Controlling tumor growth by modulating endogenous production of reactive oxygen species

Paradoxically, reactive oxygen species (ROS) can promote normal cellular proliferation and carcinogenesis, and can also induce apoptosis of tumor cells. In this report, we study the contribution of ROS to various cellular signals depending on the nature and the level of ROS produced. In nontransformed NIH 3T3 cells, ROS are at low levels and originate from NADPH oxidase. Hydrogen peroxide (H(2)O(2)), controlled by the glutathione system, is pivotal for the modulation of normal cell proliferation. In CT26 (colon) and Hepa 1-6 (liver) tumor cells, high levels of ROS, close to the threshold of cytotoxicity, are produced by mitochondria and H(2)O(2) is controlled by catalase. N-acetylcysteine, which decreases H(2)O(2) levels, inhibits mitogen-activated protein kinase and normal cell proliferation but increases tumor cell proliferation as H(2)O(2) concentration drops from the toxicity threshold. In contrast, antioxidant molecules, such as mimics of superoxide dismutase (SOD), increase H(2)O(2) levels through superoxide anion dismutation, as well as in vitro proliferation of normal cells, but kill tumor cells. CT26 tumors were implanted in mice and treated by oxaliplatin in association with one of the three SOD mimics manganese(III)tetrakis(4-benzoic acid) porphyrin, copper(II)(3,5-diisopropylsalicylate)2, or manganese dipyridoxyl diphosphate. After 1 month, the volumes of tumors were respectively 35%, 31%, and 63% smaller than with oxaliplatin alone (P < 0.001). Similar data were gained with Hepa 1-6 tumors. In conclusion, antioxidant molecules may have opposite effects on tumor growth. SOD mimics can act in synergy with cytotoxic drugs to treat colon and liver cancers.     

Photosensitization induced reactive oxygen species and oxidative damage in human erythrocytes

Generation of reactive oxygen species by photosensitization is the corner stone of photodynamic therapy of tumors. Cell damage may be mediated by free radical species and lipid peroxidation of their membranes. The effects of oxygen active species (.OH and O(2)(.-) radicals) photogenerated by the novel photosensitizer m-chloroperbenzoic acid (m-CPBA) on human erythrocyte integrity and stability were studied. The biological toxicity of the reactive oxygen species on human red blood cells (RBCs) was evident by increased osmotic fragility, spherocytosis and haemolysis. The haemolysis was increased in concentration and time dependent manner. The lipid peroxidation product thiobarbituric acid reactive substances (TBARS) was elevated in m-CPBA photosensitized RBCs indicating increased oxidative stress. This was accompanied with a depletion of erythrocyte glutathione (GSH). These effects were blunted by hydroxyl radical scavengers, thiourea and mannitol, which might indicate the production of (.)OH radical by photosensitization with m-CPBA. The antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), peroxidase (Px) and glutathione peroxidase (GSH-Px) were elevated in RBCs treated with m-CPBA in the presence and absence of hydroxyl radical scavengers, mannitol and thiourea. These results suggested that the main oxygen radical photogenerated from m-CPBA is O(2)(&z.rad;-) radical, which is transformed to (.)OH radical probably by hydrogen abstraction. This is probably the main damaging oxygen species and played an essential role in oxidative haemolysis mediated by peroxidation of membrane lipids of human erythrocytes. This study provides an investigational promising data for photodynamic therapy.     

Impact of therapeutically induced reactive oxygen species and radical scavenging by alpha-tocopherol on tumor cell adhesion

