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Detection of ROS using carboxy-H 2 DCFDA dye and MitoSox. (a) AGS cells were treated as indicated in the figure and DCF fluorescence were measured. (b) Levels of mitochondrial superoxide was quantified using MitoSox by flow cytometry and analyzed by ANOVA followed by Bonferroni's Multiple Comparison Test. (c) The formation of comets (fragmented DNA) are shown at 400Â magnifications and in a magnified view with quantittative data. (d) AGS cells were collected and processed for determination of total cell number and dead cells employing trypan blue assay. (e) DHE and (f) nitrite assays were done in AGS cells as detailed in Materials and Methods. (g) Fisetin-mediated ROS generation and DNA damage was inhibited in the presence NAC. AGS cells were treated with Fisetin (50 mM) after 6 h lysates were collected and status of H2A.X(S139) and cleaved PARP were determined by western blot analysis. Columns, mean of three independent treatments; bars, s.e.m.; the results were reproducible in two additional experiments. Data points are the means AE s.e.m. of triplcates. P < 0.001 ( ÃÃÃ ), P < 0.01 ( ÃÃ ), P < 0.05 ( Ã ). The P-value is determined by comparing each treatment with control group or indicated groups.
Source publication
The anticancer effects of fisetin, a dietary agent, are largely unknown against human gastric cancer. Herein, we investigated the mechanisms of fisetin-induced inhibition of growth and survival of human gastric carcinoma AGS and SNU-1 cells. Fisetin (25-100 µM) caused significant decrease in the levels of G1 phase cyclins and CDKs, and increased th...
Contexts in source publication
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... of less than 0.05 were considered significant. ANOVA was followed by Bonferroni's Test for Multiple Comparisons, for Figure 7b. ...
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... study the effect of fisetin on ROS generation, cells were exposed to fisetin and the changes in DCF fluorescence was measured. Fisetin increased ROS generation in a dose- dependent manner (Supplementary Figure S2) and it was blocked by an antioxidant, NAC (Figure 7a). To determine the source of ROS we further analyzed mitochondrial superoxide generation using MitoSox staining and flow cytometry analysis. ...
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... observed a significant increase in mitochondrial superoxide generation at 50 mM fisetin at 3 and 6 h; however, at 12 h ROS generation from mitochondria was dec- reased. The decrease in mitochondrial ROS might be due to the decrease in total healthy mitochondria in treated samples (Figure 7b). To find out the role of ROS in DNA damage, comet assay was performed in presence of fisetin alone and in combination with NAC (Figure 7c). ...
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... decrease in mitochondrial ROS might be due to the decrease in total healthy mitochondria in treated samples (Figure 7b). To find out the role of ROS in DNA damage, comet assay was performed in presence of fisetin alone and in combination with NAC (Figure 7c). Interestingly, NAC pre-treated cells did not show any comet tails (Figure 7c left) whereas fisetin alone showed comet tails of high intensity at 6 h of treatment which could happen only due to severe DNA damage. ...
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... find out the role of ROS in DNA damage, comet assay was performed in presence of fisetin alone and in combination with NAC (Figure 7c). Interestingly, NAC pre-treated cells did not show any comet tails (Figure 7c left) whereas fisetin alone showed comet tails of high intensity at 6 h of treatment which could happen only due to severe DNA damage. Next, we determined whether Figure 5. ...
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... generation is involved in fisetin-induced cell death. Fisetin-induced cell death was strongly pre- vented by NAC pre-treatment in both AGS (Figure 7d right) and SNU-1 cells (Supplementary Figure S2). These results suggested that fisetin- induced cell death is caused by ROS generation. ...
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... results suggested that fisetin- induced cell death is caused by ROS generation. We further analyzed the levels of cellular superoxides by DHE staining and nitrite which were strongly increased by fisetin treatment and these effects were lost when the cells were pre-treated with NAC (Figure 7e and f). We also analyzed the effects on apoptosis and DNA damage marker, cleaved-PARP, and gamma-H2A.X S139 phosphorylation, respec- tively in similar treatments. ...
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... also analyzed the effects on apoptosis and DNA damage marker, cleaved-PARP, and gamma-H2A.X S139 phosphorylation, respec- tively in similar treatments. The levels of both were increased by fisetin treatment; however, pre- treatment with NAC for 5 h inhibited the increase in the levels of both the proteins (Figure 7g), and thus suggesting that fisetin generated ROS caused DNA damage and apoptosis in gastric cancer cells. The fisetin-caused generation of ROS was further proved by using another known antioxidant, ascorbic acid, the pre-treatment of which reversed the growth inhibitory and cell death inducing effects of fisetin (Figure 8a and b). ...
