRNA expression changes were suppressed. In vivo, EGCG significantly reduced benzopyrene­DNA adduct formation and the subsequent development of precancerous lesions in rat lungs induced by cigarette smoke inhalation. Moreover, EGCG downregulated CYP1A1 overexpression, a target gene of multiple smoking­induced miRNAs, in rat lungs. EGCG may reduce the risk of lung cancer by downregulating the expression of the key gene CYP1A1, preventing the formation of smoking­induced benzopyrene­DNA adducts and alleviating smoking­induced bronchial epithelial dysplasia and heterogeneity.

The Predictive Values of Advanced Non-Small Cell Lung Cancer Patients Harboring Uncommon EGFR Mutations-The Mutation Patterns, Use of Different Generations of EGFR-TKIs, and Concurrent Genetic Alterations.

INTRODUCTION: Epidermal growth factor receptor (EGFR) 19del and L858R mutation are known as "common mutations" in non-small cell lung cancer (NSCLC) and predict sensitivities to EGFR tyrosine kinase inhibitors (TKIs), whereas 20ins and T790M mutations confer drug-resistance to EGFR-TKIs. The role of the remaining uncommon EGFR mutations remains elusive. METHODS: We retrospectively screened a group of NSCLC patients with uncommon EGFR mutations other than 20ins and T790M. The mutation patterns, use of different generations of EGFR-TKIs, and concurrent genetic alterations were analyzed. Meanwhile, a cohort of patients with single 19del or L858R were included for comparison. RESULTS: A total of 180/1,300 (13.8%) patients were identified. There were 102 patients with advanced or recurrent NSCLC that received first-line therapy of gefitinib/erlotinib/icotinib and afatinib and were eligible for analysis. The therapeutic outcomes among patients with common mutations (EGFRcm, n = 97), uncommon mutation plus common mutations (EGFRum+EGFRcm, n = 52), complex uncommon mutations (complex EGFRum, n = 22), and single uncommon mutations (single EGFRum, n = 28) were significantly different (ORRs: 76.3%, 61.5%, 54.5%, and 50.0%, respectively, p = 0.023; and mPFS: 13.3, 14.7, 8.1, and 6.0 months, respectively, p = 0.004). Afatinib showed superior efficacy over gefitinib/erlotinib/icotinib in EGFRcm (ORR: 81.0% vs. 75.0%, p = 0.773; mPFS: 19.1 vs. 12.0m, p = 0.036), EGFRum+EGFRcm (ORR: 100% vs. 54.5%, p = 0.017; mPFS: NE vs. 13.6m, p = 0.032), and single EGFRum (ORR: 78.6% vs. 21.4%, p = 0.007; mPFS: 10.1 vs. 3.0m, p = 0.025) groups. Comprehensive genomic profiling by Next Generation Sequencing encompassing multiple cancer-related genes was performed on 51/102 patients; the mPFS of patients without co-mutation (n = 16) and with co-mutations of tumor-suppressor genes (n = 31) and driver oncogenes (n = 4) were 31.1, 9.2, and 12.4 months, respectively (p = 0.046). TP53 mutation was the most common co-alteration and showed significantly shorter mPFS than TP53 wild-type patients (7.0 vs. 31.1m, p < 0.001). Multivariate analysis revealed that concurrent 19del/L858R and tumor-suppressor gene alterations independently predicted better and worse prognosis in patients with uncommon mutations, respectively. CONCLUSIONS: Uncommon EGFR mutations constitute a highly heterogeneous subgroup of NSCLC that confer different sensitivities to EGFR-TKIs with regard to the mutation patterns. Afatinib may be a better choice for most uncommon EGFR mutations. Concurrent 19del/L858R and tumor-suppressor gene alterations, especially TP53, can be established as prognostic biomarkers.

The Intrapleural Bridge Connection is One of the Reasons for Unknown Localized Pleural Adhesion.

