Antiproliferation and cell apoptosis inducing bioactivities of constituents from Dysosma versipellis in PC3 and Bcap-37 cell lines

Background Recently, interest in phytochemicals from traditional Chinese medicinal herbs with the capability to inhibit cancer cells growth and proliferation has been growing rapidly due to their nontoxic nature. Dysosma versipellis as Bereridaceae plants is an endemic species in China, which has been proved to be an important Chinese herbal medicine because of its biological activity. However, systematic and comprehensive studies on the phytochemicals from Dysosma versipellis and their bioactivity are limited. Results Fifteen compounds were isolated and characterized from the roots of Dysosma versipellis, among which six compounds were isolated from this plant for the first time. The inhibitory activities of these compounds were investigated on tumor cells PC3, Bcap-37 and BGC-823 in vitro by MTT method, and the results showed that podophyllotoxone (PTO) and 4'-demethyldeoxypodophyllotoxin (DDPT) had potent inhibitory activities against the growth of human carcinoma cell lines. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in PC3 and Bcap-37 cells, and the apoptosis ratios reached the peak (12.0% and 14.1%) after 72 h of treatment at 20 μM, respectively. Conclusions This study suggests that most of the compounds from the roots of D. versipellis could inhibit the growth of human carcinoma cells. In addition, PTO and DDPT could induce apoptosis of tumor cells.


Background
Cancer is the major cause of human deaths worldwide because of its high incidence and mortality. Thousands of people die of cancer each year despite aggressive treatment regimens that include surgery, chemotherapy and radiotherapy. Due to the infiltrative nature and the rapid recurrence of the malignant tumor, complete surgical resection of these tumors is typically not achieved [1,2], and the conventional radiation and chemotherapy are often intolerable due to the strong systemic toxicity and local irritation [3][4][5]. These factors highlight the urgent need for new therapies or therapeutic combinations to improve the survival and quality of life of cancer patients.
In recent years, interest in phytochemicals from traditional Chinese medicinal herbs has been growing rapidly due to their ability to inhibit the growth and proliferation of cancer cells [6][7][8][9]. Due to their nontoxic nature, they are often employed in medical applications. Among of them, flavonoids and lignans present in traditional Chinese medicines have been revealed having significant activities against some forms of cancer [1,10]. The lignan podophyllotoxin is a plant toxin that exerts its cytotoxicity effect by inhibiting microtubule assembly and promoting cells to die via apoptosis [11]. However, it is not used as a clinical therapeutic agent due to its serious side effects. Extensive structure modifications were performed to obtain more potent and less toxic antitumor agents, e.g. etoposide [12] and teniposide [13] are currently used in anticancer therapy. Besides, some researchers found that podophyllotoxin and its derivatives had inhibitory effect on PC3 and other cells [14][15][16]. One of the popular Chinese herbal medicines, Dysosma versipellis (Hance) M. Cheng which belongs to Berberidaceae in Dysosma Family and is used as a source of podophyllotoxin [16] is a perennial herbaceous species grown in the understory of mixed evergreen and deciduous forests of China. In some folk remedies, D. versipellis (Hance) M. Cheng has been widely used to clear sputum, kill parasites and treat epidemic encephalitis B and epidemic parotitis. Previous studies have indicated that constituents and extracts of this traditional Chinese medicinal plant have growth inhibitory activities against various tumors in vivo or in vitro [16][17][18]. However, systematic and comprehensive studies have not been performed with regard to the chemical constituents from D. Versipellis and apoptosis-inducing effect of these components.
Dysosma versipellis, an endangered and endemic Bereridaceae plant species of China, has been proved to be an important Chinese herbal medicine because of its biological activity. In this study, fifteen compounds were isolated from the rhizomes of D. versipellis which was grown in Guizhou and identified by spectroscopic analysis and physicochemical data as b-sitosterol (1), 4'demethylpodophyllotoxin (2) (9), quercetin (10), daucosterol (11), podophyllotoxone (PTO, 12), vanillic acid (13), 4'-demethyldeoxypodophyllotoxin (DDPT,14), and sucrose (15). Among them, (5), (7), (8), (12), (13) and (15) were obtained from the plants for the first time. All the compounds were then bioassayed on human prostatic carcinoma cell line PC3, human breast cancer cell line Bcap-37, human gastric carcinoma cell line BGC-823 and mouse embryonic fibroblast cell line NIH3T3 in vitro by MTT method. It was found that DDPT had the most potent inhibitory activities against the growth of human carcinoma cell lines than other compounds extracted from D. versipellis. And PTO obtained from the plants for the first time also had high antitumor activity. There are a few reports on the anticancer effects of PTO and DDPT on various tumor cells recently [19][20][21]. And it was found that the inhibitory rate of PTO (100 μg/mL) on P388 murine leukemia cell proliferation was 99.0%. Also PTO could induce HL-60 human leukemia cells apoptosis, which might be related with down-regulation of Bcl-2 expression. Other studies have found that DDPT possessed antitumor activity on A549 human lung carcinoma cell and SK-MEL-2 human melanoma cell with EC 50 of 0.023 μg/mL and 0.015 μg/mL respectively. However, no report was found on the anticancer activities of PTO and DDPT on PC3, Bcap-37 and BGC-823 cells. This prompted us to study the anticancer activities of PTO and DDPT separated from the rhizomes of D. versipellis grown in Guizhou on the three kinds of cells mentioned above and investigate their preliminarily mechanism of action as potent anticancer agents. Thus, further investigations of PTO and DDPT on the three cells lines were carried out on PC3, Bcap-37, and BGC-823 cells. The IC 50 of PTO and DDPT on the three cell lines were determined. Furthermore, experimental results of fluorescent staining and flow cytometry analysis indicated that PTO and DDPT could induce apoptosis in PC3 and Bcap-37 cells, with the apoptosis ratios of PC3 cells were 12.0% and 14.1% after 72 h of treatment at 20 μM, respectively. To the best of our knowledge, this is the first report on apoptosis inducing and antitumor activity of PTO and DDPT on PC3, Bcap-37, and BGC-823 cells.

