Alcoholic extract of Tarantula cubensis (Theranekron®) induce autophagy on gastric cancer cells

Aim: To evaluate the effects of theranekron in respect of autophagy on gastric cancer that is the fifth leading cancer type worldwide. Metods: In the present study, metastatic AGS and non-metastatic MKN-45 human gastric cell lines were used together with HEK-293 non-cancer cells as controls. Cytotoxic effect of theranekron besides appropriate treatment time was investigated through cell proliferation by using Cell Proliferation assay Kit (MTT) using different concentrations of the drug. The autophagic effect of the drug was determined using the LC3-GFP translocation assay and western blot analysis. All experiments were performed also using the ethanol since Tarantula cubensis spider was processed and diluted in 60% alcohol to generate as a drug. Results: MTT assay results demonstrated that the half maximal inhibitory concentration of theranekron was ~100 μM, its effect was found to be significant at 6 hrs, and theranekron decreased the cell viability in all cell lines without specificity in respect to the increasing concentrations. Additionally, a significantly increased GFP accumulation was detected in the autophagosomes of the cells treated with theranekron compared to non-treated cells, indicating the presence of autophagy. Conclusion: These findings were confirmed by LC3-I to LC3-II conversion with the western blot analysis. The data of ethanol experiments; however, demonstrated that ethanol also induced a cytotoxic effect and autophagic cell death. Our results suggested that theranekron results in cell death and stimulate autophagy process, but it is not specific for cancer cells since it represented similar results on non-cancer control cells. Moreover, the effect of theranekron on cell death might mostly occur through alcohol in which it is extracted

Genetic models of apoptosis-induced proliferation decipher activation of JNK and identify a requirement of EGFR signaling for tissue regenerative responses in Drosophila

Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation

(CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening

Deficiencies that modify the <i>ey>hid-p35</i>-induced AiP phenotype as suppressors or enhancers.

<p>The indicated chromosomal location is the smallest overlap of overlapping deficiencies. <i>Df(2L)TW137</i> is marked with a “?” because other overlapping deficiencies do not suppress AiP (see Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen.1004131.s008" target="_blank">Table S1</a>) indicating that the <i>Df(2L)TW137</i> chromosome carries a suppressor mutation independent of the deficiency.</p

Characterization of ‘genuine’ AiP in the eye imaginal disc: the <i>DE^ts</i>><i>hid</i> model.

<p><i>hid</i> expression was under control of <i>dorsal eye- (DE-)Gal4</i> and <i>tub</i>-<i>Gal80^ts</i> (<i>DE^ts</i>><i>hid</i>). A temperature shift (ts) to 30°C for 12 h during 2^nd larval stage induced <i>hid</i> expression (E). After the indicated recovery period (R), discs were labeled for GFP (to visualize the <i>DE</i> expression domain), Cas3* (the death domain) and ELAV (to outline the shape of the disc). (A–C) <i>DE^ts</i>><i>hid</i> experimental discs. <i>hid</i> expression induces a strong apoptotic response (A) causing strong tissue loss after 24 h recovery in some discs (panel B; R24 h, asterisk). After 72 h recovery (R72 h), the disc has fully recovered and has a normal photoreceptor pattern as judged by ELAV labeling (C). Please note the strong reduction of GFP intensity which suggests that most of the <i>GFP</i>⁺ cells have been replaced by new <i>GFP</i>⁻ cells. Arrows highlight a patch of cells that are moving to the center of the disc. (D) A control disc 72 h after <i>DE^ts</i>-induced GFP expression. Please note that GFP is a very stable protein that can still be detected 72 h after synthesis. (E) The protocol of the <i>DE^ts</i>><i>hid</i>-induced tissue ablation followed by recovery periods. (F,F′,F″,G,G′,G″) PH3-labeling of control (<i>DE^ts</i>><i>GFP</i>; F,F″) and experimental discs (<i>DE^ts</i>><i>hid</i>; G,G″). GFP marks the outline of the DE domain (F′,G′). (H) Quantification of the number of PH3-positive cells in dorsal and ventral compartments of control (F) and experimental discs (G). n = 40 for each genotype.</p

Requirement of <i>bsk</i> and <i>spi</i> for complete regeneration in the ‘genuine’ AiP model <i>DE^ts</i>><i>hid</i>.

