Metformin prevents the onset and progression of intervertebral disc degeneration: New insights and potential mechanisms (Review).

Metformin has been the go­to medical treatment for addressing type 2 diabetes mellitus (T2DM) as a frontline oral antidiabetic. Obesity, cancer and bone deterioration are linked to T2DM, which is considered a metabolic illness. Numerous diseases associated with T2DM, such as tumours, cardiovascular disease and bone deterioration, may be treated with metformin. Intervertebral disc degeneration (IVDD) is distinguished by degeneration of the spinal disc, accompanied by the gradual depletion of proteoglycans and water in the nucleus pulposus (NP) of the IVD, resulting in lower back pain. The therapeutic effect of metformin on IVDD has also attracted much attention. By stimulating AMP­activated kinase, metformin could enhance autophagy and suppress cell senescence, apoptosis and inflammation, thus effectively delaying IVDD. The present review aimed to systematically explain the development of IVDD and mechanism of metformin in the treatment and prevention of IVDD to provide a reference for the clinical application of metformin as adjuvant therapy in the treatment of IVDD.

Caffeic acid phenethyl ester surmounts acquired resistance of AZD9291 in non-small cell lung cancer cells.

Epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line therapy for EGFR mutated non-small cell lung cancer (NSCLC); however, resistance rapidly develops. The objective of this study was therefore to establish and characterize a gefitinib resistant NSCLC line (HCC827GR) and evaluate the therapeutic effects of natural products in combination with third-generation EGFR-TKI, AZD9291. The IC50 of gefitinib and AZD9291 in HCC827GR were significantly higher than those of HCC827 (p < 0.05). Furthermore, anchorage-independent colony assay indicated that HCC827GR cells were more aggressive than their predecessors. This was reflected by the gene/protein expression changes observed in HCC827GR versus HCC827 profiled by cancer drug resistance real-time polymerase chain reaction (RT-PCR) array and Western blot. Three natural products were screened and caffeic acid phenethyl ester (CAPE) exhibited the most significant combinative cytotoxic effect with AZD9291. Specifically, flow cytometry revealed that AZD9291 + CAPE considerably increased the fraction of cell in pre-G1 of the cell cycle and caspase-Glo3/7 assay showed a dramatic increase in apoptosis when compared to AZD9291 alone. Furthermore, Western blot showed significant downregulation of p-EGFR/p-AKT in HCC827GR cells treated with AZD9291 + CAPE as compared to AZD9291. Moreover, it is evident that AZD9291 + CAPE specifically resulted in a marked reduction in the protein expressions of the cell-proliferation-related genes p21, cyclin D1, and survivin. Finally, refined RT-PCR/Western blot data indicated that AZD9291 + CAPE may at least partially exert its synergistic effects via the PLK2 pathway. Together, these results suggest that CAPE is a clinically relevant compound to aid AZD9291 in treating EGFR-TKI resistant cells through modulating critical genes/proteins involved in cancer resistance/therapy.

Physiological Effects of Co-exposure to Ionizing Radiation and Noise within Occupational Exposure Limits.

ABSTRACT: Workers are frequently exposed to the occupational hazards of ionizing radiation and noise. Co-exposure to these hazards is not well understood in terms of their physiological effects. The aim of this study was to investigate the physiological effects of co-exposure to ionizing radiation and noise within the occupational limit. This study extracted the physical examination parameters of workers who met the screening criteria from the occupational health surveillance database. The workers were divided into three groups: the co-exposure (COE) group, the ionizing radiation exposure (ION) group, and the non-exposure (NON) group. The age and sex of the three groups were matched with a sample size ratio of 1:3:3. The physical examination parameters of the three groups of workers were compared. The results showed that there was no significant difference in blood pressure and blood biochemical parameters among the three groups. The COE group had higher levels of free triiodothyronine than the ION group, but there was no difference with the NON group. Moreover, the COE group had lower levels of free tetraiodothyronine than the ION group and the NON group. There was no significant difference in thyroid stimulating hormone, total triiodothyronine, and total tetraiodothyronine among the three groups. Additionally, the number of white blood cells of the COE group was lower than that of ION group and NON group. This study suggests that co-exposure to low-dose ionizing radiation and noise can cause alterations in thyroid hormone and peripheral white blood cells. These alterations are different from those observed after single exposure to low-dose ionizing radiation and require further research.

