Cytotoxic activity and apoptotic induction of some edible Thai local plant extracts against colon and liver cancer cell lines

Purpose: To evaluate eight edible Thai local plant extracts (Camellia sinensis, Careya sphaerica, Cratoxylum formosum, Eleutherococcus trifoliatus, Ficus auriculata, Persicaria odorata, Schima wallichii, and Vaccinium sprengelii) against colon and liver cancer cell lines. Methods: The 80 % ethanol plant extracts were screened for cytotoxic activity against human colon adenocarcinoma (HT-29) and human hepatocellular carcinoma (HepG2) cells by MTT assay. The 50 % cytotoxic activity concentration (CC 50 ) was then determined. Apoptotic cell death was observed by inverted microscopy and DNA fragmentation using agarose gel electrophoresis. Results: P. odorata and S. wallichii extracts showed strong cytotoxic activity, with the latter exhibiting more potent cytotoxic activity than the former. The CC 50 value of S. wallichii extract was 453 and 367 µg/mL against HT-29 and HepG2 cells, respectively. In contrast, P. odorata extract showed CC 50 value of 775 µg/mL against HT-29 and 1665 µg/mL against HepG2 cells. Microscopic observations indicate that the degree of morphological changes was concentration-dependent. The cell lines treated with both plant extracts displayed apoptosis. Conclusion: The two plant extracts have high potentials for medicinal use in colon and liver cancer management. However, further studies are needed to isolate the active compounds responsible for the cytotoxic activities.


INTRODUCTION
The numbers of cancer patients have increased every year and cancer is the second cause of death worldwide. This non-communicable disease is characterized by uncontrollable proliferation of abnormal cells. It is accompanied by uncontrolled development of cells, which have a tendency to multiply, and in some cases, to spread into surrounding tissues. Among various types of cancer, colorectal cancer and hepatocellular carcinoma are reported to be the
To alleviate these side effects, novel or alternative measures have been sought for drugs as treatments for cancer patients. One possible approach is the use of natural product therapy, a widely accepted alternative for cancer treatment. Because this therapy is based on plants or plant extracts, it usaully has less side effects, and lower costs for treatment.
Recent studies indicated that some medicinal plants may have anticancer effects. Hajiaghaalipour et al [2] found that Camellia sinensis extract exhibited anti-proliferative effects on HT-29 cells. Devika and Mohandas [3] reported that extracts of Foeniculum vulgare induced apoptosis in cervical cancer cells and had anti-proliferative effects through DNA fragmentation. Manapradit et al [4] showed that butanolic leaf extracts from Barleria strigosa exhibited the highest cytotoxicity against the P-388 cell line with a CC 50 of 127.42 µg mL.

Many plants growing in Thailand, including
Camellia sinensis, Careya sphaerica, Cratoxylum formosum, Eleutherococcus trifoliatus, Ficus auriculata, Persicaria odorata, Schima wallichii, and Vaccinium sprengelii have been reported to have high polyphenol content and antioxidant activities [5]. Therefore, they were selected to investigate their cytotoxic and apoptotic abilities in colon and liver cancer cell lines.

EXPERIMENTAL Plant materials and reagents
All plant materials were harvested from May to August in 2014 (Table 1) -2,5-diphenyltetrazolium bromide were purchased from Gibco, (El Paso,TX, USA). The blood DNA extraction kit and DNA markers were purchased from Vivantis, (Oceanside, CA, USA).

Preparation of plant extracts
Samples were dried at 50 o C in a tray dryer until the moisture content was below 10 %. After cutting the dried sample into approximately 1 cm lengths, it was ground to a powder. The sample powder (25 g) was blended with 125 mL of 80 % aqueous ethanol and extracted twice.
The samples were first shaken at room temperature for 8 h and then filtered; the residues were extracted again using a ratio of 1:3 (dried sample : solvent) for an additional 8 h. The extracts were combined and filtered using a 0.45 µm filter and then concentrated in a rotary evaporator. Dry crude extracts of 0.2 g were dissolved in 1 mL of 100 % dimethyl sulfoxide (DMSO) and 9 mL of phosphate-buffered saline (PBS) was added to give a final concentration of 20 mg/mL. The samples were kept at -20 o C until use.

Cell culture
Colon cancer (human colon adenocarcinoma, HT-29) and liver cancer (human hepatocellular carcinoma, HepG2) were obtained from the National Cancer Institute in Thailand. Cells were grown in medium (RPMI 1640 containing 8 % FBS) and gentamycin at 50 µg/mL). The cells were cultured at 37 o C in humidified air with 5 % CO 2 .

