Modulation of cancer signalling pathway(s) in two -stage mouse skin tumorigenesis by annonacin

Chemotherapy plays an important role in the treatment of many cancer type including skin cancer. The chemical agents used in chemotherapy are selectively destructive to malignant cells, but these agents can also cause damage to the healthy normal cells, which results in adverse side effects that negatively impact compliance with cancer treatment, as well as its well-being [1]. Therefore, there is an imperative need to find a strategy to resolve the problem. This eventually has led to the discovery of new plant-based chemopreventive agent as an alternative strategy as plants-based medicines are generally considered safe when it is produced and used in an appropriate manner [2, 3].

Several evidences have demonstrated that Annona muricata L possesses pleiotropic effects such as anti-inflammatory, anticancer, antiparasitic, pesticidal, antimicrobial and antiviral activities [4‐6]. Among these activities, anticancer activity has been intensively studied due to the exploration of cytotoxic compounds abundantly present in various parts of Annona muricata namely, annonaceous acetogenin [5‐8].

Annonaceous acetogenins have been reported to exert cytotoxicity in various cancer cell lines such as lung, colon, pancreatic, prostate, and breast [5, 8]. For the past 30 years, several researchers have conducted numerous studies in elucidating the underlying mechanisms of actions of these compounds. Amongst the mechanisms reported are the inhibition of mitochondrial complex 1 enzyme, induction of apoptosis, blocking of the cell cycle through the halt at certain phases and inhibition of DNA topoisomerase [5, 7, 9, 10].

Annonacin, a mono-tetrahydrofuran acetogenin is one of the major compounds consists in Annona muricata leaf and seed and in other plants from Annonaceae family. It has been priorly reported to be actively cytotoxic against various cancer cell lines [5, 8‐10]. Despite its role as a potent inhibitor of the mitochondrial complex I in the electron transport system, its other mechanisms in vivo, in tumorigenesis have yet to be extensively explored. To the best of our knowledge, the mechanisms of annonacin in modulating tumorigenesis pathways, in vitro, includes, upregulation of Bax and caspase-3 expression that lead to apoptosis [9]; inhibiting ER-α, cyclin D1 and Bcl-2 protein expressions [10]; inhibits HIF-1α and mTOR activation [11]; and downregulation of ERK protein expression [12]. These events eventually lead to cancer cell apoptosis.

Two-stage skin cancer model consists of multistage tumorigenesis, offers an investigational framework to study the basic mechanism linked with the initiation, promotion and progression stages in animal. It is also beneficial in examining chemopreventive agents that potentially affect any stages of tumorigenesis with regard to understand the molecular mechanism and evolution of cancer cells, not only in skin cancer, but in other multistage human cancers such as prostate and colon cancer [13]. Chemical-induced carcinogenesis occurs as a result of a complex multiple steps of event initiated by mutations via direct DNA damage coupled with an overproduction of reactive oxygen species [18]. A large number of evidence have demonstrated that the role of Ras/Raf/ERK and PI3K/AKT/mTOR pathways, MAPK (p38) and proto-oncogene (Src) as well as tumor suppressor gene (PTEN), in cell proliferation and apoptosis [19]. Upregulation of proteins in Ras/Raf/ERK and PI3K/AKT/mTOR pathways, MAPK (p38) and proto-oncogene (Src) as well as loss of tumor suppressor function (PTEN) have been implicated in several cancers including skin cancer [19, 20]. These signaling pathways impart cancer cells with neoplastic progression through uncontrolled cell proliferation and inhibition of apoptosis [21].

To date, all the mechanisms previously reported on annonaceous acetogenin including annonacin, were based on in vitro results. There is no study conducted in elucidating the mechanism of action of annonaceous acetogenin in any in vivo model yet. Thus, this current study was aimed to investigate the antitumor promoting effect and toxicity of annonacin, in two stage mouse skin tumorigenesis, as well as its molecular pathways in suppressing/inhibiting the skin tumorigenesis.

In this study, results showed that topical application of tumor initiator, DMBA (390 nM) followed by the promoter, TPA (1.7 nM) applied on mice, twice a week for 22 weeks produced multiple skin papillomas. Mouse skin tumorigenesis model is the best recognized in vivo models for the study of the chronological and stepwise development of tumors from initiation to progression stage [13]. To date, this model is still being a model of choice to study the antitumor promoting effect of either synthetic or plant-based antineoplastic agents [14‐16]. This due to the capability of his model mimics the features of multi-stage carcinogenesis in humans as humans are typically exposed to various doses of both carcinogens and promoting agents [17, 24]. This model also enables tumor development and progression to be monitored visually throughout the life span of the mouse.

