Targeting the PI3K/Akt/mTOR axis by apigenin for cancer prevention

Abstract

Natural products are important sources of anti-cancer lead molecules, and high dietary consumption of fruits and vegetables is associated with a reduced risk of certain cancers. Many efforts have been devoted to identifying and developing plant-derived dietary constituents as chemopreventive agents. Among them, apigenin, a naturally occurring flavonoid found in a variety of fruits and leafy vegetables, has been shown to possess remarkable anti-oxidant, anti-inflammatory and anti-carcinogenic properties. This review summarizes the anti-cancer and chemopreventive effects of apigenin at cellular and molecular levels, its chemical structure and properties, with focus on mechanism related to apigenin’s inhibition of the PI3K/Akt/mTOR signaling pathways.

1. Introduction

Cancer is a group of diseases characterized by uncontrolled growth and spread of malignant cells. In the United States alone, in addition to more than 2 million skin cancers that are diagnosed annually, a total of 1,638,910 new cancer cases and 577,190 deaths are expected to be reported in the year 2012 [1]. Cancer has become a major public health burden, and currently major treatments for cancer are still surgery, chemotherapy, radiation therapy and immunotherapy [2–4]. However, drug- and radiation-related harmful side effects are common, and not all cancers are surgically curable. In contrast, cancer chemoprevention is a rapidly growing approach that uses naturally occurring or synthetic agents to prevent, inhibit or reverse tumorigenesis or suppress the development of invasive cancer [5–7]. This approach decreases the incidence of cancer and deaths from cancer at an early stage and relies on prevention rather than cure. It is also well known that many human cancers are induced by environmental factors including chemical, radioactive and biological factors, and there are significant differences in the cancer incidence and mortality rates among different racial and ethnic groups that have different lifestyles and have been exposed to different environmental factors [1]. Epidemiologic studies have shown an inverse association between consumption of vegetables/fruits and risk of human cancers at many sites [8–11]. Some dietary components such as dietary fiber, micronutrients and polyphenolic compounds have shown inhibitory effects on human cancers [12–14].

Polyphenolic compounds are produced as the result of the secondary metabolism of plants and are frequently found attached to sugars (glycosides), although occasionally polyphenols occur in plants as aglycones [15]. More than 8000 ployphenolic structures are know so far, and polyphenols are divided into at least 10 different classes based on their structure [16, 17]. Flavonoids are the largest class of polyphenols, comprising about 5000 compounds with a common structure of diphenylpropanes (C6-C3-C6) and one or more hydroxyl substitutes. Flavonoids can be further subdivided into six major subclasses, namely flavones, isoflavones, flavonols, flavanones, flavanols (catechins), and anthocyanidins [17–19]. Flavonoids are widely distributed and ubiquitously present in foods of plant origin, such as vegetables, fruits, tea, cocoa, etc [15]. The subclasses of flavones and flavonols are structurally similar compounds, with flavonols having an extra hydroxyl substitution at the carbon-3 position, and flavone apigenin and flavonol quercetin are the frequently occurring compounds in foods [15].

Flavonoids have been the focus of a great deal of scientific interest because they exert a variety of biological effects, such as free radical scavenging, anti-oxidant activity, anti-inflammation, anti-cancer activity, as well modulating enzymatic activity, inhibiting cellular proliferation, inducing of apoptosis, inhibiting platelet aggregation and reducing plasma levels of low-density lipoproteins [19–22]. These effects may help to explain flavonoids’ potential benefit in cancer chemoprevention. In this article, we explore one common flavonoid – apigenin - and its chemical structure and properties, specifically focusing on mechanism related to apigenin’s anti-cancer and chemopreventive effects at cellular and molecular levels, particularly apigenin’s inhibition of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathways.

