Cytotoxicity of selected Cameroonian medicinal plants and Nauclea pobeguinii towards multi-factorial drug-resistant cancer cells

Background Malignacies are still a major public concern worldwide and despite the intensive search for new chemotherapeutic agents, treatment still remains a challenging issue. This work was designed to assess the cytotoxicity of six selected Cameroonian medicinal plants, including Nauclea pobeguinii and its constituents 3-acetoxy-11-oxo-urs-12-ene (1), p-coumaric acid (2), citric acid trimethyl ester (3), resveratrol (4), resveratrol β-D-glucopyranoside (5) and strictosamide (6), against 8 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. Methods The resazurin reduction assay was used to evaluate the cytotoxicity of the crude extracts and compounds, whilst column chromatography was used to isolate the constituents of Nauclea pobeguinii. Structural characterization of isolated compounds was performed using nuclear magnetic resonance (NMR) spectroscopic data. Results Preliminary experiments on leukemia CCRF-CEM cells at 40 μg/mL showed that the leaves and bark extracts from Tragia benthamii, Canarium schweinfurthii, Myrianthus arboreus, Dischistocalyx grandifolius and Fagara macrophylla induced more than 50 % growth of this cell line contrary to the leaves and bark extracts of N. pobeguinii. IC50 values below or around 30 μg/mL were obtained with leaves and bark extracts of N. pobeguinii towards two and five, respectively, of the 8 tested cancer cell lines. The lowest IC50 value was obtained with the bark extract of N. pobeguinii against HCT116 (p53−/−) colon cancer cells (8.70 μg/mL). Compounds 4 and 6 displayed selective activity on leukemia and carcinoma cells, whilst 1–3 were not active. IC50 values below 100 μM were recorded with compound 5 on all 9 tested cancer cell lines as well as with 4 against 7 out of 8 and 6 against 2 out of 8 cell lines. Collateral sensitivity was observed in CEM/ADR5000 leukemia cells, MDA-MB-231-BCRP breast adenocarcinoma cells (0.53-fold), HCT116 (p53+/+) cells, human U87MG.ΔEGFR glioblastome multiforme cells to the methanolic bark extract of N. pobeguinii, as well as in MDA-MB-231-BCRP cells and HCT116 (p53+/+) cells and U87MG.ΔEGFR cells (0.86-fold) to compound 5. Conclusions The results of this study demonstrate the cytotoxicity of six Cameroonian medicinal plants, Canarium schweinfurthii, Dischistocalyx grandifolius, Tragia benthamii, Fagara macrophylla, Myrianthus arboreus and Nauclea pobeguinii. We also demonstrated the antiproliferative potential of Nauclea pobeguinii against drug-resistant cancer cell lines. Resveratrol and its glucoside are the major cytotoxic constituents in the bark of Nauclea pobeguinii. Electronic supplementary material The online version of this article (doi:10.1186/s12906-015-0841-y) contains supplementary material, which is available to authorized users.


Background
Malignacies are still a major public concern worldwide and despite the intensive search for new chemotherapeutic agents, treatment still remains a challenging issue. The situation is more complicated by the spread of drug resistance in tumors. Continuous efforts are necessary to boost drug discovery to treat multidrug resistant (MDR) cancer cells. Many factors are involved in MDR, including the over-expression of ABC transporters, particularly breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) [1], as well as the epidermal growth factor receptor (EGFR) [2][3][4] and mutations in the p53 tumor suppressor gene [5]. MDR cancer cells are resistant to a variety of chemically unrelated drugs [6][7][8][9]. In our previous studies, we documented the cytotoxicity of several secondary metabolites from selected Cameroonian plants against MDR cancer cells [10][11][12][13][14][15]. In our continous search for potentially antineoplastic agents from Cameroonian medicinal plants, the present study was designed at investigating the cytotoxicity of (Rubiaceae). The work was extended to the isolation of the active constituents of Nauclea pobeguinii. The above plants are used in Africa to treat many different ailments (Table 1). However, it has been recommended that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases should be taken into account when selecting plants used to treat cancer, since these reflect disease states bearing relevance to cancer or cancerlike symptoms [16,17].

Chemicals
Doxorubicin 98.0 % from Sigma-Aldrich was provided by the University Pharmacy of the Johannes Gutenberg University-Mainz and dissolved in PBS (Invitrogen) at a concentration of 10 mM. Geneticin >98 % (72.18 mM) was obtained from Sigma-Aldrich.

