Article Figures & Data
Figures
- Fig. 1.
Effect of MZ on cell growth and apoptosis. A, dose-dependent inhibition of cell proliferation after MZ treatment. The H460, A549, HUVEC, and WI38 cell lines were used in this assay. B, H460 and A549 cells were treated with 0.165 μm. MZ (IC50), and a 5-day growth assay was done. C, dose-dependent DNA fragmentation analysis was done in H460 cells after 24 h of MZ treatment. Lane 1, controls; Lanes 2–4, H460 cells exposed to 0.2, 0.5, and 1.0 μm MZ. D, H460 cells treated with MZ, harvested at different time intervals, and stained with propidium iodide. The cells were processed for fluorescence-activated cell sorter analysis to determine the cell cycle phase and apoptosis. Subdiploid populations indicate the apoptotic cells. The phase-contrast photomicrographs (×40) show mitotic cells after 12 h of MZ treatment and apoptotic nuclei after 24 and 48 h of MZ treatment. Thin arrows, apoptotic nuclei; thick arrows, mitotic nuclei. E, cytochrome c detected using Western blot analysis in the cytosolic fraction of H1299 and H460 cells. A considerable increase in cytochrome c was noticed, which correlated with the MZ dose. An antibody against COX IV, a mitochondria-specific protein, was used to probe the membrane to eliminate the possibility of contamination during fractionation.
- Fig. 2.
Effect of MZ on tumor growth and angiogenesis. A, MZ inhibited H460 xenograft tumor growth in athymic nu/nu mice in a dose-dependent manner. H460 cells were injected into mice (2 × 106 cells/mouse), and mice having established tumors (3–4 mm in diameter) were fed different concentrations of MZ (T02, T04, and T08, 200, 400, and 800 μg of MZ, respectively) every other day, whereas control animals received PBS. B, significant growth inhibition was observed when nu/nu mice were fed 1 mg of MZ every other day. ○, control mice; •, MZ-treated mice. Bars, SE. C, as expected, K1735 mouse xenografts in C3H mice showed reduced tumor growth when the mice were fed with 1 mg of MZ every other day. D, tumors were excised from control and MZ-treated nu/nu mice after 4 weeks and photographed. A few mice were killed at the start of treatment, when the tumors had reached 3–4 mm in diameter. E, graphic representation of the weight (mg ± SD) of tumors in control. Co, approximate tumor weight of ∼3–5 mm diameter tumor before starting MZ treatment; C, untreated and MZ-treated mice on day 28. F, histological analysis of blood vessels in H460 xenograft tumors via immunoperoxidase detection of endothelial cells using a CD31 antibody. 1, control untreated; 2, MZ treated. G, plot of milligrams of hemoglobin/ml of tumor tissue obtained from control and treated animals after hemoglobin assay. C, control; MZ, treated. H, effect of MZ on angiogenesis in vivo. Chamber assay shows that MZ inhibited capillary formation in A549 cells. It should be noted that the control implant had a tree-like architecture of major vessels (arrow) connecting to minor branches but that the MZ-treated implants had scarce vessels. Con, control; MZ, treated. I, A549 cells prelabeled using a fluorescent cell marker were detected on the chamber membranes of control (Con) and MZ-treated (MZ) mice.
- Fig. 3.
Effect of MZ treatment on lung colony formation in an experimental metastasis. A, A549 cells formed colonies on lung surfaces when injected through the tail vein. C, control mouse received no treatment; T, MZ-treated mice received 1 mg of MZ p.o. twice a week for 3 weeks. The white spots on the lung surfaces (arrows) are colonies. B, quantitation of lung colonies in control and MZ-treated animals (P < 0.0001); the total number of lung colonies/animal were plotted. C, H&E-stained lung sections show the sizes of tumor colonies in control (Con) and MZ-treated (MZ) animals. The colonies are indicated by dotted lines.
Volume 8, Issue 9
