Antineoplastic Effect of ALK Inhibitor Crizotinib in Primary Human Anaplastic Thyroid Cancer Cells with STRN-ALK Fusion In Vitro.

Anaplastic thyroid cancer (ATC) is one of the deadliest human cancers and represents <2% of thyroid carcinomas. A therapeutic target for ATC is represented by anaplastic lymphoma kinase (ALK) rearrangements, involved in tumor growth. Crizotinib is an oral small-molecule tyrosine kinase inhibitor of the ALK, MET, and ROS1 kinases, approved in ALK-positive non-small cell lung cancer. Until now, the effect of crizotinib in "primary human ATC cells" (pATCs) with transforming striatin (STRN)-ALK fusion has not been reported in the literature. In this study, we aimed to obtain pATCs with STRN-ALK in vitro and evaluate the in vitro antineoplastic action of crizotinib. Thyroid surgical samples were obtained from 12 ATC patients and 6 controls (who had undergone parathyroidectomy). A total of 10/12 pATC cultures were obtained, 2 of which with transforming STRN-ALK fusion (17%). Crizotinib inhibited proliferation, migration, and invasion and increased apoptosis in 3/10 pATC cultures (2 of which with/1 without STRN-ALK), particularly in those with STRN-ALK. Moreover, crizotinib significantly inhibited the proliferation of AF cells (a continuous cell line obtained from primary ATC cells). In conclusion, the antineoplastic activity of crizotinib has been shown in human pATCs (with STRN-ALK) in preclinical studies in vitro, opening the way to future clinical evaluation in these patients.

Primary cell cultures for the personalized therapy in aggressive thyroid cancer of follicular origin.

Thyroid cancer (TC) is the most prevalent endocrine malignancy. More than 90 % of TC is represented by differentiated TC (DTC) arising from the follicular thyroid cells. DTC includes papillary TC (PTC), follicular TC (FTC), and Hürthle cell TC. Anaplastic TC (ATC) accounts for 1% of TC, and it represents 15-40 % of TC death. Current treatment strategies are not completely effective against aggressive DTC or ATC, and mortality is one of the most important challenges. Recently, progresses have been obtained in the understanding of the molecular/genetic basis of TC progression, and new drugs have been introduced [i.e. tyrosine kinase inhibitors (TKIs)], able to block the oncogenic or signaling kinases, associated with cellular growth. Thyroid cell lines, obtained from tumoral cells and chosen for high proliferation in vitro, have been used as preclinical models. Actually, these cells lose the characteristic features of the primary tumor, because they adapt to in vitro growth conditions. For these reasons, the use of these cell lines has important limitations, and more recently human primary cell cultures have been established as monolayer cultures, and investigated for their biological behavior. Moreover, in the past, primary TC cells could be collected only through surgical biopsies, while recently human primary cell cultures can be established also from samples of fine-needle aspiration citology from aggressive dedifferentiated DTC or ATC. Testing in vitro different TKIs in each patient can help to develop new personalized treatments, without using ineffective drugs. In conclusion, personalized medicine and precise oncology, which consider both patients and their disease features, represent the future of the treatment approach, and further progress is needed in this direction.

In Vitro Characteristics of Canine Primary Tracheal Epithelial Cells Maintained at an Air-Liquid Interface Compared to In Vivo Morphology.

Culturing respiratory epithelial cells at an air-liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents.

Nanomechanical Signatures in Glioma Cells Depend on CD44 Distribution in IDH1 Wild-Type but Not in IDH1R132H Mutant Early-Passage Cultures.

Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients' specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/- cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms.

Antineoplastic Activity of Pazopanib in Anaplastic Thyroid Cancer in Primary Culture.

