Figure 6.

4-Me treatment suppresses MDA-MB-231 orthotopic tumor growth. (a) Orthotopic tumor volumes induced in nude mice by MDA-MB-231 cells. Cells (2 × 10⁶) were inoculated into the fourth mammary fat pad of each mouse. Mice were randomly divided into two groups (n=5 per group) at day 8 post cell inoculation. Thereafter, the two groups of mice were treated intraperitoneally with either 3.2 mg/kg of 4-Me or vehicle every 3 days. Values (means±S.D.) are from three measurements of each mouse. **P<0.01; ***P<0.001. (b) Representative pictures (top panels) of vehicle control and 4-Me-treated nude mice (day 36) in (a). Higher magnifications of tumor pictures are shown in red rectangles. Bottom panels show enlarged (indicating metastasis) and normal sized lymph nodes in vehicle and 4-Me-treated mice, respectively. (c) Haematoxylin and eosin and immunofluorescence staining of tumor sections from vehicle- and 4-Me-treated nude mice. Proliferating (Ki67) and apoptotic (TUNEL) cells were identified using the indicated antibodies or assay. Sections were counterstained with DAPI (blue). Images were acquired in one z-plane using a LSM710 confocal laser scanning microscope and ZEN 2008 software (Carl Zeiss, Oberkochen, Germany). Scale bars represent 100 μm. (d) Immunoblots of proliferation (cyclin D1 and PCNA), and apoptosis (cleaved PARP and cleaved caspase-3) markers on tumor biopsies of vehicle and 4-Me-treated nude mice. Immunoblot data are from three independent experiments performed in duplicate. β-Tubulin serves as a loading and transfer control. (c and d) All experiments were performed using tumor biopsies harvested from mice described in (a) at day 36