δ-Cadinene inhibits the growth of ovarian cancer cells via caspase-dependent apoptosis and cell cycle arrest

Abstract

Ovarian cancer is one of the most common causes of mortality among all cancers in females and is the primary cause of mortality from gynecological malignancies. The objective of the current research work was to evaluate a naturally occurring sesquiterpene-δ-Cadinene for its antiproliferative and apoptotic effects on human ovary cancer (OVCAR-3) cells. We also demonstrated the effect of δ-Cadinene on cell cycle phase distribution, intracellular damage and caspase activation. Sulforhodamine B (SRB) assay was used to evaluate the antiproliferative effect of δ-cadinene on OVCAR-3 cells. Cellular morphology after δ-cadinene treatment was demonstrated by inverted phase contrast microscopy, fluorescence microscopy and transmission electron microscopy. Flow cytometry was used to analyze the effect of δ-cadinene on cell cycle phase distribution and apoptosis using propidium iodide and Annexin V-fluorescein isothiocyanate (FITC)/PI kit. The results revealed that δ-cadinene induced dose-dependent as well as time-dependent growth inhibitory effects on OVACR-3 cell line. δ-cadinene also induced cell shrinkage, chromatin condensation and nuclear membrane rupture which are characteristic of apoptosis. Treatment with different doses of δ-cadinene also led to cell cycle arrest in sub-G1 phase which showed dose-dependence. Western blotting assay revealed that δ-cadinene led to activation of caspases in OVCAR-3 cancer cells. PARP cleavage was noticed at 50 µM dose of δ-cadinene with the advent of the cleaved 85-kDa fragment after exposure to δ-cadinene. At 100 µM, only the cleaved form of PARP was detectable. Pro-caspase-8 expression remained unaltered until 10 µM dose of δ-cadinene. However, at 50 and 100 µM dose, pro-caspase-8 expression was no longer detectable. There was a significant increase in the caspase-9 expression levels after 50 and 100 µM δ-cadinene treatments.

Keywords: Ovary cancer; Sulforhodamine B; apoptosis; caspases; δ-cadinene.

Figure 1

Antiproliferative activity of δ-cadinene against human ovary cancer cells (OVCAR-3) at different doses and time intervals.

Figure 2

δ-cadinene induced morphological changes in human ovary cancer cells (OVCAR-3) as identified by phase contrast microscopy (magnification 400 ×). Cellular shrinkage and blebbing were observed in δ-cadinene-treated cells (arrows). A. Represents control (untreated cells), B-D. Represent effect of 10, 50 and 100 µM of δ-cadinene on cell morphology of OVCAR-3 cells.

Figure 3

Morphological examination of δ-cadinene-induced apoptosis with Hoechst 33258 staining at actual mag nification 200 ×. OVCAR-3 cells were treated without (A) and with δ-cadinene10 μM (B), 50 μM (C), and 100 μM (D) for 48 hours. White arrows represent Apoptotic cells exhibiting chromatin condensation and nuclear fragmentation.

Figure 4

Fluorescence microscopic study of human ovary cancer cells stained with a combination of acridine orange: ethidium bromide (1:1 ratio). (A) shows untreated Control cells, (B) shows OVCAR-3 cells treated with 10 μM dose of δ-cadinene, (C) shows OVCAR-3 cells treated with 50 μM dose of δ-cadinene and (D) shows OVCAR-3 cells treated with 100 μM dose of δ-cadinene.

Figure 5

Quantification of δ-cadinene-induced apoptosis in human ovary cancer cells (OVCAR-3). The cells were subjected to different doses of δ-cadinene (0, 10, 50 and 100 µM) for 48 h and analyzed by flow cytometry with annexin V-FITC/PI staining. The different quadrants Q1, Q2, Q3 and Q4 represent necrotic cells, late apoptotic cells, viable cells and early apoptotic cell population respectively. Percentage of apoptotic cells increases from 7.4% in control cells (A), to 24.8%, 37.8% and 71.9% in 10 µM (B), 50 µm (C) and 100 µm (D) δ-cadinene-treated cells respectively.

Figure 6

δ-Cadinene induced cellular ultrastructure alterations observed by transmission electron microscopy (TEM) in human ovary cancer cells (OVCAR-3). The cells were treated with 0, 10, 50 and 100 μM δ-Cadinene for 48 h. Clear nuclear fragmentation was observed in the treated OVCAR-3 cells. Magnification, × 2,000, Scale bar, 1.5 μm.

Figure 7

Effect of different doses of δ-cadinene on the cell cycle phase distribution of human ovary cancer cells (OVCAR-3) using flow cytometry analysis of the DNA content after staining with propidium iodide. C. Data expressed as mean ± SD from three independent experiments. A-D. Show effect of 0, 10, 50 and 100 µM dose of the compound on cell cycle respectively.

Figure 8

Activation of caspases by δ-cadinene treatment in OVCAR-3 cells. δ-cadinene induced cleavage of pro-caspase-3, -8, -9, and -2 and PARP, and XIAP. OVCAR-3 cells were treated with δ-cadinene at varying doses (0, 10, 50 and 100 µM). Whole cell lysates were subjected to Western blot analysis. Western blot analysis of β-Actin levels was included to show that equivalent amounts of protein were loaded in each lane.