Many tumor treatment modalities such as ionizing radiation or some chemotherapy induce reactive oxygen species (ROS) resulting in therapeutic cell damage. The aim of this study was to analyze whether such ROS induction may affect the mechanical stability of solid tumor tissue by degradation of the extracellular matrix proteins or by a loss of cell adhesion molecules. Additionally, the protective impact of alpha-tocopherol treatment on these processes was studied. Experimental DS-sarcomas in rats were treated with a combination of localized 44 degrees C hyperthermia, inspiratory hyperoxia and xanthine oxidase in order to induce pronounced oxidative stress. A second group of animals were pretreated with alpha-tocopherol. The in vivo expression of E- and N-cadherin, alpha-catenin, integrins alphav, beta3 and beta5 as well as the expression of the integrin dimer alphavbeta3 were assessed by flow cytometry. The activity of the matrix metalloproteinases MMP-2 and -9 and the activity of the urokinase-type plasminogen activator (uPA) were determined by zymography. The expression of E-cadherin, the alphav-, beta3-integrin and the alphavbeta3-integrin dimer was significantly reduced by ROS induction, an effect which was at least partially reversible by alpha-tocopherol. N-cadherin, alpha-catenin and the beta5-integrin expression was not affected by ROS. In addition, MMP-2, MMP-9 and uPA activities were markedly reduced immediately after hyperthermia. Whereas 24 h later the effects on MMP-2 and -9 were no longer evident, for uPA the impact of oxidative stress became even more pronounced at this time. These results show that several processes responsible for the structural stability of the tumor tissue are affected by therapeutic ROS generation. Changes in some of the markers assessed suggested a decrease in tissue stability upon ROS induction, whereas others indicated changes which could lead to a more stable tumor cell cluster. Depending on the individual tumor entity ROS may therefore influence the mechanical stability of solid tumors and by this affect metastatic behavior.     

Butein suppresses breast cancer growth by reducing a production of intracellular reactive oxygen species

Background

Butein has various functions in human diseases including cancer. While anti-cancer effects of butein have been revealed, it is urgent to understand a unique role of butein against cancer. In this study, we demonstrate that butein inhibition of reactive oxygen species (ROS) production results in suppression of breast cancer growth.

Methods

Different breast cancer cell lines were treated with butein and then subjected to cell viability and apoptosis assays. Butein-sensitive or -resistant breast cancer cells were injected into mammary fat pads of immunocompromised mice and then butein was injected. Breast cancer cells were categorized on the basis of butein sensitivity.

Results

Butein reduced viabilities of different breast cancer cells, while not affecting those of HER2-positive (HER2⁺) HCC-1419, SKBR-3 and HCC-2218 breast cancer cells. Butein reduction of ROS levels was correlated with apoptotic cell death. Furthermore, butein reduction of ROS level led to inhibitions of AKT phosphorylation. N-acetyl-L-cysteine (NAC), a free radical scavenger, also reduced ROS production and AKT phosphorylation, resulting in apoptotic cell death. In contrast, inhibitory effects of both butein and NAC on ROS production and AKT phosphorylation were not detected in butein-resistant HER2⁺ HCC-1419, SKBR-3 and HCC-2218 cells. In the in vivo tumor growth assays, butein inhibited tumor growth of butein-sensitive HER2⁺ BT-474 cells, while not affecting that of butein-resistant HER2⁺ HCC-1419 cells. Moreover, butein inhibition of ROS production and AKT phosphorylation was confirmed by in vivo tumor growth assays.

Conclusions

Our study first reveals that butein causes breast cancer cell death by the reduction of ROS production. Therefore, our finding provides better knowledge for butein effect on breast cancer and also suggests its treatment option.     

过氧化物酶体通路氧化应激基因与青蒿琥酯抗胰腺癌敏感性的相关性研究

目的：探讨过氧化物酶体通路活性氧氧化应激关键基因的筛选及其与青蒿琥酯抗胰腺癌敏感性的相关性。方法基于美国国立癌症研究所（NCI）公共数据库55株肿瘤细胞表达谱基因芯片数据,采用 Kendall 相关分析方法筛选出与青蒿琥酯抗肿瘤半抑制浓度（IC50）显著相关的过氧化物酶体通路关键基因。利用荧光定量 PCR 验证候选基因在不同青蒿琥酯敏感性胰腺癌细胞的 mRNA 表达差异,并通过 DAB 染色检测胰腺癌细胞内过氧化物酶体含量。结果13个过氧化物酶体生物合成、增殖及其活性氧氧化应激通路关键基因 mRNA 表达与青蒿琥酯抗肿瘤药敏浓度 IC50有显著相关性。与正常肝细胞 HL-7702（1．00）比较,对青蒿琥酯敏感的胰腺癌 Panc-1细胞过氧化物酶体生物合成基因CRAT（2．89±0．06）、PEX11B（1．90±0．07）、PEX16（1．35±0．07）mRNA 相对表达水平均显著增高（t ＝33．00,P ＜0．01;t ＝17．85,P ＜0．01;t ＝4．54,P ＜0．05）;其活性氧氧化应激的抗氧化基因 CAT（1．43±0．03）、SOD1（2．07±0．04）、SOD2（1．15±0．01）mRNA 相对表达水平亦显著增高（t ＝11．71,P ＜0．01;t ＝35．85,P ＜0．01;t ＝13．22,P ＜0．01）;对青蒿琥酯不敏感的胰腺癌 BXPC-3细胞的 PEX12（0．51±0．02）、CAT（0．47±0．02）、PRDX1（0．43±0．01）、SOD1（0．44±0．01）mRNA 相对表达水平显著低于正常肝细胞 HL-7702（t ＝37．53,P ＜0．01;t ＝16．52,P ＜0．01;t ＝84．20,P ＜0．01,t ＝48．24,P ＜0．01）。DAB 染色显示对青蒿琥酯敏感的胰腺癌 Panc-1细胞过氧化物酶体阳性表达率（61．5％）明显高于HL-7702细胞（43．8％）,差异有统计学意义（χ2＝16．11,P ＜0．01）。结论过氧化物酶体及其活性氧相关抗氧化酶 CAT、PRDX1、SOD 基因表达可能是影响青蒿琥酯抗胰腺癌作用敏感性的重要因素。     