MTT proliferation assay
The MTT assay has been widely used as an indirect measure to determine the viability of cells. Itis a rapid and highly accurate colorimetric test that measures the decrease in the conversion of the MTT reagent (yellow colour) by mitochondrial succinate dehydrogenase. The MTT passes into the cell mitochondria membrane, and viable cells transform the yellowcoloured MTT to a purple-coloured formazan crystal. The MTT assay followed the method of Mosmann [6] with some modifications. HT-29 and HepG2 cell lines were grown overnight in 96well tissue culture plates with 1x10 5 cells per well. Then, the extract at a final concentration of 2,000 µg/mL was added for cytotoxic screening. The cells treated with mitomycin C (cytotoxic drug) at 50 µg/mL were used as a positive control while negative control cells were treated with 0.2 % DMSO. Untreated cell cultures were used as a control and blank wells contained 100 µL of medium without cells. The cells were cultured at 37 o C for 21 h under 5 % CO 2 atmosphere. Then, 50 µL of MTT (2 mg/mL) was added to each well for further incubation for 3-4 h. MTT crystals were solubilized with 100 µL of a mixture of DMSO and absolute ethanol (ratio 1:1). The absorbance of each well was measured at 570 nm using a microplate reader. The percentage of cytotoxicity was determined using Eq 1.
where As, Ab and Ac were the absorbances of the treated sample, blank, and control samples, respectively.
Plant extracts were selected and tested for the 50 % cytotoxic concentration (CC 50 ).The final concentrations were 250, 500, 1,000, 2,000, and 4,000 µg/mL. The CC 50 values for growth inhibition were computed using GraphPad Prism 5 (La Jolla, CA, USA). All experiments were performed in triplicate.

Morphological analysis
HT-29 and HepG2 cells were treated with extracts at concentrations of 500 and 4,000 µg/mL for 24 h. Upon completion of incubation, morphological changes of cells were monitored under an inverted light microscope at 200x magnification.
Apoptotic DNA ladder assay DNA fragmentation analysis was carried out using agarose gel electrophoresis as described by Herrman et al [7] with some modifications. The HT-29 and HepG2 cells were treated with each extract and then incubated for 48 h. The treated cells were harvested by trypsinization, washed twice with PBS, and then used for DNA isolation. DNA extraction was performed using a blood DNA extraction kit as indicated in the manufacturer`s guidelines. Electrophoresis was conducted at 100 V for 30 min. The agarose gel was stained with ethidium bromide for 10 min and then rinsed in distilled water for another 10 min. The DNA bands were photographed under UV illumination. A 1 kb DNA marker was used to estimate the size of the DNA fragments.

Statistical analysis
Each experiment was run in triplicate. The results were reported as the mean ± SD. Analysis of variance was performed by ANOVA tests and significant differences between means of cytotoxicity were p < 0.05 using IBM SPSS software, version 24 (IBM Singapore Pte. Ltd., Changi, Singapore).

Cell proliferation
All the plant extracts exhibited different antiproliferative activity against HT-29 and HepG2 cells ( Table 2). The plant extracts showed higher cytotoxicity for HT-29 cells than the positive control or the cytotoxic drug, mitomycin C, at 50 µg/mL. P. odorata and S. wallichii extracts showed strong anticancer activity, particularly against HT-29 cells with cytotoxicity levels of more than 50 % cell death. In the case of the HepG2 cells, no plant extracts showed stronger anticancer activity than the positive control. However, extracts from three species inhibited HepG2 cell growth relatively effectively: V. sprengelii, P. odorata and S. wallichii.  The MTT assay data indicated that the extracts from S. wallichii and P. odorata possessed strong anti-proliferative activity in both cell lines. Therefore, they were selected for further studies to determine the CC 50 . The level of cytotoxicity generally increased gradually with increasing concentrations of the extracts (Figure 1). The S. wallichii extract had greater cytotoxic activity than the P. odorata extract in both cell lines. The CC 50 of the S. wallichii extract on HT-29 cells was 453 µg/mL, and 367 µg/mL for HepG2 cells. The CC 50 of the P. odorata extract on HT-29 cells was 775 µg/mL and 1,665 µg/mL for HepG2 cells. The CC 50 of the S. wallichii extract was 1.7 and 4.5 times greater than that of P. odorata extract against HT-29 and HepG2 cells, respectively. The results indicate that the S. wallichii extract had significantly greater cytotoxicity than the P. odorata extract against both cell lines.

Morphological changes
The effects of S. wallichii and P. odorata extracts on cell morphology of HT-29 and HepG2 cells were observed using inverted light microscopy. No morphological changes were observed in control cells; however, both extract treatments resulted in morphological changes in a concentration-dependent manner (Figure 2). The two extracts (500 µg/mL each) induced changes in morphologies of HT-29 and HepG2 cells indicating an early stage of apoptosis, including cell shrinkage. The cytoplasm was denser and the shape more tightly packed in the treated cells. As the concentration of extracts increased to 4,000 µg/mL, the loss of cell adhesion, reduced cell density, and membrane blebbing occurred.

Apoptotic DNA ladder
DNA fragmentation is an important characteristic of apoptosis. Prior to the fragmentation of the nucleus, condensation and degradation of chromatin/DNA were observed. Cheung et al [8] reported that the first marker in the apoptosis process was DNA laddering, which is a sign of cell death. Following chromatin condensation, caspase activated DNase (CAD) degrades DNA and caspase-3-treated cell lysates are observed [9]. Figure 3 (A and B) shows that the extracts induced DNA fragmentation in HT-29 and HepG2 cells. Cells treated with S. wallichii and P. odorata extracts showed a smear pattern when compared with the DNA ladder; in contrast, the smear pattern of damaged DNA was not observed in control cell samples. Apoptosis was therefore induced by the extracts in HT-29 and HepG2 cells.
Both extracts showed a concentration-dependent pattern of DNA fragmentation. In the case of HT-29 cells, S. wallichii extract was more effective than P. odorata extract ( Figure 3A). Conversely, with HepG2 cells the P. odorata extract was more effective than the S. wallichii extract ( Figure  3B). As a positive control, mitomycin C (100 µg/mL) caused more DNA fragmentation in HepG2 cells compared to HT-29 cells than the two extracts.

