Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.Peer reviewe

Identification of ε-Poly-L-lysine as an Antimicrobial Product from an Epichloë Endophyte and Isolation of Fungal ε-PL Synthetase Gene

The endophytic fungus Epichloë festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as ε-poly-L-lysine (ε-PL), which consisted of 28–34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated ε-PL showed inhibitory activity against the spore germination of the plant pathogens Drechslera erythrospila, Botrytis cinerea, and Phytophthora infestans at 1–10 μg/mL. We also isolated the fungal gene “epls” encoding ε-PL synthetase Epls. Overexpression of epls in the wild type strain E437 resulted in the enhanced production of ε-PL by 6.7-fold. Interestingly, overexpression of epls in the different strain E. festucae Fl1 resulted in the production of shorter ε-PL with 8–20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of ε-PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of vibA or/and epls genes in the Epichloë endophyte for constructing pest-tolerant plants

Primary 3D screen.

<p>PC-3 cells were cultured in 3D Matrigel ECM for 4 days and treated for 6 days with 25 betulin derivatives (from 2D high throughput screens), DMSO/vehicle control, and three reference compounds. A) Representative maximum intensity projections of confocal microscope stack images for selected compound treatments at 300 nM concentration (5× objective, scale 100 μm). B) Three graphs showing the relative impact on three morphometric parameters for 7 betulin derivatives, DMSO control, and one control compound (paclitaxel). Data scaling: displays the relative difference between median of Area/Complexity/Area Ratio, to DMSO control. Paclitaxel treatments and DMSO controls have been assigned values of -100 and 0, respectively. C) Hierarchical clustering was done using three morphological parameters derived from PC3 organoids: spheroid size (area), complexity and the number of dead cells. D) Wound healing curves of the two betulin derivatives <b>4</b> and <b>20</b>, highlighting the 50% cut-off level (orange dashed line).</p

Cytotoxicity tests performed in 2D monolayer culture.

<p><b>A</b>) Cell proliferation/cell number, <b>B</b>) programmed cell death (apoptosis), and <b>C</b>) number of dead cells were assessed by conventional assays, combined with high-content microscopy (Operetta). Cells were treated with the five most effective betulin derivatives, including paclitaxel control for 72h. Proliferation was measured as the total number of nuclei (= cells), apoptosis as ratio of caspase-3 positive versus all cells, and cell death as ethidium homodimer-2 positive nuclei versus all cells.</p

Estimated EC₅₀ values for cell proliferation.

<p>Values have been calculated using Harmony High-Content Imaging and Analysis Software.</p><p>Estimated EC₅₀ values for cell proliferation.</p

Mechanisms of action.

<p>PC-3 spheroids were exposed to two representative betulin derivatives, <b>5</b> and <b>20</b>, for 4 h (at 1 μM) and 6 days (at 0.3 μM). A) Computational quantification of signal intensities from the kinase arrays, aligned by fold changes observed (left to right). B) Western blot of total and phospho-Akt (S473) shown for both short and long-term exposure to <b>20</b> and <b>5</b>.</p

Chemical structures of the parental compound betulinic acid and its derivatives.

<p>Chemical structures of the parental compound betulinic acid and its derivatives.</p