2. Chemical structure and properties of apigenin

Apigenin (5,7,4′-trihydroxyflavone) is a naturally occurring flavonoid (Fig. (1)) belonging to the flavone subclass and is abundantly present in common fruits (oranges, apples, cherries, grapes), vegetables (onions, parsley, broccoli, sweet green pepper, celery, barley, tomatoes) and beverages (tea, wine) [15, 23, 24]. Apigenin was first structurally identified in 1900, and synthesized in 1939 [25]. In its pure form, apigenin is a yellow crystalline solid, with a melting point of 347.5 °C and molecular weight of 270.24 g/mol [23]. Apigenin is soluble in dimethylsulfoxide (DMSO) (>100 mg/mL), it is slightly soluble in acetone and alcohols such as ethanol, n-octanol, and propylene glycol (1.02–1.63 mg/mL). Apigenin is practically insoluble in highly polar solvents such as water (0.00135 mg/mL), and nonpolar solvents such as silicon fluid (0.0728 mg/mL) and safflower oil (0.0317 mg/mL) [25]. In its natural form, apigenin is present in foods mostly as glycoside conjugates, which are more water soluble than its pure form [15]. These glycosides are efficiently hydrolyzed in vivo by bacterial enzymes in the human intestinal tract to the free flavonoids [26].

Fig. 1.

Chemical structure of apigenin.

Recent reports have shown that bioavailability of specific flavonoids can be quite substantial. Dietary intakes of flavonoids in humans vary from low in Western countries (e.g. 13 mg/day in the USA) to high in Asia (e.g. 64 mg/day in Japan) [27]. Apigenin appears to be absorbable by humans after intake of parsley, an apigenin-rich food. In a randomized crossover study with two one-week intervention periods in succession, volunteers consumed a diet that included 20 g/day parsley. The urinary excretion of apigenin was significantly higher in the parsley-consumption group than in the basic diet control group. The half-life for apigenin was calculated to be on the order of 12 h, although significant individual variation in the bioavailability and excretion of apigenin was observed [28]. Apigenin derived from aqueous alcoholic extracts of chamomile flower heads was found to be concentrated in the stratum corneum within the first 2 h of dermal exposure in human subjects. After 3 h, a steady state was attained, suggesting that apigenin diffuses through deeper skin layers to be absorbed afterwards by cutaneous blood and lymph vessels [29]. Apigenin feeding by gavage to mice at a dose of 20 μg/mouse/day has been reported to achieve 0.63–0.78 μM apigenin in plasma [30, 31], and this dose is comparable to the daily consumption of flavonoid in humans as reported previously [9, 32]. Cai et al. compared tissue and plasma levels of apigenin fed to mice in diet containing apigenin, and reported that feeding 0.2% apigenin in diet for 7 days achieved steady-state tissue concentrations of 1.5 μM and 86 μM in the liver and small intestinal mucosa, respectively [33].

Compared to other flavonoids, such as quercetin, apigenin is relatively nontoxic and nonmutagenic [34, 35]. Birt et al. first reported that apigenin has anti-mutagenesis and anti-promotion properties in mouse skin [36]. Her laboratory further demonstrated that apigenin can inhibit skin cancer induced by either ultraviolet (UV) radiation [37] or chemicals [38] in mice. In these studies they found that topical application of apigenin to mouse skin resulted in inhibition of UV- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, a tumor promotion marker, and reduced cancer incidence as well as increased cancer-free survival in mice [37, 38]. These initial studies with apigenin generate further interest in the development of apigenin as a chemopreventive agent.

3. Molecular mechanisms of apigenin’s chemopreventive activity

The chemopreventive effect of apigenin is not limited to the skin carcinogenesis model. Following the reports by Birt et al.[37, 38], studies in numerous laboratories showed that apigenin displayed a wide variety of anti-carcinogenic effects in breast, prostate, colon, and cervical cancer cells, etc (ref. [39] and references therein). Table 1 shows the effect of apigenin on various human cancers. In short, apigenin’s chemopreventive activity in multiple organ sites is likely tied to its anti-mutagenic, anti-oxidant, anti-inflammatory, and anti-carcinogenic properties.

Table 1.

