Article Figures & Data
Figures
- Fig. 1.
Chemotherapy sensitivity comparison for HUVECs, MVB9 (vinblastine-resistant variant), MD22 (Adriamycin-resistant variant), and CDDP-S4 (cisplatinum-resistant variant). HUVECs were grown in monolayer, whereas cancer cells were grown as both spheroids (blue bars) and monolayer (red bars). Comparison of the effect of different chemotherapeutic agents on the proliferative capacity of each was made. In HUVECs Taxol and vinblastine were inhibitory at the lowest dose concentrations, followed by Adriamycin and cisplatinum. Tumor cell lines were consistently more resistant in spheroid culture, but even in monolayer the drug concentrations necessary for inhibition of proliferation were much higher than those for endothelial cells. The results represent two independent experiments, done in sextuplicate on two different experimental days (mean ± S.E.).
- Fig. 2.
Sensitivity of HUVECs grown in monolayer to vinblastine alone or in combination with IMC-1C11 (a monoclonal antibody to human VEGFR-2/KDR). HUVECs grown on glass slides coated with 1% gelatin, and supplemented with growth factors, were treated with either 0.5 ng/ml vinblastine or a combination of vinblastine and 1C11(25 μg/ml), a monoclonal antibody against the VEGFR-2. The culture was then fixed and stained with anti-β-tubulin/Cy-3 conjugate. The mild polymerization of tubulin evident in the cultures treated with 0.5 ng/ml vinblastine is visibly enhanced by combining the agent with IMC-1C11, and no further escalation of this effect is achieved by doubling the vinblastine dose concentration.
- Fig. 3.
In vitro inhibition of MDR of selected cancer cell lines. The MDA-MB-231 parental cell line and its drug-resistant variants were grown in monolayer, incubated with 5 μg/ml rhodamine chloride (a fluorescent Pgp pump substrate) for 30 min, and assessed for relative fluorescence by flow cytometry. The human mammary carcinoma parental cell lines MDA-MB-231 and its cisplatinum-resistant variant CDDP–S4 readily take up rhodamine chloride, shifting the curve to the right, and this uptake cannot be inhibited by cyclosporin A (a Pgp inhibitor). In contrast, its drug-resistant variants show less rhodamine chloride uptake, or efflux of rhodamine chloride from the cells, and this is, at least in vitro, readily inhibited by cyclosporin A or verapamil, suggesting a Pgp-mediated resistance.
- Fig. 4.
Growth of orthotopic human breast cancer tumor xenografts in SCID mice. Drug-resistant variants were implanted in the mammary fat pad of 4–6-week-old SCID mice, grown to 300 mm3, and treated with a low dose of the chemotherapeutic drug to which they were resistant. There is no evidence of significant tumor growth suppression in the mice treated by the low-dose chemotherapeutic regimens alone and a partial regression by the DC101 antibody alone. However, the combination of the two agents causes a reliable and reproducible tumor growth suppression/regression in all treated cell lines. Each point on the graphs is presented as mean ± S.E. and represents an average of two different experiments with five mice per group in each experiment.
- Fig. 5.
Histological evaluation of MPAHS orthotopic tumor xenografts treated with low-dose continuous vinblastine alone or in combination with DC101 for 3 weeks. Formalin-fixed, paraffin-embedded tumor tissue was stained with H&E and evaluated for general histological features (a–e), the degree of mitosis (f–j), and aggressiveness/invasion ability (k–o). This enhances the findings in Fig. 4<$REFLINK> , where at 3 weeks of treatment (day 51 on the graph), early, and only marginal, differences in tumor sizes begin to emerge. Significant and progressive increases in the numbers of pyknotic nuclei and replacement of healthy tumor cells with connective and necrotic tissue are seen with escalation of treatment (from top to bottom of each column). There is a marked decrease in viable tumor cells (e), no mitotic figures (j), and no visible vessel invasion (o) in the combination treatment group. Reference mammary tissue is included on each histological sample (left-hand edge of a–e) to avoid selection bias.
- Fig. 6.
Toxicity evaluation. Weight was plotted at regular intervals and considered a surrogate for evaluation of systemic well-being, anorexia, or failure to thrive. Mice treated with the combination of Pgp substrate (vinblastine, Taxol, and Adriamycin) and the Pgp inhibitor (verapamil or cyclosporin A), experienced significant drug toxicities despite reduction of chemotherapy doses to half. Except for this toxicity, there were minimal differences in weight loss in the vinblastine alone, DC101, or vinblastine/DC101 group MPAHS (c and MVB9). In comparison, there is significant growth retardation in the Adriamycin or cisplatinum treatment groups (MD22 and CDDP-54), with weight loss reaching up to 25% of the body weight. Note that 1 mg/kg cisplatinum twice weekly, although it has very comparable effectiveness, is significantly less toxic.
Tables
- Table 1
Differences in sensitivity of cancer cell lines and HUVECs to chemotherapeutic agents
Cell population tested Agent IC50 (nm) Monolayer Spheroid at 24 h at 72 h at 24 h at 72 h HUVECs Vinblastine 0.55 N/A N/A N/A HUVECs Adriamycin 1.65 N/A N/A N/A HUVECs Cisplatinum 4 N/A N/A N/A HUVECs Taxol 0.4 N/A N/A N/A MDA-MB-231 parental tumor Taxol 2 4.5 3,434 848 MD22 varianta Adriamycin 79.4 41.5 1,610 260 MPAHS varianta Vinblastine 15.7 7.1 1,349 >123,301 MVB9 varianta Taxol 27.4 19 10,084 1,059 CDDP-S4 varianta Cisplatinum 2581 2083 27,773 2,380 -
a All of the variants are drug-resistant sublines of MDA-MB-231 human breast carcinoma cell line.
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Volume 8, Issue 1
