Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies.

The molecular mechanisms underlying the cellular lost found in the nigrostriatal pathway during the progression of Parkinson's disease (PD) are not completely understood. Human neuroblastoma cell line SH-SY5Y challenged with 6-hydroxydopamine (6-OHDA) has been widely used as an in vitro model for PD. Although this cell line differentiates to dopaminergic neuron-like cells in response to low serum and retinoic acid (RA) treatment, there are few studies investigating the differences between proliferative and RA-differentiated SH-SY5Y cells. Here we evaluate morphological and biochemical changes which occurs during the differentiation of SH-SY5Y cells, and their responsiveness to 6-OHDA toxicity. Exponentially growing SH-SY5Y cells were maintained with DMEM/F12 medium plus 10% of fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 microM RA plus 1% of FBS during 4, 7 and 10 days in culture. We found that SH-SY5Y cells differentiated for 7 days show an increase immunocontent of several relevant neuronal markers with the concomitant decrease in non-differentiated cell marker. Moreover, cells became two-fold more sensitive to 6-OHDA toxicity during the differentiation process. Time course experiments showed loss of mitochondrial membrane potential triggered by 6-OHDA (mitochondrial dysfunction parameter), which firstly occurs in proliferative than neuron-like differentiated cells. This finding could be related to the increase in the immunocontent of the neuroprotective protein DJ-1 during differentiation. Our data suggest that SH-SY5Y cells differentiated by 7 days with the protocol described here represent a more suitable experimental model for studying the molecular and cellular mechanisms underlying the pathophysiology of PD.

Extracts of marine sponge Polymastia janeirensis induce oxidative cell death through a caspase-9 apoptotic pathway in human U138MG glioma cell line.

We have studied the apoptotic pathway activated in response to marine sponge extracts of Polymastia janeirensis. The effect on intracellular ROS production was also examined. Exposure of U138MG glioma cell line to doses higher than 5 microg/mL has decreased glioma cell viability, with an IC(50) <15 microg/mL for both aqueous and organic extracts. However, extracts at higher doses (50 and 100 microg/mL) have stronger cytotoxic effects, decreasing more than 90% of glioma cell viability. The antioxidant Trolox (100 microM) reversed the cell death percentage induced by extracts at 10 and 25 microg/mL. The type of cell death induced by such high doses was predominantly necrosis, while a high percentage of apoptotic glioma cells was found at 10 microg/mL. Moreover, inhibition of caspase-8 with Z-IETD (a caspase-8 inhibitor) had no effect on the amount of apoptosis induced by 10 microg/mL, but inhibition of caspase-9 with Z-LEHD (a caspase-9 inhibitor) decreased apoptosis. We also observed a dose-dependent increase in ROS production, and similarly to effects observed on viability of glioma cells, and on cell death, higher doses also had more severe effects. Co-treatment with Trolox significantly reduced ROS production by extracts at doses lower than 50 microg/mL. This is a first report demonstrating that marine sponge extracts of P. janeirensis induce oxidative cell death through a caspase-9 apoptotic pathway.

Brazilian marine sponge Polymastia janeirensis induces apoptotic cell death in human U138MG glioma cell line, but not in a normal cell culture.

Marine sponges have been prominently featured in the area of cancer research. Here, we examined the anti-proliferative effects of crude extracts (aqueous and organic) of the Brazilian marine sponge Polymastia janeirensis in the U138MG human glioma cell line. Moreover, we examined the effects of extracts on selective cytotoxicity in the glioma cells in comparison with a normal cell culture. Exposure of glioma cells to treatments (24 h) resulted in cell number decrease at all doses tested, with both aqueous and organic extracts (IC(50) <20 and <30 microg/ml, respectively). Parallel to this result, sponge extracts reduced glioma cell viability (IC(50) <15 microg/ml for both extracts). However, higher doses (50 and 100 microg/ml) induced a stronger cytotoxic effect when compared to the lower dose tested (10 microg/ml), inhibiting more than 80% of cellular growth and viability. Propidium iodide uptake and flow cytometry analysis further showed that sponge extracts caused necrosis in the glioma cell line at higher doses, while a high percentage of apoptotic glioma cells were observed at 10 microg/ml. Moreover, apoptosis was prevented by the pan-caspase inhibitor Z-VAD, suggesting that marine sponge extracts, at lower doses, induce caspase-dependent apoptosis in U138MG glioma cells. Surprisingly the extracts herein tested were more effective than temozolomide, a potent inductor of apoptosis used for the treatment of malignant gliomas. Furthermore, our results suggested a selectivity cytotoxic effect on glioma cell line in comparison with a normal cell culture, since the effect on viability found in glioma cells was not observed in astrocyte cultures with the lower dose (10 microg/ml). Thus, this marine sponge may be considered a good candidate for development of new cancer medicines with antitumor activity against gliomas.