3.4.2. Sulforhodamine-B Assay

The cytotoxic activity of three isolated ceramides **A** (**1**), **B** (**2**), and **C** (**3**) was determined by a Sulforhodamine-B (SRB) assay. SRB is able to bind to the intracellular proteins, providing a sensitive index of the cellular protein content. It was assessed as previously described in detail [20]. The experiment was repeated 3 times, and the IC50 values (concentration that caused a 50% decrease in cell viability) were calculated. Doxorubicin was used as a positive control with the same concentration range.

#### *3.5. In Vivo Study*

Two ceramides were chosen for further investigation in vivo: ceramide **A** (**1**) and ceramide **B** (**2**). EAC is a spontaneous murine mammary adenocarcinoma model that has extensively been used as a study model of breast cancer [88]. The effect of these ceramides against EAC and their role in regulating vascular and apoptotic factors were tested.

## 3.5.1. Tumor Induction

Human breast cancer cell line MCF-7 and EAC cells were purchased from the Tumor Biology Department at the National Cancer Institute of Cairo University. MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 μg/mL gentamycin. All cells were maintained at 37 ◦C in a fully humidified air atmosphere containing 5% CO2 and were subcultured twice a week. EAC served as the original tumor from which an ascites variant was obtained. EAC cells were suspended in normal saline (2.5 × 10<sup>6</sup> cells/100 μL). Cells were counted by a hemocytometer under the microscope. The mice were inoculated intradermally with a 100-μL EAC suspension on the lower ventral side after shaving [89].