DISCUSSION
The potent biological activities of plant extracts are associated with their phytochemical constituents. P. odorata, S. wallichii, and V. sprengelii have been reported to have high polyphenol content [5]. Polyphenols induce proapoptotic properties. Ramos [9] described the pro-apoptotic effects of dietary polyphenols on various human cancer cell lines including colon, prostate, lung, breast cancer and leukaemia.
The major phytochemicals of S. wallichii include saponins and tannins [10]. Saponins, triterpenoid glycosides, show different biological activities and have the potential for pharmaceutical applications [11]. They reportedly prevent the proliferation of cancer cells [12]. Hu et al [13] identified a compound, Nigella A, which was extracted from Nigella glandulifera (Nepenthaceae) and reported to be the major triterpene saponins.  [14].
Flavonoids were reported to be the main phytochemical responsible for anticancer activity in P. odorata [17]. The biological activities of flavonoids include anti-inflammatory, antispasmodic, and anti-allergic activities as well as protective activities for hepatic and vascular disorders [18]. Flavonoids also show anticancer activity in various cancer cells. For example, (+)catechin and (-)-epicatechin play a role in protecting liver cancer cells from DNA damage against N-nitrosodimethylamine, Nnitrosopyrrolidine and benzo (a) pyrene [19].
The CC 50 showed that the S. wallichii extract was more effective against HepG 2 cells than HT-29 cells (Figure 1), which indicated that the phytochemical in S. wallichii extracts was more sensitive and specific for HepG2 than HT-29 cells. In addition, the anticancer activity of phytochemicals mainly depends on their multitarget mechanisms of action, including antimutagenic, antioxidant and, anti-proliferative activities [20].
The cytotoxic activity assay of crude extracts showed potential against cancer cell lines, which were in accordance with Momtazi-Borojeni et al [21], that crude methanol extract of Avicennia marina potentially inhibited the viability of MDA-MB 231 cells (human breast cancer cell) with a CC 50 value of 250 µg/mL. For the rhabdomyosarcoma (RD) tumour cell line and murine fibroblast (L20B) cell line, the crude methanol extract of Nicotiana tabacum showed CC 50 values of 2,100 µg/mL for RD and 2,150 µg/mL for L20B cells after 72 h [22].
The morphological changes increased as the extract concentration increased, and were characteristics of apoptotic cell death. Apoptosis can occur via two routes, either extrinsic (activation of death receptors) or intrinsic (mitochondrial-mediated) pathways. In this study, increasing the concentration of the extracts was expected to induce the intrinsic pathway. The intrinsic signaling pathway activation occurs first, following by mitochondrial outer membrane permeabilization resulting in the release of prodeath factors into the cytosol [23].
P. odorata extract more effectively caused DNA fragmentation than the S. wallichii extract at both concentrations. The phytochemicals in the P. odorata extract may activate the caspase family of proteases better than the S. wallichii extract, and eventually lead to the degradation of chromosomal DNA in HepG2 cells.
P. odorata extract therefore has a higher potential for DNA fragmentation than S. wallichii extract in HepG2 cells. Increasing the concentration of P. odorata and S. wallichii extracts resulted in the typical DNA laddering in agarose gels, due to activation of caspase enzyme activity and oxidative stress in cells as observed in both cell lines.
The results using S. wallichii extract were similar to those of Halimah et al [24] who showed that the major compound (kaempferol-3-Orhamnoside) in the ethylacetate fraction of. S. wallichii inhibited MCF-7 cell growth via activation of caspase-9 and caspase-3, inducing apoptosis.
The effect of P. odorata extract on HepG2 cells regarding the fragmentation of DNA was opposite to the CC 50 observed from MTT results (Figure 1). S. wallichii extract was more effective than the P. odorata extract. The MTT assay is an indirect measurement of mitochondria effects. The plant extracts inhibited succinate dehydrogenase in the mitochondria, which could hinder reactions with MTT reagent to form formazan crystals. However, DNA fragmentation is associated with caspase-3-mediated cleavage releasing caspase activated DNase (CAD) and resulting in degradation of DNA into oligonucleosomal fragments [25].

CONCLUSION
The results of this study demonstrate that all the plant extracts examined in this study exert cytotoxic activity against colon cancer cells. Of the eight plant species studied, P. odorata and S. wallichii extracts possess the highest antiproliferative activity against colon and liver cell lines, and they also induce apoptosis by DNA fragmentation, thus demonstrating their potentials as anticancer chemotherapeutic agents. Further studies, however, are needed to isolate their active compounds.