Radiosensitization by Thymidine Phosphorylase Inhibitor in Thymidine Phosphorylase Negative and Overexpressing Bladder Cancer Cell Lines

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p <.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ?H2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.     

RBP2 Induces Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer

RBP2 has been found to actively participate in cancer progression. It inhibits the senescence of cancer cells, mediates cancer cell proliferation and promotes cancer metastasis. It is also essential to drug tolerance. However, the effects of RBP2 on epithelial-mesenchymal transition are still unknown. In this study, we analyzed the effects of RBP2 on epithelial-mesenchymal transition in non-small cell lung cancer. The results showed that RBP2 down-regulated the expression of E-cadherin by inhibiting the promoter activity of E-cadherin and up-regulated the expression of N-cadherin and snail via the activation of Akt signaling, and the overexpression of RBP2 induced epithelial-mesenchymal transition in non-small cell lung cancer cells. Our study further indicated thatRBP2 may be a potential target for anti-lung cancer therapy.     

Expression of Sirtuin 1 and 2 Is Associated with Poor Prognosis in Non-Small Cell Lung Cancer Patients

Background

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology.

Material and Methods

Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor.

Results

High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC.

Conclusions

Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lung cancer, promoting cell proliferation. Moreover, the expression of these proteins is associated with poor prognosis in NSCLC patients and may help to identify those NSCLC patients with high risk of recurrence that could benefit from adjuvant therapy after resection.     

A Novel Muscarinic Antagonist R2HBJJ Inhibits Non-Small Cell Lung Cancer Cell Growth and Arrests the Cell Cycle in G0/G1

Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.     

Serum APE1 Autoantibodies: A Novel Potential Tumor Marker and Predictor of Chemotherapeutic Efficacy in Non-Small Cell Lung Cancer

Apurinic/apyrimidinic endonuclease 1 (APE1), which has the dual functions of both DNA repair and redox activity, has been reported to be highly expressed in non-small cell lung cancer (NSCLC), and this appears to be a characteristic related to chemotherapy resistance. In this study, we identified serum APE1 autoantibodies (APE1-AAbs) in NSCLC patients and healthy controls by immunoblotting and investigated the expression of APE1-AAbs by indirect ELISA from the serum of 292 NSCLC patients and 300 healthy controls. In addition, serum APE1-AAbs level alterations of 91 patients were monitored before and after chemotherapy. Our results showed that serum APE1-AAbs can be detected in both NSCLC patients and healthy controls. Serum APE1-AAbs were significantly higher than those of healthy controls and closely related to APE1 antigen levels both in tumor tissues and the peripheral blood. Moreover, the change in levels of serum APE1-AAbs in NSCLC is closely associated with the response to chemotherapy. These results suggest that APE1-AAbs is a potential tumor marker and predictor of therapeutic efficacy in NSCLC.     

过表达NOLC1诱导人非小细胞肺癌细胞凋亡

人核仁磷酸化蛋白1 (Nucleolar and coiledbody phosphoprotein 1,NOLC1)在癌症的发生发展过程中起着至关重要的调控作用,为探讨NOLC1对肺癌细胞的作用,本研究通过Gateway系统构建重组NOLC1腺病毒载体,成功包装NOLC1腺病毒后,分别感染正常人类胚胎肺细胞(HEL)和非小细胞肺癌细胞(A549细胞),过表达NOLC1。通过MTT实验、AnnexinV-APC/PI双染法和线粒体膜电位实验,证明与HEL细胞相比,NOLC1的过表达对A549细胞的活性降低、凋亡增加、线粒体膜电位下降影响较为显著;通过Real-time PCR检测Caspase家族、TNF与受体家族和BCL2家族基因的表达,发现过表达NOLC1明显上调了A549细胞中促凋亡基因的表达,下调了抗凋亡基因的表达,其中两种重要的促凋亡蛋白CASP8和BAX均显著上调,但是在HEL细胞中这种影响不明显。研究结果表明过表达NOLC1蛋白通过对线粒体通路和死亡受体通路的共同作用,对非小细胞癌具有显著的抗肿瘤活性。     

Localization of Thymidine Phosphorylase in Advanced Gastric and Colorectal Cancer

