Myxobacteria versus sponge-derived alkaloids: the bengamide family identified as potent immune modulating agents by scrutiny of LC-MS/ELSD libraries.

A nuclear factor-κB (NF-κB) luciferase assay has been employed to identify the bengamides, previously known for their anti-tumor activity, as a new class of immune modulators. A unique element of this study was that the bengamide analogs were isolated from two disparate sources, Myxococcus virescens (bacterium) and Jaspis coriacea (sponge). Comparative LC-MS/ELSD and NMR analysis facilitated the isolation of M. viriscens derived samples of bengamide E (8) and two congeners, bengamide E\u27 (13) and F\u27 (14) each isolated as an insperable mixture of diastereomers. Additional compounds drawn from the UC, Santa Cruz repository allowed expansion of the structure activity relationship (SAR) studies. The activity patterns observed for bengamide A (6), B (7), E (8), F (9), LAF 389 (12) and 13-14 gave rise to the following observations and conclusions. Compounds 6 and 7 display potent inhibition of NF-κB (at 80 and 90 nM, respectively) without cytotoxicity to RAW264.7 macrophage immune cells. Western blot and qPCR analysis indicated that 6 and 7 reduce the phosphorylation of IκBα and the LPS-induced expression of the pro-inflammatory cytokines/chemokines TNFα, IL-6 and MCP-1 but do not effect NO production or the expression of iNOS. These results suggest that the bengamides may serve as therapeutic leads for the treatment of diseases involving inflammation, that their anti-tumor activity can in part be attributed to their ability to serve as immune modulating agents, and that their therapeutic potential against cancer merits further consideration

Development and validation of a rapid method for the detection of latrunculol A in plasma

Latrunculol A is a recently discovered 6,7-dihydroxy analog of the potent actin inhibitor latrunculin A. Latrunculol A has exhibited greater cytotoxicity than latrunculin A against both murine and human colon tumor cell lines in vitro. Currently, there are no reports regarding the bioavailability of latrunculol A in vivo. This study was undertaken as a prelude to pharmacokinetic assessments and it is the first work where bioavailability of latrunculol A was studied. In the present work, a simple plasma preparation and a rapid HPLC method have been developed. Mouse plasma containing latrunculol A was first treated by acetonitrile and then centrifuged at 14,000 rpm at 4 °C for 25 min. The supernatant was injected in an HPLC system comprising a Waters Symmetry NH2 column, a mobile phase of acetonitrile/water (95/5, v/v), a flow rate of 1.0 mL/min, at 220 nm. The method was validated by parameters including a good linear correlation, a limit of quantification of 9 ng/mL, and a good precision with a coefficient variation of 1.65, 1.86, and 1.26% for 20, 400, and 800 ng/mL, respectively. With this simple method, excellent separation and sensitivity of latrunculol A are achieved, thus allowing a rapid analysis of the plasma samples for absorption, distribution, and metabolism studies

Natural Products Chemistry and Taxonomy of the Marine Cyanobacterium Blennothrix cantharidosmum

A Papua New Guinea field collection of the marine cyanobacterium Blennothrix cantharidosmum was investigated for its cytotoxic constituents. Bioassay-guided isolation defined the cytotoxic components as the known compounds lyngbyastatins 1 and 3. However, six new acyl proline derivatives, tumonoic acids D−I, plus the known tumonoic acid A were also isolated. Their planar structures were defined from NMR and MS data, while their stereostructures followed from a series of chiral chromatographies, degradation sequences, and synthetic approaches. The new compounds were tested in an array of assays, but showed only modest antimalarial and inhibition of quorum sensing activities. Nevertheless, these are the first natural products to be reported from this genus, and this inspired a detailed morphologic and 16S rDNA-based phylogenetic analysis of the producing organism

Variation in RNA expression and genomic DNA content acquired during cell culture

Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14c, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. The RNA arrays also demonstrated considerable variation between the three different subcultures, with only 43% of genes expressed at the same levels in all three. Moreover, the patterns of the expressed genes did not always reflect our observed CGH aberrations. These results demonstrate extensive genomic instability and variation in RNA expression during subculture and provide supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such cultures as models of human cancer. This work also reinforces the concern that comparisons of published analyses of cultures of the same name may be dangerous

LGR5 expression is regulated by EGF in early colorectal adenomas and governs EGFR inhibitor sensitivity.

BACKGROUND LGR5 serves as a co-receptor for Wnt/β-catenin signalling and marks normal intestinal stem cells; however, its role in colorectal cancer (CRC) remains controversial. LGR5 cells are known to exist outside the stem cell niche during CRC progression, and the requirement for epidermal growth factor (EGF) signalling within early adenomas remains to be fully elucidated. METHODS Epidermal growth factor and gefitinib treatments were performed in EGF-responsive LGR5 early adenoma RG/C2 cells. 2D growth assays were measured using an IncuCyte. LGR5 or MEK1/2 silencing studies were executed using siRNA and LGR5 expression was assessed by qRT-PCR and immunoblotting. Ki67 level and cell cycle status were analysed by flow cytometry. RESULTS Epidermal growth factor suppresses expression of LGR5 at both the transcript and protein level in colorectal adenoma and carcinoma cells. Suppression of LGR5 reduces the survival of EGF-treated adenoma cells by increasing detached cell yield but also inducing a proliferative state, as evidenced by elevated Ki67 level and enhanced cell cycle progression. Repression of LGR5 further increases the sensitivity of adenoma cells to EGFR inhibition. CONCLUSIONS LGR5 has an important role in the EGF-mediated survival and proliferation of early adenoma cells and could have clinical utility in predicting response of CRC patients to EGFR therapy