Proteome derangement in malignant epithelial cells and its stroma following exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Discovering novel changes in the proteome of malignant lung epithelial cells and/or the tumor-microenvironment is paramount for diagnostic, prognostic, and/or therapy development. A time-dependent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumor model was used to screen the proteome of lung tumors. NNK-transformed human lung epithelial BEAS-2B cells were then established to evaluate the epithelial cell-specific protein changes. A duration-dependent increase of tumor burden was observed in NNK-treated mice, 2/12 (17%), 8/12 (67%), 9/12 (75%), and 10/10 (100%) at weeks 8, 12, 16, and 20 after the NNK exposure, respectively. A total of 25 differentially expressed proteins (≥ twofold change), predominantly structural, signaling, and metabolic proteins, were detected by two-dimensional difference gel electrophoresis and identified by mass spectrometry. Calregulin, ezrin, histamine releasing factor (HRF), and inorganic pyrophosphatase 1 (PPA1) exhibited changes and were further confirmed via immunoblotting. In addition, immunohistochemistry (IHC) analysis indicated upregulated E-cadherin and decreased vimentin expression in epithelial cells of tumor tissues. Acquisition of a neoplastic phenotype in NNK-transformed BEAS-2B cells was demonstrated by enhanced wound closure and increased anchorage independent colony formation. In transformed BEAS-2B cells, protein expression of E-cadherin, ezrin, and PPA1 (but not calregulin and HRF) was upregulated, as was observed in tumor tissues IHC staining using mouse lung tumor tissues further revealed that HRF upregulation was not lung epithelial cell specific. Altogether, tumorigenesis after NNK exposure may be initiated by protein dysregulation in lung epithelial cells together with proteome derangement derived from other cell types existing in the tumor-microenvironment.

Comparative Microbiomics Analysis of Antimicrobial Antibody Response between Patients with Lung Cancer and Control Subjects with Benign Pulmonary Nodules.

BACKGROUND: CT screening can detect lung cancer early but suffers a high false-positive rate. There is a need for molecular biomarkers that can distinguish malignant and benign indeterminate pulmonary nodules (IPN) detected by CT scan. METHODS: We profiled antibodies against 901 individual microbial antigens from 27 bacteria and 29 viruses in sera from 127 lung adenocarcinoma (ADC), 123 smoker controls (SMC), 170 benign nodule controls (BNC) individuals using protein microarrays to identify ADC and BNC specific antimicrobial antibodies. RESULTS: Analyzing fourth quartile ORs, we found more antibodies with higher prevalence in the three BNC subgroups than in ADC or SMC. We demonstrated that significantly more anti-Helicobacter pylori antibodies showed higher prevalence in ADC relative to SMC. We performed subgroup analysis and found that more antibodies with higher prevalence in light smokers (≤20 pack-years) compared with heavy smokers (>20 pack-years), in BNC with nodule size >1 cm than in those with ≤1 cm nodules, and in stage I ADC than in stage II and III ADC. We performed multivariate analysis and constructed antibody panels that can distinguish ADC versus SMC and ADC versus BNC with area under the ROC curve (AUC) of 0.88 and 0.80, respectively. CONCLUSIONS: Antimicrobial antibodies have the potential to reduce the false positive rate of CT screening and provide interesting insight in lung cancer development. IMPACT: Microbial infection plays an important role in lung cancer development and the formation of benign pulmonary nodules.

Pepsin enhances glycolysis to promote malignant transformation of vocal fold leukoplakia epithelial cells with dysplasia.

PURPOSE: The mechanism underlying malignant transformation of vocal fold leukoplakia (VFL) and the precise role of the expression of pepsin in VFL remain unclear. This study aimed to investigate the effects of acidified pepsin on VFL epithelial cell growth and migration, and also identify pertinent molecular mechanisms. METHODS: Immunochemistry and Western blotting were performed to measure glucose transporter type 1 (GLUT1), monocarboxylate transporters 4 (MCT4), and Hexokinase-II (HK-II) expressions. Cell viability, cell cycle, apoptosis, and migration were investigated by CCK-8 assay, flow cytometry and Transwell chamber assay, respectively. Glycolysis-related contents were determined using the corresponding kits. Mitochondrial HK-II was photographed under a confocal microscope using Mito-Tracker Red. RESULTS: It was found: the expression of pepsin and proportion of pepsin+ cells in VFL increased with the increased dysplasia grade; acidified pepsin enhanced cell growth and migration capabilities of VFL epithelial cells, reduced mitochondrial respiratory chain complex I activity and oxidative phosphorylation, and enhanced aerobic glycolysis and GLUT1 expression in VFL epithelial cells; along with the transfection of GLUT1 overexpression plasmid, 18FFDG uptake, lactate secretion and growth and migration capabilities of VFL epithelial cell were increased; this effect was partially blocked by the glycolysis inhibitor 2-deoxy-glucose; acidified pepsin increased the expression of HK-II and enhanced its distribution in mitochondria of VFL epithelial cells. CONCLUSION: It was concluded that acidified pepsin enhances VFL epithelial cell growth and migration abilities by reducing mitochondrial respiratory complex I activity and promoting metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis.

