Cytotoxicity against the D24 melanoma cells varies among the C. nutans leaves obtained from different locations
This study is the first to show variation in the cytotoxicity among the crude MeOH extracts of
C. nutans leaves collected from 11 different locations in Malaysia, Thailand and Vietnam, with diverse geographical and climatic conditions, against the D24 melanoma cells. Current findings suggest that the geographical origin of
C. nutans may not be a major determinant for its cytotoxicity, but differences in elevation and climatic conditions within a geographical area may contribute to the variation. Elevation and annual mean temperature significantly affected the bioactivity of
C. nutans leaves, suggesting that samples from higher elevations and cooler climates are likely to have a higher cytotoxic effect against the D24 cells than from samples collected from lower elevations and warmer air temperature (Additional file
2: Figure S1).
It has been proposed that environmental factors that influence growing conditions are important because they can interfere with the metabolic pathways in plants and therefore on the total concentration of bioactive compounds, which in turn affects the extent of their bioactivities [
29,
30]. The results of this study are in accordance with previous research, reporting variations in the levels of cytotoxicity of samples of different geographical origins with varying environmental conditions. Ayob et al. [
31] reported that the crude methanol extracts of
Justicia gendarussa (Acanthaceae) leaves from five different locations in Malaysia had different cytotoxic effects on MDA-MB-231 and −468 breast cancer cell lines. In another study, Basar et al. [
32] observed considerable variation in the cytotoxicity of methanol root extracts of
Glycyrrhiza glabra L. (Fabaceae) samples from nine different countries, which were tested against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver hepatocellular carcinoma (HepG2) cell lines.
The crude methanol C. nutans leaf extract exhibits selective cytotoxicity against the D24 melanoma cells
The crude MeOH extract of
C. nutans showed significant cytotoxicity against the D24 melanoma, but not the NHDF cells, which suggests that the extract is selective against cancer cells but not normal cells. The selective cytotoxic effect may be due to the genetic, molecular and biochemical differences in the mitochondria of cancer and normal cells [
33], in this case, D24 and NHDF cells, respectively. Mitochondria generate energy that is needed by the cells in the form of ATP and they are significantly involved in the regulation of apoptosis in the cells [
33]. Cancer cells, in general, have increased metabolic rates compared to their non-tumorous counterparts [
34], which may be related to changes in the mitochondrial TCA cycle [
35]. This altered metabolism may cause tumour mitochondria to be unstable [
36] and therefore, making these cells more sensitive to the
C. nutans crude MeOH extract. However, further studies on the molecular pathways are recommended to gain an insight into the differential effects of the crude MeOH leaf extract of
C. nutans has on the cell death pathways in these cells.
Results of this study are in agreement with previous findings reporting that
C. nutans leaves do have anticancer properties, although different extract and cancer cell line were used in the current study. Yong et al. [
15] tested three crude leaf extracts (chloroform, methanol and aqueous) on different human cancer cell lines i.e. HepG2, neuroblastoma (IMR-32), lung (NCI-23), gastric (SNU-1), colon adenocarcinoma (LS-174 T), HeLa, K-562, and Raji, and found that chloroform extract had the highest antiproliferative effect against the latter two cell lines. Another study by Arullappan et al. [
16] tested three crude leaf extracts of
C. nutans (petroleum ether, ethyl acetate and methanol) on HeLa and K-562 cells, and found that petroleum ether extract had the strongest cytotoxic activity against both cell lines. The cytotoxicity of the
C. nutans crude leaf extracts may be due to the presence of flavonoids, such as
C-glycosyl flavones, which have been shown in
Mimosa pudica (Fabaceae) [
37] and
Isodon lophanthoides var.
gerardianus (Lamiaceae) [
38] to have inhibitory effects on the proliferation of cancer cells. Further work, including bioassay-guided fractionation of the crude leaf extracts, purification and isolation of the bioactive compounds, is necessary to verify the specific compounds responsible for the activity.