Anaplastic thyroid cancer (ATC) is a rare and rapidly fatal human cancer. Its usual treatment includes the combination of surgery, external hyperfractionated radiation therapy, and chemotherapy. These treatments permit achieving about 6-10 months of median survival. For this reason, it is challenging to predict the ATC patient clinical therapy responsiveness. Pazopanib is a multitarget tyrosine kinase inhibitor of VEGF receptors, PDGF, and c-Kit. Until now, the effect of pazopanib in primary human ATC cells (pATC) has not been reported in the literature. The aim of our study was to evaluate in vitro the antineoplastic effect of pazopanib in pATC. Surgical thyroidal tissues were collected from five patients with ATC, from thyroid biopsy at the moment of first surgical operation. An inhibition of proliferation, migration, and invasion, and an increase in apoptosis were demonstrated upon treating pATC cells with pazopanib (p < 0.05). Moreover, pazopanib was able to significantly decrease the VEGF expression in pATC cells (p < 0.05). To conclude, in this study, we demonstrate the antineoplastic activity of the antiangiogenic inhibitor, pazopanib, in human pATC in vitro.

Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells.

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal-glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10-P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-ßIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and ßIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and ßIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal-glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.

Hydrogen Peroxide Induced Toxicity Is Reversed by the Macrocyclic IRAP-Inhibitor HA08 in Primary Hippocampal Cell Cultures.

Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.

Effects of temperature on the innate immune response on Antarctic and sub-Antarctic fish Harpagifer antarcticus and Harpagifer bispinis challenged with two immunostimulants, LPS and Poly I:C: In vivo and in vitro approach.

Rising ocean temperatures due to climate change combined with the intensification of anthropogenic activity can drive shifts in the geographic distribution of species, with the risks of introducing new diseases. In a changing environment, new host-pathogen interactions or changes to existing dynamics represent a major challenge for native species at high latitudes. Notothenioid fish constitute a unique study system since members of this group are found inside and outside Antarctica, are highly adapted to cold and particularly sensitive to temperature increments. However, data about their immune response remains scarce. Here, we aimed to evaluate the innate immune response under thermal stress in two species of Notothenioid fish, Harpagifer antarcticus and Harpagifer bispinis. Adult individuals from both species were collected on King George Island (Antarctica), and Punta Arenas (Chile), respectively. Specimens were assigned to a control group or injected with one of two agents (LPS and Poly I:C) to simulate either a bacterial or viral infection, and subjected to three different temperatures 2, 5 and 8 °C for 1 week. In parallel, we established leukocytes primary cell cultures from head kidney, which were also subjected to the immunostimulants at the same three temperatures, and incubated for 0.5, 1, 3, 6, 12, 24, and 48 h. We evaluated the relative gene expression of genes involved in the innate immune response (TLR1, TLR3, NF-kB, MYD88, IFNGR e IL-8) through real time qPCR. We found differences between species mainly in vivo, where H. antarcticus exhibited upregulation at high temperatures and H. bispinis seemed to have reached their physiological minimum at 2 °C. Although temperature had a strong effect during the in vivo assay for both species, it was negligible for primary cell cultures, which responded primarily to condition and time. Moreover, while leukocytes responded with fluctuations across time points, in vivo both species manifested strong and clear patterns of gene expression. These results highlight the importance of evaluating the effect of multiple stressors and set a precedent for future research.

The Upper Respiratory Tract of Felids Is Highly Susceptible to SARS-CoV-2 Infection.

Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air-liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.

Development of a 3D functional assay and identification of biomarkers, predictive for response of high-grade serous ovarian cancer (HGSOC) patients to poly-ADP ribose polymerase inhibitors (PARPis): targeted therapy.

