Fisetin induces G2/M phase arrest and caspase-mediated cleavage of p21Cip1 and p27Kip1 leading to apoptosis and tumor growth inhibition in HNSCC.

The anticancer potential and associated mechanisms of flavonoid fisetin are yet to be fully investigated on human head and neck squamous cell carcinoma (HNSCC). In the present study, fisetin (25-75 µM for 24-48 h) dose-dependently inhibited growth and induced death in HNSCC Cal33 and UM-SCC-22B cells, without showing any death in normal cells. Fisetin (25-50 µM) induced G2/M phase arrest via decrease in Cdc25C, CDK1, cyclin B1 expression, and an increase in p53(S15). A concentration-dependent increase in fisetin-induced DNA damage and apoptosis in HNSCC cells was authenticated by comet assay, gamma-H2A.X(S139) phosphorylation, and marked cleavage of PARP protein. Interestingly, fisetin-induced cell death occurred independently of p53 and reactive oxygen species production. The activation of JNK and inhibition of PI3K/Akt, ERK1/2, EGFR, and STAT-3 signaling were identified. Further, fisetin-induced apoptosis was mediated, in part, via p21Cip1 and p27Kip1 cleavage by caspase, which was reversed by z-VAD-FMK, a pan-caspase inhibitor. Subsequently, fisetin was also found to induce autophagy; nevertheless, autophagy attenuation exaggerated apoptosis. Oral fisetin (50 mg/kg body weight) treatment to establish Cal33 xenograft in mice for 19 days showed 73% inhibition in tumor volume (p < 0.01) along with a decrease in Ki67-positive cells and an increase in cleaved caspase-3 level in tumors. Consistent with the effect of 50 µM fisetin in vitro, the protein levels of p21Cip1 and P27Kip1 were also decreased by fisetin in tumors. Together, these findings showed strong anticancer efficacy of fisetin against HNSCC with downregulation of EGFR-Akt/ERK1/2-STAT-3 pathway and activation of JNK/c-Jun, caspases and caspase-mediated cleavage of p21Cip1 and p27Kip1.

Fisetin, a potential skin rejuvenation drug that eliminates senescent cells in the dermis.

Accumulation of senescent fibroblasts, chronic inflammation, and collagen remodeling due to aging-related secretory phenotypes have been hypothesized to cause age-related skin aging, which results in wrinkles and loss of skin elasticity, thus compromising appearance attractiveness. However, the rejuvenating effects of removing senescent cells from the human skin and the efficacy of related therapeutic agents remain unclear. Here, we investigated the effects of fisetin, a potential anti-aging component found in various edible fruits and vegetables, on senescent human dermal fibroblasts (HDFs) and aging human skin. Senescence was induced in primary HDFs using long-term passaging and treatment with ionizing radiation, and cell viability was assessed after treatment with fisetin and a control component. A mouse/human chimeric model was established by subcutaneously transplanting whole skin grafts from aged individuals into nude mice, which were treated intraperitoneally with fisetin or control a component for 30 d. Skin samples were obtained and subjected to senescence-associated-beta-galactosidase staining; the extent of aging was evaluated using western blotting, reverse transcription-quantitative PCR, and histological analysis. Fisetin selectively eliminated senescent dermal fibroblasts in both senescence-induced cellular models; this effect is attributable to cell death induction by caspases 3, 8, and 9-mediated endogenous and exogenous apoptosis. Fisetin-treated senescent human skin grafts showed increased collagen density and decreased senescence-associated secretory phenotypes (SASP), including matrix metalloproteinases and interleukins. No apparent adverse events were observed. Thus, fisetin could improve skin aging through selective removal of senescent dermal fibroblasts and SASP inhibition, indicating its potential as an effective novel therapeutic agent for combating skin aging.

Fisetin ameliorates fibrotic kidney disease in mice via inhibiting ACSL4-mediated tubular ferroptosis.

