Cell cycle arrest and apoptosis induction by methanolic leaves extracts of four Annonaceae plants

RPMI (Roswell Park Memorial Institute) medium, FBS (fetal bovine serum), Penicillin–Streptomycin, L-Glutamine, Fungizone, and 0.25% Trypsin-EDTA were purchased from Gibco-BRL, USA. Annexin V-FITC (fluorescein isothiocyanate) was purchased from ImmunoTools GmbH, Germany. HEPES was purchased from Merck Millipore, Germany. Sodium chloride, sodium bicarbonate, and calcium chloride were purchased from RCI LABSCAN, Thailand. Dimethyl sulfoxide (DMSO), Triton X-100, and propidium iodide (PI) were purchased from Sigma-Aldrich, USA. Ribonuclease A (RNase A) was purchased from Worthington Biological Corporation, USA. All solvents and chemicals used were either HPLC grade or analytical grade and were purchased commercially from Sigma Chemical Co. (St. Louis, MO), Fluka Chemical Co. (Switzerland), and Merck (Darmstadt, Germany).

The human cancer cell lines used in this study consisted of human cervical carcinoma (HeLa, SiHa, and CaSki) (a kind gift from Assoc. Prof. Tipaya Ekalaksananan, Khon Kaen University, Thailand), human hepatocellular carcinoma (HepG2 and Hep3B) (a kind gift from Prof. Duncan R. Smith, Mahidol University, Thailand), and human myeloid leukemia (K562, U937, and RAJI) (a kind gift from Prof. Sumalee Tungpradabkul, Mahidol University, Thailand). All the cell lines were maintained in the RPMI medium containing 10 mM of HEPES, 1 mM of sodium bicarbonate, 10% fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (RPMI complete media) at 37 °C in a humidified 5% CO₂ atmosphere.

Uvaria longipes (collection no.: Chaowasku 132), Dasymaschalon sp. (collection no.: Chaowasku 120), and Marsypopetalum modestum (collection no.: Chaowasku 164) were collected from private residences at coordinates 13.790384, 100.372378; and Artabotrys burmanicus (collection no.: Chaowasku 163) was collected from a private garden at coordinates 13.919300, 99.952555. All the voucher specimens were deposited in the Chiang Mai University Biology (CMUB) herbarium. It should be noted that the Dasymaschalon sp. used in our study is roughly identified as Dasymaschalon lomentaceum and is currently authenticated for its potentially new species as evidenced by both morphological and molecular data (manuscript in preparation). The collection, preparation and identification of all plant specimens used in this study was performed by Dr. Tanawat Chaowasku.

Leaves of these plants were washed, air dried at 25-30 °C and crushed to powder. Dried and powdered leaves of U. longipes, Dasymaschalon sp., A. burmanicus and M. modestum were incubated in methanol at room temperature for 24 h. Then, the solution was collected and evaporated under vacuum in a rotary evaporator. All the methanolic extracts were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml. Finally, these extracts were serially diluted into 1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml in RPMI complete media.

Human cancer cell lines (HeLa, SiHa, CaSki, HepG2, Hep3B, K562, U937, and RAJI) at 1 × 10⁵ cells/ml were cultured in 24-well plates in the presence of various concentrations of methanolic extracts (1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml) for 24 h. The quantification of the apoptotic cells was measured by Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) co-staining assay. Briefly, at the end of the 24 h incubation, the cells were harvested and centrifuged at 1800 rpm for 8 min. The pellet was resuspended in 50 μl binding buffer containing 0.5 μl Annexin V-FITC and then incubated at 4 °C for 30 min in the dark. PI (50 μg/ml) in 200 μl binding buffer was added to each of the tubes and incubated for 5 min. Finally, the cells were analyzed by flow cytometry (CyAn ADP Analyzer, Beckman Coulter, USA).

Human cancer cell lines at 1 × 10⁶ cells were cultured in 6-well plates in the presence of leaves of M. modestum methanolic extracts (1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml) for 24 h. After treatment, the cells were washed and centrifuged at 1800 rpm for 8 min. The cells were resuspended and fixed with 70% ethanol at 4 °C for 2 h. After fixing, the cells were washed with PBS and centrifuged at 1800 rpm for 8 min. The pellet was broken up by vortexing and then resuspended in 250 μl PBS containing PI (20 μg/ml), RNase A (20 μg/ml), and Triton X-100 (0.1%), and incubated for further 30 min in the dark. Finally, the cells were analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, USA).

The four crude leaf extracts were tested for constituents such as alkaloids, sterols, cardiac glycosides, anthaquinone glycosides, saponins, flavonoids, and tannins by using standard methods as described previously [23‐25]. The qualitative results are expressed as (−) for the absence and (+) for the presence of constituents.

The content of quercetin and rutin in the four crude extracts was determined by using a reversed-phase HPLC (RP-HPLC) system (Shimadzu Corporation, Japan) including LC-10AV VP pumps and SPD-10AV VP with UV detector. The column for the separation was 250 × 4.6 mm in diameter (SymmetryShield® RP18 C18; Water Co., Ltd.). The mobile phases used for the determination of quercetin and rutin consisted of 5 mM KHPO₄:acetonitrile:methanol in the ratio of 49:40:11% v/v and de-ionized H₂O:Methanol:Triethylamine in the ratio of 60:40:0.1, respectively. The flow rate and the detection wavelengths for quercetin and rutin were 0.7 and 0.5 mL/min, and 350 and 256 nm, respectively. All the crude extracts were subjected to the RP-HPLC system in parallel with known concentrations of quercetin dihydrate and rutin (GmBH, Germany). The concentrations of quercetin and rutin were calculated from the peak area using the calibration curves. The assays were performed in triplicates.

