Genetic mutations associated with lung cancer metastasis to the brain.

Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. As a barrier to metastasis, cells normally undergo an apoptotic process known as 'anoikis', in circulation. The recent technological advances in the isolation and characterisation of rare circulating tumour cells (CTCs) will allow a better understanding of anoikis resistance. Detailed molecular and functional analyses of anoikis-resistant cells may provide insight into the biology of cancer metastasis and help identify novel targets for prevention of cancer dissemination. To uncover the molecular changes that govern the transition from a primary lung tumour to a secondary metastasis and specifically the mechanisms by which CTCs survive in circulation, we carried out whole genome sequencing (WGS) of normal lung, primary tumours and the corresponding brain metastases from five patients with progressive metastatic non-small-cell lung carcinoma. We also isolated CTCs from patients with metastatic cancer and subjected them to whole genome amplification and Sanger sequencing of genes of interest. While the primary tumours showed mutations in genes associated with cell adhesion and motility, brain metastases acquired mutations in adaptive, cytoprotective genes involved in response to cellular stress such as Keap-1, Nrf2 and P300, which are key players of the Keap1-Nrf2-ARE survival pathway. Nrf2 is a transcriptional factor that upon stress translocates into the nucleus, binds to the anti-oxidant response elements (ARE) and drives the expression of anti-oxidant genes. The identified mutations affect regulatory domains in all three proteins, suggesting a functional role in providing a survival advantage to CTCs in the peripheral blood allowing their dissemination to distant organs.

Shikonin Alleviates Gentamicin-Induced Renal Injury in Rats by Targeting Renal Endocytosis, SIRT1/Nrf2/HO-1, TLR-4/NF-κB/MAPK, and PI3K/Akt Cascades.

Gentamicin causes kidney injury due to its accumulation in proximal tubule epithelial cells via the megalin/cubilin/CLC-5 complex. Recently, shikonin has been shown to have potential anti-inflammatory, antioxidant, antimicrobial, and chloride channel-inhibiting effects. The current study investigated the alleviation of gentamicin-induced renal injury by shikonin while preserving its bactericidal effect. Nine-week-old Wistar rats were administered 6.25, 12.5, and 25 mg/kg/day shikonin orally, one hour after the i.p. injection of 100 mg/kg/day gentamicin for seven days. Shikonin significantly and dose-dependently alleviated gentamicin-induced renal injury, as revealed by restoring normal kidney function and histological architecture. Furthermore, shikonin restored renal endocytic function, as indicated by suppressing the elevated renal megalin, cubilin, and CLC-5 and enhancing the reduced NHE3 levels and mRNA expressions induced by gentamicin. These potentials could be attributed to the modulation of the renal SIRT1/Nrf2/HO-1, TLR-4/NF-κB/MAPK, and PI3K/Akt cascades, which enhanced the renal antioxidant system and suppressed renal inflammation and apoptosis, as indicated by enhancements of SIRT1, Nrf2, HO-1, GSH, SOD, TAC, Iκb-α, Bcl-2, PI3K, and Akt levels and mRNA expressions, with reduction of TLR-4, NF-κB, MAPK, IL-1ß, TNF-α, MDA, iNOS, NO, cytochrome c, caspase-3, Bax levels, and Bax/Bcl-2 ratio. Therefore, shikonin is a promising therapeutic agent for alleviating gentamicin-induced renal injury.

Neuroprotective Effects of Nano-Curcumin against Cypermethrin Associated Oxidative Stress and Up-Regulation of Apoptotic and Inflammatory Gene Expression in Rat Brains.

Cypermethrin (CPM) is the most toxic synthetic pyrethroid that has established neurotoxicity through oxidative stress and neurochemical agitation in experimental rats. The toxic effects are supposed to be mediated by modifying the sodium channels, reducing Na-K ATPase, acetylcholine esterase (AchE), and monoamine oxidase (MAO). The use of curcumin nanoparticles (NC) that have potent antioxidant, anti-inflammatory and antiapoptotic properties with improved bioavailability attenuates neurotoxicity in rat brains. To test this hypothesis, animals were divided into five groups, each having six animals. Group-I control received vehicle only, while Group-II was treated with 50 mg/kg CPM. Group-III and Group-IV received both CPM and NC 2.5 mg/kg and 5 mg/kg, respectively. Group-V received 5 mg of NC alone. The CPM and NC were given by oral route. Afterwards, brain antioxidant status was measured by assessing lipid peroxidation (LPO), 4-HNE, glutathione reduced (GSH), antioxidant enzyme catalase, and superoxide dismutase (SOD) along with neurotoxicity markers Na-K ATPase, AchE, and MAO. Inflammation and apoptosis indices were estimated by ELISA, qRT-PCR, and immunohistochemistry, while morphologic changes were examined by histopathology. Observations from the study confirmed CPM-induced neurotoxicity by altering Na-K ATPase, AchE, and MAO, and by decreasing the activity of antioxidant enzymes and GSH. Oxidative stress marker LPO and the level of inflammatory interleukins IL-6, IL-1ß, and TNF-α were notably high, and elevated expressions of Bax, NF-kB, and caspase-3 and -9 were reported in CPM group. However, NC treatment against CPM offers protection by improving antioxidant status and lowering LPO, inflammation, and apoptosis. The neurotoxicity marker's enzyme successfully attenuated after NC treatment. Therefore, this study supports the administration of NC effectively ameliorated CPM-induced neurotoxicity in experimental rats.