Kidney fibrosis is the hallmark of chronic kidney disease (CKD) progression, whereas no effective anti-fibrotic therapies exist. Recent evidence has shown that tubular ferroptosis contributes to the pathogenesis of CKD with persistent proinflammatory and profibrotic responses. We previously reported that natural flavonol fisetin alleviated septic acute kidney injury and protected against hyperuricemic nephropathy in mice. In this study, we investigated the therapeutic effects of fisetin against fibrotic kidney disease and the underlying mechanisms. We established adenine diet-induced and unilateral ureteral obstruction (UUO)-induced CKD models in adult male mice. The two types of mice were administered fisetin (50 or 100 mg·kg-1·d-1, i.g.) for 3 weeks or 7 days, respectively. At the end of the experiments, the mice were euthanized, and blood and kidneys were gathered for analyzes. We showed that fisetin administration significantly ameliorated tubular injury, inflammation, and tubulointerstitial fibrosis in the two types of CKD mice. In mouse renal tubular epithelial (TCMK-1) cells, treatment with fisetin (20 µM) significantly suppressed adenine- or TGF-ß1-induced inflammatory responses and fibrogenesis, and improved cell viability. By quantitative real-time PCR analysis of ferroptosis-related genes, we demonstrated that fisetin treatment inhibited ferroptosis in the kidneys of CKD mice as well as in injured TCMK-1 cells, as evidenced by decreased ACSL4, COX2, and HMGB1, and increased GPX4. Fisetin treatment effectively restored ultrastructural abnormalities of mitochondrial morphology and restored the elevated iron, the reduced GSH and GSH/GSSG as well as the increased lipid peroxide MDA in the kidneys of CKD mice. Notably, abnormally high expression of the ferroptosis key marker ACSL4 was verified in the renal tubules of CKD patients (IgAN, MN, FSGS, LN, and DN) as well as adenine- or UUO-induced CKD mice, and in injured TCMK-1 cells. In adenine- and TGF-ß1-treated TCMK-1 cells, ACSL4 knockdown inhibited tubular ferroptosis, while ACSL4 overexpression blocked the anti-ferroptotic effect of fisetin and reversed the cytoprotective, anti-inflammatory, and anti-fibrotic effects of fisetin. In summary, we reveal a novel aspect of the nephroprotective effect of fisetin, i.e. inhibiting ACSL4-mediated tubular ferroptosis against fibrotic kidney diseases.

Fisetin Suppresses Atherosclerosis by Inhibiting Ferroptosis-Related Oxidative Stress in Apolipoprotein E Knockout Mice.

INTRODUCTION: Fisetin has been demonstrated to inhibit the occurrence of atherosclerosis; however, the mechanism of fisetin suppressing atherosclerosis remains elusive. METHODS: The function of fisetin in the inhibition of atherosclerosis was evaluated by hematoxylin and eosin and Oil Red O staining in ApoE-/- mice. Molecular biomarkers of atherosclerosis progression were detected by Western blot and qPCR. Moreover, the inhibition of atherosclerosis on oxidative stress and ferroptosis was evaluated by immunofluorescence staining, qPCR, and Western blot assays. RESULTS: The obtained results showed that serum lipid was attenuated and consequentially the formation of atherosclerosis was also suppressed by fisetin in ApoE-/- mice. Exploration of the mechanism revealed that molecular biomarkers of atherosclerosis were decreased under fisetin treatment. The level of reactive oxygen species and malondialdehyde declined, while the activity of superoxide dismutases and glutathione peroxidase was increased under the fisetin treatment. Additionally, the suppressor of ferroptosis, glutathione peroxidase 4 proteins, was elevated. The ferritin was decreased in the aortic tissues treated with fisetin. CONCLUSIONS: In summary, fisetin attenuated the formation of atherosclerosis through the inhibition of oxidative stress and ferroptosis in the aortic tissues of ApoE-/- mice.

Fisetin-loaded chitosan nanoparticles ameliorate pilocarpine-induced temporal lobe epilepsy and associated neurobehavioral alterations in mice: Role of ROS/TNF-α-NLRP3 inflammasomes pathway.

Fisetin has displayed potential as an anticonvulsant in preclinical studies yet lacks clinical validation. Challenges like low solubility and rapid metabolism may limit its efficacy. This study explores fisetin-loaded chitosan nanoparticles (NP) to address these issues. Using a murine model of pilocarpine-induced temporal lobe epilepsy, we evaluated the anticonvulsant and neuroprotective effects of fisetin NP. Pilocarpine-induced seizures and associated neurobehavioral deficits were assessed after administering subtherapeutic doses of free fisetin and fisetin NP. Changes in ROS, inflammatory cytokines, and NLRP3/IL-18 expression in different brain regions were estimated. The results demonstrate that the fisetin NP exerts protection against seizures and associated depression-like behavior and memory impairment. Furthermore, biochemical, and histological examinations supported behavioral findings suggesting attenuation of ROS/TNF-α-NLRP3 inflammasome pathway as a neuroprotective mechanism of fisetin NP. These findings highlight the improved pharmacodynamics of fisetin using fisetin NP against epilepsy, suggesting a promising therapeutic approach against epilepsy and associated behavioral deficits.

