SFE-CO2 Extract from Typhonium giganteum Engl. Tubers, Induces Apoptosis in Human Hepatoma SMMC-7721 Cells Involvement of a ROS-Mediated Mitochondrial Pathway

Typhonium giganteum Engl. (BaiFuzi) is one of the herbs commonly used in traditional Chinese medicine against cancer. In our previous studies, 37 compounds were identified the SFE-CO2 (supercritical fluid extraction with CO2) extract by GC-MS, including the four major components [β-sitosterol (40.22%), campesterol (18.45%), n-hexadecanoic acid (9.52%) and (Z,Z)-9,12-octadecadienoic acid (8.15%)]. The anti-cancer mechanisms of the SFE-CO2 extract from T. giganteum Engl. tubers have not been reported as yet. In this paper, the molecular mechanisms of the SFE-CO2 extract-mediated apoptosis in SMMC-7721 cells were further examined. SFE-CO2 extract inhibited the growth of SMMC-7721 cells in a time- and dose-dependent manner, arrested the cell cycle in the S phase and G2/M phase, and induced apoptosis. In addition, reactive oxygen species (ROS) increase, reduction of mitochondrial membrane potential, a rise in intracellular calcium levels were found in SMMC-7721 cells after treated with the extract. Western blot analysis showed that the extract caused down-regulation of Bcl-2 expression, and up-regulation of Bax expression. Moreover, caspase-3 and caspase-9 protease activity significantly increased in a dose-dependent manner. Collectively, our results showed that the SFE-CO2 extract from T. giganteum Engl. tubers induces apoptosis in SMMC-7721 cells involving a ROS-mediated mitochondrial signalling pathway.


Introduction
Apoptosis is a form of programmed cell death which occurs through activation of the cell-intrinsic suicide machinery [1] and is a hallmark of the action of many anticancer drugs [2][3][4]. Mitochondria play a pivotal role during the process of cell apoptosis which involves in a variety of key events, including loss of mitochondrial membrane potential (MMP), mitochondrial swelling and release of apoptotic proteins [5]. Activation of the apoptotic cascade results from a complex interaction of molecular events [6]. ROS, a group of highly reactive molecules, including singlet oxygen, hydroxyl radicals, superoxide anion, nitric oxide and hydrogen peroxides, have been shown to play a key role in apoptotic cell death [7]. ROS are known to induce the collapse of MMP, therefore trigger a series of mitochondria-associated events including apoptosis [8]. Excessive ROS generation can induce redox-signaling pathways, including oxidative stress, a rise in intracellular calcium levels, loss of cell function, cell cycle arrest, and apoptosis [9,10]. The mitochondria-dependent pathway for apoptosis is governed by Bcl-2-family proteins [11]. Bax/Bcl-2 regulates caspase-9 and caspase-3, which eventually leads to apoptosis [12].
The dried tuber of Typhonium giganteum Engl. is recorded in the Chinese Pharmacopoeia as a traditional Chinese medicine named Baifuzi [13]. The tuber of T. giganteum Engl. has been effectively used in Chinese folk medicine to treat cerebral apoplexy, dispel wind-phlegm, tumor-related diseases and many other illnesses [14]. It has been reported that the chemical components of T. giganteum Engl. tubers included β-sitosterol, β-sitosterol-D-glucoside, dl-inositol, cerebroside, etc. [15,16]. It also has been reported that the chemical constituents of the volatile oils from T. giganteum Engl. tubers included N-phenylbenzenamine, 2,6,10,14-tetramethylhexadecane, 6-methyl-2-phenylquinoline, etc. [17]. Several studies have reported that T. giganteum Engl. had potent anticancer activity, both in vitro and in vivo [18][19][20][21]. The aqueous extract from T. giganteum Engl. tubers induced apoptosis in SMMC-7721 cells via cell cycle arrest in S phase. The aqueous extract induces apoptosis in MCF-7 cells via cell cycle arrest in S and G2/M phase [19,20]. However, the chemical composition of the aqueous extract has not been revealed. In our previous studies, 37 compounds were identified in the SFE-CO 2 extract by GC-MS, including the four major components [β-sitosterol (40.22%), campesterol (18.45%), n-hexadecanoic acid (9.52%) and (Z,Z)-9,12-octadecadienoic acid (8.15%)] [22]. In this paper, we explored the mechanisms of the SFE-CO 2 extract from T. giganteum Engl. tubers-mediated apoptosis in human hepatoma SMMC-7721 cells. The results of this investigation might provide a scientific explanation for the traditional application of this herbal medicine in hepatic cancer therapy.