Acrylamide-induced damage to postsynaptic plasticity is CYP2E1 dependent in an SH-SY5Y co-culture system.

Acrylamide (ACR), a neurotoxic substance, is characterized by a range of industrial and population exposures. The effects of ACR on synapses have been examined, but the regulation and molecular mechanism of key proteins related to ACR and its metabolite glycidamide (GA) have not been elucidated. In this study, we constructed two co-culture systems to mimic neurons that do not express and overexpress CYP2E1. In these co-cultures, we observed the effects and relative influence of ACR and GA on cell survival as well as synaptic structural and functional plasticity. Next, we investigated the relationship between ACR-induced nerve damage and key proteins in the postsynaptic membrane. After ACR exposure, cell death and synaptic damage were significantly worse in CYP2E1-overexpressing co-culture systems, suggesting that ACR-induced neurotoxicity may be related to metabolic efficiency (including CYP2E1 activity). Moreover, with increasing doses of ACR, the key postsynaptic membrane proteins PSD-95 expression was reduced and CaMKII and NMDAR-2B phosphorylation was increased. ACR exposure also triggered a rapid dose- and time-dependent increase in intracellular Ca2+, whose changes can affect the expression of the above-mentioned key proteins. In summary, we clarified the relationship between ACR exposure, neuronal damage and postsynaptic plasticity and proposed an ACR-CYP2E1-GA: Ca2+-PSD-95-NMDAR-Ca2+-CaMKII effect chain. This information will further improve the development of an alternative pathway strategy for investigating the risk posed by ACR.

Biomarkers of exposure and effect in the serum and urine of rats or workers exposed to 1-bromopropane.

Extensively used in several industries in China as a cleaning agent, 1-bromopropane (1-BP) has significant adverse effects on the central nervous system. However, neither its mechanism of action nor sensitive biomarkers related to it have been determined thus far. In this study, animal experiments and occupational surveys were performed to explore the typical exposure and effect biomarkers of neurotoxicity induced by 1-BP. Male Wistar rats were exposed to 0, 500, or 1000 ppm of 1-BP followed by pathological and biomarker analyses. An epidemiological survey was conducted on 71 workers each from 1-BP exposed and control groups. Serum and urine samples were collected for biomarker testing. cNSE represents neuron-specific enolase (NSE) in the cerebral cortex, where as sNSE represents NSE in the serum; similar terminology applies to S-100ß, and cyclooxygenase-2 (COX-2). In rats exposed to 1000 ppm 1-BP, pathological changes were observed in Purkinje cells, lumbar gray matter, and tibiofibular nerve, while levels of cNSE, cS-100ß, cCOX-2, sS-100ß, and sCOX-2 were significantly elevated at different time checkpoints. In the 500 ppm group, cCOX-2, sNSE, and sCOX-2 levels were significantly elevated at different time checkpoints. 1-BP and N-acetyl-S-(n-propyl)-L-cysteine (AcPrCys) were detected in rat urine, and there was a correlation between the level of sNSE or sCOX-2 and AcPrCys in the 500 ppm group. In the occupational epidemiological study, a significant correlation between AcPrCys and exposure concentration was also detected. The findings of this study indicated that AcPrCys was a sensitive exposure biomarker of 1-BP in rats as well as occupational populations.

Overcoming the acquired resistance to gefitinib in lung cancer brain metastasis in vitro and in vivo.

