Cytotoxicity of Crude Extract and Isolated Constituents of the Dichrostachys cinerea Bark towards Multifactorial Drug-Resistant Cancer Cells

The effectiveness of anticancer chemotherapy is greatly impeded by the resistance of malignant cells to cytotoxic drugs. In this study, the cytotoxicity of the crude extract (DCB) and compounds isolated from the bark of Dichrostachys cinerea, namely, betulinic acid (1), glyceryl-1-hexacosanoate (2), 7-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (3), and 6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (4), was investigated. The study was extended to the assessment of the mode of induction of apoptosis by DCB and compound 1. The resazurin reduction assay was used for cytotoxicity studies. Assessments of cell cycle distribution, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed by flow cytometry. Constituents of DCB were isolated by column chromatography. Triterpenoid 1 and flavone 4 had cytotoxic effects towards the 9 tested cancer cell lines with IC50 values below 50 μM. The recorded IC50 values varied from 7.65 μM (towards multidrug-resistant CEM-ADR5000 leukemia cells) to 44.17 μM (against HepG2 hepatocarcinoma cells) for 1, 18.90 μM (CCRF-CEM leukemia cells) to 88.86 μM (against HCT116p53+/+ colon adenocarcinoma cells) for 4, and 0.02 μM (against CCRF-CEM cells) to 122.96 μM (against CEM/ADR5000 cells) for doxorubicin. DCB induced apoptosis in CCRF-CEM cells mostly mediated by MMP alteration and enhanced ROS production; compound 1 induced apoptosis through caspases activation and MMP alteration and increased ROS production. Dichrostachys cinerea is an interesting cytotoxic plant and deserves more studies leading to new antineoplastic agents to fight cancer and mostly leukemia.

. . Cytotoxicity Assay. The cytotoxicity assay performed using resazurin reduction assay was applied to the crude extract (DCB), compounds 1-4, and doxorubicin [18,20,21] with similar experimental conditions as those reported earlier [13,19,22,23]. The Infinite M2000 Pro6 plate reader (Tecan, Crailsheim, Germany) with excitation wavelength of 544 nm and an emission wavelength of 590 nm was used to read the fluorescence after 72 h incubation. IC 50 values earlier defined [13] were calculated from a calibration curve by linear regression using Microsoft Excel [24]. The degree of resistance (D.R.) was determined as the IC 50 value of the resistant cell line versus that of its sensitive congeners; meanwhile, the selectivity index (S.I.) was the IC 50 value in normal AML12 hepatocytes versus that in HepG2 hepatocarcinoma.

. . Cell Cycle Analysis and Detection of Apoptotic Cells by
Flow Cytometry and Annexin V/PI Staining. Aliquots of 1×10 6 CCRF-CEM cells were treated with the studied samples (DCB and compound 1), the reference drug (doxorubicin), or the solvent control (DMSO) at various concentrations. The distribution of CCRF-CEM cycle was analyzed as described earlier in similar experimental conditions (24 h incubation; humidified 5% CO 2 atmosphere; 37 ∘ C) [13,22,23]. The BD Accuri C6 Flow Cytometer (BD Biosciences, Heidelberg, Germany) was used to measure the propidium iodide (PI) fluorescence of individual nuclei. Assays were repeated at least three times and in triplicate. To perform the annexin V/PI staining, DCB, betulinic acid (1), and doxorubicin were used to treat an amount of 1×10 6 per 1 ml CCRF-CEM cells. The experimental conditions were similar to those earlier reported (24 h incubation; humidified 5% CO 2 atmosphere; 37 ∘ C) [13]. The BD Accuri C6 Flow Cytometer was then used to analyze apoptosis using fluorescein isothiocyanate (FITC)-conjugated annexin V/PI assay kit (eBioscience6 Annexin V; Invitrogen, San Diego, USA) similarly as reported earlier [13,22,23]; early apoptosis for cells stained with only annexin V; late apoptosis or in a necrotic stage for cells stained with both annexin V and propidium iodide [13,25,26].

. . Activation of Caspases, Integrity of MMP, and ROS
Production. Treatment of CCRF-CEM cells with DCB did not activate the activity of caspases 3/7, 8, and 9 contrary to triterpenoid 1 (Figure 4). In effect, a dose-dependent activation of caspases upon treatment with 1 was observed, with optimal effects at 8.8 M; up to 3.19-fold, 2.91-fold, and 2.37-fold increases in the activity of caspases 3/7, 8, and 9, respectively, were recorded.
The effects of DCB, betulinic acid (1), and valinomycin on integrity of MMP in CCRF-CEM are depicted in Figure 5. Both DCB and compound 1 considerably modified the MMP with up to 90.3% and 57.5% (at 2 × IC 50 ), respectively; valinomycin at 10 M induced 45.9% alteration.
The effects of DCB and compound 1 on the production of ROS in CCRF-CEM cells are given in Figure 6. The two samples dose-dependently enhanced the production of ROS in CCRF-CEM cells. The ROS level in nontreated cells was 0.2%, whilst at 2 × IC 50 , DCB caused increased ROS production by up to 61.1% and triterpenoid 1 by 53.30%. H 2 O 2 induced ROS production by 98.8% at 50 M.

