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.

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.

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.

Antiproliferative effect of bacterial cyclodipeptides in the HeLa line of human cervical cancer reveals multiple protein kinase targeting, including mTORC1/C2 complex inhibition in a TSC1/2-dependent manner.

Cervix adenocarcinoma rendered by human papillomavirus (HPV) integration is an aggressive cancer that occurs by dysregulation of multiple pathways, including oncogenes, proto-oncogenes, and tumor suppressors. The PI3K/Akt/mTOR pathway, which cross-talks with the Ras-ERK pathway, has been associated with cervical cancers (CC), which includes signaling pathways related to carcinoma aggressiveness, metastasis, recurrence, and drug resistance. Since bacterial cyclodipeptides (CDPs) possess cytotoxic properties in HeLa cells with inhibiting Akt/S6k phosphorylation, the mechanism of CDPs cytotoxicity involved was deepened. Results showed that the antiproliferative effect of CDPs occurred by blocking the PI3K/Akt/mTOR pathway, inhibiting the mTORC1/mTORC2 complexes in a TSC1/TSC2-dependent manner. In addition, the CDPs blocked protein kinases from multiple signaling pathways involved in survival, proliferation, invasiveness, apoptosis, autophagy, and energy metabolism, such as PI3K/Akt/mTOR, Ras/Raf/MEK/ERK1/2, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, Wnt/ß-catenin, HSP27, EMT, CSCs, and receptors, such as EGF/ErbB2/HGF/Met. Thus, the antiproliferative effect of the CDPs made it possible to identify the crosstalk of the signaling pathways involved in HeLa cell malignancy and to suggest that bacterial CDPs may be considered as a potential anti-neoplastic drug in human cervical adenocarcinoma therapy.

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60-250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.