Protective effects of apigenin on different human cancers and cell lines

Cancer types	Apigenin’s effects	Literature references
Breast cancer	Inhibition of proliferation, induction of apoptosis, suppression of cell invasion, anti-estrogenic activities	[40–43]
Prostate cancer	Induction of apoptosis and G1 phase arrest of cell cycle, inhibition of cell growth, suppression of HIF-1α expression, inhibition of VEGF, inhibition of FAK/src	[44–48]
Colon cancer	Inhibition of cell growth, induction of G2/M cell cycle arrest, increase in the stability of p53 protein, induction of apoptosis	[49–51]
Cervical cancer	Inhibition of cell growth through G1 cell cycle arrest and apoptosis, suppression of motility and invasion	[52, 53]
Skin cancer	Induction of autophagy, enhancement of apoptosis, inhibition of COX-2 expression, suppression of MMP-1 production	[54–57]
Lung cancer	Suppression of VEGF transcriptional activation, induction of apoptosis	[58, 59]
Ovarian cancer	Inhibition of proliferation and VEGF expression, suppression of migration and invasion	[60–62]
Liver cancer	Inhibition of cell growth, induction of apoptosis, enhancement of radiation-induced cell death	[63–65]
Pancreatic cancer	Inhibition of Focal Adhesion kinase activation, suppression of HIF-1α, VEGF and Geminin expression, induction of G2/M cell cycle arrest	[66–69]
Hematologic cancer	Inhibition of proliferation, induction of apoptosis, suppression of telomerase activity	[70, 71]

The anti-mutagenic effect of apigenin has been shown to inhibit benzo[a]pyrene and 2-aminoanthracene-induced bacterial mutagenesis [36, 72]. Apigenin was also shown to be anti-mutagenic in the Ames assay and in Chinese hamster V79 cells [36]. Apigenin significantly induces glutathione S-transferase (GST), an enzyme which protects cells against free-radical damage by increasing resistance to oxidative stress caused by hydrogen peroxide [73]. GST also plays a protective role against cancer by detoxifying xenobiotics with mutagenic potential [74]. Therefore, apigenin may both relieve oxidative stress and aid in the detoxification of mutagenic xenobiotics.

The anti-inflammatory effect of apigenin has been demonstrated in numerous studies. Apigenin suppresses TPA-mediated cyclooxygenase-2 (COX-2) expression by blocking Akt signal transduction and arachidonic acid release in human keratinocytes [56]. COX-2 is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis [75]. In mouse keratinocytes, research has shown that one pathway by which apigenin inhibits UV-induced COX-2 expression is through modulation of Upstream Stimulatory Factor (USF) transcriptional activity in the 5′ upstream region of the COX-2 gene [76]. Furthermore, two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), were found to be associated with endogenous COX-2 mRNA, and apigenin treatment increased their translocation to cell cytoplasm. More importantly, cells expressing reduced TIAR showed marked resistance to apigenin’s ability to inhibit UVB-induced COX-2 expression [77]. Taken together, these results indicate that in addition to transcriptional regulation and inhibition of Akt, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation. In another study, Nicholas et al. reported that apigenin blocked proinflammatory cytokine expression (such as IL-1beta, IL-8, and TNF) by inactivating NF-kappaB through the suppression of p65 phosphorylation [78].

The anti-carcinogenic properties of apigenin are related to its ability to modulate key targets and pathways involved in cell cycle control, apoptosis, angiogenesis, tumor cell invasion and metastasis, and signal transduction [39]. Studies have provided evidence that apigenin inhibits cell growth by inducing a reversible G₂/M arrest and that this arrest was associated, at least in part, with inhibited activity of p34(cdc2) kinase and reduced accumulation of p34(cdc2) and cyclin B1 proteins [49, 79], which was also found independent of p21/WAF1 protein [80]. In addition, apigenin treatment produced a G₁ cell cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb, and inducing the cyclin-dependent kinase (cdk) inhibitor p21/WAF1 [81]. Since p21/WAF1 is a well known downstream effector of the p53 tumor suppressor gene, it is not surprising to find that apigenin increased wild-type p53 protein expression by a mechanism involving p53 protein stabilization [82] and enhancement of p53 mRNA translation through the RNA binding protein HuR [83].

