Cytotoxicity of Plumbagin, Rapanone and 12 other naturally occurring Quinones from Kenyan Flora towards human carcinoma cells

Cancer is a major public health concern globally and chemotherapy remains the principal mode of the treatment of various malignant diseases. This study was designed to investigate the cytotoxicity of 14 naturally occurring quinones including; 3 anthraquinones, 1 naphthoquinone and 10 benzoquinones against 6 human carcinoma cell lines and normal CRL2120 fibroblasts. The neutral red uptake (NR) assay was used to evaluate the cytotoxicity of the compounds, whilst caspase-Glo assay was used to detect caspases activation. Cell cycle and mitochondrial membrane potential (MMP) were all analyzed via flow cytometry meanwhile levels of reactive oxygen species (ROS) were measured by spectrophotometry. Anthraquinone: emodin (2), naphthoquinone: plumbagin (4), and benzoquinones: rapanone (9), 2,5-dihydroxy-3-pentadecyl-2,5-cyclohexadiene-1,4-dione (10), 5-O-methylembelin (11), 1,2,4,5-tetraacetate-3-methyl-6-(14-nonadecenyl)-cyclohexadi-2,5-diene (13), as well as doxorubicin displayed interesting activities with IC50 values below 100 μM in the six tested cancer cell lines. The IC50 values ranged from 37.57 μM (towards breast adenocarcinoma MCF-7 cells) to 99.31 μM (towards small cell lung cancer A549 cells) for 2, from 0.06 μM (MCF-7 cells) to 1.14 μM (A549 cells) for 4, from 2.27 μM (mesothelioma SPC212 cells) to 46.62 μM (colorectal adenocarcinoma DLD-1 cells) for 9, from 8.39 μM (SPC212 cells) to 48.35 μM (hepatocarinoma HepG2 cells) for 10, from 22.57 μM (MCF-7 cells) to 61.28 μM (HepG2 cells) for 11, from 9.25 μM (MCF-7 cells) to 47.53 μM (A549 cells) for 13, and from 0.07 μM (SPC212 cells) to 1.01 μM (A549 cells) for doxorubicin. Compounds 4 and 9 induced apoptosis in MCF-7 cells mediated by increased ROS production and MMP loss, respectively. The tested natural products and mostly 2, 4, 9, 10, 11 and 13 are potential cytotoxic compounds that deserve more investigations towards developing novel antiproliferative drugs against human carcinoma.


Background
Cancer is a major public health problem globally killing about 3500 million people and representing 2-3% of the annual deaths [1]. Due to limited resources and other pressing public health problems, including communicable diseases such as acquired immune deficiency syndrome (AIDS), malaria, and tuberculosis, cancer continues to receive low public health priority in Africa, despite the growing burden of the disease [2]. Chemotherapy remains the principal mode of the treatment of various malignant diseases. In recent years, the search of antineoplastic compounds of natural origin has become more and more important. Many investigations are being carried out to identify new drugs or to find new lead structures from the flora of Africa to develop novel therapeutic agents for the treatment of human diseases such as cancer [3]. In our continous quest of lead molecules to fight cancer, we designed the present study to investigate the cytotoxicity of 14 quinones including 3 anthraquinones, one naphthoquinone and 10 benzoquinones, previously isolated from African medicinal plants.

Neutral red (NR) uptake assay
The cytotoxicity of samples was performed by NR uptake assay as previously described [11,12]. This method is based on the ability of viable cells to incorporate and bind the supravital dye NR in the lysosomes. The procedure is cheaper and more sensitive than other cytotoxicity tests [13]. Samples were added in the culture medium so that dimethylsufoxide (DMSO) used prior for dilution did not exceed 0.1% final concentration. Briefly, cells were detached by treatment with 0.25% trypsin/EDTA (Invitrogen) and an aliquot of 1 × 10 4 cells was placed in each well of a 96-well cell culture plate (Thermo Scientific, Germany) in a total volume of 200 μL. The cells were allowed to attach overnight and subsequently treated with different concentrations of the 14 compounds. Each of the studied samples was immediately added in varying concentrations in additional 100 μL of culture medium to obtain a total volume of 200 μL/well. After 72 h incubation in humidified 5% CO 2 atmosphere at 37°C, the medium was removed and 200 μL fresh medium containing 50 μg/mL NR was added to each well and incubation continued for an additional 3 h at 37°C in 5% CO 2 atmosphere. The dye medium was then removed and each well was then washed rapidly with 200 μL phosphate buffer saline (PBS) followed by addition of 200 μL of acetic acidwater-ethanol in water (1:49:50). The plates were kept for 15 min at room temperature to extract the dye and then shaken for a few minutes on a GFL 3012 shaker (Gesellschaft für Labortechnik mbH, Burgwedel, Germany). Absorbance was measured on ELx 808 Ultra Microplate Reader (Biotek) equipped with a 540 nm filter. Each assay was done at least three times, with three replicates each. The viability was evaluated based on a comparison with untreated cells. The IC 50 values represent the sample's concentrations required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel [14].

