CDK5 Activates Hippo Signaling to Confer Resistance to Radiation Therapy Via Upregulating TAZ in Lung Cancer.

PURPOSE: Tumor resistance to radiation therapy is a therapeutic challenge in the treatment of patients with non-small cell lung cancer. Cyclin-dependent kinase 5 (CDK5) has been proposed to participate in cell proliferation, migration and invasion, drug resistance, and immune evasion. However, the functions and regulatory mechanisms of CDK5 in lung cancer radioresistance have not been investigated. METHODS AND MATERIALS: DNA damage response and repair were measured by neutral comet assay and γ-H2AX and Rad51 foci staining. The biological functions of CDK5 in lung cancer radioresistance were investigated with clonogenic survival assays and xenograft tumor models. Small interfering RNAs and short hairpin RNAs were used to knock down CDK5 in A549 and H1299 cells. The effects of CDK5 depletion on the tumorigenic behaviors of lung cancer cells were evaluated in vitro and in vivo. Gene expression was examined by RNA-seq and quantitative real-time polymerase chain reaction. RESULTS: We report that CDK5 depletion impairs lung cancer progression and radioresistance in vitro and in vivo. Mechanistically, we identify TAZ, a component of the Hippo pathway, as a critical downstream effector of CDK5. Loss of CDK5 downregulates TAZ expression and attenuates Hippo signaling activation. Importantly, we provide evidence that TAZ is the major effector mediating the biological functions of CDK5 in lung cancer. CONCLUSIONS: These results illustrate that CDK5 activates Hippo signaling via TAZ to participate in tumorigenesis and radioresistance, suggesting that CDK5 may be a promising radiosensitization target for the treatment of lung cancer.

Weakly Supervised Deep Learning for Whole Slide Lung Cancer Image Analysis.

Histopathology image analysis serves as the gold standard for cancer diagnosis. Efficient and precise diagnosis is quite critical for the subsequent therapeutic treatment of patients. So far, computer-aided diagnosis has not been widely applied in pathological field yet as currently well-addressed tasks are only the tip of the iceberg. Whole slide image (WSI) classification is a quite challenging problem. First, the scarcity of annotations heavily impedes the pace of developing effective approaches. Pixelwise delineated annotations on WSIs are time consuming and tedious, which poses difficulties in building a large-scale training dataset. In addition, a variety of heterogeneous patterns of tumor existing in high magnification field are actually the major obstacle. Furthermore, a gigapixel scale WSI cannot be directly analyzed due to the immeasurable computational cost. How to design the weakly supervised learning methods to maximize the use of available WSI-level labels that can be readily obtained in clinical practice is quite appealing. To overcome these challenges, we present a weakly supervised approach in this article for fast and effective classification on the whole slide lung cancer images. Our method first takes advantage of a patch-based fully convolutional network (FCN) to retrieve discriminative blocks and provides representative deep features with high efficiency. Then, different context-aware block selection and feature aggregation strategies are explored to generate globally holistic WSI descriptor which is ultimately fed into a random forest (RF) classifier for the image-level prediction. To the best of our knowledge, this is the first study to exploit the potential of image-level labels along with some coarse annotations for weakly supervised learning. A large-scale lung cancer WSI dataset is constructed in this article for evaluation, which validates the effectiveness and feasibility of the proposed method. Extensive experiments demonstrate the superior performance of our method that surpasses the state-of-the-art approaches by a significant margin with an accuracy of 97.3%. In addition, our method also achieves the best performance on the public lung cancer WSIs dataset from The Cancer Genome Atlas (TCGA). We highlight that a small number of coarse annotations can contribute to further accuracy improvement. We believe that weakly supervised learning methods have great potential to assist pathologists in histology image diagnosis in the near future.

Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry.

Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.

Multisensor Imaging-From Sample Preparation to Integrated Multimodal Interpretation of LA-ICPMS and MALDI MS Imaging Data.