Plant material
The plant materials used in this study were the bark of  (Table 1).

Extraction
Air-dried plant material (3 kg for the bark of Nauclea pobeguinii and 1 kg for other samples) was powdered and extracted with methanol (MeOH; 10 L for the bark of Nauclea pobeguinii and 3 L for other samples) for two days. The organic solution was concentrated in vacuo to yield a paste (crude extract). The yield of each extract was determined (Table 1) and the samples were kept at 4°C until further use.
Antiplasmodial: against Plasmodium berghei [66]. . Similarly, a yellow solid (6, 20 mg) was obtained after filtration of sub-fractions 60-75. These sub-fractions were pooled together based on the TLC profile, after complete evaporation, the solid residue was recrystallized with acetone to give again 6 (15 mg).

Cell cultures
The cell lines used in the present work, their origins and their treatments were previously reported [18,19]. and were continuously treated with 800 ng/mL and 400 μg/mL geneticin, respectively. Normal AML12 heptocytes were obtained from the American Type Culture Collection (ATCC, USA). The above medium without geneticin was used to maintain MDA-MB-231, U87MG, HCT116 (p53 +/+ ) and AML12 cell lines. The cells were passaged twice weekly. All experiments were performed with cells in the logarithmic growth phase.

Resazurin reduction assay
The cytotoxicity of the tested samples was performed by resazurin reduction assay as we previously described

Results and discussion
Compounds were identified as 3-acetoxy-11-oxo-urs-12ene C 32 [31]. The irido-indole alkaloid strictosamide (6, 35 mg) was the major constituent of the bark extract. This is in accordance with previous studies reporting 6 as the main compound isolated from the bark methanolic extract of Nauclea pobeguinii harvested in Democratic Republic of Congo [32]. However, Xu et al. [32] also identified several other alkaloids as minor constituents of the bark extract, such as naucleidinic acid and 19-O-methyl-3,14-dihydroangustoline, naucleidinal, magniflorine, naucleofficine D, 3,14-dihydroangustoline, strictosidine, desoxycordifoline, 3a,5a-tetrahydrodeoxycordifoline lactam, and a phenol kelampayoside A. These compounds were not isolated in our sample from Cameroon. In addition, compounds 1-5 reported in this study were also not found in the plant harvested in Congo. This could either be due to the isolation techniques used or to the environmental variation that influences the concentration of the minor constituents synthesized by the Nauclea pobeguinii. strictosamide (6) was obtained as the major constituent isolated from the methanolic extract in both cases. A preliminary cytotoxicity study was first carried out on leukemia CCRF-CEM cells with crude extracts at a single concentration of 40 μg/mL for crude extracts. Doxorubicin (10 μM) served as positive control. According to the National Cancer Institute (USA), 30 μg/mL is the upper IC 50 limit considered promising for purification of a crude extract [33]. We tested a slightly higher concentration (of 40 μg/mL) in our preliminary assay. The results depicted in Fig. 2 show that more than 50 % growth was obtained if CCRF-CEM cells were treated with the methanol extracts from Tragia benthamii  Nauclea pobeguinii as well as doxorubicin (13.6 %) were able to induce more than 50 % inhibition of CCRF-CEM leukemia cell growth (Fig. 2). The IC 50 values of extracts (bark and leaves) from Nauclea pobeguinii were further determined on eight cancer cell lines and values below 30 μg/mL were obtained both extracts towards two out of eight and five out of eight cell lines, respectively ( Table 2). The lowest IC 50 value of 8.70 μg/mL was obtained with the bark extract against HCT116 (p53 −/− ) cells. Consequently, this extract was subjected to purification from which six compounds (1-6) were obtained. The best compounds (4 and 5) were less toxic towards AML12 normal hepatocytes than against cancer cells. The IC 50 threshold values of 20 μg/mL for crude extracts as well as 4 μg/mL or 10 μM for compounds [34,35] after 48 and 72 h incubation have been set by the United States National Cancer Institute (USNCI) to identify good cytotoxic phytochemicals. None of the tested compounds displayed IC 50 values below 10 μM. However, the bark extract of Nauclea pobeguinii (IC 50 value below 20 μg/mL on four out of eight tested cancer cell lines) could be considered as a promising candidate to fight cancers. The best isolated compounds (4-6) displayed rather moderate activities, suggesting possible synergistic effects of constituents in the crude extract.
The development of MDR in cancer cells either through ABC transporters [36], the epidermal growth factor receptor (EGFR) [2][3][4], or the tumor suppressor p53 gene [5] represents a major hurdle in chemotherapy. The discovery of new compounds with activity against MDR is hence very important in the ongoing fight against malignancies. In the present study, we used cell lines possessing all these resistance mechanisms to investigate multi-factorial drug resistance. The degrees of resistance were determined as the ratio of IC 50 value of the resistant cell line to that of the corresponding parental sensitive counterpart (Table 2). Compared to their corresponding sensitive cell lines, collateral sensitivity in resistant cells (hypersensitivity) was observed in P-glycoprotein-overexpressing CEM/ADR5000 cells (degree of resistance 0.80-fold), BCRP-overexpressing MDA-MB-231-BCRP cells (0.53-fold), p53 HCT116 (p53 −/− ) knockout cells (<0.54-fold) and epidermal growth factor receptor-overexpressing U87MG.ΔEGFR cells (0.47-fold) to the bark extract of N. pobeguinii. Collateral sensitivity was also found in HCT116 (p53 −/− ) cells (0.74-fold) and U87MG.ΔEGFR cells (0.86-fold) to compound 5.
The obtained data indicates that the stilbene resveratrol glucoside 5 and its aglycon 4 displayed slightly different degrees of activity on the cancer cell lines studied. This shows that glucosylation may positively (especially in leukemia cells) influence the cytotoxic activity. In fact, resveratrol glucoside 5 was more active than its aglcon 4 on the two tested leukemia cells. However, in carcinoma cell lines, the cytotoxicity of compounds 4 and 5 varied from one cell lines to other, none of two being more active than other one in all the solid cancer cell lines tested. To the best of our knowledge, this phytochemical and cytotoxicity study of the crude bark and leaf extracts as well as compounds 1-3 and 5 of Nauclea pobeguinii towards multifactorial drug resistant cancer lines is being reported here for the first time. Though strictosamide (6) is known to be the main constituent [32] of this plant, the present study suprisingly showed that it was not the most cytotoxic component of the extracts against the studied cancer cell lines. The best activities were reported with stilbenes namely resveratrol (4) and it glycoside resveratrol β-D -glucopyranoside (5). Compound 4 is a well known cytotoxic agent [37][38][39]. It is reported to suppress the proliferation of SKBR-3 breast cancer cells by inhibiting fatty acid synthase signaling pathway [38]. Compound 4 also alleviates the PI3K/Akt/ mTOR signaling in breast cancer SKBR-3cells by downregulation of Akt phosphorylation and up-regulation of PTEN expression [38]. Besides, compound 4 reportedly reverses MDR of the MCF-7/DOX breast cancer cells [39]. In the present study, compound 4 was most effective (IC 50 < 23 μM) against MDA-MB231 breast adenocarcinoma cells and their drug-resistant, MDA-MB231/ BCRP. These data are in accordance with previous reports and consolidates the potential cytotoxicity of 4 against breast cancer cells.

