In vitro ultramorphological assessment of apoptosis induced by zerumbone on (HeLa)

Abstract

Zerumbone (ZER), a potential anticancer compound, isolated from the fresh rhizomes of Zingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa), breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining), scanning and transmission electron microscopy (SEM and TEM), and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest IC(50) of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test) of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

Figure 1

Chemical structure of zerumbone (molecular weight: 218.34).

Figure 2

The colorimetric assay of caspase-3 in human cervical cancer cells treated and untreated with ZER (IC₅₀) for 72 hours. Cells were cultured in RPMI 1640 (75 mL flask) media maintained at 37°C and 5% CO₂. Independent t-test showed a significant difference (*P < .05) between treated and untreated cells in the activity of caspase-3.

Figure 3

Fluorescent micrograph of acridine orange and propidium iodide double-stained human cervical cancer cells lines (HeLa). HeLa was treated at IC₅₀ of ZER at time-dependent manner. Cells were cultured in RPMI 1640 media (25 mL flask) maintained at 37°C and 5% CO₂. (a) Untreated cells showed normal structure without prominent apoptosis and necrosis. (b) Early apoptosis features were seen after 24 hours representing intercalated acridine orange (bright green) amongst the fragmented DNA. (c) Blebbing and nuclear margination were noticed in 48-hour treatment of ZER. (d) Late apoptosis was seen in 72 hours incubated cells, whereby positive staining with orange color represents the hallmark of late apoptosis (magnification 400X).

Figure 4

Percentages of viable, apoptotic, and necrotic cells after ZER treatment. HeLa cell death via apoptosis increased significantly (*P < .05) in time-dependent manner. However, no significant (P > .05) difference was observed in the cell count of necrosis. Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C and 5% CO₂.

Figure 5

SEM micrographs of surface ultrastructural characteristics of HeLa cells treated with ZER in time-dependent manner (0, 24, 48, and 72 hours) and cultured in RPMI 1640 media maintained at 37°C and 5% CO₂. (a) The characteristic of untreated HeLa cells surface showing the restoration of a typical morphological feature of a cancer cell such as numerous microvilli (as shown in grey arrow) with several membrane connections. (b), (c), and (d) ZER-treated HeLa cells (24, 48, and 72 hours, IC₅₀: 20.41 μM) showed distinct morphological changes corresponding to typical apoptosis, including cell membrane blebbing (b), microvilli disappearance or reduction (blunt microvillus (s)), and separated apoptotic bodies (X2500).

Figure 6

(a) Electromicrograph of untreated human cervical cancer cells (HeLa) demonstrates the normal structure of HeLa cancer cell. Nucleus (N), nucleolus (NL), and the cytoplasm appeared without abnormal changes (X4600). (b) ZER-treated (24 hours) human cervical cancer cells (HeLa) demonstrate morphological features of early apoptosis: cell shrinkage, chromatin condensation (arrow), and integrity of plasma membrane (X6000). (c) ZER-treated human cervical cancer cells (HeLa) demonstrate the condensed cristae of mitochondria (MC) as a typical morphological feature in apoptosis (arrow) (X27500). (d) ZER-treated (48 hours) human cervical cancer cell line (HeLa) demonstrates morphological features of intermediate apoptosis: cell shrinkage, chromatin condensation (white arrow), and membrane blebbing (small white arrow) (X6000). (e) ZER-treated (72 hours) cervical cancer cells (HeLa) demonstrate morphological feature of late apoptosis: nuclear collapse, continuing blebbing, and apoptotic body formation (arrow) (X10 000). Cells were cultured in RPMI 1640 (25 mL flask) media maintained at 37°C and 5% CO₂.