Methylxanthines induce structural and functional alterations of the cardiac system in zebrafish embryos

Background: Zebrafish embryos are emerging as a model for pharmacological and toxicological studies. We used zebrafish embryos to study the general toxicity and cardiovascular effects of eight methylxanthines: aminophylline, caffeine, diprophylline, doxofylline, etophylline, 3-isobutyl-1-methylxanthine (IBMX), pentoxifylline and theophylline. Methods: Microinjections of the eight methylxanthines were performed in 1-2 cell stage zebrafish embryos and the general toxicity and cardiovascular effects were analyzed at different time points. Embryotoxicity and teratogenicity were evaluated to understand the general toxicity of these compounds. Structural and functional alterations of the heart were evaluated to assess the cardiovascular effects. Results: Our results showed different activity patterns of the methylxanthines drugs. Caffeine, IBMX, pentoxifylline and theophylline were highly embryotoxic and teratogenic; aminophylline, doxofylline and etophylline were embryotoxic and teratogenic only at higher doses, and diprophylline showed a minimal (< 10%) embryotoxicity and teratogenicity. Most of these drugs induced structural alteration of the heart in 20-40% of the injected embryos with the maximum dose. This structural alteration was fatal with the embryos ultimately dying within 120 hpf. All the drugs induced a transient increase in heart rate at 48 hpf which returned to baseline within 96 hpf. This functional effect of methylxanthines showed similarity to the studies done in humans and other vertebrates. Conclusion: Our results indicate the potential toxicity and teratogenicity of different methylxanthines in the embryos during embryonic development, the most sensitive period of life. Although interspecies differences need to be considered before drawing any conclusion, our study elucidated that a single exposure of methylxanthines at therapeutic range could induce cardiac dysfunction besides causing embryotoxicity and teratogenicity. Of all the drugs, diprophylline appeared to be safer, with lower degree of embryotoxicity, teratogenicity and cardiac toxicity as compared to other methylxanthines

Preclinical Evidence of Progesterone as a New Pharmacological Strategy in Human Adrenocortical Carcinoma Cell Lines

BACKGROUND Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers. METHODS NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells. CONCLUSION Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study

Biochemical correlates of synaptic plasticity in exercise

Current views of the brain are shifting towards a more changeable, plastic brain than previously envisaged. Physical training has been shown to be one of few strategies that seems able to increase the plasticity of the brain. Little is known, however, about the molecular mechanisms that regulate synaptic plasticity during training. The aim of the project is to explore training based synaptic plasticity at a molecular level, focusing primarily on the excitatory glutamatergic synapses. Most of the research done on exercise has been focused on a specific brain region. I have tried to investigate global effects of exercise on the synapse. Crude synaptosomes were prepared from trained and sedentary groups of mice, and semiquantitative western blotting was done to determine possible changes in synaptic expression of functionally crucial proteins, i.a. glutamate receptors. I found increased level of syntaxin, GluR1 AMPA receptor subunits, and the 2A/2B subunits of the NMDA receptor. There was also a decreased level of Arc in trained compared to sedentary mice. There was no significant change in the expression of the neuronal marker, beta tubulin, of the synaptic markers synaptophysin and PSD-95, or in the AMPA receptor subunit GluR2. Taken together, my results may indicate that physical exercise may lead to an increased level of ongoing hebbian plasticity, a reduction in homeostatic plasticity, an increased presynaptic release capacity, with no apparent change in the density of neurons or synapses

Zebrafish embryo as an in vivo model for behavioral and pharmacological characterization of methylxanthine drugs

Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET) test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC50) calculated in zebrafish embryos and lethal doses (LD50) in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds

Zebrafish larvae as a behavioral model in neuropharmacology.

Zebrafish larvae show a clear and distinct pattern of swimming in response to light and dark conditions, following the development of a swim bladder at 4 days post fertilization. This swimming behavior is increasingly employed in the screening of neuroactive drugs. The recent emergence of high-throughput techniques for the automatic tracking of zebrafish larvae has further allowed an objective and efficient way of finding subtle behavioral changes that could go unnoticed during manual observations. This review highlights the use of zebrafish larvae as a high-throughput behavioral model for the screening of neuroactive compounds. We describe, in brief, the behavior repertoire of zebrafish larvae. Then, we focus on the utilization of light-dark locomotion test in identifying and screening of neuroactive compound

Cisplatin Cytotoxicity in Human Testicular Germ Cell Tumor Cell Lines Is Enhanced by the CDK4/6 Inhibitor Palbociclib

