Background
Colorectal cancer or colon cancer is defined as an uncontrollable cell growth at the lower part of the digestive system that is the large intestine [
1]. Colorectal cancer is one of the main causes to high mortality and morbidity rate of cancer. Moreover, colorectal cancer is more frequent in the industrialized world than in developing countries [
2]. Colorectal cancer falls in second highest mortality and morbidity rate followed by lung cancer, gastric cancer and breast cancer. In Malaysia, colorectal cancer mostly occurs in male and third in frequency among females. Colorectal cancer is common among the age group of 50 years old and above [
3].
Plant derived anti-cancer agents play an important role in cancer chemotherapy [
4]. Additionally, plant derived anti-cancer agents are known to be safer and give less side effects when in comparison with those synthetic anti-cancer agents available [
5].
Canarium odontophyllum Miq. is a type of plant that is known as ‘dabai’ or ‘Borneo olive’. It can be found in Sarawak, Malaysia especially in Sibu, Sarikei and Kapit [
6]. It belongs in the Burseraceae family and
Canarium L. genus [
7]. The fruit of
C. odontophyllum is oval with a purplish skin and has a single seed along with a hard and thick endocarp [
8]. Almost all parts of the plant were tested for medical researches including the fruit, peel, shell of the seed, pulp, leaf and stem bark. The pulp extract from
C. odontophyllum fruit was found to inhibit the growth of
Candida glabrata [
9]. The leaf and shell extracts from
C. odontophyllum were shown to have antimicrobial activity against a wide range of pathogenic bacteria [
10] whereas both the leaf and stem bark of
C. odontophyllum demonstrated promising anticancer property [
11]. However, previous study merely reported preliminary screening of cytotoxic activity against human colorectal carcinoma HCT 116 cell line attributed to the presence of flavonoid, tannin, saponin, terpenoid and phenolic compound [
12]. Damage to DNA always occurs from endogenous and exogenous agents such as reactive oxygen species (ROS) from cellular metabolism and ultraviolet light from the sun [
13]. Chemical carcinogens, radiation and genotoxic anti-cancer agents can cause DNA damage [
14]. When there is DNA damage, the damage itself will cause cell cycle arrest where it can lead to DNA repair or cell death via apoptosis [
15]. Therefore the objective of the present study is to investigate the mechanism of cell death and to determine the genotoxic effect of extracts from the stem bark of
C. odontophyllum against HCT 116 human colorectal cancer cell line.
Discussion
The search for a potential anticancer agent has been a challenge for scientists with regards to existence of side effects and drug resistance. Plant-derived anticancer agents are likely to be more effective in the cancer treatment [
19]. In this study, evaluation of cytotoxic and genotoxic effects of extracts from stem bark of
C. odontophyllum against human colorectal cancer cell HCT 116 was done and assessment of cytotoxic effect of acetone extract from stem bark of
C. odontophyllum against normal colon cell CCD-18co was carried out.
From the MTT assay, acetone extract showed a higher cytotoxic effect compared with methanol extract whereas aqueous extract was not cytotoxic at 24 h of treatment. In the previous study, the same pattern of data was seen by all three extracts but at much higher IC
50 values [
10]. This may be because of the difference in the range of concentration used. The range of the concentration used in this study was smaller compared to the range of concentration used in the previous study. When HCT 116 cells were treated for 48 h, the acetone extract again displayed the lowest IC
50 value in contrast with methanol and aqueous extract. The IC
50 values of all three extracts obtained in 48 h were lower than the IC
50 values in 24 h and this indicated that the extracts gave a time-dependent cytotoxic activity. The longer the exposure of treatment, the lower the IC
50 value and the higher is their toxic effect [
20]. However, when the treatment was prolonged to 72 h, the same profile was observed whereby acetone exerted the lowest IC
50 value. Although the IC
50 values obtained in 72 h were slightly higher than the IC
50 values in 48 h, there was no significant difference (
p > 0.05) indicated between these values.