Cytotoxicity Assay
Cell viability was determined by the MTT assay. In our previous studies, SFE-CO 2 extract revealed different cytotoxic activities towards the seven human cancer cell lines (SMMC-7721, SGC-7901, HO-8910, A549, PC-3, MCF-7 and HCT-8). SMMC-7721 cells were the most sensitive cell line, thus, it was selected as a representative cell line for further investigation [22]. As shown in Figure 1, the growth of SMMC-7721 cells was significantly inhibited in a dose-and time-dependent manner by increasing concentrations of the extract after 24, 48 and 72 h. When SMMC-7721 cells were treated with 400 μg/mL SFE-CO 2 extract, 67.29 ± 5.48% of cells were killed after 48 h.

Analysis of Cell Cycle Distribution
The cells were treated with SFE-CO 2 extract for 48 h, and the level of cell cycle progression was monitored by flow cytometry. As shown in Figure 2, a significantly increase in S phase populations was found after treated with SFE-CO 2 extract, compared with control cells (from 20.35 ± 2.11% to 37.22 ± 2.53%). Meanwhile, a significant increase in G2/M phase was also found (from 10.34 ± 2.26% to 33.67 ± 2.38%). Hence, SFE-CO 2 extract exerted growth-inhibitory effects via S phase and G2/M phase arresting in a concentration-dependent manner. The aqueous extract from Typhonium giganteum Engl. tubers arrested the cell cycle in the S phase in SMMC-7721 cells [19], the different ingredients between the aqueous extract and the SFE-CO 2 extract showed different growth-inhibitory effects.

Cell Apoptosis Analysis
To further confirm the apoptosis induced by SFE-CO 2 extract, AnnexinV-FITC/PI staining assay was used.

Changes in Nuclear Morphology
To further investigate whether the SFE-CO 2 extract mediated cell death in SMMC-7721 cells due to an apoptotic mechanism, the morphological changes were observed under inverted fluorescence microscope by Hoechst 33258 staining. Figure 4 shows that the nuclei of untreated control SMMC-7721 cells were stained in less bright blue and homogeneous color, but the cells treated with the 200 μg/mL extract for 48 h displayed typical apoptotic features including chromatin condensation and nuclear fragmentation [23]. White arrows pointed at the condensed chromatin. All of these changes suggested that the extract could induce apoptosis toward SMMC-7721 cells.

SFE-CO 2 Extract Decreases Mitochondrial Membrane Potential( MMP) in SMMC-7721 Cells
The disruption of mitochondrial integrity is one of the early events leading to apoptosis. Loss of MMP is an important event during the mitochondrial pathway of apoptosis [24][25][26], so we investigated whether SFE-CO 2 extract could induce the loss of MMP in SMMC-7721 cells. As shown in Figure 5, the MMP decreased to 74.35% ± 3.01, 67.17 ± 3.28% and 50.36 ± 2.79% in cells treated with the extract at 50, 100 and 200 μg/mL, respectively. These results demonstrated that SFE-CO 2 extract induced mitochondria damage and diminished MMP in SMMC-7721 cells in a concentrationdependent manner.

