mithramycin-sk and Colonic-Neoplasms

HCT116 (p21(-/-)) human colon carcinoma cells treated with mithramycin SK (MSK), a novel analog of the antitumor antibiotic mithramycin A (MTA), were transiently arrested in G2/M, with some cells entering a faulty mitotic cycle without cytokinesis that resulted in G1-like cell arrest, which consisted of post-mitotic aneuploid G1 cells. Some of these cells synthesized DNA and elicited an apoptotic response. The absence of p21(WAF1) made HCT116 cells more sensitive to MSK than to the related MTA. MSK also showed higher antiproliferative activity than MTA on HCT116 cells with different genetic backgrounds, including those lacking the p53 gene. Apoptosis in MSK-treated p21(-/-) cells involved caspase 2 rather than caspase 3. Untreated HCT116 (p21(-/-)) cells presented a little caspase 3 activity, which increased slightly after treatment with MSK. The apoptotic response in p21(-/-) cells comprised caspase 2 acting as an executor caspase together with a loss of mitochondrial membrane potential that may be initiated by caspase 2. In contrast, caspase 3 was activated in wild-type HCT116 after treatment with MSK.

Topics: Apoptosis; Caspase 2; Cell Cycle; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; G2 Phase; HCT116 Cells; Humans; Mitosis; Plicamycin; Tumor Cells, Cultured

Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A(3) (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles.

Topics: Antibiotics, Antineoplastic; Cell Line, Tumor; Chromomycin A3; Chromomycins; Colonic Neoplasms; Deoxyribonucleases, Type II Site-Specific; DNA Restriction Enzymes; Female; Flow Cytometry; Humans; Male; Ovarian Neoplasms; Plicamycin; Prostatic Neoplasms; Structure-Activity Relationship

During a normal cell cycle, polyploidy and aneuploidy can be prevented by several checkpoints, which are mainly p53 dependent. Here, we show that treatment of HCT-116 (p53+/+) colon carcinoma cells with the novel antitumor antibiotic mithramycin SK (MSK) results in polyploidization and mitotic catastrophe, which occurs after a transient halt in G1 phase followed by the overtaking of the G2-M checkpoint when treated cells are incubated in a fresh drug-free medium. Cells reentering aberrant mitosis mainly died by necrosis, although active caspase-3 was observed. Our results indicate that a decrease in p53 RNA and protein levels, together with concomitant changes in the expression of other proteins such as p21WAF1, were involved in MSK-induced polyploidy. Furthermore, the effects of MSK on HCT-116 (p53+/+) cells cannot be attributed exclusively to the down-regulation of p53 by MSK, because these effects differed from those observed in MSK-treated HCT-116 (p53-/-) cells. The p53(-/-) cells died mainly from G2-M through early p53-independent apoptosis, which appeared to be mediated by caspase-2, although secondary necrosis was also observed.

Topics: Blotting, Western; Cell Death; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytokinesis; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Mitosis; Necrosis; Neoplasm Proteins; Plicamycin; Polyploidy; Transcription, Genetic; Tumor Stem Cell Assay; Tumor Suppressor Protein p53