Discussion
Phytochemicals isolated from the bark of Dichrostachys cinerea were one triterpenoid 1, one ester of fatty acid 2, and two flavone-type flavonoids 3 and 4. Previous phytochemical investigation of the bark of Dichrostachys cinerea led to the isolation of meroterpene derivatives, dichrostachines A-R [10] which were not isolated in this study, probably due to the isolation procedure used or the fact that the plant was harvested in different geographic locations.
Drug resistance of malignant cells seriously hampers the chemotherapy of cancer. In the search for cytotoxic compounds, scientists should take into consideration the ability of these cells to rapidly develop drug resistance. This is possible when investigations also consider resistant phenotypes of malignant cells. In the present study, we have used several models of MDR cancer cell lines including ATPbinding cassette (ABC)-transporter-overexpressing MDRmediating P-glycoprotein (P-gp; ABCB1/MDR1) or breast cancer resistance protein (ABCG2/BCRP), a p53 knockout      cell line, and a mutation-activated EGFR gene (ΔEGFR) cell line. The resistant P-gp overexpressing CEM/ADR5000 cells treated with the crude extract DCB were collaterally sensitive [5] compared to their sensitive parental subline CCRF-CEM cells ( Table 1). Hypersensitivity of all resistant cell lines to betulinic acid as compared to their respective sensitive counterparts was also observed; for flavones 3 and 4, the hypersensitivity or otherwise normally sensitive (D.R. below or around 1) of at least three resistant cell lines was also recorded. Generally, the D.Rs. recorded upon treatments with DCB, compounds 1, 3, and 4 were lower than with doxorubicin (Table 1). Previous studies also reported the hypersensitivity of CEM/ADR5000 leukemia cells to compound 1 as compared to its sensitive congener CCRF-CEM cells [34]. These data are indications that Dichrostachys cinerea and its constituents have the potential to combat cancer multidrug resistance. According to the National Cancer Institute USA (NCI), good botanicals should exert their cytotoxicity with IC 50 values below 20 g/ml upon 48 h or 72 h incubation [11], while this set point is 10 M for phytochemicals [11,12]. Also, NCI recommends that botanicals yielding IC 50 values below or around 30 g/ml should undergo purification to isolate cytotoxic molecules [35]. In this work, IC 50 values as low as 4.69 g/ml and 4.13 g/ml were recorded with the crude extract DCB, on both sensitive and resistant leukemia cells, respectively (Table 1). Selective and lower IC 50 values were recorded with DCB on carcinoma cells, clearly indicating that this plant could likely be used to combat leukemia. This was also the case with betulinic acid (1), as IC 50 values below 10 M were also recorded towards leukemia cells, and higher values obtained in carcinoma cells. Though flavones 3 and 4 had cytotoxic effects in several cell lines including leukemia and carcinoma phenotypes, all IC 50 values obtained were above 10 M. This confirms the hypothesis that this plant and its constituents could mostly be used in the fight against leukemia. The good S.I. (>2) of compound 1 also indicates that it can be used in chemotherapy (Table 1). In effect, the low cytotoxicity of betulinic acid towards the normal PBL peripheral blood lymphoblast was also reported [36]. However, its lower S.I. as compared to that of doxorubicin, clinically associated with many adverse effects to patients (despite higher S.I.), clearly indicates that further studies on the toxicity of this compound as well as the crude extract will be necessary.
To the best of our knowledge, this is the first intensive study on cytotoxicity of Dichrostachys cinerea and its constituents 3 and 4 against MDR cancer cell lines. However, preliminary antiproliferative effects of this plant were reported towards DU145 and 22Rv1 prostate cancer cells and HeLa cervical cancer cells, with the lowest IC 50 values of 8.04 g/ml recorded in 22Rv1 cells [7]. Also, betulinic acid is a well-known cytotoxic compound [34]. Its effects have been reported towards several cancer cell lines including sensitive and resistant phenotypes such as CCRF-CEM cells and CEM/ADR5000 leukemia cells, MDA-MB-231-pcDNA and MDA-MB-231/BCRP breast adenocarcinoma cells, HEK293 and HEK293/ABCB embryonic kidney cells, and U87.MG and U87.MGΔEGFR glioblastoma cells with IC 50 values Evidence-Based Complementary and Alternative Medicine 9 ranging from 15.1 M (against HEK293 cells) to 29.4 M (towards CCRF-CEM cells) [34,36].
In this study, the crude extract DCB and triterpenoid 1 had the best cytotoxic effects on the two leukemia cells with IC 50 values below 10 M. They were consequently selected for further cellular mechanistic studies towards CCRF-CEM cells, such as induction of apoptosis, caspases activation, and alteration of MMP as well as the production of ROS [37]. DCB and compound 1 induced apoptosis in CCRF-CEM cells (Figures 2 and 3). Induction of apoptosis by DCB was mediated by MMP alteration and increased ROS production, while that induced by triterpenoid 1 was mediated by caspases activation (Figure 4), MMP alteration ( Figure 5), and increased ROS production ( Figure 6). Previous studies on the molecular mechanism of the cytotoxic action of compound 1 showed that it inhibited P-gp, BCRP, and ABCB5 and mutation activated EGFR overexpressing cells. Besides, various genes significantly correlated to its activity on cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination, and proteasome degradation [34].

Conclusions
The present study indicated that Dichrostachys cinerea is a potential cytotoxic plant and should be further explored to develop new antineoplastic agents to fight recalcitrant cancers. The crude extract DCB induced apoptosis in CCRF-CEM cells mostly mediated by MMP alteration and enhanced ROS production; compound 1 induced apoptosis through caspases activation and MMP alteration and increased ROS production.