BACKGROUND: Simple signs of local pleural adhesion are often found in people during a physical examination. In the present study, we aimed to clarify whether the merely localized pleural adhesion was just caused by previous pleural inflammation or physiological variation. MATERIALS AND METHODS: Chest X-ray image materials were collected to analyze the incidence of simple pleural adhesions. Moreover, the causes of these simple pleural adhesions were further analyzed using thoracoscopy under direct vision and biopsy data. RESULTS: In all 2218 chest X-ray images, 68 cases were found to have pleural lesions (3.07%), including 15 cases of localized pleural adhesion only. Subsequently, we analyzed the characteristics of 70 cases of pleural lesions using thoracoscopy. In two lung cancer patients with pleural metastasis, we found an unusual pleural junction. This connective strip was smooth and free of inflammation, resembling the normal pleura. CONCLUSION: Some of these purely localized pleural adhesions might be attributed to previous inflammation. However, there was still at least a possibility that there must be a physiological pleural junction, which could be the cause of the purely localized pleural adhesion shown in the chest radiograph.

Serum IL-6 as a vital predictor of severe lung cancer.

BACKGROUND: Recent clinical studies have reported that some cytokines are associated with lung cancer prognosis and mortality. However, the relationship between cytokines and clinical outcomes in severe lung cancer patients was unclear. IL-6 as an important cytokine in inflammation, expression level in severe lung cancer patients was unknown. METHODS: A cohort of 55 severe lung cancer patients were enrolled retrospectively in this study. The clinical characteristics, including performance status (PS), therapeutic effect, and patients' adverse effects, were recorded. The association of cytokines and the concerned clinical outcomes were assessed by logistic regression analysis. The area under the curve (AUC) was assessed to evaluate the strength of prediction. RESULTS: The mean age of the patients was 59.8, and 42 patents were males. Increased IL-6 levels were associated with worse PS. Logistic regression analysis demonstrated that higher IL-6 was associated with an increased risk of progressive disease (PD) (OR =1.03, 95% CI: 1.0-1.06). The area under the ROC curve (AUC) of the model used for predicting PD was 0.821. CONCLUSIONS: Increased IL-6 levels are correlated with worse PS and are an essential predictor for PD in severe lung cancer patients. Monitoring the IL-6 level may represent an essential strategy in improving the prognosis of patients with severe lung cancer.

Bronchial Fistula: Rare Complication of Treatment with Anlotinib.

BACKGROUND: Anlotinib is a newly developed small molecule multiple receptor tyrosine kinase (RTK) inhibitor that was approved for the treatment of patients with lung cancer in China. We aim to report 3 cases of rare complication of anlotinib-bronchial fistula (BF) during the treatment of lung cancer patients and summarize the possible causes. METHODS: We collected three patients who developed BF due to anlotinib treatment, and conducted a search of Medline and PubMed for medical literature published between 2018 and 2020 using the following search terms: "anlotinib," "lung cancer," and "fistula." RESULTS: Our literature search produced two case reports (three patients) which, in addition to our three patients. We collated the patients' clinical characteristics including demographic information, cancer type, imaging features, treatment received, risk factors for anlotinib related BF, and treatment-related outcomes. The six patients shared some common characteristics: advanced age, male, concurrent infection symptoms, diabetes mellitus (DM), advanced squamous cell and small cell lung cancers, centrally located tumors, tumor measuring ≥5 cm in longest diameter, and newly formed tumor cavitation after multi-line treatment especially after receiving radiotherapy. Fistula types included broncho-pericardial fistula, broncho-pleural fistula, and esophago-tracheobronchial fistula. Six patients all died within 6 months. CONCLUSIONS: Although anlotinib is relatively safe, it is still necessary to pay attention to the occurrence of BF, a rare treatment side effect that threatens the quality of life and overall survival of patients. Anlotinib, therefore, requires selective use and close observation of high-risk patients.

Poor prognosis of pulmonary sarcomatoid carcinoma with KRAS mutation and ALK fusion.

Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of poorly differentiated non-small-cell lung cancer (NSCLC), and no effective treatment is available in clinical practice currently. In the present report, a 61-year-old male patient was hospitalized due to cough, dyspnea, and right chest pain. Computed tomography (CT) showed spot- and piece-shaped shadows. The patient became very weak and had breathing difficulty after preliminary anti-pneumonia treatment with cefoperazone-sulbactam. Physical examination revealed dull sound by percussion and decreased breath sounds in the right lateral lung areas by auscultation. A second CT scan revealed a large amount of pleural effusion, and the patient was diagnosed with bloody pleural effusion through pleural space puncture. Multiple nodular lesions were found in the right pleural cavity under thoracoscopy. PSC was confirmed by biopsy and histopathology in combination with immunohistochemistry (IHC). Single-photon emission CT (SPECT) scan indicated multiple bone metastases. KRAS exon 2 mutation and EML4-ALK fusion were identified in carcinoma tissue by IHC and amplification refractory mutation system (ARMS)-PCR. The patient received one cycle of first-line combination chemotherapy of cisplatin and paclitaxel liposomes. However, the patient did not respond to the platinum-based combination chemotherapy within 3 weeks and was thus administered oral crizotinib instead of chemotherapy. Unfortunately, he still had rapid disease progression and died 2 weeks after the initiation of crizotinib treatment. Collectively, our results suggest that a PSC patient with coexistent KRAS mutation and ALK rearrangement would not benefit from chemotherapy and tyrosine kinase inhibitor (TKI) treatment.

A comparison between lung carcinoma and a subcutaneous malignant tumor induced in rats by a 3,4-benzopyrene injection.

Lung cancer is one of the most common carcinomas worldwide. It is of value to know whether lung is more vulnerable to carcinogens than other tissues. In this study we compared the carcinogenic potential of 3,4-benzopyrene administered by intrapulmonary injection or subcutaneous injection. Ninety rats were randomly divided into three groups (n=30/group). Rats under deep anesthesia were treated with 3,4-benzopyrene by intrapulmonary injection or scapular subcutaneous injection, or with the vehicle by subcutaneous injection. The Rats were sacrificed when they developed advanced somatic sarcomas or severe dyspnea and the rats without severe phenotypes were sacrificed after 1 year. The tumors were isolated and examined with H&E staining. The expression of Bcl-2, CYP1A1, and NF-κB mRNA and protein in somatic sarcoma and lung carcinoma tissues was examined by in situ hybridization, immunohistochemistry, and Western blot. No tumor development was observed in the control rats. Fifteen of the 30 rats receiving an intrapulmonary injection of 3,4-benzopyrene developed lung carcinomas, whereas all 30 rats treated with subcutaneous injection developed a malignant neoplasm under the skin. Positive Bcl-2, CYP1A1, and NF-κB protein staining was observed in lung carcinoma and subcutaneous malignant neoplasm but Bcl-2 protein expression was much stronger in subcutaneous malignant neoplasms than in lung carcinoma. The expression pattern of Bcl-2, CYP1A1, and NF-κB mRNA in lung carcinoma and subcutaneous malignant neoplasms was consistent with its protein expression. Our results indicated that the lung is not more vulnerable to carcinogens than other tissues. The lung may acquire a protective mechanism against lung carcinogenesis through regulation of Bcl-2 expression.

Tyrosine kinase inhibitors show different anti-brain metastases efficacy in NSCLC: A direct comparative analysis of icotinib, gefitinib, and erlotinib in a nude mouse model.

