Figure 6
In-vivo anti-tumor activities of 3-AWA.
(A,B) 1.5 million 4T1 cells in normal saline were implanted into the mammary pad of each female Balb/c mice. After tumor cell implantation mice were injected i.p. with either normal saline or 3-AWA at 5 mg/kg, 10 mg/kg and positive control 5-FU, 22 mg/kg body weight every alternate day for two weeks. Animals were sacrificed the tumors were dissected and weighed to check the tumor growth inhibition. (C,D) Metastatic lung nodules were counted using a dissecting microscope. (E,F) With the help of 70 μm cell strainer the single cell suspension was obtained from the lungs and serially diluted. Each dilution was resuspended in an appropriate medium containing 6-thioguanine, used for the selection of 4T1 cells. 4T1 cells were allowed to grow and colonies were counted and photographed under fluorescent inverted microscope. (G) Tumors excised from controls and treated animals were homogenized and sonicated in lysis buffer. Lysates were boiled with gel loading buffer and loaded on an SDS-polyacrylamide gel and processed for western blotting as shown. (H) For immunohistochemical detection of the Phospho-eIF4E and Total eIF4E, paraffin embedded sections from the tumours of treated and untreated mice were incubated with respective primary antibodies and were exposed to 3,3′-diaminobenzidine (DAB) substrate, counterstained with hematoxylin and finally dehydrated and mounted for confocal microscopy. Data are mean ± S.D. of three similar experiments. *P < 0.05; **P < 0.01. One-way ANOVA with SAS 9.2 was carried out to compare the mean of wet tumor weight and number of lung nodules between the six experimental groups.