Apigenin has been shown to induce apoptosis in different types of cells [46, 70, 84, 85]. In human keratinocytes and organotypic keratinocyte cultures, apigenin treatment enhanced UVB-induced apoptosis more than 2-fold. In addition, apigenin stimulated changes in Bax localization, and increased the release of cytochrome c from the mitochondria. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in apigenin-induced apoptosis, demonstrating that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways [55]. In human prostate cancer cells, apigenin treatment has been shown to alter the Bax/Bcl-2 ratio in favor of apoptosis [46]. In human promyelocytic leukemia HL-60 cells, apigenin induced caspase-3 activity and cleavage of poly-(ADP-ribose) polymerase (PARP), reduced mitochondrial transmembrane potential, released mitochondrial cytochrome c into the cytosol, and subsequently induced procaspase-9 processing [70]. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. Apigenin breaks TRAIL resistance by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 [51].

Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths, supplying nutrients and oxygen and removing waste products [86]. Given the role of angiogenesis in tumor growth and progression, apigenin has been tested for inhibition of angiogenesis. Apigenin has been reported to be a potent angiogenesis inhibitor through its inhibitory effect on the inflammatory cytokine IL-6/STAT3 pathway. Apigenin also modulated the activation of extracellular signal-regulated kinase-1/2 (ERK) signaling triggered by IL-6, as well markedly reducing proliferation, migration and morphogenic differentiation of endothelial cells. More interestingly, it also modulated the expression of IL-6 signal transducing receptor (IL-6Rα) and the secretion of the extracellular matrix degrading enzyme MMP-2 [87]. Platelet-derived growth factor (PDGF)-dependent recruitment of mural cells such as pericytes and smooth muscle cells plays a central role in the maturation and stabilization of newly formed vasculature during angiogenesis. Lamy et al. demonstrated that apigenin interfered with this event through its inhibitory effect on PDGF-dependent phosphorylation of PDGF receptor beta (PDGFR-beta) [88]. In another study, Fang et al. showed that apigenin inhibits tumor angiogenesis through decreasing hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in different types of cancer cells via the PI3K/Akt/p70S6K1 and HDM/p53 pathways [61].

Most cancer deaths are attributed to metastatic disease rather than primary, organ-confined cancers. Apigenin inhibits migration and invasion in breast cancer cells [89] and melanoma cells [90]. To study the effect of apigenin via oral administration on tumor growth and metastasis, He et al. developed an orthotopic ovarian tumor model in nude mice, and found that apigenin inhibited the micrometastasis of cancer cell by blocking MMP-9 expression, which was mediated by the Akt/p70S6K1 pathway [91]. Apigenin also inhibited expression of focal adhesion kinase (FAK) and migration and invasion of human ovarian cancer A2780 cells [62]. Similarly, Franzen et al. also observed that apigenin inhibited FAK activation and altered cell cytoskeleton in human prostate cancer cells (PC3-M). Overexpression of constitutively active Src blunted the effect of apigenin on cell motility and cytoskeleton remodeling [48].

Apigenin has also been shown to counteract tumor promoter-mediated inhibition of intercellular communication. When exposed to apigenin and either TPA or butylated hydroxytoluene (BHT), rat liver epithelial cells exhibited an increase in gap junctional intercellular communication (GJIC), reversing the GJIC inhibition mediated by the tumor promoters [92].

As mentioned above, anti-carcinogenic properties of apigenin have been attributed to its ability to inhibit UV- and chemical-induced ODC activity [37, 38]. In addition, apigenin has been found to modulate other enzymatic activity related to tumorigenesis. For example, Le Bail et al. demonstrated that apigenin was an effective inhibitor of aromatase (human estrogen synthetase) and 17β-hydroxysteroid dehydrogenase activities in human placental microsomes, suggesting that it may be beneficial in treatment of human breast cancer [93]. In keratinocyte and colon carcinoma cell lines, apigenin induced a dose-dependent phosphorylation of both (ERK) and p38 kinase but had little effect on the phosphorylation of c-jun amino terminal kinase (JNK), and immunoprecipitation-coupled kinase assays showed that apigenin increased the kinase activity of ERK and p38 but not JNK [94]. Apigenin has further been shown to inhibit casein kinase (CK)-2 in both breast and prostate cancer cells [95, 96]. In addition, apigenin significantly inhibited protein kinase C (PKC) activity [97], and in the human anaplastic thyroid carcinoma cell line (ARO), apigenin treatment decreased the expression of EGF receptor tyrosine kinase as well as MAP kinase [98]. Taken together, these studies could provide mechanistic insight into developing novel strategies for cancer chemoprevetion by apigenin.

