Background
Plant-derived compounds have been extensively used in the pharmaceutical industry for the treatment of several human diseases [
1‐
3]. According to the world health organization (WHO), traditional medicine constitutes more than 80% of the total world’s population primary health care needs [
4]. Various phytochemicals such as alkaloids, flavonoids, lactones, terpenoids as well as terpenes are known to be key immunomodulators specifically as effective anti-inflammatory and anticancer agents [
5‐
8].
One family of plants that has extensive traditional use is the Annonaceae.
Annona, which is a genus of flowering plants in this sugar apple family, includes approximately 166 species and is considered the second largest genus in this family. The origin of the generic name is anόn, a Hispaniolan Taίno name for the fruit [
9,
10].
Several Annona species were found to exhibit anti-parasitic [
11], anti-inflammatory [
12], and anti-tumor effects. An extract from
A. glabra, rich in two diterpenoids displayed a cytotoxic effect on liver cancer cell lines by up-regulating the Bax to Bcl-2 expression ratio [
13] and on human leukemia cell lines in vitro [
14]
. A. squamosa chloroform seed extract also showed antitumor and pro-apoptotic effects on murine and human tumor cells through the induction of Reactive Oxygen Species (ROS) [
15].
A. muricata ethyl acetate leaf extract exhibited a mitochondrial-mediated apoptosis on colon cancer cell lines [
16] in vitro, on pancreatic cancer cells [
17] in vitro and in vivo, and on breast cancer cell lines [
18,
19] by upregulating Bax, p53 and downregulating Bcl-2 proteins. In addition, ethanolic and aqueous extracts from leaves, twigs and roots of
A. muricata showed a strong anti-proliferative potential and pro-apoptotic effect through G0/G1 cycle arrest [
20,
21].
A. cherimola, an edible subtropical fruit-bearing species is an evergreen low branched spreading tree [
22] that belongs to the Annonaceae family and is commercially cultivated for its edible fruits and traditional uses [
23]. Cherimoya, the large green fruit of the tree [
24] has an exceptional taste and is reported to have been used as an antioxidant [
25,
26] and in phytotherapy for the treatment of several ailments such as stomachache, pancreatic ulcers, skin disease [
22,
24]. The various phytochemicals present in
A. cherimola such as flavonoids, tannins, alkaloids, phytosterols, and terpenoids are traditionally utilized in the treatment of diabetes, nervous disorders and even cancer [
25,
27]. Furthermore, annomolin and acetogenins, isolated from
A. cherimola seed extracts [
28], demonstrated a cytotoxic and pro-apoptotic effect in human prostate [
29], breast [
30], and colon [
30] cancer cell lines. Moreover,
A. cherimola leaves are sold and consumed by people to improve their health, such as in the treatment of hypercholesterolemia in Azores [
31]. Other studies on ethanolic leaf extracts revealed an antitumor activity in human larynx epidermoid carcinoma cells in vitro [
32].
The current study aims to explore the anti-cancer and anti-proliferative effects of a terpene-rich A. cherimola ethanolic leaf extract on acute myeloid leukemia cell lines in vitro.
Discussion
A correlation between diet and cancer prevention has been demonstrated by the implementation of many plant extracts which exhibited anti-cancerous effects as part of the human diet [
10]. Many species classified under the
Annona genus have shown antitumor effects against several types of cancers, including cervical, breast, prostate, lung, leukemia, colorectal, renal, pancreatic cancers [
38]. Many studies have focused on the anti-proliferative effects of
Annona muricata [
16,
18,
20,
21], and
Annona squamosa [
39]. Moreover, most research conducted on
Annona cherimola leaves has focused on its anti-hyperglycemic [
40], and antiprotozoal activity [
41], with few studies conducted to assess its anti-proliferative activity. A recent study performed in our laboratory has reported the anti-proliferative effects of
A. cherimola seed extract via activation of both intrinsic and extrinsic pro-apoptotic pathways in AML cells [
35].
The aim of this study was to investigate the mechanism of action of AELE in the apoptotic pathways of the AML cell lines used (Monomac-1 and KG-1), whereby the results suggested a dose- and time-dependent anti-proliferative effect within the 24 h treatment, with an IC
50 of 333.4 μg/mL and 254.5 μg/mL for Monomac-1 and KG-1, respectively, as well as within the 48 h and 72 h treatment, with a significant time-dependent decrease in the IC
50 value, and no inhibitory effect on normal MNCs from human BM. Interestingly, AELE exhibited positive effects on the proliferation of normal cells, similar to what has been reported in the literature for other anti-carcinogenic plant extracts such as
Angelica sinensis [
42].
According to Najmuddin et al., crude leaf extracts from
Annona muricata Linn exhibited anti-cancer effects on breast cancer cell lines, with IC
50 values at 72 h post-treatment, comparable to the ones reported in this study at 24 h post-AELE treatment, thus suggesting the effectiveness of AELE [
18].
