In vitro activity: Streptozotocin directly methylates DNA and is highly genotoxic, producing DNA strand breaks, alkali-labile sites, unscheduled DNA synthesis, DNA adducts, chromosomal aberrations, micronuclei, sister chromatid exchanges, and cell death. Free radicals are involved in the production of DNA and chromosome damage by Streptozotocin. Streptozotocin is toxic to pancreatic beta cell. Exposed to 15 mM Streptozotocin for 1 hr followed by a 24 hrs recovery period induces apoptosis in murine pancreatic beta cell line, INS-1. Streptozotocin (30 mM) causes the cells to undergo necrosis (22%) as well as apoptosis (17%).

Kinase Assay: Streptozocin is a potent DNA-methylating agent, with IC50s of 11.7, 904 and 1024 μg/mL in HL60, K562 and C1498 cells respectively.

Cell Assay: Human and murine cell lines are cultured in triplicate in 96-well plates at a density of 2×104 cells/well in the absence (untreated control) or presence of various concentrations of ALX (20-3000 μg/mL) or STZ (1-3000 μg/mL) for 48 h at 37°C under a humidified atmosphere containing 5% CO2. Cells incubated in complete media including dH2O in a final concentration of 0.1% served as control for solvent toxicity and cells incubated in complete medium are used as a control for the experiments. The effects of the tested drugs on tumor cell growth or viability are determined employing the MTT assay in accordance with the manufacturer's instructions. The IC50values (drug concentration that induces 50% inhibition of the cell growth) are calculated using the GraphPad Prism 4 program.

Rats: Thirty rats underwent oophorectomy to induce menopausal status. Oophorectomy is performed under sterile conditions via a midline incision after anesthetization using intraperitoneal injection of ketamine hydrochloride (90 mg/kg) and xylazine (10 mg/kg). The incisions are closed with 3.0 polyglactin sutures. The remaining 10 rats formed the control group (Group 1). Ten of the 30 rats that underwent oophorectomy constituted the menopause control group (Group 2). Beginning 1 week after the operation, methylprednisolone (1 mg/kg) is injected daily until the end of the study to mimic steroid treatment for rheumatologic diseases in 10 rats (Group 3). Group 4 consisted of rats that received intraperitoneal administration of Streptozocin (50 mg/kg) to induce diabetes mellitus (DM) 1 week after the oophorectomy. Blood glucose level is checked 3 days after Streptozocin administration, and values>250 mg/dL are considered as positive for DM.