Thymidine phosphorylase (TP) is known to be more concentrated in human cancer tissues than in adjacent normal tissue based on findings using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. However, the ultrastructural localization of TP in cancer tissues has not previously been demonstrated. We investigated the localization of TP in gastric cancer and colorectal cancer tissue by ELISA, immunohistochemistry, and immunoelectron microscopy. Between April 1997 and May 2000, we obtained surgically resected specimens from 42, 46, and 36 cases of advanced gastric, colon, and rectal cancer, respectively. ELISA demonstrated that the TP level was higher in cancer tissues than in adjacent normal tissue. Immunohistochemically, cancer cells were positive for the enzyme in some cases. However, in a number of cases immunopositive inflammatory cells were also present in cancerous tissues. At the electron microscope level, TP was diffusely distributed in the cytoplasm of cancer cells and in the mitochondria of the neutrophil in gastric cancer tissue. In rectal cancer tissues, cytoplasmic granules in macrophages in cancer tissues were immunoreactive for the TP. These findings suggest that TP is produced by macrophages and exists in neutrophils and cancer cells.     

Folate Receptor-Positive Circulating Tumor Cells as a Novel Diagnostic Biomarker in Non-Small Cell Lung Cancer

The study aims to determine the efficacy and feasibility of a novel folate receptor (FR)-based circulating tumor cell (CTC) detection method in the diagnosis of non-small cell lung cancer (NSCLC). CTCs were collected from 3 ml of blood based on negative enrichment by immunomagnetic beads and then labeled by a conjugate of a tumor-specific ligand folate and an oligonucleotide. After washing off redundant conjugates, the bound conjugates were removed and analyzed by quantitative polymerase chain reaction. The captured cells were validated as tumor cells by immunofluorescence staining. In the evaluation of clinical utility, the results showed that the CTC levels of 153 patients with NSCLC were significantly higher than the controls (49 healthy donors and 64 patients with benign lung diseases; P < .001). With a threshold of 8.64 CTC units, the method showed a sensitivity of 73.2% and a specificity of 84.1% in the diagnosis of NSCLC, especially a sensitivity of 67.2% in stage I disease. Compared with the existing clinical biomarkers such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cyfra21-1, and squamous cell carcinoma antigen (SCC Ag), the method showed the highest diagnostic efficiency (area under the curve, 0.823; 95% confidence interval, 0.773–0.874). Together, our results demonstrated that FR-positive CTCs were feasible diagnostic biomarkers in patients with NSCLC, as well as in early-stage tumors.     

Expression of THOP1 and Its Relationship to Prognosis in Non-Small Cell Lung Cancer

Background

The study was designed to detect the expression level of thimet oligopeptidase (THOP1) protein in non-small cell lung cancer (NSCLC) and investigate its correlation with clinicopathologic features and prognosis.

Methods

Immunohistochemical staining was used to determine the expression of THOP1 protein in 120 NSCLC specimens and 53 distant normal lung tissues. Quantitative real-time PCR and western blotting were employed to measure the expression of THOP1 in 16 pairs of primary NSCLC and corresponding normal tissues.

Results

Analysis of immunohistochemical staining suggested low THOP1 expression was found in 71 (59.2%) of the 120 NSCLC specimens and significantly correlated with positive lymph node metastasis (P = 0.048). However, low THOP1 expression was found in 22 (41.5%) of the 53 normal lung tissues. Chi-square test suggested that the expression of THOP1 was significantly higher in the normal lung tissues than that in the NSCLC specimens (P = 0.032). Real-Time PCR and western blotting showed that NSCLC specimens had decreased THOP1 mRNA and protein expression compared to corresponding normal tissues. Univariate analysis demonstrated that low THOP1 expression significantly predicted decreased 5-year disease-free survival (P = 0.038) and overall survival (P = 0.017). In addition, positive lymph node metastasis (P = 0.025) and advanced TNM stage (P = 0.009) significantly predicted decreased 5-year overall survival. However, multivariate Cox regression analysis showed that only low THOP1 expression retained its significance as an independent prognostic factor for unfavorable 5-year disease-free survival (P = 0.046) and overall survival (P = 0.021).

Conclusions

THOP1 may have clinical potentials to be employed as a promising biomarker to identify individuals with better prognosis and a novel antitumor agent for therapy of patients with NSCLC.     

The Prognostic Value of SOX2 Expression in Non-Small Cell Lung Cancer: A Meta-Analysis

Objective

To investigate the association of SOX2 expression in tumor with clinicopathological features and survival of non-small-cell lung carcinoma (NSCLC) patients.

