Fisetin: An anticancer perspective

Many efforts have been made on researching potential anticancer agents in last decades. Natural products were among the popularly investigated agents. They are considered to have wider targeting pathways in comparison with synthetic drugs. Fisetin as a polyphenol with pleiotropic pharmacological properties showed promising anticancer activity in a wide range of cancers

1) is generally recognized as a bioactive plant, which has been used as potential drug against various free radical mediated as well as human cancers (Sengupta, Banerjee, & Sengupta, 2005). Fisetin is frequently present in vegetables (onions and cucumbers), fruits (persimmon, apples, strawberries), wine, and nuts, and their concentrations are varied from 2 to 160 mg/g with an average daily intake estimate of 0.4 mg.
Nutritional supplements are also prepared by fisetin by using higher concentrations and exhibited different pharmacological activities, including antioxidant and anti-inflammatory activity acting. Due to scavenging and neutralizing effects of fisetin, it also induces apoptosis, exhibits cell cycle hold, and suppresses the cyclin-dependent kinases (CDKs) in human cancer cell lines. Fisetin also has been known to modulate the lipid kinase and protein kinase pathways (Inkielewicz-Stepniak, Radomski, & Wozniak, 2012;Liao, Shih, Chao, Lee, & Chiang, 2009;Park et al., 2008).
In human multiple myeloma U266 cells, fisetin stimulated the production of free radical species that led to apoptosis signaling as well as showed AMP-activated protein kinase signaling (Jang et al., 2012). Multiple studies also authenticated the anticancer role of fisetin through various signaling pathways such as blocking of mammalian target of rapamycin (PI3K/Akt/mTOR)/phosphatidylinositol-3-kinase/protein kinase B, mitogen-activated protein kinases (MAPK)-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and p38, respectively, whereas these cellular processes have significantly led to malignancy (Mukhtar, Adhami, Sechi, & Mukhtar, 2015;Ren et al., 2020).

| Breast cancer
Epigenetic regulation of human epidermal growth factor receptor 2 (HER2) is observed in breast cancer. Fisetin has shown the effects on human epidermal growth factor receptor 2 (HER2)/neu-overexpressing breast cancer cell lines. Fisetin caused induction through inactivating the receptor, inducing the degradation of the proteasomes, reducing its half-life, reducing phosphorylation enolase, and altering the phosphatidylinositol 3-kinase/Akt signaling (Guo, Dong, & Shi, 2018). In triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and BT549 cells, fisetin in dose-dependent concentration has been found to suppress the cell proliferation, migration, and invasion. In addition, mutation of epithelial-to-mesenchymal transition (EMT), inhibition of phosphoinositide 3-kinase (PI3K)-Akt-GSK-3β signaling pathway, and up-regulation of expression of PTEN mRNA and protein were reported after fisetin treatment .
In breast cancer cells (4T1 and JC cells), fisetin increased HO-1 mRNA and protein expressions, elevated Nrf2 expression, and abrogated the HO-1 expression, whereas HO-1 expression was mediated by up-regulation of the transcription factor Nrf2. In addition, fisetin reduced MMP-2 and MMP-9 enzyme activity and gene expression for both mRNA levels and protein (Tsai et al., 2018). Different mechanisms are linked with preventive role of fisetin against the proliferation of breast cancer cell lines such as MDA-MB-231, MCF-7, and 4T1. Fisetin, a natural flavonoid, was found to prevent the various stages of cancer (migration and invasion). In in vitro study of mice, the effects of fisetin led to prohibition of metastasis and invasiveness, induction of the apoptosis, and regulation of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway, whereas suppression of growth of breast tumors and enhancement of tumor cell apoptosis were observed in serum of tumor-bearing mice (Sun et al., 2018).
Many recent reports elaborated the potential and significant role of fisetin against human breast cancer MCF-7 cell proliferation, and they found that fisetin decreased the growth and development and also induced apoptotic cell death (Pawar, Singh, Rajalakshmi, Shaikh, & Bothiraja, 2018). Fisetin evidently suppressed the tumor burden, migration, and volume in MCF-7 breast cancer cells as observed by Wang, Zhang, and Wang (2017). The mechanism of growth inhibition of MDA-MB-468 and MDA-MB-231 TNBC cells by fisetin treatment was explored; it was shown that fisetin prevented the activity of the estrogen receptor (MCF-7 cells), colony formation, human epidermal growth factor receptor 2-overexpressing (SK-BR-3 cells), cell division (TNBC cells), and induced apoptosis. In addition, fisetin treatment further led to permeabilization of mitochondrial membrane, activation of caspase-8 and caspase-9, as well as the cleavage of poly(ADP-ribose) polymerase 1. Moreover, it also induced caspase-dependent apoptosis, lowered phosphorylation of histone H3 at serine 10, caused G2/M phase of the cell cycle, and suppressed the Aurora B kinase, respectively, in above-mentioned human cancer cell lines (Smith, Murphy, Doucette, Greenshields, & Hoskin, 2016). Our data showed that significant attenuation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell invasion, suppression of activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways, and reduction in NF-κB activation were reported after fisetin treatment. However, these changes led to the down-regulation of matrix metalloproteinase (MMP)-9 expression in MCF-7 human breast cancer cells (Noh et al., 2015). In human breast cancer MCF-7, we validated the preventive role of fisetin against cancer proliferation in MDA-MB-231 cells through previous findings. They examined that fisetin showed numerous apoptotic characteristics such as breakup of plasma membrane, mitochondrial depolarization, caspase (−7, −8, and −9) activation and PARP cleavage, p53 activation, and autophagy reduction. In contrast, fisetin also performed neither other features nor such fragmentation of DNA and phosphatidylserine (PS) externalization (Yang, Tseng, Peng, Chen, & Chiu, 2012).