<p>(A, A′) <i>DE^ts</i>><i>hid</i> discs treated following the protocol in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g006" target="_blank">Figure 6E</a> fully recover after 72 h (R72H). n = 30. (A′) shows the ELAV-only channel. (B, B′) About 35% of <i>DE^ts</i>><i>hid</i> discs expressing <i>UAS</i>-<i>bsk</i> RNAi do not completely recover after 72 h. n = 25. The arrow in (B′) highlights the incomplete ELAV pattern on the dorsal half of the disc indicating that the regeneration response was partially impaired by reduction of <i>bsk</i> activity. Please note that this disc has also been labeled for GFP. (C, C′) A control eye disc expressing <i>UAS</i>-<i>spi</i> RNAi under <i>DE^ts</i>-control following the protocol in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g006" target="_blank">Figure 6E</a>. After 72 h recovery, the obtained ELAV pattern in the dorsal half of the eye disc is largely normal (red in C, gray in C′). n = 20. (D, D′) An experimental <i>DE^ts</i>><i>hid</i> eye disc that was simultaneously treated with <i>spi</i> RNAi. The arrow in (D′) highlights the incomplete ELAV pattern on the dorsal half of the disc indicating that the regeneration response was partially impaired by reduction of <i>spi</i> activity. 30 out of 30 discs show incomplete regeneration. Please note that this disc has also been labeled for GFP. (E, E′) An experimental <i>DE^ts</i>><i>hid</i> eye disc that was heterozygous for <i>spi⁰¹⁰⁶⁸</i>. Similar to (D), the ELAV pattern is incomplete on the dorsal half of the disc (E′, arrow). n = 20. (F, F′, G, G′) <i>spi-lacZ</i> pattern in control (<i>DE^ts</i>><i>GFP</i>; red in F, grey in F′) and experimental discs (<i>DE^ts</i>><i>hid</i>; red in G, grey in G′) at 24 h after recovery. The arrow in (G′) points to the increased β-Gal pattern in the dorsal half of the disc. Blue is Cas3*. (H, H′, I, I′) <i>kek-lacZ</i> pattern in control (<i>DE^ts</i>><i>GFP</i>; red in H, grey in H′) and experimental discs (<i>DE^ts</i>><i>hid</i>; red in I, grey in I′) at 30 h recovery. The arrow in (I′) points to the increased β-Gal pattern in the dorsal half of the disc. Blue is Cas3*.</p

The <i>ey>hid-p35</i> model induces hyperplastic overgrowth and displays markers of apoptosis-induced proliferation.

<p>In this and the following figures, anterior is to the left. White dotted lines indicate the anterior portion of the eye imaginal discs. ELAV labels photoreceptor neurons and is used to mark the developing eye field posterior to the morphogenetic furrow (MF). (A,A′) An <i>ey>p35</i> control eye disc labeled with PH3 as proliferation marker (red in A; grey in A′) and ELAV (green in A). (B,B′) An <i>ey>hid-p35</i> experimental disc labeled with PH3 (red in B; grey in B′) and ELAV (green in B). Please note the increase in size of the region anterior to the MF at the expense of the posterior region (green). (C,D,E,F) Dorsal views of heads (C,D) and eyes (E,F) of <i>ey>p35</i> control (C,E) and <i>ey>hid-p35</i> experimental flies (D,F). Enlarged head cuticle with additional ocelli and bristles (arrows) is observed in <i>ey>hid-p35</i> flies (D), while eyes are reduced in size (F). (G,G′,H,H′) Increased expansion of <i>wg</i> expression (<i>wg</i>-<i>lacZ</i>, red in G,H; grey in G′,H′) in <i>ey>hid-p35</i> discs (H, arrow) compared to <i>ey>p35</i> control discs (G). (I,I′) In <i>ey>p35</i> control discs, <i>puc</i>-<i>lacZ</i> expression (β-Gal; red in I, gray in I′) as marker of Bsk/JNK activity is low anterior to the MF and induced posterior to the MF. (J,J′) <i>puc</i>-<i>lacZ</i> expression (β-Gal; red in J, gray in J′) as marker of Bsk/JNK activity is strongly induced anterior to the MF in <i>ey>hid-p35</i> eye discs (arrows). Note the reduction in the posterior eye field as visualized by ELAV labeling (green).</p

Modification of the <i>ey>hid-p35</i> phenotype by JNK pathway components.

<p>(A–E) <i>dronc</i> (A) and <i>ark</i> (C) heterozygosity strongly suppresses the <i>ey>hid-p35</i> phenotype (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g001" target="_blank">Figure 1D</a>). RNAi targeting <i>dronc</i> (B) and <i>bsk</i> (E) also strongly suppresses it. Double RNAi targeting <i>dcp-1</i> and <i>drICE</i> (D) has no effect. (F) Results of the suppression of <i>ey>hid-p35</i> using RNAi targeting components of the Bsk/JNK pathway in <i>Drosophila</i>. Only select members of the Bsk/JNK pathway (<i>dTraf2</i>, <i>Rho1</i>, <i>dTAK1</i>, <i>dMKK4</i>, <i>Bsk</i> and to a weaker extent <i>hep</i>, <i>Jra</i> and <i>kay</i>) show suppression. Each RNAi analysis was repeated at least twice with scoring more than 50 <i>ey</i>><i>hid</i>-<i>p35</i>/<i>dsRNA</i> adult flies. (G) Schematic summary of the suppression analysis of the Bsk/JNK pathway. Pathway components highlighted in red show RNAi-mediated suppression and are thus required for <i>ey>hid-p35</i>-induced proliferation. (H–J) The <i>GMR</i>><i>eiger</i>-induced eye ablation phenotype (H) is strongly suppressed by <i>dTRAF2</i> RNAi (I), but not by <i>Rho1</i> RNAi (J). (K) <i>GMR-Gal4</i> driven RNAi targeting <i>Rho1</i> does not cause an eye ablation phenotype. This control experiment shows that failure of <i>Rho1</i> RNAi to suppress <i>GMR</i>><i>eiger</i> (J) is not due to a secondary effect.</p

Suppression of <i>ey>hid-p35</i> by <i>spi</i> and <i>Egfr</i> inactivation.