Methods

Publications assessing the clinicopathological characteristics and prognostic significance of SOX2 in NSCLC were identified up to May 2013. A meta-analysis of eligible studies was performed using standard statistical methods to clarify the association between SOX2 expression and these clinical parameters.

Results

A total of eight studies met the inclusion criteria. Analysis of these data showed that SOX2 expression was positively associated with squamous histology, (pooled OR = 5.26, 95% CI: 1.08–25.6, P = 0.040). Simultaneously, we also found that SOX2 expression was positively associated with overall survival (pooled HR = 0.65, 95% CI: 0.47–0.89, P = 0.007, random-effect).

Conclusions

SOX2 expression in tumor is a candidate positive prognostic biomarker for NSCLC patients.     

Overexpression of JAM-A in Non-Small Cell Lung Cancer Correlates with Tumor Progression

The objective of the current study was to determine the clinical significance of junctional adhesion molecule A (JAM-A) in patients with non-small cell lung cancer (NSCLC) and the biological function of JAM-A in NSCLC cell lines. We showed that JAM-A is predominantly expressed in cell membranes and high expression of JAM-A occurred in 37% of lung tumor specimens compared to corresponding normal tissues. High expression of JAM-A was significantly correlated with TNM stage (P = 0.021), lymph node metastasis (P = 0.007), and decreased overall survival (P = 0.02), In addition, we observed that silencing JAM-A by small interfering RNA inhibited tumor cell proliferation and induced cell cycle arrest at the G1/S boundary. Western blotting analysis revealed that knockdown of JAM-A decreased the protein levels of cyclin D1, CDK4, 6, and P-Rb. Thus, JAM-A plays an important role in NSCLC progression.     

Involvement of TRPC Channels in Lung Cancer Cell Differentiation and the Correlation Analysis in Human Non-Small Cell Lung Cancer

The canonical transient receptor potential (TRPC) channels are Ca²⁺-permeable cationic channels controlling the Ca²⁺ influx evoked by G protein-coupled receptor activation and/or by Ca²⁺ store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca²⁺ imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca²⁺ influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy.     

GINS2基因对非小细胞肺癌的作用及功能分析

GINS2在多种侵袭性肿瘤中上调,然而,其在非小细胞肺癌(NSCLC)中的作用及功能仍有待阐明.本研究主要对GINS2在非小细胞肺癌中的作用及功能进行分析,通过肿瘤基因组图谱(TCGA)数据库分析发现,GINS2在NSCLC中显著上调.为了研究GINS2在NSCLC细胞中的作用及其功能,首先,采用小干扰RNA技术设计了该基因的siRNA,以此来沉默GINS2的mRNA,细胞水平转染后,该基因在非小细胞肺癌细胞系NCI-H292和A549中的表达降低;随后,采用实时细胞分析仪(real time cell analyzer,RTCA)和Tran-swell小室实验对细胞的增殖、迁移和侵袭进行测定,最后采用流式细胞仪分析细胞的周期以及细胞凋亡.实验结果显示:在NCI-H292和A549两个细胞系中,用siRNA沉默GINS2的表达后,NCI-H292和A549细胞的生长能力、迁移能力和侵袭能力均会受到明显的抑制;此外,细胞的周期凋亡实验显示,在NCI-H292细胞系中,阻碍了细胞G1期向S期的转变;在A549细胞系中,阻碍了细胞S期向G2期的转变,同时在两个细胞系中均表现出促进细胞凋亡等功能.综上所述,敲低GINS2可抑制NCI-H292和A549细胞的增殖、迁移和侵袭,并且能阻滞细胞周期促进细胞凋亡等.因此,研究该基因在NSCLC中的功能对于后续研究分子机制具有很大的意义,可能是未来的特异性治疗靶点.     

Trametinib with or without Vemurafenib in BRAF Mutated Non-Small Cell Lung Cancer

