Ripening-induced chemical modifications of papaya pectin inhibit cancer cell proliferation

Abstract Papaya (Carica papaya L.) is a fleshy fruit with a rapid pulp softening during ripening. Ripening events are accompanied by gradual depolymerization of pectic polysaccharides, including homogalacturonans, rhamnogalacturonans, arabinogalactans, and their modified forms. During intermediate phases of papaya ripening, partial depolymerization of pectin to small size with decreased branching had enhanced pectin anti-cancer properties. These properties were lost with continued decomposition at later phases of ripening. Pectin extracted from intermediate phases of papaya ripening markedly decreased cell viability, induced necroptosis, and delayed culture wound closing in three types of immortalized cancer cell lines. The possible explanation for these observations is that papaya pectins extracted from the third day after harvesting have disrupted interaction between cancer cells and the extracellular matrix proteins, enhancing cell detachment and promoting apoptosis/necroptosis. The anticancer activity of papaya pectin is dependent on the presence and the branch of arabinogalactan type II (AGII) structure. These are first reports of AGII in papaya pulp and the first reports of an in vitro biological activity of papaya pectins that were modified by natural action of ripening-induced pectinolytic enzymes. Identification of the specific pectin branching structures presents a biological route to enhancing anti-cancer properties in papaya and other climacteric fruits

Aged gp78 knockout mice develop obesity and gp78 is unregulated upon ER stress.

<p>(<b>A</b>) Schematic of gp78-targeting strategy. The gp78 allele disrupted by the insertion of a gene trap vector (OmniBank Vector 76) in the first intron. Genomic DNA isolated from WT, heterozygous and homozygous was genotyped by PCR. Primer set including double reverse primers was designed for internal PCR quality. (<b>B</b>) Total RNA and lysates were prepared in embryo and adult liver and were analyzed in RT-PCR (Top), both lysates were immunoprecipitated with rabbit anti-gp78 antibodies (epitope: 524–537) and then immunoblotted with monoclonal anti-gp78 antibodies (epitope: 451–551) to identify gp78 protein (bottom). (<b>C</b>) Immunohistochemistry (IHC) of liver with monoclonal anti-gp78 antibody. Arrow indicates gp78 positive stain. PV, portal vein. Original image, x400. (<b>D</b>) Upregulation of gp78 in response to ER stress. Immortalized THLE-3 and cancerous HepG2 liver cells were treated with tunicamycin (1ug/ml) for 24 hrs. and lysates were immunoprecipitated with anti-gp78 antibody and immunoblotted. GRP78/BIP, a chaperon of UPR pathways. (<b>F</b>) Photography of abdomen of 1-year-old mice (left). Comparison of body weight of WT and gp78-KO mice at 3 months (n = 25), 6 months (n = 25), 12 months (n = 25) old (right). Asterisk indicates a significance determined by Student’s test (*, p < 0.05).</p

Gp78-KO mice spontaneously develop hepatic steatosis with inflammatory infiltrates.

<p>(<b>A</b>) Livers of 1-year-old gp78 KO mice, grown with normal diet were stained with H&E and visualized as indicated magnification. (a) Steatohepatitis shows swelling cytoplasm with lipid droplets (black arrow) and infiltrating cells. (b) Mild lipid droplets and infiltrating cells (Red arrow). (c) Infiltrate cells gather in the absence of lipid droplets. (<b>B</b>) Oil red stain. Arrow indicates lipid droplets. (<b>C</b>) Trichrome stain was preformed to identify blue colored collagen fibers. PV; portal vein, FL; fatty liver area. (<b>D</b>) Percentage of incidence including mild fatty liver, inflammation in 1-year-old KO mice (n = 25 mice per each group).</p

Gp78-KO mice with acute ER stress progresses to severe fatty liver through UPR-driving SREBP-1 activation.

<p>(<b>A</b>) 1mg/kg body weight of tunicamycin (TM) was intraperitoneally injected to 6 months-old gp78-KO mice. Body weight is represented after normalization to the starting weight as the meant ± SE (n = 3 per group). (<b>B</b>) Radish livers are brown colored after TM injection. Liver of gp78-KO at 11 days was not recovered. (<b>C</b>) Acute ER stress potentiates entire fatty liver of gp78-KO. H&E stain at 3 days after TM injection. Ballooned cells (b) are typically bigger size than WT hepatocytes (a). Cytoplasmic lipid droplets (arrow) (40x). (<b>D</b>) Prolonged fatty liver and fibrosis in gp78-KO. H&E, oil red and Trichrome stains at 11 days after TM injection. White ballooned cells (arrows) were maintained on H&E (200x). Frozen tissues were stained with oil-red O. Irregular fibrosis was extended from connective tissue of portal vein surrounding accumulated lipid droplets (white spots) at pinkish gp78-KO liver (100x). (<b>E</b>) Persistent SREBP-1 activation along with UPR up regulation is responsible for fatty liver of gp78-KO. TM-injected mice were scarified respectively (n = 3). Liver extracts at indicated days were subjected to immunoblots. GRP78, Glucose-Regulated Protein; PDI, Protein Disulfide Isomerase, SERBP; Sterol Regulatory Element Binding Transcription Factor; Insig, Insulin Induced Gene. (<b>F</b>) Chop-mediated apoptosis. Cell survival was analyzed with viable counting in gp78-KO mouse embryonic fibroblast (MEF) cells treated with TM (1μg/ml) as indicated times (top). Induction of UPR was analyzed in immunoblots and gp78 expression was visualized after its immunoprecipitation (bottom). Chop; ER stress-mediated apoptosis marker.</p

