Stigmalactam from Orophea Enterocarpa Induces Human Cancer Cell Apoptosis Via a Mitochondrial Pathway

Orophea enterocarpa is in Annonaceae family, found endemically in southern and eastern parts of Thailand. For other species of Orophea, O. uniflora is endemic in India whereas O. palawanensis is found in Philippines. Various bioactive alkaloids are extracted from medicinal herbs such as nitidine chloride from Zanthoxylum nitidum with anticancer effect against breast cancer cells (Sun et al., 2014). N-norchelerythrine and dihydrosanguinarine, from Broussonetia papyrifera (L.) Vent. (Moraceae), a traditional Chinese medicinal herb, are cytotoxic to human cervical cancer HeLa and hepatocellular carcinoma BEL7402 cells (Pang et al., 2014). Camptothecin, a potent alkaloid drug with an anticancer effect, is extracted from the bark and stem of Camptotheca acuminata, which contains the mechanism of action to inhibit topoisomerase I activity (Efferth et al., 2007; Ulukan and Swaan, 2002). There has been a report of aqueous extract of C. acuminata fruit containing antitumor effects on human endometrial carcinoma cells via the accumulation of cyclin-A2 and -B1, then activation of caspase-3 and -7, similar to the effect of camptothecin (Lin et al., 2014). There are various attempts to search for a new source of camptothecin from other kinds of plants. There has been


Introduction
Orophea enterocarpa is in Annonaceae family, found endemically in southern and eastern parts of Thailand.For other species of Orophea, O. uniflora is endemic in India whereas O. palawanensis is found in Philippines.Various bioactive alkaloids are extracted from medicinal herbs such as nitidine chloride from Zanthoxylum nitidum with anticancer effect against breast cancer cells (Sun et al., 2014).N-norchelerythrine and dihydrosanguinarine, from Broussonetia papyrifera (L.) Vent.(Moraceae), a traditional Chinese medicinal herb, are cytotoxic to human cervical cancer HeLa and hepatocellular carcinoma BEL-7402 cells (Pang et al., 2014).Camptothecin, a potent alkaloid drug with an anticancer effect, is extracted from the bark and stem of Camptotheca acuminata, which contains the mechanism of action to inhibit topoisomerase I activity (Efferth et al., 2007;Ulukan and Swaan, 2002).There has been a report of aqueous extract of C. acuminata fruit containing antitumor effects on human endometrial carcinoma cells via the accumulation of cyclin-A2 and -B1, then activation of caspase-3 and -7, similar to the effect of camptothecin (Lin et al., 2014).
There are various attempts to search for a new source of camptothecin from other kinds of plants.There  a first report of camptothecin alkaloids from Meliaceae family, i.e., Dysoxylum binectariferum bark, to be a source for camptothecin production.Rohitukine, camptothecin and 9-methoxy-camptothecin can be isolated from D. binectariferum (Jain et al., 2014).
As mentioned, some alkaloids from Broussonetia papyrifera fruits containing isoquinonline alkaloids, viz., N-norchelerythrine and dihydrosanguinarine; are cytotoxic to human cancer cells, whereas nitidine, broussonpapyrine and chelerythrine from the same plant are also toxic to non-cancer cells.This leads to further modification of structure-activity relationship to achieve anticancer effect without cytotoxicity to normal cells (Pang et al., 2014).
Stigmalactam, a known alkaloid isolated from Orophea enterocarpa, is cytotoxic to human colon adenocarcinoma (HCT15) cell line with IC 50 concentration of 1.32 µM (Nayyatip et al., 2012).However, the mechanism of cancer cell cytotoxicity remains elusive.The aims of this study were to investigate the mode of cancer cell death and the mechanism involved.The human hepatocellular carcinoma HepG2 and human breast cancer MDA-MB-231 cell lines were used in the present study as models for anticancer study.It was found that stigmalactam was cytotoxic to both cancer cell lines and induced HepG2 to undergo apoptosis with the reduction of mitochondrial transmembrane potential and the induction of caspase-9 and -3 activities.However, the compound acted as anti-oxidant in the HepG2 cells with the evidence of decreased 2',7'-dichlorofluorescein fluorescence intensity employing flow cytometric technique, implying the response to apoptotic death induction.

