Cold Atmospheric Plasma Modification of Amyloid β

Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind the CAP-induced structural perturbations of biomacromolecules. Here, we investigated the potential effects of CAP irradiation of amyloid β (Aβ), an amyloidogenic protein associated with Alzheimer’s disease. Using nuclear magnetic resonance spectroscopy, we observed gradual spectral changes in Aβ after a 10 s CAP pretreatment, which also suppressed its fibril formation, as revealed by thioflavin T assay. As per mass spectrometric analyses, these effects were attributed to selective oxidation of the methionine residue (Met) at position 35. Interestingly, this modification occurred when Aβ was dissolved into a pre-irradiated buffer, indicating that some reactive species oxidize the Met residue. Our results strongly suggest that the H₂O₂ generated in the solution by CAP irradiation is responsible for Met oxidation, which inhibits Aβ amyloid formation. The findings of the present study provide fundamental insights into plasma biology, giving clues for developing novel applications of CAP.     

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen,Hydrogen Peroxide,Nitric Oxide and Nitrite/Nitrate

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H₂O₂ present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.     

Effects of Acidification on Dewaterability and Aluminum Concentration of Alum Sludge

This study utilizes the Terzaghi-Voigt model to characterize the effects of acidification on the dewaterability of alum sludge. Alum sludge, which was obtained from the sedimentation basins of a water treatment plant using poly aluminum chloride as coagulant, was acidified to different pH levels with sulfuric acid. The dewaterability of the acidified sludge was characterized by expression tests. The results show dewaterability enhancement was insignificant until pH was below 4. Further improvement in dewaterability can be achieved by polymer conditioning. The Terzaghi-Voigt model can be applied to explain the difference in dewaterability between acidified and original sludges. Results also show dissolved aluminum concentrations were controlled by minerals in the influent rather than amorphous aluminum hydroxide.     

A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin,Tested Over a Wide Range of Concentrations

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10⁻¹¹–10⁻² M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10⁻⁹–10⁻⁵ M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high—but not low—melatonin concentrations, although the mechanism of action still remains to be elucidated.     

浮选光度法测定饮用水中亚硝酸根

基于亚硝酸根在弱酸性的条件下重氮化反应 ,重氮化盐再与 N,N-二甲基苯胺进行偶合反应 ,生成黄色甲基橙 ,酸化后甲基橙变成红色 ,用十二烷基硫酸钠浮选富集 ,在正丁醇中收集浮选液 ,进行分光光度测定 ,从而求得亚硝酸根的含量。摩尔吸光系数ε为 9.7× 1 0 4L/ mol· cm(正丁醇为溶剂 ) ,亚硝酸根的浓度在 0 .0 4 1～ 0 .0 0 4 1μg/ m L与吸光度有良好的线性关系。该法可检出纯净水中痕量亚硝酸根的含量     

Sustained nitrite accumulation in a membrane‐assisted bioreactor (MBR) for the treatment of ammonium‐rich wastewater

A membrane‐assisted bioreactor (MBR) for sustained nitrite accumulation is presented, treating a synthetic wastewater with total ammonium nitrogen (TAN) concentrations of 1 kg N m^?3 at a hydraulic retention time down to 1 day. Complete biomass retention was obtained by microfiltration with submerged hollow fibre membranes. A membrane flux up to 189.5 dm³ day^?1 m^?2 could be maintained at a suction pressure below 100 kPa. Nitrification was effectively blocked at the nitrite stage (nitritation), and nitrate concentration was less than 29 g N m^?3. The rate of aeration was reduced to obtain a mixture of ammonium and nitrite, and after adjusting this rate the TAN/NO₂‐N ratio in the reactor effluent was kept around unity, making it suitable for further treatment by anaerobic oxidation of ammonium with nitrite. After increasing again the rate of aeration, complete nitrification to nitrate recovered after 11 days. It is suggested that nitrite accumulation resulted from a combination of factors. First, the dissolved oxygen (DO) concentration in the reactor was always limited with concentrations below 0.1 g DO m^?3, thereby limiting nitrification and preventing significant nitrate formation. The latter is attributed to the fact that ammonium‐oxidising bacteria cope better with low DO concentrations than nitrite oxidisers. Second, the MBR was operated at a high ammonia concentration of 7–54 g N m^?3, resulting in ammonia inhibition of the nitrite‐oxidising microorganisms. Third, a temperature of 35 °C was reported to yield a higher maximum growth rate for ammonium‐oxidising bacteria than for nitrite‐oxidising bacteria. Nitrite oxidisers were always present in the MBR but were out‐competed under the indicated process conditions, which is reflected in low concentrations of nitrate. Oxygen limitation was shown to be the most important factor to sustain nitrite accumulation. Nevertheless, nitritation was possible at ambient temperature (22–24 °C), lower ammonia concentration (<7 g N m^?3) and when using raw nitrogenous wastewater containing some biodegradable carbon. Overall, application of the MBR for nitritation was shown to be a reliable technology. © 2003 Society of Chemical Industry     

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGlo^TM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.     