| Prostate cancer
The combining effect of fisetin (20μmol/L) and cabazitaxel (5 nmol/L) significantly decreased cell viability and metastatic properties of different cancer cell lines such as PC-3M-luc-6, 22Rν1, and C4-2 on normal prostate epithelial cells. Additionally, both compounds inhibited the cancer stages (proliferation, tumor growth, and invasion) and induced apoptosis in in vivo xenograft mouse models (Mukhtar, Adhami, Siddiqui, Verma, & Mukhtar, 2016). Fisetin treatment resulted in prostate cancer (PCa) of in vitro and in vivo xenograft animals caused down-regulation of intracellular and secreted hyaluronan levels and suppressed the hyaluronan (HA) synthesis and degradation enzymes (Lali et al., 2016). Multiple pathways are involved to prevented from the proliferation of human prostate cancer cell lines through fisetin administration. The changes occurred are inhibition of viability and colony formation, robust up-regulation of microtubule-associated proteins (MAP)-2 and MAP-4, suppression of the migration and invasion stages, and inhibition of protein Nudc, which is linked with microtubule motor dynein complex that regulates microtubule dynamics (Mukhtar et al., 2015).
Epithelial-to-mesenchymal transition (EMT) and transcription/ translation regulatory Y-box binding protein-1 (YB-1) have imperative role in association with cancer metastasis. In human prostate cancer tissues, enhancement of YB-1 expression was linked with tumor grade, while it exhibited an inverse relationship with E-cadherin. The induction of mesenchymal morphology via forced YB-1 expression was linked with epithelial markers that are controlled down. In PCa cells, ridiculing of YB-1 reversed mesenchymal characteristics and reduced cell proliferation, migration, and invasion. Within the cold shock domain (CSD), YB-1 is stimulated directly through Akt-mediated phosphorylation at Ser102. Computational docking and molecular dynamics indicated that fisetin binds on CSD residues from β1 to β4 strands, inhibiting the interaction between Akt and YB-1. Calculated free binding energy ranged from −11.9845 to −9.6273 kcal/mol. Fisetin binds to YB-1 with an average affinity of 35 μM, in both slow association and dissociation. Fisetin also prevented EGF-induced YB-1 phosphorylation and EMT markers both in vitro and in vivo (Adhami, Syed, Khan, & Mukhtar, 2012;Khan, Afaq, Syed, & Mukhtar, 2008). A study conducted by Szliszka, Helewski, Mizgala, and Krol (2011) investigated that fisetin treatment in prostate cancer cell lines (LNCaP, DU145, and PC3) sensitized the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant HepG2 cell. The augmentation of TRAIL-mediated cytotoxicity and apoptosis through engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways was observed after fisetin treatment. Furthermore, it also lowered the activity of NF-κB, caused significant activation of caspase-8 and caspase-3 and disruption of ΔΨm, and enhanced the levels of TRAIL-R1, respectively (Szliszka et al., 2011). A study performed by Haddad et al., 2010 ex-plored that fisetin dose-dependently in PC3 and LNCaP cells at the rate of 1-50 microM significantly decreased the cell viability along with enhancement in apoptotic cell death as well as alterations of gene expressions. Moreover, the alteration in chromosome organization, apoptosis, and stress response was also observed (Haddad et al., 2010).
The targeting of mammalian target of rapamycin (mTOR) kinase and PTEN/PI3K/Akt are an important strategy to prevented from the proliferation of prostate cancer. Being a chemopreventive role against different cancer cell lines, fisetin has been found to suppress the PTEN/PI3K/Akt and mTOR signaling pathway. The down-regulation of PRAS40, Rictor, Raptor, and GbetaL, which further led to loss of mTOR complex (mTORC)1/2 formation, activation of mTOR repressor TSC2 by suppressing Akt and activating AMPK, suppression of Cap-dependent translation, and hypophosphorylation of 4EBP1 as well as induction of autophagic-programmed cell death, was reported after fisetin treatment (Suh et al., 2010). A peer group of researchers and investigators explored that multiple processes are involved to prevented from the proliferation of prostate cancer such as suppression of MMP-2 and MMP-9 activities, inhibition of phosphorylation of c-Jun N-terminal kinases 1 and 2 (JNK1/2) and Akt, and reduction in c-Fos, NF-kB, and c-Jun, as well as showed binding abilities of NF-kappaB and activator protein-1 (AP-1), respectively (Chien, Shen, Huang, Ko, & Shih, 2010). Therefore, data

