Rebecca J. Whelan

MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress

Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3–5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer.

Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC50 of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC50 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.

Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing.

AbstractThe development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process—and not only its endpoint—to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach. Graphical AbstractA population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.

Short-chain carboxylic acids from gray catbird (Dumetella carolinensis) uropygial secretions vary with testosterone levels and photoperiod

The uropygial gland of birds produces secretions that are important in maintaining the health and structural integrity of feathers. Non-volatile components of uropygial secretions are believed to serve a number of functions including waterproofing and conditioning the feathers. Volatile components have been characterized in fewer species, but are particularly interesting because of their potential importance in olfactory interactions within and across species. We used solid-phase microextraction headspace sampling with gas chromatography-mass spectrometry to detect and identify volatiles in uropygial secretions of gray catbirds (Dumetella carolinensis), a North American migratory bird. We consistently detected the following carboxylic acids: acetic, propanoic, 2-methylpropanoic, butanoic, and 3-methylbutanoic. We tested for the effect of lengthened photoperiod and/or exogenous testosterone on volatile signal strength and found a negative effect of lengthened photoperiod on the signal strength of propanoic, 2-methylpropanoic, and butanoic acids, suggesting a trade-off between their production and heightened night-time activity associated with lengthened photoperiod. Signal strength of propanoic and 2-methylpropanoic acids was lower in birds treated with exogenous testosterone than in birds treated with placebos. Sex did not affect signal strength of any of the volatile compounds.

Volatile and Semivolatile Compounds in Gray Catbird Uropygial Secretions Vary with Age and Between Breeding and Wintering Grounds

The uropygial secretions of some bird species contain volatile and semivolatile compounds that are hypothesized to serve as chemical signals. The abundance of secretion components varies with age and season, although these effects have not been investigated in many species. We used solid-phase microextraction headspace sampling and solvent extraction coupled with gas chromatography–mass spectrometry to detect and identify volatile and semivolatile chemical compounds in uropygial secretions of gray catbirds (Dumetella carolinensis). We identified linear and branched saturated carboxylic acids from acetic (C2) through hexacosanoic (C26); linear alcohols from decanol (C10) through docosanol (C22); one aromatic aldehyde; one monounsaturated carboxylic acid; two methyl ketones; and a C28 ester. We tested for the effect of age on signal strength and found that juvenile birds produced greater amounts of volatile C4 through C7 acids and semivolatile C20 through C26 acids, although the variation among individuals was large. Adult birds displayed small concentrations and minimal individual variation among volatile compounds, but produced significantly higher levels of long-chain linear alcohols than juvenile birds. We tested for the effects of season/location by sampling adult catbirds at their Ohio breeding grounds and at their Florida wintering grounds and found that the heaviest carboxylic acids are significantly more abundant in secretions from birds sampled during winter at the Florida site, whereas methyl ketones are more abundant in birds sampled during summer on the Ohio breeding grounds. We observed no effect of sex on semivolatile compounds, but we found a significant effect of sex on levels of carboxylic acids (C4 through C7) for juvenile birds only.

Modulation of oxidative stress and subsequent induction of apoptosis and endoplasmic reticulum stress allows citral to decrease cancer cell proliferation.

The monoterpenoid, citral, when delivered through PEG-b-PCL nanoparticles inhibits in vivo growth of 4T1 breast tumors. Here, we show that citral inhibits proliferation of multiple human cancer cell lines. In p53 expressing ECC-1 and OVCAR-3 but not in p53-deficient SKOV-3 cells, citral induces G1/S cell cycle arrest and apoptosis as determined by Annexin V staining and increased cleaved caspase3 and Bax and decreased Bcl-2. In SKOV-3 cells, citral induces the ER stress markers CHOP, GADD45, EDEM, ATF4, Hsp90, ATG5, and phospho-eIF2α. The molecular chaperone 4-phenylbutyric acid attenuates citral activity in SKOV-3 but not in ECC-1 and OVCAR-3 cells. In p53-expressing cells, citral increases phosphorylation of serine-15 of p53. Activation of p53 increases Bax, PUMA, and NOXA expression. Inhibition of p53 by pifithrin-α, attenuates citral-mediated apoptosis. Citral increases intracellular oxygen radicals and this leads to activation of p53. Inhibition of glutathione synthesis by L-buthionine sulfoxamine increases potency of citral. Pretreatment with N-acetylcysteine decreases phosphorylation of p53 in citral-treated ECC-1 and OVCAR-3. These results define a p53-dependent, and in the absence of p53, ER stress-dependent mode of action of citral. This study indicates that citral in PEG-b-PCL nanoparticle formulation should be considered for treatment of breast and other tumors.