To investigate the anti-cancer activity of the crude extracts on eight human cancer cell lines, we employed Annexin V and PI co-staining to examine possible induction of cell death (necrosis and/or apoptosis). The percentages of cells analyzed by flow cytometry following Annexin V and PI staining could be classified into four categories. The populations of cells residing in the Annexin V+/PI- and the Annexin V+/PI+ quadrants were determined as early and late apoptotic cells, respectively. The Annexin V−/PI - and the Annexin V−/PI+ quadrants were determined as living cells and necrotic cells, respectively. Both the untreated control (UT-C) and the DMSO-treated cells (DMSO-C) showed the percentages of the apoptotic cells as ranging from 3% to 12% after 24 h of incubation (Table 1). All treatments of crude extracts (leaves from Uvaria longipes: LUL, Dasymaschalon sp.: LD, Artabotrys burmanicus: LAB, and Marsypopetalum modestum: LMM) showed varying degrees of percentages of apoptotic cells (the percentages of the early apoptotic cells and the late apoptotic cells combined) depending on the type of crude extract. Consistently, changes in cell morphology were observed under an inverted microscope. This was corroborated by the decreases in the intensity of the forward light scatter when analyzed by flow cytometry (data not shown). Interestingly, the apoptotic effects from individual crude extracts exhibited activity in a cell-type specific manner. For example, the percentages of apoptotic HepG2 cells treated with LUL were significantly higher than those of SiHa cells treated with the same extract (Table 1). A dose-dependent manner was also observed in some crude extracts in particular cell types, for example, RAJI cells treated with LAB and LMM (Table 1 and Fig. 1). In our study, the most prominent apoptotic effect on cancer cell lines was observed in LMM and LAB crude extracts (Table 1), particularly against the RAJI cell line (Fig. 1). The half maximal inhibitory concentrations (IC50) of all crude extracts on cell death are shown in Table 2.

Table 1. Cancer cell lines were incubated for 24 h in the presence of 125–1000 μg/ml of crude extracts (leaf of U. longipes: LUL, leaf of Dasymaschalon sp.: LD, leaf of A. burmanicus: LAB, and leaf of M. modestum: LMM). Untreated cells (UT-C) and DMSO-treated cells (DMSO-C) were used as the controls. Statistical comparisons were analyzed by the one-way ANOVA with Tukey’s HSD post hoc test.

Table 2. IC₅₀ values of leaf crude extract against cancer cell lines was calculated from all types of cell death (Annexin V+/PI-, Annexin V−/PI+ and the Annexin V+/PI+ quadrants).

To test whether the apoptotic cell death was due to the inhibitory effect of the crude extracts on the cell cycle process, cell cycle analysis was then carried out. Although both LMM and LAB showed maximum potent effect on most of the cancer cell lines, LMM was chosen for the cell cycle analysis since there have been no reports on its anti-cancer activity, whereas the crude extract from A. siamensis (closely related to A. burmanicus) was reported having inhibitory effect on three other cell lines [26]. Furthermore, A. burmanicus is a rare species in the genus Artabotrys and resources for raw material were limited, making it difficult to study for therapeutic uses in the long run. Cancer cell lines were treated with various concentrations of LMM for 24 h, stained with propidium iodide, and subsequently analyzed by flow cytometry. All the cancer cell lines except for SiHa and Hep3B showed an increase in the DNA fragmentation in a dose-dependent manner as reflected by the increase in the percentages of cells in the sub G1 phase compared to the untreated or the DMSO-treated cells (Table 3 and Fig. 2).

Table 3. Cancer cell lines were cultured in the presence of 125–1000 μg/ml of crude extract from the leaves of M. modestum for 24 h, stained with PI, and then analyzed by flow cytometry. Statistical comparisons were analyzed by the one-way ANOVA with Tukey’s HSD post hoc test.

All four leave-derived crude extracts were tested for phytochemicals. The results showed the presence of tannins and flavonoids in all the crude extracts (Table 4). Alkaloids were found in U. longipes and A. burmanicus, and saponins were found in U. longipes and Dasymaschalon sp. The phytochemicals in the Annonaceae family observed in our study was consistent with that in a previous report [27].

Quercetin and rutin have been reported for their numerous biological activities, such as anti-inflammatory, free radical scavenging, immunomodulatory, and cancer chemoprevention [28, 29]. Interestingly, quercetin and rutin have been reported as present in a number of species of the Annonaceae family [30]. Therefore, all the methanolic leaves extracts were analyzed for quercetin and rutin by reversed-phase HPLC. The HPLC chromatogram demonstrated that the leaf methanolic extracts of U. longipes (Fig. 3b) and Dasymaschalon sp. (Fig. 3c) contain rutin amounting to 3.29 ± 0.22 mg/g extract and 2.05 ± 0.10 mg/g extract, respectively. While, the leaf methanolic extract of U. longipes and M. modestum contained quercetin amounting to 1.52 ± 0.13 mg/g extract and 0.43 ± 0.11 mg/g extract, respectively (Fig. 4b and c).