| Colon
In in vitro and in vivo studies, fisetin works as an effective anticancer agent via inhibiting cell viability and tumor growth, inducing apoptosis, promoting caspase-8 and cytochrome c expressions, and suppressing the aberrant activation of IGF1R and AKT proteins in LoVo cells of athymic nude mouse xenograft model (Jeng et al., 2018). Various in vivo studies showed that fisetin alone performed anticancer activities against human colon cancer cell lines (CT-26 and HCT116) and markedly down-regulated the level of the oncoprotein securin in a p53-independent manner, whereas fisetin in combination with 2-Gy radiation did not significantly suppress securin-null HCT116 tumor growth compared with normal HCT116 tumors (Leu et al., 2016). Similarly, a study investigated after treatment of fisetin against CT26 cells concluded that fisetin exhibited multiple processes including suppression of tumor growth and proliferation, and enhancement of survival time, apoptotic cell death, and antiangiogenesis activities, respectively (Chen, Wu, et al., 2015;Wu, Lien, Shen, Yang, & Chen, 2014). Researchers also found that fisetin in combination with geldanamycin or radicicol in vivo study in human COLO205 colon cancer cells significantly increased expressions of caspase-3 and caspase-9 activities, modified the Bcl-2 but not Bax protein or Bcl-XL, and decreased the and DNA synthesis, exhibited G(2)/M phase arrest, lowered cell number, the activities of CDK-2,4), cyclin E and D1, enhanced the p21(CIP1/WAF1) levels as well as shifting of phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated, decreased and an increase in. In fisetin-treated cells, it also decreased cell division cycle protein levels (CDC)2 and CDC25C, and CDC2 activity (Lu et al., 2005).

| Liver
Fisetin has been known to prevented from the liver cancer cell line proliferation in vivo study of mice via inhibiting the migration and invasion, inducing apoptosis, activating caspase-3, suppressing TGF-β1, lowering epithelial-mesenchymal transition (EMT), ameliorating cancer progression, enhancing survival time, and down-regulating the expressions of p-ERK1/2, vascular endothelial growth factor receptor 1(VEGFR1), p38, and pJNK, respectively (Liu, Long, Miao, Liu, & Yao, 2017). A study addressed by Chen et al. (2002) determined the cytotoxic effect of fisetin against hepatocellular carcinoma cell SK-HEP-1. Fisetin (80 microM) showed dose-dependently caused DNA fragmentation, induced cellular swelling and apoptotic death, and showed characteristics of apoptosis. Moreover, fisetin also induced caspase-3/CPP32 activity, but not of caspase-1 activity. The cleavage of caspase-3 substrates including D4-GDI protein and poly(ADP-ribose) polymerase (PARP) reduction in pro-caspase-3 proteins, and enhancement of p53 protein were reported after fisetin treatment in SK-HEP-1 cells . Fisetin treatment at the dose of 0.1 to 10 microM in dose-and time-dependent manner in cells significantly induced quinone oxidoreductase (QR) activity associated with QR mRNA expression and activated the ARE/ EpRE (Hou et al., 2001).