Laterally resolved chemical analysis (chemical imaging) has increasingly attracted attention in the Life Sciences during the past years. While some developments have provided improvements in lateral resolution and speed of analysis, there is a trend toward the combination of two or more analysis techniques, so-called multisensor imaging, for providing deeper information into the biochemical processes within one sample. In this work, a human malignant pleural mesothelioma sample from a patient treated with cisplatin as a cytostatic agent has been analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). While LA-ICPMS was able to provide quantitative information on the platinum distribution along with the distribution of other elemental analytes in the tissue sample, MALDI MS could reveal full information on lipid distributions, as both modes of polarity, negative and positive, were used for measurements. Tandem MS experiments verified the occurrence of distinct lipid classes. All imaging analyses were performed using a lateral resolution of 40 µm, providing information with excellent depth of details. By analyzing the very same tissue section, it was possible to perfectly correlate the obtained analyte distribution information in an evaluation approach comprising LA-ICPMS and MALDI MS data. Correlations between platinum, phosphorus, and lipid distributions were found by the use of advanced statistics. The present proof-of-principle study demonstrates the benefit of data combination for outcomes beyond one method imaging modality and highlights the value of advanced chemical imaging in the Life Sciences.

Effect of tumor properties on energy absorption, temperature mapping, and thermal dose in 13.56-MHz radiofrequency hyperthermia.

Computational techniques can enhance personalized hyperthermia-treatment planning by calculating tissue energy absorption and temperature distribution. This study determined the effect of tumor properties on energy absorption, temperature mapping, and thermal dose distribution in mild radiofrequency hyperthermia using a mouse xenograft model. We used a capacitive-heating radiofrequency hyperthermia system with an operating frequency of 13.56 MHz for in vivo mouse experiments and performed simulations on a computed tomography mouse model. Additionally, we measured the dielectric properties of the tumors and considered temperature dependence for thermal properties, metabolic heat generation, and perfusion. Our results showed that dielectric property variations were more dominant than thermal properties and other parameters, and that the measured dielectric properties provided improved temperature-mapping results relative to the property values taken from previous study. Furthermore, consideration of temperature dependency in the bio heat-transfer model allowed elucidation of precise thermal-dose calculations. These results suggested that this method might contribute to effective thermoradiotherapy planning in clinics.

Activation of an AKT/FOXM1/STMN1 pathway drives resistance to tyrosine kinase inhibitors in lung cancer.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) have demonstrated clinical benefits in the treatment of several tumour types. However, the emergence of TKI resistance restricts the therapeutic effect. This study uses non-small cell lung cancer (NSCLC) to explore the mechanisms contributing to TKI resistance in tumours. METHODS: Biological phenotypes and RNA microarray expression data were analysed in NSCLC cells with and without TKI pretreatment. Specific inhibitors and siRNAs were used to validate the direct involvement of an AKT/FOXM1/STMN1 pathway in TKI resistance. Patients' tissues were analysed to explore the clinical importance of FOXM1 and STMN1. RESULTS: In vitro and in vivo studies showed that TKIs induced the enrichment of cancer stem cells (CSC), promoted epithelial to mesenchymal transition (EMT), and conferred multidrug resistance on NSCLC cells in a cell type- and TKI class-dependent manner. Mechanistically, TKIs activated an AKT/FOXM1/STMN1 pathway. The crucial role of this pathway in TKI-induced enrichment of CSC and drug resistance was verified by silencing FOXM1 and STMN1 or blocking the AKT pathway. Additionally, overexpression of STMN1 was associated with upregulation of FOXM1 in advanced NSCLC patients, and STMN1/FOXM1 upregulation predicted a poor outcome. CONCLUSIONS: Our findings elucidate an additional common mechanism for TKI resistance and provide a promising therapeutic target for reversing TKI resistance in NSCLC.

Targeted Therapy as an Alternative to Whole-Brain Radiotherapy in EGFR-Mutant or ALK-Positive Non-Small-Cell Lung Cancer With Brain Metastases.

CLINICAL QUESTION: Is up-front whole-brain radiotherapy required to treat multiple brain metastases from non-small-cell lung cancer when highly active targeted therapies are available? CLINICAL APPLICATION: Patients with EGFR-mutant or ALK-positive non-small-cell lung cancer with brain metastases now have the potential to achieve a prolonged survival. Through use of highly active targeted therapies, whole-brain radiotherapy can be safely postponed, diminishing toxic effects that could impair quality of life.

Antimetastatic pectic polysaccharide from Decalepis hamiltonii; galectin-3 inhibition and immune-modulation.