Analysis and Instruments
1 H NMR, 13 C NMR, and DEPT spectra were measured on a JOEL-ECX 500 MHz NMR spectrometer in CDCl 3 , CD 3 OD, or DMSO-d 6 using tetramethylsilane (TMS) as an internal standard. The IR spectra were obtained in the KBr pellet using a SHIMADZU-IR Prestige-21 spectrometer. Melting points were determined on an XT-4 digital microscope (Beijing Tech Instrument Co.) Analytical TLC was performed on silica gel GF254 (400 mesh), and column chromatographic operations were performed on Silica gel (100-200 or 200-300 mesh, Qingdao Haiyang Chemical Co.). In addition, Sephadex LH-20 column chromatographic instrument (Beijing Huideyi Tech Instrument Co.) was employed for the extraction and purification of chemical composition.

Plant material
The rootstalk of D. versipellis was collected in Qingzhen, Guizhou Province, China, in the month of May 2008. The voucher specimen was identified as D. versipellis (hance.) M.Cheng by Qing-De Long, the Dean of teaching-research section, School of Pharmacy of Guiyang Medical University, and was submitted to our laboratory for further investigation.

Extraction and isolation
The dried roots of D. versipellis (20 kg) from Guizhou Province were powdered and extracted by maceration in 80% industrial alcohol (50 L, each 4d) for three times. The extracts which filtered and washed with alcohol each time were combined and reduced in vacuo to afford 4 kg crude extract. A part of crude extract (950 g) was further extracted with petroleum ether, chloroform and ethyl acetate to obtain three fractions of 40 g, 2 kg and 280 g respectively.

Anticancer activity bioassay Cell culture
Human prostate cancer cell line PC3, breast cancer cell line Bcap-37, and gastric cancer cell line BGC-823 were purchased from Institute of Biochemistry and Cell Biology, China Academy of Science and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Mouse embryonic fibroblast cell line NIH 3T3 was also obtained from the same place and cultured in DMEM supplemented with 10% FBS. All cell lines were maintained at 37°C in a humidified 5% carbon dioxide and 95% air incubator.