| Kidney
Being a potent anticancer agent, fisetin administration in in vitro and in vivo studies in kidney renal stem cells (HuRCSCs) effectively inhibited cancer cell stages such as proliferation, cell division, and invasion as well as lowered the TET1 expression levels. It also inhibits 5hmC modification levels at the CpG islands in cyclin Y (CCNY) and CDK16 and reduces the transcription and activity (Si et al., 2019). A study has shown that fisetin treatment prevented from the prolif-  In another study reported by Jang et al. (2005), they found that fisetin administration to human osteosarcoma (HOS) cells significantly inhibited growth, induced apoptosis, cleavaged PARP, activated the caspase-8 and Bax, suppressed the Bcl-2 levels, and released cytochrome c (Jang et al., 2005).

| Oral
Fisetin enhances the suppression of autophagy and induces apoptotic cell death in human tongue squamous cell line Ca9-22 (Park et al., 2019). Considering anticancer role, fisetin has been found to exhibit effectual role against different human oral squamous cell including Tca-8113 and UM-SCC-23 cancer cell lines through multiple pathways, that is, suppression of Met/Src signaling pathways, inhibition of level of a disintegrin and metalloproteinase 9 (ADAM9) protein, and reduction in basal expression of Met and Src protein, respectively (Li, Qin, & Dai, 2017). In in vitro study of HSC3 human oral cancer cell lines, the administration of fisetin was associated with induction of apoptotic cell death via enhancement of reactive oxygen species and Ca 2+ , caspase-3, caspase-8, and caspase-9 activities along with the reduction in mitochondrial membrane potential.
The reduction in antiapoptotic protein such as BCL-X and BCL-2; increment of apoptotic-associated protein expressions like BCL2associated X (BAX), antagonist/killer (BAK), and B-cell lymphoma 2 (BCL2); and enhancement of apoptosis-inducing factor (AIF), caspase-3, caspase-8, and caspase-9 (cleaved forms), and cytochrome c, and endonuclease G (ENDO G) from mitochondria into the cytoplasm, respectively, were reported after fisetin treatment (Shih et al., 2017). Multiple mechanisms are involved to prevented from the human oral cancer SCC-4 cells after administration of fisetin in in vitro study. These mechanisms include induction of cell death via morphological changes, promotion of ROS and Ca 2+ production, reduction in expressions of ΔΨ m , and enhancement of caspase-3, caspase-8, and caspase-9 activities, and caused G2/M phase arrest, respectively. It also caused lowering in levels of Bcl-2 (antiapoptotic proteins) and enhancement in expressions of pro-apoptotic proteins (Bax and Bid) as well as enhancement in the AIF, cytochrome c, and Endo G secreted from mitochondria. Furthermore, cell death in SCC-4 cells was done through endoplasmic reticulum stress (Su et al., 2017). The role of fisetin on TU212 cell proliferation was reported by Zhang and Jia (2016), and they investigated that fisetin induced apoptosis, suppressed cell proliferation, and promoted caspase-3 expressions by regulating PI3K/AKT/NF-κB and inhibiting the activation of PI3K/AKT-regulated mTOR. Moreover, fisetin administration in in vivo study markedly lowered tumor volume and weight in nude mice (Zhang & Jia, 2016).  (Li, Park, & Kim, 2015).

| Stomach
Fisetin bioactive compound exerts anticancer activity against SGC7901 cancer and GES-1 normal cells by acting on cellular events.
Resultantly, different doses of fisetin at the rate of 1, 5, 10, 15, and 20 µM for 48h in dose-dependent manner significantly decreased the proliferation, enhanced the apoptosis, and reduced the activation of ERK 1/2, accordingly (Yan, Chen, Zhao, & Ye, 2018). Fisetin was also reported to prevented from the human gastric carcinoma AGS and SNU-1 cells by applying different concentrations (25-100 μM). It markedly increases the levels of p53 and its S15 phosphorylation, enhances apoptotic cells, lowers the levels of G1 phase cyclins and CDKs, exhibits mitochondrial membrane depolarization, and enhances the expressions of p53 and its S15 phosphorylation (Sabarwal, Agarwal, & Singh, 2017).

