Potential Anticancer Properties and Mechanisms of Action of Formononetin

Nature, a vast reservoir of pharmacologically active molecules, has been most promising source of drug leads for the cure of various pathological conditions. Formononetin is one of the bioactive isoflavones isolated from different plants mainly from Trifolium pratense, Glycine max, Sophora flavescens, Pycnanthus angolensis, and Astragalus membranaceus. Formononetin has been well-documented for its anti-inflammatory, anticancer, and antioxidant properties. Recently anticancer activity of formononetin is widely studied. This review aims to highlight the pharmacological potential of formononetin, thus providing an insight of its status in cancer therapeutics. Formononetin fights progression of cancer via inducing apoptosis, arresting cell cycle, and halting metastasis via targeting various pathways which are generally modulated in several cancers. Although reported data acclaims various biological properties of formononetin, further experimentation on mechanism of its action, medicinal chemistry studies, and preclinical investigations are surely needed to figure out full array of its pharmacological and biological potential.


Introduction
Natural products have served as an infinite reservoir of various diversified chemical compounds, driving pharmaceutical industries for many years [1]. In the discipline of cancer therapeutics, natural products hold a great potential. It has been reported that, from 1981 to 2014, about 1562 drugs were approved out of which 1211 drugs were derived from small molecules that are nonsynthetic [2]. About 50% of the medicines and 48.6% of anticancer drugs are derivatives of natural products [3]. Chinese encyclopedia that dates back to 2000 BC has reported that 1898 herbal products have been used as medicines [4]. In accordance to World Health Organization, about 80% of population depends upon plant-derived traditional medicines. This drug discovery approach exhibits far-reaching domain where large-scale research could provide novel leads against cellular or molecular targets [5].
Triterpenoid saponins and isoflavones belong to family of amphiphilic glycosides which are naturally present in medicinal plants, herbs, and marine organisms. Saponins and isoflavones have major role in folk medicine due to their biological and pharmacological properties [9]. These secondary metabolites possess various anticancer, anti-inflammatory, and antioxidant properties. Epidemiological studies recommend that intake of food enriched with isoflavonoids reduces the risk of various cancers [9].

Biological Activities of Formononetin and Mechanisms of Its Action
The biological and pharmacological activities of bioactive compound "Formononetin" are well-documented as antitumor activity, [23] antiproliferative activity [14], growth inhibitory activity [55], vasorelaxant action [56], neuroprotective effect [57], antiapoptotic activity [48], cardioprotective activity [58], mammary gland proliferative activity [59], antioxidant activity [60], antimicrobial activity, and anti-inflammatory activity [15] as provided in Figure 2. Numerous in vivo/in vitro investigations have been done on Formononetin to uncover its biological attributes and mechanisms of actions. 3.1. Anticancer Activity. Cancer is uncontrolled proliferation of cells which occurs due to genetic or epigenetic modifications and signaling defects in cells [61]. Different synthetic drugs are used for the treatment of this deadly disease [2]. Due to the limited success of synthetic drugs, it is necessary to identify novel natural products having the ability to induce apoptotic cell death and arrest cell cycle in tumor cells without toxic effect on normal cells [62]. Various studies have declared that formononetin can block, delay, or inhibit the initiation and progression of cancer. This review intends to focus on the studies relevant to anticancer potential of Formononetin which will allow the researchers to further investigate this novel chemical entity as a potential anticancer drug candidate. Currently, it is documented that about 60% drugs that are used for cure of cancer are derived from natural sources [63]. Secondary metabolites isolated from natural products encompassing terpenes, alkaloids, isoflavones, and polyphenols have been reported as anticancer agents [64,65]. Anticancer properties of formononetin have been documented     against several types of cancer [66] such as breast [67], colon [41], glioma [54], osteosarcoma [68], multiple myeloma [52], adrenal medulla [51], nasopharyngeal [50], prostate [49], bladder [45], laryngeal [23], lung [43], and cervical cancer [42] (Figure 3).