BACKGROUND: Poly(ADP-ribose) polymerase inhibitors (PARPis) specifically target homologous recombination deficiency (HRD) cells and display good therapeutic effect in women with advanced-stage BRCA1/2-mutated breast and epithelial ovarian cancer (EOC). However, about 50% of high grade serous ovarian cancers (HGSOC) present with HRD due to epigenetic BRCA1 inactivation, as well as genetic/epigenetic inactivation(s) of other HR genes, a feature known as "BRCAness". Therefore, there is a potential for extending the use of PARPis to these patients if HR status can be identified. METHODS: We have developed a 3D (spheroid) functional assay to assess the sensitivity of two PARPis (niraparib and olaparib) in ascites-derived primary cell cultures (AsPCs) from HGSOC patients. A method for AsPCs preparation was established based on a matrix (agarose), allowing for easy isolation and successive propagation of monolayer and 3D AsPCs. Based on this method, we performed cytotoxicity assays on 42 AsPCs grown both as monolayers and spheroids. RESULTS: The response to PARPis treatment in monolayer AsPCs, was significantly higher, compared to 3D AsPCs, as 88% and 52% of the monolayer AsPCs displayed sensitivity to niraparib and olaparib respectively, while 66% of the 3D AsPCs were sensitive to niraparib and 38% to olaparib, the latter being more consistent with previous estimates of HRD (40%-60%) in EOC. Moreover, niraparib displayed a significantly stronger cytotoxic effect in both in 3D and monolayer AsPCs, which was confirmed by consecutive analyses of the HR pathway activity (γH2AX foci formation) in PARPis-sensitive and resistant AsPCs. Global gene expression comparison of 6 PARPi-resistant and 6 PARPi-sensitive 3D AsPCs was indicative for the predominant downregulation of numerous genes and networks with previously demonstrated roles in EOC chemoresistance, suggesting that the PARPis-sensitive AsPCs could display enhanced sensitivity to other chemotherapeutic drugs, commonly applied in cancer management. Microarray data validation identified 24 potential gene biomarkers associated with PARPis sensitivity. The differential expression of 7 selected biomarkers was consecutively confirmed by immunohistochemistry in matched EOC tumor samples. CONCLUSION: The application of this assay and the potential biomarkers with possible predictive significance to PARPis therapy of EOC patients now need testing in the setting of a clinical trial.

Electropermeabilization of metastatic chondrosarcoma cells from primary cell culture.

Primary cell cultures are challenging, but reliable model reflecting tumor response in vitro. The study was designed to examine if the increased electropermeabilization can overcame initial drug insensitivity in chondrosarcoma cells from lung metastasis. We established a primary cell culture and evaluated the cytotoxic impact of four drugs-cisplatin (CDDP), camptothecin, 2-methoxyestradiol, and leucovorin calcium (LeuCa). After determination of parameters allowing for electropermeabilization, we performed electrochemotherapy in vitro with the least toxic drugs-CDDP and LeuCa. Although combining CDDP and leucovorin together increased their toxicity and supported apoptosis, application of pulsed electric fields (PEFs) brought no advantage for their efficacy. The study emphasizes the need for introduction of primary cell cultures into studies on pulse electric fields as model frequently less sensitive to PEF-based treatments than continuous cell lines.

The Protective and Restorative Effects of Growth Hormone and Insulin-Like Growth Factor-1 on Methadone-Induced Toxicity In Vitro.

Evidence to date suggests that opioids such as methadone may be associated with cognitive impairment. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are suggested to be neuroprotective and procognitive in the brain and may therefore counteract these effects. This study aims to explore the protective and restorative effects of GH and IGF-1 in methadone-treated cell cultures. Primary cortical cell cultures were harvested from rat fetuses and grown for seven days in vitro. To examine the protective effects, methadone was co-treated with or without GH or IGF-1 for three consecutive days. To examine the restorative effects, methadone was added for the first 24 h, washed, and later treated with GH or IGF-1 for 48 h. At the end of each experiment, mitochondrial function and membrane integrity were evaluated. The results revealed that GH had protective effects in the membrane integrity assay and that both GH and IGF-1 effectively recovered mitochondrial function and membrane integrity in cells pretreated with methadone. The overall conclusion of the present study is that GH, but not IGF-1, protects primary cortical cells against methadone-induced toxicity, and that both GH and IGF-1 have a restorative effect on cells pretreated with methadone.

The aurora kinase inhibitor VX-680 shows anti-cancer effects in primary metastatic cells and the SW13 cell line.

