Recently, a large number of literature reports on the potential use of plant extracts to inhibit cancer cell proliferation by targeting components of the PI3K/AKT/mTOR signaling pathway [50, 51, 52, 53, 54] indicating how this is an important field of research in continuous expansion. The PI3K/AKT/mTOR signaling pathway is one of the major intracellular pathways, tightly regulated under normal physiological conditions, and it is recognized to regulate both normal and malignant hematopoiesis [11]. Hyperactivation of the PI3K-AKT signaling axis drives uncontrolled cell proliferation and survival in several types of cancer while its inhibition is considered the most important factor in determining the response to chemotherapy and the outcome of AML [55]. It has been reported that patients with AML and hyperactivated AKT signaling in AML cells exhibit a worse prognosis and shorter survival time compared with patients with normal levels of activated AKT [55, 56]. Drugs targeting this pathway may prove useful in the treatment of different types of malignancy. Moreover, AML treatment remains a major challenge due to poor efficacy of the current chemotherapeutics [57]. Therefore, development of novel and more effective treatment strategies is urgently needed. Herbal remedies have been widely used in traditional medicine to support the treatment of different types of cancer. Moreover, the use of plant-derived products in the treatment of cancer may contribute to reduce adverse and toxic side effects, often observed using specific synthetic drugs. For this reason, many plants showing very promising anticancer activities in vitro and in vivo are studied, but most of the times their active anticancer principle have yet to be evaluated. Therefore, investigating plants as potential resources for antitumor agents is an increasingly important topic in cancer research. This underlines the growing interest related to the use of plant extracts as support therapeutics in this regard. For instance, flavonoids and isoflavones extracted from Tephroseris kirilowii and Astragalus membranaceus, well-known herbs used in Chinese traditional medicine, resulted in induced apoptosis by inhibiting the PI3K/AKT/mTOR pathway in various types of human breast cancer cells [50, 51]. More recently, rosemary (Rosmarinus officinalis) total extracts resulted in inhibited phosphorylation/activation of AKT and mTOR, being indicated as having potent anticancer properties [53].
In this study, the apoptotic effect of a total extract and some compounds found at relevant concentrations in Rhus tripartita, was tested in a THP-1 cell cultures. Rhus tripartita is a local pre-Saharan Tunisian plant that often grows in non-agricultural regions and it is widely used in food industry and in modern and traditional medicine. Antimalarial, antiviral, antimicrobial, antitumorigenic and atherosclerosis properties have been reported for this species [58]. Few articles report on the phytochemical composition of this plant [59] but there are still no studies that investigate which compounds are responsible for the biological activity or possible mechanisms involved. On the other side, it is complicated to compare the biochemical profiles of the Rhus tripartita described in literature, due to the different maturity stage and the climatic and environmental conditions that often influence the compositional quality and the quantity of several metabolites of the plant. In this work, the main phytochemicals present in Rhus tripartita extracts were determined and quantified. In particular, relevant concentrations of rutin and catechins were found; also, the percentage of saponins resulted to be significantly high. These compounds, and in general many of the flavonoids present in the total extract, play an important role in inhibition of the PI3K/AKT/mTOR signaling pathways. In instance, rutin, present in several aromatic and medicinal plants, is an active ingredient and has significant activity against breast, hepatic and colon cancer [36, 38]. Among the observed activities, rutin is also able to exert inhibitory effect on AKT and induce apoptosis in cancer cells. Likewise, catechins have been reported to induce apoptosis in many cancer cell lines, in some of which downregulating PI3K and AKT [60]. Data shown in the present article confirm this evidence. All treatments induced apoptosis in a concentration‑dependent manner; the number of late apoptotic cells was found to increase for rutin, kaempferol and mix of catechins according to the used concentrations. Similarly, total plant extract (RTE) and saponin fraction extract (SE) induced late apoptosis in the THP-1 cells after 48 hours, suggesting a synergistic effect of the different compounds found in the plant extract. To investigate if Rhus tripartita induction of apoptosis affects the expression of genes related to the PI3K/AKT/mTOR signaling pathway (PI3K, AKT1, PTEN, RPS6KB, TSC1, eIF4E, and mTOR), a qRT-PCR analysis was carried in treated/untreated THP-1 cells. Analysis of the PI3K, AKT1 and mTOR apoptosis-linked genes demonstrated that treatment with rutin (R), at the highest concentration, and CEEG mixture were able to significantly downregulate the expression of these genes whereas eIF4E and TSC1 genes were upregulated in THP-1 cells treated with these extracts. All the treatments downregulate the mTOR gene expression level in a concentration-dependent manner. Except for saponins and kaempferol at the highest concentration, all the treatments strongly decrease the expression level of AKT1, especially the total plant extract (RTE) and the highest concentrations of rutin and catechins mix. This agrees with other studies showing downregulation of PI3K/AKT/mTOR in response to treatments with plant phytochemicals extracted from Ricinus communis [61], Alpinia nantoensis [62], Taraxacum officinale [63]. Moreover, the eIF4E and TSC1 genes, acting as negative regulators of mTOR [64], were upregulated in our study, thus strengthening once more the inhibitory effect that Rhus tripartita extracts have on THP-1 cell proliferation. In fact, accordingly with the observed results regarding the mTOR downregulation, almost all the treatments, especially EA, CEEG mix and SE, sharply increase the expression level of TSC1. To further explore the effect of Rhus tripartita extracts and active compounds on the mTOR signaling pathway, the expression of AKT protein, S6 and their phosphorylated forms was investigated. It was observed that, with exception of SE, which decrease the phosphorylated form of the protein, western blotting analysis confirms that the highest concentrations of the CEEG mix and R, together with EA and RTE, inhibit the AKT phosphorylation. Total plant extract seems to decrease the expression of p-AKT and p-S6, indicating that plant extract may exert anticancer effects partly by inhibition of the PI3K/AKT/mTOR signaling pathway. Herein, to the best of our knowledge, it was demonstrated for the first time that Rhus tripartita total extract can inhibit cell proliferation and induce apoptosis in THP-1 cells by altered expression of PI3K, AKT, mTOR and PTEN and inhibition of AKT and S6 phosphorylation. These results suggest that the compounds contained in the total plant extracts, more than the single compound, are effective to suppressing the proliferation of AML THP-1 cells and that this may be partially mediated by the downregulation of the PI3K/AKT/mTOR signaling pathway. In conclusion, Rhus tripartita extracts represents useful candidates for development of supporting anticancer therapies, specifically in the treatment of AML, due to its ability to kill the cells and then positively regulate apoptotic mechanism.