Effect of SFE-CO 2 Extract on Intracellular ROS in SMMC-7721 Cells
Since a loss of MMP is associated with the generation of ROS [27], we detected the level of ROS in SMMC-7721 cells treated with various concentrations of SFE-CO 2 extract for 48 h with the cellular oxidation of H 2 DCFDA, a probe that is oxidized to green fluorescent DCF by various peroxide-like ROS and nitroxide-derived reactive intermediates [28].
As shown in Figure 6, the level of ROS in cells treated with SFE-CO 2 extract was increased in a concentration-dependent manner, the level of ROS fluorescence increased to 12.71 ± 1.45%, 24.93 ± 3.64% and 42.68 ± 3.22% in cells treated with SFE-CO 2 extract at 50, 100 and 200 μg/mL, respectively. These data demonstrated that SFE-CO 2 extract significantly increased ROS production in SMMC-7721 cells. ROS production may promote mitochondrial dysfunction and trigger mitochondria-mediated apoptosis.

Effect of SFE-CO 2 Extract on Intracellular Calcium Concentration ([Ca 2+ ] c ) in SMMC-7721 Cells
Some studies reveal that ROS inactivates some transporters, which lead to a rise in [Ca 2+ ] c and subsequent cell dysfunction [10]. We determined the intracellular Ca 2+ concentration using flow cytometry. The fluorescence intensities of the control and treated groups were not coincident, and the intensity of the treated group was shifted to the right [29]. The [Ca 2+ ] c increased to 15.67 ± 2.01%, 24.64 ± 2.75% and 42.42 ± 3.81% in cells treated with the extract at 50, 100 and 200 μg/mL, respectively (Figure 7). The results indicated that SFE-CO 2 extract-induced [Ca 2+ ] c increase in a dose-dependent manner in SMMC-7721 cells.

Activation of Caspase-3 and -9 by SFE-CO 2 Extract
Caspases, which are a family of cysteine proteases, play key roles in executing the apoptotic process. Once activated, caspases activate downstream caspases, leading to apoptosis [30,31]. In the untreated SMMC-7721 cells, OD value of caspase-3 was 0.064 ± 0.007, and the OD value of caspase-9 was 0.119 ± 0.005. After treatment with SFE-CO 2 extract, caspase-3 and -9 protease activity significantly increased in a dose-dependent manner. The highest activities of caspase-3 and caspase-9 were found upon exposure to 100 μg/mL SFE-CO 2 extract. The OD values were 0.124 ± 0.012 and 0.154 ± 0.022, respectively, and were significantly higher than those in the control group (Figure 8).

SFE-CO 2 Extract-Mediated Up-Regulation of Bax and Down-Regulation of Bcl-2
Bcl-2 family proteins have a central role in controlling the mitochondrial pathway. The Bcl-2 family significantly regulates apoptosis either as an activator (e.g., Bax) or as an inhibitor (e.g., Bcl-2), Therefore, it has been suggested that the Bax/Bcl-2 ratio was a key factor in regulation of the apoptotic process [32,33]. We used Western blotting to measure the expression of the Bcl-2 family members. As shown in Figure 9, Western blot analyses revealed that the levels of pro-apoptotic Bax were significantly increased, whereas the levels of anti-apoptotic Bcl-2 were decreased in a concentrationdependent manner. These results indicate that apoptosis induced by SFE-CO 2 extract is related to the mitochondrial pathway. Figure 9. Effects of the SFE-CO 2 extract on apoptosis-related proteins expression analyzed by Western blot.

Plant Material and Extraction
T. giganteum Engl. tubers were purchased from a commercial source (Typhonium giganteum Engl. Planting and Development Base Company, Jilin, China) and authenticated by Prof. Shao-Quan Nie (Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Harbin, China). SFE-CO 2 extract from T. giganteum Engl. tubers was prepared in our laboratory [22]. Briefly, the tubers were sliced and air-dried at room temperature to a final moisture content below 0.5%. Air-dried tubers were pulverized in a milling machine and then sieved (40 mesh). The SFE-CO 2 extraction experiments were performed using an HA121-50-01 SFE device (Hua'an Supercritical Fluid Extraction Corp., Nantong, China). The extraction vessel pressure and temperature were 30 MPa and 45 °C, separation vessel pressure and temperature were 5 MPa and 50 °C, and CO 2 flow rate of 15 kg·h −1 . After the scheduled time, the extraction vessel was depressurized and the oil was collected from the separation vessel. The extract was concentrated on a vacuum rotary evaporator under reduced pressure, and stored at −20 °C.