Parthenolide induces significant apoptosis and production of reactive oxygen species in high-risk pre-B leukemia cells

We investigated whether parthenolide, the principal bioactive component of the herb feverfew (Tanacetum parthenium) induced apoptosis in pre-B acute lymphoblastic leukemia (ALL) lines, including cells carrying the t(4;11)(q21;q23) chromosomal translocation. Parthenolide induced rapid apoptotic cell death distinguished by loss of nuclear DNA, externalization of cell membrane phosphatidylserine, and depolarization of mitochondrial membranes at concentrations ranging from 5 to 100 microM. Using reactive oxygen species (ROS)-specific dyes, an increase in nitric oxide and superoxide anion was detected in the cells by 4 h after exposure to parthenolide. Parthenolide-induced elevation of hypochlorite anion was observed only in the two t(4;11) lines. These data suggest parthenolide may have potential as a potent and novel therapeutic agent against pre-B ALLs.     

Induction of apoptosis in leukemic cells by homovanillic acid derivative, capsaicin, through oxidative stress: implication of phosphorylation of p53 at Ser-15 residue by reactive oxygen species

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a homovanillic acid derivative found in pungent fruits. Several investigators have reported the ability of capsaicin to inhibit events associated with the promotion of cancer. However, the effects of capsaicin on human leukemic cells have never been investigated. We investigated the effects of capsaicin on leukemic cells in vitro and in vivo and further examined the molecular mechanisms of capsaicin-induced apoptosis in myeloid leukemic cells. Capsaicin suppressed the growth of leukemic cells, but not normal bone marrow mononuclear cells, via induction of G(0)-G(1) phase cell cycle arrest and apoptosis. Capsaicin-induced apoptosis was in association with the elevation of intracellular reactive oxygen species production. Interestingly, capsaicin-sensitive leukemic cells were possessed of wild-type p53, resulting in the phosphorylation of p53 at the Ser-15 residue by the treatment of capsaicin. Abrogation of p53 expression by the antisense oligonucleotides significantly attenuated capsaicin-induced cell cycle arrest and apoptosis. Pretreatment with the antioxidant N-acetyl-L-cystein and catalase, but not superoxide dismutase, completely inhibited capsaicin-induced apoptosis by inhibiting phosphorylation of Ser-15 residue of p53. Moreover, capsaicin effectively inhibited tumor growth and induced apoptosis in vivo using NOD/SCID mice with no toxic effects. We conclude that capsaicin has potential as a novel therapeutic agent for the treatment of leukemia.     