A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs

The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TSQ) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27kip1 compared to proliferating spheroids (TSP). The quiescent status of the TSQ was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TSQ retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TSP. However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TSQ. In contrast, 5-FU and vinblastine did not produce cell death in the TSQ. In summary, TSQ show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours

Exosomes derived from mesenchymal stem cells enhance radiotherapy-induced cell death in tumor and metastatic tumor foci

We have recently shown that radiotherapy may not only be a successful local and regional treatment but, when combined with MSCs, may also be a novel systemic cancer therapy. This study aimed to investigate the role of exosomes derived from irradiated MSCs in the delay of tumor growth and metastasis after treatment with MSC + radiotherapy (RT). The tumor cell loss rates found after treatment with the combination of MSC and RT and for exclusive RT, were: 44.4% % and 12,1%, respectively. Concomitant and adjuvant use of RT and MSC, increased the mice surviving time 22,5% in this group, with regard to the group of mice treated with exclusive RT and in a 45,3% respect control group. Moreover, the number of metastatic foci found in the internal organs of the mice treated with MSC + RT was 60% less than the mice group treated with RT alone. We reasoned that the exosome secreted by the MSC, could be implicated in tumor growth delay and metastasis control after treatment. Our results show that exosomes derived form MSCs, combined with radiotherapy, are determinant in the enhancement of radiation effects observed in the control of metastatic spread of melanoma cells and suggest that exosome-derived factors could be involved in the bystander, and abscopal effects found after treatment of the tumors with RT plus MSC. Radiotherapy itself may not be systemic, although it might contribute to a systemic effect when used in combination with mesenchymal stem cells owing the ability of irradiated MSCs-derived exosomes to increase the control of tumor growth and metastasis.This work was supported by CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brasil, Junta de Andalucía, project of Excellence from Junta de Andalucía P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2015-70520-R to FJO and JMRdA, RTICC RD12/0036/0026 and CIBER Cáncer ISCIII CB16/12/00421 to FJO

Protein expression profiles indicative for drug resistance of non-small cell lung cancer

Data obtained from multiple sources indicate that no single mechanism can explain the resistance to chemotherapy exhibited by non-small cell lung carcinomas. The multi-factorial nature of drug resistance implies that the analysis of comprising expression profiles may predict drug resistance with higher accuracy than single gene or protein expression studies. Forty cellular parameters (drug resistance proteins, proliferative, apoptotic, and angiogenic factors, products of proto-oncogenes, and suppressor genes) were evaluated mainly by immunohistochemistry in specimens of primary non-small cell lung carcinoma of 94 patients and compared with the response of the tumours to doxorubicin in vitro. The protein expression profile of non-small cell lung carcinoma was determined by hierarchical cluster analysis and clustered image mapping. The cluster analysis revealed three different resistance profiles. The frequency of each profile was different (77, 14 and 9%, respectively). In the most frequent drug resistance profile, the resistance proteins P-glycoprotein/MDR1 (MDR1, ABCB1), thymidylate-synthetase, glutathione-S-transferase-π, metallothionein, O6-methylguanine-DNA-methyltransferase and major vault protein/lung resistance-related protein were up-regulated. Microvessel density, the angiogenic factor vascular endothelial growth factor and its receptor FLT1, and ECGF1 as well were down-regulated. In addition, the proliferative factors proliferating cell nuclear antigen and cyclin A were reduced compared to the sensitive non-small cell lung carcinoma. In this resistance profile, FOS was up-regulated and NM23 down-regulated. In the second profile, only three resistance proteins were increased (glutathione-S-transferase-π, O6-methylguanine-DNA-methyltransferase, major vault protein/lung resistance-related protein). The angiogenic factors were reduced. In the third profile, only five of the resistance factors were increased (MDR1, thymidylate-synthetase, glutathione-S-transferase-π, O6-methylguanine-DNA-methyltransferase, major vault protein/lung resistance-related protein)

Identification of molecular mechanisms for cellular drug resistance by combining drug activity and gene expression profiles

Acquired drug resistance is a major problem in cancer treatment. To explore the genes involved in chemosensitivity and resistance, 10 human tumour cell lines, including parental cells and resistant subtypes selected for resistance against doxorubicin, melphalan, teniposide and vincristine, were profiled for mRNA expression of 7400 genes using cDNA microarray technology. The drug activity of 66 cancer agents was evaluated on the cell lines, and correlations between drug activity and gene expression were calculated and ranked. Hierarchical clustering of drugs based on their drug–gene correlations yielded clusters of drugs with similar mechanism of action. Genes correlated with drug sensitivity and resistance were imported into the PathwayAssist software to identify putative molecular pathways involved. A substantial number of both proapoptotic and antiapoptotic genes such as signal transducer and activator of transcription 1, mitogen-activated protein kinase 1 and focal adhesion kinase were found to be associated to drug resistance, whereas genes linked to cell cycle control and proliferation, such as cell division cycle 25A and signal transducer of activator of transcription 5A, were associated to general drug sensitivity. The results indicate that combined information from drug activity and gene expression in a resistance-based cell line panel may provide new knowledge of the genes involved in anticancer drug resistance and become a useful tool in drug development