Novel GC-rich DNA-binding compound produced by a genetically engineered mutant of the mithramycin producer Streptomyces argillaceus exhibits improved transcriptional repressor activity: implications for cancer therapy

Abstract

The aureolic acid antibiotic mithramycin (MTM) binds selectively to GC-rich DNA sequences and blocks preferentially binding of proteins, like Sp1 transcription factors, to GC-rich elements in gene promoters. Genetic approaches can be applied to alter the MTM biosynthetic pathway in the producing microorganism and obtain new products with improved pharmacological properties. Here, we report on a new analog, MTM SDK, obtained by targeted gene inactivation of the ketoreductase MtmW catalyzing the last step in MTM biosynthesis. SDK exhibited greater activity as transcriptional inhibitor compared to MTM. SDK was a potent inhibitor of Sp1-dependent reporter activity and interfered minimally with reporters of other transcription factors, indicating that it retained a high degree of selectivity toward GC-rich DNA-binding transcription factors. RT–PCR and microarray analysis showed that SDK repressed transcription of multiple genes implicated in critical aspects of cancer development and progression, including cell cycle, apoptosis, migration, invasion and angiogenesis, consistent with the pleiotropic role of Sp1 family transcription factors. SDK inhibited proliferation and was a potent inducer of apoptosis in ovarian cancer cells while it had minimal effects on viability of normal cells. The new MTM derivative SDK could be an effective agent for treatment of cancer and other diseases with abnormal expression or activity of GC-rich DNA-binding transcription factors.

Pomalidomide and Lenalidomide Induce p21WAF-1 Expression in Both Lymphoma and Multiple Myeloma through a LSD1-Mediated Epigenetic Mechanism

“…This is suggested by the fact that the treatment of Namalwa cells by MTM, which inhibits all transcription factors binding to GC-rich regions (37), abolished the up-regulation of p21 WAF-1 . Sp1, whose binding to DNA is inhibited by MTM (31), is involved in the up-regulation of p21 WAF-1 in several cell types (14,38), as well as in the repression of this gene (39)(40)(41)(42).…”

Nanoparticulate formulations of mithramycin analogs for enhanced cytotoxicity

“…[9][10][11][12][13] In order to address these issues, extensive combinatorial biosynthesis has been performed on the MTM pathway and has resulted in several novel analogs. [14][15][16] The present authors have shown that the inactivation of the mtmW gene, which codes for the last enzyme in the MTM biosynthetic pathway, yields the most favorable of the new analogs, MTM SK (SK) and MTMSDK (SDK). 15,16 Both SK and SDK exhibited higher anticancer activity than the native MTM, 15,16 yet their short plasma retention time and low accumulation in tumors remained to be improved.…”

“…15 S. argillaceus cells of the M7W1 mutant strain were plated on R5A agar and allowed to grow until spores were formed. After 2 days, colonies began to appear on the plates; after 3 days, spores began to form; and after 4 days, the majority of the cells were present as spores.…”

“…15 Cells of S. argillaceus M7W1 were plated on R5A agar and grown for 4 days. After 4 days, the spores were used to inoculate 100 mL of TSB for 24 hours in an orbital shaker at 28°C, 250 rpm.…”

Dynamic transcriptional regulatory complexes including BORIS, CTCF and Sp1 modulate NY-ESO-1 expression in lung cancer cells

“…Initial experiments were undertaken to ascertain the effects of mithramycin A, a compound that competitively inhibits binding of Sp1 to target GC boxes (Albertini et al, 2006) on NY-ESO-1 promoter activity in Calu-6 cells. As shown in Figure 1a, mithramycin A (2 Â 10 À6 M) reduced the activity of pGL3/NY762 to 30% of its original activity; in fact, luciferase activity of the promoter-reporter construct in sequential DAC/ DP-treated Calu-6 cells following mithramycin exposure was nearly as low as that observed in vector controltransfected Calu-6 cells, which were exposed to chromatin remodeling agents.…”