Caspase-Glo 3/7 and caspase-Glo 9 assay
Caspases activity in MCF-7 cells was detected using Caspase-Glo 3/7 and Caspase-Glo 9 Assay kits (Promega, Mannheim, Germany) as previously reported [15][16][17]. Cells were treated with compounds 4 and 9 at their 2 × IC 50 and IC 50 values with DMSO as solvent control for 6 h. Luminescence was measured using an BioTek Synergy™ HT multi-detection microplate reader. Caspase activity was expressed as percentage of the untreated control.
Subsequently, cells were measured in a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). The JC-1 signal was measured at an excitation of 561 nm (150 mW) and detected using a 586/ 15 nm band-pass filter. The signal was analyzed at 640 nm excitation (40 mW) and detected using a 730/ 45 nm bandpass filter. Cytographs were analyzed using BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson). All experiments were performed at least in triplicate.

Results
The fourteen investigated compounds included three anthraquinones; chrysophanol C 15

Cytotoxicity
The cytotoxicity of the 14 quinones and doxorubicin was determined by the NR uptake assay and the recorded IC 50 values are summarized in Table 1. The selectivity index was determined as the ratio of IC 50 value in the CRL2120 normal fibroblast divided by the IC 50 in the cancer cell line. Compounds 2, 4, 9, 10, 11 and 13 as well as doxorubicin displayed IC 50 values below 100 μM in the six tested cancer cell lines. Compounds 3, 5 and 12 were not active with IC 50 values above 120 μM in all cancer cell lines meanwhile 1, 6, 7,

Cell cycle analysis and apoptosis
Naphthoquinone 4 and benzoquinone 9 were analyzed for their ability to alter the distribution of the cell cycle of MCF-7 breast cancer cells (Fig. 2). It was observed that the two compounds induced concentrationdependent cell cycle modifications with progressive increase of sub-G0/G1 phase cells. Compounds 4 and 9 induced cell cycle arrest between G0/G1 and S phases. MCF-7 cells treated with the compounds 4 and 9 progressively underwent apoptosis, with increase of sub-G0/ G1 cells from 10.4% (¼ IC 50 ) to 20.4% (IC 50 ) for 4 and from 34.8% (¼ IC 50 ) to 43.2% (IC 50 ) for 9. The positive control, doxorubicin also caused up to 60% sub-Go/G1 phase with IC 50 treatment in comparison to only 3.1% in non-treated cells.

Caspases activities
Upon treatment of MCF-7 cells with naphthoquinone 4 and benzoquinone 9 with equivalent (eq.) to the IC 50 and 2-fold IC 50 for 6 h, no modification of the activity of caspase 3/7 and caspase 9 was observed (data not shown).

MMP breakdown
Treatment of MCF-7 cells with compounds 4 and 9 with eq. to the 1/4 × IC 50 , 1/2 × IC 50 and IC 50 values for 72 h induced concentration-dependent depletion of MMP (Fig. 3). More pronounced effect was observed with 9 with up to 88.1% depletion of MMP at eq. to IC 50 while 4 caused 12.2% MMP loss at IC 50 . In similar experimental condition, doxorubicin caused 26% loss of MMP meanwhile only 4.3% was observed with non-treated control.

ROS production
After treatment of MCF-7 cells with naphthoquinone 4 and benzoquinone 9 at eq. to the 1/4 × IC 50 , 1/2 × IC 50 and IC 50 values for 24 h, the production of ROS in cells was analyzed (Fig. 4). Naphthoquinone 4 induced increased ROS levels of more than 3-fold (at IC 50 ) as compared with non-treated cells meanwhile the increase was lesser (less than 2-fold) after treatment with benzoquinone 9. In similar experimental condition doxorubicin also induced more than 2-fold increase in ROS production in MCF-7 cells at eq. to IC 50 .

