Autophagic cell death induced by a novel class of substituted pyrroles in the breast tumor cell Free

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Introduction: Two major drawbacks to the effectiveness of cancer chemotherapy are the limited selectivity of many drugs for the tumor versus normal host tissues and the development of drug resistance. Our current work is directed towards the development of substituted pyrroles for the treatment of breast cancer, based on their structural similarity to agents derived from marine organisms. The substituted pyrroles are readily synthesized and their structural elements easily manipulated in terms of the attachment and removal of functional groups from the ring backbone structure. Preliminary studies suggest that these compounds act through inhibition of microtubule polymerization, thereby interfering with mitotic activity in the tumor cell.

Experimental Design: Our lead compound, JG-03-14, was screened against MCF-7 (p53 wild type), MDA-MB 231 (mutant p53), and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay and the MTT assay. Apoptosis was evaluated by TUNEL and cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on β-galactosidase staining and cell morphology.

Results: Continuous exposure to the substituted pyrrole JG-03-14, at a concentration of 500 nM, induces time dependent cell death in the MCF-7 and MDA-MB 231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no evidence of a surviving senescent population following treatment of MDA-MB 231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in MCF-7 cells. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (significantly < 10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis ( but likely not > 20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in MCF-7 cells and quite pronounced in the MDA-MB 231 cells. Cell-cycle analysis indicated that JG-03-14 induced a substantial early G2M arrest 24 hours following treatment in MCF-7 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in the MCF-7/ADR cell line that is resistant to Adriamycin.

Conclusions: Substituted pyrroles are promising agents, in part, because of their ease of synthesis and susceptibility to structural modification. Furthermore, preliminary studies in vivo using a prostate tumor xenograft model indicate that these compounds are relatively nontoxic and therefore may prove to be relatively selective in terms of targeting the tumor. The observation that the substituted pyrroles retain activity in breast tumor cells expressing the multidrug resistance pump indicates that these novel microtubule poisons hold promise in the treatment of breast cancer.