SID-1 Domains Important for dsRNA Import in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that Caenorhabditis nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2–7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport. Among these missense alleles, we identify and characterize a sid-1 allele, qt95, which causes tissue-specific silencing defects most easily explained as a systemic RNAi export defect. However, we conclude from genetic and biochemical analyses that sid-1(qt95) disrupts only import, and speculate that the apparent export defect is caused by the cumulative effect of sequentially impaired dsRNA import steps. Thus, consistent with previous studies, we fail to detect a requirement for sid-1 in dsRNA export, but demonstrate for the first time that SID-1 functions in the intestine to support environmental RNAi (eRNAi)

SID-1 Domains Important for dsRNA Import in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) spreads systemically to cause gene silencing throughout the organism and its progeny. We confirm that Caenorhabditis nematode SID-1 orthologs have dsRNA transport activity and demonstrate that the SID-1 paralog CHUP-1 does not transport dsRNA. Sequence comparison of these similar proteins, in conjunction with analysis of loss-of-function missense alleles, identifies several conserved 2–7 amino acid microdomains within the extracellular domain (ECD) that are important for dsRNA transport. Among these missense alleles, we identify and characterize a sid-1 allele, qt95, which causes tissue-specific silencing defects most easily explained as a systemic RNAi export defect. However, we conclude from genetic and biochemical analyses that sid-1(qt95) disrupts only import, and speculate that the apparent export defect is caused by the cumulative effect of sequentially impaired dsRNA import steps. Thus, consistent with previous studies, we fail to detect a requirement for sid-1 in dsRNA export, but demonstrate for the first time that SID-1 functions in the intestine to support environmental RNAi (eRNAi)

Quantification of an Antibody-Conjugated Drug in Fat Plasma by an Affinity Capture LC-MS/MS Method for a Novel Prenyl Transferase-Mediated Site-Specific Antibody–Drug Conjugate

The novel prenyl transferase-mediated, site-specific, antibody–drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2), with the equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 19.17~958.67 ng/mL for acDrug. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions (short-term: 1 day, long-term: 10 months, Freeze/Thaw (F/T): 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats

The Natural Anticancer Agent Plumbagin Induces Potent Cytotoxicity in MCF-7 Human Breast Cancer Cells by Inhibiting a PI-5 Kinase for ROS Generation

<div><p>Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast <em>Schizosaccharomyces pombe</em>, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI₅₀; 10 µM) and induced cell elongation and septum formation in wild-type <em>S. pombe</em>. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in <em>S. pombe</em>. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in <em>S. pombe</em> deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.</p> </div

ROS-dependent growth inhibition of wild-type <i>S. pombe</i> (SP286) by plumbagin.

<p>(A) Wild-type <i>S. pombe</i> cells were plated onto 96-well plates at 1×10⁵ cells/well, followed by treatment with various concentrations of plumbagin and incubation at 30°C for 14 h. Then, OD₆₀₀ was measured using a microplate reader. Data represent the mean ± standard error (n = 3). (B) <i>S. pombe</i> cells were left untreated or were treated with 10 µM plumbagin and incubated for 6 h. Then, cells were photographed. The scale bar below the right figure represents 10 µm. (C) Cells were treated with 0 or 10 µM plumbagin for 6 h and were stained with 4 µM DHE, an ROS staining reagent, for 30 min. Then, the level of ROS was measured by fluorescence microscopy as indicated in Materials and Methods. (D) Wild-type <i>S. pombe</i> cells were treated with 10 µM plumbagin in the absence or presence of NAC (2 mM) for 14 h, then the cell mass was measured as OD₆₀₀ in a microplate reader. Data represent the mean ± standard error (n = 3). ***<i>P</i><0.001 compared with the untreated samples.</p

Comparative analysis of DIH by plumbagin between an its3-deletion mutant and a PI3K-deletion mutant.

