Migrated cells attached on the under surface were fixed with 4% paraformaldehyde for 10 min and then stained with a crystal violet solution for 10 min. Cells were counted under a microscope at 200 magnification. Subcutaneous and orthotopic xenografts in SCID mice SCID mice were purchased from HFK Bioscience Ltd. Animal experiments were especially performed in accordance with relevant institutional and national regu lations, and research protocols were approved by the relevant authorities. AsPC 1 cells suspended in a 100 ul mixture of equal volumes of medium and matri gel were implanted subcutaneously into the right flank of 6 week old female SCID mice. When the tumors had reached a volume of about 50 70 mm3, the mice were then randomly divided into two groups.

The treatment group received an intraperitoneal injection of RocA, whereas the vehicle control group received olive oil alone. These treatments were carried out once daily for 48 days. Tumor volumes and the body weight of animals were measured twice a week. Tumor volumes were calculated with the following formula V LS2 2. At the end of experiment, the mice Inhibitors,Modulators,Libraries were sacrificed and the tumors were harvested, fixed in formalin, and em bedded in paraffin for tissue sectioning and immunohis tochemistry. For orthotopic metastasis assays, AsPC 1 cells were orthotopically injected into the pan creas of mice as described previously. At 1 week post implantation, RocA or the vehicle was administrated via intraperitoneal injection daily for 3 weeks. Then, these mice were sacrificed to evaluate metastasis to the organs such as the liver and lung.

The metastatic nodules in the right lung and liver were quantified under a dissecting microscope. An other ten mice were subjected to Inhibitors,Modulators,Libraries the same treatment. The survival time of these mice in each group was monitored. Immunohistochemistry Immunohistochemical analysis was performed as described previously with antibodies against PHB, Ki 67, and cyclin D1. Statistics Data are representative of at least three Inhibitors,Modulators,Libraries independent experiments or multiple independent mice as indicated. Statistical analyses were performed by Students t tests and analysis of variance followed by post hoc compari sons. Kaplan Meier survival Inhibitors,Modulators,Libraries data were reanalyzed using the log rank test. Background Melanoma is the leading cause of fatal skin cancer, and in recent years, the incidence and mortality of melanoma have increased.

Prior to the recent advances in therapy for pa tients Inhibitors,Modulators,Libraries with stage IV disease, the prognosis of metastatic melanoma was very poor with a median survival of less than 12 months. One of the most significant full read advances in recent years was the elucidation of the etiological role of the mitogen activated protein kinase pathway in melanomagenesis, particularly the roles of mutant B RAF and N RAS.