Inhibition of ERK oscillations by ionizing radiation and reactive oxygen species

The shuttling of activated protein kinases between the cytoplasm and nucleus is an essential feature of normal growth factor signaling cascades. Here we demonstrate that transforming growth factor alpha (TGFα) induces oscillations in extracellular signal regulated kinase (ERK) cytoplasmic‐nuclear translocations in human keratinocytes. TGFα‐dependent ERK oscillations mediated through the epidermal growth factor receptor (EGFR) are inhibited by low dose X‐irradiation (10 cGy) and low concentrations of hydrogen peroxide (0.32–3.26 µM H₂O₂) used as a model reactive oxygen species (ROS). A fluorescent indicator dye (H2‐DCFDA) was used to measure cellular ROS levels following X‐irradiation, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) and H₂O₂. X‐irradiation did not generate significant ROS production while 0.32 µM H₂O₂ and TPA induced significant increases in ROS levels with H₂O₂ > TPA. TPA alone induced transactivation of the EGFR but did not induce ERK oscillations. TPA as a cotreatment did not inhibit TGFα‐stimulated ERK oscillations but qualitatively altered TGFα‐dependent ERK oscillation characteristics (amplitude, time‐period). Collectively, these observations demonstrate that TGFα‐induced ERK oscillations are inhibited by ionizing radiation/ROS and perturbed by epigenetic carcinogen in human keratinocytes. © 2010 Wiley‐Liss, Inc.     

Autophagy is required during cycling hypoxia to lower production of reactive oxygen species

Background and purpose

Human tumors are characterized by the presence of cells that experience periodic episodes of hypoxia followed by reoxygenation. These cells are exposed to reactive oxygen species (ROS) upon reoxygenation and require adaptation to this stress by lowering ROS production or enhancing ROS-clearance for their survival. We hypothesized that autophagy, a lysosomal degradation pathway, may be involved in reducing ROS during periodic hypoxia through removal of ROS producing species.

Materials and methods

Human tumor cells (MCF-7, HT29, U373) were exposed to cycles of hypoxia (O₂ < 0.02%) and reoxygenation in the absence or presence of the autophagy inhibitor chloroquine (CQ). Clonogenic survival, ROS production and mitochondrial-DNA content were assessed. In addition, A549 cells overexpressing wild-type or K63-mutated ubiquitin (K63R) were analyzed for ROS production.

Results

Our data indicate that CQ treatment sensitizes cells to cycling hypoxia, due to increased production of ROS, associated with an incapacity to reduce mitochondrial content. Addition of the ROS-scavenger N-acetyl-cysteine increased cell viability and neutralized CQ-effects. Additionally, genetic prevention of K63-linked ubiquitin chains that are required for the removal of toxic protein aggregates by autophagy, resulted in increased ROS production.

Conclusions

Inhibition of autophagy substantially increases cell death induced by cycling hypoxia through increased ROS production, providing an opportunity to decrease the hypoxic fraction within tumors and enhance tumor therapy.     

Generation of reactive oxygen species by human mesothelioma cells.

Malignant mesothelioma cells contain elevated levels of manganese superoxide dismutase (MnSOD) and are highly resistant to oxidants compared to non-malignant mesothelial cells. Since the level of cellular free radicals may be important for cell survival, we hypothesized that the increase of MnSOD in the mitochondria of mesothelioma cells may alter the free radical levels of these organelles. First, MnSOD activity was compared to the activities of two constitutive mitochondrial enzymes; MnSOD activity was 20 times higher in the mesothelioma cells than in the mesothelial cells, whereas the activities of citrate synthase and cytochrome c oxidase did not differ significantly in the two cell lines. This indicates that the activity of MnSOD per mitochondrion was increased in the mesothelioma cells. Superoxide production was assayed in the isolated mitochondria of these cells using lucigenin chemiluminescence. Mitochondrial superoxide levels were significantly lower (72%) in the mesothelioma cells compared to the mesothelial cells. Oxidant production in intact cells, assayed by fluorimetry using 2',7'-dichlorodihydrofluorescein as a fluorescent probe, did not differ significantly between these cells. We conclude that mitochondrial superoxide levels are lower in mesothelioma cells compared to nonmalignant mesothelial cells, and that this difference may be explained by higher MnSOD activity in the mitochondria of these cells. Oxidant production was not different in these cells, which may be due to the previously observed increase in H2O2-scavenging mechanisms of mesothelioma cells.     

