Phenolic Compounds Isolated from Caesalpinia coriaria Induce S and G2/M Phase Cell Cycle Arrest Differentially and Trigger Cell Death by Interfering with Microtubule Dynamics in Cancer Cell Lines

Abstract

Caesalpinia coriaria (C. coriaria), also named cascalote, has been known traditionally in México for having cicatrizing and inflammatory properties. Phytochemical reports on Caesalpinia species have identified a high content of phenolic compounds and shown antineoplastic effects against cancer cells. The aim of this study was to isolate and identify the active compounds of a water:acetone:ethanol (WAE) extract of C. coriaria pods and characterize their cytotoxic effect and cell death induction in different cancer cell lines. The compounds isolated and identified by chromatography and spectroscopic analysis were stigmasterol, ethyl gallate and gallic acid. Cytotoxic assays on cancer cells showed different ranges of activities. A differential effect on cell cycle progression was observed by flow cytometry. In particular, ethyl gallate and tannic acid induced G2/M phase cell cycle arrest and showed interesting effect on microtubule stabilization in Hep3B cells observed by immunofluorescence. The induction of apoptosis was characterized by morphological characteristic changes, and was supported by increases in the ratio of Bax/Bcl-2 expression and activation of caspase 3/7. This work constitutes the first phytochemical and cytotoxic study of C. coriaria and showed the action of its phenolic constituents on cell cycle, cell death and microtubules organization.

Keywords: Caesalpinia coriaria; apoptosis; cancer; cell cycle; cell death; ethyl gallate; gallic acid; microtubules; phenolic compounds; tannic acid.

Figure 1

Compounds isolated from the WAE extract of C. coriaria.

Figure 2

Effect of the WAE extract of C. coriaria, GA, EG and TA on cell cycle progression in cancer cells lines. (A) PC3 (prostate); (B) Hep3B and (C) HepG2 (hepatocellular carcinoma); (D) HeLa and 2E) Caski (cervical cancer). Podophillotoxin (PDX) was used as a positive control (0.005 µM). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Figure 2

Effect of the WAE extract of C. coriaria, GA, EG and TA on cell cycle progression in cancer cells lines. (A) PC3 (prostate); (B) Hep3B and (C) HepG2 (hepatocellular carcinoma); (D) HeLa and 2E) Caski (cervical cancer). Podophillotoxin (PDX) was used as a positive control (0.005 µM). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Figure 3

Effect of isolated compounds on stabilization of microtubules in Hep3B cells by immunofluorescence of α-Tubulin and confocal microscopy. EG; GA; TA; Podophillotoxin (Microtubules destabilizing agent) and Taxol (Microtubules stabilizing agent).

Figure 4

Effect of WAE extract of C. coriaria extract and pure compounds on cell death in Hep3B cells by epifluorescence microscopy. (A) Negative control; (B) C. coriaria extract; (C) GA; (D) EG; (E) TA; (F) Podophillotoxin 0.005 µM (positive control); (G) H₂O₂ apoptosis positive control; (H) Necrosis control.

Figure 5

Effect of TA, GA and EG on mRNA expression levels of Bcl-2 and Bax in Hep3B cells after 72 h treatment. GAPDH was used as an internal control.

Figure 6

Caspase 3/7 activity after treatment with GA; EG; TA and taxol (TX) in Hep3B cells. * p < 0.05, ** p < 0.01, *** p < 0.001 were obtained when compared to the negative control.