Melanoma is a malignant neoplasm of major concern because of its high mortality rate and failure of chemotherapy. Previously we have shown that galectin-3, a galactose specific lectin, plays a pivotal role in the initiation of metastasis. It was hypothesized that blocking galectin-3 with galactose rich dietary pectic polymer would inhibit metastasis. The current study analyzes the preventive effect and mode of action of a pectic polymer from Swallow Root (Decalepis hamiltonii) in a preventative study of B16F10 cells lung colonization. Matrix metalloproteinase (MMPs) activity was assayed by zymography. Apoptotic/proliferative markers and cytokines were analyzed by immunoassay. Results indicated ~88% inhibition of lung colonization by SRPP as compared to 60% by CPP and only 7% by GRPP. Further molecular analysis revealed that galectin-3 blockade was associated with down regulation of MMPs and NFκB. Activation of caspases supported the apoptotic effect of SRPP. Infiltration of inflammatory cells into the lung was evidenced by presence of CD11b+ cells and release of the pro-inflammatory cytokine-IL-17, indicating inflammation during the cancer cell colonization process. SRPP enhanced the release of IL-12 that enables the reduction of inflammation. Our data for the first time indicate the effective anti-metastatic effect of SRPP due to both galectin-3 blockade and immunomodulation.

The aqueous extract of Brucea javanica suppresses cell growth and alleviates tumorigenesis of human lung cancer cells by targeting mutated epidermal growth factor receptor.

As a practical and safe herbal medicine, the seeds of Brucea javanica (L.) Merr., were used to cure patients suffering from infectious diseases such as malaria. Recent advances revealed that the herb could also be a useful cancer therapy agent. The study demonstrated that aqueous B. javanica (BJ) extract attenuated the growth of human non-small-lung cancer cells bearing mutant L858R/T790M epidermal growth factor receptor (EGFR). The reduced cell viability in H1975 cells was attributed to apoptosis. Transfection of EGFR small hairpin RNA reverted the sensitivities. When nude mice were fed BJ extract, the growth of xenograft tumors, as established by H1975 cells, was suppressed. Additional histological examination and fluorescence analysis of the resected tissues proved that the induced apoptosis mitigated tumor growth. The work proved that the BJ extract exerted its effectiveness by targeting lung cancer cells carrying mutated EGFR while alleviating tumorigenesis. Aqueous BJ extract is a good candidate to overcome drug resistance in patients undergoing target therapy.

Orai3 is a predictive marker of metastasis and survival in resectable lung adenocarcinoma.

Orai3 channel has emerged as important player in malignant transformation. Indeed, its expression is increased in cancer and favors cell proliferation and survival by permitting calcium influx. In this study, Orai3 was overexpressed in lung adenocarcinoma as compared to their matched non-tumour samples and was associated with tumoural aggressiveness. Moreover, its expression was associated with estrogen receptor alpha (ERα) expression and visceral pleural invasion in multivariate analysis. Furthermore, both the overall survival (OS) median and the metastasis free survival (MFS) median of tumors with high Orai3 expression were lower than in low Orai3 expression regardless of cancer stage (35.01 months vs. 51.11 months for OS and 46.01 months vs. 62.04 months for MFS). In conclusion, Orai3 protein level constitutes an independent prognostic marker in lung adenocarcinoma, and a novel prognostic marker that could help selecting the patients with worst prognosis to be treated with adjuvant chemotherapy in resectable stage.

Anodic alumina coating for extraction of volatile organic compounds in human exhaled breath vapor.

The objective of the study is to develop a facile and highly sensitive solid phase microextraction-gas chromatography/mass spectrometry method for the analysis of volatile organic compounds in human exhaled breath vapor. For the purpose, a highly ordered nanoporous anodic alumina coating was prepared by a two-step anodic oxidization method based on aluminum substrate. To have a good knowledge of the fiber, some features were characterized and the results indicate that the coating has several advantages, including excellent chemical and thermal stability, high mechanical strength, large surface area and good extraction performance. In addition, some parameters related to extraction efficiency were also studied. Under the optimal conditions, the coating was used to quantitatively extract volatile organic compounds. Good linearity and wide linear range were obtained with correlation coefficients (R(2)) ranging from 0.9933 to 0.9999. The detection limits of benzene homologues, aldehydes and ketones were between 0.7 and 3.4 ng L(-1). Relative standard deviations (n=5) ranged from 1.8 to 15.0%. Satisfied recovery (89-115%) was obtained at two spiked concentration levels. Finally, the developed method was successfully applied for the analysis of volatile organic compounds in human exhaled vapor samples of lung cancer patients and the controls, and the results were statistically analyzed with Independent-Sample T Test. The proposed method exhibits some outstanding merits, including convenience, non-invasion, low cost and sensitivity. It provides a potential tool for rapid detection of volatile organic compounds in human exhaled breath.

