Chemosensitivity of BRCA1-Mutated Ovarian Cancer Cells and Established Cytotoxic Agents.

OBJECTIVE: Serous adenocarcinomas that arise in patients with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are initially well treatable with platinum/paclitaxel. For recurrent disease in patients with BRCA1 or BRCA2 mutations, olaparib treatment is available. To study additional therapeutic regimens, a better understanding of the cellular and molecular mechanisms of the tumors in in vitro models is important. METHODS/MATERIALS: From a high-grade serous ovarian tumor of a BRCA1 mutation carrier, we established 3 distinct cell line subclones, OVCA-TR3.1, -2, and -3. Immunohistochemical characterization, flow cytometric analyses, chemosensitivity, and somatic mutation profiling were performed. RESULTS: The cell lines expressed AE1/AE3, Pax8, WT-1, OC125, estrogen receptor (ER), and p53, comparable to the primary tumor. Synergism could be shown in the combination treatment eremophila-1-(10)-11(13)-dien-12,8ß-olide (EPD), with cisplatin, whereas combination with olaparib did not show synergism. Eremophila-1-(10)-11(13)-dien-12,8ß-olide, a sesquiterpene lactone, is a novel chemotherapeutic agent. The inherited BRCA1 c.2989_2990dupAA mutation was confirmed in the cell lines. Loss of heterozygosity of BRCA1 was detected in each cell line, as well as a homozygous TP53 c.722C>A mutation. Flow cytometry showed that all cell lines had a distinct DNA index. CONCLUSIONS: Three new isogenic ovarian cancer cell lines were developed from a patient with a germ line BRCA1 mutation. Chemosensitivity profiling of the cell lines showed high tolerance for olaparib. Treatment with EPD proved synergistic with cisplatin. The effects of EPD will be further investigated for future clinical efficacy.

Synergistic effects of the sesquiterpene lactone, EPD, with cisplatin and paclitaxel in ovarian cancer cells.

BACKGROUND: Ovarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed. METHODS: Previously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit. RESULTS: In JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis. CONCLUSIONS: Our results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.

Proliferation patterns reflect architectural dedifferentiation: a study of nodular basal cell carcinoma.

The distribution of proliferating cells in basal cell carcinoma (BCC) may be related to lesion type and architecture. Single proliferation indexes may not be representative. We aimed to establish the distribution of cell proliferation in BCC as related to architecture. We studied an unselected, consecutive series of 45 resection specimens of nodular BCC from patients in the age range of 25-95 years using MIB-1 staining and systematically reviewed the cases. These lesions included nodular (n=32) and non-nodular (n=9) BCC. Within the nodular BCC, two patterns were recognised, not related to age or gender. In small nodular patterns with well developed peripheral palisading and central parallel streaming of small, elongated nuclei, proliferation is limited to the basal palisading cells in a clustered distribution. In large nodular patterns, proliferation is absent at the basal membrane (BM) and distributed in single random cells throughout the lesion. Both patterns preclude accurate quantitation. Many lesions contained both patterns in a side-by-side, unmixed manner. These pattern differences suggest a loss of differentiation in nodular BCC. Perhaps a single mutation results in the loss of BM associated cell architecture and proliferation control related to tumor-stroma interactions. As a result, the lesion reverts to a low frequency, non-regulated proliferation, diffusely distributed throughout the lesion. The two patterns may exist side-by-side in a single lesion, further supporting the concept of polyclonality. This hypothesis explains perilesional clefting and previously reported variations in intra-lesional laminin synthesis. Based on our findings, representation of tumor cell proliferation activity by a single value is not justified. Nodular BCC exists in one of two dedifferentiation-mutation-determined patterns of cell proliferation; many lesions clearly demonstrate bi-clonality.

Significance of MiB-1 staining in smears with atypical glandular cells.

MiB-1 immunostaining may facilitate recognition of developing adenocarcinoma in cervical smear screening. In a retrospective analysis of prospectively collected data of 170 patients with atypical endocervical glandular cells and with repeat smears, archival Papanicolaou-stained smears were restained for MiB-1 and classified for the presence of preneoplastic changes of MiB-1 positive epithelial fragments. The results of classification based on MiB-1 positive epithelial fragments corresponded "roughly" with cytomorphological diagnoses. Of the 38 patients in which the primary smear was found to be MiB-1-positive, 12 of these persisted in the repeat smear. Thirty-eight of the 132 repeat smears of patients originally negative for MiB-1 pathology were positive. Of the total 50 MiB-1 positive repeat smears, four showed adenocarcinoma in situ on cytomorphological grounds. MiB-1 staining enhances detection of (pre)neoplastic changes. This approach does not destroy the morphology of the original smear and can be applied to routine material.

Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers.

BACKGROUND: Ovarian cancer remains the leading cause of death from gynaecological malignancy. More than 60% of the patients are presenting the disease in stage III or IV. In spite of combination of chemotherapy and surgery the prognosis stays poor for therapy regimen. METHODS: The leaves of a plant endemic to Australia, Calomeria amaranthoides, were extracted and then fractionated by column chromatography. In vitro cytotoxicity tests were performed with fractions of the plant extract and later with an isolated compound on ovarian cancer cell lines, as well as normal fibroblasts at concentrations of 1-100 µg/mL (crude extract) and 1-10 µg/mL (compound). Cytotoxicity was measured after 24, 48 and 72 hours by using a non-fluorescent substrate, Alamar blue.In vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test. RESULTS: Two compounds were isolated by chromatographic fractionation and identified by 1H-NMR, 13C-NMR and mass spectrometry analyses, EPD, an α-methylene sesquiterpene lactone of the eremophilanolide subtype, and EPA, an α-methylene carboxylic acid.Cytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 µg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 µg/mL (95% confidence interval 4.3 to 6.5 µg/mL).Both, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 µg/mL and 5 µg/mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects.Changes in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13. CONCLUSIONS: For the first time both crude plant extract from Calomeria amaranthoides and EPD have been shown to have potent anti-cancer effects against ovarian cancer.