A priori activation of apoptosis pathways of tumor (AAAPT) technology: Development of targeted apoptosis initiators for cancer treatment

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. Amongst many, the principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously and selectively in cancer cells sparing normal cells. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (lung cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro when used as a combination. At higher doses, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AAAPT leading molecules as neoadjuvant to chemotherapy to achieve better efficacy and reduced off-target toxicity compared to the current treatments

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

<div><p>MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume <i>Medicago truncatula</i>. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen <i>Fusarium graminearum in vitro</i> at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β₂ and β₃ strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA. </p> </div

MtDef4 variants, MtDef4^{RGFRRR/AAAARR} and MtDef4^{RGFRRR/RGFRAA}, are less efficient in permeabilizing <i>F</i>.

<div><p><b><i>graminearum</i> membrane compared to MtDef4 or MtDef4^{RGFRRR/RGAARR}</b>. </p> <p>Quantitative measurement of fluorescence emitted by hyphae treated with different concentrations of MtDef4 or its variants plus 0.5 µM of SYTOX Green. Values are means of three replications. Error bars indicate standard deviations. </p> <p>A and B. Fluorescence measurement at 30 min and 8 hr, respectively.</p></div

Surface characteristics of MtDef4.

<div><p>A. In this diagram, the surface potential (calculated by DelPhi) is mapped onto the molecular surface of MtDef4 as a semi-transparent surface. The structure of MtDef4 is represented by a ribbon diagram that is colored blue to red as the chain extends from the amino-terminus to carboxy terminus. The disulfide bonds and the side chains of the arginine and lysine residues in the structure are also shown. Note that the overall surface is strongly basic. </p> <p>B. Surface accessibility of the atoms in the MtDef4 structure. The semi-transparent molecular surface is colored blue to red for the least to most accessible atoms. Note that the very hydrophobic F37 on the β2-β3 loop, which is highly conserved among defensin proteins, is significantly exposed. The residues in this β2-β3 loop region are labeled.</p></div

Immunogold detection of MtDef4 in treated cells (3 µm, 3 hours) of <i>F</i>.

<div><p><b><i>graminearum</i></b>. </p> <p>Scale bar = 1 µm. Section was not post-stained. Of the four cells shown in this section, the two dead cells (arrows) have significantly higher cytoplasmic labeling than the two living cells.</p></div

Number of gold particles found in the cytoplasm and cell walls of live and dead cells of <i>F</i>.

<div><p><b><i>graminearum</i></b>. </p> <p>The error bars represent the standard errors of six replicates.</p></div

Immunogold label distribution in MtDef4-treated cells.

<div><p>Scale bars = 250 nm (A, B), 1 µm (C). A, B are of post-stained sections, C is not. </p> <p>(A) In dead cells cytoplasmic label is associated with electron dense aggregated cytoplasm of undetermined cellular structure. Vacuole (asterisk) does not label. In these cells cytoplasmic label density is 95 particles per µm² while that of cell wall is 312 particles per µm². </p> <p>(B) Cells not yet killed by MtDef4 show a small amount of MtDef4 in the cytoplasm (circled gold particles) but much more in the cell wall. </p> <p>(C) Control cells treated with water alone are not labeled (two gold particles circled are background). Label density over the cytoplasm was the same as on resin alone, 0.04 particles per µm² (+ /-) 0.04).</p></div

Tetramethyl rhodamine-labeled 16-mer peptide (TMR-GMA4-C) corresponding to the C-terminus of MtDef4 enters <i>F</i>.

<div><p><b><i>graminearum</i> but a variant peptide (TMR-GMA4-CM) with AAAA replaced for RGFR motif does not</b>. </p> <p>Fluorescence images of <i>F</i>. <i>graminearum</i> hyphae incubated with 96 µM TMR-GMA-C or TMR-GMA-CM for up to 4 h.</p> <p>A. TMR-GMA4-C entered the cytoplasm.</p> <p>B. Cross section fluorescent image of selective hyphae from (A) showing that TMR-GMA-C is present in the cytoplasm. </p> <p>C. TMR-GMA4-CM faintly bound to the surface layer but with much lower intensity. </p> <p>Scale Bar = 5 μm.</p></div

The RGFRRR loop present in the γ-core motif strongly regulates the antifungal activity of MtDef4.

<div><p>A. Sequence of MtDef4 and its variants. The γ-core motif is indicated in larger font. RGFRRR sequence of MtDef4 is indicated in bold and the conserved amino acids are listed underneath with highly conserved amino acids on the top followed by less conserved ones. RGFR sequence which closely resembles the RXLR motif of the fungal and oomycete effectors is italicized. </p> <p>B. Images showing the inhibition of <i>F</i>. <i>graminearum</i> PH-1 conidial germination and hyphal growth at different concentrations of MtDef4 or its variants. Images were taken after 16 hours of incubation of conidia with defensins. Bar = 50 μm. </p> <p>C. Quantitative assessment of the <i>in </i><i>vitro</i> antifungal activity of MtDef4 or its variants at 4 days after incubation of PH-1 conidia with defensins. Values are means of thee replications. Error bars indicate standard deviations.  </p> <p>D. Images showing the growth of PH-1 strain after 6 days in the presence of MtDef4 or its variants.</p></div

DyLight 550-labeled MtDef4 and MtDef4^{RGFRRR/RGAARR} but not MtDef4^{RGFRRR/AAAARR} and MtDef4^{RGFRRR/RGFRAA} enter <i>F</i>.

<div><p><b><i>graminearum</i> cytoplasm</b>. </p> <p><i>F</i>. <i>graminearum</i> conidia were incubated with indicated concentrations of DyLight 550-labeled proteins and confocal fluorescence images were taken at various time intervals for up to 6 h.</p> <p>A. Within 15 min, DyL-MtDef4, DyL-MtDef4^{RGFRRR/RGAARR} and DyL-MtDef4^{RGFRRR/RGFRAA} bound to the surface of conidia whereas DyL-MtDef4^{RGFRRR/AAAARR} did not.</p> <p>B. At 2 h, DyL-MtDef4 and DyL-MtDef4^{RGFRRR/RGAARR} bound to the surface of germ tubes but DyL-MtDef4^{RGFRRR/AAAARR} and DyL-MtDef4^{RGFRRR/RGFRAA} did not.</p> <p>C. DyL-MtDef4 entered selective hyphae by 4 h. </p> <p>D. By 6 h, DyL-MtDef4^{RGFRRR/RGAARR} entered hyphae but not all hyphae were affected. </p> <p>E. DyL-MtDef4^{RGFRRR/AAAARR} did not enter the hyphae even after 6 h. </p> <p>F. DyL-MtDef4^{RGFRRR/RGFRAA} bound to the surface of conidial cells but did not bind to hyphal surface. </p> <p>Scale Bar = 10 µm. </p></div