TPA induces the production of superoxide free radicals that have been implicated as the causative factor in various diseases including skin cancer [27]. In comparison to the initiation stage which is an irreversible process, the promotion stage occurs over to an extended period of time that may be reversible during the tumorigenesis process [28]. Upon induction by the tumor promoter, several key events have been recognized in skin tumor promotion including epidermal hyperplasia, keratinocyte proliferation, inflammation and oxidative stress [19].

In the current study, the application of annonacin 85 nM was significantly reduced the tumor incidence and tumor volume in DMBA/TPA-induced tumor on mice skin. This finding was in agreement with previous findings where annonaceous acetogenins including annonacin have been reported to be responsible for the potent anticancer activities in many cancer cell lines through the inhibition of mitochondrial complex I enzyme, to cause energy depletion [7, 8]. The dose of 85 nM used was in accordance with many previous established reports that employing similarly in vivo model using bioactive compound in the antitumor promoting study [29‐31].

In another study by Wang et al. [32], annonacin (10 mg/kg) was able to inhibit mouse lung cancer by 57.9% in an in vivo model using hybrid mice (BDF-1) when administered orally. In addition, several studies on the antitumoral effect of Annona muricata leaves extract (AML) were conducted in various in vivo model [33‐35]. Recently, the antitumoral effect of Annona muricata was shown to be primarily due to the activity of annonacin [35].

Histopathological analysis revealed that the appearance of the tumor in each Group II, III and V were morphologically identified as papilloma, which is characterized as a small benign solid, with a clear-cut border growing exophytically from the epidermal layer of the skin. Representative microphotographs of H&E sections from each group were depicted in Fig. 1. Notably, the current regimen in our study did not lead to the formation of SCC. This may be possibly due to the low DMBA/TPA dosage applied, shorter period of observation and the type of mouse strain used in this experiment, which are accounted as crucial factors for the development of SCC [13].

Sustained cell proliferation is one of the major characteristics in skin tumor promotion [13, 19]. The occurrence of this event is a response to the repetitive application of tumor promoter, preceded by inflammation and followed by skin papillomatosis that eventually leads to malignant transformation (SCC) [19]. As shown in Fig. 1b, severe increased keratinocyte proliferation depicted via epidermal thickening was noted in Group II (carcinogen control), in which this event is caused by the upregulation of AKT, ERK, p38, mTOR and Src [18, 19]. Interestingly, treatments with 85 nM annonacin (Fig. 1c) and 10 mg/kg curcumin (equivalent with 6.2 mM) have significantly reduced keratinocyte proliferation prior to the induction with DMBA/TPA for 22 weeks, respectively (Fig. 1d).

During skin tumorigenesis, single and multiple applications of tumor promoter leads to the activation of the Ras/Raf/MAPK/ERK and PI3K/AKT/mTOR pathways [19, 37] In this study, once EGFR is activated by the tumor initiator (DMBA) it will phosphorylate the upper stream targets in PI3K/AKT/mTOR and Ras/Raf/MAPK pathways which in majorly consisted of kinases protein. Protein kinases are responsible for the mechanism of phosphorylation. They are activated by phosphorylation which in turn activates a cascade of events leading to the phosphorylation of downstream effectors including regulatory protein than controlling gene expression [38].

Phosphorylation is one of the most important processes in post - translational modification of protein. Additional of phosphate group into the protein structure will eventually change the protein conformation and thus alter its activity when interacting with other molecules. This process plays a crucial role in many cellular processes including protein activity, subcellular localization and transmits signal downstream to the reaction path. Phosphorylation may act in two different ways, either activates or inactivate the protein [38].

Phosphorylation is a major event in activation of PI3K/AKT/mTOR and Ras/Raf/MAPK pathways. Both pathways play an important role in cell survival, cell proliferation, apoptosis and protein synthesis through AKT activation that phosphorylates their downstream proteins Bad, Bax, Caspase and mTOR. Cell proliferation and inflammation are also activated through RAS activation through cascade phosphorylation of Raf, MEK, ERK, p38 and NFκB (Additional file 1: Fig. S1) [19, 37]. Therefore, the capability of annonacin to inhibit the activation of AKT, ERK, mTOR and p38 will halt the cascade phosphorylation of other downstream proteins that involve in PI3K/AKT/mTOR and Ras/Raf/MAPK pathways.