The Antimicrobial, Antioxidant, and Anticancer Activity of Greenly Synthesized Selenium and Zinc Composite Nanoparticles Using Ephedra aphylla Extract.

The current work aimed to synthesize selenium and zinc nanoparticles using the aqueous extract of Ephedra aphylla as a valuable medicinal plant. The prepared nanoparticles were characterized by TEM, zeta potential, and changes in the phytochemical constituents. Hence, the phenolic, flavonoid, and tannin contents were reduced in the case of the prepared samples of nanoparticles than the original values in the aqueous extract. The prepared extract of Ephedra aphylla and its selenium and zinc nanoparticles showed high potency as antioxidant agents as a result of the DPPH• assay. The samples were assessed as anticancer agents against six tumor cells and a normal lung fibroblast (WI-38) cell line. The selenium nanoparticles of Ephedra aphylla extract revealed very strong cytotoxicity against HePG-2 cells (inhibitory concentration (IC50) = 7.56 ± 0.6 µg/mL), HCT-116 cells (IC50 = 10.02 ± 0.9 µg/mL), and HeLa cells (IC50 = 9.23 ± 0.8 µg/mL). The samples were evaluated as antimicrobial agents against bacterial and fungal strains. Thus, selenium nanoparticles showed potent activities against Gram-negative strains (Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli), Gram-positive strains (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Staphylococcus epidermidis), and the fungal strain Candida albicans. In conclusion, the preparation of nanoparticles of either selenium or zinc is crucial for improved biological characteristics.

Exosomes as a Surrogate Marker for Autophagy in Peripheral Blood, Correlative Data from Phase I Study of Chloroquine in Combination with Carboplatin/Gemcitabine in Advanced Solid Tumors.

BACKGROUND: Autophagy is a catabolic process, utilized constitutionally by body cells to recycle nutrients and to remove unwanted/damaged intracellular constituents. It is enhanced during periods of stress, such as starvation and hypoxia, aiding in cell survival and it is linked to major cellular processes, such as apoptosis and antigen expression. The process has been extensively studied in vitro models or tumor tissue samples with rare application on human subjects. METHODS: Plasma samples from 24 advanced solid tumor patients were collected at different time points before and after chemotherapy. Their exosomes were isolate and blotted for microtubule-associated protein-1 light chain-3 (LC-3B) protein as a marker for autophagy. All the subjects received a standard chemotherapy regimen of carboplatin- gemcitabine with chloroquine (CQ)/ hydroxychloroquine (HCQ) in chronic doses throughout their treatment period as an autophagy modulator. CQ/HCQ was given in 50 mg increments as guided by their tolerability to treatment. RESULTS: A total of 267 plasma samples were obtained for the 24 patients and processed. Each sample corresponds to a single time point. The first group included 6 patients, all received 50 mg of CQ with chemotherapy. LC-3B I was detected in their isolated exosomes, while LC3-BII was not detected in their samples. The second cohort of patients included 3 subjects who re-ceived 100mg of HCQ. They demonstrated both LC3-BI and II on day 15 after chemotherapy in one patient, and on third cycle after 24 hours in the second patient. The third cohort included 3 subjects who received 150 mg of HCQ. All cases demonstrated LC3-BI and II on first cycle of treatment after less than 24 hours. The last cohort included 8 subjects, who received a fixed dose of 100 mg of HCQ with treatment. In this cohort, we were able to detect both LC3-B isoforms on advanced time points of second and third cycles. CONCLUSION: Detection of autophagy protein LC3-B in exosomes serves as a dynamic method to monitor autophagy. It can be utilized to study the effects of anti-neoplastic agents on autophagy and mechanisms of drug resistance, however, to standardize our results a larger specimen of patients should be included.

ROS1 amplification mediates resistance to gefitinib in glioblastoma cells.

Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed EGFR, however, tumors treated with tyrosine kinase inhibitors (TKIs) will inevitably recur highlighting the need to identify signalling pathways involved in GBM resistance to these drugs. To this end, we treated GBM cells that overexpress EGFR with increasing concentrations of gefitinib and isolated resistant clones. These resistant clones were subject to RNAseq and the expression of several genes was found to be upregulated. These genes are mainly tyrosine kinase receptors and include ROS1, DDR1 and PDGFRA and are known to control several downstream targets of EGFR. The upregulation of ROS1 and DDR1 was confirmed at the protein level by western blot. Treatment with a potent and highly specific pyrazole ROS1 inhibitor in ROS1 overexpressing clones led to a sensitization of these cells to low concentrations of gefitinib. Combined treatment with gefitinib and ROS1 inhibitor induces massive cell death by apoptosis following a prolonged S phase cell cycle arrest. Our current study led to the discovery of alternative pathways used by GBM cells to evade cell death following treatment with gefitinib and identifies new therapeutic targets to prevent GBM cell resistance to the drug.