Rapid diffusion-state switching underlies stable cytoplasmic gradients in the Caenorhabditis elegans zygote.

Protein concentration gradients organize cells and tissues and commonly form through diffusion away from a local source of protein. Interestingly, during the asymmetric division of the Caenorhabditis elegans zygote, the RNA-binding proteins MEX-5 and PIE-1 form opposing concentration gradients in the absence of a local source. In this study, we use near-total internal reflection fluorescence (TIRF) imaging and single-particle tracking to characterize the reaction/diffusion dynamics that maintain the MEX-5 and PIE-1 gradients. Our findings suggest that both proteins interconvert between fast-diffusing and slow-diffusing states on timescales that are much shorter (seconds) than the timescale of gradient formation (minutes). The kinetics of diffusion-state switching are strongly polarized along the anterior/posterior (A/P) axis by the PAR polarity system such that fast-diffusing MEX-5 and PIE-1 particles are approximately symmetrically distributed, whereas slow-diffusing particles are highly enriched in the anterior and posterior cytoplasm, respectively. Using mathematical modeling, we show that local differences in the kinetics of diffusion-state switching can rapidly generate stable concentration gradients over a broad range of spatial and temporal scales.

RNA ligation in neurons by RtcB inhibits axon regeneration.

Activity of the RNA ligase RtcB has only two known functions: tRNA ligation after intron removal and XBP1 mRNA ligation during activation of the unfolded protein response. Here, we show that RtcB acts in neurons to inhibit axon regeneration after nerve injury. This function of RtcB is independent of its basal activities in tRNA ligation and the unfolded protein response. Furthermore, inhibition of axon regeneration is independent of the RtcB cofactor archease. Finally, RtcB is enriched at axon termini after nerve injury. Our data indicate that neurons have co-opted an ancient RNA modification mechanism to regulate specific and dynamic functions and identify neuronal RtcB activity as a critical regulator of neuronal growth potential.

DNA-Binding, Photocleavage, and Photodynamic Anti-cancer Activities of Pyridyl Corroles.

The DNA-binding, photocleavage, and antitumor activity of three free base pyridyl corroles 1, 2, and 3 have been investigated. The binding affinity toward CT-DNA decreases with increasing number of pentafluorophenyl, whereas the photocleavage activity toward pBR322 DNA becomes more efficient. Singlet oxygen was demonstrated as active species responsible for DNA cleavage. These corroles exhibited high cytotoxicity against three tested cancer cells (Hela, HapG2, and A549) and the cytotoxicity could be further enhanced under irradiation. Intracellular reactive oxygen species level was also monitored using HeLa Cells upon the combined treatment of corroles and light. These corroles could be absorbed by HeLa cells at low concentration. They can induce the decrease of mitochondrial membrane potential and apoptosis of tumor cells under irradiation.

Synthesis, Molecular Structure, DNA/Protein Binding, Cytotoxicity, Apoptosis, Reactive Oxygen Species, and Mitochondrial Membrane Potential of Dibenzoxanthenes Derivatives.

Two dibenzoxanthene isomers 3 and 4 were synthesized and characterized. The crystal structures of the two compounds were solved by single-crystal X-ray diffraction. Binding of two compounds with calf thymus DNA (CT DNA) and BSA (bovine serum albumin) has been thoroughly investigated by UV-Vis and fluorescence spectroscopy. The DNA-binding constants were determined to be 2.51 (± 0.09) × 10(3) for compound 3 and 4.55 (± 0.10) × 10(3) for compound 4. Two compounds can cleave pBR322 DNA upon irradiation. Significant nuclear damages of BEL-7402 cells were observed with compound treatment in a comet assay. The cytotoxicity in vitro was investigated by MTT method. These compounds have been found to induce nuclear condensation and fragmentation in BEL-7402 cells. The two compounds can enhance intracellular reactive oxygen species and decrease the mitochondrial membrane potential. The compounds activated caspase-3 and caspase-7, down-regulated the expression levels of anti-apoptotic protein Bcl-2, and up-regulated the expression levels of pro-apoptotic protein Bax. These compounds induce apoptosis of BEL-7402 cells through an ROS-mediated mitochondrial dysfunction pathway.

Multifunctional chitosan-based lanthanide luminescent hydrogel with stretchability, adhesion, self-healing, color tunability and antibacterial ability.