All experiments showed that AELE exhibited a dose-dependent increase in apoptosis in the two AML cell lines used. These findings were supported by an increase in DNA fragmentation, as well as the double positive Annexin V/PI staining, indicating the translocation of phosphatidylserine moieties to the outer surface of cell membrane which is a hallmark of apoptosis.
After assessing the anti-proliferative and pro-apoptotic effects of AELE, we moved to decipher the underlying molecular mechanism by which apoptosis was triggered. The results obtained revealed that AELE induces apoptosis through a Bax/Bcl2 dependent mechanism, in concordance with previous studies performed on
Annona muricata leaves. Dinardo et al. suggested the effectiveness of a selective Bcl2-inhibitor, venetoclax in treating relapsed and refractory AML patients [
43]. On the other hand, Reyna et al. developed a pharmacologically optimized Bax activator called BTSA1, which suppressed human AML xenografts, overcoming apoptosis resistance, thus suggesting that direct Bax activation is a possible treatment strategy in AML [
44]. Movement of Bax from the cytosol to the mitochondria, through the Bax pores at the mitochondrial membrane, is critical in triggering DNA damage-mediated apoptosis [
45,
46], which was observed through the dose-dependent increase in DNA fragmentation detected in cell death Elisa
. Hence, upregulation of the pro-apoptotic protein Bax detected at 346 μg/ml (at 24 h), accompanied by the downregulation of the anti-apoptotic protein Bcl2, is critical in disrupting the mitochondrial membrane potential, a hallmark of apoptosis. The effect of adding Bax/Bcl2 inhibitors was not further explored since the efficacy of available inhibitors is still controversial [
47].
Furthermore, the routine repair of DNA damage is normally controlled by PARP, which adds poly (ADP ribose) polymers in response to a variety of cellular stresses [
48]. The increase in PARP cleavage, that was observed upon AELE treatment, will lead to its inactivation, coinciding with its inability to repair DNA damage. This is in line with the dose-dependent increase in DNA fragmentation observed in Cell Death ELISA, further confirming that the cytotoxicity of AELE is indeed apoptosis-triggered.
Upon analysis of the composition of the extract, Terpinolene was found to be the major compound. Terpinolene is one of the most abundant monoterpenes, which is known for its sedative [
8]
, antifungal [
49]
, anticancer, antioxidant [
6], apoptotic [
50] activities, as well anti-inflammatory and anti-nociceptive activities in association with diclofenac [
7]. Interestingly, terpinolene, which is a main constituent of the essential oil of
Protium heptaphyllum, exhibited an anti-mutagenic activity, suggesting its potential use as a chemo-preventive agent for cancer [
51]. α-Pinene, another bicyclic monoterpene, was also found to induce cell cycle arrest in mice Xenograft models, and promote apoptosis in human prostate cancer [
52].
The second most common compound in the extract was the sesquiterpene Germacrene D. This compound was previously identified by Bomfim et al., who reported the presence of various sesquiterpenes in the essential oil extracted from
Annona vepretorum leaves. This extract exhibited in vitro antitumor effects in B16-F10 (mouse melanoma), HL-60 (human promyelocytic leukemia), K562 (human CML), and HepG2 (human hepatocellular carcinoma) cells, as well as in vivo activity [
53]. According to Shakeri et al., germacrene D was also found to be the most abundant component in
Nepeta ucrainica L. spp.
kopetdaghensis, which was found to be cytotoxic in human ovarian carcinoma A2780 and human breast adenocarcinoma MCF-1 cell lines in vitro [
54]. Furthermore, terpenes are the second most common abundant components of
Decatropis bicolor leaf extracts, which triggered apoptosis in MDA-MB-231 breast cancer cell line, through a Bax/Bcl2 dependent mechanism, translated by a dose-dependent upregulation of Bax, and downregulation of Bcl2 [
55]
, similar to what was observed in our study.
A third major component in AELE was Alpha-tocopherol, an isoform of vitamin E. Zulkapli et al. demonstrated its antitumor activity in oral squamous carcinoma cells ORL-48, whereby accumulation of cells at the sub-G0 phase, along with cell shrinkage and apoptotic bodies were reported [
56].
Another identified compound in AELE was β-sitosterol. A study by Zhao et al. reported its ability to inhibit cell growth and trigger apoptosis in SGC-7901 human stomach cancer cells in vitro, in a Bax/Bcl2 and caspase dependent manner [
57]. Similar findings were observed on U937 AML cells, involving caspase 3 activation, and an increase in the Bax/Bcl2 ratio [
58]. Other studies suggest the anti-inflammatory capacity of β-sitosterol [
59], as well as its antihyperglycemic and insulin-releasing activities [
60]. Other compounds in AELE remain unidentified and require further investigation. A study performed by Díaz-de-Cerio E. et al reports the presence of polar compounds in
Annona cherimola leaves using a combined approach of MS and NMR techniques, as well as amino acids, carbohydrates, organic acids, phenolic acids and derivatives, cholines, flavonoids and phenylpropanoids [
61].
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