In our previous work, PC-9-Br, a PC-9 brain seeking line established via a preclinical animal model of lung cancer brain metastasis (LCBM), exhibited not only resistance to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib in vitro, but also chemotherapy regimens of cisplatin plus etoposide in vivo. Using this cell line, we investigated novel potential targeted therapeutics for treating LCBM in vitro and in vivo to combat drug resistance. Significant increases in mRNA and protein expression levels of Bcl-2 were found in PC-9-Br compared with parental PC-9 (PC-9-P), but no significant changes of Bcl-XL were observed. A remarkable synergistic effect between EGFR-TKI gefitinib and Bcl-2 inhibitors ABT-263 (0.17 ± 0.010 µM at 48 h and 0.02 ± 0.004 µM at 72 h), or ABT-199 (0.22 ± 0.008 µM at 48 h and 0.02 ± 0.001 µM at 72 h) to overcome acquired resistance to gefitinib (> 0.5 µM at 48 h and 0.10 ± 0.007 µM at 72 h) in PC-9-Br was observed in MTT assays. AZD9291 was also shown to overcome acquired resistance to gefitinib in PC-9-Br in MTT assays (0.23 ± 0.031 µM at 48 h and 0.03 ± 0.008 µM at 72 h). Western blot showed significantly decreased phospho-Erk1/2 and increased cleaved-caspase-3 expressions were potential synergistic mechanisms for gefitinib + ABT263/ABT199 in PC-9-Br. Significantly decreased protein expressions of phospho-EGFR, phospho-Akt, p21, and survivin were specific synergistic mechanism for gefitinib + ABT199 in PC-9-Br. In vivo studies demonstrated afatinib (30 mg/kg) and AZD9291 (25 mg/kg) could significantly reduce the LCBM in vivo and increase survival percentages of treated mice compared with mice treated with vehicle and gefitinib (6.25 mg/kg). In conclusion, our study demonstrated gefitinib + ABT263/ABT199, afatinib, and AZD9291 have clinical potential to treat LCBM.

Identification of anti-Epstein-Barr virus (EBV) antibody signature in EBV-associated gastric carcinoma.

BACKGROUND: Around 10% of gastric carcinomas (GC) contain Epstein-Barr virus (EBV) DNA. We characterized the GC-specific antibody response to this common infection, which may provide a noninvasive method to detect EBV-positive GC and elucidate its contribution to carcinogenesis. METHODS: Plasma samples from EBV-positive (n = 28) and EBV-negative (n = 34) Latvian GC patients were immune-profiled against 85 EBV proteins on a multi-microbial Nucleic Acid Programmable Protein Array (EBV-NAPPA). Antibody responses were normalized for each sample as ratios to the median signal intensity (MNI) across all antigens, with seropositivity defined as MNI ≥ 2. Antibodies with ≥ 20% sensitivity at 95% specificity for tumor EBV status were verified by enzyme-linked immunosorbent assay (ELISA) and validated in independent samples from Korea and Poland (n = 24 EBV-positive, n = 65 EBV-negative). RESULTS: Forty anti-EBV IgG and eight IgA antibodies were detected by EBV-NAPPA in ≥ 10% of EBV-positive or EBV-negative GC patients, of which nine IgG antibodies were discriminative for tumor EBV status. Eight of these nine were verified and seven were validated by ELISA: anti-LF2 (odds ratio = 110.0), anti-BORF2 (54.2), anti-BALF2 (44.1), anti-BaRF1 (26.7), anti-BXLF1 (12.8), anti-BRLF1 (8.3), and anti-BLLF3 (5.4). The top three had areas under receiver operating characteristics curves of 0.81-0.85 for distinguishing tumor EBV status. CONCLUSIONS: The EBV-associated GC-specific humoral response was exclusively directed against lytic cycle immediate-early and early antigens, unlike other EBV-associated malignancies such as nasopharyngeal carcinoma and lymphoma where humoral response is primarily directed against late lytic antigens. Specific anti-EBV antibodies could have utility for clinical diagnosis, epidemiologic studies, and immune-based precision treatment of EBV-positive GC.

Helicobacter pylori Immunoproteomic Profiles in Gastric Cancer.