Cold Atmospheric Plasma Apoptotic and Oxidative Effects on MCF7 and HCC1806 Human Breast Cancer Cells

Breast cancer (BC) is a malignant neoplasia with the highest incidence and mortality rates in women worldwide. Currently, therapies include surgery, radiotherapy, and chemotherapy, including targeted therapies in some cases. However, treatments are often associated with serious adverse effects. Looking for new options in BC treatment, we evaluated the therapeutic potential of cold atmospheric plasma (CAP) in two cell lines (MCF7 and HCC1806) with distinct histological features. Apoptosis seemed to be the most prevalent type of death, as corroborated by several biochemical features, including phosphatidylserine exposure, the disruption of mitochondrial membrane potential, an increase in BAX/BCL2 ratio and procaspase 3 loss. Moreover, the accumulation of cells in the G2/M phase of the cell cycle points to the loss of replication ability and decreased survival. Despite reported toxic concentrations of peroxides in culture media exposed to plasma, intracellular peroxide concentration was overall decreased accompanying a reduction in GSH levels shortly after plasma exposure in both cell lines. In HCC1806, elevated nitric oxide (NO) concentration accompanied by reduced superoxide levels suggests that these cells are capable of converting plasma-derived nitrites into NO that competes with superoxide dismutase (SOD) for superoxide to form peroxinitrite. The concomitant inhibition of the antioxidative activity of cells during CAP treatment, particularly the inhibition of cytochrome c oxidase with sodium azide, synergistically increased plasma toxicity. Thus, this in vitro research enlightens the therapeutic potential of CAP in the treatment of breast cancer, elucidating its possible mechanisms of action.     

nirS基因重组工程菌降解亚硝酸盐的初步研究

通过基因工程手段增加厌氧氨氧化菌亚硝酸盐还原酶(nitrite reductase,nirS)的表达量,运用质粒载体p GEM-T克隆nirS基因。琼脂糖凝胶电泳检测显示,nirS基因重组工程菌在440 bp处有明显的目的条带;nirS基因重组工程菌扩大培养7~8 h后即达到生长曲线稳定期,引入外加氮源后,菌体生长情况更优。通过不同菌液投加量以及处理不同初始浓度的亚硝酸钠溶液,检测nirS基因重组工程菌的性能。结果表明,当nirS基因重组工程菌投加30 m L(细菌数为2.3×107个/m L),亚硝酸盐初始质量浓度为40 mg/L时,亚硝酸盐去除率达到90%以上。nirS基因重组工程菌可适用于亚硝酸盐废水的处理。     

Acidification of Harbor Sediment and Removal of Heavy Metals Induced by Water Splitting in Electrodialytic Remediation

Abstract

Harbor sediments are often contaminated with heavy metals, which can be removed by electrodialytic remediation. Water splitting at the anion exchange membrane in contact with the contaminated material in electrodialytic remediation is highly important for the removal of heavy metals. Here it was investigated how acidification caused by water splitting at the anion exchange membrane during electrodialytic remediation of contaminated harbor sediment and hence the metal removal, was influenced by different experimental conditions. Two different experimental cells were tested, where the number of compartments and ion exchange membranes differed. Totally, 14 electrodialytic experiments were made, with varying remediation time, current densities, and liquid to solid ratio (L/S). pH in the sediment decreased slightly after 1 day of remediation, even if the sediment had a high buffering capacity, suggesting that water splitting at the anion exchange membrane started early in the remediation process. An increase in the voltage over the cell and a decrease in the electrical conductivity in the sediment suspension also indicated that the water splitting started within 1 day of remediation. When the sediment was acidified, the voltage decreased and electrical conductivity increased. After 5 days of remediation the sediment was acidified at the chosen current density (1 mA/cm²) and the main metal removal was observed shortly after. Thus it was crucial for the metal removal that the sediment was fully acidified. Lower metal removal was seen in an experimental cell with three compartments compared to five compartments, due to increased sensitivity of pH changes in the cell.     