<p>The hyperplastic phenotype of <i>ey>hid-p35</i> flies can be grouped in three categories, severe, moderate and weak. Flies were scored as severe when the head cuticle was strongly overgrown without discernible patterning and eyes were absent or strongly reduced in size. A moderate phenotype was scored when the head cuticle was overgrown, but recognizably patterned with duplicated ocelli and bristles. A weak phenotype was scored when size of head cuticle and eyes was almost normal with very few ectopic ocelli or bristles occasionally observed. (A–H) Representative pictures of <i>ey>hid-p35</i> fly head cuticles scored in different categories. Completely suppressed <i>ey>hid-p35</i> phenotype (wild-type-like head cuticles) by <i>spi</i> or <i>Egfr</i> heterozygotes are not shown here. Arrows indicate ectopic ocelli or bristles. (A,B) About 50% of <i>ey>hid-p35</i> flies show severe hyperplastic overgrowth of the head cuticle (A), while the remaining 50% display a moderate phenotype (B). (C–H) Heterozygosity of <i>spi¹</i>, <i>spi⁰¹⁰⁶⁸</i> and <i>Egfr^f2</i> almost completely eliminated the severe overgrowth phenotype of <i>ey>hid-p35</i> flies and largely extends the population of flies with a weak phenotype. (I) Summary of the suppression of the <i>ey>hid-p35</i> overgrowth phenotype in <i>spi</i>, <i>egfr</i>, <i>dRas</i> and <i>rolled (rl)</i> heterozygous condition. Pink indicates severe, orange indicates moderate and green indicates weak phenotypes. Mutant alleles are indicated.</p

Epistasis analysis of <i>spi</i> and <i>bsk</i>.

<p>Arrowheads indicate the morphogenetic furrow (MF) which separates the anterior (left) from the posterior eye tissue visualized by ELAV labeling. (A) <i>spi-lacZ</i> pattern (β-Gal; red in A; gray in A′) in <i>ey>p35</i> control discs. Note there is little expression anterior to the MF. (B,C) Because the <i>spi-lacZ</i> allele (<i>spi⁰¹⁰⁶⁸</i>) is a suppressor of <i>ey>hid-p35</i> adult overgrowth phenotype (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g004" target="_blank">Figure 4I</a>), there is variation in the β-Gal pattern. About 25% of the eye discs show strong induction of <i>spi</i>-<i>lacZ</i> in the anterior portion of the eye disc (B,B′; arrows) with a strong reduction of the eye field (ELAV). The remaining 75% of the eye discs show a suppressed, largely normal β-Gal and ELAV pattern in <i>ey>hid-p35</i> larvae (C,C′). This ratio corresponds to the suppression of the adult overgrowth phenotype (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g004" target="_blank">Figure 4I</a>). (D,E) Strong induction of <i>kek</i>-<i>lacZ</i> (β-Gal; red in D,E; gray in D′,E′) in <i>ey>hid-p35</i> eye discs (E; arrows) compared to <i>ey>p35</i> control discs (D). (F,F′) <i>kek</i>-<i>lacZ</i> (β-Gal; red in F, gray in F′) is preferentially induced in patches of tissue adjacent to areas with high levels of active caspases (arrows, Cas3* in green). (G,G′) Heterozygosity of <i>spi</i> normalizes the eye field (ELAV, green), but does not suppress ectopic <i>puc</i>-<i>lacZ</i> expression (β-Gal; red in G, gray in G′) in <i>ey>hid-p35</i> eye discs (arrows, compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004131#pgen-1004131-g001" target="_blank">Figure 1J</a>). Dotted white lines outline the region anterior to the MF. (H,H′) Expression of <i>bsk^RNAi</i> in <i>ey>hid-p35</i> discs normalizes the eye field (ELAV, green) and suppresses ectopic increase of <i>spi</i>-<i>lacZ</i> expression (β-Gal; red in H, gray in H′). This pattern was observed in all experimental discs (n = 30). (I,I′) Expression of <i>bsk^RNAi</i> in <i>ey>hid-p35</i> discs normalizes the eye field (ELAV, green) and suppresses ectopic <i>kek</i>-<i>lacZ</i> expression (β-Gal; red in I, gray in I′; compare to E). The analysis in G, H and I strongly suggests that <i>spi</i> acts genetically downstream of <i>bsk</i>.</p