Brain metastasis is an increasing problem in non-small cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKIs), including gefitinib, erlotinib, and icotinib, are reported to be effective in patients with brain metastases. However, direct comparative studies of the pharmacokinetics and efficacy of these three drugs in treating brain metastases are lacking. In the present investigation, we found that gefitinib penetrated the blood-tumor barrier and was distributed to brain metastases more effectively than erlotinib or icotinib in a nude mouse model. The 1-h ratio of brain metastases to plasma concentration for gefitinib, erlotinib, and icotinib was 9.82±1.03%, 4.83±0.25%, and 2.62±0.21%, respectively. The 2-h ratio of brain metastases to plasma concentration for gefitinib, erlotinib, and icotinib was 15.11±2.00%, 5.73±1.31%, and 2.69±0.31%, respectively. Gefitinib exhibited the strongest antitumor activity (pgefitinib vs. erlotinib=0.005; pgefitinib vs. icotinib=0.002). Notably, erlotinib exhibited a better treatment efficacy than icotinib (p=0.037). Consistently, immunohistochemical data showed that TKIs differentially inhibit the proliferation of metastatical tumor cells. Gefitinib and erlotinib markedly inhibited the proliferation of tumor cells, while there were more ki-67-positive tumor cells in the icotinib group. Additionally, gefitinib inhibited the phosphorylation of EGFR better than the other drugs, whereas pEGFR expression levels in erlotinib groups were lower than levels in the icotinib group (pgefitinib vs. erlotinib=0.995; pgefitinib vs. icotinib=0.028; perlotinib vs. icotinib=0.042).Altogether, our findings suggest that gefitinib and erlotinib can inhibit the growth of PC-9-luc brain tumors. Gefitinib demonstrated better antitumor activity and penetration rate in brain metastases than erlotinib or icotinib.

HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche.

High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/ß-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to 'build' an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis.

Snail1-expressing cancer-associated fibroblasts induce lung cancer cell epithelial-mesenchymal transition through miR-33b.

Lung cancer has a high propensity for metastasis. Cancer-associated fibroblasts (CAFs) are the main type of stromal cells in cancer tissue, are activated by tumor cells, and play a significant role in tumor development. However, whether CAFs induce lung cancer cell metastasis, as well as pathway involved in CAF-induced lung cancer cell metastasis, is uncertain. Snail1 is a transcriptional factor whose expression in the stroma is associated with lower survival rates in patients with cancer. However, how Snail1 regulates the crosstalk between stromal cells and tumor cells when it is expressed in the stroma has not been determined. Altered microRNA (miRNA) expression is correlated with lung cancer metastasis. Our previous study of microRNAs showed that miR-33b levels were clearly reduced in lung cancer cell lines and lung cancer tissues, and miR-33b suppressed tumor cell epithelial-mesenchymal transition (EMT) when its expression was elevated. In this study, we found that co-culturing CAFs with lung cancer cells induced miR-33b downregulation and promoted epithelial cells EMT. Moreover, we found that miR-33b overexpression in lung cancer cells counteracted CAF-induced EMT. Interestingly, Snail1 expression in fibroblasts activate the inductive effects of CAFs on lung cancer cell EMT. Hence, understanding the molecular mechanism underlying the communication between stromal cells and tumor cells mediated by miR-33b may lead to the identification of novel targets for the treatment of lung cancer. Additionally, understanding the role of Snail1 driving CAFs to induce lung cancer cell EMT may provide with a new perspective on the treatment of lung cancer.

EGCG induces lung cancer A549 cell apoptosis by regulating Ku70 acetylation.

Lung cancer is the leading cause of cancer-related death worldwide. (-)-Epigallocatechin-3-gallate (EGCG) is a potential chemopreventive and therapeutic agent for lung cancer. Induction of apoptosis was examined using Annexin V/PI double staining flow cytometry. Western blot analysis detected the protein expression of cleaved caspase-3, Bax and Bcl-xL. Co-immunoprecipitation was used to detect the interaction of Ku70-Bax and the acetylation status of Ku70. Treatment of A549 cells with EGCG-induced apoptosis via increased expression of cleaved caspase-3 and Bax, but decreased expression of Bcl-xL. EGCG upregulated the K70 acetylation status of A549 cells and downregulated the interaction of Bax-Ku70 in a concentration- and time-dependent manner. The apoptosis-promoting effect of EGCG on A549 cells was obviously weakened, along with strengthening of the Bax-Ku70 interaction, after pCDNA3.1(+)-Ku70 plasmid and pCDNA3.1(+)-Ku70539/542R plasmid transfection. Our results established a role of EGCG in inducing cell apoptosis by suppressing Bax activity. Regulating Ku70 acetylation by EGCG, that block the interaction between Ku70 and Bax, will result in lung cancer cell apoptosis.