4. Inhibition of PI3K/Akt and mTOR signaling pathways by apigenin

The PI3K/Akt and the mTOR signaling pathways are two interdependent pathways that play important roles in the regulation of cell growth, proliferation, and survival. Aberrant activation of these pathways has been linked to cancer development and is frequently detected in malignancies. PI3K are lipid kinases divided into three classes and characterized by their substrate specificity and lipid products [99]. Class IA PI3K is composed of a catalytic p110 subunit and a regulating p50/p55/p85 subunit, and converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) [100]. Mammalian cells also express three Akt protein kinases (Akt1-3), which share >80% amino acid sequence identity and are encoded by different genes [101]. Akt binds to PIP3 via its pleckstrin homology domain and translocates to the plasma membrane, where 3-phosphoinositide-dependent kinase-1 (PDK1) phosphorylates Akt on T308 and activates Akt [102]. Once activated, Akt phosphorylates a broad range of proteins involved in apoptosis, cell cycle regulation, growth and survival. The PI3K/Akt pathway is negatively controlled by several phosphatases. Among them, the 5′-phosphatase and tensin homologue (PTEN) lipid phosphatase antagonizes the PI3K action on PIP3, and genetic inactivation of PTEN leads to constitutive activation of the PI3K/Akt pathway in many cancer cells [103].

In mammalian cells, mTOR functions as two distinct complexes, mTORC1 and mTORC2. Besides mTOR, mTORC1 contains the regulatory-associated protein of mTOR (Raptor), mLST8, and proline-rich Akt substrate 40 kDa (Pras40). The mTORC1 is sensitive to rapamycin, and regulates cell growth by controlling ribosome biogenesis, protein and lipid synthesis, and autophagy. The two best-known substrates of mTORC1 are the ribosomal protein S6 kinase (P70S6K) and the eukaryotic initiation factor 4E binding protein (4E-BP1). The mTORC1 pathway can be activated by the tuberous sclerosis complex (TSC1/TSC2/Rheb) axis [104]. mTORC2 is composed of mTOR, mLST8, mSIN1, and the rapamycin-insensitive companion of TOR protein (Rictor) [104]. mTORC2 has a PDK2 kinase activity and phosphorylates Akt on S473 that is required for its full activation [105]. mTORC2 also stabilizes Akt by constitutive phosphorylation of its turn motif on T450. mTORC2 is generally thought to be rapamycin-insensitive although a long exposure time to high concentrations of this drug may prevent its assembly and inhibit Akt phosphorylation on S473 [106].

The PI3K/Akt and mTOR signaling pathways are closely interdependent. Akt positively regulates mTORC1 by acting at different levels: First, Akt inactivates TSC1/TSC2 by phosphorylating TSC2, a negative regulator of mTORC1 [107]. Second, Akt inhibits Pras40, another negative regulator of mTORC1 [108]. Third, Akt regulates intracellular ATP levels and suppresses AMP-activated protein kinase (AMPK), which inhibits mTORC1 [109]. On the other hand, mTORC1 activation induces a negative feedback loop toward the PI3K/Akt pathway: Phosphorylated P70S6K, a downstream substrate of mTORC1, phosphorylates insulin receptor substrate 1 (IRS-1), leading to their proteasomal degradation and limits PI3K activation [104]. In addition, as mentioned above, PDK1 phosphorylates Akt on T308, but full activation of Akt kinase activity requires its phosphorylation on S473 by mTORC2 [110].

