Methanol Extract of Flacourtia indica Aerial Parts Induces Apoptosis via Generation of ROS and Activation of Caspases in Human Colon Cancer HCT116 Cells

The World Health Organization (WHO) reports that about 80% of the population in developing countries relies on the traditional system of medication for primary health care. Traditional medicines in the form of standardized herbal preparations are also gaining popularity in many developed countries (Pal and Shukla, 2003). A broad spectrum of secondary metabolites present in herbs is effective in the treatment and/or prevention of various chronic diseases, including cancer (Chun et al., 2013; Kundu and Surh, 2005; Shukla and Pal, 2004). Colorectal cancer ranks as the fourth most common cancer in men and the third most common in women worldwide, accounting for about 8% of all cancer-related deaths (Kamangar et al., 2006; Moghimi-Dehkordi and Safaee, 2012). A wide variety of plants extracts as well as purified phytochemicals have been shown to prevent colon carcinogenesis (Chun et al., 2013; Kim et al., 2008; Vayghan et al., 2014). Flacourtia indica (Burm. F) Merr. (familyFlacourtiaceae; Bengali Name Baichi, ‘Madagascar plum’ in English) is a medium-sized, bushy, thorny tree available in the rural areas of Bangladesh and India


Introduction
The World Health Organization (WHO) reports that about 80% of the population in developing countries relies on the traditional system of medication for primary health care. Traditional medicines in the form of standardized herbal preparations are also gaining popularity in many developed countries (Pal and Shukla, 2003). A broad spectrum of secondary metabolites present in herbs is effective in the treatment and/or prevention of various chronic diseases, including cancer Kundu and Surh, 2005;Shukla and Pal, 2004). Colorectal cancer ranks as the fourth most common cancer in men and the third most common in women worldwide, accounting for about 8% of all cancer-related deaths (Kamangar et al., 2006;Moghimi-Dehkordi and Safaee, 2012). A wide variety of plants extracts as well as purified phytochemicals have been shown to prevent colon carcinogenesis Kim et al., 2008;Vayghan et al., 2014).
Flacourtia indica (Burm. F) Merr. (family-Flacourtiaceae; Bengali Name -Baichi, 'Madagascar plum' in English) is a medium-sized, bushy, thorny tree available in the rural areas of Bangladesh and India 1 (Kaou et al., 2010). Different parts of this plant have long been used in Ayurvedic medicine. Previous studies have demonstrated the antibacterial (Eramma and Devaraja, 2013) antimalarial (Kaou et al., 2010), hepatoprotective (Nazneen et al., 2009) and anti-inflammatory (Kundu et al., 2013) activities of methanol extract of F. indica (FIM). Moreover, phytochemical analysis of the plant revealed the presence of several bioactive constituents, such as coumarins (Nazneen et al., 2002) and phenolic glycosides (Kaou et al., 2010). Since plant polyphenols possess anticancer properties, the present study was aimed at investigating the possible anticancer activity of FIM in human colon cancer HCT116 cells and to elucidate its underlying mechanisms. One of the hallmarks of cancer is the evasion of tumor cells from apoptosis (Hanahan and Weinberg, 2011). Numerous plant products have been reported to induce apoptosis in various cancer cells, thereby eliciting anticancer properties (Kundu and Surh, 2005). Here, we report that FIM induces apoptosis in human colon cancer (HCT116) cells through the generation of reactive oxygen species (ROS), activation of caspases and the inhibition of proliferative markers.

Preparation of plant extract
The aerial parts of Flacourtia indica were collected from Sripur of Gazipur district of Bangladesh and a voucher specimen has been deposited in the Herbarium of Department of Botany, University of Dhaka, Bangladesh. The plant was sun-dried for fifteen days and then pulverized. The coarse powder (700 g) was extracted with methanol by cold extraction process. All the extracts obtained were filtered off and evaporated to dryness in vacuuo at low temperature and reduced pressure by rotary evaporator. The bioactivity of only the methanol extract was investigated in the present study. FIM was dissolved in dimethylsulfoxide (DMSO) immediately before treatment of cells.

Cell culture and treatment
HCT116 cells were obtained from American Type Culture Collections and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin) at 37°C in a humidified incubator containing 5% CO 2 and 95% air. In all the experiments, cells were seeded at 2×10 5 cells/ml and incubated with FIM at 50-60% confluence. All chemicals were dissolved in DMSO keeping its final concentration at less than 1%.