Formononetin and Cell Cycle
Arrest. Uncontrolled divisions of cells are key trait of cancer cells [69]. Natural compounds have ability to prohibit the functions of cyclin dependent kinases and various other cell cycle regulatory proteins, thus causing cell cycle arrest [70]. Formononetin has been documented to arrest the cell cycle at various stages [44]. In human ovarian cancer, Formononetin leads cancer cells towards apoptosis and arrested cell cycle at G0/G1 phase in ES2 and OV90 cell lines [10]. Formononetin downregulated cyclin D1 which further arrested the cell cycle at the phase of G0/G1 in HCT-116 cells [41]. Formononetin arrested cells at G0/G1 phase in HepG2 cancerous cells [23]. In lung cancer cells, it caused the arresting of cell cycle at G1 phase via alteration of the p21, cyclin A, and cyclin D expression [44]. In PC-3 and DU145 cells, this compound has the ability to arrest the cells at G0/G1 phase via reducing AKT/cyclin D1/CDK4 expression [49] (Figure 4). It can be summarized that Formononetin causes the arrest the cells at G0/G1 phase but it needs to be investigated whether it has arrested the cellular cycle at G0 or G1 phase. Furthermore, it is interesting for researchers to investigate whether Formononetin could arrest the cells at G2/M or Sphase. Thus, further mechanistic investigations are yet obligatory to understand the mechanisms by which Formononetin arrests the cell cycle in various cancers.

Formononetin and Apoptosis.
Apoptosis is a systematic and synchronized way of cell death which is peculiarized by different morphological features including formation of blebs on cell membrane, condensation of chromosomes, and fragmentation of nucleus [71,72]. It is reported that certain cellular signals are necessary to regulate the growth of cells but mutation in these signals prevent the cells to undergo apoptosis [73,74]. Accumulated data indicated that various chemopreventive agents have been identified that can lead cancer cells towards apoptosis [75,76]. Formononetin has the ability to inhibit or block the growth of cancerous cells via intrinsic or extrinsic apoptosis pathways [49].
(1) Formononetin and MAPK and PI3K/AKT Pathway. MAPK, mitogen-activated protein kinase pathway, performs significant function in cell division, differentiation, proliferation, and cellular apoptosis [77]. MAPK pathway has four distinct signaling domains such as ERK, JNK, BMK-1, and p38 MAPK. Extracellular signals conducted by these kinases regulate the process of cellular apoptosis and growth [78,79]. MAPK pathway has been reported as novel target to combat cancer. The PI3K/AKT pathway also performs a significant role in tumor development. This activated pathway is affiliated with several cancer types. Therefore, targeting this pathway has the ability to combat cancer [80].
Isoflavonoids have been documented as anticancer agents that inhibit cancer cell proliferation and have antimetastatic effects [35,81]. Formononetin has ability for the induction of apoptosis in breast cancerous cells via activating Ras/p38 MAPK pathway in a dose dependent mode [40].
Formononetin reduced the p-AKT, p-P90RSK, p-S6, p-ERK1/2, and p-P70S6K proteins as well as enhancing the levels of p-p38 MAPK to mediate cellular proliferation and apoptosis in OV90 and ES2 cells [10]. Treatment with formononetin enhanced the levels of ER and p-AKT in HUVEC and MCF-7 cancer cells [37]. This natural compound Formononetin suppresses proliferation and invasive capability of cells by inhibiting MMP-2/-9 via inactivation of phosphorylation of AKT and PI3K in HCT-116 and SW1116 cells [37,41]. Formononetin also exerts its anticancer effects due to downmodulation of p-AKT, miR-21 expression, and upregulation of PTEN in T24 cells [45]. Formononetin inhibited the proliferation via inactivation of PI3K/ AKT pathway, enhancing the Bax, and downmodulating the Bcl-2 expression [68]. Formononetin and its derivatives such as dithiocarbamate with IC 50 value of 1.9 M possess inhibitory potential against PC3 cells [46] (Table 2).
(2) Formononetin and JAK/STAT Signaling Pathway. JAK/STAT pathways mediate the transduction of various signals involving cell division, immunity, tumor development, and cell death. Disruption in JAK/STAT signaling pathways may cause several diseases including cancer and immune disorders [82]. Formononetin decreased the activation of STAT5 and STAT3 by suppressing the nuclear translocation of p-STAT5 and p-STAT3 and also inhibited the activation JAK1 and JAK2 in U266 cells. Formononetin also inhibited the IL-6-induced STAT3 activity which ultimately inhibits the cell viability and activates apoptosis [52]. Formononetin suppresses the cellular invasion and proliferation by inhibition of MMP-2/-9 via inactivation of p-STAT3 pathway in colon carcinoma cells [41].