活性氧与肿瘤研究进展

目的：探讨活性氧与肿瘤的发生、发展和治疗之间的关系。方法：应用PubMed和CNKI期刊全文数据库检索系统,以“活性氧和肿瘤”为关键词,检索2000-01-2013-06的相关文献。共检索到英文文献29条,中文文献330条,纳入标准：1）活性氧与肿瘤发生;2）活性氧与肿瘤转移;3）活性氧与肿瘤治疗。根据纳入的标准,最后分析文献27篇。结果：肿瘤患者体内氧化还原失衡,表现为氧化应激水平增高,国内外在胃肠道肿瘤、舌癌和乳腺癌等的研究中均发现了氧化应激状态改变。肿瘤患者活性氧增多的机制主要集中在如下几个方面：1）遗传分子生物学的改变,包括转入因子Nrf2及其抑制蛋白Keap1、RAS途径的相关突变,癌基因蛋白（比如Raf、MOS、MEK和Myc）的过度表达,抑癌基因（如p53）的沉默;2）肿瘤细胞处于高代谢状态,患者正常营养素摄取减少,引起活性氧的堆积;3）免疫系统非特异性的慢性激活,产生过多的前炎性因子;4）抗肿瘤药物特别是多柔比星和顺铂等的使用。活性氧与肿瘤生物学特性密切相关。一方面,它通过脂质过氧化、DNA损伤和蛋白质破坏等参与肿瘤的形成;另一方面,活性氧也参与肿瘤的转移,这不仅表现在其清除剂可以降低细胞转移能力,也包括其可以调节肿瘤细胞迁移和侵袭;再者,活性氧和转录因子Snial相互作用可以诱导上皮细胞间充质转化的产生。活性氧的作用与其浓度有关,高浓度的活性氧可能导致细胞凋亡,而低浓度可致细胞增殖和癌变,国内外研究发现许多抗肿瘤药物通过增加细胞内活性氧的产生来诱导肿瘤细胞凋亡,如乙烷硒啉、三氧化二砷、顺铂、柔红霉素和5-FU等。结论：活性氧不仅影响肿瘤的生物学特性,而且与肿瘤的治疗有密切关系,寻找合适的活性氧浓度,将为肿瘤的防治提供帮助。     

Constitutive histone H2AX phosphorylation on Ser-139 in cells untreated by genotoxic agents is cell-cycle phase specific and attenuated by scavenging reactive oxygen species

DNA damage, particularly when it involves formation of double-strand breaks (DSBs), triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX has been named gammaH2AX, and induction of gammaH2AX in cells exposed to genotoxic agents is considered a sensitive and specific reporter of DNA damage. However, in untreated normal cells as well in the cells of various tumor lines cells, a fraction of histone H2AX molecules remain phosphorylated. In the present study, we observed that the extent of this constitutive H2AX phosphorylation varies depending on the cell type (line) and on cell cycle phase and, in most cell types, S and G(2)/M phase cells exhibit greater levels of H2AX phosphorylation than do cells in the G(1) phase. Furthermore, constitutive H2AX phosphorylation in human pulmonary carcinoma A549, lymphoblastoid TK6, and in normal bronchial epithelial cells was reduced following cell exposure to N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates; the reduction was most pronounced for G(2)M cells. Growth of A549 cells in the presence of buthionine sulfoximine, an inhibitor of glutathione synthetase, amplified the level of constitutive H2AX phosphorylation in A549 cells. The observed constitutive H2AX phosphorylation may be a reflection of the ongoing DNA damage mediated by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle, leading to formation of DSBs during the S phase. Because cumulative DNA damage in proliferating cells mediated by ROS is considered the key mechanism for cell ageing, the present approach to estimate the degree of attenuation of constitutive H2AX phosphorylation by antioxidants may provide a convenient tool to assess the DNA-protective and possible anti-ageing properties of other agents.     

Autophagy induction by the 30-100kDa fraction of areca nut in both normal and malignant cells through reactive oxygen species