Plant materials
The twigs and leaves of Orophea enterocarpa were collected from Prajeenburi Province, Thailand, in March 2009.The plant was identified by Narong Nuntasaen, with the voucher specimen (BKF no.151499), deposited at the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok, Thailand.
Stigmalactam was obtained from the following procedure.Air-dried twigs and leaves (2.7 kg) were ground and extracted according to the protocol (Nayyatip et al., 2012).Briefly, the extraction was performed with hexane and subsequently with methanol-dichloromethane (3:1) successively.The crude methanol-dichloromethane extract (211.18g) was separated by using silica gel chromatography technique.The gradient elution was conducted with hexane, enriched with ethyl acetate, followed by gradually increasing amount of methanol in ethyl acetate and finally with methanol to obtain eleven fractions, F1-F11.Then fraction F4 (16.6 g) was rechromatographed by silica gel with the gradient system using hexane, ethyl acetate and methanol to afford 4 subfractions, A1-A4.The subfraction A3 (0.38 g) underwent the purification process to obtain four subfractions, B1-B4.The yellowish precipitate in subfraction B2 was crystallized from ethanol to give a purified compound, stigmalactam (0.07 g), and the structure is shown in Figure 1.

Cell lines
Human

Cell culture and treatment
HepG2, MDA-MB-231 and NIH3T3 cells (50,000 cells/well) were cultured in the absence or presence of stigmalactam at various concentrations, incubated at 37°C in 5% CO 2 atmosphere for 24 h.The cell viability was determined by employing MTT assay (Wudtiwai et al., 2011).Briefly, sterile MTT solution (stock solution of 5 mg/ml) was added to cell suspension at the final concentration of 100 μg/ml and incubated for 4h at 37°C in a humidified 5% CO 2 atmosphere.The medium was then removed and the crystal was dissolved with DMSO for 30 min.The absorbance of the cell lysate was measured at 540 nm with reference wavelength of 630 nm using microtiter plate reader (Biotek, Winooski, VT, USA) at 570 nm.The percentage of cell viability was calculated and 10, 20 and 50% inhibitory concentrations (IC 10 , IC 20 and IC 50 ) were determined and used for further experiments.

Determination of reactive oxygen species (ROS) production
HepG2 cells were treated with stigmalactam at C 10 , IC 20 and IC 50 for 4 h, then the cells were washed with phosphate-buffered saline twice before 2',7'-dichlorohydrofluorescein diacetate (DCFH-DA) was added to the cell suspension with the final concentration of 5 μM.ROS production was determined and analyzed by flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) (Banjerdpongchai and Khaw-On, 2013).

Determination of mitochondrial transmembrane potential
A f t e r H e p G 2 c e l l t r e a t m e n t , 3,3'-dihexyloxacarbocyanine iodide (DiOC 6 ) was added to the media to the final concentration of 40 nM for mitochondrial transmembrane potential (MTP) determination.Cells were analyzed by a FACScan equipped with a 488 nm argon laser using CellQuest software (Becton-Dickinson, Franklin Lakes, NJ, USA).Data were depicted as histograms and analyzed for percentage of cells with loss of MTP (Banjerdpongchai and Wilairat, 2005).