In Vivo Studies and Biosafety Profile
Formononetin with IC 50 value 2-6 M has the ability to promote the expression of p-AKT, miR-375, and Bcl-2 in in vivo mice model [37]. Treatment with Formononetin suppresses growth of tumor in in vivo tumor mouse model at the dose of 60 mg/kg [49]. An in vivo investigation demonstrated that Formononetin combined with other compounds reduced allergic inflammation in mice model via downregulating NF-B activation [83]. Administration of Formononetin reduced the size, volume, and weight of HeLa tumor in in vivo mice model induced by injection of HeLa cells in the posterior flanks of mice [84]. As Formononetin is the imperative components of Chinese folk medicines Radix Astragalus and Fukeqianjin which are mostly used as antioxidant and anticancer agents, respectively, therefore, it might serve as safe chemotherapeutic drug candidate [85,86]. Formononetin turns out to be a fascinated bioactive entity as it combines active chemotherapeutic effect with less toxicity in comparison to other isoflavones. However, safe doses and effectiveness of formononetin in targeted therapeutic fields still need to be executed in future.

Other Biological Functions
Formononetin is extracted from different plants such as Dalbergia odorifera in which Formononetin along with other compounds showed antiallergic and anti-inflammatory activities [87]. Formononetin together with other known compounds acts as active inhibitor of EV-A71 infection [88]. Brazilian Red propolis extract containing isoflavonoids such as Formononetin exhibits anti-inflammatory potential in a rat model of inflammation [89]. Isoflavones such as Formononetin isolated from soy bean possess antimicrobial and antioxidant activities with IC 50 values from 10.6 to 22.6 g/mL [15]. A study indicates that a methoxy Isoflavone, Formononetin, has potential for bone healing process in mouse model and has promising role in fracture-repair process [90]. Hydroalcoholic extract of Red propolis containing Red propolis has the ability to repair axon after sciatic nerve injury in rat model [91].

Conclusion and Future Perspectives
In this review article, we have suggested that Formononetin is a good drug candidate with promising pharmacological activities. Various researches have documented the potential applications of Formononetin both in vivo and in vitro. Being an important bioactive constituent of edible foods such as soybean, chickpeas, and alfalfa beans, Formononetin might turn up as a safe chemotherapeutic drug candidate. Many studies have revealed that formononetin is an inducer of  Downregulation↓, Upregulation ↑, Activation Act , Inhibition⊥, Bax: Bcl-2 associated x protein, Bcl-2: B-cell lymphoma, JNK: c-Jun-N-terminal kinase, MAPK: mitogen activated protein kinase, mTOR: mammalian target of rapamycin, Cdks: cyclin dependent kinases, EKR: extracellular signal-regulated kinase.
apoptosis in many cancerous cells, but still mechanism of its action is not fully clarified. After the analysis of data, we have found that Formononetin is most active against nasopharyngeal cell line CNE2 with IC 50 value of 1 M; so, further mechanistic studies should be conducted in future because nasopharyngeal carcinomas are one of the most prevalent cancers in Asia. This review also elucidates the potential role of Formononetin for the cure of several other diseases. Reported studies acclaim multiple biological properties of Formononetin but further experimentations on mechanism of its action, medicinal chemistry, and preclinical researches are yet necessary to figure out full array of its biological and pharmacological potential.

Conflicts of Interest
Authors declare that there are no conflicts of interest.

Authors' Contributions
Dongjun Jiang and Rabia Batool made contribution to writing different parts of the manuscript. Azhar Rasul and Ghulam Hussain have made substantial contribution to integration of the data and drafting of the manuscript. Muhammad Mateen Tahir has contributed to acquisition of data. Iqra Sarfraz and Tian Qin designed and generated figures of manuscript. Zeliha Selamoglu and Muhammad Ali have reviewed the manuscript. Xiaomeng Li and Jiang Li have read and approved the final manuscript.