Selection of DNA Aptamers for Ovarian Cancer Biomarker CA125 Using One-Pot SELEX and High-Throughput Sequencing

CA125 is a mucin glycoprotein whose concentration in serum correlates with a womans risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient samples is of great clinical relevance. We suggest that powerful new diagnostic tools may be enabled by the development of nucleic acid aptamers with affinity for CA125. Here, we report on our use of One-Pot SELEX to isolate single-stranded DNA aptamers with affinity for CA125, followed by high-throughput sequencing of the selected oligonucleotides. This data-rich approach, combined with bioinformatics tools, enabled the entire selection process to be characterized. Using fluorescence anisotropy and affinity probe capillary electrophoresis, the binding affinities of four aptamer candidates were evaluated. Two aptamers, CA125_1 and CA125_12, both without primers, were found to bind to clinically relevant concentrations of the protein target. Binding was differently influenced by the presence of Mg2+ ions, being required for binding of CA125_1 and abrogating binding of CA125_12. In conclusion, One-Pot SELEX was found to be a promising selection method that yielded DNA aptamers to a clinically important protein target.

Synthesis and structural characterization of the peptide epitope of the ovarian cancer biomarker CA125 (MUC16)

A highly conserved region of 21 amino acids flanked by cysteine residues, contained within a larger repeated domain, has been proposed to be the antibody-binding site in the ovarian cancer biomarker CA125 (MUC16). In this study solid-phase peptide synthesis with Fmoc protection chemistry was used to assemble a 21-mer peptide corresponding to the most frequently occurring antibody binding sequence in CA125. Potentially significant sequence variants were also synthesized. Peptide secondary structure was investigated using Fourier transform infrared spectroscopy, revealing the consensus sequence peptide to be largely unstructured at physiological pH whether the cysteine residues were reduced or were oxidized to form an intramolecular disulfide bond. Substitution of serine for proline at position 8 (P8S) results in β-sheet formation in peptides involved in intramolecular disulfide bonds. This β-sheet structure does not persist in peptides incapable of intramolecular disulfide bonding because of sequence nor in peptides treated with the reducing agent dithiothreitol. In CA125, P8S is predicted to occur in ∼25% of repeat domains, suggesting that this structural motif is a non-negligible contributor to overall structure and function. These findings suggest that future structural characterization efforts of CA125 should be especially mindful of the amino acid sequence and oxidation state of the protein.

Attraction of Culex pipiens to uropygial gland secretions does not explain feeding preference for American robins

ABSTRACT: Culex pipiens, the endemic mosquito vector of West Nile virus in eastern North America, is responsible for maintenance of the virus in avian reservoir hosts, the most important of which appears to be the American robin. One reason for the greater involvement of robins is believed to be the feeding preference of Cx. pipiens, however, the basis of this preference is not understood. We tested the hypothesis that the species-specific chemical profile of avian uropygial gland secretions are used by Cx. pipiens as cues to locate birds and, therefore, may contribute to the observed feeding preferences. We used gas chromatography-mass spectrometry to identify the semi-volatile components of the uropygial gland secretions of American robins and two other common reservoir host species, the house sparrow and European starling. We found that the chemical composition of the robin secretions was different from those of the sparrows and starlings. Through behavioral choice trials conducted in a dual-port olfactometer, we also found that Cx. pipiens did not prefer the secretions of robins over the other two species. Surprisingly, however, we found that Cx pipiens were more often attracted to live starlings over robins and to the secretions of starlings over those of robins.

Attraction of Culex pipiens to House Sparrows Is Influenced by Host Age but Not Uropygial Gland Secretions

Culex pipiens serves as the endemic vector of West Nile virus (WNV) in eastern North America, where house sparrows (HOSP, Passer domesticus) serve as a reservoir host. We tested the hypotheses that: (1) Attraction of Cx. pipiens to HOSP is influenced by bird age and (2) that age-specific variation in chemical profiles of bird uropygial gland secretions informs this choice. We conducted mosquito choice trials in an olfactometer and found that Cx. pipiens were more often attracted to adult sparrows over nestlings, however, they demonstrated no preference for adults over fledglings. Using gas chromatography-mass spectrometry we observed age-specific differences in the semi-volatile chemical profiles of house sparrow uropygial gland secretions. Contrary to our hypothesis, we found no significant difference in mosquito feeding preference between the secretions of adults and those of either nestlings or fledglings. We suggest that other chemical cues influence the feeding preference of Cx. pipiens, either independently of uropygial gland secretions, or synergistically with them.