Novel Antimicrobial Peptides with High Anticancer Activity and Selectivity

<div><p>We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.</p></div

Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

<div><p>Altered metabolism is one of the hallmarks of cancers. Deregulation of ribose-5-phosphate isomerase A (RPIA) in the pentose phosphate pathway (PPP) is known to promote tumorigenesis in liver, lung, and breast tissues. Yet, the molecular mechanism of RPIA-mediated colorectal cancer (CRC) is unknown. Our study demonstrates a noncanonical function of RPIA in CRC. Data from the mRNAs of 80 patients’ CRC tissues and paired nontumor tissues and protein levels, as well as a CRC tissue array, indicate RPIA is significantly elevated in CRC. RPIA modulates cell proliferation and oncogenicity via activation of β-catenin in colon cancer cell lines. Unlike its role in PPP in which RPIA functions within the cytosol, RPIA enters the nucleus to form a complex with the adenomatous polyposis coli (APC) and β-catenin. This association protects β-catenin by preventing its phosphorylation, ubiquitination, and subsequent degradation. The C-terminus of RPIA (amino acids 290 to 311), a region distinct from its enzymatic domain, is necessary for RPIA-mediated tumorigenesis. Consistent with results in vitro, RPIA increases the expression of β-catenin and its target genes, and induces tumorigenesis in gut-specific promotor-carrying RPIA transgenic zebrafish. Together, we demonstrate a novel function of RPIA in CRC formation in which RPIA enters the nucleus and stabilizes β-catenin activity and suggests that RPIA might be a biomarker for targeted therapy and prognosis.</p></div

RPIA expression is positively correlated with β-catenin protein levels and stability in HCT116 cells.

<p>(A) Knockdown of RPIA reduced β-catenin protein levels and overexpression of RPIA increased β-catenin protein levels in both the cytoplasmic and nuclear fractions of HCT116 cells. (B) Knockdown of RPIA did not decrease ERK and pERK protein levels, which were measured by western blotting in total protein analysis (up panel) in HCT116. Conversely, overexpression of RPIA did not increase ERK and pERK protein levels (up panel). In the lower panel, both cytoplasmic and nuclear fraction showed that ERK and pERK protein levels did not up-regulate in HCT116. (C) Scatter plots show a positive correlation between RPIA and β-catenin expression in the colon tissue or nucleus. (D) To determine the half-life of β-catenin protein, western blots were used to measure the abundance of β-catenin at different time points following the addition of 10 μg/ml of the protein synthesis inhibitor CHX to HCT116 cells transfected with either control siRNA or RPIA-siRNA. The lower panels show plots of the relative β-catenin protein level, expressed as a percentage as a function of time after CHX treatment. (E) RPIA-ΔD lost the ability to stabilize β-catenin. Relative β-catenin protein levels as measured by quantification of western blot are shown in HCT116 cells. (F) The reduced β-catenin levels by RPIA knockdown were rescued by 5 μM of MG132 treatment (left panel). Inhibition of RPIA stimulated ubiquitination of β-catenin (right panel). β-Catenin was precipitated by specific antibody. Coprecipitated ubiquitin levels were examined via western blot with antiubiquitin antibody. (G) Phosphorylated β-catenin (at Ser33/Ser37) versus total β-catenin was elevated upon RPIA knockdown. Gel images are shown in the up panel. (H) Overexpression of nondegradable β-catenin can overcome the growth inhibition induced by RPIA knockdown in HCT116 cells. The proliferation fold is compared to pMCV6 transfected control cell at first day. (I) The elevated viability induced by expression of RPIA was decreased upon ICRT14 (β-catenin inhibitor) treatment. Dose-dependent effects were revealed in HCT116 cells. (J) pGSK3β^Ser9 protein expression levels were up-regulated in the cytoplasmic extract upon overexpression of RPIA-WT but not upon RPIA-ΔD in HCT116 cells. (K) Cell proliferation was measured in RPIA knockdown HCT116 cells combined with 2.5 mM LiCl or 5 μM CHIR99021, respectively. The statistical significance was calculated with the Student <i>t</i> test (*** <i>P</i> < 0.001). Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003714#pbio.2003714.s011" target="_blank">S3 Data</a>. CHX, cycloheximide; ERK, extracellular signal-regulated kinase; LiCl, lithium chloride; MG132, proteasome inhibitor; pcDNA, pcDNA3 vector control; pERK, phosphorylated-ERK; Rel, relative; RPIA-ΔD, RPIA deletion domain D mutant; RPIA, ribose-5-phosphate isomerase A; RPIA-WT, RPIA wild type; si-NC; negative control siRNA; siRNA, small interfering RNA; si-RPIA, RPIA small interfering RNA.</p

RPIA regulates colon cell proliferation through β-catenin expression in HCT116 cells.