From the present study, the methanol and acetone extract at 200 μg/ml produced higher percentage of cell viability than at 100 μg/ml. Exogenous antioxidants that contain in the extract may give both good and bad outcome in redox condition. In addition, a few studies showed that exogenous antioxidants gave debatable results especially at high doses. The type, concentration and matrix of exogenous antioxidant from a natural compound are the features that affect the balance of the benefits [
21].
Out of all three extracts, acetone extract exhibited the most potent cytotoxic activity with a significantly higher cytotoxic effect than aqueous extract but the cytotoxicity between acetone and methanol extract showed no significant difference. This finding correlates with the previous study which reported that acetone extract from the stem bark of
C. odontophyllum [
12] displayed the highest cytotoxic effect against HCT 116 cells and was also in agreement with acetone extract from the leaves of
C. odontophyllum [
11]. The cytotoxic activity of a plant against cancer cells is based on their phytochemical properties [
22]. The phytochemical screening of aqueous, methanol and acetone extracts from stem bark of
C. odontophyllum showed that all three extracts contain flavonoid, saponin, tannin, terpenoid and phenolic compounds [
12]. The potent cytotoxic effect exerted by acetone extract may be caused by non-polar terpenoid compound. For example, a few terpenoid derivatives from
Rhizoma curcumae were found to have antiproliferative properties against cancer cell lines [
23]. Polyphenolic compounds such as flavonoid may also contribute to the antitumor activity. Flavonoids are known to have beneficial biological effects that include anti-inflammatory, anti-allergic, antimicrobial, anticarcinogenic and antitumor effects [
24]. Based from the results obtained, acetone extract was chosen to proceed with the rest of the experiments as it is considered to be the most potent out of all extracts tested.
In general, effective anticancer agents have to execute or halt the cancer cells to live and at the same time will not exert any toxic effect towards the normal cells [
25]. As example, extract from
Eugenia jambolana Lam. are able to inhibit the growth of breast cancer cell line MCF-7aro and MDA-MB-231 but did not inhibit the growth of normal breast cells MCF10A [
26]. Generally, antitumor agents show cytotoxicity against cells with higher growth activity by mechanisms such as the inhibition or suppression of increasing nucleic acid synthesis and metabolic pathways than in normal cells [
27]. Selective killing towards cancer cells can be achieved by anticancer agents because the characteristics of cancer cells are not the same as the normal cells. Cancer cells are said to be under pressure and are destined to die. They depend highly on abnormalities of apoptosis signalling pathways to stay viable [
28]. However, when treatment of acetone extract from stem bark of
C. odontophyllum was prolonged to 72 h, CCD-18co cells proliferated at the lower doses. This may be due to the presence of compounds that contribute to the mitogenic activity of the cells [
29].
Although the IC
50 value obtained for CCD-18co cells was higher than the IC
50 obtained for HCT 116 cells, the toxicity of menadione are general to both cells with its Selective Index (SI) value of 2.5. The SI can be achieved by calculating the ratio of IC
50 value of normal cell and IC
50 value of cancer cell. SI values that are higher than three are considered as selective toxicity which means that the compound gives selective toxicity to cancer cells but gives no harm or minimal toxicity to normal cells [
30]. Hence, we can say that acetone extract from stem bark of
C. odontophyllum exhibited selective toxicity towards colorectal cancer cell line HCT 116.
Mode of cell death assessment of acetone extract from stem bark of
C. odontophyllum using flow cytometry Annexin V-FITC/PI labelling assay revealed that the primary cell death of HCT 116 cells was via apoptosis after 48 h treatment. Our findings demonstrated an increase of apoptotic cells and a decrease of viable cells with increasing concentration of acetone extract from stem bark of
C. odontophyllum. It is said that plant-derived polyphenolic compounds act as antitumor compounds and have apoptosis-inducing properties in cancer cells [
31]. Based on the previous study [
10], acetone extract from stem bark of
C. odontophyllum was found to have phenolic compounds and other phytochemicals such as saponin, terpenoid and tannin. A study found that acetone extract from stem bark of
Cephaltaxus griffithii Hook f. induced apoptosis towards HeLa cells [
32]. In addition, the mode of cell death of HL-60 cells after treatment with ethyl acetate extract from stem bark of
Cudrania tricuspidata was via apoptosis [
33]. Apoptosis was also the primary cell death of HCT 116 cells after treatment with ethanol extract from sporophyll of
Undaria pinnatifilda [
34].
