Cytotoxicity of four Aframomum species (A. arundinaceum, A. alboviolaceum, A. kayserianum and A. polyanthum) towards multi-factorial drug resistant cancer cell lines

Chemotherapy remains the major treatment of cancers but often fails due to cells multidrug resistance (MDR) [1, 2]. MDR is displayed by many cancer cells to withstand increasingly higher doses of antineoplastic compounds [3]. Investigation for naturally occurring molecules as potential cytotoxic drugs has yielded promising candidates [3‐7]. However, MDR is still considered a major hurdle for patients receiving chemotherapy [8, 9]. Various Cameroonian dietary plants including those from the family Zinziberaceae are used in traditional medicine to manage various ailments [5, 10‐13]. The genus Aframomum, belonging to the Zingiberaceae family have 40 species and is most common in tropical and subtropical regions [14]. Twenty species are found in Cameroon, where they are widely used as spices and in traditional medicine [14]. The Seeds of Aframomum arundinaceum K. Schum are used as laxative and as anti-helmintic. The fresh juice of the rhizomes is used against body odor. The rhizomes are used against toothache and the crushed seeds against fungal infections [10]. The decoction of the leaves Aframomum melegueta K. Schum together with the leaves of Momordica charantia and Sorghum arundinaceum cereal in local dry gin (alcohol) is recommended to be taken one dose daily against cholera [15]. Several Aframomum species such as Aframomum angustifolium, A. danielli, A. sanguineum, and A. sulcatum are also traditionally used to treat fevers in Africa [16], and recently, the antiplasmodial activity of some labdanes from A. sceptrum and A. latifolium was demonstrated [17]. The antibacterial activities of Aframomum kayserianum[12] and Aframomum polyanthum[13] were also reported on Gram-negative multidrug-resistant phenotypes. The cytotoxicity of other Afromomum species such as A. citratum and A. melegueta towards leukemia CCRF-CEM and ADR5000 cell lines was also reported [5]. The present study was designed to investigate the cytotoxicity of four dietary Aframomum species commonly used as spices in Cameroon, Aframomum alboviolaceum (Ridl.) K. Schum, A. arundinaceum (Oliver & Hanbury) K. Schum, Aframomum kayserianum K. Schum and Aframomum polyanthum K. Schum towards sensitive and multi-factorial drug resistant cancer cell lines. The study was extended to the identification of the bioactive constituents of A. arundinaceum.

The structures of the compounds isolated from Aframomum arundinaceum were established using spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HMQC, HMBC, and direct comparison with published information and with authentic specimens obtained in our research group for some cases. The compounds isolated from the fruits of A. arundinaceum (Figure 1) were identified as Aframodial C₂₀H₃₀O₃ (1; m/z 318.2) [27], 8(17),12-labdadien-15,16-dial C₂₀H₃₀O₂ (2; m/z 302.2) [17], galanolactone C₂₀H₃₀O₃ (3; m/z 318.2) [27], galanal A C₂₀H₃₀O₃ (4; 15 mg, m/z 318.2) [28], and galanal B C₂₀H₃₀O₃ (5; m/z 318.2) [29], 1-p-menthene-3,6-diol C₁₀H₁₈O₂ (6; m/z 170.1; m.p:165-167°C) [30], 1,4-dihydroxybenzene C₆H₆O₂ (7; m/z 110.0) [31], naringenin C₁₅H₁₂O₅ (8; m/z 272.0; 245-248°C) [32] and kaempferol-3,7,4’-trimethylether C₁₈H₁₆O₆ (9; m/z 328.0; 157-158°C) [33]. The cytotoxicity of compounds 1-9 as well as the crude extracts was determined towards drug sensitive and resistant cancer cell lines.

