The HGF/Met Receptor Mediates Cytotoxic Effect of Bacterial Cyclodipeptides in Human Cervical Cancer Cells.

BACKGROUND: Human cervix adenocarcinoma (CC) caused by papillomavirus is the third most common cancer among female malignant tumors. Bioactive compounds such as cyclodipeptides (CDPs) possess cytotoxic effects in human cervical cancer HeLa cells mainly by blocking the PI3K/Akt/mTOR pathway and subsequently inducing gene expression by countless transcription regulators. However, the upstream elements of signaling pathways have not been well studied. METHODS: To elucidate the cytotoxic and antiproliferative responses of the HeLa cell line to CDPs by a transcriptomic analysis previously carried out, we identified by immunochemical analyses, differential expression of genes related to the hepatocyte growth factor/mesenchymal-epithelial transition factor (HGF/MET) receptors. Furthermore, molecular docking was carried out to evaluate the interactions of CDPs with the EGF and MET substrate binding sites. RESULTS: Immunochemical and molecular docking analyses suggest that the HGF/MET receptor participation in CDPs cytotoxic effect was independent of the protein expression levels. However, protein modulation of downstream Met-targets occurred due to the inhibition of phosphorylation of the HGF/MET receptor. Results suggest that the antiproliferative and cytotoxicity of CDPs in HeLa cells involve the HGF/MET receptor upstream of PI3K/Akt/mTOR pathway; assays with the human breast cancer MCF-7 and MDA-MB-231cell lines supported the finding. CONCLUSION: Data provide new insights into the molecular mechanisms involved in CDPs cytotoxicity and antiproliferative effects, suggesting that the signal transduction mechanism may be related to the inhibition of the phosphorylation of the EGF/MET receptor at the level of substrate binding site by an inhibition mechanism similar to that of Gefitinib and foretinib anti-neoplastic drugs.

Linolenic Acid Plus Ethanol Exacerbates Cell Death in Saccharomyces cerevisiae by Promoting Lipid Peroxidation, Cardiolipin Loss, and Necrosis.

Polyunsaturated fatty acids (PUFA) hypersensitize yeast to oxidative stress. Ethanol accumulation during fermentation is another factor that induces oxidative stress via mitochondrial dysfunction and ROS overproduction. Since this microorganism has raised growing interest as a PUFA factory, we have studied if the combination of PUFA plus ethanol enhances yeast death. Respiration, ROS generation, lipid peroxidation, mitochondrial cardiolipin content, and cell death were assessed in yeast grown in the presence of 10% ethanol (ETOH) or linolenic acid (C18:3), or ethanol plus C18:3 (ETOH+C18:3). Lipid peroxidation and cardiolipin loss were several-fold higher in cells with ETOH+C18:3 than with C18:3. On the contrary, ETOH tended to increase cardiolipin content without inducing changes in lipid peroxidation. This was consistent with a remarkable diminution of cell growth and an exacerbated propidium iodide staining in cells with only ETOH+C18:3. The respiration rate decreased with all the treatments to a similar degree, and this was paralleled with similar increments in ROS between all the treatments. These results indicate that PUFA plus ethanol hypersensitize yeast to necrotic cell death by exacerbating membrane damage and mitochondrial cardiolipin loss, independent of mitochondrial dysfunction and ROS generation. The implications of these observations for some biotechnological applications in yeast and its physiology are discussed.

Transcriptomics Reveals the Mevalonate and Cholesterol Pathways Blocking as Part of the Bacterial Cyclodipeptides Cytotoxic Effects in HeLa Cells of Human Cervix Adenocarcinoma.

The incidence of human cervix adenocarcinoma (CC) caused by papillomavirus genome integration into the host chromosome is the third most common cancer among women. Bacterial cyclodipeptides (CDPs) exert cytotoxic effects in human cervical cancer HeLa cells, primarily by blocking the PI3K/Akt/mTOR pathway, but downstream responses comprising gene expression remain unstudied. Seeking to understand the cytotoxic and anti-proliferative effects of CDPs in HeLa cells, a global RNA-Seq analysis was performed. This strategy permitted the identification of 151 differentially expressed genes (DEGs), which were either up- or down-regulated in response to CDPs exposure. Database analysis, including Gene Ontology (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG), revealed differential gene expression on cancer transduction signals, and metabolic pathways, for which, expression profiles were modified by the CDPs exposure. Bioinformatics confirmed the impact of CDPs in the differential expression of genes from signal transduction pathways such as PI3K-Akt, mTOR, FoxO, Wnt, MAPK, P53, TGF-ß, Notch, apoptosis, EMT, and CSC. Additionally, the CDPs exposure modified the expression of cancer-related transcription factors involved in the regulation of processes such as epigenetics, DNA splicing, and damage response. Interestingly, transcriptomic analysis revealed the participation of genes of the mevalonate and cholesterol biosynthesis pathways; in agreement with this observation, total cholesterol diminished, confirming the blockage of the cholesterol synthesis by the exposure of HeLa cells to CDPs. Interestingly, the expression of some genes of the mevalonate and cholesterol synthesis such as HMGS1, HMGCR, IDI1, SQLE, MSMO1, SREBF1, and SOAT1 was up-regulated by CDPs exposure. Accordingly, metabolites of the mevalonate pathway were accumulated in cultures treated with CDPs. This finding further suggests that the metabolism of cholesterol is crucial for the occurrence of CC, and the blockade of the sterol synthesis as an anti-proliferative mechanism of the bacterial CDPs, represents a reasonable chemotherapeutic drug target to explore. Our transcriptomic study supports the anti-neoplastic effects of bacterial CDPs in HeLa cells shown previously, providing new insights into the transduction signals, transcription factors and metabolic pathways, such as mevalonate and cholesterol that are impacted by the CDPs and highlights its potential as anti-neoplastic drugs.

