In Vitro

In vitro activity: SC66 is a novel, potent and allosteric inhibitor of AKT, it displays a dual-inhibitory function toward AKT activity with IC50 values of 0.77, 2.85 and 0.47 μg/ml in HepG2, Huh7 and Hep3B cells, respectively. SC66 reduces cell viability in a dose- and time-dependent manner, it also inhibits colony formation and induces apoptosis in hepatocellular carcinoma (HCC) cells. SC66 treatment led to a reduction in total and phospho-AKT levels. SC66 induced the production of reactive oxygen species (ROS) and DNA damage. SC66 significantly potentiated the effects of both conventional chemotherapeutic and targeted agents, doxorubicin and everolimus, respectively. In vivo, SC66 inhibited tumor growth of Hep3B cells in xenograft models, with a similar mechanism observed in the in vitro model. SC66 had antitumor effects on HCC cells.

Cell Assay: Cells are seeded into each well (5 ×103/well) of 96-well microtiter plates and then incubated overnight. At time 0, the medium is replaced with fresh complete medium and different doses of SC66 are added. Cells are cultured for 24, 48 and 72 hours. At the end of treatment, MTS assays are performed using the CellTiter Aqueous OneSolution kit. Cell viability is expressed as a percentage of the absorbance measured in the control cells. Values are expressed as means±SD of three separate experiments, each performed in triplicate

In Vivo

Male nude athymic mice (Fox1 nu/nu) aged 4 weeks were obtained from Harlan and allowed to acclimatize for 1 week. Suspensions of 10 × 106 Hep3B cells in 0.2 ml of PBS were inoculated into the right flank of the animal. When tumors became palpable (around 150 mm3), the mice were randomly divided into three groups of 6 animals each, with the various tumor volumes equally distributed among the three groups. Two groups of mice were treated twice a week with 15 and 25 mg/Kg SC66 suspended in DMSO, further diluted in a solution of 25% ethanol and administered via i.p. injection. The control group received the vehicle alone. Tumor volumes were determined twice a week using calipers. Primary tumor volumes were calculated with the formula: v = length × (width)2/2. Mice were euthanized by cervical dislocation when the tumor burden exceeded 10% of animal body weight, or when tumor ulcerated or other conditions of morbidity were ascertained, in conformity with institutional guidelines which are in compliance with national (D.L., 116 G.U., Suppl.40; 18 February 1992) and international laws and policies (ECC Council Directive 86/609, OJ L358.1, 12 December 1987). This study was authorized by the Italian Ministry of Health (D.M. n. 39/2014-B).

Animal model

Male nude athymic mice

Formulation & Dosage

References

Oncotarget. 2015 Jan 30;6(3):1707-22.