Fig. 2.

Overexpression of Hpa2 attenuates the pro-tumorigenic characteristics of gastric carcinoma cells. (A) Cell proliferation. Control (Vo) and Hpa2 overexpressing MKN-45 cells (2 × 10³/well) were seeded in a 96-well plate and relative cell numbers were examined over time as described under “Materials and Methods” (upper panel). The relative number of Hpa2 cells at day 3 is shown graphically vs control (Vo) cells set arbitrarily to a value of 1 (lower panel). (B) Cell migration. Control (Vo) and Hpa2 overexpressing MKN-45 cells were seeded on fibronectin-coated inserts and cell migration was examined 16 hours (upper panels) and 24 hours (lower panels) afterward. Shown are representative images taken at x20 magnification. The number of migrating cells is shown graphically in the right panels. (C) Colony formation. Control (Vo) and Hpa2 overexpressing MKN-45 (upper panels), SGC-7901 (middle panels), and BGC-832 (lower panels) cells were grown in soft agar as described under “Materials and Methods” After 3 to 4 weeks, dishes were fixed with formalin and cell colonies were stained with Crystal violet. Representative photomicrographs are shown in the left panels (original magnifications x10). Quantification of the number of colonies per dish is shown graphically in the right panels. (D, E) Survival times and tumor growth. Control (Vo) and Hpa2 overexpressing MGC-803 cells (0.5 × 10⁶) were injected intraperitoneal (i.p) into NOD/SCID mice (n = 7) and survival times recorded (D). Control and Hpa2 MGC-803 cells were similarly inoculated ip into NOD/SCID mice (n = 7). After 14 days mice were sacrificed and all the tumor lesions from each mouse were collected, weighed (E, left) and photographed. Shown are representative images of the tumor lesions collected from mice implanted with control (Vo) and Hpa2 cells (E, right). Hpa2, heparanase 2.