Maintenance of Human Cancer Cell Lines
Human hepatoma cancer SMMC-7721 cell line was purchased from China Center for Type Culture Collection (Wuhan, China). All the cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/L streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C.

Measurement of ROS Generation
ROS generation was monitored by flow cytometry using DCFHDA [28]. Briefly, SMMC-7721 cells (1 × 10 6 cells/well) were seeded in a 6-well plate for 24 h and then exposed to SFE-CO 2 extract (0, 50, 100, 200 μg/mL). After incubation for 48 h, cells were collected and suspended with DCFH-DA (10 μM) at 37 °C for 30 min. Fluorescence generation was due to the hydrolysis of DCFHDA to dichlorodihydrofluorescein (DCFH) by non-specific cellular esterases, and the subsequent oxidation of DCFH by peroxides was measured by means of flow cytometry.

Intracellular Calcium Analysis
The concentration of calcium was measured using Ca 2+ indicator Fluo-3/AM as described previously [39]. SMMC-7721 cells (1 × 10 6 cells/well) were seeded in a 6-well plate. After adherence for 24 h, the cells were treated with different concentrations of SFE-CO 2 extract (0, 50, 100, 200 μg/mL) for 48 h. Fluo-3/AM (Sigma, 5 μM) was added to the treated cells for 30 min at 37 °C. The cells were then analyzed immediately by flow cytometry.

Measurement of Caspase-3 and Caspase-9 Activities
Activity of caspase-3 and caspase-9 were determined with a colorimetric kit (Nanjing kaiji Bio-Tek Corporation, Nanjing, China) [40]. SMMC-7721 cells were treated with SFE-CO 2 extract (0, 50, 100 μg/mL) for 48 h, respectively. The cells (1 × 10 6 cells/mL) were harvested and washed once with PBS. After the SMMC-7721 cells were lysed, reaction buffer was added to the SMMC-7721 cells followed by the additional 5 μL of caspase-3 or caspase-9 colorimetric substrate (DEVD-pNA) and incubated in a 96-well plate for 4 h at 37 °C in a CO 2 incubator. The plate was then measure with an ELISA reader at an absorbance of 405 nm. Activities of caspase-3 and caspase-9 were expressed relative to theoretical density value (OD).

Statistical Analysis
Data are expressed as the mean ± SD. Statistical analysis of group differences was performed using Student's t-test. A value of p < 0.05 was considered statistically significant.

Conclusions
In summary, the present study showed that SFE-CO 2 extract from T. giganteum Engl. tubers could induce ROS production, lead to loss of MMP, increase the ratios of Bax/Bcl-2, activate stress-responsive caspase-9 and caspase-3 in SMMC-7721 cells. These results suggested that SFE-CO 2 extract induced apoptosis involving a ROS-mediated mitochondrial pathway in SMMC-7721 cells. T. blumei and T. flagelliforme are other Typhonium species. It has been reported that T. blumei induced A549 cells apoptosis via the mitochondrial pathway [41]. T. flagelliforme induced CEMss cells apoptosis via the mitochondrial pathway [42]. These dates were consistent with the results of our study. The SFE-CO 2 extract included the four major components (β-sitosterol, campesterol, n-hexadecanoic acid and (Z,Z)-)9,12-octadecadienoic acid [22]. We presume that the four major compounds play a major role in apoptosis in SMMC-7721 cells. Further studies are in progress about activity of SFE-CO 2 extract towards SMMC-7721 xenograft tumors in nude mice.