MTT asssy
All tested compounds were dissolved in DMSO (1-100 μM solution) and subsequently diluted in the culture medium before treatment of the cultured cells. Tested cells were plated in 96-well plates at a density 2×10 3 cells/well/100 μL of the proper culture medium and treated with the compounds at 1 to 100 μM for 72 h. In parallel, the cells treated with 0.1% DMSO served as control. An MTT assay (Roche Molecular Biochemicals, 1465-007) was performed 30 h later according to the instructions provided by Roche. This assay was based on the cellular cleavage of MTT into formazane which is soluble in cell culture medium. Any absorbance caused by formazan was measured at 595 nm with a microplate reader (BIO-RAD, model 680), which is directly proportional to the number of living cells in culture. The experiment was performed in triplicate. The percentage cytotoxicity was calculated using the formula.

AO/EB staining
Cells were seeded at a concentration of 5×10 4 cell/ml in a volume of 0.6 mL on sterile cover slip in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and supplemented with podophyllotoxone and 4'demethyldeoxypodophyllotoxin (20 μM). After the treatment period, the cover slip with monolayer cells was inverted on the glass slide with 20 μL of AO/EB stain (100 μg/mL). The fluorescence was read on an IX71SIF-3 fluorescence microscope (OLYPUS Co., Japan).

Hoechst 33258 staining
Cells grown on sterile cover slip in 6-well tissue culture plates were treated with podophyllotoxone and 4'demethyldeoxypodophyllotoxin (20 μM) for a certain range of treatment time. The culture medium containing compounds was removed and the cells were fixed in 4% paraformaldehyde for 10 min. After washing twice with PBS, cells were stained with 0.5 mL of Hoechst 33258 staining (Beyotime) for 5 min. After washing twice with PBS, stained nuclei were observed under an IX71SIF-3 fluorescence microscope by using 350 nm excitation and 460 nm emission.

TUNEL assays
TdT-UTP nick end labeling (TUNEL) assays were performed with colorimetric TUNEL apoptosis assay kit according to the manufacturer's instructions (Beyotime). Cells grown in 6 well culture clusters were treated as mentioned in mitochondrial depolarization assay. In short, Bcap-37 cells grown in 6-well tissue culture plates were washed with PBS and fixed in 4% paraformaldehyde for 40 min. After washing once with PBS, cells were permeabilized with immunol staining wash buffer (Beyotime) for 2 min on ice. Cells were washed once with PBS again and incubated in 0.3% H 2 O 2 in methanol at room temperature for 20 min to inactivate the endogenous peroxidases after which the cells were washed for three times with PBS. Thereafter, the cells were incubated with 2 μL of TdT-enzymea and 48 μL of Biotin-dUTP per specimen for 60 min at 37°C. After termination for 10 min, cells were incubated with streptavidin-HRP (50 μL per specimen) conjugate diluted at 1:50 in diluent of streptavidin-HRP for 30 min. After washing three times with PBS, cells were incubated with diaminobenzidine (DAB) solution (200 μL per specimen) for 10 min. This was again followed by washing with PBS for two times and the result was imaged under an XDS-1B inverted biological microscope (Chongqing photoelectric device CO.).

Flow cytometry analysis
Prepared PC3 cells (1×10 6 /mL) were washed twice with cold PBS and then re-suspended gently in 500 μL binding buffer. Thereafter, cells were stained in 5 μL Annexin V-FITC and shaked well. Finally, 5 μL PI was added to these cells and incubated for 20 min in a dark place, analyzed by FACS Calibur, Becton Dickinson.

Statistical analysis
All statistical analyses were performed with SPSS 10.0. Data were analyzed by one-way analysis of variance (ANOVA). Mean separations were performed using the least significant difference method (LSD test). Each experiment had three replicates and all experiments were run three times with similar results. Measurements from all the replicates were combined and treatment effects analyzed.

