The future potential of Annona muricata L. extract and its bioactive compounds as radiation sensitizing agents: proposed mechanisms based on a systematic review

Introduction Notwithstanding technological advances in cancer treatment modalities, cancer is still a challenging disease for medical experts. According to GLOBOCAN 2018, more than 18 million new cases and 9.6 million mortalities were estimated in 2018. One among five men and one among six women are diagnosed with cancer throughout life. Moreover, one among eight men and one among eleven women die from cancer (1). Many efforts have been made to increase the ratio between the probability of tumor control and the chance of normal tissue damage, known as a therapeutic ratio. Although radiotherapy technology is developing rapidly and has brought along new techniques able to increase therapeutic ratio, tumor radio-resistance is still a big challenge for oncologists. Many studies have reported the anticancer effect of Annona muricata L. (AM) extract and its bioactive compounds either using in vitro or in vivo preclinical studies. However, so few studies observed the combination of radiation and AM in cancer cell lines that not enough evidence is available to conclude the role of AM in altering cell lines radio-sensitivity. Therefore, we separate our searching strategy. In this review, we collected the anticancer mechanisms of AM and then further correlated them to radio-sensitizing possibility. Methods Search strategy We performed a search in PubMed, Cochrane, EBSCO, http://www.herbmedpharmacol.com doi: 10.34172/jhp.2021.18

and Scopus database using keywords "(Annona muricata) AND ((molecular target) OR mechanism OR pathway) AND (cancer OR tumor OR neoplasm OR malignancy OR antiproliferative), which resulted 285 articles in PubMed, 1 article in Cochrane, 22 articles in EBSCO, and 58 articles in Scopus). After initial searching, the articles were filtered to exclude duplicated studies, conference abstracts, reviews/systematic reviews, letters/editorials, cases, and other irrelevant studies.
After that, we assessed the remaining articles to determine whether they met our inclusion criteria, using titles and abstracts. Our inclusion criteria were: (1) in vitro (cell lines) or in vivo (animal study) trials; (2) studies using AM extracts or its bioactive compounds; (3) studies analyzing/observing pathways or proteins occupied by AM to take effect. We chose to rule out articles which not discuss the working mechanism of AM specifically. We also did not include studies using molecular docking methods in order to observe the actual anticancer mechanisms of AM thoroughly.
We made a flow diagram in agreement with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement on systematic review reporting (see Figure 1) (2). Thenceforth, we did a full-text reading on 20 articles, and made a critical appraisal. Since there is no standard appraisal tool for in vitro studies, we used a reporting standard of Science in Risk Assessment and Policy (SciRAP) and Systematic Review Center for Laboratory Animal Experimentation (SYRCLE). No article was ruled out in this critical appraisal and eventually we generated a summary. We then further analyzed and discussed the role of the pathways in enhancing cancer cell radio-sensitivity. Cellular response to ionizing radiation: an overview Numerous cellular signaling pathways are induced after the cell was exposed to ionizing radiation, which will eventually generate responses like apoptosis, cellular senescence, and cell cycle checkpoint activation along with DNA repairing (3). Radiation, unfortunately, can also induce apoptosis-suppressing pathways like cell cycle arrest and DNA repair, which give the cell chance for rehabilitation (4,5). They protect cancer cells from the killing effect of radiation, which significantly causes radio-resistance (6).
If DNA repair after a single-strand break (SSB) and double-strand break (DSB) fails, the intrinsic apoptotic pathway is activated (7). Longer and stronger activation of p53 has been correlated with higher chances of apoptosis rather than growth arrest (8). The intrinsic apoptotic pathway is regulated by the B-cell lymphoma (Bcl-2) proteins family, which consists of proapoptotic and antiapoptotic members (9).
Released Bcl-2 associated X protein (BAX) induces outer mitochondrial membrane permeabilization and consecutive cytochrome c discharge (10,11). Accordingly,  the release of cytochrome c into the cytosol will lead to the formation of cytochrome c/apoptotic protease activating factor 1 (APAF 1)/caspase-9 containing apoptosome complex (12). After that, caspases-3 and -7 as effector caspases will be activated, and will further lead to postmitochondrial-mediated caspase cascade (13). The extrinsic apoptotic pathway, on the other hand, is regulated by the Fas-associated death domain (FADD) (14). CD 95 (well known as Fas) belongs to the tumor necrosis factor (TNF)-receptor superfamily. Its ligand CD95L (known as FasL or CD95L or CD178) is a transmembrane cytokine belonging to the TNF family (15). Radiation-induced p53 transactivates CD95, DR5, and the Fas ligand (14,16).
FADD bridges Fas-receptor to procaspase-8, which interacts with the death effector domain of FADD, forming the death-inducing signaling complex, and ultimately activates procaspase-3 and procaspase-7. Other downstream of CD95 could also activate caspases, which results in mitochondria-dependent mechanisms (17).

