Tandem MS-Based Metabolite Profiling of 19,20-Epoxycytochalasin C Reveals the Importance of a Hydroxy Group at the C7 Position for Biological Activity

Abstract

Seven cytochalasins, 19,20-epoxycytochalasin N, cytochalasin P1, deacetyl 19,20-epoxycytochalasin C, 19,20-epoxycytochalasin D, 19,20-epoxycytochalasin C, cytochalasin D, and cytochalasin C, were isolated from a fungal (Rosellinia sanctae-cruciana) crude extract. A cytotoxicity assay (sulforhodamine B) was performed on a series of cancer cell lines: HT-29, A-549, PC-3, HCT-116, SW-620, and MCF-7. Simultaneously, the liquid chromatography-mass spectrometry (LC-MS)/MS profile of 19,20-epoxycytochalasin C-treated cell lines revealed that 19,20-epoxycytochalasin C (m/z 524.25) oxidized to a metabolite of m/z 522.25 Da (-2 Da (-2H) from 19,20-epoxycytochalasin C). Further chemical oxidation of 19,20-epoxycytochalasin C using the Dess-Martin reagent produced an identical metabolite. It has been noticed that the parent molecule (19,20-epoxycytochalasin C) showed an IC₅₀ of 650 nM (on HT-29), whereas for the oxidized metabolite (m/z 522.24) of 19,20-epoxycytochalasin C, the IC₅₀ was >10 μM. It is clear that the parent molecule had 16 times higher cytotoxic potential as compared to the oxidized metabolite. The spectroscopic investigation indicated that the oxidation of the hydroxyl (-OH) group occurred at the C7 position in 19,20-epoxycyctochalsin C and led to the inactivation of 19,20-epoxycytochalasin C. Further, cell cycle analysis and histopathological evidence support the findings, and CDK2 could be a possible target of 19,20-epoxycyctochalasin C.

Figure 1

LC–ESI–MS/MS profiles and tandem MS spectra of different cell line extracts: (a) HT-29, (b) SW-620, and (c) PC-3 peak at m/z 524 eluted at t_R = 18.8 min, corresponding to 19,20-epoxycytochalasin C; peak at m/z 522 eluted at t_R = 18.8 min, corresponding to oxidized 19,20-epoxycytochalasin C. The tandem MS spectra of m/z 522 give a common daughter ion at m/z 91.0 and 120.08 as 19,20-epoxycytochalasin C. The common daughter ion and the loss of 2H confirm the oxidation in 19,20-epoxycytochalasin C (Figure S2).

Figure 2

LC–MS chromatograms and mass spectra: (a) +ESI-base peak chromatogram of the standard mixture, (b) +EIC of m/z 524.25, 19,20-epoxycytochalasin C of the standard mixture, (c) +EIC of m/z 522.25, oxidized 19,20-epoxycytochalasin C of the standard mixture, (d) +ESI-base peak chromatogram (A-549 cell line), (e) +EIC of m/z 524.25, 19,20-epoxycytochalasin C (A-549 cell line), and (f) +EIC of m/z 522.25 oxidized 19,20-epoxycytochalasin C (A-549 cell line). Note: The chemically oxidized 19,20-eopxycytochalasin C eluted at t_R = 14.7 min and in the chromatogram of A-549 the peak at m/z 522.25 oxidized 19,20-epoxycytochalasin C also eluted at t_R = 14.7 min, the chromatographic separation and the common daughter ion is a confirmation of structural similarity (Figure S2). The chemical structure of 19,20-epoxycytochalasin C and oxidized 19,20-epoxycytochalasin C elucidated by NMR (Figures S11–S19).

Figure 3

Imaging of platinum-coated HT-29 cells: (A) control HT-29 cells, (B) HT-29 cells treated with 50 nM paclitaxel, (C) HT-29 cells treated with 650 nM of 19,20-epoxycytochalasin C, (D) HT-29 cells treated with 1.3 μM 19,20-epoxycytochalasin C, and (E) HT-29 cells treated with 3 μM 19,20-epoxycytochalasin C. (HT-29 cells were fixed for 1 h with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at RT and then incubated at 4 °C overnight). Subsequently, the glutaraldehyde solution was extracted and the cells were washed with PBS. After complete dehydration via a gradient ethanol series, samples were suspended in 100% ethanol, placed onto a Nanopercolator (JEOL Ltd., Tokyo, Japan), dried up, then coated with a platinum layer using an MSP-1S sputter coater (Shinku Device, Ibaraki, Japan), and investigated as well as photographed using a Phenom ProX desktop scanning electron microscope (Phenom-World BV, Eindhoven, The Netherlands).

Figure 4

19,20-Epoxycytochalasin C dose–responses curve against 25 ng of CDK2/cyclin A2 to determine its inhibitory potency (19,20-epoxycytochalasin C, IC₅₀ = 866 ± 0.176 nM; Flavopiridol as a positive control, IC₅₀ = 290 ± 0.035 nM).

Figure 5

Cross examination of the sections of the colon of BALB/c mice exposed to AOM and stained with H&E. The control group received 0.9% normal saline, the positive control received 5-Fluorouracil (20 mg/kg, ip), and the test group received 19,20-epoxycytochalasin C (1 mg/kg, ip).