Apigenin has been shown to inhibit Akt function in different cell types by directly suppressing PI3K activity through blocking the ATP-binding site of PI3K, and subsequently inhibiting Akt kinase activity [111]. Recently, Zhao et al. demonstrated that apigenin inhibited CK2 activity, reduced phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/kinase client complex, and thus induced degradation of multiple kinase clients including Akt [112]. Many of the chemoprevetive effects of apigenin are related to its inhibition of Akt activity. It is well known that PI3K/Akt signaling plays an important role in inhibiting apoptosis for cancer cell survival and growth, and apigenin has been reported in numerous studies in vitro as well as in vivo to enhance apoptosis by inactivation of Akt [113, 114]. Apigenin has also been demonstrated to inhibit ovarian tumor metastasis through down-regulation of MMP-9, which is mediated by Akt signaling [91]. Apigenin also inhibits metastasis in breast cancer cells by blocking PI3K/Akt pathway [89]. In addition, apigenin has been shown to inhibit cancer angiogenesis by suppressing HIF-1α and VEGF expression, which is also related to the inhibition of Akt by apigenin [47, 58]. Deregulation of insulin-like growth factor (IGF)-I signaling has been implicated in the development and progression of prostate cancer. Shukla et al reported that apigenin substantially reduced the levels of IGF-I in the serum and in the dorso-lateral prostate, and this modulation of IGF-I was associated with the inhibition of Akt [115].

Compared to numerous reports in the literature that apigenin inhibits Akt activity, there are only a few reports demonstrating apigenin’s effect on mTOR activity. Tong et al. recently demonstrated that apigenin induced AMPK activation in human keratinocytes (both cultured HaCaT cell line and primary normal human epidermal keratinocytes) [54]. They also found that the activation of AMPK by apigenin was independent of upstream kinase LKB1. Instead, calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ), another upstream kinase of AMPK, was required for apigenin-induced AMPK activation. Apigenin-induced AMPK activation further inhibited mTOR activity (AMPK phosphorylates TSC2 and enhances its ability to turn off mTOR activity [116]. AMPK can also directly phosphorylate the mTOR binding partner Raptor on two well-conserved serine residues, and this phosphorylation further inhibited mTOR activity [117]) and induced autophagy in human keratinocytes [54]. At early stages of tumorigenesis, autophagy acts as a tumor suppressor. It prevents tumorigenesis by removing damaged organelles and proteins, reducing chromosome instability, and inducing type II programmed cell death [118–120]. Therefore, pharmacological induction of autophagy may provide a new strategy for cancer chemoprevention. Another study by Turktekin and coworkers also demonstrated that apigenin reduced mTOR expression in colon cancer cells [121]. However, in serum-starved malignant neuroblastoma cells, apigenin alone or combined with synthetic retinoid N-(4-hydroxyphenyl) retinamide activated Akt/mTOR signaling pathwaty and the molecular mechanism for the activation is not clear [122].

Since apigenin has been demonstrated to inhibit Akt activity, and Akt has been reported to inhibit AMPK activation, one would wonder whether the activation of AMPK by apigenin is dependent on its inhibition of Akt? The answer is no, at least in human keratinocytes, because neither inhibition of Akt activity nor overexpression of constitutively active Akt had any effect on AMPK activation induced by apigenin [54]. However, the same study also suggested that apigenin treatment could inhibit the mTOR activity by two different mechanisms, either by activation of AMPK and subsequent activation of TSC2, or by direct inhibition of Akt, which is a negative regulator of both TSC2 and Pras40 (Fig. (2)) [54].

Fig. 2.

Apigenin inhibits both Akt and mTOR signaling pathways. Apigenin activates AMPK through CaMKKβ, further blocking mTOR activity. Apigenin also inhibits Akt, which activates mTOR by negatively regulating TSC2 and Pras40.

mTOR has emerged as a promising target for cancer treatment, and since the discovery of rapamycin as the first mTOR inhibitor, many rapamycin analogs have been developed to treat cancer. However, the clinical results are modest in many cancers, and it has been suggested that these mTOR inhibitors could activate Akt due to the loss of feedback inhibition of the PI3K pathway by preventing mTORC1-mediated P70S6K activation [123]. Recently, development of the second generation of mTOR inhibitors is underway, and the new mTOR/PI3K dual inhibitors are supposed to be more effective but also more toxic than rapamycin [110]. The ability of apigenin to inhibit both PI3K/Akt and mTOR signaling pathways makes it a unique and distinctive chemopreventive agent, and an added benefit is that apigenin is associated with very little toxicity, making it more attractive for cancer chemoprevention.