Article Figures & Data
Figures
- Fig. 1.
a, antiangiogenic versus conventional scheduling of cyclophosphamide for drug-resistant Lewis lung carcinoma. ▵, control saline; ○, conventional schedule [150 mg/kg every other day for three doses (white arrows, total 450 mg/kg) every 21 days]; •, antiangiogenic schedule (170 mg/kg every 6 days, CTX, thin black arrows); ▪, antiangiogenic schedule of cyclophosphamide and TNP-470 (170 mg/kg cyclophosphamide and 12.5 mg/kg TNP-470 administered on the same day of the 6-day cycle for seven cycles, CTX + TNP, thick black arrows). The inset (top right) has magnified axes for the first 21 days of therapy (n = 6 mice/group). All control and conventional schedule-treated mice died with large tumor burdens. Therapy was discontinued on the antiangiogenic schedule of cyclophosphamide alone after two of six mice died with pulmonary inflammation, accompanied by high peripheral leukocyte counts. No mouse on either schedule had visibly detectable pulmonary metastases at time of death. Therapy was discontinued on the antiangiogenic schedule of cyclophosphamide plus TNP-470 after seven cycles, three cycles beyond the point at which tumors were no longer visible. This graph depicts the long-term survival of five of six mice treated with the antiangiogenic schedule of cyclophosphamide and TNP-470 in one of five separate experiments. The arrow and Note on the graph reflect the recurrence of one of six drug-resistant tumors at 18 days off therapy in this experiment. b, antiangiogenic versus conventional scheduling of cyclophosphamide for drug-sensitive Lewis lung carcinoma. ▵, control saline; ○, conventional schedule [150 mg/kg every other day for 3 doses (white arrows, total 450 mg/kg) every 21 days]; •, antiangiogenic schedule (170 mg/kg every 6 days, thin black arrows). The inset (top right) reveals magnified axes for the first 21 days of therapy (n = 6 mice/group). Therapy on the antiangiogenic schedule was discontinued after seven cycles, three cycles beyond the point at which tumors were no longer visible. Three separate experiments produced similar results. Two mice from two separate experiments were reinoculated with Lewis lung carcinoma >500 days after the eradication of the original tumor. The growth of these second Lewis lung carcinomas was similar to that of tumors in untreated mice, an outcome not consistent with immune-mediated regression of the primary Lewis lung tumor by cyclophosphamide.
- Fig. 2.
a, in vitro antiendothelial effects of activated cyclophosphamide on bovine capillary endothelial cell migration, survival and proliferation, apoptosis, and cell cycle distribution. 4-Hydroperoxycyclophosphamide, which spontaneously converts to 4-HC in aqueous solution, was added at the indicated concentrations (conc.). Values shown are the mean ± SE. Relative cell number refers to the remaining, adherent endothelial cells (>600 fl) from an initial plating of 12,500 cells. Apoptosis was determined as a percentage of 10,000 intact (gated) cells by fluorescence flow cytometry. Cell cycle analysis was determined similarly using ModFit LT software. Without the stimulation of S phase (14% to 74%) produced by bFGF, activated cyclophosphamide (4-HC) was 1.5-fold less inhibitory of proliferation and caused 3-fold less induction of apoptosis (data not shown). b, antiangiogenic activity of systemic cyclophosphamide against bFGF-induced corneal neovascularization. Treatment was initiated 24 h after pellet implantation, when limbal dilation and vascular sprouts first appear. Left panel, saline-treated control; right panel, 170 mg/kg cyclophosphamide administered once 24 h after pellet implantation in a 6-day assay (8) . The area of inhibition was calculated in c using the formula 0.2 × π × neovessel length × clock hours of neovessels. The percentage of inhibition was calculated by normalizing the area of neovascularization in treated mice to the area of neovascularization in saline-treated controls (n = 8 mice/group). The experiment was repeated three times with similar results. c, relative inhibition of the area of corneal neovascularization and schedule-dependent antiangiogenic efficacy of cyclophosphamide and other chemotherapeutic agents as detailed in “Materials and Methods.” Continuous infusion of the antimetabolites (5-fluorouracil and 6-mercaptopurine ribose phosphate) demonstrated superior inhibition of growth factor-induced angiogenesis when compared with equivalent bolus injections. Likewise, the pegylated liposomal formulation of doxorubicin enhanced the antiangiogenic efficacy of an equivalent dose of doxorubicin (see “Discussion”).
- Fig. 3.