Effects of fisetin on ethanol-induced rewarding properties in mice.

Background: Alcohol use disorder (AUD) is a chronic relapsing disorder associated with compulsive drinking of alcohol. Natural flavonoid fisetin affects a variety of transmitter systems relevant to AUD, such as aminobutyric acid, N-methyl-D-aspartate, and dopamine, as well as peroxisome proliferator-activated receptors.Objectives: This study investigated fisetin's impact on the motivational properties of ethanol using conditioned place preference (CPP) in mice (n = 50).Methods: Mice were conditioned with ethanol (2 g/kg, i.p.) or saline on alternating days for 8 consecutive days and were given intragastric (i.g.) fisetin (10, 20, or 30 mg/kg, i.g.), 45 min before ethanol conditioning. During extinction, physiological saline was injected to the control and ethanol groups, and fisetin was administered to the fisetin groups. To evaluate the effect of fisetin on the reinstatement of ethanol-induced CPP, fisetin was given 45 min before a priming dose of ethanol (0.4 g/kg, i.p.; reinstatement test day).Results: Fisetin decreased the acquisition of ethanol-induced CPP (30 mg/kg, p < .05) and accelerated extinction (20 and 30 mg/kg, p < .05). Furthermore, fisetin attenuated reinstatement of ethanol-induced CPP (30 mg/kg, p < .05).Conclusions: Fisetin appears to diminish the rewarding properties of ethanol, as indicated by its inhibitory effect and facilitation of extinction in ethanol-induced CPP. These findings imply a potential therapeutic application of fisetin in preventing ethanol-seeking behavior, promoting extinction, and reducing the risk of relapse.

Mechanochemical Approach to Obtaining a Multicomponent Fisetin Delivery System Improving Its Solubility and Biological Activity.

In this study, binary amorphous solid dispersions (ASDs, fisetin-Eudragit®) and ternary amorphous solid inclusions (ASIs, fisetin-Eudragit®-HP-ß-cyclodextrin) of fisetin (FIS) were prepared by the mechanochemical method without solvent. The amorphous nature of FIS in ASDs and ASIs was confirmed using XRPD (X-ray powder diffraction). DSC (Differential scanning calorimetry) confirmed full miscibility of multicomponent delivery systems. FT-IR (Fourier-transform infrared analysis) confirmed interactions that stabilize FIS's amorphous state and identified the functional groups involved. The study culminated in evaluating the impact of amorphization on water solubility and conducting in vitro antioxidant assays: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-ABTS, 2,2-diphenyl-1-picrylhydrazyl-DPPH, Cupric Reducing Antioxidant Capacity-CUPRAC, and Ferric Reducing Antioxidant Power-FRAP and in vitro neuroprotective assays: inhibition of acetylcholinesterase-AChE and butyrylcholinesterase-BChE. In addition, molecular docking allowed for the determination of possible bonds and interactions between FIS and the mentioned above enzymes. The best preparation turned out to be ASI_30_EPO (ASD fisetin-Eudragit® containing 30% FIS in combination with HP-ß-cyclodextrin), which showed an improvement in apparent solubility (126.5 ± 0.1 µg∙mL-1) and antioxidant properties (ABTS: IC50 = 10.25 µg∙mL-1, DPPH: IC50 = 27.69 µg∙mL-1, CUPRAC: IC0.5 = 9.52 µg∙mL-1, FRAP: IC0.5 = 8.56 µg∙mL-1) and neuroprotective properties (inhibition AChE: 39.91%, and BChE: 42.62%).

Fisetin Alleviates Inflammation and Oxidative Stress in Deep Vein Thrombosis via MAPK and NRF2 Signaling Pathway.