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... mitochondria-derived ROS by fisetin is supported by our findings including the fisetin- mediated ROS generation is significantly attenuated in the presence of MRC complex I inhibitors, rotenone, and DPI but not in the presence of complex III inhibitor, myxothiazol (Figure 8a). The ROS scavenger, NAC, significantly blocked cell death caused by fisetin in both gastric cancer cell lines, AGS and SNU-1 cells (Figure 7d, Supplementary Figure S3). Further, NAC pre-treatment also blocked the activation of PARP and gamma-H2A.X S139 phosphorylation which sug- gested that fisetin-mediated DNA damage and subse- quent apoptosis was likely triggered by ROS. ...
Citations
... Studies performed on pancreatic cancer cells indicate the PI3K/AKT signaling cascade as a possible candidate in the initiation of apoptosis [60,63,66]. Additionally, Sabarwal et al. found that FIS caused an increase in total p53 in gastric cancer cells and is activated by phosphorylation at the S15 position, indicating the likelihood of its involvement in DNA damage [61].Subsequent studies involving gastric cancer cells with FIS showed a reduction in ERK activation 1/2 in a concentration-dependent manner, suggesting the involvement of another pathway in the initiation of apoptosis [62]. ...
The complex and multi-stage processes of carcinogenesis are accompanied by a number of phenomena related to the potential involvement of various chemopreventive factors, which include, among others, compounds of natural origin such as flavonols. The use of flavonols is not only promising but also a recognized strategy for cancer treatment. The chemopreventive impact of flavonols on cancer arises from their ability to act as antioxidants, impede proliferation, promote cell death, inhibit angiogenesis, and regulate the immune system through involvement in diverse forms of cellular death. So far, the molecular mechanisms underlying the regulation of apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis occurring with the participation of flavonols have remained incompletely elucidated, and the results of the studies carried out so far are ambiguous. For this reason, one of the therapeutic goals is to initiate the death of altered cells through the use of quercetin, kaempferol, myricetin, isorhamnetin, galangin, fisetin, and morin. This article offers an extensive overview of recent research on these compounds, focusing particularly on their role in combating cancer and elucidating the molecular mechanisms governing apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis. Assessment of the mechanisms underlying the anticancer effects of compounds in therapy targeting various types of cell death pathways may prove useful in developing new therapeutic regimens and counteracting resistance to previously used treatments.
... Fisetin also showed cytotoxic effects on normal NIH/3T3 cells by reducing their viability, in a study that investigated the anti-angiogenic and anti-cancer effects of this natural flavonoid [34]. Fisetin has previously demonstrated cytotoxicity against various human cancer cell lines, such as breast cancer cells (MCF7) [35], human leukemia cells (HL60) [36], gastric cancer cells (AGS, SNU-1, and FHs74int) [37], and colon cancer cells (HT29) [38]. Morin has also been proven to have cytotoxic effects on many human cancer cell lines such as human melanoma cells (G361, SK-MEL-2) [39], ovarian cancer cells (OVCAR3, SKOV3) [40], bladder cancer cells (EJ) [41] and lung cancer cells (A549) [42]. ...
... Fisetin also showed cytotoxic e on normal NIH/3T3 cells by reducing their viability, in a study that investigated the angiogenic and anti-cancer effects of this natural flavonoid [34]. Fisetin has previ demonstrated cytotoxicity against various human cancer cell lines, such as breast c cells (MCF7) [35], human leukemia cells (HL60) [36], gastric cancer cells (AGS, SNU-1 FHs74int) [37], and colon cancer cells (HT29) [38]. Morin has also been proven to cytotoxic effects on many human cancer cell lines such as human melanoma cells ( SK-MEL-2) [39], ovarian cancer cells (OVCAR3, SKOV3) [40], bladder cancer cells (EJ and lung cancer cells (A549) [42]. ...