Areca nut (AN) is an addictive carcinogen used by about 200-600 million people worldwide. Some AN components are shown to induce apoptosis; however, we previously demonstrated that AN extract (ANE) and the 30-100kDa fraction of ANE (ANE 30-100K) induced autophagy-like responses, such as swollen cell morphology, empty cytoplasm, acidic vesicles, and LC3-II accumulation, in an oral cancer cell line, OECM-1. To further assess the responses of other cell types to ANE 30-100K, we used both normal and malignant cells as the targets of ANE 30-100K and found that normal oral fibroblasts (CMT415), peripheral blood lymphocytes (PBLs), Jurkat leukemia T cells, and esophageal carcinoma cells (CE81T/VGH) exhibited similar responses after ANE 30-100K challenge. ANE 30-100K drastically increased acidic vesicle-containing PBLs isolated from two independent donors (from 0.1% to 92.1% and 2.9% to 64.2%). Furthermore, both ANE- and ANE 30-100K-induced LC3-II accumulation in CMT415 and CE81T/VGH was further increased in the presence of the lysosomal protease inhibitors (pepstatin A, E64d, and leupeptin). On the other hand, ANE 30-100K also increased the level of intracellular reactive oxygen species (ROS), and the ROS scavengers, N-acetylcysteine (NAC) and Tiron, inhibited ANE 30-100K-induced cell death and LC3-II accumulation. Collectively, these results suggest the existence of an autophagy-inducing AN ingredient (AIAI) in ANE 30-100K, which renders ANE as an autophagic flux inducer through ROS in both normal and malignant cells.     

ACCELERATED PAPER: Relationship between the intracellular reactive oxygen species and the induction of oxidative DNA damage in human neutrophil-like cells

To clarify the mechanisms of intracellular induction of oxidativeDNA damage, we have investigated the concentrations of intracellularreactive oxygen species and the amounts of 8-hydroxydeoxyguanosine(8OHdG), a mutagenic oxidative DNA damage, in human neutrophil-likecells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). Wedetermined intracellular concentrations of hydrogen peroxideand superoxide by flow cytometry with dichlorofluorescein diacetateand hydroethidine, respectively. We determined the 8OHdG amountswith an electrochemical detector connected to HPLC after anaerobicsample processing. DMSO-HL60 releases superoxide upon stimulationwith phorbol myristate acetate, and the released superoxidedismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with100 nM phorbol myristate acetate increased intracellular hydrogenperoxide, superoxide and 8OHdG (control). Addition of 1000 U/mlcatalase decreased hydrogen peroxide (313% of control) and 8OHdG(20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%)and 8OHdG (41.6%). Addition of 1 mM deferoxamine decreased 8OHdG(30.4%), but increased hydrogen peroxide (129.6%). Additionof 200 µM 4-acetamido-4-isothiocyanostilbene-2, 2'-disulfonicacid decreased superoxide (59.9%) and 8OHdG (42.0%). Additionof 0.4% ethanol had no effect on superoxide concentration (102.2%),but tended to decrease hydrogen peroxide (83.5%) and 8OHdG (84.3%).Pretreatment of DMSO-HL60 with 0.1 mM FeSO₄ increased 8OHdG(1173%), but decreased hydrogen peroxide(75.8%). These findingsindicate that the extracellularly released superoxide and hydrogenperoxide diffuse into the cell, but that such reactive oxygenspecies are not the direct molecules to induce 8OHdG. Our resultssuggest that 8OHdG is induced by the hydroxyl radical whichis generated from intracellular hydrogen peroxide and superoxide-reducedFe.     

Auranofin induces urothelial carcinoma cell death via reactive oxygen species production and synergy with cisplatin

Urothelial carcinoma (UC) is one of the most common cancer types of the urinary tract. UC is associated with poor 5-year survival rate, and resistance to cisplatin-based therapy remains a challenge for invasive bladder cancer treatment. Therefore, there is an urgent need to develop new drugs for advanced UC therapy. Auranofin (AF) was developed over 30 years ago for the treatment of rheumatoid arthritis and has been reported to exert an antitumor effect by increasing the level of reactive oxygen species (ROS) in cancer cells. The aim of the present study was to examine the effects of AF on cancer cell proliferation, cell cycle and apoptosis, either alone or in combination with cisplatin. AF induced cell death in two separate cell lines, HT 1376 and BFTC 909, in a concentration- and time-dependent manner by inducing cell cycle arrest. However, the distribution of cells in different phases of the cell cycle differed between the two cell lines, with G₀/G₁ cell cycle arrest in HT 1376 cells and S phase arrest in BFTC 909 cells. In addition, AF induced apoptosis in HT 1376, as well as redox imbalance in both HT 1376 and BFTC 909 cells. Cell viability was rescued following treatment with N-acetyl-L-cysteine, a ROS scavenger. Furthermore, AF treatment synergistically increased the cytotoxicity of HT 1376 and BFTC 909 cells when combined with cisplatin treatment. These findings suggest that AF may represent a potential candidate drug against UC and increase the therapeutic effect of cisplatin.