Discussion
Neoplastic diseases are one of the leading causes of mortality worldwide and the number of cancer cases are increasing regularly [1]. In general, leukemia cells are clinically more sensitive to chemotherapy than tumors [18,19]. In the present study we focused on carcinoma cells involved in lung, colon, breast and liver cancers. In regards of the broad diversity of phytochemicals, the search of anticancer agents from plants represents an attractive strategy [20]. Molecules having IC 50 values around or below 4 μg/mL or 10 μM [3,21,22] have been recognized as potential cytotoxic substances. IC 50 values below 10 μM were observed with naphthoquinone 4 in all the six cancer cell lines. Interestingly, IC 50 values below 1 μM were obtained with this compound in 4 of the 6 cancer cell lines, highlighting its good cytotoxic potential. In addition, the IC 50 values obtained with 4 towards Caco-2 cells and MCF-7 cells were lower than that of the reference compound, doxorubicin. Other compounds such as 8, 9 and 10 against SPC212 cells as well as 13 towards MCF-7 also displayed IC 50 values below 10 μM, suggesting that they can be useful in the management of human carcinoma. Moreover, they were more toxic towards carcinoma cells than towards normal CRL2120 fibroblast (selectivity index > 1), indicating their good selectivity. The good activity obtained with naphthoquinone 4 is in accordance with previous studies. In fact, 2-acetylfuro-1,4-naphthoquinone previously displyed good cytotoxicity with IC 50 values below 10 μM against a panel of cancer cell lines such as PF-382 leukemia T-cells, MiaPaCa-2 pancreatic cells, U87MG glioblastoma-astrocytoma cells, Colo-38 skin melanoma cells, HeLa and Caski cervical carcinoma cells [8]. Also, compound 4 is well known for its remarkable anticancer activities [6]. The present study therefore provides additional data on the anticancer potential of this compound and highlights the role of naphthoquinones as good cytotoxic compounds. The moderate cytotoxic effects of some anthraquinones such as damnacanthal, damnacanthol, 3-hydroxy-2-hydroxymethyl anthraquinone and schimperiquinone B on a panel of cancer cell lines was documented [9]. In the present study, the moderate to low activities obtained with anthraquinones 1-3 are also in accordance with such results. Induction of apoptosis is recognized as an efficient strategy for cancer chemotherapy and a useful indicator for cancer treatment and prevention. In the present study, it was found that compounds 4 and 9 induced apoptosis in MCF-7 cells (Fig. 2). Hence, further investigations of the mode of induction of apoptosis were performed. Caspases regulate apoptosis by cleaving cellular proteins at specific aspartate residues [23]. The activity of initiator caspase 9 and effector caspases 3/7 were investigated in MCF-7 cells treated with 4 and 9. However, it was found that caspase-dependent cell death may not be one of the pathways of induction of apoptosis by 4 and 9 in MCF-7 cells. Loss of MMP is also classical evidence for apoptosis, occuring during the early stage of apoptosis before the cell morphology changes. The disruption of MMP was suggested to be very strong at percentages above 50%, and strong between 20 and 50% [3]; Up to 88.1% (at IC 50 ) MMP depletion was obtained, when MCF-7 cells were treated with IC 50 concentrations of 9, suggesting that MMP depletion is involved in apoptotic pathway induced by this compound. ROS levels between 20 and 50% are considered as high [3]; More than 3-fold increase in ROS production was also obtained as results of treatment of MCF-7 with compound 4. However, only 12.2% MMP depletion was obtained with this compound, suggesting that increase in ROS production the likely mode of apoptosis induced by naphthoquinone 4. The tetraprenylquinone, sargaquinoic acid was shown to  [7]. However, it was demonstrated in this study that the related compound 9 induced apoptosis mediated by MMP loss but did not induced increase in the activity of caspases 3 and 9. Herein, it was also shown that compound 4 induced MMP loss in MCF-7 cells. Nonetheless, the induction was moderate. Compound 4 was also reported to induce apoptosis in PC-3 and DU145 cells, mediated by MMP loss [24]; this corroborates the results obtained in this work.
Regarding the structure-activity relationship, it appears that the naphthoquinone 4 was more potent in all six tested cancer cell lines than anthraquinones (1-5) and benzoquinone (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Within anthraquinones, the substitution of hydroxyl (−OH) group in C8 (1 and 2) by a methyl group (3) significantly reduced the cytotoxic activity meanwhile the presence of − OH group in both C8 and C6 (2) seems to increase the activity. Consequently, IC 50 values were obtained with 2 on all tested cancer cells lines and 1 on 2/6 ( Table 1). Within benzoquinones, the degree of activity seems to increase with the size of the lateral chain in C2, the best effects being obtained between n = 10 (11) to n = 14 (10). However, compound 9 with n = 12 displayed better cytotoxic effects than 10 and 11 in all tested cancer cell lines (Table 1), most probably because of its higher lipophilicity.