Erybraedin C and bitucarpin A, two structurally related pterocarpans purified from Bituminaria bituminosa, induced apoptosis in human colon adenocarcinoma cell lines MMR- and p53-proficient and -deficient in a dose-, time-, and structure-dependent fashion

Abstract

Pterocarpans, the second group of natural isoflavonoids, have received considerable interest on account of their medicinal properties. These drugs are employed as antitoxins, but display antifungal, antiviral and antibacterial properties as well. Erybraedin C and bitucarpin A are two new structurally related pterocarpans recently purified and characterized. Bitucarpin A differs from erybraedin C for the absence of a prenyl group in 5′ position and the presence of a methoxylate hydroxyl group in 7, 4′ positions. These compounds proved not to be clastogens in human lymphocytes per se but displayed anticlastogenic activity against mytomicin C and bleomycin C. Here we extended the study of their antiproliferative and apoptosis-inducing mechanism on human cell lines. Two human adenocarcinoma cell lines, LoVo and HT29, as examples of slow-growing solid tumors, proficient and deficient in mismatch repair system (MMR), p53 and Bcl-2, were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle, measured by flow cytometry. Erybraedin C similarly affects the survival of HT29 (MMR +/+, p53 −/− and Bcl-2 +/+) and LoVo (MMR −/−, p53 +/+ and Bcl-2 −/−) cells (LD 50: 1.94 and 1.73 μg/ml, respectively). By contrast, bitucarpin A exhibits a differential cytotoxicity in the cell lines (LD 50: 6.00 μg/ml, HT29, and 1.84 μg/ml, LoVo). The cell cycle distributions of the LoVo and HT29 cells treated with erybraedin C lacked a specific checkpoint arrest, whereas they underwent a characteristic sub-G 1 peak, time- and drug-concentration dependent. So that apoptotic process induced by erybraedin C in both adenocarcinoma cell lines is independent of cell cycle arrest and of phenotypic status of the cells as well. By contrast, bitucarpin A affects cell cycle progression on both cell lines, inducing a transient block in G 0/G 1 along 24–96 h, and induces apoptosis with a cell density and treatment time dependency. Similar results were obtained with the positive control drug etoposide. The programmed cellular death on human adenocarcinoma cell lines may be efficiently activated, via a topoisomerase II poison pattern, by erybraedin C, the drug containing regio-specific hydroxyl and prenyl groups. The apoptotic effect induced by the methoxylated bitucarpin A proved to be conditioned by cell density and required higher dose (5-fold-LD 50) and longer treatment time. The present study provides evidences that erybraedin C may act as a potent growth inhibitory compound, at low and high cell density, comparable to other clinically important antineoplastic natural drugs including etoposide, on human colon adenocarcinoma cells. Bitucarpin A proved less active because it was conditioned by cell density effect, but this finding may represent a clinical advantage against early micrometastatic diseases.