<p>(A) Knockdown of RPIA significantly reduced cell proliferation and RPIA overexpression enhanced cell proliferation in HCT116 cells. Co-treatment of si-RPIA and pcDNA-RPIA rescued the reduction of cellular proliferation upon knockdown of RPIA in HCT116. Cell viability assays were performed by measuring the cells at the second, third, fourth, and fifth days and the proliferation fold is compared to control cell at the first day. Control: Co-transfect with scramble RNA and pcDNA empty vector as negative control. (B) RPIA knockdown significantly reduced colony formation ability, and RPIA overexpression enhanced colony formation ability in HCT116 cells. si-NC: Transfect with scramble siRNA as negative control. Representative images of colonies were shown on top of the quantification result. (C) Knockdown of RPIA reduced β-catenin protein levels as measured by western blotting (left panel) and quantified using Image J (middle panel) but did not significantly alter mRNA levels of β-catenin as measured by qPCR (right panel) in HCT116 cells. (D) RPIA overexpression increased β-catenin protein levels (left and middle panels) but did not affect β-catenin mRNA levels (right panel) in HCT116 cells. (E) Knock down of RPIA reduced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (F) Overexpression of RPIA induced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (G) Knockdown of RPIA decreased the mRNA levels of the β-catenin target genes <i>CCND1</i>, <i>CCNE2</i>, and <i>AXIN2</i> in HCT116 cells. (H) Overexpression of RPIA increased the mRNA levels of the β-catenin target genes <i>CCND1</i>, <i>CCNE2</i>, and <i>AXIN2</i> in HCT116 cells. The statistical significance was calculated with Student <i>t</i> test (* 0.01 < <i>P</i> < 0.05, ** 0.001 < <i>P</i> < 0.01, and *** <i>P</i> < 0.001). Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003714#pbio.2003714.s010" target="_blank">S2 Data</a>. <i>AXIN2</i>, <i>Axis inhibition protein 2</i>; <i>CCND1</i>, <i>Cyclin D1</i>; <i>CCNE2</i>, <i>Cyclin E2</i>; <i>CTNNB1</i>, <i>CATENIN BETA 1</i>; pcDNA, pcDNA3 vector control; qPCR, quantitative PCR; RPIA, ribose-5-phosphate isomerase A; si-NC, negative control small interfering RNA; si-RPIA, RPIA small interfering RNA; TCF, T-cell transcription factor.</p

RPIA is highly expressed in different stages of CRC.

<p>(A) Representative RPIA IHC staining at different stages of colon cancer is shown. Scale bar: 500 μm. (B) The average IRS for RPIA staining showed significantly increased RPIA expression from stage I to IVB and the M. Stage III was divided into IIIB and IIIC, and stage IV was divided into IVA and IVB based on their subcategories. The statistical significance was calculated with Student <i>t</i> test (*** <i>P</i> < 0.001). (C) The average RPIA mRNA fold change in paired tissues (tumor tissue versus the surrounding normal tissue) from CRC patients at stages I to IV. The box plot indicates the median (central horizontal line), 75th percentile (the top of box), 25th percentile (the bottom of box), maximum value (the top end), minimum value (the bottom end), and the outlier (the point). Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003714#pbio.2003714.s009" target="_blank">S1 Data</a>. CRC, colorectal cancer; IHC, immunohistochemistry; IRS, immunoreactive score; M, metastasis stage; N, normal colorectal tissue; RPIA, ribose-5-phosphate isomerase A.</p

RPIA localizes in the nucleus and interacts with APC and β-catenin in HCT116 cells.

<p>(A) Nuclear localization of RPIA was detected by immunostaining with an anti-RPIA antibody (green) in HCT116 cells with and without overexpression of RPIA. DAPI was used to counterstain nuclei (blue). Scale bar: 50 μm. (B) Left panels: The cell lysates were precipitated by anti-APC, anti-β-catenin, and anti-RPIA antibodies in HCT116 cells. The APC, β-catenin, and RPIA interaction can be increased by RPIA-WT but not by RPIA-ΔD. Right panels: Protein loading input for IP assay of HCT116 cells. The orange boxes indicate the signals were enhanced by RPIA-WT but not in RPIA-ΔD. (C) Model of RPIA-β-catenin-APC interaction in HCT116 cell line. APC, adenomatous polyposis coli; Cyt, cytoplasm; IgA, immunoglobulin A; IP, immunoprecipitation; pcDNA, pcDNA3 vector control; Nu, nucleus; RPIA-ΔD, RPIA deletion domain D mutant; RPIA, ribose-5-phosphate isomerase A; RPIA-WT, RPIA wild type.</p

K4R2-Nal2-S1 treatment attenuates xenograft tumor growth.

<p>(a) Dorsal sides of male nude mice s.c. injected with PC9 human lung cancer cells and i.v. treated with K4R2Nal2-S1 (right) or PBS control (left) at the 46^th day after cancer cell implantation (5 days for tumor growth, 40 days for treatment, photographed on the 46^th day). (b) Mice body weight (left) and tumor volume (right) from (a) were monitored over the time period as indicated. (c) Exposed tumors (mice were sacrificed at the 46^th day after cancer cell implantation) of mice treated with K4R2Nal2-S1 (upper) or PBS (lower). 12 exposed tumors were found in the PBS group (6 mice x 2 side implantation), but only 7 exposed tumors were found in the K4R2Nal2-S1 treatment group. Total tumor weight for both groups were measured and shown in right.</p

Excised xenografted tumors from the mice in Fig 3 were subjected to H&E staining and immunohistochemical assay for cleaved PARP expression to monitor cellular apopotosis.

<p>Scale bars = 500 μm, 100 μm, and 50 μm (40X, 200X, and 400X), respectively.</p

Plots of the activities of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1 against OECM-1, C9, SAS, A549, PC9, PC9-G cancer cell lines.

<p>Plots of the activities of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1 against OECM-1, C9, SAS, A549, PC9, PC9-G cancer cell lines.</p

Hemolysis and cytotoxicity to human red blood cells (hRBCs) and human fibroblast (HFW) cells of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1.

<p>Hemolysis and cytotoxicity to human red blood cells (hRBCs) and human fibroblast (HFW) cells of S1, Nal2-S1, K4R2-Nal2-S1, and K6-Nal2-S1.</p