The development of plant derived anticancer drug plays a vital role in the treatment of cancer [
35]. Many synthetic drugs such as alkylating and antimetabolite agent can affect normal cell and produce side effects to cancer patient [
36]. Apoptosis or programmed cell death has been the aim for treatment of cancer at many level of tumour development [
37]. During apoptosis, apoptotic bodies undergo phagocytosis and will not submit itself to the inflammation process and may not cause disturbance towards nearby cell [
38]. Apoptosis signalling pathways are divided into two mechanisms which are those that involved the mitochondria known as the intrinsic pathway or those that signals through death receptors namely the extrinsic pathway [
29]. The specific pathway of apoptosis induced by acetone extract from stem bark
C. odontophyllum is not fully investigated and understood yet.
Based from the outcomes of MTT assay and flow cytometry Annexin V-FITC/PI assay, the percentage of viable cells obtained were different in these two approaches. This may be due to the difference in the endpoint measurement of these assays [
39]. MTT assay is based on the involvement of active mitochondria in living cell to produce succinate dehydrogenase enzyme in order to reduce MTT salt to formazan whereas flow cytometry Annexin V-FITC/PI assay is based on the detection of exposed phosphatidylserine on the outer part of the membrane in dead cells [
16,
17]. Besides that, MTT assay can only measure living cell but not dead cell. Flow cytometry Annexin V-FITC/PI assay is able to measure percentage of both living and dead cell in a known amount of cell. The differences between these two assays may be contributing to the deviation of result obtained.
Cytotoxic effect induced by acetone extract from stem bark of
C. odontophyllum towards HCT 116 cells may be caused by DNA damage and the detected genotoxicity might be the early mechanism of cell death via apoptosis. Alkaline comet assay was used to detect genotoxic effect of tested compound by measuring its DNA damage at single cell that can be observed under a fluorescent microscope whereas a comet head (nucleus) and its tail (DNA fragments) can be seen [
40]. The concentrations used in this study were at IC
10 and IC
25 values that were obtained from the graph of cell viability versus concentration for acetone extract. A lower and non-cytotoxic concentration was used to dodge any false positive result of dying or dead cells [
41]. Among the frequently used comet parameters, percentage of DNA in tail and tail moment could offer the most precise result for the degree of damage [
42].
In this study, IC
10 and IC
25 of acetone extract from stem bark of
C. odontophyllum showed significant (
p < 0.05) DNA damage in HCT 116 cells after 30 min of treatment. DNA is the key target by most cytotoxic anticancer drugs whether it acts directly through reactive metabolites or indirectly through the incorporation into DNA nucleotide analogues or by blockade of DNA-metabolizing functions such as DNA polymerase or topoisomerase. Cancer cells divide more repeatedly than normal cells and this cell division becomes the aim for anticancer agents where the most vital cell cycle phase is DNA replication. Most anticancer agents cause highly damaged-DNA in cancer cells and these cytotoxic agents have different mechanism of action and different types of DNA lesion [
43]. Damage may cause disruption of transcription or replication of the DNA where it can lead to cell death or aging [
44]. An example of genotoxic agent that binds to DNA and abrupts the replication is doxorubicin. Doxorubicin binds to DNA through intercalation between specific bases and thus prevents DNA synthesis [
45]. Nonetheless, the mechanism and type of DNA lesion of HCT 116 cells induced by acetone extract from stem bark of
C. odontophyllum need further investigation to truly understand the mechanism of DNA damage that leads to apoptosis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DFB designed, analysed and supervised the study. CKM supervised the study. ZA performed all the experiments, data analysis and drafted the manuscript. All authors read and approved the final manuscript.