The Natural Product ß-Escin Targets Cancer and Stromal Cells of the Tumor Microenvironment to Inhibit Ovarian Cancer Metastasis.

The high mortality of OvCa is caused by the wide dissemination of cancer within the abdominal cavity. OvCa cells metastasize to the peritoneum, which is covered by mesothelial cells, and invade into the underlying stroma, composed of extracellular matrices (ECM) and stromal cells. In a study using a three-dimensional quantitative high-throughput screening platform (3D-qHTS), we found that ß-escin, a component of horse chestnut seed extract, inhibited OvCa adhesion/invasion. Here, we determine whether ß-escin and structurally similar compounds have a therapeutic potential against OvCa metastasis. Different sources of ß-escin and horse chestnut seed extract inhibited OvCa cell adhesion/invasion, both in vitro and in vivo. From a collection of 160 structurally similar compounds to ß-escin, we found that cardiac glycosides inhibited OvCa cell adhesion/invasion and proliferation in vitro, and inhibited adhesion/invasion and metastasis in vivo. Mechanistically, ß-escin and the cardiac glycosides inhibited ECM production in mesothelial cells and fibroblasts. The oral administration of ß-escin inhibited metastasis in both OvCa prevention and intervention mouse models. Specifically, ß-escin inhibited ECM production in the omental tumors. Additionally, the production of HIF1α-targeted proteins, lactate dehydrogenase A, and hexokinase 2 in omental tumors was blocked by ß-escin. This study reveals that the natural compound ß-escin has a therapeutic potential because of its ability to prevent OvCa dissemination by targeting both cancer and stromal cells in the OvCa tumor microenvironment.

High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer.

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras/Raf/ERK1/2 signaling and hnRNP H1 in castration-resistant prostate cancer cells.

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells.

AKT1 mediates bypass of the G1/S checkpoint after genotoxic stress in normal human cells.

Certain forms of hexavalent chromium [Cr(VI)] are human carcinogens. Our recent work has shown that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV), abrogated both Cr(VI)-induced growth arrest and clonogenic lethality. Notably, SOV enhanced Cr(VI) mutation frequency, ostensibly through forced survival of genetically damaged cells. In the present study, co-treatment with this PTP inhibitor bypassed the Cr(VI)-induced G(1)/S checkpoint arrest in diploid human lung fibroblasts (HLF). Moreover, the PTP inhibitor abrogated the Cr(VI)-induced decrease in the expression of key effectors of the G(1)/S checkpoint [Cyclin D1, phospho Ser 807/811 Rb (pRB), p27]. Cr(VI)-induced G(1) arrest was associated with the cytoplasmic appearance of pRb and the nuclear localization of p27, both of which were reversed by the PTP inhibitor. The PTP inhibitor's reversal of G(1)/S checkpoint effector localization after Cr exposure was found to be Akt1-dependent, as this was abrogated by transfection with either akt1 siRNA or an Akt1-kinase dead plasmid. Furthermore, Akt1 activation alone was sufficient to induce G(1)/S checkpoint bypass and to prevent Cr(VI)-induced changes in pRb and p27 localization. In conclusion, this work establishes Akt1 activation to be both sufficient to bypass the Cr(VI)-induced G(1)/S checkpoint, as well as necessary for the observed PTP inhibitor effects on key mediators of the G(1)/S transition. The potential for Akt to bypass G(1)/S checkpoint arrest in the face of genotoxic damage could increase genomic instability, which is a hallmark of neoplastic progression.

Bypass of hexavalent chromium-induced growth arrest by a protein tyrosine phosphatase inhibitor: enhanced survival and mutagenesis.

Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na(2)CrO(4)), the soluble oxyanionic dissolution product of certain particulate chromates, which are well-documented human respiratory carcinogens. In vitro soluble Cr(VI) induces a wide spectrum of DNA damage, in both the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate (SOV). Notably, SOV abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after SOV treatment was predominantly due to a bypass of cell cycle arrest, as there was no effect of the PTP inhibitor on Cr-induced apoptosis. Moreover, the SOV effect was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by SOV was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in Cr(VI)-induced expression of cell cycle promoting genes. Importantly, SOV resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of certain types of DNA damage may lead to increased genomic instability, via bypass of cell cycle checkpoints.