Helmut Schönenberger

Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity

SummaryConventionally in vitro cytotoxicity assays are performed as single-end-point determinations. To compensate for the diversity of growth rates among different cell lines in this report we describe a computerized kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. As a prerequisite four human breast cancer cell line (MDA-MB-231, MCF-7, T-47-D and ZR-75-1) were characterized with regard to oestrogen and progesterone receptor content, modal chromosome number and proliferation kinetics depending on the number of passages in culture. With prolonged time in culture for ZR-75-1 exposed to various concentrations of cisplatinum a dose-related increase in drug effect was observed. Owing to a correction of the T/C values for the initial cell mass (at the time when drug is added) a sharp distinction between cytostatic and cytocidal drug effects becomes obvious in plots of corrected T/C values versus time of incubation. The influence of the untreated control on the corrected T/C values and possible time courses of theoretical inhibition profiles (reflecting cytostatic, transient cytotoxic or cytocidal drug effects as well as development of resistance) and their relationship to the corresponding growth curves of drugtreated cells are discussed. Chemosensitivity assays with diethylstilbestrol dipropionate, tamoxifen, melphalan, cisplatinum, vinblastine, Adriamycin and 5-fluorouracil prove the theoretical considerations to be true for MDA-MB-231, MCF-7, T-47-D and ZR-75-1 human breast cancer cell lines in practice.

Computerized Determination of Growth Kinetic Curves and Doubling Times from Cells in Microculture

In this paper we describe the microcomputer-aided determination of cell proliferation kinetics and doubling times utilizing a crystal violet assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in microtitration plates. The analysis of spectrophotometric data provides the doubling times at any time of incubation. Plots of doubling time versus time of incubation give reproducible information on the exact duration of the logarithmic growth phase. This method is applicable to anchorage-dependent as well as anchorage-independent cells when colorimetric or fluorometric data are accessible.

Hyaluronidase enhances the activity of Adriamycin in breast cancer models in vitro and in vivo

SummaryThe effect of hyaluronidase and a combination of hyaluronidase with Adriamycin was investigated on several breast cancer models in vitro and in vivo. In vitro enzyme treatment (using concentrations up to 80 000 IU/l) of murine (MXT−, MXT±, and MXT+) and human (MCF-7, ZR-75-1 and T-47-D) breast cancer cell lines did not inhibit tumour cell proliferation (measured by a kinetic crystal violet assay) in either case. Although highdose hyaluronidase (1.2×106 IU/kg) was ineffective, when administered peritumourally to the MXT M3.2 mammary carcinoma of the B6D2F1 mouse, it is remarkable that five “megadoses” were excellently tolerated. However, the antineoplastic activity of Adriamycin against the oestrogen-receptor-positive variant of the MXT tumour was significantly enhanced by combination with concentrations of hyaluronidase that were inactive per se, both in vitro and in vivo. Interestingly, the enhancement of the in vivo antitumour activity was not compromised by toxic side-effects.

Investigations on the Antiproliferative Effects of Amino Acid Antagonists Targeting for Aminoacyl-tRNA Synthetases Part I - The Antibacterial Effect

Amino acid antagonists with proven or potentially inhibitory activities on aminoacyl‐tRNA synthetases were tested for their antiproliferative effect against E. coli B. The compounds 4‐ and 6‐fluorotryptophan, 5‐methyltryptophan, selenocystine and ß‐(2‐thienyl)alanine gave strong growth inhibition in minimal medium, which disappeared after addition of structurally related natural amino acids or in an enriched broth. The inhibitory effect on amino‐acyl‐tRNA synthetases and the minimal inhibitory concentration for growth inhibition in minimal medium could not be correlated.

Hyaluronidase significantly enhances the efficacy of regional vinblastine chemotherapy of malignant melanoma

The regional chemotherapy of the human malignant melanomas (SK-MEL-2, -3, -5, -24) implanted in NMRInu/nu mice with a combination of the hyaluronic-acid-cleaving enzyme hyaluronidase (HYase) and vinblastine is a very effective therapeutic procedure. In three out of four melanoma models (SK-MEL-2,-3, -5) the weekly peritumoral administration of high-dose HYase (100 000 IU/kg) 4 h prior to the injection of 0.3 mg/kg vinblastine in the vicinity of the tumor (seven weekly therapeutic cycles) caused marked antitumor effects, while HYase and vinblastine were inactive when given alone. The pretreatment with HYase, which is well tolerated by the test animals, prevented local inflammation reactions commonly seen after subcutaneous vinblastine administration. Tumor growth and metastatic behavior of the melanomas used were neither increased nor reduced by HYase after peritumoral administration without subsequent vinblastine injection. The curative activity of the regional chemotherapy with HYase/vinblastine could be demonstrated on the SK-Mel-3 melanoma. After an observation time of 18 weeks tumor cells could no longer be detected in the subcutaneous region of the former lesion. Only macrophages, which had abundantly incorporated melanin, gave evidence of previously growing tumors. In contrast to the controls, no metastases could be observed in the axillary lymph nodes of the test animals.