Oxidative stress and inflammation play pivotal roles in the progression of deep vein thrombosis (DVT). Fisetin has demonstrated promising pharmacological features; however, its underlying mechanisms in DVT remain elusive. In our study, we investigated the effects and underlying mechanisms of Fisetin on a DVT mouse model. The protective effects of Fisetin on DVT were evaluated by comparing the size of thrombosis and detecting the mRNA expression levels of pro-inflammatory cytokines. After that, the biological processes were studied via transcriptomics after Fisetin administration. The antioxidant effect was evaluated and explained via NRF2 signaling pathway. Finally, the anti-inflammatory effect was explained according to KEGG analysis and the final mechanism was verified via Western blot. Our results found that the mRNA expression levels of pro-inflammatory cytokines were inhibited by Fisetin. Moreover, transcriptomic studies suggested that MAPK signaling pathway may be associated with the anti-inflammatory activity of Fisetin. Then, we confirmed that Fisetin administration significantly inhibited the activation of typical pro-inflammatory signaling pathways via Western blot. Finally, the results of Western blot showed that Fisetin significantly activated NRF2 signaling pathway and induced the expression of downstream antioxidant enzymes. Our findings suggested that Fisetin exhibits potential therapeutic effects on DVT through its ability to attenuate inflammation and oxidative stress. The underlying mechanism may involve the suppression of MAPK-mediated inflammatory signaling pathway and activation of NRF2-mediated antioxidant signaling pathway.

Aspirin-Fisetin Combinatorial Treatment Exerts Cytotoxic and Anti-Migratory Activities in A375 Malignant Melanoma Cells.

Background and Objectives: Malignant melanoma (MM) remains one of the most aggressive cancers worldwide, presenting a limited number of therapeutic options at present. Aspirin (ASA), a broadly used non-steroid anti-inflammatory medicine, has recently emerged as a candidate for repurposing in cancer management, due to its therapeutic potential in the treatment of several neoplasms which include MM. Fisetin (FIS) is a flavonoid phytoestrogen instilled with multispectral pharmacological activities, including a potent anti-melanoma property. The present study aimed to assess the potential improved anti-neoplastic effect resulting from the association of ASA and FIS for MM therapy. Materials and Methods: The study was conducted using the A375 cell line as an experimental model for MM. Cell viability was assessed via the MTT test. Cell morphology and confluence were evaluated using bright-field microscopy. The aspect of cell nuclei and tubulin fibers was observed through immunofluorescence staining. The irritant potential and the anti-angiogenic effect were determined on the chorioallantoic membrane of chicken fertilized eggs. Results: The main findings related herein demonstrated that the ASA 2.5 mM + FIS (5, 10, 15, and 20 µM) combination exerted a higher cytotoxicity in A375 MM cells compared to the individual compounds, which was outlined by the concentration-dependent and massive reduction in cell viability, loss of cell confluence, cell shrinkage and rounding, apoptotic-like nuclear features, constriction and disruption of tubulin filaments, increased apoptotic index, and suppressed migratory ability. ASA 2.5 mM + FIS 20 µM treatment lacked irritant potential on the chorioallantoic membrane and inhibited blood-vessel formation in ovo. Conclusion: These results stand as one of the first contributions presenting the anti-melanoma effect of the ASA + FIS combinatorial treatment.

Fisetin induces apoptosis in colorectal cancer cells by suppressing autophagy and down-regulating nuclear factor erythroid 2-related factor 2 (Nrf2).