Statistical analysis
All experiments were performed in triplicates and thrice as a representative for the three with the same results.The data were presented with mean±S.D. analyzed with One Way ANOVA and comparison between two groups of data were considered significant when p<0.05, by using Student's t-test.
Purification of extracts from Fissistigma balansae  C.
It has been reported of the cytotoxicity of stigmalactam against colon cancer cell lines (HCT15) (Nayyatip et al., 2012).However, the bioactivities on mode of cell death and death-inducing mechanisms of the compound remain unknown.This study involves the apoptotic inducing activity in liver cancer HepG2 cells of stigmalactam, which is an aristolactam-type alkaloid extracted from Orophea enterocarpa.
Stigmalactam was cytotoxic to human hepatocellular carcinoma HepG2, breast cancer MDA-MB-231 and murine fibroblast NIH3T cells dose-dependently with the IC50 levels of 22.98±2.67,33.23±4.54and 24.44±6.75µM, respectively as shown in Figure 2 and Table 1.The compound is toxic to HepG2 > NIH3T3 > MDA-MB-231 with the range of IC 50 levels between 23-33 µM.Stigmalactam needs to be modified in its structure to make it less toxic to normal cells but still very toxic to cancer cells.It also suggests that the metabolizing enzymes or detoxifying enzymes in the hepatocellular carcinoma HepG2 cells may metabolize the compound (stigmalactam) to be a more active metabolite and make it cytotoxic to liver cancer HepG2 cells more than MDA-MB-231 cells which do not contain metabolizing enzymes.
Due to the cell sensitivity, HepG2 cells were selected for further study of the mechanism of cell death.Stigmalactam could decrease the peroxide radical production in HepG2 cells as shown in Figure 3 significantly.However, apoptotic cells usually generate ROS.The antioxidant activity of the stigmalactam may be induced to respond to the ROS production, which might be a compensatory effect to the stigmalactam treatment or it might be indicated that stigmalactam acted as an antioxidant.However, the decreased ROS might be the response to cell death-inducing effect.
Stigmalactam induced HepG2 cells to undergo apoptosis with the evidence of significant increase in caspase-9 and -3 activities (Figure 4A, 4C), whereas the capase-8 activity did not change statistically significantly (Figure 4B).There was also a reduction of mitochondrial transmembrane potential (MTP) and percentage of cells with MTP loss significantly increased in a dose dependent manner as shown in Figure 5.
In conclusion, stigmalactam was cytotoxic to hepatocellular carcinoma HepG2 and breast cancer MDA-MB-231 cells together with normal fibroblast NIHT3T cells, with HepG2 > NIH3T3 > MDA-MB-231 cells.Stigmalactam induced human hepatocellular carcinoma HepG2 cell apoptosis via the mitochondrial pathway, with the activation of caspase-9 and -3, and a decrease in MTP.The ROS production was decreased in apoptotic cells, indicating the antioxidant activity or cell death effect of stigmalactam.

Figure 5 .
Figure 5. Loss of Mitochondrial Transmembrane Potential (MTP) of Stigmalactam-treated Human Hepatocellular Carcinoma HepG2 Cells.Percentage of treated-cells with loss of MTP was increased in a dose dependent manner.The value compared to control is marked with asterisk, p<0.05

Figure 4 .
Figure 4. Caspase-9, -8 and -3 Activities Induced by Stigmalactam in HepG2 cells.The caspase activities were determined as described in Materials and Methods.(A) Caspase-9, (B) caspase-8 and (C) caspase-3 activities were shown as relative fluorescence intensity compared to control (fold).The significant enhanced activities of caspase-9 and -3 were induced in HepG2 cells compared to control, with asterisk, p<0.05

Table 1 . Cytotoxicity of Stigmalactam on Human Hepatocellular Carcinoma HepG2, Breast Cancer MDA-MB-231 Cells and Murine Fibroblast NIH3T3 Cells with IC 10 , IC 20 and IC 50 of Each Cell Line
hepatocellular carcinoma HepG2 and human breast cancer MDA-MB-231 cells were gifts from Assoc.Prof. Dr. Prachya Kongtawelert at Excellent Center of Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, whereas murine fibroblast NIH3T3 cell line was from Prof. Dr. Usanee Vinitketkumneun at Department of Biochemistry, Faculty of Medicine, Chiang Mai University.HepG2 and NIH3T3 cells were cultured