Synthesis and Uptake of Fluorescence-Labeled Combi-molecules by P-Glycoprotein-Proficient and -Deficient Uterine Sarcoma Cells MES-SA and MES-SA/DX5

Abstract

Here, we report on the first synthesis of fluorescent-labeled epidermal growth factor receptor-DNA targeting combi-molecules, and we studied the influence of P-glycoprotein status of human sarcoma MES-SA cells on their growth inhibitory effect and cellular uptake. The results showed that 6, bearing a longer spacer between the quinazoline ring and the dansyl group, was more stable and more cytotoxic than 4. In contrast to the latter, it induced significant levels of DNA damage in human tumor cells. Moreover, in contrast to doxorubicin, a drug known to be actively effluxed by P-gp, the more stable combi-molecule 6 induced almost identical levels of drug uptake and DNA damage in P-gp-proficient and -deficient cells. Likewise, in contrast to doxorubicin, 4 and 6 exerted equal levels of antiproliferative activity against the two cell types. The results in toto suggest that despite their size, the antiproliferative effects of 4 and 6 were independent of P-gp status of the cells.

Subcellular Distribution of a Fluorescence-Labeled Combi-Molecule Designed to Block Epidermal Growth Factor Receptor Tyrosine Kinase and Damage DNA with a Green Fluorescent Species

“…We observed the blue fluorescence associated with the aminoquinazoline FD105 (451 nm) and the green fluorescence with the DNA damaging dansylated moiety (525 nm) in the cytoplasm and in the vicinity of the nucleus. Whereas the entire molecule can still fluoresce in green, the detection of blue FD105 fragment is indicative of a degradation of the molecule, which as reported elsewhere has a half-life of 22 minutes (30). Whether the combi-molecule was partially or completely decomposed, the green fluorescence was consistently localized in the perinuclear area (Fig.…”

“…The methods used for AL237 and JDA41 complete synthesis were described elsewhere (22,30). N-dansylaziridine was purchased from Biomol, and Temozolomide and Iressa were purchased from the hospital pharmacy and extracted from pills in our laboratory.…”

Search for cytotoxic agents in multiple Laurencia complex seaweed species (Ceramiales, Rhodophyta) harvested from the Atlantic Ocean with emphasis on the Brazilian State of Espírito Santo

“…For comparative purposes, the ID50 for doxorubicin was 0.05 µM for the wild type cell line (MES-SA), but almost two orders of magnitude higher (i.e., 3.4 µM; see Figure 2, panel A) for the MDR mutant (MES-SA/Dx5). Analogous differences in the response of these cell lines to doxorubicin have been reported by others authors (Larroque-Lombard et al, 2010).…”

“…24 h), show desirable morphology, are large enough to facilitate counting/analysis of drug-induced mechanisms of damage via microscopic analysis, are highly susceptible to a large variety of anticancer drugs (i.e., relatively easy to perform in vitro assays of mortality), and are also easy to manipulate and maintain (Larroque-Lombard, 2010;Lacerda et al, 2005;Belostotsky et al, 2011). The susceptibility of MES-SA cells to doxorubicin is analogous to that observed for HeLa cells (compare panels A and B in Figure 2).…”

“…These cells were grown in modified McCoy's 5A medium supplemented with 10% FBS, amphotericin B (0.63 µg/mL), penicillin (100 units/mL) and streptomycin (100 µg/mL). The medium used to grow the MES-SA/Dx5 mutants also contained doxorubicin (0.5 µM), as required for the maintenance of the drug-resistance phenotype (Harker, 1983;Harker & Sikic, 1985;Larroque-Lombard et al, 2010). HeLa (CCL-2™) human cervix adenocarcinoma cells were kindly provided by Dr. Mari Cleide Sogayar (Departamento de Bioquímica, Universidade de São Paulo) and grown in DMEM medium, supplemented as described above for the McCoy's medium used to grow the MES-SA cells.…”