Conclusion
In conclusion, we demonstrate the cytotoxic potential of six Cameroonian medicinal plants, Canarium schweinfurthii, Dischistocalyx grandifolius, Tragia benthamii, Fagara macrophylla, Myrianthus arboreus and Nauclea pobeguinii. We also demonstrated the cytotoxic potential of leaves and bark of Nauclea pobeguinii against sensitive and MDR cancer cell lines. We further identified resveratrol and its β-glucoside as major cytotoxic constituents of the bark of Nauclea pobeguinii. The bark and leaves extracts of Nauclea pobeguinii are potential (*): The degree of resistance was determined as the ratio of IC 50 value in the resistant divided by the IC 50 in the sensitive cell line; NPB: extract from the bark of Nauclea pobeguinii; NPL: extract from the leaves of Nauclea pobeguinii; Compounds isolated from NPB [1: 3-acetoxy-11-oxo-urs-12-ene; 2: p-coumaric acid; 3: citric acid trimethyl ester; 4: resveratrol; 5: resveratrol β-D -glucopyranoside; 6: strictosamide]; n.a.: not applicable. IC 50 value are mean ± SD of at least two experiments with six replicates each cytotoxic botanicals that deserves more investigations to develop novel cytotoxic phytomedicines against drugresistant cancers.

Additional files
Additional file 1: Supporting information S1. Data on compounds isolated from Nauclea pobeguiinii (DOC 860 kb)