<i>miR-655</i> Is an EMT-Suppressive MicroRNA Targeting <i>ZEB1</i> and <i>TGFBR2</i>

<div><p>Recently, the epithelial-to-mesenchymal transition (EMT) has been demonstrated to contribute to normal and disease processes including cancer progression. To explore EMT-suppressive microRNAs (miRNAs), we established a cell-based reporter system using a stable clone derived from a pancreatic cancer cell line, Panc1, transfected with a reporter construct containing a promoter sequence of <i>CDH1/E-cadherin</i> in the 5′ upstream region of the <i>ZsGreen1</i> reporter gene. Then, we performed function-based screening with 470 synthetic double-stranded RNAs (dsRNAs) mimicking human mature miRNAs using the system and identified <i>miR-655</i> as a novel EMT-suppressive miRNA. Overexpression of <i>miR-655</i> not only induced the upregulation of E-cadherin and downregulation of typical EMT-inducers but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift toward the epithelial phenotype. In addition, we found a significant correlation between <i>miR-655</i> expression and a better prognosis in esophageal squamous cell carcinoma (ESCC). Moreover, <i>ZEB1</i> and <i>TGFBR2</i>, which are essential components of the TGF-b signaling pathway, were identified as direct targets of <i>miR-655</i>, suggesting that the activation of the TGF-b-ZEB1-E-cadherin axis by aberrant downregulation of <i>miR-655</i> may accelerate cancer progression.</p></div

EMT-suppressive effects of <i>miR-655</i> on mesenchymal-like cancer cells having phenotypic plasticity at EMT/MET.

<p><b><i>A</i></b>, TaqMan real-time RT-PCR analysis of <i>CDH1/E-cadherin</i> and <i>miR-655</i> in a panel of 23 pancreatic cancer cell lines and a breast cancer cell line, MDA-MB-231. Relative expression levels of transcripts of <i>CDH1/E-cadherin</i> and <i>miR-655</i> were quantified in comparison to <i>GAPDH</i> and <i>RNU6B</i>, respectively, to normalize the initial input of total RNA. Bar graphs show the ratio of the expression level in these cell lines to that in normal pancreas (Ambion). <b><i>B</i></b>, Representative results of phase contrast images (<b><i>Upper</i></b>) and <i>CDH1/E-cadherin</i> protein expression level detected by immunofluorescence staining (<b><i>Lower</i></b>) in Panc1, KP1N, KP4-4 and MDA-MB-231 cells 96 hours after transfection with 10 nM of ds-<i>NC</i> or dsRNA mimicking <i>miR-655</i> (ds-<i>miR-655</i>) (Ambion). <b><i>C</i></b>, TaqMan real-time RT-PCR analysis (<b><i>Upper</i></b>) and Western blot (<b><i>Lower</i></b>) analysis of mRNA and protein levels of <i>CDH1/E-cadherin</i>, respectively, in Panc1, KP1N, KP4-4 and MDA-MB-231 cells 96 hours after transfection of 10 nM of ds-<i>NC</i> or ds-<i>miR-655</i>. Asterisks (*), statistical analysis with the Mann-Whitney <i>U</i> test. <b><i>D</i></b>, Growth curves in Panc1, KP1N, KP4-4 and MDA-MB-231 cells after transfection of 10 nM of ds-<i>NC</i> or ds-<i>miR-655</i>. Each data point represents the mean of duplicate determinations (bars, SD) in these experiments. Asterisks (*), statistical analysis with the Mann-Whitney <i>U</i> test. <b><i>E</i></b>, Representative phase micrographs of Panc1, KP1N, KP4-4 and MDA-MB-231 cells transiently transfected with 10 nM of ds-<i>NC</i> or ds-<i>miR-655</i> in cell migration and invasion assays <i>in vitro</i> using uncoated and Matrigel-coated transwell-chamber culture systems (Becton Dickinson), respectively. At 48 hours after transfection of dsRNA, cells were transferred into the upper chamber of the transwell (4×10⁴ cells per well). The migrating or invading cells on the lower surface of filters were fixed and stained with the Diff-Quik stain 48 hours after cell transfer. <b><i>F</i></b>, Quantification of the cell migration (<b><i>Left</i></b>) and invasion (<b><i>Right</i></b>) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062757#pone-0062757-g003" target="_blank">Figure 3B</a>. Bar graphs show the percentage (%) of <i>miR-655</i>-transfectants migrating (<b><i>Left</i></b>) or invading (<b><i>Right</i></b>) through uncoated or Matrigel-coated filters, respectively, relative to control-transfectants. Asterisks (*), statistical analysis with the Mann-Whitney <i>U</i> test.</p

Expression analysis of <i>miR-655</i> in primary ESCC and OSCC cases.