New therapeutic targets are needed to fight cancer. Aurora kinases (AK) were recently identified as vital key regulators of cell mitosis and have consequently been investigated as therapeutic targets in preclinical and clinical studies. Aurora kinase inhibitors (AKI) have been studied in many cancer types, but their potential capacity to limit or delay metastases has rarely been considered, and never in adrenal tissue. Given the lack of an effective pharmacological therapy for adrenal metastasis and adrenocortical carcinoma, we assessed AKI (VX-680, SNS314, ZM447439) in 2 cell lines (H295R and SW13 cells), 3 cell cultures of primary adrenocortical metastases (from lung cancer), and 4 primary adrenocortical tumor cell cultures. We also tested reversan, which is a P-gp inhibitor (a fundamental efflux pump that can extrude drugs), and we measured AK expression levels in 66 adrenocortical tumor tissue samples. Biomolecular and cellular tests were performed (such as MTT, thymidine assay, Wright's staining, cell cycle and apoptosis analysis, Western blot, qRT-PCR, and mutation analysis). Our results are the first to document AK overexpression in adrenocortical carcinoma as well as in H295R and SW13 cell lines, thus proving the efficacy of AKI against adrenal metastases and in the SW13 cancer cell model. We also demonstrated that reversan and AKI Vx-680 are useless in the H295R cell model, and therefore should not be considered as potential treatments for ACC. Serine/threonine AK inhibition, essentially with VX-680, could be a promising, specific therapeutic tool for eradicating metastases in adrenocortical tissue.

A comprehensive characterization of cell cultures and xenografts derived from a human verrucous penile carcinoma.

This study aimed to establish and characterize primary cell cultures and xenografts derived from penile carcinoma (PeCa) in order to provide experimental models for cellular processes and efficacy of new treatments. A verrucous squamous cell carcinoma (VSCC) was macrodissected, dissociated, and cultivated in KSFM/DF12 medium. Cell cultures were evaluated at passage 5 (P5) using migration and invasion assays and were serially propagated, in vivo, in BALB/c nude mice until passage 3 (X1-X3). Immunophenotypic characterization of cultures and xenografts was performed. Genomic (CytoScan HD, Affymetrix) and transcriptomic profiles (HTA 2.0 platform, Affymetrix) for VSCC, cell cultures, and xenografts were assessed. P5 cells were able to migrate, invade the Matrigel, and produce tumors in immunodeficient mice, demonstrating their malignant potential. The xenografts unexpectedly presented a sarcomatoid-like carcinoma phenotype. Genomic analysis revealed a high similarity between the VSCC and tumor-derived xenograft, confirming its xenograft origin. Interestingly, a subpopulation of P5 cells presented stem cell-related markers (CD44(+)CD24(-) and ALDH1(high)) and sphere-forming capacity, suggesting their potential xenograft origin. Cell cultures and xenografts retained the genomic alterations present in the parental tumor. Compared to VSCC, differentially expressed transcripts detected in all experimental conditions were associated with cellular morphology, movement, and metabolism and organization pathways. Malignant cell cultures and xenografts derived from a verrucous penile carcinoma were established and fully characterized. Nevertheless, xenograft PeCa models must be used with caution, taking into consideration the selection of specific cell populations and anatomical sites for cell/tumor implantation.

IGF-I and IGF-II effects on local IGF system and signaling pathways in gilthead sea bream (Sparus aurata) cultured myocytes.