Modulation of autophagy is evolving as a relevant strategy in cancer pathogenesis and therapeutic intervention and hence, needs to be examined as a target for the promising anticancer agents. Fisetin, a dietary flavanol, is emerging as a potent anticancer agent, however, its tumour-specific pharmacological targets remain largely unexplored. This article describes correlative profiles of autophagy and apoptotic markers versus nuclear factor erythroid 2-related factor 2 (Nrf2) and reactive oxygen species (ROS) in the colorectal cancer (CRC) cell line SW-480. As compared to the untreated cells, significantly less number of fluorescent detected autophagic vacuoles (AVOs) in the fisetin-treated cells coincided with a similar decline of the autophagy flux markers, Beclin 1 and microtubule-associated protein-1 light chain-3 and accumulation of p62 in those cells. The significantly increased number of annexin-V/propidium iodide (+/+) positive and acridine orange/ethidium bromide-stained apoptotic cells coincided with the enhanced signals for the cleaved caspase 3 and nuclear PARP-1 in those fisetin-treated cells. This was consistent with the collapse of mitochondrial membrane potential and release of cytochrome c. The fisetin-treated cells showed increased ROS level and a significant decline in nuclear Nrf2 immunosignal versus recovery in nuclear Nrf2 due to the treatment with curcumin and resveratrol (Nrf2 activators) and thus, suggesting a role of Nrf2 suppression in fisetin-mediated apoptosis in SW-480 cells. The effect of chloroquine, an autophagy inhibitor, resulted into declined number of AVOs and enhanced apoptosis, similar to that of the fisetin effect. Also, regaining of AVOs number and reduced apoptosis of CRC cells due to the treatment with rapamycin, an autophagy inducer, could be observed. These loss and gain of functions experiments thus suggested a correlation between fisetin-mediated autophagy suppression and apoptotic induction in a colorectal cell line.

DNA hypomethylation by fisetin preserves mitochondria functional genes and contributes to the protection of I/R rat heart.

Myocardial I/R can alter the expression of different sets of cardiac genes that negatively influence the I/R outcome via epigenetic modifications. Fisetin is known to be cardioprotective against I/R, but its underlying epigenetic mode of action is not known and is addressed in the present study. Male Wistar rats were subjected to I/R by using the Langendorff perfusion system. Fisetin (20 mg/kg; i.p.) was administered before I/R induction, followed by the measurement of cardiac injury, hemodynamics, physiological indices, the differential expression of genes that regulate DNA methylation, and the function of mitochondria were performed. Fisetin administered I/R rat heart significantly reduced the global DNA hypermethylation and infarct size with an improved physiological recovery, measured via RPP (81%) and LVDP (82%) from the I/R control. Additionally, we noted decreased expression of the DNMT1 gene by 35% and increased expression of the TET1, TET2, and TET3 genes in fisetin-treated I/R rat hearts. Molecular docking analysis data reveals that the fisetin inhibits DNMT1 at the substrate binding site with minimum binding energy (- 8.2 kcal/mol) compared to the DNMT1 inhibitor, 5-azacytidine. Moreover, fisetin-treated I/R heart reversed the expression of the I/R-linked declined expression of bioenergetics genes (MT-ND1, MT-ND2, MT-ND4, MT-Cyt B, MT-COX1, MT-COX2, MT-ATP6), mitochondrial fission gene (Fis1), replication control genes PGC-1α, POLG, and TFAM to near-normal level. Based on the above findings, we demonstrated that fisetin possesses the ability to modulate the expression of different mitochondrial genes via influencing the global DNA methylation in cardiac tissue, which contributes significantly to the improved contractile function and thereby renders cardioprotection against I/R.

Fisetin-loaded grape-derived nanoparticles improve anticancer efficacy in MOLT-4 cells.

PURPOSE: Fisetin (FIS) is a natural flavonoid with anti-proliferative and anti-apoptotic effects on different human cancer cell lines and can be considered a therapeutic agent for ALL treatment. However, FIS has little aqueous solubility and bioavailability, limiting its therapeutic applications. Thus, novel drug delivery systems are needed to improve solubility and bioavailability of FIS. Plant-derived nanoparticles (PDNPs) could be considered a great delivery system for FIS to the target tissues. In this study, we investigated the anti-proliferative and anti-apoptotic effect of free FIS and FIS-loaded Grape-derived Nanoparticles (GDN) FIS-GDN in MOLT-4 cells. MATERIALS/METHODS: In this study, MOLT-4 cells were treated with increasing concentration of FIS and FIS-GDN and viability of cells were assessed by MTT assay. Additionally, cellular apoptosis rate and related genes expression were evaluated using flow cytometry and Real Time-PCR methods, respectively. RESULTS: FIS and FIS-GDN decreased cells viability and increased cells apoptosis dose-dependently, but not time dependently. Treatment of MOLT-4 cells with increasing concentrations of FIS and FIS-GDN considerably increased the expression of caspase 3, 8 and 9 and Bax level, and also decreased the expression of Bcl-2. Results indicated an increased apoptosis after increased concentration of FIS and FIS-GDN at 24, 48 and 72 h. CONCLUSIONS: Our data proposed that FIS and FIS-GDN can induce apoptosis and have antitumor properties in MOLT-4 cells. Furthermore, compared to FIS, FIS-GDN induced more apoptosis in these cells by increasing the solubility and efficiency of FIS. Additionally, GDNs increased FIS effectiveness in proliferation inhibition and apoptosis induction.