MicroRNA-33b inhibits lung adenocarcinoma cell growth, invasion, and epithelial-mesenchymal transition by suppressing Wnt/ß-catenin/ZEB1 signaling.

Altered expression of microRNA (miRNA) is associated with lung carcinogenesis and metastasis. Our previous study of lung cancer miRNAs using the gene chip assay demonstrated altered miR-33b expression in lung adenocarcinoma. The present study further investigated miR-33b expression, function, and gene regulation in lung cancer cells in vitro and in nude mouse xenografts. Our data showed that the level of miR-33b expression was dramatically decreased in lung adenocarcinoma cell lines and tissues and that the reduced miR-33b expression was associated with tumor lymph node metastasis. Furthermore, restoration of miR-33b expression inhibited lung adenocarcinoma cell proliferation, migration, and invasion and tumor cell epithelial-mesenchymal transition (EMT) in vitro. Luciferase assay revealed that miR-33b bound to ZEB1 3'-UTR region and inhibited ZEB1 expression, while expression of ZEB1 mRNA and miR-33b was inversely associated with lung adenocarcinoma cell lines and tissues. Subsequently, we found that miR-33b suppressed the activity of WNT/ß-catenin signaling in lung adenocarcinoma cells and in turn suppressed tumor cell growth and EMT in vitro and in vivo nude mouse xenografts. In conclusion, the present study provided novel insight into the molecular mechanism of lung adenocarcinoma progression. MicroRNA-33b should be further investigated as a potential therapeutic target in human lung adenocarcinoma.

[Clinical Characteristics and Prognostic Analysis of 38 Patients 
with Pulmonary Sarcomatoid Carcinoma].

BACKGROUND AND OBJECTIVE: Pulmonary sarcomatoid carcinoma is a rare histologic subtype of non-small cell lung cancer. The effective treatment for this disease has not well defined due to its extremely low morbidity. This study explores the clinicopathological characteristics and prognosis of 38 patients with PSC, so as to provide some clues for its diagnosis and treatment. METHODS: The study enrolled 38 patients with PSC that were diagnosed with histology or cytology in our hospital between January 2000 and December 2013. We retrospectively analyzed general clinical characteristics, smoking history, tumor size, TNM staging, pathology, immunohistochemistry, diagnostic method, treatment and prognosis. We used SPSS 19.0 statistical software and Kaplan-Meier method to analyze our data. RESULTS: Patients in this study were aged from 26 to 76 years old (the median age was 57.5 years old). Among all of them, the male to female ratio was 4:1, and 81.6% of patients had smoking history. Cough and hemoptysis were the most common primary symptoms. The median survival was 21 months, while one-year survival rate, three-year survival rate and five-year survival rate were 68.4%, 31.6% and 18.4% respectively. Tumor size, TNM staging, distant metastasis and surgery therapy were associated with the prognosis of patients. CONCLUSIONS: Patients with PSC present with no special symptoms generally. According to our study, factors that affect patients' prognosis include tumor size, TNM staging, distant metastasis and surgery. Complete resection is the key treatment for PSC patients, but comprehensive chemoradiotherapy needs further exploration in evidence-based medicine. Biological target therapy may give new insight into treatment for PSC.
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Theanine improves the function of dendritic cells via the downregulation of cyclooxygenase-2 expression.