<p>All <i>S. pombe</i> cells, including the wild-type and mutants indicated, were treated under the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045023#pone-0045023-g003" target="_blank">Figure 3</a> with various doses of plumbagin for 14 h, and the relative cell mass was analyzed by measuring OD₆₀₀. Data represent the mean ± standard error (n = 3).</p

Induction of drug-induced haploinsufficiency by plumbagin in an its3-deleted heterozygous mutant.

<p>(A) Wild-type <i>S. pombe</i> (SP286) and an its3-deleted heterozygous mutant (SPAC19G12.14; its3::KanMX) were treated with 10 µM plumbagin under the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045023#pone-0045023-g003" target="_blank">Figure 3</a>. Cultures were incubated at 30°C, and OD₆₀₀ was recorded every 2 h for 14 h using a microplate reader. (B) Relative cell mass at 14 h after the treatment in (A) was measured as OD₆₀₀. Data represent the mean ± standard error (n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 compared with the untreated samples. Cells grown in the presence of 10 µM plumbagin for 14 h were either serially four-fold diluted, spotted onto YES plates, and incubated for 3 days and photographed when colonies appeared (C) or observed under microscope (D). (E) After the treatment in D, mRNA was prepared and the levels of mRNAs of its3 and Act1 were measured using RT-PCR analysis. RT-PCR analysis was performed according to the following procedures. Total RNA was isolated by lysis with glass beads in the presence of Accuzol (BIONEER). RT-PCR amplification of total RNA (1 µg/reaction) was performed using <i>its3</i> -specific primers (forward, 5′-GATGGCATTCCCCCCGATATTG-3; reverse, 5′-TCGTCGAGTTCCCTTCCTAGGG-3′) and <i>act1</i>-specific primers (forward, 5′- CACCCTTGCTTGTTGACTGAGGC-3; reverse, 5′-AGCTTCAGGGGCACGGAAACGC-3′) in a 20 -µl reaction using the Accupower RT/PCR PreMix (Bioneer, Daejeon, Korea). PCR amplification was performed as follows: one cycle at 42°C for 1 h, 94°C for 5 min, 30 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, and one final extension cycle at 72°C for 5 min. PCR reaction products were analyzed by agarose gel electrophoresis.</p

Its <i>3</i>-dependent ROS generation by plumbagin in <i>S. pombe</i>.

<p>(A) Wild-type and the its3-deletion mutant were treated with 10 µM plumbagin and incubated for each time indicated. Then, they were stained with 4 µM DHE for 30 min, and the level of ROS was observed by fluorescence microscopy. (B) The level of ROS in the treated cells was measured by fluorescence microscopy as indicated in Materials and Methods. Data represent the mean ± standard error (n = 3). <i>P</i> values compared with wild type (SP286) cells.</p

Downregulation of PI5K-1B by plumbagin and functional role of PI5K-1B for cell viability and ROS generation in MCF-7 human breast cancer cells.

<p>(A) MCF-7 cells were cultured in a 6-well plate as described in Materials and Methods and then either left untreated or treated with 5 µM plumbagin for 24 h. Then, cells were harvested, and the levels of proteins in the cell lysates were analyzed by western blotting (panel A). (B) PI5K-1B knockdown in MCF-7 cancer cells was carried out a described in Materials and Methods using PI5K-1B-specific siRNA (5′-GUCCUCAAUUAGCCAGGAA(dTdT)-3′) or a control siRNA (5′-CUUACGCUGAGUACUUCGA(dTdT)-3′) for 48 h. Then, knockdown of PI5K-1B was validated by either RT-PCR (panel Ba) or western blotting (panel Bb) as described in Materials and Methods. (C) Cell viability after siRNA transfection for 48 h was measured by the WST-1 assay. Data represent the mean ± standard error (n = 3) (panel Ca). ***<i>P</i><0.001 compared with the untreated samples. The cells after siRNA transfection for 48 h were stained with 4 µM DHE for 30 min, and then the ROS level was observed using a fluorescence microscope (excitation 518 nm, emission 605 nm) (panel Cb).</p

Diagram depicting mode of action of plumbagin.

<p>Diagram depicting mode of action of plumbagin.</p