V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) mutated lung cancer is relatively aggressive and is resistant to currently available therapies. In a recent phase II study for patients with BRAF-V600E non-small cell lung cancer (NSCLC), BRAF V600E inhibitor demonstrated evidence of activity, but 30% of this selected group progressed while on treatment, suggesting a need for developing alternative strategies. We tested two different options to enhance the efficacy of vemurafenib (BRAF V600E inhibitor) in BRAF mutated NSCLC. The first option was the addition of erlotinib to vemurafenib to see whether the combination provided synergy. The second was to induce MEK inhibition (downstream of RAF) with trametinib (MEK inhibitor). We found that the combination of vemurafenib and erlotinib was not synergistic to the inhibition of p-ERK signaling in BRAF-V600E cells. Vemurafenib caused significant apoptosis, G1 arrest and upregulation of BIM in BRAF-V600 cells. Trametinib was effective as a single agent in BRAF mutated cells, either V600E or non-V600E. Finally, the combination of vemurafenib and trametinib caused a small but significant increase in apoptosis as well as a significant upregulation of BIM when compared to either single agent. Thus, hinting at the possibility of utilizing a combinational approach for the management of this group of patients. Importantly, trametinib alone caused upregulation of p-AKT in BRAF non-V600 mutated cells, while this effect was nullified with the combination. This finding suggests that, the combination of a MEK inhibitor with a BRAF inhibitor will be more efficacious in the clinical setting for patients with BRAF mutated NSCLC.     

非小细胞肺癌放疗所致并发症的影响因素分析

目的:分析非小细胞肺癌放疗所致并发症的影响因素.方法:选择2008年6月至2011年10月我院收治的原发非小细胞肺癌150例,所有病人接受精确放疗治疗.对各因素和并发症发生率的关系采用统计学分析.结果:本文150例病例治疗3个月后随访确认较严重的并发症16例(10.7％).单因素分析显示年龄及放射剂量与并发症相关,而病灶位置、肿瘤大小与并发症的发生无关.以上相关因素带入多因素Logistic回归模型,结果提示只有同时放射剂量指标为独立的相关因素,年龄与并发症的发生可能相关,但无显著性意义.结论:肺癌患者接受精确放疗治疗并发症与放射剂量密切相关,并发症发生于患者年龄也可能相关.放疗剂量的合理控制有助于增加放疗的安全性,这在高龄患者更尤为重要.     

Causal Effects of Time-Dependent Treatments in Older Patients with Non-Small Cell Lung Cancer

Background

Treatment selection for elderly patients with lung cancer must balance the benefits of curative/life-prolonging therapy and the risks of increased mortality due to comorbidities. Lung cancer trials generally exclude patients with comorbidities and current treatment guidelines do not specifically consider comorbidities, so treatment decisions are usually made on subjective individual-case basis.

Methods

Impacts of surgery, radiation, and chemotherapy mono-treatment as well as combined chemo/radiation on one-year overall survival (compared to no-treatment) are studied for stage-specific lung cancer in 65+ y.o. patients. Methods of causal inference such as propensity score with inverse probability weighting (IPW) for time-independent and marginal structural model (MSM) for time-dependent treatments are applied to SEER-Medicare data considering the presence of comorbid diseases.

Results

122,822 patients with stage I (26.8%), II (4.5%), IIIa (11.5%), IIIb (19.9%), and IV (37.4%) lung cancer were selected. Younger age, smaller tumor size, and fewer baseline comorbidities predict better survival. Impacts of radio- and chemotherapy increased and impact of surgery decreased with more advanced cancer stages. The effects of all therapies became weaker after adjustment for selection bias, however, the changes in the effects were minor likely due to the weak selection bias or incompleteness of the list of predictors that impacted treatment choice. MSM provides more realistic estimates of treatment effects than the IPW approach for time-independent treatment.

Conclusions

Causal inference methods provide substantive results on treatment choice and survival of older lung cancer patients with realistic expectations of potential benefits of specific treatments. Applications of these models to specific subsets of patients can aid in the development of practical guidelines that help optimize lung cancer treatment based on individual patient characteristics.     

Overexpression of MTA3 Correlates with Tumor Progression in Non-Small Cell Lung Cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of MTA3 in patients with non-small cell lung cancer (NSCLC). The expression profile of MTA3 in NSCLC tissues and adjacent noncancerous lung tissues was detected by immunohistochemistry. MTA3 was overexpressed in 62 of 108 (57.4%) human lung cancer samples and correlated with p-TNM stage (p<0.0001), nodal metastasis (p = 0.0009) and poor prognosis (p<0.05). In addition, the depletion of MTA3 expression with small interfering RNAs inhibited cell growth and colony formation in the A549 and H157 lung cancer cell lines. Moreover, MTA3 depletion induced cell cycle arrest at the G1/S boundary. Western blotting analysis revealed that the knockdown of MTA3 decreased the protein levels of cyclin A, cyclin D1 and p-Rb. These results indicate that MTA3 plays an important role in NSCLC progression.