Background: Cisplatin-based chemotherapy is the mainstay of pharmacological treatment of testicular germ cell tumors (TGCTs) that, together with early diagnosis, surgery, and/or radiotherapy, has dramatically improved the prognosis. However, under the pressure of such pharmacological therapy (both classical cytotoxic drugs and targeted therapy), cancer cells may develop resistance. Thus, combination therapy that may include cytotoxic drugs and targeted therapy could offer an advantage to curing cancers. Here, we investigated the in vitro and in vivo antitumor activity of cisplatin, as a single-agent or in combination with palbociclib. Patients and methods: The cell viability of Ntera-2/cl.D1 (NT2/D1) and 833K after exposure to palbociclib and/or cisplatin was evaluated by MTT dye reduction assay and by ATPLite Luminescence Assay. Gene and protein expression was evaluated by quantitative reverse transcription polymerase chain reaction and by western blot. Flow cytometric cell-cycle analysis was performed, as well. The in vivo experiments were conducted on NT2/D1 xenografts in AB zebrafish embryos exposed to the drugs. Results: Palbociclib and cisplatin decreased TGCT cell viability both in vitro and in vivo. This effect was additive when cells were exposed to the drug combination. In the NT2/D1 cell lines, the drug combination also exerted a positive effect with regard to delaying cell recovery after the toxic insult. In the combination experiments, cisplatin-induced cell accumulation in G2/M was predominant compared with the palbociclib effect. Conclusions: These results could provide the rationale for developing further studies to improve the pharmacological treatment of TGCTs, but they must be demonstrated in a dedicated clinical trial

Preclinical Evidence of Progesterone as a New Pharmacological Strategy in Human Adrenocortical Carcinoma Cell Lines

Background: Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers. Methods: NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry. Results: Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells. Conclusion: Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study

Cytotoxic effects of targeted agent alone or with chemotherapy in the treatment of adenoid cystic carcinoma: a preclinical study

Adenoid cystic carcinoma (ACC) is a rare malignancy characterized by high incidence of relapse. When relapsing, ACC has an indolent but relentless behaviour, thus leading to a poor long-term prognosis. The treatment of choice of relapsing ACC remains surgery followed by radiotherapy, whenever feasible. Therapeutic weapons are limited to systemic drugs. The most widely used chemotherapy regimen is the combination of cisplatin and doxorubicin, however with low response rate and not long lasting; there is also a lack of alternatives for second line therapies in case of disease progression. Therefore, a more comprehensive strategy aimed at identifying at preclinical level the most promising drugs or combination is clearly needed. In this study, the cytotoxic effects of two standard chemotherapy drugs, cisplatin and doxorubicin, and of five targeted therapy-drugs was tested in vitro, on an h-TERT immortalized ACC cell line, and in vivo, on zebrafish embryos with ACC tumoral cell xenograft. Then, combinations of one standard chemotherapy drug plus one targeted therapy drug were also evaluated, in order to find the best treatment strategy for ACC. Data obtained demonstrated that both vorinostat and olaparib significantly increased the standard chemotherapy cytotoxic effects, suggesting new interesting therapeutic options for ACC

Trabectedin impairs invasiveness and metastasis in adrenocortical carcinoma preclinical models

The pharmacological approach to adrenocortical carcinoma (ACC) is based on mitotane with/without etoposide, doxorubicin, and cisplatin, according to the disease stage. Considering the limited efficacy and toxicity of this treatment, new strategies are required. Trabectedin is a marine-derivated antitumoral agent that inhibits oncogenic transcription. We have already demonstrated trabectedin cytotoxic activity at sub-nanomolar concentrations in ACC cells. Here, we expanded the investigation of trabectedin effect on ACC preclinical models, evaluating whether trabectedin could affect ACC cells' invasiveness and metastasis formation. NCI-H295R, MUC-1, and TVBF-7 cell lines were used. Cell tumor xenografts in Danio rerio embryos were performed. The tumor mass areas and the number of embryos with metastasis were evaluated. The in vitro invasiveness of cells was evaluated. Effects of trabectedin of MMP2, TIMP1, and TIMP2 were evaluated at gene level qRT-PCR. MMP2 secreted in the cell medium was evaluated by Western blot and by zymography. Xenograft experiments demonstrated that trabectedin significantly reduced the tumor area in each ACC cell model and metastasis formation in embryos injected with metastasis-derived cell lines. Trabectedin treatment reduced the invasiveness of ACC cells across the matrix, which was greater at baseline for the metastatic models. In metastatic cell models, protein analysis demonstrated a reduction of MMP2 secretion and activity in the culture medium after treatment. Our results indicate that trabectedin interferes with invasiveness and metastasis processes, both dramatic features of ACC. Furthermore, these results support those previously published in providing the rationale for a clinical evaluation of the efficacy of trabectedin in ACC patients