Flavonoids share a common structural framework that serves as a hallmark indicative of their biological activity. In this study, we investigated the effects of two structurally similar flavonoids, fisetin and morin, through independent and combined in vitro assessments on embryonic mouse cells overexpressing the human 70 kDa heat shock protein (Hsp70) (Tg/Tg) and normal mouse fibroblast cell line (NIH/3T3). The primary objectives were to evaluate the biocompatibility and potential cytotoxicity of these flavonoids, along with assessing the cytoprotective role of Hsp70 in these cellular environments. To address these objectives, we conducted dose- and time-dependent cell survival tests. Additionally, we utilized flow cytometry to detect intracellular reactive oxygen species (ROS) production and to analyze apoptosis and the cell cycle. Throughout the experimental procedures, a notable observation was made: NIH/3T3 normal cells exhibited greater susceptibility compared to Tg/Tg cells when exposed to fisetin and morin. This difference in susceptibility is likely attributed to the robust cytoprotective effect of Hsp70 in Tg/Tg cells. Importantly, both cell lines demonstrated increased sensitivity to fisetin toxicity in comparison to morin, leading to significantly lower cell survival rates. These findings shed light on the differential responses of cell lines to flavonoid exposure, emphasizing the influence of Hsp70 and the distinct impact of fisetin and morin on cell viability.
... Since fisetin is used as a food supplement, it is important to know whether fisetin is safe under consumption conditions. Although our investigation along with reports from other groups (16,(55)(56)(57) clearly indicates that fisetin has a genotoxic potential, it is unlikely that the harmful concentrations can be reached in vivo. In animal experiments, the peak plasma level 15 min after i.p. administration of free fisetin (223 mg/kg BW) was 3 mg/mL and 4 h later 0.3 mg/ml (51), which corresponds to 10 and 1 μM, respectively. ...
Background/aim:
Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells.
Materials and methods:
We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis.
Results:
Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 μM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells.
Conclusion:
Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.
... The Comet assay was conducted following the previously described procedure [24]. After the desired treatment, cells were harvested and mixed with 1% low melting point agarose. ...
... This was followed by gel electrophoresis, and staining with ethidium bromide. Cells were visualized by fluorescence microscope and comet tail length was analyzed, as described previously [24]. ...
Simple Summary
Head and neck cancers arise in the mouth, pharynx, salivary glands, and larynx, and most of these cancers are squamous cell carcinomas. Causative agents for head and neck squamous cell carcinoma (HNSCC) include tobacco-derived carcinogens, alcohol consumption, and HPV infection. Currently, the survival rate is poor, and available chemotherapeutic agents are associated with toxicities and chemoresistance largely due to cancer stem cells (CSC). Therefore, the development of agents that are nontoxic to normal cells, and inhibit the growth of both head and neck cancer cells as well as CSC are desired. Epidemiological studies showed that dietary intake of Allium vegetables lowered the risk of various cancers, including head and neck cancer. Several studies have reported processed-garlic-constituent diallyl trisulfide (DATS) as a promising compound that induced growth arrest and apoptosis in prostate and breast cancer. In the present study, DATS decreased cell viability, and induced growth arrest and apoptotic cell death involving DNA damage and reactive oxygen species generation in HNSCC cells. DATS also reduced CD133high/CD44high CSC fraction, spheroid formation and aldehyde dehydrogenase 1 (ALDH1) activity, and downregulated Oct4 and SOX2 expression. Further, DATS inhibited HNSCC tumor growth and CSC fraction in the tumor xenograft model. Thus, DATS could be a potential anticancer agent against head and neck cancer.
Abstract
Despite advances in therapeutic approaches, the five-year survival rate for head and neck squamous cell carcinoma (HNSCC) patients is still less than fifty percent. Research has indicated that the consumption of Allium vegetables or processed garlic containing diallyl trisulfide (DATS) can lower the risk of multiple types of cancer. Nevertheless, the effectiveness and underlying mechanisms of DATS against HNSCC have not been thoroughly explored until the current study. In this research, it was found that DATS notably curtailed the growth and viability of HNSCC cells. Additionally, DATS triggered a significant G2/M cell cycle arrest in these cells, accumulating cyclin B1, Cip1/p21, and Ser-10 phospho-histone H3—this was indicative of mitotic arrest attenuated by NAC pretreatment, suggesting the role of reactive oxygen species (ROS) induction. The production of ROS induced by DATS led to DNA damage and apoptosis, a process associated with elevated levels of cleaved caspase-3 and cleaved PARP, along with reduced XIAP. When HNSCC cells were exposed to pharmacological concentrations of DATS, it resulted in the suppression of cancer stem cell (CSC) populations, as indicated by a decrease in the CD133high/CD44high cell fraction, reduced aldehyde dehydrogenase 1 (ALDH1) activity, inhibited spheroid formation and downregulated SOX2 and Oct4 expression. Furthermore, the administration of DATS to tumor xenografts demonstrated its in vivo capacity to hinder CSCs. Further, DATS treatment inhibited the growth of UMSCC-22B head and neck cancer tumor xenograft in immunocompromised mice. Overall, DATS inhibited cell proliferation; induced cell cycle mitotic arrest and apoptosis involving DNA damage through ROS generation; reduced the CSC fraction and spheroid formation; and downregulated SOX2 and Oct4 expression. More importantly, DATS inhibited HNSCC tumor growth and CSC fraction in vivo. Thus, DATS could be a potential anticancer agent that can be used against head and neck cancer.