Anticancer activity
The potential effect of the extracts from D. versipellis was investigated on the viability of PC3, Bcap-37, BGC-823 and NIH3T3 cells using MTT assay at the concentration of 20 μM, with ADM (adriamycin) [39] being used as the positive control [40,41]. MTT [3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay is a common method of measuring the proliferation of cells. The results are summarized in Table 1. It could be seen that PTO and DDPT possess potent activities against the three human cancer cell lines tested. The inhibitory ratios of PTO and DDPT at 72 h after treatment were 52.0% and 67.1% against PC3 cells, 42.1% and 56.6% on Bcap-37 cells, 47.9% and 60.7% on BGC-823 cells, and 43.7% and 59.8% on NIH 3T3 cells. Further experiments found that proliferation of these three carcinoma cells were significantly inhibited by PTO and DDPT in a concentration-dependent manner, as shown in Figures 2 and 3. The IC 50 values of PTO on PC3, Bcap-37 and BGC-823 cells were (17.8±1.0) All the values are expressed as mean ± SD (n = 6). * P < 0.05, ** P < 0.01 compared with control. To determine whether the growth inhibitory activity of PTO and DDPT were related to the induction of apoptosis, the morphological character changes of PC3 and Bcap-37 cells were investigated using the AO/EB staining and Hoechst 33258 staining under fluorescence microscopy.
Since acridine orange (AO) was a vital dye that could stain nuclear DNA across an intact cell membrane and ethidium bromide (EB) could only stain cells that had lost membrane integrity. Thus, live cells will be uniformly stained green, early apoptotic cells will be densely stained as green yellow or displayed green yellow fragments, while late apoptotic cells will be densely stained as orange or displayed orange fragments, and necrotic cells will be stained with orange with no condensed chromatin could be found by the AO/EB doubly staining. After tumor cells were treated with PTO and DDPT (20 μM) for 24, 48 h, the morphological changes were analyzed. As shown in Figure 4, green live PC3 and Bcap-37 cells with normal morphology were seen in the negative control group ( Figure 4A and 4A'). In contrast, early apoptotic cells with yellow green dots in PC3 cell nuclei and late apoptotic cells with orange dots in Bcap-37 cell nuclei could be seen in the positive control group ( Figure 4D and 4D'), meanwhile bright green early apoptotic cells with nuclear margination and chromatin condensation occurred in the experimental group with 24 h of treatment ( Figure 4B and 4E) and orange colored apoptotic   cells with apoptotic bodies and chromatin fragmentation could be seen when PTO and DDPT were applied for 48 h ( Figure 4E and 4F). Similarly, morphological changes of Bcap-37 cell apoptosis were also observed under microscope ( Figure 4B', 4C', 4E', and 4F'). The results suggested that PTO and DDPT were able to induce apoptosis in PC3 and Bcap-37 cells.
Hoechst 33258 staining was also carried out to investigate the apoptosis induction of PTO and DDPT (20 μM) on PC3 and Bcap-37 cells. Membrane-permeable Hoechst 33258 was a blue fluorescent dye and stained the cell nucleus. When cells were treated with Hoechst 33258, live cells with uniformly light blue nuclei were observed under fluorescence microscope, while apoptotic cells exhibited bright blue because of karyopyknosis and chromatin condensation, and the nuclei of dead cells could not be stained. The experimental results were shown in Figure 5. Compared with the negative control ( Figure 5A and 5A'), a part of cells with smaller nuclei and condensed staining appeared in the positive control group ( Figure 5D and 5D'). After treated with PTO and DDPT for a given time, some PC3 cell nuclei became pyknotic (shrunken and dark), as shown in Figure 5B, 5C, 5E and 5F. Besides, Bcap-37 cells treated with PTO and DDPT for 24 h had no obvious morphologic changes ( Figure 5B' and 5E'), but most cell nuclei appeared to be highly condensed (brightly stained) after 48 h of treatment ( Figure 5C' and F'). The results once again indicated that PTO and DDPT could induce apoptosis in PC3 and Bcap-37 cells.
TUNEL assay was further carried out to confirm the cell apoptosis inducing activities of PTO and DDPT.
TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) is a popular method for identifying apoptotic cells in situ by detecting DNA fragmentation. Due to degradation of DNA that resulted from the activation of Ca/Mg dependent endonucleases in apoptotic cells, DNA cleavage occurred and led to breaking of strand within the DNA. These strand breaks of cleaved DNA could be identified by terminal deoxynucleotidyl transferase (TdT) that catalyzed the addition of biotin-dUTP. The biotin-labeled cleavage sites were then detected by reaction with streptavidin-HRP and visualized by DAB indicating a brown color. As shown in Figure 6, most nuclei were stained as a discernible brown in the treatment groups with HCPT ( Figure 6C and 6C'), PTO ( Figure 6B and 6B'), and DDPT ( Figure 6D and 6D') compared with the control ( Figure 6A and 6A').
In addition, the apoptosis ratios induced by PTO and DDPT caused apoptosis in tumor cells was quantitatively assessed by flow cytometry. In the early stages of apoptosis, phosphatidylserine (PS) was translocated from the inside of the cell membrane to the outside. Annexin V, a calcium dependent phospholipid-binding protein associated with a high affinity for phosphatidylserine, was used to detect early apoptotic cells. PI (Propidine Iodide) was a red fluorescent dye and stained cells that had lost membrane integrity. So, cells stained with FITC-annexin V and PI were discriminated necrotic cells (Q1, Annexin -/PI + ), late apoptotic cells (Q2, Annexin + /PI + ), intact cells (Q3, Annexin -/PI -) and early apoptotic cells (Q4, Annexin + /PI -). As shown in Figure 7, PTO and DDPT (20 μM) could induce apoptosis of PC3 cells, and highest apoptosis ratios, 12.0% and 14.1% for PTO and DDPT respectively, were obtained after 72 h of treatment at a concentration of 20 μM. Furthermore, as shown in Figure 8, the early (Q4) and late (Q2) apoptosis of PC3 cells which treated with PTO and DDPT increased gradually in a time-dependent manner. The late apoptotic ratio of cells increased to approximately 11.0% at 72 h after treatment of PTO (20 μM), which was close to that of positive control HCPT (11.8%). And the highest rate of early apoptosis was 7.0% when cells were treated with DDPT at the concentration of 20 μM for 72 h. In summary, these results indicated that inhibitive effects observed in response to PTO and DDPT were associated with induction of apoptotic cell death.

