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Molecular Mechanism of Action of 5‐Azacytidine and its Resistance in MDA‐MB 435 Cells
Author(s) -
DiNatale Anthony,
Hellreich Jaclyn,
O'Donnell Robert
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.897.22
Subject(s) - cytotoxic t cell , cell culture , gene , biology , microbiology and biotechnology , cancer cell , cancer research , cancer , in vitro , genetics
DNA methyltransferase inhibitors such as 5‐Azacytidine (5‐Aza) have shown promise in cancer treatment because of their ability to allow re‐expression of tumor suppressor genes in cancer cells. Preliminary studies showed that high doses of 5‐Aza are clearly cytotoxic to MDA‐MB 435 tumor cells. However, we have begun treating the MDA‐MB 435 cells with a low dose so that the majority of the cells survive. A resistant cell line has been developed through continuous growth in the presence of 5‐Aza. Tests were done to determine if the resistance inhibited growth in a methylcellulose assay. The number of colonies decreased in the 5‐Aza resistant cell line compared to the control. Control 435 cells were found to have an average colony count of 969±332 but when 5‐Aza was added the colony count dropped to 464±31. In order to look at the mechanism of 5‐Aza and its resistance, quantitative reverse transcriptase PCR was done to measure the expression of various genes. Between the control cells and the control cells plus 5‐Aza there was a five‐fold decreased expression of Mammaglobin B, a member of the secretoglobin family associated with chemoresistance, while the other genes tested did not have any change (TP53, RB1, BCL‐2, Myc). When the same tests were done on the resistant cell line the colony counts showed little change, 287±129 and 357±46, respectively, nor was there a difference between the resistant and resistant plus 5‐Aza in terms of Mammaglobin B expression. However, the resistant and the resistant plus 5‐aza both showed a six‐fold decrease in Mammaglobin B expression compared to the control cells. Future experiments will investigate a phenotypic role for this gene in the resistance.