Lanthanide luminescent hydrogels have broad application prospects in various fields. However, most of lanthanide hydrogels possess relatively simple functions, which is not conducive to practical applications. Therefore, it is becoming increasingly urgent to develop multifunctional hydrogels. Herein, a multifunctional chitosan-based lanthanide luminescent hydrogel with ultra-stretchability, multi-adhesion, excellent self-healing, emission color tunability, and good antibacterial ability was prepared by a simple one-step free radical polymerization. In this work, our designed lanthanide complexes [Ln(4-VDPA)3] contain three reaction sites, which can be copolymerized with N-[tris(hydroxymethyl) methyl] acrylamide (THMA), acrylamide (AM), and diacryloyl poly(ethylene glycol) (DPEG) to form the first chemical crosslinking network, while hydroxypropyltrimethyl ammonium chloride chitosan (HACC) interacts with the hydroxyl and amino groups derived from the chemical crosslinking network through hydrogen bonds to form the second physical crosslinking network. The structure of the double network as well as the dynamic hydrogen bond and lanthanide coordination endow the hydrogel with excellent stretchability, adhesion and self-healing properties. Moreover, the introduction of lanthanide complexes and chitosan makes the hydrogel exhibit outstanding luminescence and antibacterial performances. This research not only realizes the simple synthesis of multifunctional luminescent hydrogels, but also provides a new idea for the fabrication of biomass-based hydrogels as intelligent and sustainable materials.

Focus on the molecular mechanisms of cisplatin resistance based on multi-omics approaches.

Cisplatin is commonly used in combination with other cytotoxic agents as a standard treatment regimen for a variety of solid tumors, such as lung, ovarian, testicular, and head and neck cancers. However, the effectiveness of cisplatin is accompanied by toxic side effects, for instance, nephrotoxicity and neurotoxicity. The response of tumors to cisplatin treatment involves multiple physiological processes, and the efficacy of chemotherapy is limited by the intrinsic and acquired resistance of tumor cells. Although enormous efforts have been made toward molecular mechanisms of cisplatin resistance, the development of omics provides new insights into the understanding of cisplatin resistance at genome, transcriptome, proteome, metabolome and epigenome levels. Mechanism studies using different omics approaches revealed the necessity of multi-omics applications, which provide information at different cellular function levels and expand our recognition of the peculiar genetic and phenotypic heterogeneity of cancer. The present work systematically describes the underlying mechanisms of cisplatin resistance in different tumor types using multi-omics approaches. In addition to the classical mechanisms such as enhanced drug efflux, increased DNA damage repair and changes in the cell cycle and apoptotic pathways, other changes like increased protein damage clearance, increased protein glycosylation, enhanced glycolytic process, dysregulation of the oxidative phosphorylation pathway, ferroptosis suppression and mRNA m6A methylation modification can also induce cisplatin resistance. Therefore, utilizing the integrated omics to identify key signaling pathways, target genes and biomarkers that regulate chemoresistance are essential for the development of new drugs or strategies to restore tumor sensitivity to cisplatin.

Recent advances in drug delivery systems for targeting brain tumors.

Brain tumor accounts for about 1.6% of incidence and 2.5% of mortality of all tumors, and the median survival for brain tumor patients is only about 20 months. The treatment for brain tumor still faces many challenges, such as the blood-brain barrier (BBB), blood-brain tumor barrier (BBTB), the overexpressed efflux pumps, the infiltration, invasion, high heterogeneity of tumor cells, drug resistance and immune escape caused by tumor microenvironment (TME) and cancer stem cells (CSC). This review attempts to clarify the challenges for multi-functional nano drug delivery systems (NDDS) to cross the BBB and target the cancer cells or organelles, and also provides a brief description of the different types of targeted multi-functional NDDS that have shown potential for success in delivering drugs to the brain. Further, this review also summarizes the research progress of multi-functional NDDS in the combination therapy of brain tumors from the following sections, the combination of chemotherapy drugs, chemotherapy-chemodynamic combination therapy, chemotherapy-immunization combination therapy, and chemotherapy-gene combination therapy. We also provide an insight into the recent advances in designing multi-functional NDDS for combination therapy.

Dual-action gallium-flavonoid compounds for combating Pseudomonas aeruginosa infection.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) causes infections that are difficult to treat, which is due to the bacterial natural resistance to antibiotics. The bacterium is also able to form a biofilm that protects the bacterium from clearance by the human immune system and leads to chronic infection. Herein, we synthesized and characterized a novel gallium compound that interferes with both the iron metabolism and quorum sensing system of P. aeruginosa to achieve a significant bactericidal activity. The compound could substantially reduce the secretion of bacterial virulence factors as well as eliminate biofilm formation. Integrative omics analysis indicates that this compound can significantly disturb the gene transcription and metabolism of P. aeruginosa. The effectiveness of the gallium compound was further validated in mammalian cell and murine skin infection models. Our study offers a new strategy to design new gallium-based antimicrobials to combat P. aeruginosa infection.

