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Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

ABSTRACT: Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.

SUBMITTER: Musrap N

PROVIDER: S-EPMC4498061 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

Musrap Natasha N Tuccitto Alessandra A Karagiannis George S GS Saraon Punit P Batruch Ihor I Diamandis Eleftherios P EP

The Journal of biological chemistry 20150524 28

Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving ...[more]

PMID: 26004777

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Project description:The transmembrane 16 (TMEM16) family contains 10 subtypes, and the function of each protein is different. TMEM16A is a calcium-activated chloride channel involved in physiological and pathological situations. Liquiritigenin is an aglycone derived from Glycyrrhiza glabra, and it is generated via the metabolism of enterobacterial flora. It has been known that liquiritigenin reduces pain sensation involving TMEM16A activation in primary sensory neurons. In addition, other pharmacological effects of liquiritigenin in physiological functions involving TMEM16A have been reported. However, the relationship between TMEM16A and liquiritigenin is still unknown. Therefore, we hypothesized that TMEM16A is inhibited by liquiritigenin. To confirm this hypothesis, we investigated the effect of liquiritigenin on TMEM16A currents evoked by intracellular free calcium in HEK293T cells transfected with TMEM16A. In this study, we found that liquiritigenin inhibited the mouse and human TMEM16A currents. To further confirm its selectivity, we also investigated its pharmacological effects on other ion channels, including transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), which are non-selective cation channels involved in pain sensation. However, liquiritigenin did not inhibit the currents of TRPV1 and TRPA1 induced by capsaicin and allyl isothiocyanate, respectively. Therefore, our findings indicate that selective TMEM16A inhibition could be one molecular mechanism that explains liquiritigenin-induced pain reduction. Additionally, we also investigated the inhibitory effects of estrogens on TMEM16A because liquiritigenin reportedly binds to the estrogen receptor. In this study, a pregnancy-dependent estrogen, estriol, significantly inhibited TMEM16A. However, the efficacy was weak. Although there is a possibility that TMEM16A activity could be suppressed during pregnancy, the physiological significance seems to be small. Thus, the inhibitory effect of estrogen might not be significant under physiological conditions. Furthermore, we investigated the effect of dihydrodaidzein, which is an analog of liquiritigenin that has a hydroxyphenyl at different carbon atom of pyranose. Dihydrodaidzein also inhibited mouse and human TMEM16A. However, the inhibitory effects were weaker than those of liquiritigenin. This suggests that the efficacy of TMEM16A antagonists depends on the hydroxyl group positions. Our finding of liquiritigenin-dependent TMEM16A inhibition could connect the current fragmented knowledge of the physiological and pathological mechanisms involving TMEM16A and liquiritigenin.

Dataset Information

Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

Publications

Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

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