Chronic Helicobacter pylori infection is the major risk factor for gastric cancer (GC). However, only some infected individuals develop this neoplasia. Previous H. pylori serology studies have been limited by investigating small numbers of candidate antigens. Therefore, we evaluated humoral responses to a nearly complete H. pylori immunoproteome (1527 proteins) among 50 GC cases and 50 controls using Nucleic Acid Programmable Protein Array (NAPPA). Seropositivity was defined as median normalized intensity ≥2 on NAPPA, and 53 anti-H. pylori antibodies had >10% seroprevalence. Anti-GroEL exhibited the greatest seroprevalence (77% overall), which agreed well with ELISA using whole-cell lysates of H. pylori cells. After an initial screen by H. pylori-NAPPA, we discovered and verified that 12 antibodies by ELISA in controls had ≥15% of samples with an optical reading value exceeding the 95th percentile of the GC group. ELISA-verified antibodies were validated blindly in an independent set of 100 case-control pairs. As expected, anti-CagA seropositivity was positively associated with GC (odds ratio, OR = 5.5; p < 0.05). After validation, six anti-H. pylori antibodies showed lower seropositivity in GC, with ORs ranging from 0.44 to 0.12 (p < 0.05): anti-HP1118/Ggt, anti-HP0516/HsIU, anti-HP0243/NapA, anti-HP1293/RpoA, anti-HP0371/FabE, and anti-HP0875/KatA. Among all combinations, a model with anti-Ggt, anti-HslU, anti-NapA, and anti-CagA had an area under the curve of 0.73 for discriminating GC vs. controls. This study represents the first comprehensive assessment of anti-H. pylori humoral profiles in GC. Decreased responses to multiple proteins in GC may reflect mucosal damage and decreased bacterial burden. The higher prevalence of specific anti-H. pylori antibodies in controls may suggest immune protection against GC development.

Brain-derived neurotrophic factor protects against acrylamide-induced neuronal and synaptic injury via the TrkB-MAPK-Erk1/2 pathway.

Acrylamide has been shown to be neurotoxic. Brain-derived neurotrophic factor (BDNF) can alleviate acrylamide-induced synaptic injury; however, the underlying mechanism remains unclear. In this study, dibutyryl-cyclic adenosine monophosphate-induced mature human neuroblastoma (NB-1) cells were exposed with 0-100 µg/mL acrylamide for 24-72 hours. Acrylamide decreased cell viability and destroyed synapses. Exposure of co-cultured NB-1 cells and Schwann cells to 0-100 µg/mL acrylamide for 48 hours resulted in upregulated expression of synapsin I and BDNF, suggesting that Schwann cells can activate self-protection of neurons. Under co-culture conditions, activation of the downstream TrkB-MAPK-Erk1/2 pathway strengthened the protective effect. Exogenous BDNF can increase expression of TrkB, Erk1/2, and synapsin I, while exogenous BDNF or the TrkB inhibitor K252a could inhibit these changes. Taken together, Schwann cells may act through the BDNF-TrkB-MAPK-Erk1/2 signaling pathway, indicating that BDNF plays an important role in this process. Therefore, exogenous BDNF may be an effective treatment strategy for acrylamide-induced nerve injury. This study was approved by the Laboratory Animal Welfare and Ethics Committee of the National Institute of Occupational Health and Poison Control, a division of the Chinese Center for Disease Control and Prevention (approval No. EAWE-2017-008) on May 29, 2017.

Chronic electronic cigarette use elicits molecular changes related to pulmonary pathogenesis.

The relative safety of chronic exposure to electronic cigarette (e-cig) aerosol remains unclear in terms of lung pathogenesis. Therefore, this study aims to evaluate gene/protein biomarkers, which are associated with cigarette-induced pulmonary injury in animals chronically exposed to nicotine containing e-cig aerosol. C57BL/6 J mice were randomly assigned to three exposure groups: e-cig, tobacco cigarette smoke, and filtered air. Lung tissues and/or paraffin embedded slides were used to evaluate gene and/or protein expressions of the CYP450 metabolism (CYP1A1, CYP2A5, and CYP3A11), oxidative stress (Nrf2, SOD1), epithelial-mesenchymal transition (E-cadherin and vimentin), lung pathogenesis (AhR), and survival/apoptotic pathways (p-AKT, BCL-XL, p53, p21, and CRM1). Expressions of E-cadherin and CRM1 were significantly decreased, while CYP1A1, AhR, SOD1 and BCL-XL were significantly upregulated in the e-cig group compared to the control (p < 0.05). Nuclear sub-cellular localization of p53, evaluated by immunohistochemistry staining, in bronchiolar tissues was higher in the e-cig group (25.3 ± 2.7%) as compared to controls (12.1 ± 1.8%) (p < 0.01). Although the biomarkers responses were not identical, in general, the responses had similar qualitative trends between the e-cig and cigarette groups. As these related molecular changes are involved in the pathogenesis of cigarette-induced lung injury, the possibility exists that e-cigs can produce a similar outcome. Although further investigation is warranted, e-cigs are unlikely to be considered as safe in terms of pulmonary health.

Synergistic effects of Bcl-2 inhibitors with AZD9291 on overcoming the acquired resistance of AZD9291 in H1975 cells.