Conclusions
Dysosma versipellis (Hance) M. Cheng, an important medicinal plant species, is considered 'endangered' by the China Species Red List and has been considered as vulnerable by the IUCN due to its rapid decline [42,43]. Therefore, studies on the chemical constituents from D. versipellis and their biological activities have assumed significance for the rational development and utilization of this plant. In our study, fifteen compounds were extracted and identified from D. versipellis grown in Guizhou province, and the cell growth inhibition effects of these constituents on PC3, Bcap-37, and BGC-823 cells were carried out by MTT assay. Among these compounds, PTO, which was extracted from D. versipellis for the first time, together with DDPT showed potent activities on PC3, Bcap-37, and BGC-823 cells in a dose-dependent manner. And the IC 50 values of PTO and DDPT on three cell lines were (17.8±1.0) μM, (21.1 ±1.8) μM, (19 ±1.6) μM and (10.6±1.5) μM, (13.2±0.5) μM, (11.5±0.6) μM, respectively.
The apoptosis inducing activities of PTO and DDPT on PC3 and Bcap-37 cells were investigated through AO/EB staining, Hoechst 33258 staining, TUNEL and flow cytometry analysis assay. The results demonstrated that PTO and DDPT from D. versipellis have potential to be employed in adjuvant therapy for treating human prostate and breast tumors. Further studies of the   specific mechanisms of these compounds on human malignant tumors are currently underway.