Background
Malignant diseases still constitute one of the major health problems worldwide, despite considerable progress in cancer therapy in recent years. Globally, about 14.1 million new cancer cases and 8.2 million deaths were reported in 2012 [
1,
2], with lung, breast, colon, prostate and liver cancers being the most occurring neoplasia [
3]. The mortality related to breast cancer reached 459,000 victims in 2008 [
4] meanwhile colorectal cancer was reported as second killing cancer [
5]. Every year, lung adenocarcinoma also kills more than one million persons and appears in top in cancer-related death [
6]. The mortality assigned to liver cancer was 696,000 deaths in 2008 [
7]. The fight against these types of cancers as well as many others is therefore of interest and should be intensified. The use of natural products has been and continues to be one of the most effective ways to fight cancers. Various compounds from African flora displayed prominent cytotoxic effects in vitro in many cancer cell lines [
8,
9]. Within our drug discovery research program, the present study was planned to investigate the cytotoxicity of a panel of 14 natural products previously isolated from the Cameroonian medicinal plant
Garcinia epunctata Stapf (Guttiferae) [
10],
Ptycholobium contortum (
N.E.Br.) Brummitt (Leguminosae) from Botswana [
11,
12], and freshly isolated from
Synsepalum zenkeri Engl. ex Aubrév. & Pellegr. (Sapotaceae) harvested in Cameroon. The study was extended to the analysis of the mode of action of the two most active samples, namely the flavonoid thonningiol and the pterocarpan isoflavonoid, seputhecarpan D. Various flavonoids and pterocarpan isoflavonoids from African medicinal plants previously showed antiproliferative effects. Some prominent anticancer flavonoids of the flora of Africa previously identified in our cancer research program include isobavachalcone, 4-hydroxylonchocarpin [
13,
14], 6,8-diprenyleriodictyol [
13], cycloartocarpesin [
14] and gancaonin Q [
13] meanwhile examples of good cytotoxic pterocarpan isoflavonoids are sophorapterocarpan A and isoneorautenol [
15,
16].
Discussion
In the present investigation, we assessed the ability of 14 phytochemicals from African medicinal plants to prevent the proliferation of four cancer cell lines namely breast, colon, lung and liver cells. They are amongst the frequently diagnosed cancers globally [
3]. The threshold recognized for a good phytochemical is the IC
50 values around or below 4 μg/mL or 10 μM as defined by the National Cancer Institute (NCI) [
8,
30,
31]. Amongst the 14 tested compounds, six (
3, 9–13) had recordable IC
50 values in all the four tested cancer cell lines.
To the best of our knowledge, the cytotoxicity of compounds
9 and
11 is being reported for the first time here. The triterpenoid16
β-hydroxylupeol (
3) is a hydroxylated derivative of the known cytotoxic compound, lupeol. In effect, the cytotoxicity of lupeol has been reported in several cell lines including colorectal cancer, gastric cancer or liver cancer cells [
32‐
35]. The cytotoxicity of compound
3 is therefore in accordance with published literature and this work provides more information on the activity of lupeol derivatives. The cytotoxicity of compounds
10, 12 and
13 was reported against A549 and SPC-212 lung cancer cells [
12]. The present work brings additional data on the cytotoxic potential of these compounds. In this study, IC
50 values below 10 μM were obtained with compounds
9 and
13 in 4/4 and 3/4 tested cell lines respectively. This is an indication that these two molecules have promising cytotoxic potential.
Cysteine-aspartic proteases commonly known as caspases are protease enzymes essential for programmed cell death and inflammation [
36]. In this study, it was found that phytochemicals
9, 13 as well as doxorubicin induce apoptotic cell death in MCF-7 cells with increase in caspases 3/7 and 9 activities (Figs.
2 and
3). These data indicate that activation of caspases is one of the modes of action of compounds
9 and
13. Flavonol such as isorhamnetin was also shown to induce apoptosis in cancer cells by activation of caspases [
37,
38], consolidating the results obtained with compound
9 belonging to the class of dihydroflavonol. However, isorhamnetin rather exerted cell cycle arrest in G2/M in HCT116 colon cancer cells contrary to compound
9 that exerted arrest in G0/G1 as observed in this study [
38]. Also, pterocarpan isoflavonoids such as sophorapterocarpan A and isoneorautenol previously induced apoptosis in CCRF-CEM leukemia cells through activation of caspases 3/7, 8 and 9 as well as the loss of MMP and increased ROS production [
15,
16]. In this study, it was also found that phytochemicals
9 and
13 slightly induced MMP alteration but enhanced ROS production in MCF-7 cells (Figs.
4 and
5). The activation of caspases 3/7 (effector caspases) and 9 (initiator caspases) (Fig.
3) as well as the low alteration of MMP, probably due to the low concentrations of compounds tested (¼ × IC
50, ½ × IC
50 and IC
50), is an indication that intrinsic mitochondrial pathway could be involved in the cytotoxic effect of compounds
9 and
13 [
39]. Mitochondria play a central role in cellular metabolism as main ATP source, and during ATP biosynthesis, ROS are generated. Mitochondria-targeting compounds kill cancer cells due to their ability to initiate mitochondrial outer membrane permeabilization [
9,
40]. This also indicates that ROS production is another mode of apoptotic cell death induced these two phytochemicals.
Regarding the structure-activity relationship, it appears that the best spectra of activity were achieved with triterpenoid
3, flavonol
9 and the 4 tested isoflavonoids
10–13. Five terpenoids including one steroid (
1) and four trierpenoids (
2–5) were tested. The steroid
1 as well as triterpenoids
2,
4 and
5 had low and selective cytotoxic effects (Table
1). Only triterpenoid
3 with two hydroxyl (-OH) group in C3 and C16 was active towards the four tested cancer cell lines. Hydroxylation of triterpenoids therefore seems to increase the cytotoxic effect. However, steroid
1 with only one –OH group in C3 had poor activity. Flavonoids tested herein included glycosylated flavonols
7 and
8 as well as non glycosylated flavonol
9. Amongst them, compounds
7 and
8 had low and selective cytotoxicity, with a recordable IC
50 values obtained only towards MCF-7 cells. In contrast, dihydroflavonol
9 had significant cytotoxic effect (IC
50 values below 10 μM) in 4/4 tested cancer cell lines (Table
1). It can therefore be deduced that glycosylation of flavonoids in C3 significantly reduces their cytotoxic effect. The four tested isoflavonoids (
10–13) were active in all cancer cell lines. Amongst them, pterocarpan
13 had the best activity with IC
50 values below 10 μM against 3/4 cancer cells. Contrary to pterocarpans
11 and
12, pterocarpan
13 do not have an additional furo- cycle; this is an indication that additional furo- cycle of pterocarpans reduces the degree of activity. Between pterocarpans
11 and
12, the substitution of 4’-OH by 4’-OCH
3 seems not to significantly influence their activity (Table
1). The only tested xanthone (
14) displayed selective cytotoxicity. However, this effect was significant towards Caco-2 cells (IC
50 value of 3.15 μM).