The crude methanol C. nutans leaf extract induces apoptosis in the D24 melanoma cells
This study is the first to examine the modes of cell death in the D24 melanoma cells treated with the crude MeOH extract in vitro. Muse cytofluorometric analysis using the characterisation of biochemical features (Annexin V to detect apoptotic cells with expressed phosphatidylserine externalisation on the cell surface and 7-AAD to distinguish dead cells) revealed a significant increase in the percentage of late apoptotic/necrotic and a significant decrease in viable cell populations, particularly at the highest test concentration (2 mg/mL) and longest treatment time (72 h), compared to the untreated controls. Besides, visual assessment of the confocal images of the Annexin V/PI double-stained D24 cells treated with the leaf extract confirmed the presence of late apoptotic/necrotic cells, although a few early apoptotic cells were observed at the lowest test concentration. Therefore, results of this study suggest that the extract induced late apoptotic/necrotic cell death in the D24 cells in a dose- and time-dependent manner.
Although utilisation of Annexin V, 7-AAD and PI is a standard procedure to observe the progression of apoptosis, this method is incapable of distinguishing between late apoptotic and primary necrotic cells, since both groups were positive to Annexin V and 7-AAD/PI [
39], as shown in the Muse cytofluorometric and confocal analyses results. Therefore, to further determine the mode of cell death in D24 induced by the crude MeOH
C. nutans extract, detection of changes in cell morphology using other types of microscopy techniques, including phase-contrast and transmission electron were used.
Optical and electron microscopy have been used to detect morphological changes that occur during apoptosis, although the latter gives better definition of subcellular changes [
40]. Phase contrast images and electron micrographs of the treated D24 cells revealed classical morphological features of apoptosis, including cell shrinkage as a result of condensation of organelles and the density of cytoplasm; chromatin condensation peripherally at the nuclear membrane; fragmented and/or lobulated nucleus; and extensive blebbing of plasma membrane, budding into apoptotic bodies consisting of cytoplasm with tightly packed organelles with or without nuclear fragments [
40‐
43]. Distinguishing apoptosis from necrosis is difficult most of the time, especially using conventional histology, and both events can happen simultaneously depending on factors like the concentration and exposure time of stimulus, the degree of ATP depletion and the availability of caspases [
44]. Nevertheless, some of the typical features of necrosis include cell swelling, highly vacuolated cytoplasm and disrupted cell membrane that becomes permeable, resulting in the release of cellular contents [
40,
42]. However, the treated D24 cells did not show the necrosis features when observed under a transmission electron microscope and hence, results of this study may suggest that apoptosis as the most likely type of cell death in the D24 cells, although the compound or component of the crude MeOH leaf extract of
C. nutans that induced apoptosis is unknown. Further studies are necessary to confirm that the crude MeOH extract activates the apoptotic pathway in these cells.
Several medicinal plant species of the family Acanthaceae have been reported to induce apoptotic cell death in different cancer cell lines. For example,
Justicia spicigera (Acanthaceae) leaves have shown to induce apoptosis in mouse fibroblasts (3 T3), human cervical carcinoma (CALO and INBL) [
45] and HeLa [
46] cells, while crude leaf extracts of
Ruellia tuberosa (Acanthaceae) and
Andrographis paniculata (Acanthaceae) exhibited potent apoptogenic activity on HepG2 [
47] and human oropharyngeal cancer cells (KB) [
48] cells, respectively. It has been described that a synergistic activity of interferon (IFN)-γ and tumour necrosis factor (TNF)-α strongly induces apoptosis in HaCaT keratinocyte cells [
49]. However, Thongrakard and Tencomnao [
50] showed that the crude ethanol
C. nutans leaf extract significantly inhibited the induction of apoptosis by IFN-γ/TNF-α in HaCaT cells. Nonetheless, it should be noted that the stereotype outcome either as apoptosis or necrosis cannot always be expected. This is because induction or inhibition of cell death modes depends on a number of factors, such as the plant species, preparation methods (crude extracts, fractions and isolated compounds), concentrations and exposure durations of stimuli, cell types and the nature of the cell death signal [
44,
51‐
53].