Cell proliferation assay

In order to evaluate the in vitro antitumor activity of the prodrugs, compounds 20 and 21 were incubated at varied concentrations with human T-lineage acute lymphoblastic leukemia MOLT-3 cells in the presence or absence of ImM of PGA. The data from the cell proliferation assays are presented in Fig. 5.15. 

Although a number of cell lines were shown to be sensitive to inhibition by PatA (Low et al., 2005), we selected RKO cells (IC50 of 0.4 nM in cell proliferation assay) to prepare lysates for the isolation and identification of target protein(s). We often select RKO cells for target identification of small molecules using biotin-conjugates, because they appear to be particularly suitable for target protein isolation. 

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

Okubo T, Suzuki T, Yokoyama Y, Kano K, Kano I (2003) Estimation of estrogenic and antiestrogenic activities of some phthalate diesters and monoesters by MCF-7 cell proliferation assay in vitro. Biol Pharm Bull 26 1219-1224... 

BaF3 cell assays were used to study the effect of AP23464 against the most relevant Abl mutations. In a cell proliferation assay with BaF3 cells expressing... 

FIGURE 6.3 Comparison of MTT tetrazolium and tritiated thymidine incorporation assays to measure effects of hGM-CSF on proliferation of TF-1 cells. A no-cell blank absorbance of 0.065 was subtracted from all MTT values before plotting. Similar ED50 values are shown for both assays. (Source Modified from Promega Corporation Technical Bulletin 112. CellTiter 96 Non-Radio active Cell Proliferation Assay.)... 

I. Human Umbilical Vein Endothelial Cells Proliferation Assay... 

All experimental agents exhibited activity in the 72-hour cell proliferation assay and inhibited cell proliferation in one or more of the cell lines listed with an IC50 less than or equal to about IOjjlM. 

Several compounds derived from these libraries were identified as Hsp70 modulators and were further evaluated in cell proliferation assays such as the SK-BR-3 and MCF-7 breast cancer cell lines and the HT29 colon cancer cell line (Fig. 6) [26]. Compounds 22-24 were potent against all three cell lines. The dihydro-pyrimidinone libraries were also tested for replication inhibition of the malarial parasite, Plasmodium falciparum, where Hsp70 chaperones are believed to play an important role in the parasite s homeostasis [24]. Nine compounds that inhibit replication of the parasite were identified. Among them, compounds 25 and 26 were the most potent (Fig. 7) and were prepared by direct couplings of dihydropyrimidone acids 19 with a pyrrolo amine. 

In the neutral red (cell viability) and total protein (cell proliferation) assays, cells are treated with various concentrations of a test substance in petri or multiwell dishes after a period of exposure, the substance is washed out of the medium. (An analytical reagent is added in the case of protein measurements.) Neutral red is a supravital dye, which accumulates in the lysosomes of viable, uninjured cells, and it can be washed out of cells, which have been damaged. In the protein test, Kenacid blue is added and reacts with cellular protein. Controlled cells are dark blue killed cells are lighter colored. The IC50 (the concentration which inhibits by 50%) is determined the test can be rapidly performed with automation. However, materials must be solubilized into the aqueous cell media for analysis. For many test materials this will require large dilutions which eliminate properties of the materials which cause irritation. 

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