Cell proliferation assay
The anti-proliferative effect of FIM against HCT116 cells was measured by using a solution of tetrazolium compound 3-(4, 5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl) -2 -(4-sulfophenyl) -2H-tetrazolium, inner salt (MTS) (Promega, WI, USA). Briefly, cells (2×10 3 ) were incubated in triplicate in a 96-well plate in presence or absence of FIM in a final volume of 0.1 ml for different time intervals at 37°C. Thereafter, 20 μl of MTS solution was added to each well and incubated for 60min. The number of viable cells was measured in a 96-well plate at an optical density of 492 nm on a microplate reader (Tecan Trading AG, Switzerland). Cell viability was described as the relative percentage of control.

Annexin V staining
Annexin V staining was performed using FITC-Annexin V staining kit (BD-Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Briefly, cells incubated with or without FIM were washed with PBS and resuspended in binding buffer containing Annexin V and propidium iodide. Flourescence intensity was measured using flow cytometry (BD Biosciences, San Jose, CA, USA).

Western blot analysis
Cells were harvested and lysed with RIPA buffer, and collected protein samples were quantified by using bichinconinic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis was done according to the protocol described earlier (Kundu et al., 2012). Immunoblot membranes were incubated with Super-signal pico-chemiluminescent substrate or dura-luminol substrate (Thermo Scientific, MA, USA) according to manufacturer's instruction and visualized with imagequant TM LAS 4000 (Fujifilm Life Science, Japan).

Measurement of ROS accumulation
Cells were treated with FIM in the presence or absence of NAC for 24h and then loaded with 25 μM of DCF-DA. After incubation for 30 min at 37°C in a 5% CO 2 incubator, cells were washed twice with HBSS solution, suspended in the complete media and were examined under a fluorescence microscope to detect the intracellular accumulation of ROS. Fourescence of oxidized DCF was also measured at an excitation wave length of 480 nm and emission wavelength of 525 nm using a flow cytometer.

Statistical analysis
When necessary, data were expressed as mean ± SD of at least three independent experiments, and statistical analysis for single comparison was performed using the Student's t-test. *p<0.01 and **p<0.001 as compared to control.

Treatment with FIM inhibits cell growth and induces apoptosis in HCT116 cells
We initially examined the effect of FIM on the viability of HCT116 cells by MTS assay. Incubation of cells with FIM (100, 200 or 500 µg/ml) reduced the cell viability in a time-and concentration-dependent manner ( Figure  1A). Annexin V staining of cells treated with indicated concentrations of FIM showed a concentration-and timedependent induction of apoptosis ( Figure 1B).

Effects of FIM on the expression of apoptotic markers
To elucidate the mechanisms underlying growth inhibitory effects of FIM, the expression of several major apoptosis regulating proteins were measured by the Western blot analysis. Caspases are important mediators of apoptosis and contribute to the overall apoptotic morphology by cleavage of various cellular substrates. PARP, a known caspase substrate, is a 116 kDa nuclear protein that is specifically cleaved by active caspase-3 into an 85 kDa (Janicke et al., 1998). Incubation of the HCT116 cells with FIM for 24h increased the expression level of caspase-3, cytochrome c and induced the cleavage of PARP in HCT116 cells ( Figure 2A). As shown in Figure  2B, FIM treatment led to a concomitant decrease in the level of Bcl-2 and Bcl-xl in a concentration-dependent manner, whereas the expression of Bax remained unchanged. In addition, treatment of cells with FIM for 24h decreased the expression of survivin, a cell survival protein known to inhibit apoptosis (Altieri, 2003).

Generation of ROS is important in FIM-induced growth inhibition and apoptosis in HCT116 cells
Accumulation of intracellular ROS induces cell death. We examined the effect FIM on ROS generation. Treatment of cells with FIM generated ROS in concentration dependent manner as revealed by immunofluorescence analysis upon DCF-DA staining ( Figure 3A) as well as by FACS analysis ( Figure 3B). As shown in Figure 4A and 4B, FIM-induced ROS generation was abrogated by pretreatment of cells with NAC ( Figure 4A and 4B). Moreover, cells treated with NAC abrogated FIM-induced apoptosis of HCT116 cells as revealed by Annexin V staining method ( Figure 4C).