The insulin-like growth factors (IGFs) have a fundamental role in a vast range of functions acting through a tyrosine-kinase receptor (IGF-IR). IGFs in muscle can affect the expression of components of the local IGF system, myogenic regulatory factors (MRFs), proliferating (proliferating cell nuclear antigen, PCNA) or differentiating molecules (myosin heavy chain, MHC) and, lead to the activation of different signaling pathways. The response of all these genes to IGFs incubation at two different times in day 4 cultured myocytes of gilthead sea bream was analyzed. Both IGFs increased the expression of IGF-I and IGFBP-5, but showed different effects on the receptors, with IGF-I suppressing the expression of both isoforms (IGF-IRa and IGF-IRb) and IGF-II up-regulating only IGF-IRb. Moreover, the protein levels of PCNA and target of rapamycin (TOR) increased after IGF-II incubation, although a decline in Myf5 and a rise in MHC gene expression was caused by IGF-I. Taken together, these results provide evidence for the importance of IGFs on controlling muscle development and growth in gilthead sea bream and suggest that each IGF may be preferentially acting through a specific IGF-IR. Moreover, the data support the hypothesis that IGF-II has a more important role during proliferation, whereas IGF-I seems to be relevant for the differentiation phase of myogenesis.

In vitro effects of somatostatin on the growth hormone-insulin-like growth factor axis in orange-spotted grouper (Epinephelus coioides).

Growth in vertebrates is mainly mediated by the growth hormone (GH)-insulin-like growth factor (IGF) axis, and somatostatin (SRIF) inhibits growth by decreasing GH release at the pituitary level and antagonizing the release and action of GHRH in the hypothalamus. However, the effects of SRIF on the regulation of growth at levels other than GH release from the pituitary gland are less well known. In the present study, we comprehensively examined the pituitary and peripheral actions of SRIF on the GH-IGF axis in grouper using a primary pituitary and hepatocyte cell culture system. Our results showed that SRIF inhibited GH release at the pituitary level, but had no influence on GH mRNA expression. Basal hepatic GH receptor 1 (GHR1), IGF-I and IGF-II mRNA levels declined over time, whereas GHR2 mRNA levels remained stable throughout the culture period. GH stimulated the hepatic expression of GHR and IGF mRNAs in a dose-dependent manner, while SRIF suppressed both basal and GH-stimulated expression of GHR and IGF mRNAs in primary cultured hepatocytes. The inhibition of GHR and IGF mRNA levels by SRIF was not attributed to the rate of mRNA degradation. To the best of our knowledge, we demonstrated the effects of SRIF on basal and GH-stimulated IGF-II mRNA levels in teleosts for the first time. These results indicate that SRIF regulates growth at the level of the pituitary and peripheral liver.

Migration of phthalates on culture plates - an important challenge to consider for in vitro studies.

Phthalates are endocrine disruptors of the reproductive system and suspected to influence many other organ and hormone systems. They are also semi-volatile organic compounds present in the gas phase in the environment. Their mode of action has been investigated in numerous in vitro studies. Multi-well culture plates are typically used to study phthalates in cell cultures. In a pilot study, we observed evidence of phthalate migration in 24-well culture plates. As this has not previously been described, we investigated the phenomenon in more detail. Primary human thyroid epithelial cell cultures (n = 8 cultures) were exposed to either di-ethyl phthalate (DEP), di-n-butyl phthalate (DnBP), mono-n-butyl phthalate (MnBP) or di-(2-ethylhexyl) phthalate (DEHP). Measurement of phthalate metabolites by mass spectrometry demonstrated that the short-branched DEP was able to migrate to adjacent wells when added to cell culture plates. DnBP also seemed to be able to migrate, unlike the long-branched DEHP or the monoester MnBP which did not seem to have this ability. High background levels of phthalate metabolites were also observed, which might compromise results from low dose phthalate studies. In conclusion, the migration of phthalates which is probably caused by their volatile properties might lead to false interpretation of study results.

First steps for integrating sex and gender considerations into basic experimental biomedical research.

In recent decades there has been an increasing recognition of the need to account for sex and gender in biology and medicine, in order to develop a more comprehensive understanding of biological phenomena and to address gaps in medical knowledge that have arisen due to a generally masculine bias in research. We have noted that as basic experimental biomedical researchers, we face unique challenges to the incorporation of sex and gender in our work, and that these have remained largely unarticulated, misunderstood, and unaddressed in the literature. Here, we describe some of the specific challenges to the incorporation of sex and gender considerations in research involving cell cultures and laboratory animals. In our view, the mainstreaming of sex and gender considerations in basic biomedical research depends on an approach that will allow scientists to address these issues in ways that do not undermine our ability to pursue our fundamental scientific interests. To that end, we suggest a number of strategies that allow basic experimental researchers to feasibly and meaningfully take sex and gender into account in their work.