BACKGROUND: Tumor cells can reduce the number of dendritic cells (DCs) in the tumor environment and cause DC dysfunction through autocrine or paracrine pathways. We sought to measure cyclooxygenase-2 (COX-2) expression in bombesin-inhibited DCs treated with theanine in vitro and to explore the protection and activation effects of theanine on DCs. METHODS: Enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were used to analyze the effects of theanine on COX-2 expression and interleukin (IL)-12/IL-10 secretion of bombesin-treated DCs. RESULTS: DCs acquired an impaired phenotype as a result of bombesin treatment. Theanine increased the expression of mature DC surface molecules. The number of cell apoptosis with the treatment of bombesin and theanine significantly decreased, accounting for 15.9%, compared with 26.1% of cell apoptosis with bombesin. COX-2 expression in bombesin-treated DCs was inhibited by theanine in a dose-dependent manner. Theanine promoted DC secretion of IL-12. IL-12 levels reached (137.4 ± 4.9) pg/ml with theanine at 200 µmol/L. However, theanine inhibited the secretion of IL-10 in a dose-dependent manner. IL-10 levels were only (58.4 ± 6.9) pg/ml with theanine at 200 µmol/L. CONCLUSION: Theanine inhibits the transcription and translation of COX-2 and regulates the balance of IL-10/IL-12 secretion in bombesin-inhibited DCs, leading to the recovery of a state of activation in DCs.

Biological characteristics and epidermal growth factor receptor tyrosine kinase inhibitors efficacy of EGFR mutation and its subtypes in lung adenocarcinoma.

Mutation of epidermal growth factor receptor (EGFR) gene has been reported to be present in lung adenocarcinoma (LAC). In this study, we extensively investigated the impact of patients' biological characteristics on EGFR mutation and the impact of EGFR mutation subtypes on targeted therapy of advanced LAC. We examined EGFR exons18to21status in169 LAC patients by direct sequencing to study the impact of patients' biological characteristics on the EGFR mutational spectrum. And then, 59 patients with advanced LAC harboring EGFR exon 19 deletions(del 19) or exon 21 point mutation(L858R) mutations received first-line treatment of gefitinib or erlotinib, the efficacy of treatment, and the progression-free survival (PFS) of these patients were recorded. The frequency of the EGFR mutation and its subtypes and the variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using all samples (n = 169). The EGFR mutation was significantly associated with well-differentiated tumor and excessive household cooking fumes(P < 0.05). The deletions in exon 19 were more frequently associated with well-differentiated tumor (P < 0.05). The overall frequency of the EGFR mutation was 49 %. Then the impact of EGFR mutation subtypes on targeted therapy were investigated by the retrospective analysis on 59 advanced LAC patients with del 19 or L858R mutations and treated first-line with erlotinib or gefitinib. The deletions in exon 19 got longer PFS (P < 0.05). But there were no differences in PFS between erlotinib therapy and gefitinib therapy. EGFR mutations were more frequently in high tumor differentiation and excessive household cooking fumes LAC. The del 19 mutation rate is relatively high with a high differentiation degree in advanced lung adenocarcinoma. The deletions in exon 19 may benefit more from first-line targeted therapy of advanced LAC compared with exon 21 point mutation L858R. There was no significant difference between the efficacy of gefitinib and erlotinib treatments associated with EGFR mutation and its subtypes.

Inhibition of nuclear factor-κB activity enhanced chemosensitivity to cisplatin in human lung adeno-carcinoma A549 cells under chemical hypoxia conditions.

BACKGROUND: Tumor hypoxia, one of the features of solid tumors, is associated with chemo-resistance. Recently, nuclear factor-κB (NF-κB) was found to be activated during hypoxia. However, the impact of NF-κB activation on chemo-resistance during hypoxia remains unknown. METHODS: Human lung adenocarcinoma A549 cells were transfected with NF-κB p65siRNA and treated with cobalt chloride (CoCl2) to mimic hypoxia in the presence or absence of cisplatin. NF-κB expression was measured by Western blotting, immune-fluorescence and real-time PCR. Hypoxia-inducible factor-1α (HIF-1α) and Bcl-2 expression were determined by Western blotting. Cell apoptosis and survival with half-maximum inhibitory concentration (IC50) of cisplatin were determined by Annexin V-FITC/PI and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), respectively. RESULTS: Exposure of A549 cells to CoCl2 increased nuclear HIF-1a protein expression, and enhanced NF-κB p65 protein nuclear accumulation (the mark of NF-κB activation) in a time and dose dependant manner. CoCl2 did not promote apoptosis in A549 cells; on the contrary, it reduced cisplatin-induced apoptosis and increased the IC50 of cisplatin. However, when we inhibited CoCl2-induced activation of NF-κB through NF-κB p65siRNA, cisplatin-induced apoptosis was increased and IC50 of cisplatin was reduced to levels similar to those in control cells. Meanwhile, CoCl2-induced Bcl-2 overexpression was down-regulated in the presence of cisplatin when NF-κB activity was inhibited. CONCLUSION: Up-regulating Bcl-2 might be involved in NF-κB activation induced resistance to cisplatin in A549 cells under CoCl2-induced chemical hypoxia.