... The growth inhibitory and cell death effects of HU on cancer cells were determined by trypan blue dye exclusion assay as described earlier (Punia et al., 2017;Sabarwal et al., 2017). Briefly, 1× 10 5 cells were seeded in 6-well plates and after 24 hours, cells were treated with 50-400 µg/ml concentrations of HU. ...
Objective:
A polyherbal medicine, Habb-e-Ustukhuddus (HU), is used for its anti-inflammatory properties. However, the anticancer and chemopreventive properties of HU were not known, and Therefore, investigated in the present study.
Methods:
Cancer cells were treated with 50-400 µg/ml HU and MTT, trypan blue, and clonogenic assays were performed. Propidium iodide (PI) staining, annexin V-FITC assay, and JC-1 staining were done for cell cycle progression, apoptosis, and mitochondrial membrane potential, respectively, using flow cytometry. Immunoblotting, cell migration and invasion assays were performed. Chemical characterization of HU was done through GC-MS and HPLC analyses. C57BL/6 mice were used to assess the in vivo toxicity of HU.
Results:
While evaluating the anticancer activity, the methanolic extract of HU (50-400 µg/ml) strongly inhibited the growth and survival (P<0.05-0.001) of lung and breast cancer cells and increased the cell population in the sub-G1 phase of the cell cycle. HU caused apoptotic death of cancer cells (P<0.05-0.001), which was associated with the depolarization of mitochondrial membrane potential (Δψ) (P<0.001) and an increase in Bax to Bcl-2 protein ratio. Further, HU inhibited the invasion and migration of cancer cells, which was accompanied by an increase in the epithelial marker, E-cadherin, and a decrease in the mesenchymal marker, vimentin. The HU characterization by GC-MS and HPLC analyses showed the abundance of bioactive compounds including flavonoids and alkaloids. In the chemopreventive study, the oral administration of methanolic extract of the formulation HU (50 and 100 mg/kg body weight) to mice did not cause any toxicity and significantly increased the specific activities of hepatic drug metabolizing phase I and phase II enzymes, which suggested for its detoxification potential of xenobiotic compounds.
Conclusion:
Together, these results demonstrated the anticancer potential HU, without any apparent toxicity in mice, and thus HU could be further explored for its clinical utility in cancer control.
... All these mechanisms were observed in a various panel of cancer cell lines ( Fig. 3), such as human multiple myeloma U266 cells, human chronic myeloid leukaemia K562 cells, human non-small cell lung cancer NCI-H460 cells, human gastric cancer AGS and SNU-1 cells, human osteosarcoma U2 cells, human cervical cancer HeLa cells, human epidermoid cancer A431 cells, triple-negative human breast cancer MDA-MB-468, MDA-MB-231 cells, human Burkitt's lymphoma Raji cells, human colon cancer COLO205 cells, human acute promyelocytic leukaemia HL60, human liver adenocarcinoma SK-HEP-1 cells, androgen-dependent LNCaP, and androgen-independent DU145 and PC3 prostate cancer cells (Adan and Baran 2015; Chen et al. 2002;Kang et al. 2015Kang et al. , 2016Khan et al. 2008;Kim et al. 2010Kim et al. , 2015Kim et al. , 2016Li et al. 2015;Lim and Park 2009;Lim et al. 2015;Pal et al. 2013;Sabarwal et al. 2017;Smith et al. 2016;Suh et al. 2009;Wu et al. 2013;Ying et al. 2012). ...