<p><b><i>A</i></b>, TaqMan real-time RT-PCR analysis of endogenous <i>miR-655</i> in 22 normal human tissues (Ambion and Clontech). Marked upregulation of <i>miR-655</i> expression (>2-fold increase compared with pancreas) was observed in brain, cervix, esophagus and placenta. <b><i>B</i></b>, Expression profiles of <i>miR-655</i> in a panel of paired tumorous and non-tumorous tissues from primary ESCC and OSCC cases. Bar graphs show the ratio of the expression level in tumors (T) to those in their paired normal mucosae (N). <b><i>C</i></b>, Kaplan-Meier survival curves for high and low <i>miR-655</i> groups based on TaqMan real-time RT-PCR. In univariate analyses of overall and non-recurrent survival with log-rank tests, a high level of <i>miR-655</i> expression was significantly associated with a much better survival rate among patients with ESCC (<i>P</i> = 0.0359, log-rank test).</p

Function-based screening of EMT-suppressive miRNAs using reporter system for investigating <i>CDH1/E-cadherin</i>-promoter activity in Panc1 cells.

<p><b><i>A</i></b>, Map of the promoter region of the <i>CDH1/E-cadherin</i> gene. To construct a reporter plasmid, 1,058 bp promoter sequences indicated by the closed arrow in this map was introduced into a promoterless pZsGreen1-1 vector with the <i>ZsGreen1</i> gene as a reporter gene. A cell-based reporter system was established by isolation of a stable clone with the limiting dilution method after transfection of the construct into Panc1 cells. <b><i>B</i></b>, Confirmation of the expression of the ZsGreen1 protein in the cell-based reporter system following transfection of <i>miR-200a</i> or <i>-200b</i>. A stable cell clone with the reporter plasmid was evaluated 96 hours after transient transfection of 10 nM of dsRNA mimicking <i>miR-200a</i> or <i>-200b,</i> or control non-specific miRNA (ds-<i>miR-200a</i>, ds-<i>miR-200b</i> or ds-<i>NC</i>) (Ambion). <b><i>Upper</i></b><b>,</b> Detection of ZsGreen1 in these transfectants using fluorescence micrographs. <b><i>Lower</i></b><b>,</b> Quantification of fluorescence intensity in these transfectants (<b><i>Left</i></b>). Results of the TaqMan real-time RT-PCR analysis (<b><i>Middle</i></b>) and Western blot analysis (<b><i>Right</i></b>) for expression of the <i>CDH1/E-cadherin</i> transcript and protein, respectively, in these transfectants. <b><i>C</i></b>, Results of the function-based screening of EMT-suppressive miRNAs in a cell-based reporter system using Pre-miR™ miRNA Precursor Library-Human V3 (Ambion) containing 470 dsRNAs mimicking human mature miRNAs. The fluorescence intensity of ZsGreen1 was evaluated by fluorescence microplate reader in duplicate. The relative fluorescence intensity in each transfectant was calculated by normalization of each result to the fluorescence intensity in control cells transfected with non-specific miRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062757#pone-0062757-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062757#pone.0062757.s011" target="_blank">Table S2</a>). The lower closed arrow indicates the 470 miRNAs examined. <b><i>D</i></b>, Western blot analysis of E-cadherin protein levels in parental Panc1 cells 96 hours after transient transfection with 10 nM of ds-<i>NC</i> or 10 nM of ds-miRNAs mimicking <i>miR-96-5p, -132-3p, -183-5p, -139-5p, -217, -520d-3p, -526b-3p, -629-3p, -655</i> and <i>-200b-3p</i>. Because <i>miR-200b</i> has already been confirmed to induce expression of the <i>CDH1/E-cadherin</i> transcript and protein in this study (Fig. 1B) and multiple previous studies, ds-<i>miR-200b</i> was used as a positive control in this analysis.</p

Summary of 17 miRNA genes selected as candidates for EMT-suppressive miRNAs in functional-based screening using a stable Panc1 clone transfected with a reporter construct containing a promoter sequence of <i>CDH1/E-cadherin</i> in the 5′ upstream region of the <i>ZsGreen1</i> reporter gene and Pre-miR™ miRNA Precursor Library - Human V3 (Ambion).

*<p>The ratio of fluorescence intensity of ZsGreen1 (RFI) in cells 4 days after transfection with each dsRNA was normalized to that in control transfectants (Pre-miRTM Negative Control #1, Ambion).</p>**<p>The ratio of growth level (RG) of viable cells assessed by WST8 assay 4 days after transfection with dsRNAs. WST-8 assay was employed to normalize the number of viable cells relative to the control transfectants.</p