Immunofluorescence analysis of tumor cell and endothelial cell apoptosis in drug-resistant Lewis lung carcinoma. a, endothelial cell versus tumor cell apoptosis in cyclophosphamide-resistant Lewis lung carcinoma treated on the conventional schedule (white arrows). Endothelial cell apoptosis (○, dashed line) precedes and subsides before peak drug-resistant tumor cell apoptosis (•, solid line). Tumors were analyzed on days 1, 3, 5, 7, 10, 13, 17, and 21. Day 0 reflects the analysis of two control tumors harvested at tumor volumes of 100–200 mm3. Note that tumor cell apoptosis falls to background levels just as similar tumors begin to regrow on the conventional schedule (see Fig. 1 <$REFLINK> a, inset). b, endothelial cell versus tumor cell apoptosis in cyclophosphamide-resistant Lewis lung carcinoma treated on the antiangiogenic schedule (black arrows). Endothelial cell (○, dashed line) apoptosis precedes drug-resistant tumor cell (•, solid line) apoptosis after each of four cycles of cyclophosphamide delivered on the antiangiogenic schedule. Tumors were analyzed on days 1, 2, 4, 6, 6.5, 7, 8, 10, 12, 14, 16, 19, and 21. Day 0 reflects the analysis of two control tumors harvested at tumor volumes of 100–200 mm3. In contrast to one broad wave on the conventional schedule, the antiangiogenic schedule of cyclophosphamide induces repetitive waves of drug-resistant tumor cell apoptosis, and this schedule prevents net drug-resistant tumor growth for 36 days (see Fig. 1a <$REFLINK> ). Note that from day 13 through day 21, the antiangiogenic schedule results in a nearly 3-fold increase in drug-resistant tumor cell apoptosis over the background level in tumors treated on the conventional schedule (a). The second cycle (days 6–12) shows that endothelial apoptosis occurs within 12 h (day 6.5) of a dose of cyclophosphamide on the antiangiogenic schedule and precedes maximum drug-resistant tumor cell apoptosis by 3.5 days (on day 10). c, representative immunofluorescence (von Willebrand factor/TUNEL) of drug-resistant Lewis lung carcinoma from control (left panel, day 0), 12 h (middle panel, day 6.5), and 4 days (right panel, day 10) after cyclophosphamide administration on the antiangiogenic schedule. Microvessels are stained fluorescent red, and apoptotic tumor cell nuclei are stained fluorescent green. The white arrow marks an apoptotic endothelial cell nucleus (yellow).
- Fig. 4.
a, growth of drug-resistant Lewis lung carcinoma in p53+/+ (dashed line) versus p53−/− (solid line) C57Bl6/J mice treated on the antiangiogenic schedule of cyclophosphamide (170 mg/kg every 6 days, black arrows). Mice were treated as described in the Fig. 1 <$REFLINK> legend, except that therapy was discontinued after three cycles in the p53−/− mice to prevent the development of cardiopulmonary toxicity. The p53−/− mice showed no evidence of primary Lewis lung carcinoma regrowth or pulmonary metastases and died of spontaneous tumors (expected in p53−/− mice; Ref. 23 ) within 2 months after therapy. Four separate experiments produced similar results. In the fourth experiment, mice were reinoculated with drug-resistant Lewis lung carcinoma 2 days after the third dose on the antiangiogenic schedule of cyclophosphamide. The first (cyclophosphamide-treated) drug-resistant Lewis lung carcinoma regressed completely, whereas these second (untreated) tumors grew in four of four mice (data not shown). This outcome is not compatible with an anamnestic response from immune-mediated regression of the primary tumor in these congenic p53−/− C57Bl6/J mice. b, immunofluorescence (von Willebrand factor/TUNEL) of drug-resistant Lewis lung carcinoma in p53−/− mice treated with the antiangiogenic schedule of cyclophosphamide. Left panel, a representative field of the growing tumor on day 1 after cyclophosphamide administration, which remained unchanged (e.g., days 0, 1, 2, and 4) until the second dose of cyclophosphamide was administered on day 6. Middle panel, within 20 min after the second dose of cyclophosphamide on day 6, extensive endothelial cell apoptosis was manifested, without an increase in tumor cell apoptosis. The three white arrows mark apoptotic endothelial cell nuclei (yellow). By 180 min after the second dose of cyclophosphamide on day 6, 70–90% of this cyclophosphamide-resistant tumor underwent massive, central necrosis. Only a thin, cortical rim of identifiable tumor tissue (approximately 330-μm thick) remained on H&E-stained sections. Right panel, a representative area of this rim of identifiable tumor tissue that displayed massive endothelial cell and tumor cell apoptosis (described in the Fig. 3 <$REFLINK> legend).
- Fig. 5.
a, antiangiogenic versus conventional scheduling of cyclophosphamide for the drug-resistant breast cancer EMT-6/CTX (7) . ▵, control saline; ○, conventional schedule [150 mg/kg every other day for three doses (white arrows, total 450 mg/kg) every 21 days]; •, antiangiogenic schedule (170 mg/kg every 6 days, CTX, black arrows); n = 12 in two experiments. All control and conventional schedule-treated mice died with large tumor burdens. Therapy was discontinued on the antiangiogenic schedule of cyclophosphamide alone after two of six mice died similarly to mice described in the Fig. 1a <$REFLINK> legend. b, antiangiogenic versus conventional scheduling of cyclophosphamide for the drug-sensitive leukemia cell line L1210 (9) . ▵, control saline; ○, conventional schedule [150 mg/kg every other day for three doses (white arrows, total 450 mg/kg) every 21 days]; •, antiangiogenic schedule (170 mg/kg every 6 days, black arrows); n = 10 in two experiments. Therapy on the antiangiogenic schedule was discontinued after nine cycles, five cycles beyond the point at which tumors were no longer visible. Three of 10 mice developed recurrent tumor toward the end or immediately after the discontinuation of therapy on the antiangiogenic schedule (small crosses). Seven of 10 mice (70%) are long-term, tumor-free survivors 170 days after the initiation of therapy at this writing.
Volume 60, Issue 7
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- Article
- Abstract
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Drug-resistant Tumor Growth Inhibition by Cyclophosphamide Is Linked to Endothelial Cell p53
- Eradication of Drug-sensitive Lewis Lung Carcinoma and L1210 Leukemia by the Antiangiogenic Schedule of Cyclophosphamide
- Eradication of Drug-resistant Lewis Lung Carcinoma by Adding a Second Angiogenesis Inhibitor (TNP-470) to the Antiangiogenic Schedule of Cyclophosphamide
- DISCUSSION
- Footnotes
- References
- Figures & Data
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