... G 0 /G 1 phase arrest was achieved via decrease in cyclins D1, D2, and E and their activating partner CDK2, 4, and 6, with concomitant induction of WAF1/p21 and KIP1/p27. These effects were noticed in human gastric cancer AGS and SNU-1 cells, human urinary bladder cancer T24 cells, human endometrial cancer EJ cells, prostate cancer LNCaP and PC-3 cells, and colorectal cancer HT-29 cells (Khan et al. 2008;Li et al. 2015;Sabarwal et al. 2017). ...
Fisetin is a flavonol present in various fruits (strawberries, apples, grapes) and
vegetables (onions, cucumbers). Biotechnology or chemosynthesis tools are also
used for its synthesis. Numerous studies have presented fisetin as an important
anticancer, neuroprotective (senolytic, anti-Alzheimer, anti-Parkinson, etc.), antidiabetic, and anti-inflammatory agent. These effects have been demonstrated both
in in vitro and in experimental animal models. Furthermore, the ongoing or
upcoming clinical trials are assessing the health-promoting effects in elderly
populations or the anti-inflammatory effects in osteoarthritis and COVID-19.
Fisetin is marketed worldwide as a drug ingredient, oral dietary supplement, or
medical food and possesses a good safety profile. The chemical features of fisetin,
biosynthesis, pharmacokinetics, as well as the latest aspects in relation to the
molecular mechanisms of action, pharmacology in animals, toxicology, clinical
trials, patents, and marketed products are summarized in this chapter
... In human osteosarcoma cells, fisetin caused apoptosis by activating the hippo pathway and the JNK-ERK-AP-1 axis [14]. Fisetin has been shown to have anti-cancer effects against various cancer types, including lung, breast, renal, and gastric [15][16][17][18]. Flavonoids, such as apigenin and luteolin, exert anti-tumor effects through the activation of the endoplasmic reticulum (ER) stress response and the unfolded protein response (UPR) in various cancers [19,20]. ...
Fisetin, a well-known plant flavonol from the natural flavonoid group, is found in traditional medicines, plants, vegetables, and fruits. Fisetin also has anti-oxidant, anti-inflammatory, and anti-tumor effects. This study investigated the anti-inflammatory effects of fisetin in LPS-induced Raw264.7 cells and found that fisetin reduced the LPS-induced production of pro-inflammation markers, such as TNF-α, IL-1β, and IL-6, demonstrating the anti-inflammatory effects of fisetin. Furthermore, this study investigated the anti-cancer effects of fisetin and found that fisetin induced apoptotic cell death and ER stress through intracellular calcium (Ca2+) release, the PERK-ATF4-CHOP signaling pathway, and induction of GRP78 exosomes. However, the suppression of PERK and CHOP inhibited the fisetin-induced cell death and ER stress. Interestingly, fisetin induced apoptotic cell death and ER stress and inhibited the epithelial-mesenchymal transition phenomenon under radiation in radiation-resistant liver cancer cells. These findings indicate that the fisetin-induced ER stress can overcome radioresistance and induce cell death in liver cancer cells following radiation. Therefore, the anti-inflammatory agent fisetin, in combination with radiation, may be a powerful immunotherapy strategy to overcome resistance in an inflammatory tumor microenvironment.
... Fisetin played a role in the induction of apoptosis, independently of p53, and increased mitochondrial ROS generation. Overall, these findings confirmed that fisetin holds anticancer potential via ROS creation, most probably via the MRC complex I causing gastric carcinoma cells apoptosis [95]. Gastric cancer cells such as GES-1 and SGC7901 cells were exposed with various concentrations of fisetin (1 to 20 µM) and suggestively decreased the proliferation rate of SGC7901 cells from 98% to 11%, correspondingly, as compared to the control (100%) after 48 h. ...
... Fisetin substantially decreases G1 phase cyclins and CDKs levels, and the levels of p53 increased. [95] Fisetin treatment with various concentrations suggestively decreased the proliferation rate of SGC7901 cells. [96] Pancreatic cancer ...
