Doxorubicin resistant choriocarcinoma cell line derived spheroidal cells exhibit stem cell markers but reduced invasion.

Cell cycle-specific cancer chemotherapy is based on the ability of a drug to halt, minimise or destroy rapidly dividing cells. However, their efficacy is limited by the emergence of a self-renewing cell pool called "cancer stem cells" (CSC). Choriocarcinoma is a tumour of trophoblastic tissue. We, in this study, analysed whether spheroids generated from doxorubicin-treated and non-treated choriocarcinoma cell lines exhibit markers of stem cells. Two choriocarcinoma cell lines, namely JEG-3 and BeWo, were used in this study. Spheroids were generated from doxorubicin-treated cells and the non-treated cells under non-adherent condition, followed by analysis of stem-cell markers' expression, namely NANOG, OCT4 and SOX2. Immunofluorescence analysis suggested a general increase in the markers' concentration in spheroids relative to the parental cells. RT-qPCR and immunoblots showed an increase in the stem-cell marker expression in spheroids generated from doxorubicin-treated when compared to non-treated cells. In spheroids, Sox2 was significantly upregulated in doxorubicin-treated spheroids, whereas Nanog and Oct4 were generally downregulated when compared to non-treated spheroids. Both 2D and 3D invasion assays showed that the spheroids treated with doxorubicin exhibited reduced invasion. Our data suggest that choriocarcinoma cell lines may have the potential to produce spheroidal cells, yet the drug-treatment affected the invasion potential of spheroids. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03243-x.

Analyses of selected tumour-associated factors expression in normotensive and preeclamptic placenta.

INTRODUCTION: Human placenta is often considered a controlled-tumour because of shared properties such as invasion and angiogenesis. We assessed the status of a few selected tumour-associated factors (TAFs) in late onset pre-eclamptic (PE) and normotensive (NT) placentae, to understand their involvement in trophoblast invasion. These molecules include aldehyde dehydrogenase (ALDH3A1), aurora kinases (AURK-A/C), platelet derived growth factor receptor-α (PDGFRα), jagged-1 (JAG1) and twist related protein-1 (TWIST1). METHODS: The expression of TAF was compared in 13 NT and 11 PE (late onset) placentae using immunoblotting/immunohistochemistry. We then used a novel spheroidal cell model developed from transformed human first trimester trophoblast cell lines HTR8/SVneo and TEV-1 to determine the expression and localization of these six factors during invasion. We also compared the expression of these TAFs during migration and invasion. RESULTS: Our results suggest that expressions of ALDH3A1, AURK-A, PDGFRα, and TWIST1 are significantly upregulated in PE placentae (p < 0.05) when compared to NT placentae, whereas AURK-C and JAG1 are down-regulated (p < 0.05). The protein expression pattern of all the six factors were found to be similar in spheroids in comparison to their parental counterparts. The invasive potential of the spheroids was also enhanced when compared with the parental cells. DISCUSSION: Collectively, data from our present study suggests that these TAFs are involved in placental invasion and their altered expressions may be regarded as a compensatory mechanism against reduced invasion.

The effects of flavonoids on human first trimester trophoblast spheroidal stem cell self-renewal, invasion and JNK/p38 MAPK activation: Understanding the cytoprotective effects of these phytonutrients against oxidative stress.

Adequate invasion and complete remodelling of the maternal spiral arteries by the invading extravillous trophoblasts are the major determinants of a successful pregnancy. Increase in oxidative stress during pregnancy has been linked to the reduction in trophoblast invasion and incomplete conversion of the maternal spiral arteries, resulting in pregnancy complications such as pre-eclampsia, intrauterine growth restriction, and spontaneous miscarriages resulting in foetal/maternal mortality. The use of antioxidant therapy (vitamin C and E) and other preventative treatments (such as low dose aspirin) have been ineffective in preventing pre-eclampsia. Also, as the majority of antihypertensive drugs pose side effects, choosing an appropriate treatment would depend upon the efficacy and safety of mother/foetus. Since pre-eclampsia is mainly linked to placental oxidative stress, new diet-based antioxidants can be of use to prevent this condition. The antioxidant properties of flavonoids (naturally occurring phenolic compounds which are ubiquitously distributed in fruits and vegetables) have been well documented in non-trophoblast cells. Therefore, this study aimed to investigate the effects of flavonoids (quercetin, hesperidin) and their metabolites (Quercetin 3-O-ß-glucuronide and hesperetin), either alone or in combination, on first trimester trophoblast cell line HTR-8/SVneo during oxidative stress. The data obtained from this study indicate that selected flavonoids, their respective metabolites significantly reduced the levels of reduced glutathione (p < 0.0001) during HR-induced oxidative stress. These flavonoids also inhibited the activation of pro-apoptotic kinases (p38 MAPK and c-Jun N-terminal kinase) during HR-induced phosphorylation. In addition, they enhanced spheroid stem-like cell generation from HTR8/SVneo cells, aiding their invasion. Our data suggest that dietary intake of food rich in quercetin or hesperidin during early pregnancy can significantly improve trophoblast (placenta) health and function against oxidative stress.

Identification and characterisation of NANOG+/ OCT-4high/SOX2+ doxorubicin-resistant stem-like cells from transformed trophoblastic cell lines.

Treatment of gestational trophoblastic diseases (GTD) involves surgery, radiotherapy and chemotherapy. Although, these therapeutic approaches are highly successful, drug resistance and toxicity remain a concern for high risk patients. This Chemoresistance has also been observed in the presence of cancer stem cells that are thought to be responsible for cases of cancer recurrence. In this study, we report the presence of previously unknown populations of trophoblastic stem-like cells (SLCs) that are resistant to the chemotherapeutic drug doxorubicin. We demonstrate that these populations express the stem cell markers NANOG and Sox2 and higher levels of OCT-4 (NANOG+/OCT-4high/SOX2+). Although chemoresistant, we show that the invasive capacity of these trophoblastic SLCs is significantly inhibited by doxorubicin treatment. To better characterise these populations, we also identified cellular pathways that are involved in SLCs-chemoresistance to doxorubicin. In summary, we provide evidence of the presence of NANOG+/OCT-4+/SOX2+ trophoblastic SLCs that are capable to contribute to the susceptibility to GTD and that may be involved in Chemoresistance associated with drug resistance and recurrence in high risk GTDs' patients. We propose that targeting these populations could be therapeutically exploited for clinical benefit.