In a preliminary investigation of the four studied Aframomum species and compounds isolated from A. arundinaceum, we tested a single concentration of 40 μg/mL for each sample and 20 μg/mL for doxorubicin against the sensitive CCRF-CEM leukemia cell line (Figure 2). The extracts from A. kayserianum and A. alboviolaceum were less active and induced respectively 50.33% and 54.36% growth proliferation of CCRF-CEM cells. Compounds 1, 2, 3, 6 and 7 also induced more than 50% growth of this cell line. The extracts from A. polyanthum (36.28%) and A. arundinaceum (24.68%) as well as compounds 4 (47.78%), 5 (49.81%), 8 (38.49%) and 9 (39.58%) displayed less than 50% growth proliferation of CCRF-CEM cells. The IC₅₀ values of the above samples were further determined on nine cancer cell lines, including both sensitive and MDR phenotypes (Table 1). Aframomum. arundinaceum extract as well as compound 8 and doxorubucin induced less than 50% proliferation of all tested cancer cell lines, with IC₅₀ values below 40 μg/mL. A. polyanthum extract, compounds 9, 4 and 5 showed selective activities, the IC₅₀ values <40 μg/mL being obtained on 5/9, 4/9, 2/9 and 1/9 tested cell lines respectively (Table 1). According to the National Cancer Institute (USA), 30 μg/mL is the upper IC₅₀ limit considered promising for purification of a crude extract [34]. We therefore, tested a slightly higher concentration of 40 μg/mL in our preliminary assay. Also, the IC₅₀ threshold value of 4 μg/ml or 10 μM [35, 36] after 48 and 72 h incubations has been set to identify good cytotoxic compounds. Considering these thresholds, the IC₅₀ values below or around 30 μg/mL were recorded with A. arundinaceum extract against eight of the nine tested cancer cell lines (Table 1) explaining why it was considered further for purification. Nonetheless, the extract from A. polyanthum also showed activities with IC₅₀ values <30 μg/mL on four of the nine tested cancer cell lines. Though Compound 8 was active on all the tested cancer cell lines, no IC₅₀ below 4 μg/ml was recorded, the lowest values being 7.86 μg/mL against CEM/ADR5000 cells. Interestingly, none of the selected extracts and compounds was more toxic towards AML12 normal hepatocytes (IC₅₀ > 40 Mg/mL) than cancer cell lines, suggesting their good selectivity. Importantly, collateral sensitivity (hypersensitivity) was also observed with A. arundinaceum extract towards CEM/ADR5000 cells (degree of resistance of 0.76) and U87MG.ΔEGFR (degree of resistance of 0.95) compared to their respective sensitive counterparts CEM/CEM and U87MG. This extract was also more active against hepatocarcinoma HepG2 as compared to AML12 normal hepatocytes, confirming its selectivity to cancer cells (Table 1). Despite the fact that compound 8 showed moderate activities, it also displayed better collateral sensitivity of MDR cell lines compared to doxorubicin. The use of natural products to fight multidrug resistance is an attractive strategy in chemotherapy [37‐39]. P-gp-expressing CEM/ADR5000 as well as p53 knock out HCT116 (p53 ^-/- ) and BCRP- expressing U87MG.ΔEGFR cells were less cross-resistant towards the best samples namely A. arundinaceum and compound 8 than towards the positive drug, doxorubicin, highlighting their possible therapeutic potential in the fight against multidrug resistance. This report also highlights the importance of the plants of the genus Aframomum as potential source of cytotoxic compounds. The results obtained collaborate with previous investigations. In effect, Aframomum melegueta previously inhibited the proliferation of the leukemia ADR5000 cell lines with a reported IC₅₀ value of 7.80 μg/mL [5]. Also, naringenin (8) has shown cytotoxicity in various human cancer cell lines and induced apoptosis via a transient induction of caspase-3/CPP32 activity, in the human promyeloleukemia cell line HL-60 [40‐42]. The moderate cytotoxicity of galanals A (4; IC₅₀ of 18 μM or 5.62 μg/mL) and B (5; IC₅₀ of 32 μM or 12.21 μg/mL) towards human T lymphoma Jurkat cells was also reported [29].

Finally, this work provides further evidence of the cytotoxic potential of Aframomum species and highlights the good activity of Aframomum arundinaceum on sensitive and drug-resistant cancer cell lines. Bioactive constituents of this plant include galanals A and B, naringenin and kaempferol-3,7,4’-trimethylether. Aframomum arundinaceum could be explored in more detail in the future to develop novel anticancer drugs against sensitive and resistant phenotypes.