Results
Many published studies reported various pathways. We briefly summarised them in Table 1 according to the reporting author. We chose several main pathways, categorized them, and searched the radio-sensitivity evidence relating to the pathway and discussed them.
Increased reactive oxygen species (ROS) formation Three studies have reported an increased ROS formation after AM administration (18)(19)(20). Among them, Moghadamtousi et al also explicitly reported that cytochrome c was released from mitochondria upon apoptosis (20). As we know, radiation also induces apoptosis and cell damage via ROS formation. Any increased ROS, as opposed to decreased ROS formation in hypoxic cell conditions, will increase radiation efficacy (21).
Inhibition at G2-M phase The cell cycle phase has been shown to determine the relative radio-sensitivity of cells. The cells will be the most radio-sensitive in the G2-M phase, less sensitive in the G1 phase, and the least sensitive at the late S phase (22). AM has shown an inhibiting effect on the cell cycle G2-M phase (23), although there are different reports by other authors (see Discussion section). This cell cycle inhibition effect could enhance the radio-sensitivity of the cancer cells.
Regulation of Bcl-2 family proteins a. Upregulation of Bax and downregulation of Bcl-2 proteins Being known as the death triangle, the Bcl-2 family consists of 3 types of members (24). Antiapoptotic and proapoptotic Bcl-2 homologs occupy two corners among them: Bcl-2, Bcl-xL, and Mcl-1 as antiapoptotic protein members; and Bax and Bak as proapoptotic protein members. The third corner is occupied by indirect/direct activator BH-3 only-proteins (25).
The mitochondrial pathway of cell death is controlled by the Bcl-2 protein family. The family is subdivided into an antiapoptotic group consisting of Bcl-2 itself, Bcl-xL, and myeloid cell leukemia sequence 1 (Mcl-1); and a proapoptotic group comprising Bax, Bak, Bok, and several Bcl-2 homology-domain 3 (BH3)-only containing proteins (26).
Bcl-2 has been associated with increased radioresistance (27)(28)(29), and inhibiting Bcl-2 proteins using different strategies has been proven to increase radiosensitivity (30). While there is no direct application of activating Bax protein to increase sensitivity to radiation, Bax's overexpression has been correlated to radiosensitivity in head and neck cancer (31).
Loss of mitochondrial membrane potential (MMP) Pieme et al (38), Moghadamtousi et al (20), and Kuete et al (39) reported respectively in HL-60, A549, and CCRF-CEM leukemia cells that AM leaves extract has an antiproliferative effect through a loss of MMP. MMP is provoked by an asymmetric distribution of protons and other ions on both sides of the inner mitochondrial membrane. Some authors have reported that some pathways induce radioresistance by increasing MMP or inhibiting its deterioration. MMP is regulated by growth differentiation factor-15 (GDF15), MEK/ERKsignaling pathway, and histone deacetylase inhibitor. Therefore, MMP is a potential target for radio-sensitivity improvement (40).
Li et al proved the involvement of GDF15, a transforming growth factor beta (TGF-β superfamily), in head and neck cancer radio-resistance, by triggering MMP and inhibiting intracellular ROS production (41). The effect of MEK-ERK on MMP and the developed resistance has been successfully countered using MEK-specific inhibitor. Table 1. Anticancer effects of Annona muricata (AM) and its reported pathways of mechanism Author Annona muricata L. plant component/bioactive compound