Cancer is a main culprit and the second-leading cause of death worldwide. The current mode of treatment strategies including surgery with chemotherapy and radiation therapy may be effective, but cancer is still considered a major cause of death. Plant-derived products or their purified bioactive compounds have confirmed health-promoting effects as well as cancer-preventive effects. Among these products, flavonoids belong to polyphenols, chiefly found in fruits, vegetables and in various seeds/flowers. It has been considered to be an effective antioxidant, anti-inflammatory and to play a vital role in diseases management. Besides these activities, flavonoids have been revealed to possess anticancer potential through the modulation of various cell signaling molecules. In this regard, fisetin, a naturally occurring flavonoid, has a confirmed role in disease management through antioxidant, neuro-protective, anti-diabetic, hepato-protective and reno-protective potential. As well, its cancer-preventive effects have been confirmed via modulating various cell signaling pathways including inflammation, apoptosis, angiogenesis, growth factor, transcription factor and other cell signaling pathways. This review presents an overview of the anti-cancer potential of fisetin in different types of cancer through the modulation of cell signaling pathways based on in vivo and in vitro studies. A synergistic effect with anticancer drugs and strategies to improve the bioavailability are described. More clinical trials need to be performed to explore the anti-cancer potential and mechanism-of-action of fisetin and its optimum therapeutic dose.
... Fisetin is reported to exhibit various biological activities such as anti-oxidant, 7 anti-inflammatory, 8 and anti-cancer activities in preclinical models. [9][10][11][12] Previously, we have reported that fisetin possesses anticancer activity against non-small cell lung cancer cells by inhibiting metastasis and epithelial to mesenchymal transition. 13 Fisetin, in combination with doxorubicin, showed synergistic effects at inhibiting lung cancer cell growth and proliferation, 14 through mechanisms that involve inhibition of cell cycle arrest and induction of apoptosis. ...
... (***). many chemotherapeutic agents induce cell cycle arrest to inhibit cancer cell proliferation 12,26 Previously, fisetin was demonstrated to induce cell cycle arrest and apoptosis 12,43,44 ; thus, we investigated the effect of 4′-Br fisetin analogues in this regard. Consistent with the earlier findings, fisetin induced G 2 /M phase cell cycle arrest and apoptosis in A549 lung cancer cells. ...
... (***). many chemotherapeutic agents induce cell cycle arrest to inhibit cancer cell proliferation 12,26 Previously, fisetin was demonstrated to induce cell cycle arrest and apoptosis 12,43,44 ; thus, we investigated the effect of 4′-Br fisetin analogues in this regard. Consistent with the earlier findings, fisetin induced G 2 /M phase cell cycle arrest and apoptosis in A549 lung cancer cells. ...
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 G2M 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.
... Cells were seeded and treated with silibinin and IR as desired. After respective treatment time points, cells were harvested and whole cell lysate preparation, protein quantification and immunoblotting were performed as detailed in the earlier study [22]. ...
Emergence of radioresistance in prostate cancer (PCa) cells is a major obstacle in cancer therapy and contributes to the relapse of the disease. EGF receptor (EGFR) signaling plays an important role in the development of radioresistance. Herein, we have assessed the modulatory effects of silibinin on radiation-induced resistance via DNA repair pathways in EGFR-knockdown DU145 cells. shRNA-based silencing of EGFR was done in radioresistant human PCa DU145 cells and effects of ionizing radiation (IR) and silibinin were assessed using clonogenic and trypan blue assays. Furthermore, radiosensitizing effects of silibinin on PCa in context with EGFR were analyzed using flow cytometry, comet assay, and immunoblotting. Silibinin decreased the colony formation ability with an increased death of DU145 cells exposed to IR (5 Gray), with a concomitant decrease in Rad51 protein expression. Silibinin (25 μM) augmented the IR-induced cytotoxic effect in EGFR-knockdown PCa cells, along with induction of G2/M phase cell cycle arrest. Further, we studied homologous recombination (HR) and non-homologous end joining (NHEJ) pathways in silibinin-induced DNA double-strand breaks in EGFR-knockdown DU145 cells. Silibinin down-regulated the expression of Rad51 and DNA-dependent protein kinase proteins without any considerable effect on Ku70 and Ku80 in IR-exposed EGFR-knockdown PCa cells. The pro-survival signaling proteins, phospho-extracellular signal-regulated kinases (ERK)1/2, phospho-Akt and phospho-STAT3 were decreased by silibinin in EGFR-deficient PCa cells. These findings suggest a novel mechanism of silibinin-induced radiosensitization of PCa cells by targeting DNA repair pathways, HR and NHEJ, and suppressing the pro-survival signaling pathways, ERK1/2, Akt and STAT3, in EGFR-knockdown PCa cells.