Non-small cell lung cancer (NSCLC) patients with epithermal growth factor receptor (EGFR) mutations can be treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs), however, development of acquired resistance could significantly limit curative effects of EGFR-TKIs. Different mechanisms of acquired resistance to first-generation and second-generation EGFR TKIs have been widely reported, but there were few reports on the resistant mechanism of third-generation EGFR-TKI such as osimertinib (AZD9291). In the present study, significant upregulation of Bcl-2 was found in AZD9291-resistant H1975 cells (H1975AR) compared with H1975, which may constitute an important resistant mechanism of acquired resistance to AZD9291. More importantly, our study showed that synergism between AZD9291 and Bcl-2 inhibitor ABT263 (0.25 µM) or ABT199 (1 µM) could effectively overcome the acquired resistance of AZD9291 in H1975AR in vitro. Flow cytometry analyses demonstrated that AZD9291 + ABT263/ABT199 caused a significantly different cell cycle distribution and produced significantly more apoptosis compared with either AZD9291 or ABT263/ABT199 treatment alone. Further multiscreen/Western blot analyses revealed that NF-κB was significantly downregulated in AZD9291 + ABT263/ABT199 treatment groups compared with AZD9291 or ABT263/ABT199 treatment alone, with a more significant reduction of NF-κB in AZD9291 + ABT199 compared with AZD9291 + ABT263. It is also noticeable that AZD9291 + ABT263 specifically caused a significantly reduced expression of p21 compared with AZD9291 or ABT263 treatment alone while AZD9291 + ABT199 specifically caused significantly reduced expressions of SQSTM1 and survivin, but increased expression of autophagosome marker LC3-II compared with AZD9291 or ABT199 treatment alone. Furthermore, cytotoxicity of AZD9291 + ABT199 could be partially reversed by autophagy inhibitor chloroquine. These results suggest that ABT263 and ABT199 may work through different signaling pathways to achieve synergistic cytotoxicity with AZD9291 in H1975AR. These findings suggest that Bcl-2 inhibitor may provide an effective option in combination therapy with EGFR-TKIs to treat NSCLC with EGFR-TKI acquired resistance.

Drug resistance occurred in a newly characterized preclinical model of lung cancer brain metastasis.

BACKGROUND: Cancer metastasis and drug resistance have traditionally been studied separately, though these two lethal pathological phenomena almost always occur concurrently. Brain metastasis occurs in a large proportion of lung cancer patients (~ 30%). Once diagnosed, patients have a poor prognosis surviving typically less than 1 year due to lack of treatment efficacy. METHODS: Human metastatic lung cancer cells (PC-9-Br) were injected into the left cardiac ventricle of female athymic nude mice. Brain lesions were allowed to grow for 21 days, animals were then randomized into treatment groups and treated until presentation of neurological symptoms or when moribund. Prior to tissue collection mice were injected with Oregon Green and 14C-Aminoisobutyric acid followed by an indocyanine green vascular washout. Tracer accumulation was determined by quantitative fluorescent microscopy and quantitative autoradiography. Survival was tracked and tumor burden was monitored via bioluminescent imaging. Extent of mutation differences and acquired resistance was measured in-vitro through half-maximal inhibitory assays and qRT-PCR analysis. RESULTS: A PC-9 brain seeking line (PC-9-Br) was established. Mice inoculated with PC-9-Br resulted in a decreased survival time compared with mice inoculated with parental PC-9. Non-targeted chemotherapy with cisplatin and etoposide (51.5 days) significantly prolonged survival of PC-9-Br brain metastases in mice compared to vehicle control (42 days) or cisplatin and pemetrexed (45 days). Further in-vivo imaging showed greater tumor vasculature in mice treated with cisplatin and etoposide compared to non-tumor regions, which was not observed in mice treated with vehicle or cisplatin and pemetrexed. More importantly, PC-9-Br showed significant resistance to gefitinib by in-vitro MTT assays (IC50 > 2.5 µM at 48 h and 0.1 µM at 72 h) compared with parental PC-9 (IC50: 0.75 µM at 48 h and 0.027 µM at 72 h). Further studies on the molecular mechanisms of gefitinib resistance revealed that EGFR and phospho-EGFR were significantly decreased in PC-9-Br compared with PC-9. Expression of E-cadherin and vimentin did not show EMT in PC-9-Br compared with parental PC-9, and PC-9-Br had neither a T790M mutation nor amplifications of MET and HER2 compared with parental PC-9. CONCLUSION: Our study demonstrated that brain metastases of lung cancer cells may independently prompt drug resistance without drug treatment.