In vitro cell lines/in vivo models Reported pathway or molecular target and results Conclusion
Najmuddin et al (49) • Crude leaves extract • Induced apoptosis in 4 T1 breast cancer cells • Annona muricata leaf and stem extract dose-dependently suppress UW-BCC1 and A431 cell growth, motility, wound closure, and clonogenicity.

Moghadamtousi et al (50)
• Annomuricin E • HT-29 • Down-regulation of proliferating cell nuclear antigen and Bcl-2 proteins • Upregulation of Bax protein • The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction. • Leakage of cytochrome c from the mitochondria.
• Activation of caspase 3/7 and caspase 9 • Annomuricin E as one of the contributing compounds in the anticancer activity of AM leaves.

Yiallouris et al (51)
• Ethanol extract of Graviola leaf • Inhibition of multiple signaling pathways that regulate metabolism, cell cycle, survival, and metastatic properties in pancreatic cancer cells
• Annonacin is a potential therapeutic compound targeting tumor-promoting stage in skin tumorigenesis by modulating multiple genes and protein in cancer signaling pathways without apparent toxicity. Yap et al (48) • Annonacin (seeds)

Han et al (19)
• Annonaceous acetogenins AA005 • SW620 • AA005 induces cell death through a caspase-3 independent pathway • ROS mediates AA005-induced cell death of SW620 cells • RIP1 is required for AA005-induced cell death • AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway

Daddiouaissa et al (56)
• IL-GFE • MCF-7 • Growth inhibition of the cells by extracts was associated with cell cycle arrest at the G0/ G1 phase, and phosphatidylserine externalization confirms the antiproliferation through apoptosis.
• IL-GFE affect the cytokinetics behavior of MCF-7 cells by reducing cell viability, induce apoptosis and cell cycle arrest at the G0/G1 phase • Annonacin induced growth arrest and apoptosis in ERαrelated pathways in MCF-7 cells.

