IGF-1 levels are higher in the medium of cells transfected with the IGF-TD fusion protein

GraphPad Prism Software version 6.0. Results 3.1 Cytotoxicity of 1,3,4-thiadiazolium derivatives on HepG2 and rat hepatocytes The viability of HepG2 cells was determined after 24 h of treatment with derivatives at 5, 25 and 50 M, by both MTT and LDH-release assays. As observed in Fig 2, upper panel, MI-J, MI-4F and MI-2,4diF reduced HepG2 cells viability by about 50% at 25 M when analyzed by MTT. MI-D only reduced by 28% the cell viability, requiring 50 M to reach 50%. The results of the LDH-release assay also demonstrated the reduction of cell viability by MI-J, MI-4F and MI-2,4diF treatments. The enzymatic activity of the culture medium was increased by 55, 24 and 16%, respectively, for MI-J, MI-4F and MI-2,4diF at 25 M, in comparison to controls without mesoionic derivative. MI-D, on the contrary, did not significantly affect the LDH activity. The viability of primary hepatocytes was also determined in order to verify the selectivity of derivatives for tumor cells. As observed in Fig 3, no cytotoxicity was observed in MTT assays, except for MI-2,4diF producing a 36% effect. However, no increase in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736355 LDH activity was observed with any derivative. Interestingly, MI-D induced a reduction of LDH activity. 3.2 Apoptosis induction by 1,3,4-thiadiazolium derivatives in HepG2 cancer cells but not in control hepatocytes Considering the significant toxicity of the derivatives on HepG2 cells, we evaluated the induction of apoptosis in these cells by DNA fragmentation, a key event of cells undergoing 6 / 17 Selective Cytotoxicity of Mesoionic Derivatives on Hepatocarcinoma Fig 2. Cytotoxic effects of 1,3,4-thiadiazolium derivatives on HepG2 cells. A. MTT assay. The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 5, 25 or 50 M for 24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay. Under the same treatment conditions, as described above, LDH activity was measured in supernatants. Data represent means of four Peretinoin different experiments in quadruplicate The results were expressed as % of viability in comparison to control. and denote values significantly different from PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19737072 the control or between the different treatments at P< 0.05 and P< 0.0001, respectively. doi:10.1371/journal.pone.0130046.g002 apoptosis. After 24 h of treatment with MI-J, MI-4F and MI-2,4diF, approximate increases of 12%, 9% and 8%, respectively, were observed, as evidenced by the higher number of cells in sub-G1 region. MI-D under the same conditions did not promote any significant alteration in DNA fragmentation, but increased the number of cells in G2/M phase. The G1/G0 and G2/M phases were not significantly changed by the other derivatives. To further investigate the induction of apoptosis by mesoionic derivatives, HepG2 cells were simultaneously stained with FITC-conjugated annexin V and PI, and analyzed by flow 7 / 17 Selective Cytotoxicity of Mesoionic Derivatives on Hepatocarcinoma Fig 3. Cytotoxic effects of 1,3,4-thiadiazolium derivatives on hepatocytes. A. MTT assay The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 for 1824 h. The results were expressed as % of viability in comparison to control. B. LDH release assay. Under the same treatment conditions described above, LDH activity was measured in the supernatants. Data represent means of four different experiments in quadruplicate. The results were expressed as % of viability in comparison to control. and denotes values significantly