Treatment of diffuse malignant peritoneal mesothelioma (DMPM) by cytoreductive surgery and HIPEC.

AIM: Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and locally aggressive tumor with poor prognosis, related in most cases to asbestos exposure. It is increasing in frequency, but currently no standard therapy is available. The biology of this disease is still poorly understood. Several highly specialized centers have recently reported improved survival by means of an innovative local-regional approach. The purpose of this article is to evaluate the survival benefit and the morbidity rate of patients affected by DMPM treated at our institution by cytoreductive surgery (CRS) associated with hyperthermic intraperitoneal perioperative chemotherapy (HIPEC). METHODS: This study includes 42 patients affected by DMPM treated by an uniform approach consisting of cytoreductive surgery associated with HIPEC using cisplatin and doxorubicin. The primary end point was overall survival and morbidity rate. The secondary end point was evaluation of prognostic variables for overall survival. RESULTS: The median follow-up period was 72 months (range 1-235 months). Thirty-five patients (83.3%) presented epithelial tumors and 7 were affected by multicystic mesothelioma. The mean peritoneal cancer index (PCI) was 13. Thirty-eight patients (90.4%) had complete cytoreduction (CC-0/1). The overall morbidity rate was 35.7% associated to a perioperative mortality of 7.1%. Median overall survival rate was 65 months with a 1- and 5-year survival rates of 63% and 44%, respectively. CONCLUSION: The treatment of DMPM by CRS+HIPEC in selected patients is a feasible technique that allows to achieve encouraging results in terms of overall survival rate, with an acceptable morbidity rate. Further investigations are needed to clarify the role and the timing of this promising technique.

Membrane carbonic anhydrase IX expression and relapse risk in resected stage I-II non-small-cell lung cancer.

BACKGROUND: Adjuvant chemotherapy reduces recurrences of non-small-cell lung cancer (NSCLC). To determine which patients need adjuvant chemotherapy, we assessed factors associated with time to relapse (TTR). METHODS: In 230 resected stage I-II NSCLCs, we correlated immunohistochemistry scores for factors associated with cell growth rate, growth regulation, hypoxia, cell survival, and cell death with TTR. RESULTS: With a median follow-up of 82 months (1-158) for those alive and relapse free at last follow-up, median time to recurrence was not reached. The 2- and 5-year probabilities of maintaining freedom from recurrence were 80.7% (95% confidence interval, 75.3%, 86.4%) and 74.6% (95% confidence interval, 68.6%, 81.2%), respectively. TTR curves flattened at an apparent cure rate of 70%. In multicovariate Cox models, factors correlating with shorter TTR were membranous carbonic anhydrase IX (mCAIX) staining (any versus none, hazard ratio = 2.083, p = 0.023) and node stage (N1 versus N0, hazard ratio = 2.591, p = 0.002). mCAIX scores correlated positively with tumor size, grade, squamous histology, necrosis, mitoses, Ki67, p53, nuclear DNA methyltransferase 1, and cytoplasmic enhancer-of-split-and-hairy-related protein, and they correlated inversely with papillary histology, epidermal growth factor receptor mutation (trend), copper transporter-1, and cytoplasmic hypoxia-inducible factor-1α, vascular endothelial growth factor, DNA methyltransferase 1, and excision repair cross-complementing rodent repair deficiency, complementation group 1. CONCLUSION: Nodal stage and mCAIX immunohistochemistry were the strongest independent predictors of shorter TTR in resected NSCLCs. mCAIX correlated with tumor size, markers of tumor proliferation and necrosis, and tumor genetic characteristics, and it paradoxically correlated inversely with the hypoxia markers, hypoxia-inducible factor-1α and vascular endothelial growth factor. Presence of mCAIX could help determine patients with high risk of recurrence who might require adjuvant chemotherapy.

Application of proteomics to identify the target molecules involved in Lonicera japonica-induced photokilling in human lung cancer CH27 cells.

