Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Potencial herramienta en la personalización del tratamiento oncológico: Casos clínicos / In vitro cell response to chemotherapeutic agents, to personalize ovarian cancer treatment: Report of two cases

Our laboratory has implemented an in vitro assay to estimate the response to chemotherapy in ovarian cancer cells pertaining to individual patients. In two selected patients, we determined the correlation between an in vitro assay of cells from suspected ovarian cancer ascites, with the clinical chemotherapy response. Cancer cells isolated from peritoneal fluid with suspected ovarian cancer were tested for cytotoxicity with corresponding chemotherapy regimens. Circulating Cal25 levels and attending physician consultation determined clinical course and response to chemotherapy. The in vitro assay result correlated with Cal25 levels, progression free survival and attending physician evaluation. The assay predicted correctly the failure of two successive chemotherapy regimes in the first patient, while predicting a favorable clinical response in the second subject.

In vivo and in vitro estrogenic and progestagenic actions of Tibolone

Estrogen and progestin combination in hormone replacement therapy (HRT) increases the incidence of breast cancer, but decreases the endometrial cancer risk of unopposed estrogen. Therefore, a SERM such as Tibolone, that delivers the beneficial, but not the adverse side effects, of steroid hormones would be clinically advantageous. However, data from the Million Women Study suggests that Tibolone increases the risk of both breast and endometrial cancer. Herein, we assessed the estrogenic and progestagenic actions of Tibolone using transvaginal sonography studies and an in vitro model of breast (ZR-75, MCF7) and endometrial cancer (Ishikawa). The known cancer associated proteins (ER, EGFR, STAT5, tissue factor and Bcl-xL) were selected for study. Transvaginal sonography demonstrated that postmenopausal women treated with Tibolone displayed a thinner endometrium than in the late proliferative phase, but had a phenotype characteristic of the secretory phase, thus demonstrating the estrogenic and progestagenic actions of this SERM. In vitro, Tibolone acted as an estrogen in downregulating ER and upregulating Bcl-xL, yet as progesterone, increasing STAT5 and tissue factor in breast cancer cells. The increase in tissue factor by Tibolone correlated with its coagulative potential. Interestingly, EGFR was up-regulated by progesterone in the breast and by estrogen in endometrial cells, while Tibolone increased protein levels in both cell types. In conclusion, this study further demonstrates the estrogenic and progestagenic nature of Tibolone. The pattern of regulation of known oncogenes in cells of breast and endometrial origin dictates caution and vigilance in the prescription of Tibolone and subsequent patient monitoring.

The action of ovarian hormones in cardiovascular disease

The incidence of cardiovascular disease (CAD) differs between men and women, in part because of differences in risk factors and hormones. This sexual dimorphism means a lower incidence in atherosclerotic diseases in premenopausal women, which subsequently rises in postmenopausal women to eventually equal that of men. These observations point towards estrogen and progesterone playing a lifetime protective role against CAD in women. As exogenous estrogen and estrogen plus progesterone preparations produce significant reductions in low-density lipoprotein (LDL) cholesterol levels and significant increases in high-density lipoprotein (HDL) cholesterol, this should in theory lower the risk of CAD. However, results from oral contraceptive (OC) use and combined estrogen and progesterone hormone replacement therapy (HRT) have suggested that hormone replacement regimes do not provide cardiovascular protection. In fact, depending on the preparation and the presence or absence of genetic risk factors, an increased risk of cardiovascular diseases such as venous thrombosis, myocardial infarction (MI) and stroke have been observed. Interestingly, in the majority of studies the increase in risk was highest in the first year, after which an increase in risk was not observed, and in some studies a lower risk of CAD was evident after four or five years of exogenous hormone administration. While the debate continues about the merits of HRT, and several good reviews exist on the statistics of CAD in relation to exogenous hormones, we have decided to review the literature to piece together the physiological actions of estrogen and progesterone preparations on the individual mechanistic components leading to CAD; namely, the altered endothelium and the haemostatic balance between coagulation and fibrinolysis. We present possible mechanisms for how HRT and OCs protect against MI in the absence of cardiovascular risk factors but increase the incidence of MI in their presence. We also speculate on the roles played by hormones on the short- and long-term risks of cardiovascular disease.

Parto prematuroaasociado a colestasia intrahepaica del embarazo: rol del acidoocolico en la modulacion de la via ocitocina-receptor de ot en el miometrio

Se ha reportado una asociación entre CIE y distrés fetal, muerte fetal in utero y síntomas de parto prematuro/parto prematuro. Con la hipótesis que en la CIE el aumento en los ácidos biliares, en particular ácido cólico, activa la vía ocitocina-receptor de ocitocina (OT-ROT) en el miometrio, generando un inicio prematuro del trabajo de parto, evaluamos la sensibilidad miometrial a OT in vitro e in vivo, y la expresión de mRNA en pacientes CIE y controles y la capacidad de ácido cólico para regular la activación de la vía OT-ROT. Las pacientes con CIE mostraron una alta sensibilidad a OT in vivo e in vitro comparado con controles. Al incubar segmentos/células en cultivo de miometrio con ácido se observó una marcado aumento de la sensibilidad contráctil para la OT y un aumento de la expresión celular del mRNA y los niveles de la proteína del ROT. La activación de la vía OT-ROT parece ser el resultado de un aumento de la síntesis/función de RsOT mediada por el ácido cólico. Esto resultados entregan las bases celulares/moleculares que podrían explicar la asociación entre CIE y parto prematuro.

Intrahepatic cholestasis of pregnancy is associated with preterm labor and delivery. We hypothesize that in the course of this disease, bile acids activate the oxytocin receptor pathway leading to the premature onset of labor. We evaluated in vitro and in vivo myometrial sensitivity to oxytocin and the oxytocin-receptor expression in patients with cholestasis and control. Furthermore, we evaluated the ability of cholic acid to mediate the activation of the oxytocin-receptor pathway. Patients with cholestasis of pregnancy, compared to controls, required lower doses of oxytocin to elicit 3 uterine contractions in 10 minutes (1.3 ± 0.6 vs 3.6 ± 0.8 Units; p < 0.05) and had lower in vitro ED50 (1.6 x 10_10 M vs 1.0 x 10_8 M; p < 0.05), reflecting higher oxytocin sensitivity. The long-term (24 h) incubation of myometrial strips from control patients with cholic acid (20 µM) markedly increased the sensitivity to oxytocin, but not to endothelin-1 or prostaglandin F2a. The 24 h incubation of cultured myometrial cells with cholic acid increased the expression of oxytocin-receptor at mRNA (northern blot) and protein levels (western blot and immunohistochemistry). Our results demonstrate that in the setting of intrahepatic cholestasis of pregnancy, an activation of the oxytocin-receptor pathway occurs. The activation of such pathways seems to be the result of a cholic acid mediated increase in receptor expression, which in turn could explain the increase in the preterm labor and delivery rate observed in this disease.