| Blood
The effect of fisetin was analyzed on human Burkitt's lymphoma Raji  . Therefore, fisetin also induces apoptosis, enhances the fraction of the cells with sub-G1 content, up-regulates the Bax, Bim, and Bad, activates the caspase-3, down-regulates the Bcl-2 and Mcl-1(L), activates the adenosine monophosphateactivated protein kinase (AMPK) along with its substrate acetyl-CoA carboxylase (ACC), stimulates the production of ROS, and decreases the phosphorylation of AKT and mTOR, respectively.
A group of researchers and investigators found that fisetin has inhibitory effect on different cancer stages (adhesion, migration, and invasion) of human lung adenocarcinoma A549 cells through multiple pathways such as down-regulation of levels of matrix metalloproteinase-2 (MMP-2), urokinase-type plasminogen activator (uPA), and suppression of phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) at both the protein and mRNA levels.
Likewise, reduction in nuclear levels of nuclear factor kappa B (NF-kappaB), c-Jun, and c-Fos, and prevention from the binding abilities of NF-kappaB and activator protein-1 (AP-1) were done after fisetin administration.

| Bladder
The induction of bladder cancer in experimental animals (rat) was done via intravesical N-methyl-N-nitrosourea (MNU), whereas administration of fisetin bioactive compound prevented from the bladder cancer cell proliferation through inducing apoptosis, down-regulating the NF-κB pathway activity, up-regulating the p53 pathway activity, and exhibiting changes in the ratio of pro-and antiapoptotic proteins (Li, Qu, et al., 2014). Moreover, a group of re-

| Ovarian
Dose-dependently, fisetin in a xenograft mouse model carrying SKOV3 cells significantly induced increased tumor apoptosis, proliferation suppression, and antiangiogenesis activities (Xiao et al., 2018). The previous findings reported by Meng et al. n in vitro and in vivo studies observed that fisetin-treated cells (ovarian cancer cell line SKOV3) markedly decreased the tumor volume, tumor mass, and Bcl-2 levels and increased the Bax expressions in concentration-dependent manner in athymic rude rat model (Meng et al., 2016). The effects of fisetin bioactive ingredient in SKOV-3/ PAX cells in a dose-dependent manner are associated with suppressing tumor growth, the cleavage of caspase-9, caspase-8, and caspase-3, and PARP as well as enhanced the sub-G1 phase and lowered AKT phosphorylation (Choi et al., 2016). In

| Cervical
The combination of fisetin and sorafenib synergistically induced apoptosis in HeLa cells followed by enhancement in loss of mitochondrial membrane potential, activation of caspase-3 and caspase-8, and increment of Bax/Bcl-2 ratio; exhibited cleavage of PARP level; and disrupted the potential of mitochondrial membrane (Lin et al., 2016).

| Skin
Malignant melanoma is causative around 75% of deaths, which is associated with skin cancer. Fisetin has significant effect on production of free fatty acids, tumor incidence, IL-1α, TNF-α, and antioxidant enzymes (glutathione and catalase contents) in ultravoilet-induced skin cancer rats (Moolakkadath et al., 2019). YB-1 leads to cell proliferation and invasion through promoting epithelial-to-mesenchymal transition. The p90 ribosomal S6 kinase (RSK) activates YB-1 to proliferate melanoma growth. Multiple pathways are involved such as YB-1 dephosphorylation, suppression of mesenchymal markers, reduction in transcript levels and matrix-metalloproteinases, inhibition of RSK activity through binding to the kinase, inhibition of YB-1/ RSK signaling independent of its effect on ERK, and reduction in MDR1 levels in melanoma cells (Sechi et al., 2018).

| Other cancers
A study described by Chen, Wu, et al. (2015) assessed that noncytotoxic dose of fisetin bioactive compound in GBM8401 cells markedly inhibited the cell migration and invasion, and phosphorylated the ERK1/2 that suppressed the levels of ADAM9 protein and mRNA. In another study reported by Kim and their coworkers, they explored that fisetin in combination with gemcitabine has anticancer role on human cholangiocarcinoma (CCA) cell line SNU-308. Both compounds suppress the survival rate of cells via phosphorylating ERK and induce apoptotic cell death, and also decreased the levels of cellular proliferative markers (myelocytomatosis and phospho-p6) .