BACKGROUND: The Lonicera japonica has been used as natural and healthy drink for its anti-inflammatory effect and pleasant odor in China and Taiwan. METHODS: 2D electrophoresis was used to analyze the proteins involved in photoactivated Lonicera japonica-induced CH27 cell apoptosis. The fluorescent dyes MitoTracker Red CMXRos, calcein AM and JC-1 were used to elucidate mitochondrial function. The protein expression was performed by Western blotting. Fluorescent image of endoplasmic reticulum was accomplished by using ER-Tracker Green. This study used fluorescent dye CM-H2DCFDA to detect intracellular generation of reactive oxygen species. RESULTS: The identified proteins can be classified into three major groups, which include proteins involved in mitochondrial function, cytoskeleton-related proteins and proteins associated with endoplasmic reticulum (ER) stress. Photoactivated Lonicera japonica caused a significant effect on the mitochondrial function and ER stress in CH27 cells. The reactive oxygen species producing was found to be involved in photoactivated Lonicera japonica-induced CH27 cell apoptosis. CONCLUSION: Mitochondria and endoplasmic reticulum are the integral targets in photoactivated Lonicera japonica-induced CH27 cell apoptosis. We also demonstrated that ethyl acetate fraction of Lonicera japonica extracts caused photocytotoxicity in a dose-dependent manner in CH27 cells. This could explain the fact that the ethyl acetate fraction of Lonicera japonica extracts may contain compounds which exhibit the photosensitizing activity in CH27 cells.

Feasibility of preemptive biomarker profiling for personalised early clinical drug development at a Comprehensive Cancer Center.

PURPOSE: Multiple investigational drugs are currently explored in cancer patient populations defined by specific biomarkers. This demands a new process of patient selection for clinical trials. PATIENTS AND METHODS: Starting January 1, 2012, preemptive biomarker profiling was offered at the West German Cancer Center to all patients with advanced non-small-cell lung (NSCLC) or colorectal cancer (CRC), who met generic study inclusion criteria. Tumour specimens were subjected to prespecified profiling algorithms to detect 'actionable biomarkers' by amplicon sequencing, in situ hybridisation and immunohistochemistry. The clinical course was closely monitored to offer trial participation whenever applicable. RESULTS: Within 12 months, 267 patients (188 NSCLC, 79 CRC) were profiled. Estimated additional cost for biomarker profiling was 219615.51 EUR excluding histopathology workup and administration. The most prevalent biomarkers in pulmonary adenocarcinoma were KRAS mutations (29%), loss of PTEN expression (18%), EGFR mutations (9%), HER2 amplification (5%) and BRAF mutations (3%), while the prevalence of ALK translocations and PIK3CA mutations was extremely low. In pulmonary squamous cell carcinoma FGFR1 amplifications were found in 15%, PTEN expression was lost in 20% and DDR2 was mutated in a single case. KRAS mutations (41%) predominated in CRC, followed by loss of PTEN expression (16%), PIK3CA (5%) and BRAF (5%) mutations. So far 13 patients (5%) have entered biomarker-stratified clinical trials. Therapeutic decisions for approved drugs were guided in another 45 patients (17%). CONCLUSION: Preemptive biomarker profiling can be implemented into the diagnostic algorithm of a large Comprehensive Cancer Center. Substantial investments in diagnostics and administration are required.

Primo vessel inside a lymph vessel emerging from a cancer tissue.

Primo vessels were observed inside the lymph vessels near the caudal vena cava of a rabbit and a rat and in the thoracic lymph duct of a mouse. In the current work we found a primo vessel inside the lymph vessel that came out from the tumor tissue of a mouse. A cancer model of a nude mouse was made with human lung cancer cell line NCI-H460. We injected fluorescent nanoparticles into the xenografted tumor tissue and studied their flow in blood, lymph, and primo vessels. Fluorescent nanoparticles flowed through the blood vessels quickly in few minutes, and but slowly in the lymph vessels. The bright fluorescent signals of nanoparticles disappeared within one hour in the blood vessels but remained much longer up to several hours in the case of lymph vessels. We found an exceptional case of lymph vessels that remained bright with fluorescence up to 24 hours. After detailed examination we found that the bright fluorescence was due to a putative primo vessel inside the lymph vessel. This rare observation is consistent with Bong-Han Kim's claim on the presence of a primo vascular system in lymph vessels. It provides a significant suggestion on the cancer metastasis through primo vessels and lymph vessels.

