Meayamycin inhibits pre-messenger RNA splicing and exhibits picomolar activity against multidrug-resistant cells

Abstract

FR901464 is a potent antitumor natural product that binds to the splicing factor 3b complex and inhibits pre-mRNA splicing. Its analogue, meayamycin, is two orders of magnitude more potent as an antiproliferative agent against human breast cancer MCF-7 cells. Here, we report the picomolar antiproliferative activity of meayamycin against various cancer cell lines and multidrug-resistant cells. Time-dependence studies implied that meayamycin may form a covalent bond with its target protein(s). Meayamycin inhibited pre-mRNA splicing in HEK-293 cells but not alternative splicing in a neuronal system. Meayamycin exhibited specificity toward human lung cancer cells compared with nontumorigenic human lung fibroblasts and retained picomolar growth-inhibitory activity against multidrug-resistant cells. These data suggest that meayamycin is a useful chemical probe to study pre-mRNA splicing in live cells and is a promising lead as an anticancer agent.

Figure 1

Structures of FR901464, pladienolide D and their synthetic analogs.

Figure 2

(a) Degradation of meayamycin (10 μM) in phosphate buffers and 10% FBS in RPMI at 37 °C. Benzoic acid and rhodamine were used as an internal standard in phosphate buffers (pH 5, 6, 7 and 7.4) and culture media, respectively. The quantity of meayamycin relative to each of these internal standards was monitored by reversed phase HPLC. ◆: pH 5, R² = 0.952. ■: pH 6, R² = 0.9408. σ: pH 7, R² = 0.968. 5: pH 7.4, R² = 0.978. Culture media: R² = 0.989. (b) Exposure time-dependent growth inhibition of A549 cells in the presence of meayamycin at 2 nM for the indicated periods of time. Cell density was measured on day 4. (c) Growth inhibition of A549 cells treated with meayamycin for 96 hours (triangles), and for 8 hours with meayamycin followed by an 88-hour incubation without meayamycin (squares).

Figure 3. Meayamycin's activity against multi-drug resistant VCRd-5L cells

DC3F (A) or VCRd5L (B) cells were seeded into 384-well plates and treated for 72 hours with the indicated concentrations of paclitaxel (PTX, closed squares), vincristine (VCR, closed triangles), meayamycin (open diamonds) or FR901464 (open triangles). Cells were stained with Hoechst 33342 and nuclei enumerated on an ArrayScan II high-content reader. Nuclei counts were normalized to wells treated with vehicle (0.1% DMSO). Data represent the averages ± SE of quadruplicate determinations from a single representative experiment that has been repeated at least three times.

Figure 4. Inhibition of pre-mRNA splicing by meayamycin

(Left) Denaturing gel of HeLa nuclear extracts. Lane 1: DMSO. Lane 2: [Isoginkgetin] = 50 μM. Lanes 3–8: [1] = 50 pM, 500 pM, 5 nM. 50 nM, 100 nM, 500 nM. Lanes 9–11: [5] = 100 nM, 1 μM, 10 μM. (Middle) Native gel to analyze spliceosomal complex. Lane 1: [1] = 50 nM. Lane 2: DMSO. (Right) RT-PCR analysis of 1 (10 nM)-treated HEK-293 cells.

Figure 5. Analysis of the effects of meayamycin on alternative splicing in cultured neurons

(A) RT-PCR analysis of RNA transcripts after 24-hour treatment with a final concentration of 0, 10, and 20 pM meayamycin in the culture medium. Exons tested include alternative cassette exons from the NMDA R1 receptor (GRIN1, C1 cassette), KCNQ2 potassium channel (KCNQ2, E8), and hnRNP H3 (HNRPH3, E3) transcripts. Constitutively included exons (ACTIN, E2 and HNRPH3, E8) and the intronless histone transcript (H1F0) are included as controls. Assays were negative for the accumulation of intronic regions indicative of splicing inhibition for the calcium channel CAV1.3 and two regions of the GRIN1 transcript (CAV1.3, E2-E3; GRIN1, E2-N1 and intron 2-N1). RT-PCR products were resolved on 1% agarose gels containing ethidium bromide. Schematics represent the structures of the RNA products amplified; boxes, exons; shaded boxes represent alternatively spliced exon; lines, introns. Lanes M, 100 base pair DNA ladder. (B) Neurons were photographed in culture after treatment ± meayamycin; left panel of each pair. Results of trypan blue staining are shown; right panel of each pair. Trypan blue, which stains dead cells, shows similar results for the two samples.

Figure 6. High-content analysis of A549 and IMR-90 cells treated with meayamycin

A549 (A) or IMR-90 cells (B) were plated at high density (10,000 cells per well), treated with vehicle or ten two-fold serial dilutions meayamycin for 24 hours and analyzed for cell density, chromatin condensation, nucleus area, p53 induction, and caspase cleavage on the ArrayScan II high-content reader. (A-C) Concentration dependence. Meayamycin caused non-cell loss (closed squares) and induced p53 (open circles) in A549 but not IMR-90 cells. Neither cell line underwent apoptosis as indicated by the absence of nuclear condensation (open squares) or caspase cleavage (close circles). Data are the averages ± SE of quadruplicate wells from a single representative experiment that has been repeated at least twice. D. Magnitude of response at 10 nM meayamycin. Data are the averages ± SE of multiple independent experiments performed in quadruplicate (n=6 for A549 and n=3 for IMR-90) at the highest concentration of meayamycin used (10 nM). Cell loss, percent of cells lost during treatment; nuc_cond, % cells with condensed nuclei; nuc_area, % increase in nuclear size compared with vehicle; p53, % p53 positive cells; caspase cleavage, % of cleaved caspase positive cells.