Linking unfolded protein response to ovarian cancer cell fusion.

BACKGROUND: Polyploid giant cancer cells (PGCCs) have been observed in epithelial ovarian tumors. They can resist antimitotic drugs, thus participating in tumor maintenance and recurrence. Although their origin remains unclear, PGCC formation seems to be enhanced by conditions that trigger the unfolded protein response (UPR) such as hypoxia or chemotherapeutic drugs like paclitaxel. Hypoxia has been shown to promote the formation of ovarian PGCCs by cell fusion. We thus hypothesized that the UPR could be involved in EOC cell fusion, possibly explaining the occurrence of PGCCs and the aggressiveness of EOC. METHODS: The UPR was induced in two ovarian cancer cell lines (SKOV3 and COV318). The UPR activation was assessed by Western blot and polyploidy indexes were calculated. Then, to confirm the implication of cell fusion in PGCC formation, two populations of SKOV3 cells were transfected with plasmids encoding for two distinct nuclear fluorescent proteins (GFP and mCherry) associated with different antibiotic resistance genes, and the two cell populations were mixed in co-culture. The co-culture was submitted to a double-antibiotic selection. The resulting cell population was characterized for its morphology, cyclicity, and proliferative and tumorigenic capacities, in addition to transcriptomic characterization. RESULTS: We demonstrated that cell fusion could be involved in the generation of ovarian PGCCs and this process was promoted by paclitaxel and the UPR activation. Double-antibiotic treatment of PGCCs led to the selection of a pure population of cells containing both GFP- and mCherry-positive nuclei. Interestingly, after 3 weeks of selection, we observed that these cells were no longer polynucleated but displayed a single nucleus positive for both fluorescent proteins, suggesting that genetic material mixing had occurred. These cells had reinitiated their normal cell cycles, acquired an increased invasive capacity, and could form ovarian tumors in ovo. CONCLUSIONS: The UPR activation increased the in vitro formation of PGCCs by cell fusion, with the newly generated cells further acquiring new properties. The UPR modulation in ovarian cancer patients could represent an interesting therapeutic strategy to avoid the formation of PGCCs and therefore limit cancer relapse and drug resistance.

PLGA Nanoparticles for the Intraperitoneal Administration of CBD in the Treatment of Ovarian Cancer: In Vitro and In Ovo Assessment.

The intraperitoneal administration of chemotherapeutics has emerged as a potential route in ovarian cancer treatment. Nanoparticles as carriers for these agents could be interesting by increasing the retention of chemotherapeutics within the peritoneal cavity. Moreover, nanoparticles could be internalised by cancer cells and let the drug release near the biological target, which could increase the anticancer efficacy. Cannabidiol (CBD), the main nonpsychotropic cannabinoid, appears as a potential anticancer drug. The aim of this work was to develop polymer nanoparticles as CBD carriers capable of being internalised by ovarian cancer cells. The drug-loaded nanoparticles (CBD-NPs) exhibited a spherical shape, a particle size around 240 nm and a negative zeta potential (-16.6 ± 1.2 mV). The encapsulation efficiency was high, with values above 95%. A controlled CBD release for 96 h was achieved. Nanoparticle internalisation in SKOV-3 epithelial ovarian cancer cells mainly occurred between 2 and 4 h of incubation. CBD antiproliferative activity in ovarian cancer cells was preserved after encapsulation. In fact, CBD-NPs showed a lower IC50 values than CBD in solution. Both CBD in solution and CBD-NPs induced the expression of PARP, indicating the onset of apoptosis. In SKOV-3-derived tumours formed in the chick embryo model, a slightly higher-although not statistically significant-tumour growth inhibition was observed with CBD-NPs compared to CBD in solution. To sum up, poly-lactic-co-glycolic acid (PLGA) nanoparticles could be a good strategy to deliver CBD intraperitoneally for ovarian cancer treatment.

Effect of local aromatase inhibition in endometriosis using a new chick embryo chorioallantoic membrane model.

Endometriosis is an oestrogen-dependent, inflammation-driven gynaecologic disorder causing severe disability. Endometriosis implants are characterized by unbalanced local oestrogen metabolism leading to hyperoestrogenism and aromatase up-regulation is one of main mechanism involved. Aromatase inhibitors such as letrozole or anastrozole use in young women are associated with severely side effects limiting their long-term clinical use. An endometriosis-targeted inhibition of local aromatase could be a viable alternative, although the role of the local inhibition of this enzyme is still unclear. Using a new chick embryo allantoic membrane (CAM) model incorporating xenografted human endometriosis cyst, we showed that topical treatment with anastrozole reduced lesion size, although oestrogens produced by CAM female embryo blunted this effect. Xenografted human endometriosis CAM is a new efficient model for the screening of new drugs targeting endometriosis tissue.

Oestradiol enhances apoptosis in bovine mammary epithelial cells in vitro.

Ovarian steroids, oestradiol and progesterone, are required for normal mammary growth at puberty and during pregnancy. They contribute to mammary parenchyma development by stimulating mammary epithelial cell (MEC) proliferation. However several studies demonstrate that oestradiol negatively affects milk production during the declining phase of lactation, but the oestradiol effect on MEC in lactating mammary gland remains unclear. The objective of this study was to investigate the differential effect of oestradiol on bovine MECs mimicking two physiological statuses: active and early apoptotic MECs. We demonstrated that oestradiol has a major effect on early apoptotic MECs and might accelerate MEC apoptosis by activation of caspases rather than by inducing apoptosis in active MECs. Early apoptotic MECs could be compared with senescent cells in the late-lactation mammary gland. These results suggest that the negative effect of oestradiol on milk production during the declining phase of lactation would be due to an enhancement of apoptotic processes in MECs.

Suppression of ovarian secretions before puberty strongly affects mammogenesis in the goat.

The objective of this study was to provide insight into the biological mechanisms underlying mammary development and the role of the ovaries in prepubertal caprine mammogenesis using a serial ovariectomy approach. Young Alpine goats were ovariectomized (Ovx) or sham-operated (Int) at three periods before puberty (G1=1 month, G2=2 month and G3=3 months of age) and one after puberty (G7=7 months of age). The goats were slaughtered at 9 months of age and mammary glands were removed. Ovariectomy performed at 1, 2 and 3 months of age caused a 50% reduction in DNA concentration, in mammary tissue taken from the parenchyma-stroma border region. Morphological analysis of mammary tissue sections indicated that the parenchymal structures of Ovx goats were negatively affected by ovariectomy. Goats ovariectomized before 2 months of age (Ovx-1 and Ovx-2) showed a significant decrease in the percent of cells proliferating in mammary glands of 9-month old goats (proliferating cell nuclear antigen expression and antigen Ki67-positive cell number). Also, goats ovariectomized at 1 and 2 months of age had reduced matrix metalloprotease 2 activity at 9 months of age. E-cadherin was strongly decreased in goats ovariectomized before 2 months of age (80 and 85% in Ovx-1 and Ovx-2 goats, respectively). Quantitative PCR analysis of transcripts encoding for oestrogen (ERα) and progesterone receptors (PR) and immunodetection of ERα showed that ovariectomy at 1 and 2 months of age strongly inhibited the transcription of ERα and PR in the mammary gland. We conclude that ovariectomy before 3 months of age markedly impaired parenchymal development. These findings suggest that prepubertal mammogenesis in goats depends on the ovaries to initiate mammary epithelial cell proliferation and mammary gland remodelling.