Besides, according to Ko et al. [10], the molecular mechanism of annonacin in the regulation of gene protein expressions can be studied through binding assay and transcriptional activation assays. Downregulation of ERK, JNK and STAT3 protein expression by annonacin in the study [10] is due to the regulation of its transcriptional activity trough phosphorylation inhibition. Similar result was also reported by Chung et al. [38], where they found that reduced phosphorylation of ERK protein in endometrial cancer cell lines (HEC-1A, EC6-ept, and EC14-ept) treated with annonacin resulted with low expression of ERK.

It is ubiquitously known, overexpression of ERK, AKT, mTOR, p38 and proto-oncogene (Src) as well as the loss of tumor suppressor function (PTEN) proteins have been implicated in wide number of cancers in human including skin cancer [36]. In addition, curcumin has been reported to exert remarkable effects in modulating various protein kinases and antiapoptotic proteins that are crucial for cell growth such as AKT, ERK, p38, mTOR and Src either through inhibition or activation of phosphorylation [39‐42]. As curcumin has been known effectively to hit several deviant targets simultaneously in many cancer pathways and models, therefore, annonacin, is suggested to exert the similar mechanism like curcumin in regulating gene and protein expressions.

In addition, the downregulation and upregulation of protein in this study could be explained through ATP usage during phosphorylation. Annonacin also known of its anticancer property as an inhibitor of complex I enzyme, to cause energy depletion [7]. The phosphorylation process itself require phosphate group that derive from the hydrolysis of ATP due to enzymatic activity of kinase [38]. Since annonacin causes ATP depletion in tumor cells, thus phosphorylation will be interfered and eventually will change the protein activity and signal transduction process. This was explained by the study done by Liu et al. [43]. The study has shown that, polyether mimicking acetogenin (AA005) decrease the phosphorylation of mTOR through suppression of ATP production in colon cancer cells lines [37].

It is also widely known that, there is a strong association between inflammation and tumorigenesis/carcinogenesis, where NF-κB, a family of transcription factors plays an essential role in inflammation as well as in immunity [44], which was later identified to be responsible for the various steps in cancer initiation and progression [19]. Annona muricata has been known to act effectively as an anti-inflammatory [45, 46], where inflammation has also been known to be implicated in tumorigenesis through modulation of NF-κB signaling pathways that commonly associates with ERK, p38 MAPK, Ras, and PI3K/AKT pathways [47]. Moreover, Annona muricata was reported to inhibit the inflammatory process through inhibition of both COX-1 and COX-2 [45]. Consistently, the complement cascade activation of COX-2 by NF-κB has been shown to activate PGE₂ in which, it has been shown to play a role in inducing keratinocyte proliferation through crosstalk via multiple upper stream signaling pathways including PI3K/AKT, ERK and EFGR [19, 48].

Likewise, the downregulation of p38, AKT and ERK expression by annonacin in this study, is also suggested to affect COX-2 suppression. Moreover, ERK and p38 have been shown to modulate NF-κB and COX-2 activation [48, 49].

On the other hand, COX-2 activity can also be modulated under the influence of Src activity [50]. Src can activate most of the crucial cascades such as PI3K/AKT as well as Ras/Raf/MAPK that function in cell proliferation, cell cycle progression and survival [50, 51]. Matsumoto et al. [22], also reported that, activation of Src is a crucial event in epidermal hyperplasia in skin tumor promotion. Moreover, reduced activity of Src in this study could be explained by inhibition of phosphorylation caused by insufficiency of ATP as annonacin inhibits the production of ATP in the mitochondria [7, 8]. Therefore, annonacin is suggested to undergo the similar mechanism in preventing the activation of ERK and AKT pathways through inhibition of Src activation. This event eventually inhibits keratinocyte proliferation, which is a crucial process during the promotion stage in tumorigenesis.

A study has demonstrated the restoration of PTEN and simultaneous inhibition of P13K expression resulted in the antitumor effect of selected natural compounds [52]. In addition, PTEN has been identified to regulate many cellular functions including growth, adhesion, migration, invasion and apoptosis [53]. The role of PTEN in apoptosis is well perceived. Restoration of PTEN expression has also been described to downregulate PI3K signaling, thus causing tumor cell death and cell cycle arrest at the G1 stage due to the cleavage of PIP3 [54]. Modulation of prominent tumor suppressor gene, PTEN by annonacin, has been suggested to be linked with AKT expression as evidence in this study (Fig. 2), where the level of PTEN expression was inversely correlated with AKT phosphorylation. This trend was in accordance with previous studies that shown AKT Inhibitor VIII blocked phosphorylation of AKT and Bad, but not PTEN [55]. This could explain the increased expression pattern of compared to AKT in Group III (Fig. 2).