Activation of caspase 3/7 and caspase 9
Caspase plays a vital role in apoptosis, with caspase-8 activated via the extrinsic pathway and caspase-9 triggered by cytochrome c release from mitochondria. Both intrinsic and extrinsic pathways will eventually progress through the executioner caspase-3 and caspase-7 (and also possibly caspase-6). Several reports have shown that caspase-3 reconstitution in caspase-3 deficiency cancer cells could significantly enhance radiation-induced apoptosis (44). By increasing caspase protein, especially caspase-3 (19,20,(45)(46)(47)(48), it is expected that AM could sensitize cancer cells to radiation.
Downregulation of molecules related to hypoxia and glycolysis (HIF-1 α, GLUT1, GLUT4, HKII, LDHA) Hypoxia and glycolysis have been reported to be related to cancer cell radio-sensitivity. Many studies reported that hypoxia-inducible factor (HIF-1) regulates adaptive cellular responses to hypoxia. However, HIF-1 could also be activated and give rise to radio-resistance through cancer-specific genetic alterations and the disproportion of intermediate metabolites. HIF-1 has been reported recently to reduce cancer cell radio-sensitivity through glucose metabolism alteration and overproduction of antioxidants. In case that AM extract and its bioactive compounds could downregulate HIF-1α expression, there is a prospect of reducing the cancer cell radio-resistance (60,61). GLUT expression was also reported in many studies to be correlated with radio-resistance (62)(63)(64)(65)(66)(67), supposing that the AM can suppress the GLUT expression. This is a chance for radio-sensitizing the cancer cells. Hexokinase-II (HK-II) was observed as a crucial glycolytic enzyme that initiates the first essential step of glucose metabolism. Zhong et al noted that HK-II could play a role in radioresistance of laryngeal carcinoma and remark the possibility of inhibiting the HK-II signaling pathway, which conceivably will enhance the radio-sensitivity (68).
Lactate dehydrogenase (LDH) plays a vital role in the tumor microenvironment as it converts pyruvate to lactate and vice versa (69). It is composed of two different subunits, encoded in humans by LDHA and LDHB genes (70). Di et al reported that inhibiting this gene using siRNA mediated knockdown of LDHA will make cells more sensitive to radiation and chemotherapy in glioblastoma multiforme (GBM) (71). A similar finding has also been put forward in prostate cancer cells by Hao et al. When they used siRNA to inhibit the LDHA, the cells became more sensitive to radiotherapy (72). It is expected that AM could be a good radio-sensitizer by downregulating LDHA.
Following radiation, ERK1/2 is activated through dual tyrosine and threonine phosphorylation by MEK1/2 and will further phosphorylate 160 substrates (77). The best characterized antiapoptotic transcription factors targeted by ERK1/2 signaling are the cyclic AMPresponsive element-binding protein (CREB) and CAAT/ enhancer-binding protein β (C/EBP-β). In response to radiation, ERK1/2 phosphorylates p90 rsk kinase, which in turn activates CERB and C/EBP-β, thereby inducing the expression of a number of antiapoptotic proteins including Bcl-xL, Mcl-1, and c-FLIPs (77)(78)(79)(80). Thus, by increasing the expression of antiapoptotic proteins and inhibiting the activity of proapoptotic proteins, the net effect of radiation-induced ERK1/2 signaling activation is the suppression of apoptosis in irradiated cells. Supposing that, by downregulating the ERK and Akt, AM may improve the radio-sensitivity.
Janus kinase (JAK), especially JAK2 and signal transducer and activator of transcription, chiefly STAT3, was reported by Park et al to be activated in radioresistant CRC tissues, which grow persistently after delivered irradiation. It was also observed that cyclin D2 transcription, which is vital for maintaining intact cell cycle and proliferation despite DNA damage, is increased due to the direct binding of STAT3 to cyclin D2 (CCND2) promoter (82). Furthermore, Xie et al also reported that STAT3 and ERK1/2 dual blockage would resensitize GBM to radiotherapy (83).
Cyclin D1 is an important regulator, which is aided by cyclin-dependent kinase (CDK), controls the progression of the cell cycle at the G1/S transition (84,85). Cyclin D1 has been reported in several studies to have a role in regulating radio-sensitivity and is an excellent target to increase cancer cell radio-sensitivity (84)(85)(86)(87)(88).
Suppressed hedgehog signaling Chamcheu et al reported that AM extract could suppress the hedgehog signaling pathway (46). The hedgehog signaling pathway has been reported to be associated with radio-resistance in head and neck cancer, and also cervical cancer (89,90). That opens an opportunity for AM to radiosensitize the cancer cells.
Downregulation of mammalian target of rapamycin (mTOR) Integration between intracellular and extracellular signals is performed by mTOR. Moreover, mTOR also regulates cell metabolism, growth, proliferation, and survival. Many trials have concluded that the inhibition of mTOR could increase radio-sensitivity (91)(92)(93). Uniquely the opposite also applies: when the mTOR signal was enhanced, cancer became radio-resistant (94).