A novel 4'-brominated derivative of fisetin induces cell cycle arrest and apoptosis and inhibits EGFR/ERK1/2/STAT3 pathways in non-small-cell lung cancer without any adverse effects in mice.

The therapeutic toxicity and resistance to currently available treatment options are major clinical challenges for the management of lung cancer. As a novel strategy, we synthesized analogues of a known flavonol, fisetin, which has shown anti-tumorigenic potential against cancer in cell culture with no adverse effects in animal models. We studied the synthetic analogues of fisetin for their anti-cancer potential against lung cancer cells, toxicity in mice and efficacy in a xenograft model. Brominated fisetin analogues were screened for their effects on the viability of A549 and H1299 lung cancer cells, and three analogues (3a, 3b, 3c), showed improved activity compared to fisetin. These analogues were more effective in restricting lung cancer cell proliferation, inducing G2 M phase cell cycle arrest and apoptosis. The fisetin analogues also downregulated EGFR/ERK1/2/STAT3 pathways. Fisetin analogue-induced apoptosis was accompanied by a higher Bax to Bcl-2 expression ratio. Based on the in vitro studies, the most effective fisetin analogue 3b was evaluated for in vivo toxicity, wherein it did not show any hepatotoxicity or adverse health effects in mice. Furthermore, analogue 3b showed greater antitumor efficacy (p < .001) as compared to its parent compound fisetin in a human lung cancer cell xenograft study in athymic mice. Together, our data suggest that the novel fisetin analogue 3b is more effective in restricting lung cancer cell growth, both in vitro as well as in vivo, without any apparent toxicity, supporting its further development as a novel anti-lung cancer agent.

Fisetin-Loaded Nanostructured Lipid Carriers: Formulation and Evaluations against Advanced and Metastatic Melanoma.

Fisetin (Fis), a natural flavonoid with anticancer effects, suffers from delivery constraints. Fisetin-nanostructured lipid carriers (NLCs) were developed for better efficacy against metastatic melanoma, employing the design of experiment (DoE) approach. The optimized NLCs depict a particle diameter of 135.0 ± 5.5 nm, a polydispersity index (PDI) of 0.176 ± 0.035, and an entrapment efficiency of 78.16 ± 1.58%. The formulation was stable over a period of 60 days and demonstrated sustained release of the drug (74.79 ± 3.75%) over 96 h. Fis-NLCs depicted at least ∼3.2 times lower IC50 value and ∼1.8 times higher drug uptake at 48 h in A-375 and B16F10 cells compared to that of Fis. It also inhibited the mobility of melanoma cells and induced cell cycle arrest at the G1/S phase. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot results show enhanced expression of Nrf2/NQO1 genes and an apoptotic effect by the upregulation of BAX mRNA expression. The protein levels of BAX and p53 were ∼2-fold higher compared with that of pure Fis. In-vivo studies demonstrated 5.9- and 10.7-fold higher inhibition in melanoma-associated metastasis in the lungs and liver, respectively. The outcomes from this study demonstrated Fis-NLCs as an effective tool against melanoma.

Study of quercetin and fisetin synergistic effect on breast cancer and potentially involved signaling pathways.

Naturally-derived drugs have drawn much attention in recent decades. Efficiency, lower toxicity, and economic reasons are some of their advantages that justify this broad range of administration for different diseases, including cancer. If we can find a specific combination that boosts the effects of their single therapy, leading to synergism effect, increased efficiency, and decreased toxicity, they can act even better. Quercetin and fisetin, two well-known flavonoids, have been used to fight against various cancers. In this study, we investigated their possible synergism quercetin and fisetin on MCF7, MDA-MB-231, BT549, T47D, and 4T1 breast cancer cell lines. Then the optimum combined dose was used to study their impacts on wound healing abilities and clonogenic properties. The real-time qPCR was used to study the expression of their validated downstream effectors in predicted pathways. A significant synergism effect (p < .01, combination index: <1) was observed for all cell lines. Combination therapy was significantly more effective in colony formation (p < .0001) and wound healing assays (p < .001) compared to single therapies. The expression level of potential effectors was also showed a greater change. In vivo study confirmed the in vitro results and showed how significantly (p < .001) their synergism promotes their singular function in inhibiting cancer progression. The breast cancer mouse models receiving combined therapy lived longer with higher average body weight and smaller tumor sizes. These results exhibit that quercetin and fisetin inhibit cancer cell proliferation, migration and colony formation synergistically, and matrix metalloproteinase signaling and apoptotic pathways are relatively responsible for inhibitory activities.