Participation of Acyl-Coenzyme A Synthetase FadD4 of Pseudomonas aeruginosa PAO1 in Acyclic Terpene/Fatty Acid Assimilation and Virulence by Lipid A Modification.

The pathogenic bacterium Pseudomonas aeruginosa possesses high metabolic versatility, with its effectiveness to cause infections likely due to its well-regulated genetic content. P. aeruginosa PAO1 has at least six fadD paralogous genes, which have been implicated in fatty acid (FA) degradation and pathogenicity. In this study, we used mutagenesis and a functional approach in P. aeruginosa PAO1 to determine the roles of the fadD4 gene in acyclic terpene (AT) and FA assimilation and on pathogenicity. The results indicate that fadD4 encodes a terpenoyl-CoA synthetase utilized for AT and FA assimilation. Additionally, mutations in fadD paralogs led to the modification of the quorum-sensing las/rhl systems, as well as the content of virulence factors pyocyanin, biofilm, rhamnolipids, lipopolysaccharides (LPS), and polyhydroxyalkanoates. In a Caenorhabditis elegans in vivo pathogenicity model, culture supernatants from the 24-h-grown fadD4 single mutant increased lethality compared to the PAO1 wild-type (WT) strain; however, the double mutants fadD1/fadD2, fadD1/fadD4, and fadD2/fadD4 and single mutant fadD2 increased worm survival. A correlation analysis indicated an interaction between worm death by the PAO1 strain, the fadD4 mutation, and the virulence factor LPS. Fatty acid methyl ester (FAME) analysis of LPS revealed that a proportion of the LPS and FA on lipid A were modified by the fadD4 mutation, suggesting that FadD4 is also involved in the synthesis/degradation and modification of the lipid A component of LPS. LPS isolated from the fadD4 mutant and double mutants fadD1/fadD4 and fadD2/fadD4 showed a differential behavior to induce an increase in body temperature in rats injected with LPS compared to the WT strain or from the fadD1 and fadD2 mutants. In agreement, LPS isolated from the fadD4 mutant and double mutants fadD1/fadD2 and fadD2/fadD4 increased the induction of IL-8 in rat sera, but IL1-ß cytokine levels decreased in the double mutants fadD1/fadD2 and fadD1/fadD4. The results indicate that the fadD genes are implicated in the degree of pathogenicity of P. aeruginosa PAO1 induced by LPS-lipid A, suggesting that FadD4 contributes to the removal of acyl-linked FA from LPS, rendering modification in its immunogenic response associated to Toll-like receptor TLR4. The genetic redundancy of fadD is important for bacterial adaptability and pathogenicity over the host.

Bacterial Cyclodipeptides Target Signal Pathways Involved in Malignant Melanoma.

Melanoma is an aggressive cancer that utilizes multiple signaling pathways, including those that involve oncogenes, proto-oncogenes, and tumor suppressors. It has been suggested that melanoma formation requires cross-talk of the PI3K/Akt/mTOR and Ras-ERK pathways. This pathway cross-talk has been associated with aggressiveness, drug resistance, and metastasis; thus, simultaneous targeting of components of the different pathways involved in melanoma may aid in therapy. We have previously reported that bacterial cyclodipeptides (CDPs) are cytotoxic to HeLa cells and inhibit Akt phosphorylation. Here, we show that CDPs decreased melanoma size and tumor formation in a subcutaneous xenografted mouse melanoma model. In fact, CDPs accelerated death of B16-F0 murine melanoma cells. In mice, antitumor effect was improved by treatment with CDPs using cyclodextrins as drug vehicle. In tumors, CDPs caused nuclear fragmentation and changed the expression of the Bcl-2 and Ki67 apoptotic markers and promoted restoration of hyperactivation of the PI3K/Akt/mTOR pathway. Additionally, elements of several signaling pathways such as the Ras-ERK, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, epithelial-mesenchymal transition, and cancer stem cell pathways were also modified by treatment of xenografted melanoma mice with CDPs. The findings indicate that the multiple signaling pathways implicated in aggressiveness of the murine B16-F0 melanoma line are targeted by the bacterial CDPs. Molecular modeling of CDPs with protein kinases involved in neoplastic processes suggested that these compounds could indeed interact with the active site of the enzymes. The results suggest that CDPs may be considered as potential antineoplastic drugs, interfering with multiple pathways involved in tumor formation and progression.