The surgeon's role in molecular biology.

The biomolecular era is rapidly becoming shaped around the supreme interest in targeted therapy for patients with non-small cell lung cancer. Tissue analysis has become crucial in the definition of biomarkers and genomic signatures able to predict the response to treatment or even survival. Lung screening programs and minimally invasive thoracic surgery are jointly aimed at increasing the quantity and quality of specimens of non-small cell lung cancer caught at the earliest stages with the attendant, significant, effect on patient survival. In addition, biomolecular researchers are disclosing an ever-increasing cohort of patients with specific genetic mutations that make their cancer susceptible to individualized treatment. When needed for immunohistochemical characterization, investigators are ready to request "research biopsies" to consolidate tissue availability for clinical trials, translational research, and in biobanks. With unique and diverse tools in the surgical armamentarium, the thoracic surgeon plays a central role in this new multidisciplinary professional environment, actively participating in creating the foundations of the biomolecular era.

High expression of the transcriptional co-activator p300 predicts poor survival in resectable non-small cell lung cancers.

PURPOSE: To investigate the correlation between p300 (a transcriptional co-activator) expression and clinical/prognostic characteristics in surgically resected NSCLC patients for the purpose of identifying patients with increased risk of cancer recurrence and providing them with tailored therapy. METHODS: One hundred and sixty-nine completely resected NSCLC patients were included in this study. Paraffin-embedded primary tumour tissues of patients were supplied to produce a tissue microarray, and immunohistochemistry was used for the evaluation of p300 expression. The clinical/prognostic significance of p300 expression was analysed for statistical significance. Survival was calculated by the Kaplan-Meier method, and the log-rank test was used to assess differences in survival between the groups. The prognostic impact of clinicopathologic variables and p300 expression was evaluated using a Cox proportional hazards model. RESULTS: High expression of p300 was associated with poor disease-free survival (p = 0.027) and overall survival (p = 0.006) in NSCLC patients. Further analysis suggested that this difference in overall survival also existed in patients with T2 (p = 0.040), positive lymph nodes (p = 0.023), stage IIIA (p = 0.003), adenocarcinoma (p = 0.021), and a well-differentiated histological grade score (p = 0.011). The multivariate Cox regression analysis showed that low p300 expression is an independent marker of better disease-free survival (relative risk = 0.628, p = 0.047) and overall survival (relative risk = 0.545, p = 0.024) in operable NSCLC patients. CONCLUSIONS: Low p300 expression is an independent prognostic marker of better survival in operable NSCLC patients. The combination of clinicopathological TNM staging classification with p300 expression may be useful in identifying patients with increased risk of cancer recurrence to provide them with tailored therapy.

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28.

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 µg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 µg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 µg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 µg/ml to 15 µg/ml with a GI(50) of 0.58 µg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 µg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.

The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells.

Despite a lack of scientific authentication, Scutellaria baicalensis is clinically used in Chinese medicine as a traditional adjuvant to chemotherapy of lung cancer. In this study, cytotoxicity assays demonstrated that crude ethanolic extracts of S. baicalensis were selectively toxic to human lung cancer cell lines A549, SK-LU-1 and SK-MES-1 compared with normal human lung fibroblasts. The active compounds baicalin, baicalein and wogonin did not exhibit such selectivity. Following exposure to the crude extracts, cellular protein expression in the cancer cell lines was assessed using 2D gel electrophoresis coupled with MALDI-TOF-MS/Protein Fingerprinting. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity. These observations were supported by PI staining cell cycle analysis using flow cytometry and Annexin-V apoptotic analysis by fluorescence microscopy of cancer cells treated with the crude extract and pure active compounds. Moreover, specific immunoblotting identification showed the decreased expression of cyclin A results in the S phase arrest of A549 whereas the G(0)/G(1) phase arrest in SK-MES-1 cells results from the decreased expression of cyclin D1. Following treatment, increased expression in the cancer cells of key proteins related to the enhancement of apoptosis was observed for p53 and Bax. These results provide further insight into the molecular mechanisms underlying the clinical use of this herb as an adjuvant to lung cancer therapy.