In Vitro Anticancer Potential of Jasione montana and Its Main Components against Human Amelanotic Melanoma Cells

Jasione montana L. (Campanulaceae) is used in traditional Belarusian herbal medicine for sleep disorders in children, but the chemical composition and biological activity have not been investigated. In this study, the activities of J. montana extracts, their fractions and main compounds were evaluated in amelanotic melanoma C32 (CRL-1585) cells and normal fibroblasts (PCS-201-012). The extracts and fractions were analyzed using liquid chromatography–photodiode array detection–electrospray ionization–mass spectrometry (LC–PDA–ESI–MS/TOF) to characterize 25 compounds. Further, three major and known constituents, luteolin (22) and its derivatives such as 7-O-glucoside (12) and 7-O-sambubioside (9) were isolated and identified. The cytotoxic activities against fibroblasts and the amelanotic melanoma cell line were determined using the fixable viability stain (FVS) assay. The influence of diethyl ether (Et₂O) fraction (JM4) and 22 on apoptosis induction was investigated using an annexin V binding assay. The obtained results showed significant cytotoxicity of JM4 and 22 with IC₅₀ values of 119.7 ± 3.2 and 95.1 ± 7.2 μg/mL, respectively. The proapoptotic potential after 22 treatment in the C32 human amelanotic melanoma cell line was comparable to that of vinblastine sulfate (VLB), detecting 29.2 ± 3.0% apoptotic cells. Moreover, 22 displayed less necrotic potential against melanoma cells than VLB. In addition, the influences of JM4 and 22 on the dysfunction of the mitochondrial membrane potential (MMP), cell cycle and activity of caspases 3, 8, 9, and 10 were established. The effects of JM4 on MMP change (74.5 ± 3.0% of the cells showed a reduced MMP) corresponded to the results obtained from the annexin V binding assay and activation of caspase-9. JM4 and 22 displayed a significant impact on caspase-9 (40.9 ± 2.4% of the cells contained active caspase-9 after JM4 treatment and 16.6 ± 0.8% after incubation with 22) and the intrinsic (mitochondrial) apoptotic pathway. Moreover, studies have shown that JM4 and 22 affect the activation of external apoptosis pathways by inducing the caspase-8 and caspase-10 cascades. Thus, activation of caspase-3 and DNA damage via external and internal apoptotic pathways were observed after treatment with JM4 and 22. The obtained results suggest that J. montana extracts could be developed as new topical preparations with potential anticancer properties due to their promising cytotoxic and proapoptotic potential.     

Chikusetsu Saponin V Attenuates MPP+-Induced Neurotoxicity in SH-SY5Y Cells via Regulation of Sirt1/Mn-SOD and GRP78/Caspase-12 Pathways

Studies have shown that saponins from Panax japonicus (SPJ) possess neuroprotective effects. However, whether Chikusetsu saponin V (CsV), the most abundant member of SPJ, can exert neuroprotective effects against 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity is not known. In this study, we aimed to investigate the neuroprotective effects of CsV on MPP⁺-induced cytotoxicity in human neuroblastoma SH-SY5Y cells and explore its possible mechanisms. Our results show that CsV attenuates MPP⁺-induced cytotoxicity, inhibits ROS accumulation, and increases mitochondrial membrane potential dose-dependently. We also found that levels of Sirt1 protein and Mn-SOD mRNA significantly decreased in MPP⁺-treated group but were restored with CsV treatment in a dose-dependent manner. Furthermore, GRP78 protein and Caspase-12 mRNA levels were elevated by MPP⁺ exposure but reversed by CsV treatment. CsV inhibited the MPP⁺-induced downregulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. Overall, these results suggest that Sirt1/Mn-SOD and GRP78/Caspase-12 pathways might be involved in the CsV-mediated neuroprotective effects.     

Evaluation of heterotrophic nitrite removal by a sulphide and acetate oxidizing mixed culture originated from an oil reservoir

BACKGROUND: Shortcut biological nitrogen removal (SBNR) has attracted much attention in recent years due to lower aeration and chemical oxygen demand (COD) requirements, shorter residence time and smaller biomass production. In this work an oil reservoir denitrifying culture, with the ability to function under autotrophic and heterotrophic conditions was used for heterotrophic denitritation. Using freely suspended cells, effects of nitrite concentration (10–50 mmol L^?1) and temperature (15–35 °C) on the kinetics of denitritation were investigated and a kinetic model was developed. Potential for enhancement of nitrite removal rate, and impacts of nitrite concentration and loading rate were investigated in a continuous biofilm reactor. RESULTS: Nitrite did not impose any inhibitory effect, even at the highest applied concentration of 50 mmol L^?1. Increase of temperature in the range 15–35 °C enhanced the reduction rate significantly. Fitting the experimental data into the model developed, values of biokinetic coefficients (µ_max?NO2, K_S?NO2, Y_X?NO2, Y_X?Ace?NO2 and Eµ‐_NO2) were determined. In the biofilm reactor increases in nitrite loading rate (through flow rate or feed nitrite concentration) led to a linear increase of nitrite removal rate, with the highest removal rate of 140.6 mmol L^?1 h^?1 achieved with a residence time of 0.19 h. CONCLUSION: The enrichment culture used in this study is not only a superior biocatalyst for simultaneous removal of sulphide, nitrate and BOD, it could also be used effectively in the denitritation step of an SBNR process. The kinetic model developed would certainly have beneficial applications in the design, operation and control of the SBNR process. Copyright © 2011 Society of Chemical Industry     

Norketamine,the Main Metabolite of Ketamine,Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca2+-Regulated ERK1/2-Activating Pathway

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01–0.5 mM) exhibited greater cytotoxicity than ketamine (0.1–3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca²⁺]_i). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca²⁺ chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca²⁺]_i in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca²⁺]_i-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca²⁺]_i/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.     