Exogenous proline enhances susceptibility of NSCLC to cisplatin via metabolic reprogramming and PLK1-mediated cell cycle arrest.

The occurrence of cisplatin resistance is still the main factor limiting the therapeutic effect of non-small cell lung cancer (NSCLC). It is urgent to elucidate the resistance mechanism and develop novel treatment strategies. Targeted metabolomics was first performed to detect amino acids' content in cisplatin-resistant cancer cells considering the relationship between tumour metabolic rearrangement and chemotherapy resistance and chemotherapy resistance. We discovered that levels of most amino acids were significantly downregulated, whereas exogenous supplementation of proline could enhance the sensitivity of NSCLC cells to cisplatin, evidenced by inhibited cell viability and tumour growth in vitro and xenograft models. In addition, the combined treatment of proline and cisplatin suppressed ATP production through disruption of the TCA cycle and oxidative phosphorylation. Furthermore, transcriptomic analysis identified the cell cycle as the top enriched pathway in co-therapy cells, accompanied by significant down-regulation of PLK1, a serine/threonine-protein kinase. Mechanistic studies revealed that PLK1 inhibitor (BI2536) and CDDP have synergistic inhibitory effects on NSCLC cells, and cells transfected with lentivirus expressing shPLK1 showed significantly increased toxicity to cisplatin. Inhibition of PLK1 inactivated AMPK, a primary regulator of cellular energy homeostasis, ultimately leading to cell cycle arrest via FOXO3A-FOXM1 axis mediated transcriptional inhibition in cisplatin-resistant cells. In conclusion, our study demonstrates that exogenous proline exerts an adjuvant therapeutic effect on cisplatin resistance, and PLK1 may be considered an attractive target for the clinical treatment of cisplatin resistance in NSCLC.

Ultra-specific genotyping of single nucleotide variants by ligase-based loop-mediated isothermal amplification coupled with a modified ligation probe.

Specific and accurate detection of single nucleotide variants (SNVs) plays significant roles in pathogenic gene research and clinical applications. However, the sensitive but ultra-specific detection of rare variants in biological samples still remains challenging. Herein, we report a novel, robust and practical SNV assay by integrating the outstanding features of high selectivity of an artificial mismatched probe, and the powerful loop-mediated isothermal amplification. In this strategy, we rationally introduce artificial mismatched bases into the 3'-terminal regions of the probe located in the ligation region to reduce the risk of nonspecific ligation, which can dramatically improve the specificity for the SNV assay. The proposed method can discern as little as 0.01% mutant DNA in the high background of wild-type DNA with high sensitivity (10 aM). In virtue of its outstanding performance, the artificial mismatched probe may also be employed and expanded in various DNA and RNA genetic analyses with ligase-assisted approaches, showing great potential in biomedical research, clinical diagnostics, and bioanalysis.

SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors.

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3'UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3'UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

Single-molecule dynamics of the P granule scaffold MEG-3 in the Caenorhabditis elegans zygote.

During the asymmetric division of the Caenorhabditis elegans zygote, germ (P) granules are disassembled in the anterior cytoplasm and stabilized/assembled in the posterior cytoplasm, leading to their inheritance by the germline daughter cell. P granule segregation depends on MEG (maternal-effect germline defective)-3 and MEG-4, which are enriched in P granules and in the posterior cytoplasm surrounding P granules. Here we use single-molecule imaging and tracking to characterize the reaction/diffusion mechanisms that result in MEG-3::Halo segregation. We find that the anteriorly enriched RNA-binding proteins MEX (muscle excess)-5 and MEX-6 suppress the retention of MEG-3 in the anterior cytoplasm, leading to MEG-3 enrichment in the posterior. We provide evidence that MEX-5/6 may work in conjunction with PLK-1 kinase to suppress MEG-3 retention in the anterior. Surprisingly, we find that the retention of MEG-3::Halo in the posterior cytoplasm surrounding P granules does not appear to contribute significantly to the maintenance of P granule asymmetry. Rather, our findings suggest that the formation of the MEG-3 concentration gradient and the segregation of P granules are two parallel manifestations of MEG-3's response to upstream polarity cues.

Combination of gallium(iii) with acetate for combating antibiotic resistant Pseudomonas aeruginosa.