Naringenin in combination with quercetin/fisetin shows synergistic anti-proliferative and migration reduction effects in breast cancer cell lines.

INTRODUCTION & AIM: Breast cancer is one of the most common cancers with a high mortality rate among women worldwide. Quercetin/fisetin and naringenin, three well-known flavonoids, have been used to fight against various cancers. The aim of the present study was to investigate the possible synergism of quercetin/fisetin with naringenin on MCF7 and MDA-MB-231 breast cancer cell lines. METHODS: In this study, cultured MCF7 and MDA-MB-231 cells were treated with different concentrations of quercetin/fisetin individually and in combination with naringenin. MTT assay and scratch assay was employed to determine cell viability and migration respectively. Real-time PCR was used to study the expression level of apoptosis genes and miR-1275 (tumor suppressor miRNA) and mir-27a-3p (oncogenic miRNA). RESULTS: A synergism effect of quercetin/fisetin and naringenin (CI < 1) was observed for both cell lines. Combination therapies were significantly more effective in cell growth reduction, migration suppression and apoptosis induction than single therapies. Gene expression analysis revealed the upregulation of miR-1275 and downregulation miR-27a-3p. CONCLUSION: Our results indicate that quercetin/fisetin enhances the anti-proliferative and anti-migratory activities in combination with naringenin in MCF7 and MDA-MB-231 human breast cancer cell lines. Therefore, the combination of Que/Fis and Nar can be proposed as a promising therapeutic strategy for further investigations.

Effect and mechanism of Fisetin on myocardial damage induced by Patulin.

OBJECTIVES OF THE STUDY: The aim of this study is to investigate whether fisetin can effectively reduce the myocardial damage induced by patulin. This study also aims to reveal the mechanism and target of fisetin in inhibiting myocardial damage. MATERIALS AND METHODS: Network pharmacology was used to screen the targets of fisetin on myocardial damage and the regulatory network of active ingredients-drug targets was constructed. GO and KEGG enrichment analyses were performed to screen out the key pathways and targets of fisetin on myocardial damage. Patulin induced apoptosis in H9c2 cardiomyocytes to verify the key targets. The mechanism of fisetin in inhibiting myocardial damage was determined. RESULTS: FIS can reduce the apoptosis of cardiomyocytes by protecting cardiomyocytes from PAT injury. According to the results of network pharmacology analysis, combined with enzyme activity detection and WB experiment, it was found that the mechanism of FIS to reduce myocardial damage may be related to the P53 signaling pathway, Caspase3/8/9 and Bax/Bcl-2. CONCLUSION: FIS plays a protective role in PAT-induced myocardial damage. On the one hand, FIS inhibits the protein overexpression of P53, Caspase-9 and Bax. On the other hand, FIS enhances the protein expression of Bcl-2.

Fisetin treatment alleviates kidney injury in mice with diabetes-exacerbated atherosclerosis through inhibiting CD36/fibrosis pathway.