Discussion
There is a lack of trials that directly investigate the role of Annona muricata in combination with radiation. One of few works that we found is a publication from Mansour et al (95), which compared the effect of the addition of sole AM, with radiation only, or in combination with AM, and with placebo in Ehrlich ascites carcinoma bearing mice. They reported that a combination of AM and radiation reduced tumor volume more effectively, and the survival in the combination group was better than the irradiation alone group. Nevertheless, there is still controversy regarding the antioxidant role of AM, which was shown by significantly attenuated serum lipid profiles, decreased malondialdehyde and total nitrate/nitrite levels, DNA fragmentation, and significantly increased caspase-3 and superoxide dismutase activity, glutathione content, and expression of glutathione peroxidase in the lung and kidney tissues compared with an irradiated group (95).
In the previous section, we discussed various pathways that could be effectively influenced (induced or inhibited) by AM extract or bioactive compounds, which in the end will increase cancer cell radio-sensitivity. We also found several pathways or proteins whose role has not been well studied, e.g., eotaxin, AIF, RIP-1. These pathways or proteins might be an exciting topic for further study.
Besides that, we also found distinct findings amongst different authors. Inhibition at the G2-M phase was reported by Magadi et al (23) (20,38,46,49,54,56,58). Neither of these two contrasting observations might be wrong, as they perform different observations in different cell lines. Several other findings are too general that we have some difficulties in finding the specific role of those findings. Among them are induced apoptosis, which was reported by Syed Najmuddin et al, Yiallouris et al, and Li et al (49,51,54).
Nevertheless, we also found several pathways that could contrarily reduce radio-sensitivity. As we have discussed before, Fas-ligand plays an important role in the extrinsic apoptotic pathway (14)(15)(16). When Annona muricata reduces the Fas-ligand (49), in line with the study by Reap et al, it will reduce the radio-sensitivity of the cells (96). The way that AM upregulates PERK-eIF2α (57) will also decrease the sensitivity of cells to radiation as PERK-eIF2α signaling could give protection against ROS (97), and has been reported to correlate with radioresistance in oropharyngeal carcinoma (98). However, the role of PERK-eIF2α, including its downstream CCAAT/ enhancer-binding protein homologous protein (CHOP) and its initiator binding-immunoglobulin-protein (BIP) in potentiating radiation might not be studied clearly as this endoplasmic reticulum stress pathway may serve prosurvival or proapoptotic function depends on the severity of stress conditions (99).
Notch is a transmembrane protein (100) that regulates self-renewal and cell fate determination in normal stem cells (101). Annonaceous acetogenins from AM have been noted for showing an increased Notch expression in several cell lines (54). As Notch signaling pathway is correlated to cell radio-resistance by many authors, this will eventually result in reduced radio-sensitivity (100)(101)(102)(103)(104)(105). Similarly, increasing TNF-α has been associated with the enhanced antitumor effect of radiotherapy. When AM decreased the TNF-α (49), radio-sensitivity will be lowered.
Another example is the downregulation of c-JUN N-terminal kinase (JNK) by AM (59). Activation of the JNK is involved in damage response after ionizing radiation (106,107). This pathway is initiated by mitogenactivated protein/extracellular signal-regulated kinase (MAP/ERK) (which is recognized as MEKK1) and requires MEKK4, JNK, and JUN to be consequently activated (108). This proapoptotic pathway of JNK could also be activated after membrane-derived signals, and subsequently releases ceramide (106,108) and deathassociated protein 6 (DAXX), a CD95 binding protein (109). There is no direct study reporting the correlation of JNK and radio-resistance, but since JNK is proapoptotic, the downregulation of this protein might reduce the cancer cells killed by radiation.

Conclusion
Through our systematic review and further analysis of several main selected pathways, we revealed the future potential of AM as a promising radiation sensitizing agent. There are significant reported pathways or proteins that hypothetically could be used by either extract or bioactive compounds of AM in enhancing the radiosensitivity, compared to few pathways and mechanisms that conversely could reduce the radio-sensitivity. What we theoretically assume might be different in clinical applicability and reality. One of the challenges is, for example, the fundamental question of what effect this extract or bioactive compounds could induce to healthy cells surrounding the tumor. Hence, further in vitro or in vivo studies are needed to establish the evidence of this radio-sensitizing potential.

Authors' contributions
All of the authors participated in the planning of this study. DAW, ML, and AT were responsible for the critical appraisal process. HW, TBMP, H, EN, HK, SAG were responsible for the interpretation and discussion of the appraisal results. All of the authors read and agreed to the final manuscript for publication.