Gallium(iii) has been widely used as a diagnostic and therapeutic agent in clinics for the treatment of various diseases, in particular, Ga-based drugs have been exploited as antimicrobials to combat the crisis of antimicrobial resistance. The therapeutic properties of Ga(iii) are believed to be attributable to its chemical similarity to Fe(iii). However, the molecular mechanisms of action of gallium remain unclear. Herein, by integrating metalloproteomics with metabolomics and transcriptomics, we for the first time identified RpoB and RpoC, two subunits of RNA polymerase, as Ga-binding proteins in Pseudomonas aeruginosa. We show that Ga(iii) targets the essential transcription enzyme RNA polymerase to suppress RNA synthesis, resulting in reduced metabolic rates and energy utilization. Significantly, we show that exogenous supplementation of acetate could enhance the antimicrobial activity of Ga(iii), evidenced by the inhibited growth of persister cells and attenuated bacterial virulence. The effectiveness of co-therapy of Ga(iii) and acetate was further validated in mammalian cell and murine skin infection models, which is attributable to enhanced uptake of Ga(iii), and reduced TCA cycle flow and bacterial respiration. Our study provides novel insights into the mechanistic understanding of the antimicrobial activity of Ga(iii) and offers a safe and practical strategy of using metabolites to enhance the efficacy of Ga(iii)-based antimicrobials to fight drug resistance.

PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo.

In the C. elegans embryo, the germline lineage is established through successive asymmetric cell divisions that each generate a somatic and a germline daughter cell. PIE-1 is an essential maternal factor that is enriched in embryonic germline cells and is required for germline specification. We estimated the absolute concentration of PIE-1::GFP in germline cells and find that PIE-1::GFP concentration increases by roughly 4.5 fold, from 92 nM to 424 nM, between the 1 and 4-cell stages. Previous studies have shown that the preferential inheritance of PIE-1 by germline daughter cells and the degradation of PIE-1 in somatic cells are important for PIE-1 enrichment in germline cells. In this study, we provide evidence that the preferential translation of maternal PIE-1::GFP transcripts in the germline also contributes to PIE-1::GFP enrichment. Through an RNAi screen, we identified Y14 and MAG-1 (Drosophila tsunagi and mago nashi) as regulators of embryonic PIE-1::GFP levels. We show that Y14 and MAG-1 do not regulate PIE-1 degradation, segregation or synthesis in the early embryo, but do regulate the concentration of maternally-deposited PIE-1::GFP. Taken together, or findings point to an important role for translational control in the regulation of PIE-1 levels in the germline lineage.

Polo-like Kinase Couples Cytoplasmic Protein Gradients in the C. elegans Zygote.

Intracellular protein gradients underlie essential cellular and developmental processes, but the mechanisms by which they are established are incompletely understood. During the asymmetric division of the C. elegans zygote, the RNA-binding protein MEX-5 forms an anterior-rich cytoplasmic gradient that causes the RNA-binding protein POS-1 to form an opposing, posterior-rich gradient. We demonstrate that the polo-like kinase PLK-1 mediates the repulsive coupling between MEX-5 and POS-1 by increasing the mobility of POS-1 in the anterior. PLK-1 is enriched in the anterior cytoplasm and phosphorylates POS-1, which is both necessary and sufficient to increase POS-1 mobility. Regulation of POS-1 mobility depends on both the interaction between PLK-1 and MEX-5 and between MEX-5 and RNA, suggesting that MEX-5 may recruit PLK-1 to RNA in the anterior. The low concentration of MEX-5/PLK-1 in the posterior cytoplasm provides a permissive environment for the retention of POS-1, which depends on POS-1 RNA binding. Our findings describe a novel reaction/diffusion mechanism in which the asymmetric distribution of cytoplasmic PLK-1 couples two RNA-binding protein gradients, thereby partitioning the cytoplasm.

Multi-omics and temporal dynamics profiling reveal disruption of central metabolism in Helicobacter pylori on bismuth treatment.

Integration of multi-omics enables uncovering cellular responses to stimuli or the mechanism of action of a drug at a system level. Bismuth drugs have long been used for the treatment of Helicobacter pylori infection and their antimicrobial activity was attributed to dysfunction of multiple proteins based on previous proteome-wide studies. Herein, we investigated the response of H. pylori to a bismuth drug at transcriptome and metabolome levels. Our multi-omics data together with bioassays comprehensively reveal the impact of bismuth on a diverse array of intracellular pathways, in particular, disruption of central carbon metabolism is systematically evaluated as a primary bismuth-targeting system in H. pylori. Through temporal dynamics profiling, we demonstrate that bismuth initially perturbs the TCA cycle and then urease activity, followed by the induction of oxidative stress and inhibition of energy production, and in the meantime, induces extensive down-regulation in H. pylori metabolome. The present study thus expands our knowledge on the inhibitory actions of bismuth and provides a novel systematic perspective of H. pylori in response to a clinical drug that sheds light on enhanced therapeutic methodologies.

Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway.

In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5±1.2µM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Synthesis, characterization, in vitro cytotoxicity and anticancer effects of ruthenium(II) complexes on BEL-7402 cells.

Four new ruthenium(II) polypyridyl complexes [Ru(dmb)2(DQTT)](ClO4)2 (1) (DQTT=12-(1,4-dihydroquinoxalin-6-yl)-4,5,9,14-tetraazabenzo[b]triphenylene, dmb=4,4'-dimethyl-2,2'-bipyridine), [Ru(bpy)2(DQTT)](ClO4)2 (2) (bpy=2,2'-bipyridine), [Ru(phen)2(DQTT)](ClO4)2 (3) (phen=1,10-phenanthroline) and [Ru(dmp)2(DQTT)](ClO4)2 (4) (dmp=2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized by elemental analysis, ESI-MS, (1)H NMR and (13)C NMR. The cytotoxic activity in vitro of the complexes was evaluated against human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines by MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) method. The IC50 values of complexes 1-4 against BEL-7402 cells are 31.8 ± 1.0, 35.8 ± 1.6, 29.0 ± 0.8 and 25.0 ± 0.9 µM, respectively. The morphological apoptosis was investigated with AO/EB (acridine orange/ethidium bromide) and Hoechst 33258 staining methods. The DNA damage was assayed by comet assay. The inhibition of cell migration was evaluated by the wound healing assay. The levels of ROS (reactive oxygen species) and the changes of mitochondrial membrane potential were studied under fluorescent microscope. The percentages in the cells of apoptotic and necrotic cells and the cell cycle arrest were determined by flow cytometry. The expression of Bcl-2 family proteins was investigated by western blot analysis. The results show that the complexes induce BEL-7402 cells apoptosis through a ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulation of the expression of Bcl-2 family proteins.

Protein binding and anticancer activity studies of ruthenium(II) polypyridyl complexes toward BEL-7402 cells.

Four new ruthenium(II) polypyridyl complexes [Ru(dmb)2(dqtbt)](ClO4)2 (1) (dqtbt=12-(2,3-diphenyl-quinoxalin-6-yl)-4,5,10,13-tetraazabenzo[b]triphenylene, dmb=4,4'-dimethyl-2,2'-bipyridine), [Ru(bpy)2(dqtbt)](ClO4)2 (2) (bpy=2,2'-bipyridine), [Ru(phen)2(dqtbt)](ClO4)2 (3) (phen=1,10-phenanthroline) and [Ru(dmp)2(dqtbt)](ClO4)2 (4) (dmp=2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized. The cytotoxicity in vitro of the complexes was evaluated against human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. These complexes are sensitive to BEL-7402 cells, the IC50 values are 4.9±0.5, 4.6±0.4, 7.7±1.8 and 1.9±0.3µM toward BEL-7402 cells. The complexes can increase the levels of reactive oxygen species and induce the decrease of mitochondrial membrane potential. Morphological and comet assay studies show that the complexes can effectively induce apoptosis in BEL-7402 cells. Complexes 1-4 inhibit the cell growth at G0/G1 phase in BEL-7402 cell line. The complexes can downregulate the expression of Bcl-2 and Bcl-x proteins and upregulate the levels of Bid protein in BEL-7402 cells. The results show that the complexes induce BEL-7402 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway. In addition, the complexes show strong protein-binding affinities.

The induction of apoptosis in HepG-2 cells by ruthenium(II) complexes through an intrinsic ROS-mediated mitochondrial dysfunction pathway.

Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(dhbn)](ClO4)2 (N-N = dmb: 4,4'-dimethyl-2,2'-bipyridine 1; bpy = 2,2'-bipyridine 2; phen = 1,10-phenanthroline 3; dmp = 2,9-dimethyl-1,10-phenanthroline 4) were synthesized and characterized. The cytotoxicity in vitro of the ligand and complexes toward HepG-2, HeLa, MG-63 and A549 were assayed by MTT method. The IC50 values of the complexes against the above cells range from 17.7 ± 1.1 to 45.1 ± 2.8 µM. The cytotoxic activity of the complexes against HepG-2 cells follows the order of 4 > 2 > 3 > 1. Ligand shows no cytotoxic activity against the selected cell lines. Cellular uptake, apoptosis, comet assay, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest, and the expression of proteins involved in apoptosis pathway induced by the complexes were investigated. The results indicate that complexes 1-4 induce apoptosis in HepG-2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway.