Liposomes: versatile and biocompatible nanovesicles for efficient biomolecules delivery. points and in different cell-lines, to assess drug-and formulation-induced cytotoxic effects is due to a greater stability of Caelyx?. [29]. The cytotoxic effect of ceramide could potentially become mediated through AMPK since Empty-C6-Lip enhanced its phosphorylation. Open in a separate window Number 6 Effects of ceramide and doxorubicin on cell death signaling(A) HeLa cells were incubated with numerous concentrations (1-30 M) of DOX-loaded liposomes and Free-DOX. Pan-caspase inhibitor zVADfmk (10 or 30 M) was added to address the effect of caspase-activity on cell viability measured from the MTT assay after 24 h. Pub graphs display mean ideals from three self-employed experiments Rabbit Polyclonal to Histone H2A (phospho-Thr121) and standard deviations. (B) Immunoblotting of HeLa cells were performed to investigate influence of ceramide and DOX on cellular signaling pathways. HeLa cells treated with either Free-DOX D-AP5 (0.1 – 10 M), Empty-Lip-C6 (0.3 – 30 M) or DOX-Lip-C6 (0.3 C 30 M) were lysed, the lysates separated on SDS-PAGE and immunoblotted against PARP, phosphorylated (Ser473) AKT, GAPDH, phosphorylated (Thr172) AMPK and gamma-tubulin in duplicate. Untreated cells, cells treated with Empty-Lip or Staurosporin (1 M) were used as regulates. Ceramide does not enhance the effect of DOX on tumor growth inside a mouse model The effect of DOX-containing liposomes D-AP5 on tumor growth was analyzed by intravenous injection of a liposomal formulation related to a DOX dose of 8 mg/kg to mice bearing MAS98.12 patient-derived breast tumor xenografts (Number ?(Figure7).7). Two weeks after treatment all DOX-additions reduced the tumor volume compared to that acquired with the bare liposomes (bad control). Although not statistically significant, ceramide comprising liposomes seem to have a slightly better effect on tumor growth than Free-DOX, and Caelyx? seems to have the best effect (Number ?(Figure7).7). The tumor growth was equal for all the bare liposome treatments (Empty-Lip-C6, Empty-Lip-C12 and Empty-Lip), indicating no effect of ceramide only, regardless of chain size (C6 or C12). Little difference was observed for systemic toxicity between the different DOX-containing liposomes, albeit Free-DOX was more harmful than DOX-Lip-C6 and Caelyx? (Supplementary Number 4). Open in a separate window Number 7 Effect of ceramide liposomes on tumor growth in mice bearing MAS9812 breast tumor xenografts. The tumor quantities were measured from day time 22, i.e. one day prior to injection day (arrow mark) and up to day time 47, i.e. 24 days after intravenous injection of DOX-containing liposomes or Free-DOX (8 mg/kg DOX) or a similar amount of bare liposomes. Tumor quantities are demonstrated as relative to the tumor quantities at start of treatment. Data show mean ideals and standard deviations (n = 7-11 tumors). Conversation cell toxicity studies revealed the selected assays resulted in different readout of the cellular toxicity. The cell proliferation assay, measuring incorporation of [3H]thymidine, did not reveal any significant effect of ceramide only after 24 h (Number ?(Figure2),2), while such an effect was obvious when using the MTT cell viability assay (Supplementary Figure 3B). Screening the harmful effects on cells after numerous incubation instances may reveal important variations in the cellular response, such as the delay here reported for Caelyx? toxicity. Therefore, to understand the mechanisms of added medicines, and especially when trying combinatorial methods, different types of assays are important. studies The different liposome preparations were intravenously injected in mice with breast tumor xenografts (MAS98.12) to study the effect on tumor growth. These studies showed large effects within the tumor growth of all DOX-containing formulations, but did not show any significant difference between Free-DOX and CER-Lip-DOX. This may be due to insufficient ceramide concentration in the liposomes, since our data do not reveal any effect of ceramide only, in contrast to earlier studies where 20-30x higher final ceramide concentrations were used [36C38]. Fonseca of Caelyx? compared to our liposomes is due to a greater stability of Caelyx?. If true, different stabilities may be due to the presence of ceramide in our liposomes or the presence of cholesterol in Caelyx?. Although, we did not observe an increased therapeutic effect by adding ceramide to our liposomes, we can D-AP5 of course not exclude the.