Antifungal and Cytotoxic 2‑Acylcyclohexane-1,3-diones from <i>Peperomia alata</i> and <i>P. trineura</i>

Bioactivity-guided fractionation of the separate CH₂Cl₂ extracts from the aerial parts of <i>Peperomia alata</i> and <i>P. trineura</i> yielded seven polyketides: alatanone A [3-hydroxy-2-(5′-phenylpent-4′<i>E</i>-enoyl)­cyclohex-2-en-1-one, <b>1a</b>] and alatanone B [3-hydroxy-2-(3′-phenyl-6′-methylenedioxy­propanoyl)­cyclohex-2-en-1-one, <b>2a</b>] from <i>P. alata</i> and trineurone A [3-hydroxy-2-(11′-phenylundec-10′<i>E</i>-enoyl)­cyclohex-2-en-1-one, <b>1b</b>], trineurone B [3-hydroxy-2-(15′-phenyl-18′-methylenedioxy­pentadecanoyl)­cyclohex-2-en-1-one, <b>2b</b>], trineurone C [3-hydroxy-2-(17′-phenyl-20′-methylenedioxy­heptadecanoyl)­cyclohex-2-en-1-one, <b>2c</b>], trineurone D [3-hydroxy-2-(hexadec-10′<i>Z</i>-enoyl)­cyclohex-2-en-1-one, <b>3a</b>], and trineurone E [(6<i>R</i>)-(+)-3,6-dihydroxy-2-(hexadec-10′<i>Z</i>-enoyl)­cyclohex-2-en-1-one, <b>3b</b>] from <i>P. trineura</i>. The isolated compounds were evaluated for antifungal activity against <i>Cladosporium cladosporioides</i> and <i>C. sphaeospermum</i> and for cytotoxicity against the K562 and Nalm-6 leukemia cell lines

SB225002 and <i>GLIPR1</i> knockdown effects on ROS generation in ALL cells.

<p><b>(A)</b> Reactive oxygen species production in B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) cells treated with DMSO (vehicle; 0.1%) and SB225002 [5 μM and 10 μM]. Cells were treated for 24 h. <b>(B)</b> ROS generation in <i>GLIPR1</i>-knockdown (G-KD) <i>versus</i> control scramble (S) cells treated with DMSO (vehicle; 0.1%) or SB225002 [10 μM] for REH and RS4;11 or SB225002 [5 μM] for Jurkat and TALL-1 for 24 h. <b>(C)</b> Effect of N-Acetyl Cysteine (NAC; a ROS scavenger) pre-treatment on the survival of <i>GLIPR1</i>-knockdown (G-KD) <i>versus</i> control scramble (S) ALL cell lines upon SB225002 treatment for 48 h. Cells were pre-incubated or not with NAC [10 mM] for 3h prior to the SB225002 treatment. SB225002 was used at [10 μM] (REH and RS4;11) or [5 μM] (Jurkat and TALL-1). S = scramble transfection control; G-KD = cells infected with <i>GLIPR1</i>-shRNA lentiviral particles (Sigma-Aldrich). P values were calculated using two-tailed Student’s t-test.</p

Modulation of <i>CX3CR1</i> and <i>GLIPR1</i> expression in ALL cells upon SB225002 treatment.

<p><b>(A)</b><i>CX3CR1</i> and <b>(B)</b><i>GLIPR1</i> gene expression analysis by quantitative PCR (Q-PCR) and Western blot in Jurkat cells treated with SB225002 [IC₅₀] or DMSO (vehicle control; 0.1%). Treatments were performed for 3h, 6 h, 9 h or 12 h, as indicated. In the Q-PCR analysis, expression values were calculated considering vehicle control (DMSO) as 100%. β-actin was used as loading control in Western blot analysis. Control = DMSO (vehicle control); SB = SB225002 treatment.</p

SB225002 induces cell death in ALL cell lines.

<p>Effect of SB225002 [100 to 1.5625 μM] on the survival and proliferation of <b>(A)</b> B-ALL and T-ALL cell lines. <b>(B)</b> Effect of SB225002 [5 and 10 μM] on the survival and proliferation of normal PHA-stimulated human lymphocytes. <b>(C)</b> Cell cycle analysis of B-ALL (REH and RS4;11), T-ALL (Jurkat and TALL-1) and normal human PHA-stimulated lymphocytes treated with DMSO (vehicle; 0.1%) and the following concentrations of SB225002: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]; PHA-stimulated lymphocytes [10 μM]. Representative PI-staining histograms of cells treated with vehicle (clear area) or SB225002 (shaded area) are shown. <b>(D)</b> Annexin-V and propidium iodide flow cytometry analyses of B-ALL (REH and RS4;11) and T-ALL (Jurkat and TALL-1) treated with DMSO (vehicle; 0.1%) and SB225002 [10 μM]. Cells were treated for 24 h (for cell cycle and Annexin-V analyses) and 48 h (for MTT analysis). ALL = acute lymphoblastic leukemia; PI = propidium iodide; Lym = PHA-stimulated lymphocytes; C or Ctr = DMSO (vehicle control); SB = SB225002 treatment.</p

Connectivity Map and Ingenuity Pathway Analysis using the SB225002-derived gene expression signature.

<p><b>(A)</b> Connectivity Map (C-Map) analysis using the gene expression signature of Jurkat cells treated with SB225002 [IC₅₀] for 9 h. Compounds colored as black bars in each respectively C-Map plot. Compounds are color-coded as follows: blue, PI3K/mTOR inhibitors; green, HSP90 inhibitors; red, tubulin inhibitors. <b>(B)</b> Signaling pathways activated in Jurkat cells in response to 6 h of SB225002 [IC₅₀] treatment. The statistical threshold (line without boxes) represents the cut-off for significance on the log scale (<i>y</i>-axis, left side). The ratio (line with boxes) of the number of significant genes from the data set that mapped to a pathway divided by the total number of genes from the pathway is also shown (<i>y</i> axis, right side). <b>(C)</b><i>JUN</i>, <b>(D)</b><i>p53</i> and <b>(E)</b><i>TNF</i> pathways are modulated in Jurkat cells after 6 h of SB225002 [IC₅₀] treatment. Analyses were performed using the Ingenuity Pathways Analysis package (Ingenuity Systems).</p