Analysis of Mutations in K-ras and p53 Genes in Sputum and Plasma Samples.

Sputum and plasma can provide noninvasive materials to investigate biomarkers for cancer detection and diagnosis. Mutations in the K-ras oncogene and p53 suppressor gene have been frequently found in sputum and plasma samples collected not only from lung cancer patients but also in those of patients prior to presenting clinical symptoms of lung cancer, suggesting that they may also provide useful biomarkers from early lung cancer diagnosis. However, the detection of these mutations has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells, and of cell-free DNA present in plasma. This chapter describes methods to isolate low fraction epithelial cells from sputum and cell-free DNA from plasma samples obtained from lung cancer patients and to identify low fraction K-ras and p53 mutations in these samples.

Oxidative stress induced by ultrafine carbon black particles can elicit apoptosis in vivo and vitro.

Although carbon black (CB) particles have potential hazards to human health, the toxicological studies on CB are still limited. The purpose of this study was to investigate the effect of oxidative stress induced by ultrafine CB particles on apoptosis in vivo and vitro. Male C57BL/6 mice were inhalation exposed to CB for 28 days, and 16HBE cells were treated by CB particles and also added antioxidant (NAC). Antioxidant enzymes activities (CAT, SOD, GSH-Px) and ROS in the lungs and cells were evaluated. Apoptosis-related proteins (Bcl-2, Bax, Cleaved Caspase-3, pro-Caspase-3, Caspase-7, Caspase-8, Caspase-9, PARP-1) were tested by Western blot (WB), immunohistochemistry (IHC), and real-time PCR. The reduction of antioxidant enzymes activities and the addition of ROS in CB exposure groups were observed, and the gene and apoptosis-related proteins levels were increased in CB exposure mice. The results of CB-treated 16HBE cells were consistent with those of mice, and apoptosis rate was increased in CB-treated 16HBE cells. When the cells were treated with NAC, ROS induced by CB decreased, SOD and CAT activities of CB-treated 16HBE cells were increased. Apoptosis rate of 16HBE cells treated with NAC and CB was significantly decreased, and the expression of C-Caspase-3 was also decreased. Therefore, oxidative stress induced by ultrafine CB particles can elicit apoptosis in vivo and vitro. Antioxidants can significantly reduce oxidative damage and apoptosis induced by CB.

Overcoming acquired resistance of gefitinib in lung cancer cells without T790M by AZD9291 or Twist1 knockdown in vitro and in vivo.

The T790M mutation is recognized as a typical mechanism of acquired resistance to first generation of epithermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib in non-small cell lung cancer (NSCLC) patients who are commonly treated by third generation of EGFR-TKI AZD9291 (osimertinib). However, the therapeutic strategy for overcoming acquired resistance to EGFR-TKIs in NSCLC patients without T790M remains to be definitively determined. In the present study, gefitinib-resistant H1650 (H1650GR) or AZD9291-resistant H1975 (H1975AR) was generated by exposing NSCLC cell line H1650 or H1975 to progressively increased concentrations of gefitinib or AZD9291 over 11 months. The cytotoxic effects of gefitinib or AZD9291 in vitro were evaluated via the half maximal inhibitory concentrations (IC50s) determined by the MTT assay. IC50 of gefitinib in H1650GR (50.0 ± 3.0 µM) significantly increased compared with H1650 (31.0 ± 1.0 µM) (p < 0.05). Similarly, the IC50 of AZD9291 in H1975AR (10.3 ± 0.9 µM) significantly increased compared with H1975 (5.5 ± 0.6 µM) (p < 0.05). However, IC50 of AZD9291 on H1650GR (8.5 ± 0.5 µM) did not increase compared with H1650 (9.7 ± 0.7 µM). On the other hand, IC50 of AZD9291 on gefitinib-resistant A549 (A549GR established in our previous study) (12.7 ± 0.8 µM) was significantly increased compared with A549 (7.0 ± 1.0 µM) (p < 0.05). AZD9291 induced caspase 3/7 activation in A549, H1650, and H1650GR, but not in A549GR. Western blot analyses showed that p-Akt played a key role in determining the sensitivities of A549, A549GR, H1650, and H1650GR to gefitinib or AZD9291. Additionally, increased expression of Twist1 was observed in all cells with acquired EGFR-TKI resistance and knockdown of Twist1 by shRNA was found to significantly enhance the sensitivity of A549GR to gefitinib or AZD9291 via reversing epithelial-mesenchymal transition and downregulating p-Akt, but not of H1975AR to AZD9291. The enhanced cytotoxic effect of AZD9291 on A549GR by Twist1 knockdown in vitro was further validated by in vivo studies which showed that Twist1 knockdown could lead to significantly delayed tumor growth of A549GR xenograft with increased sensitivity to AZD9291 treatment in nude mice without any observed side toxic effects. In summary, our study demonstrated that the mechanisms of acquired resistance in different NSCLC cell lines treated by even the same EGFR-TKI might be quite different, which provide a rationale for adopting different therapeutic strategies for those NSCLC patients with acquired EGFR-TKI resistance based on different status of heterogeneous mutations.

Ultrafine CB-induced small airway obstruction in CB-exposed workers and mice.

The potential threat of superfine carbon black (CB) particles to human health has received attention, but there are few human toxicological data available. The purpose of this study was to investigate the relationships between serum CC16 and SP-A with small airway related pulmonary functions in CB workers. Ninety-nine male CB packers and 115 non-CB-exposed healthy male workers were recruited. Serum CC16 and/or SP-A and pulmonary function tests were evaluated, and the relationship between them were also analyzed. To further assess pulmonary damage induced by CB particles in target organs, an animal inhalation exposure study was conducted. Male C57BL/6 mice were exposed to 15 and 30 mg/m3 CB for 6 h per day for 28 days. Levels of CC16 and SP-A were evaluated by ELISA and immunohistochemical staining (IHC). The results showed a 20% decreased in median CC16 and a 15% increase in median SP-A among CB-exposed workers. FEV1%, FEV/FVC, MMEF%, FEF25%, and FEF75% were also decreased in CB-exposed workers (P < 0.05). A significant positive correlation was observed between serum CC16 concentration and FEV1/FVC, although a negative correlation was found between serum SP-A concentration and FEV1/FVC. Serum CC16 was significantly reduced by 72% in mice with high CB-exposure, and serum SPA was 1.65x and 1.17x higher than CB-unexposed control mice in low and high CB-exposed mice, respectively. Lung CC16 and serum CC16 levels were positively correlated in mice (P = 0.024). Long-term exposure to ultrafine CB particles is associated with a decrease in CC16 and an increase in SP-A in the peripheral blood of CB-exposed workers. In conclusion, superfine CB particles have the potential to cause small airway obstruction.

Polycyclic aromatic hydrocarbons in breast milk of obese vs normal women: Infant exposure and risk assessment.

Biomonitoring of human breast milk is one of the best ways to identify body burdens of contaminants and associated risk estimation. The objectives of the current study were to evaluate milk concentrations of persistent organic pollutants (POPs), mainly polycyclic aromatic hydrocarbons (PAHs), associated exposure estimation, and the role of body mass index (BMI) in their bioaccumulation. A total of 45 breast milk samples were collected from 24 women with BMI > 30 (obese) and 21 women with BMI < 25 (18.5-24.9, normal) from 14 different counties surrounding Lubbock in west Texas/New Mexico (age range: 18-34 years). Samples were analyzed using high resolution gas chromatography coupled with mass spectrometry. A total of 31/45 (69%) of samples tested positive for PAHs. Phenanthrene was the most frequently detected PAH followed by pyrene and fluoranthene. The mean of individual PAH concentration for all samples ranged from 0 to 25.1 ng/g milk fat; the sum of all means of individual PAHs was 146.9 ng/g milk fat. The mean concentration of total PAHs in the BMI > 30 group was 224.8 ng/g milk fat, which was approximately 4 times the mean concentration of total PAHs in the BMI 18.5-24.9 group (57.9 ng/g milk fat). None of the samples from the BMI 18.5-24.9 group contained higher molecular weight (5-6 rings) PAHs, while in the BMI >30 group, a total of 11 PAHs including listed EPA priority pollutants were observed. In this study, benzo(b)fluoranthene was found to contribute the highest percentage of carcinogenic PAHs (32.08%), yet it was not detected in any samples from the BMI 18.5-24.9 group. The estimated total PAHs intakes by infants via obese and normal mothers' milk were 1.26 and 0.32 (µg/kg/day), which are 0.049 and 0.003 (µg/kg/day) B[a]P equivalent, respectively. These findings suggest that breastfed babies from obese mothers are potentially at higher risk of exposure to carcinogenic PAHs.