Supplementary MaterialsSupplementary Document 1. for the bioactivity of crambescidins indicate that crambescidin 816 (C816) and crambescidin 800 (C800) possess cytotoxic, antifungal, antioxidative, antiviral and antimicrobial actions [18,19,20,21,22,23,24]. C816 exerts a powerful Ca2+ antagonist activity also, even more intense than nifedipine actually, a ROM1 selective blocker of l-type Ca2+ stations . Moreover, we’ve previously examined the cytotoxic activity of C816 over many human tumor cell types and characterized some of the cellular mechanisms responsible of the anti-proliferative effect of C816 on human liver-derived tumor cells . While the biological effects of crambescidins have been widely investigated, in the case of crambescins very few data are available. In order to tackle this lack of knowledge and to establish if these compounds could have interest as drugs leads, we examined the effect of crambescin-C1 (CC1) and crambescin-A1 (CA1) on human tumor hepatocarcinoma cells HepG2. According to this, comparative gene expression profiles following CC1 treatment were firstly performed. Obtained results showed that PCI-32765 cost up-regulation of PCI-32765 cost metallothionein mRNA was one of the major cellular responses to CC1. Besides this, effects on cell cycle progression and cellular antioxidant response were also observed. Comparative transcriptome analysis results were then backed up with assays which confirmed the biological effects inferred from them. 2. Results and Discussion 2.1. CC1 Inhibits Cell Proliferation and Induces Cell Death at High Doses In order to establish the appropriate concentrations to perform transcriptome analysis, we initially assayed the effects of CC1 and CA1 (Figure 1A) on HepG2 cells growth and viability. Open in a separate window Figure 1 (A) Structure of crambescin C1 (CC1) and crambescin A1 (CA1); (B) Proliferation of HepG2 cells after CC1 treatment for PCI-32765 cost 24 and 48 h; (C) Proliferation of HepG2 cells after CA1 treatment for 24 h and 48 h. In both cases cellular growth was determined by the 3-(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazolium bromide (MTT) method. * Significant differences respect to controls, 0.05, = 3. The 3-(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazolium bromide (MTT) assays showed that after 24 h CC1 reduced cell viability by approximately 33% only at the highest concentration tested PCI-32765 cost (Figure 1B). While no effect was observed after 24 h, an inhibition percentage of 22% was caused by 5 M CC1 after 48 h (Figure 1B). Similar doses of CA1 did not reduce cell proliferation whatever the length of the exposure (Figure 1C). Interestingly, CA1s lack of ability to reduce cellular growth refuted the possibility of a broad crambescin family effect in this regard. CC1 induced apoptosis in HepG2 cells as determined by Annexin V and propidium iodide (IP) staining. While no apoptosis was detected after 24 h treatment with 1 M and 5 M CC1, 10 M induced phosphatidylserine translocation. Hook increase from the apoptotic inhabitants was detected after 48 h contact with 5 M CC1 also. As a result, CC1 induced HepG2 cell apoptosis as one factor of your time and dosage publicity (Body 2). Open up in another window Body 2 Apoptosis recognition by confocal microscopy after 24 h and 48 h remedies with 1, 5 and 10 M crambescin C1 (CC1). Representative photos of control and treated cells are proven. Fluorescein isothiocyanate (FITC) was useful for phosphatidylserine translocation recognition (green) and propidium iodide (IP) was useful for nuclei staining of loss of life cells (reddish colored). Acquiring these total outcomes into consideration, cC1 was selected to execute transcriptome evaluation just. Concentrations of just one 1 M,.