Diabetes-related vascular complications include diabetic cardiovascular diseases (CVD), diabetic nephropathy (DN) and diabetic retinopathy, etc. DN can promote the process of end-stage renal disease. On the other hand, atherosclerosis accelerates kidney damage. It is really an urge to explore the mechanisms of diabetes-exacerbated atherosclerosis as well as new agents for treatment of diabetes-exacerbated atherosclerosis and the complications. In this study we investigated the therapeutic effects of fisetin, a natural flavonoid from fruits and vegetables, on kidney injury caused by streptozotocin (STZ)-induced diabetic atherosclerosis in low density lipoprotein receptor deficient (LDLR-/-) mice. Diabetes was induced in LDLR-/- mice by injecting STZ, and the mice were fed high-fat diet (HFD) containing fisetin for 12 weeks. We found that fisetin treatment effectively attenuated diabetes-exacerbated atherosclerosis. Furthermore, we showed that fisetin treatment significantly ameliorated atherosclerosis-enhanced diabetic kidney injury, evidenced by regulating uric acid, urea and creatinine levels in urine and serum, and ameliorating morphological damages and fibrosis in the kidney. In addition, we found that the improvement of glomerular function by fisetin was mediated by reducing the production of reactive oxygen species (ROS), advanced glycosylation end products (AGEs) and inflammatory cytokines. Furthermore, fisetin treatment reduced accumulation of extracellular matrix (ECM) in the kidney by inhibiting the expression of vascular endothelial growth factor A (VEGFA), fibronectin and collagens, while enhancing matrix metalloproteinases 2 (MMP2) and MMP9, which was mainly mediated by inactivating transforming growth factor ß (TGFß)/SMAD family member 2/3 (Smad2/3) pathways. In both in vivo and in vitro experiments, we demonstrated that the therapeutic effects of fisetin on kidney fibrosis resulted from inhibiting CD36 expression. In conclusion, our results suggest that fisetin is a promising natural agent for the treatment of renal injury caused by diabetes and atherosclerosis. We reveal that fisetin is an inhibitor of CD36 for reducing the progression of kidney fibrosis, and fisetin-regulated CD36 may be a therapeutic target for the treatment of renal fibrosis.

Fisetin attenuates doxorubicin-induced cardiotoxicity by inhibiting the insulin-like growth factor II receptor apoptotic pathway through estrogen receptor-α/-ß activation.

Doxorubicin (DOX), an effective chemotherapeutic drug, has been used to treat various cancers; however, its cardiotoxic side effects restrict its therapeutic efficacy. Fisetin, a flavonoid phytoestrogen derived from a range of fruits and vegetables, has been reported to exert cardioprotective effects against DOX-induced cardiotoxicity; however, the underlying mechanisms remain unclear. This study investigated fisetin's cardioprotective role and mechanism against DOX-induced cardiotoxicity in H9c2 cardiomyoblasts and ovariectomized (OVX) rat models. MTT assay revealed that fisetin treatment noticeably rescued DOX-induced cell death in a dose-dependent manner. Moreover, western blotting and TUNEL-DAPI staining showed that fisetin significantly attenuated DOX-induced cardiotoxicity in vitro and in vivo by inhibiting the insulin-like growth factor II receptor (IGF-IIR) apoptotic pathway through estrogen receptor (ER)-α/-ß activation. The echocardiography, biochemical assay, and H&E staining results demonstrated that fisetin reduced DOX-induced cardiotoxicity by alleviating cardiac dysfunction, myocardial injury, oxidative stress, and histopathological damage. These findings imply that fisetin has a significant therapeutic potential against DOX-induced cardiotoxicity.

Fisetin, a dietary flavonoid, increases the sensitivity of chemoresistant head and neck carcinoma cells to cisplatin possibly through HSP90AA1/IL-17 pathway.

Cisplatin (DDP) resistance is a bottleneck in the treatment of head and neck cancer (HNC), leading to poor prognosis. Fisetin, a dietary flavonoid, has low toxicity and high antitumor activity with unclear mechanisms. We intended to predict the targets of fisetin for reversing DDP-resistance and further verify their expressions and roles. A network pharmacology approach was applied to explore the target genes. The hub genes were screened out and subjected to molecular docking and experimental verification (in vivo and in vitro). Thirty-two genes common to fisetin and DDP-resistance were screened, including three hub genes, namely HSP90AA1, PPIA, and PTPRS. Molecular docking suggested that fisetin and the candidate proteins could bind tightly. HSP90AA1 was identified as the key gene. Administration of fisetin increased the sensitivity of chemoresistant cells (Cal27/DDP and FaDu/DDP) to DDP, accompanied by the downregulation of HSP90AA1 and IL-17. HSP90AA1 silencing increases the sensitivity of DDP-resistant cells to DDP, which was mediated by IL-17. In summary, fisetin might inhibit the chemoresistance of HNC cells to DDP by targeting the HSP90AA1/IL-17 pathway. Several hub genes might be the targets of fisetin for reversing DDP-resistance in HNC cells and might also serve as prognostic factors and therapeutic targets for HNC.