https://pubs.rsc.org/en/content/articlepdf/2019/NP/C8NP00092A

Marine natural products.

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

https://cancerres.aacrjournals.org/content/canres/48/3/589.full.pdf

Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay

We describe here the development and implementation of a pilot-scale, in vitro, anticancer drug screen utilizing a panel of 60 human tumor cell lines organized into subpanels representing leukemia, melanoma, and cancers of the lung, colon, kidney, ovary, and central nervous system. The ultimate goal of this disease-oriented screen is to facilitate the discovery of new compounds with potential cell line-specific and/or subpanel-specific antitumor activity. In the current screening protocol, each cell line is inoculated onto microtiter plates, then preincubated for 24-28 hours. Subsequently, test agents are added in five 10-fold dilutions and the culture is incubated for an additional 48 hours. For each test agent, a dose-response profile is generated. End-point determinations of the cell viability or cell growth are performed by in situ fixation of cells, followed by staining with a protein-binding dye, sulforhodamine B (SRB). The SRB binds to the basic amino acids of cellular macromolecules; the solubilized stain is measured spectrophotometrically to determine relative cell growth or viability in treated and untreated cells. Following the pilot screening studies, a screening rate of 400 compounds per week has been consistently achieved.

Feasibility of a High-Flux Anticancer Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell Lines

Perp. punya vol. X. Flowering plant, eudicots : sapindales, cucurbitales, myrtaceae. Perp.punya: 1eks.

The Families And Genera Of Vascular Plants

The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.

/pdf/sulforhodamine-b-colorimetric-assay-for-cytotoxicity-261l8uir8v.pdf

Sulforhodamine B colorimetric assay for cytotoxicity screening

We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

https://www.researchgate.net/file.PostFileLoader.html?id=50d0b614e24a467d68000010&assetKey=AS%3A273569009864704%401442235342217

New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening

Eight new coumarin compounds (1-8) were isolated by anti-HIV bioassay-guided fractionation of an extract of Calophyllum lanigerum. The structures of calanolide A (1), 12-acetoxycalanolide A (2), 12-methoxycalanolide A (3), calanolide B (4), 12-methoxycalanolide B (5), calanolide C (6) and related derivatives 7 and 8 were solved by extensive spectroscopic analyses, particularly HMQC, HMBC, and difference NOE NMR experiments. The absolute stereochemistry of calanolide A (1) and calanolide B (4) was established by a modified Mosher's method. Calanolides A (1) and B (4) were completely protective against HIV-1 replication and cytopathicity (EC50 values of 0.1 microM and 0.4 microM, respectively), but were inactive against HIV-2. Some of the related compounds also showed evidence of anti-HIV-1 activity. Studies with purified bacterial recombinant reverse transcriptases (RT) revealed that the calanolides are HIV-1 specific RT inhibitors. Moreover, calanolide A was active not only against the AZT-resistant G-9106 strain of HIV-1 but also against the pyridinone-resistant A17 strain. This was of particular interest since the A17 virus is highly resistant to previously known HIV-1 specific, non-nucleoside RT inhibitors (e.g., TIBO; BI-RG-587; L693,593) which comprise a structurally diverse but apparently common pharmacologic class. The calanolides represent a substantial departure from the known class and therefore provide a novel new anti-HIV chemotype for drug development.

James B. McMahon

Papers

New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening

The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum.

Initial studies on the cellular pharmacology of 2′,3′-dideoxycytidine, an inhibitor of HTLV-III infectivity

Circulins A and B. Novel human immunodeficiency virus (HIV)-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia.

The guttiferones, HIV-inhibitory benzophenones from Symphonia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia rosea