The isc gene cluster expression ethanol tolerance associated improves its ethanol production by organic acids flux redirection in the ethanologenic Escherichia coli KO11 strain.

Fossil fuels consumption impacts the greenhouse gas emissions. Biofuels are considered as alternative renewable energy sources to reduce the fossil fuels dependency. Bioethanol produced by recombinant microorganisms is a widely suggested alternative to increase the yield in fermentation processes. However, ethanol and acetate accumulation under the fermentation process had been described as important stressors for the metabolic capabilities of the microorganisms, stopping the fermentation process and affecting the ethanol yield. Ethanol tolerance is a determining factor in the improvement of fermentative properties of microorganisms; however understanding of ethanol tolerance is limited. The engineered Escherichia coli KO11 strain has been studied in detail and used as an ethanologenic bacteria model. The strain is capable of using glucose and xylose for an efficient ethanol yield. In the current work, the effect of the iron-sulfur cluster (ISC) over-expression in the KO11 strain, on its tolerance and ethanol yield, was evaluated. Fatty acids profiles of membrane phospholipids in the E. coli KO11 were modified under ethanol addition, but not due to the hscA mutation. The hscA mutation provoked a decrease in ethanol tolerance in the Kmp strain when was grown with 2% ethanol, in comparison to KO11 parent strain. Ethanol tolerance was improved in the mutant Kmp complemented with the recombinant isc gene cluster (pJC10 plasmid) from LD50 2.16% to LD50 3.8% ethanol. In batch fermentation on 1 L bioreactor using mineral medium with glucose (120 g/L), the KO11 strain showed ethanol production efficiencies of ~ 76.9%, while the hscA mutant (Kmp) ~ 75.4% and the transformed strain Kmp(pJC10) showed ~ 92.4% efficiency. Ethanol amount increase in the engineered Kmp(pJC10) strain was correlated with less organic acids (such as acetate and lactate) production in the fermentation medium (2.3 g/L), compared to that in the KO11 (17.05 g/L) and the Kmp (16.62 g/L). Alcohol dehydrogenase (ADH) activity was increased ~ 350% in the transformed Kmp(pJC10) strain, whereas in the Kmp mutant, the phosphoglycerate kinase (PGK), pyruvate kinase (PYK), and ADH activities were diminished, comparing to KO11. The results suggest that the isc system over-expression in the ethanologenic E. coli KO11 strain, increases ethanol yield mainly by improving ethanol tolerance and ADH activity, and by redirecting the metabolic flux from acetate synthesis to ethanol.

Over-expression of Isu1p and Jac1p increases the ethanol tolerance and yield by superoxide and iron homeostasis mechanism in an engineered Saccharomyces cerevisiae yeast.

The ethanol stress response in ethanologenic yeast during fermentation involves the swishing of several adaptation mechanisms. In Saccharomyces cerevisiae, the Jac1p and Isu1p proteins constitute the scaffold system for the Fe-S cluster assembly. This study was performed using the over-expression of the Jac1p and Isu1p in the industrially utilized S. cerevisiae UMArn3 strain, with the objective of improving the Fe-S assembly/recycling, and thus counteracting the toxic effects of ethanol stress during fermentation. The UMArn3 yeast was transformed with both the JAC1-His and ISU1-His genes-plasmid contained. The Jac1p and Isu1p His-tagged proteins over-expression in the engineered yeasts was confirmed by immunodetection, rendering increases in ethanol tolerance level from a DL50 = ~ 4.5% ethanol (v/v) to DL50 = ~ 8.2% ethanol (v/v), and survival up 90% at 15% ethanol (v/v) comparing to ~ 50% survival in the control strain. Fermentation by the engineered yeasts showed that the ethanol production was increased, producing 15-20% more ethanol than the control yeast. The decrease of ROS and free-iron accumulation was observed in the engineered yeasts under ethanol stress condition. The results indicate that Jac1p and Isu1p over-expression in the S. cerevisiae UMArn3.3 yeast increased its ethanol tolerance level and ethanol production by a mechanism that involves ROS and iron homeostasis related to the biogenesis/recycling of Fe-S clusters dependent proteins.