The effect of salinity on nitrite accumulation in a down‐flow hanging sponge reactor

BACKGROUND: In this study, the inductive effect of salinity on nitrite accumulation in a down‐flow hanging sponge (DHS) reactor, developed as a novel and cost‐effective wastewater treatment process, was evaluated by conducting a long‐term continuous experiment lasting more than 1400 days. RESULTS: The influent salinity was controlled by adding NaCl at concentrations ranging from 0 to 25 g Cl^? L^?1. The effluent nitrite increased with increases in salinity, i.e. the fraction of nitrite to total nitrogen in the effluent increased from 1.6% at 0 g Cl^? L^?1 to 87.6% at 25 g Cl^? L^?1. Fluorescence in situ hybridization (FISH) analysis revealed that as salinity increased, the nitrifying bacterial community in the DHS changed markedly at the species level. In particular, the dominant nitrite‐oxidizing bacteria changed from Nitrospira‐sublineage I at 0 g Cl^? L^?1 to Nitrobacter spp. at 15 g Cl^? L^?1. At 25 g Cl^? L^?1, no nitrite‐oxidizing bacteria were detected. CONCLUSION: Our findings suggest that the DHS reactor is suitable for cost‐effective nitrite production processes and that salinity control using NaCl is an effective method for inducing nitrite accumulation. Copyright © 2012 Society of Chemical Industry     

Molecular Mechanisms of Parthanatos and Its Role in Diverse Diseases

Differential evolution of apoptosis, programmed necrosis, and autophagy, parthanatos is a form of cell death mediated by poly(ADP-ribose) polymerase 1 (PARP1), which is caused by DNA damage. PARP1 hyper-activation stimulates apoptosis-inducing factor (AIF) nucleus translocation, and accelerates nicotinamide adenine dinucleotide (NAD⁺) and adenosine triphosphate (ATP) depletion, leading to DNA fragmentation. The mechanisms of parthanatos mainly include DNA damage, PARP1 hyper-activation, PAR accumulation, NAD⁺ and ATP depletion, and AIF nucleus translocation. Now, it is reported that parthanatos widely exists in different diseases (tumors, retinal diseases, neurological diseases, diabetes, renal diseases, cardiovascular diseases, ischemia-reperfusion injury...). Excessive or defective parthanatos contributes to pathological cell damage; therefore, parthanatos is critical in the therapy and prevention of many diseases. In this work, the hallmarks and molecular mechanisms of parthanatos and its related disorders are summarized. The questions raised by the recent findings are also presented. Further understanding of parthanatos will provide a new treatment option for associated conditions.     

Insights into the in vitro Anticancer Effects of Diruthenium‐1

The in vitro anticancer activity of the dinuclear trithiolato‐bridged arene ruthenium complex diruthenium‐1 (DiRu‐1) was evaluated against a panel of human cancer cell lines used as in vitro models for hepatocellular carcinoma (HepG2 cells), estrogen‐responsive breast adenocarcinoma (MCF‐7 cells), and triple‐negative breast adenocarcinoma (MDA‐MB‐231 cells). DiRu‐1 is highly cytotoxic to these cell lines, demonstrating half‐maximal inhibitory concentrations (IC₅₀) in the low‐nanomolar range (77±1.4 to 268.2±4.4 nm ). The main molecular mechanisms responsible for the high cytotoxicity of DiRu‐1 against the most responsive MCF‐7 cell line (IC₅₀=77±1.4 nm) were investigated on the basis of the capacity of DiRu‐1 to induce oxidative stress, apoptosis, and DNA damage, and to inhibit the cell cycle and proliferation. The results show that DiRu‐1 triggers caspase‐dependent apoptosis in MCF‐7 cells on both the intrinsic and extrinsic pathways. Moreover, the Ru complex also causes necrosis, mitotic catastrophe, and autophagy. DiRu‐1 increases the intracellular levels of reactive oxygen species (ROS), which play a significant role in its cytotoxicity and pro‐apoptotic activity. An important mechanism of the anticancer activity of DiRu‐1 appears to be the induction of DNA lesions, mainly due to apoptotic DNA fragmentation and cell‐cycle arrest at the G₂/M checkpoint. These changes are correlated with the concentration of DiRu‐1, the duration of the cell treatment, and the post‐treatment time.