Stability and cellular studies of [rac-1,2-bis(4-fluorophenyl)- ethylenediamine][cyclobutane-1,1-dicarboxylato]platinum(II), a novel, highly active carboplatin derivative

Abstract The synthesis of the diastereomeric [1, 2-bis(4-fluorophenyl)ethylenediamine][cyclobutane-1, 1-dicarboxylato]platinum(II) complexes, rac- and meso-4F-Pt(CBDC), the evaluation of their structures, their tumor-inhibiting properties and their stability in physiological environment are described (reference complexes: the dichloro- and sulfatoplatinum(II) analogues, carboplatin and cisplatin). The most interesting diastereomer, rac-4F-Pt(CBDC), equals cisplatin and surpasses carboplatin in its effect on human breast cancer cell lines (MCF-7 and MDA-MB-231). Rac-4F-Pt(CBDC) is largely insensitive against attack of nucleophiles e.g. Cl−, a prerequisite for sufficient stability in vivo and for fewer side effects. In accordance with this, in vitro studies on the binding of rac-4F-Pt(CBDC) to albumin, the main plasma protein, show that the free, non-protein-bound fraction is relatively high, coming close to that of carboplatin. These properties are of importance for the transferability of the promising effects found in the cell culture experiments to in vivo conditions. The distinctly better anti-breast cancer activity of rac-4F-Pt(CBDC) than of carboplatin has been attributed to its ability to accumulate in the tumor cells. The human ovarian cancer cell line NIH-OVCAR-3 is also strongly inhibited by rac-4F-Pt(CBDC).

Entwicklung neuer Antiöstrogene vom Typ des 3,3′-Dihydroxy-α,β-diäthylstilbens und ihre Prüfung am DMBA-induzierten, hormonabhängigen Mammacarcinom der SD-Ratte

SummaryThe displacement of the phenolic OH-group of diethylstilbestrol into the 3,3′-position (trans-3,3′-dihydroxy-α,β-diethylstilbene compd. III) leads to a strong decrease of the estrogenic effect under conservation of the receptor affinity. In vitro, III inhibits the estradiol-receptor-interaction competitively and, in vivo, antagonises the uterotropic effect of estrone in the mouse. In tests with the DMBA-induced, hormone-dependent mammary carcinoma of the rat a dose-dependent strong decrease of tumor size and yield is achieved under the influence of III, due to the antiestrogenic, properties of III. The replacement of the α,β-bound ethyl groups in III by other alkyl chains leads to no further increase of the antiestrogenic and antitumor activity.ZusammenfassungDie Verlagerung der phenolischen OH-Gruppen des Diäthylstilböstrols in die 3,3′-position (trans-3,3′-Dihydroxy-α,β-diäthylstilben, Verb. Nr. III) führt unter Erhaltung der Rezeptoraffinität zu einer starken Abnhme der östrogenen Wirkung. III hemmt in vitro die östradiol-Rezeptor-Wechselbeziehung kompetitiv und antagonisiert in vivo bei der Maus die uterotrope Wirkung des Östrons. In Versuchen am DMBA-induzierten, hormonabhängigen Mammacarcinom der Ratte kommt es, unter III-Einwirkung dosisabhägig zu einer starken Abnahme von Tumorgröße und-zahl, die durch die antiöstrogenen Eigenschaften von III bedingt, ist. Der Austausch der α,β-ständigen Äthylreste in III durch andere Alkylketten führt zu keiner weiteren, Steigerung der antiöstrogenen und tumorhemmenden, Wirkung.

Chemosensitivity of human MCF-7 breast cancer cells to diastereoisomeric diaqua(1,2-diphenylethylenediamine) platinum(II) sulfates and specific platinum accumulation

SummaryCisplatin, raceme-diaqua[1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) sulfate (compound I), meso-diaqua[1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) sulfate (compound II), and meso-diaqua[1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]platinum(II) sulfate (compound III) were compared with regard to their effect on the MCF-7 breast cancer cell line in vitro. At equimolar concentrations (5 μM), cisplatin, compound I, and compound II were equiactive after 231 h drug exposure, whereas compound III was ineffective. Although compounds I and II showed markedly greater inactivation than did cisplatin after 6 h incubation with culture medium, compound I (but not compound II) exhibited antitumor activity equivalent to that of cisplatin when cells were exposed to the drugs for 6 h. Platinum measurements by neutron-activation analysis revealed that compound I was selectively and rapidly accumulated by MCF-7 cells, resulting in a high degree of DNA platination within the first few hours of drug exposure. However, when the drug-exposure period was long enough, platinum enrichment was not reflected in an overall difference in the cytotoxicity of compound I vs cisplatin. Nevertheless, compound I should be superior to cisplatin in vivo, provided that effective plasma levels can be maintained for about 6 h.

Synthesis and antitumor activity of platinum(II) complexes containing substituted ethylenediamine ligands

Abstract The synthesis of substituted ethylenediamines, their reactions with K 2 PtCl 4 to give the dichloroplatinum(II) complexes, and the exchange of the chloro ligands for other leaving groups are described. The new compounds have been tested as antitumor agents both in vitro using the hormone independent human mammary carcinoma cell line MDA-MB 231 as well as in vivo using the lymphocytic P388 leukemia of the CD 2 F 1 -mouse. In the P388 test, 53 of the 55 tested complexes fulfill the minimum activity of 125% T/C required for a substance to be active.

Influence of ring substituents on the antitumor effect of dichloro(1,2-diphenylethylenediamine)platinum(II) complexes

SummaryDiastereomeric para-substituted dichloro-(1,2-diphenylethylenediamine)platinum(II) complexes were synthesized and tested for their antitumor activity on the human MDA-MB 231 breast cancer cell line and the P 388 leukemia of the mouse. An interaction with the DNA was demonstrated by UV difference spectroscopy. The D,L configurated, 4-fluoro-substituted complex was the most active.