Discussion
Extractives of the medicinal plant Flacourtia indica have been reported to exert a broad range of pharmacological activities, such as antimalarial, hepatoprotective, analgesic, diuretic and anti-inflammatory activities (Eramma and Devaraja, 2013;Kaou et al., 2010;Kundu et al., 2013;Nazneen et al., 2009). Phytochemical investigation of the plant has revealed the presence of a wide variety of bioactive phytochemicals including coumarins (Nazneen et al., 2002) and phenolic glycosides (Kaou et al., 2010), of which a coumarin compound scoparone has been reported to possess anti-inflammatory (Jang et al., 2005) and anticancer activities . Because of the causal link between inflammation and cancer, numerous plant extractives as well as purified plant constituents retaining anti-inflammatory properties have been shown to possess anticancer activity (Abdull Razis and Noor, 2013;Sehitoglu et al., 2014). We, therefore, examined the anticancer potential of methanolic extracts of the aerial parts of F. indica in the present study.
Our study revealed that FIM elicited significant cytotoxic effect in HCT116 colon cancer cells in a timeand concentration-dependent manner. In an attempt to elucidate the underlying mechanism of FIM-induced colon cancer cell death, we examined the effect of FIM on cellular apoptotic markers. The findings that the incubation with FIM induced the cleavage of caspase-3 and PARP confirm the ability of this plant extract to induce cancer cell death. Since the activation of caspase-3, which brings Flacourtia indica Induces Apoptosis in HCT116 Cells with ROS about catastrophic degradation of various intracellular proteins, depends on the destabilization of mitochoindrial membrane potential and release of cytochrome c (Earnshaw et al., 1999), our findings that FIM upregulates cytosolic protein expression of cytochrome c indicates that FIM induced apoptosis in a mitochondria-mediated mechanism.
Although the understanding of whether FIM can decrease mitochondrial membrane potential is still unclear, our study provides convincing evidence in support of a mitochondria-mediated cell death induced by this plant extract. It was previously shown that the reduced expression of Bcl-2 leads to the dysfunction of mitochondria resulting in the release of intermembrane protein cytochrome c that functions in activation of caspase-9, which subsequently cleaves procaspase-7 and -3 (Borner, 2003). So the upregulated expression of cytochorome c by FIM may lead to the activation of caspase-9. Moreover, the increased mitochondrial membrane attachment of Bax, a proapoptotic protein, can promote cytochrome c release . We, therefore, examined the effect of FIM on the expression of several Bcl-2 family proteins and found that FIM markedly diminished the expression of antiapoptotic protein Bcl-2 and Bcl-xl without altering that of the proapoptotic protein Bax. Since Bcl-2 is overexpressed in colon cancer (Hasan et al., 2011) and the Bcl-2-mediated inhibition of apoptosis restores the tumorigenecity of spontaneously regressed colon tumors in vivo (Bonnotte et al., 1998), the inhibitory effect of FIM on Bcl-2 and Bcl-xl expression, thus, provides a mechanistic basis of its colon cancer preventive effects. FIM also attenuated the expression of survivin, another cell proliferation marker, which has been reported to inhibit Fas-mediated apoptosis in cancer cells (Asanuma et al., 2004). Thus, the downregulation of survivin expression by FIM suggests that FIM may induce apoptosis by activating Fas-mediated signaling, which merits further investigation.
One of the triggers of mitochondrial dysfunction is the accumulation of intracellular ROS. Many of the alcoholic extracts of medicinal plants are reported to induce ROSdependent apoptosis in cancer cells Yeh et al., 2012). We examined whether FIM can induce ROS generation as a mechanism of its cytotoxic effects. Our finding that FIM induced ROS generation, which was abrogated by pretreatment with ROS scavenger NAC, suggests that the cytotoxic effect of FIM is dependent on its ability to generate ROS. This speculation has been confirmed by the finding that when cells were pre-incubated with NAC, the cytotoxic effect of FIM was reverted.
Despite the remarkable apoptotic effect of FIM, it still remains elusive which specific component(s) are responsible for this effect. However, scoparone, present in F. indica (Nazneen et al., 2002), elicited antitumor activity in DU145 prostate cancer cells via down regulation of Stat3 signaling pathway . Thus, the observed cytotoxic effect of FIM may be partly attributed to scoparone present in this plant. In conclusion, the present study demonstrates for the first time that FIM induced generation of ROS, activation of caspases and downregulation of Bcl-2, Bcl-xl and survivin, thereby inducing apoptosis in HCT116 cells.