In terms of toxicity, organ weight and/or with ratio to body weight has been widely accepted for evaluation of test substance-associated toxicities [56, 57]. Increased in body weight due to toxicity has been linked with the increased weight of several vital organs including liver, kidney, heart, brain and spleen [56]. This study shown that, non-induced mice treated with annonacin alone (Group IV) showed significant changes in body weight gain compared to vehicle control mice (Group I). However, no significant changes in the liver and kidney weights were observed in both groups as mentioned earlier, indicating the non-influential effect of weight gain with either toxicity or adverse effect. This generalization is supported by the studies conducted by Chan [58] and Hoffman [59], who explained the possible toxicity is due to the significant changes in weights of both liver and kidney. Meanwhile, our current results found that annonacin failed to show any hyperplasic and cytomegalic changes in both organs when examined grossly and microscopically. Both features are known to cause body weight gain due to toxicity [58, 59]. Interestingly, no other trace of hepatocyte toxicity features, i.e. irreversible injury (necrosis) was seen in both liver and kidney in all groups. No degeneration and sinusoidal dilatation were found in the liver of mice treated with annonacin (Group IV, treatment control) indicating its non-toxicity when topically applied for 22 weeks.

Current results also showed a very mild score of inflammation and microvesicular steatosis observed in the liver (Group IV) as compared to the carcinogen control (Group II). The weak occurrences of inflammation scattered in few areas of both liver and kidney tissues in all groups are considered normal condition [60, 61]. In addition, very mild occurrence of microvesicular steatosis in the liver observed in mice treated with annonacin alone (Group IV) is also deduced as a normal condition, mainly regarded as an adaptive mechanism towards the accumulation of toxic compound. Accumulation of toxic substance will lead to oxidative stress which eventually causes mitochondrial dysfunction along with altered oxidative phosphorylation and impairment of mitochondrial β oxidation [62]. Nevertheless, increased concentrations of intracellular fatty acids may be directly toxic to hepatocytes [63]. According to Kaplowitz [64], liver cell damage may also be due to precipitation of metabolites from the toxic compound. Therefore, this may explain, that annonacin showed no significant sign of toxicity manifested by analysis of body and liver weight and microscopic liver histopathology. The insignificant marked inflammation and microvesicular steatosis are considered as normal adaptation mechanisms of hepatocytes due to the exposure of xenobiotic. Our findings were also in line with the study done by Hansra et al. [65], where they found treatment using Annona muricata decoction (10–12 dry leaves in water) for 5 years to a breast cancer patient, managed to stabilize the disease with no side effects observed.

Moreover, induction of DMBA/TPA in carcinogen control (Group II) mice irreversibly affected the kidney’s toxicity, manifested by significant increase of glomerular cellularity, Bowman’s capsule dilation and mild number of tubular cell degeneration, respectively. Our findings were in agreement with the previous study, where croton oil (a crude form that contain TPA) has also been reported to cause glomerulonephritis, thus led to an increase in cellularity (hypercellularity) and inflammation [66]. Glomerular hypercellularity is commonly marked with an increase of the number of endothelial, mesangial, and inflammatory cells [67]. Interestingly, there is no toxicity features such as Bowman‘s capsule dilatation, increase in glomerular cellularity and degeneration were observed in both Group III (annonacin) and IV (treatment control) as shown in Table 3. In addition, most of the histopathological observations of these tissues resemble the features characterized in Group I (vehicle control).

Collectively, the current findings provide new insight of annonacin’s mechanism of action in suppressing tumorigenesis. Annonacin demonstrates down-regulation of AKT, ERK, mTOR, p38 and Src, whilst simultaneously prevents PTEN down-regulation at gene and protein levels. Modulation of these genes eventually promotes cell apoptosis in the skin tumor cells via inhibition of PI3K/AKT/mTOR signaling pathway in vivo. This is evidenced by delayed papilloma development, incidence as well as its reduction of volume. On the other hand, annonacin was found to be non-toxic to the liver and kidney and was able to protect skin from further development of neoplastic changes featured by the absence of mitotic activity, mild hyperplasia and mild hyperkeratosis. Finally, this study suggests that annonacin may serve as a potential therapeutic compound for the prevention and treatment of skin cancer.