Tea polyphenols prevent lung from preneoplastic lesions and effect p53 and bcl-2 gene expression in rat lung tissues.

Lung cancer is one of the cancers that have the highest incidence and the highest mortality rate, and it is of great interest to identify ways to prevent its occurrence. We had established an animal model by using 3,4-benzopyrene intra-pulmonary injection in our previous study, and had observed that the rats lung carcinoma incidence and multiplicity were significantly reduced by green tea administration. This study further investigated the effect of tea polyphenols on rat lung preneoplastic lesions using the lung carcinoma model established by 3,4-benzopyrene intra-pulmonary injection. Sprague-Dawley rats of the same age were randomly divided into 10 groups and treated with 3,4-benzopyrene by intra-pulmonary injection. Five groups were given 0.3% solution of tea polyphenols (equivalent to 1.2% of green tea) in drinking water, while the other 5 groups were given pure drinking water. The rats were sacrificed at 0, 1, 4, 8 and 16 weeks after carcinogen treatment. In the control groups of rats, local bronchial inflammation were observed at 1 week after 3,4-benzopyrene treatment. From 4 weeks to 16 weeks after carcinogen treatment, hyperplasia, cell hyperproliferation, heterogeneity were observed in the bronchial epithelium. Meanwhile, the expression of p53 mRNA and protein, as well as the level of bcl-2, increased in the bronchial epithelial lesion. Tea polyphenols treatment significantly alleviated the bronchial epithelial lesions. At the same time, tea polyphenols treatment enhanced p53 expression, but reduced bcl-2 expression. These results indicated that tea polyphenols may have preventive effect against lung preneoplasm lesions, possibly through regulating the expression of some critical genes such as p53 and bcl-2.

Good response of malignant pleural effusion from carcinoma of unknown primary site to the anti-tuberculosis therapy: a case report.

UNLABELLED: Malignant pleural effusion in patients with cancers or malignant pleural mesothelioma may often appear at the late stage of disease and significantly affect the patients' life quality and survival. However, there is still no very effective treatment to control malignant pleural effusion. Here we report that malignant pleural effusion in one patient was completely relieved for 15 months by the anti-tuberculosis therapy. CASE PRESENTATION: A 54-year-old female patient complained of cough, dyspnea, chest pain, night sweat and light fever in the afternoon. Computed tomography (CT) of the chest revealed bilateral pleural effusion. But no tumor was found in the lung, pleura and in other sites. Blood test revealed serum carcinoembryonic antigen (CEA) level at 300 ng/mL. One week after we tried anti-tuberculosis combined therapy with isoniazid, pyrazinamide, rifapentine and ethambutol. The pleural effusion in patient was eliminated, along with decreasing CEA. But the CEA increased gradually again when the anti-tuberculosis treatment was forced to discontinuation. Sixteen months after anti-tuberculosis treatment, the symptoms of cough and breathing difficulty relapsed. Chest CT revealed left pleural effusion, pleural thickness and pericardium nodules. Thoracoscopy and biopsy were conducted. The pleural nodules specimen was pathologically diagnosed as squamous cell carcinoma. CONCLUSION: We reported a rare case of successfully treating malignant pleural effusion caused by squamous cell carcinoma of unknown primary site with the anti-tuberculosis combined. This report provides useful evidences for that the anti-tubercular agents may have potential anticancer activity in some carcinomas.