The antitumor effect of metformin with and without carboplatin on primary endometrioid endometrial carcinoma in vivo.

OBJECTIVES: New treatment options for advanced and recurrent endometrial carcinoma (EC) are necessary. Epidemiological studies showed that diabetic patients using metformin have reduced risks of endometrial cancer (EC) incidence. Moreover, pre- and clinical studies demonstrated an antitumor effect by metformin, with and without additional treatments, for different solid malignancies. However, cancer cell-autonomous effects of metformin on EC have not been fully characterized yet. The aim of this study was to investigate the effect of metformin, with or without carboplatin, on patient-derived primary endometrioid EC cells xenografted in nude mice, to assess its ability to reduce or impair growth in already established tumors. METHODS: Two xenograft models were established by subcutaneous inoculation of primary endometrioid EC cell suspensions. Tumors were allowed to grow and then mice were treated with metformin (250 mg/kg, daily, p.o.), carboplatin (50 mg/kg, 1×/week, i.p.), or the combination of both compounds at the same concentration as single treatment, for three weeks. Effects of metformin treatment on the tumor mass were determined by tumor growth follow-up. Metformin influences on AMPK/mTOR cell signaling were evaluated by investigating AKT, AMPK and S6 phosphorylation levels. RESULTS: In vivo, metformin did not affect the growth of EC tumors established from patient-derived primary cultures and the phosphorylation of AKT, AMPK and S6. In addition, no enhanced antitumor effect was determined by combining metformin and carboplatin treatments. CONCLUSIONS: Metformin, at clinically relevant concentrations, did not show effects on the growth of already established tumors. Adding metformin to carboplatin did not have synergistic effects.

Multiple treatments with L-3,4-dihydroxyphenylalanine modulate dopamine biosynthesis and neurotoxicity through the protein kinase A-transient extracellular signal-regulated kinase and exchange protein activation by cyclic AMP-sustained extracellular signal-regulated kinase signaling pathways.

Multiple treatments with L-3,4-dihydroxyphenylalanine (L-DOPA; 20 µM) induce neurite-like outgrowth and reduce dopamine biosynthesis in rat adrenal pheochromocytoma (PC) 12 cells. We therefore investigated the effects of multiple treatments with L-DOPA (MT-LD) on cell survival and death over a duration of 6 days by using PC12 cells and embryonic rat midbrain primary cell cultures. MT-LD (10 and 20 µM) decreased cell viability, and both types of cells advanced to the differentiation process at 4-6 days. MT-LD induced cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylation and exchange protein activation by cAMP (Epac) expression at 1-3 days, which led to transient extracellular signal-regulated kinase (ERK1/2) phosphorylation in both cells. In these states, MT-LD activated cAMP-response element binding protein (CREB; Ser133) and tyrosine hydroxylase (Ser40) phosphorylation in PC12 cells, which led to an increase in intracellular dopamine levels. In contrast, MT-LD induced prolonged Epac expression at 4-5 days in both cells, which led to sustained ERK1/2 phosphorylation. In these states, the dopamine levels were decreased in PC12 cells. In addition, MT-LD induced c-Jun N-terminal kinase1/2 phosphorylation and cleaved caspase-3 expression at 4-6 days in both cells. These results suggest that MT-LD maintains cell survival via PKA-transient ERK1/2 activation, which stimulates dopamine biosynthesis. In contrast, at the later time period, MT-LD induces differentiation via both prolonged Epac and sustained ERK1/2 activation, which subsequently leads to the cell death process. Our data demonstrate that L-DOPA can cause neurotoxicity by modulating the Epac-ERK pathways in neuronal and PC12 cells.