STAT3-induced NCK1 elevation promotes migration of triple-negative breast cancer cells via regulating ERK1/2 signaling.

BACKGROUND: Noncatalytic region of tyrosine kinase 1 (NCK1) plays a key role in extracellular matrix degradation, which is required for the metastasis of triple-negative breast cancer (TNBC). However, the role NCK1 plays in the metastatic progression of TNBC is unknown. METHODS AND RESULTS: Based on online databases, NCK1 was found to be highly expressed in TNBC as compared to normal breast-like subjects, which was also confirmed using TNBC cells and a tissue microarray. NCK1 expression gradually decreased with increased tumor stage. High NCK1 expression displayed a poor prognosis in lymph node-positive metastatic TNBC patients, but not in lymph node-negative patients. Using transwell assays and immunoblotting, we confirmed that NCK1 overexpression promoted, while NCK1 downregulation inhibited migration capabilities, as well as the expression of matrix metalloproteinases (MMP2/9), uridylyl phosphate adenosine, and plasminogen activator inhibitor-1 in TNBC cells. Mechanistically, NCK1 upregulation mediated the activation of MMP2/9 through ERK1/2 activity. Signal transducer and activator of transcription 3 (STAT3) was positively correlated with NCK1. STAT3 could directly bind to the promoter region of NCK1 to promote its expression and was accompanied by the elevation of MMP2/9 and ERK1/2 signaling, which were partially abolished by the knockdown of NCK1 in TNBC cells. CONCLUSIONS: NCK1 may serve as a diagnostic and prognostic marker of metastatic TNBC. STAT3 upregulation promoted the expression of NCK1, which subsequently induced the migration and activity of MMPs in a ERK1/2 signaling-dependent manner in TNBC cells. NCK1 is a promising target for improving TNBC migration.

Single-crystal structure and intracellular localization of Zn(II)-thiosemicarbazone complex targeting mitochondrial apoptosis pathways.

Tracking of drugs in cancer cells is important for basic biology research and therapeutic applications. Therefore, we designed and synthesised a Zn(II)-thiosemicarbazone complex with photoluminescent property for organelle-specific imaging and anti-cancer proliferation. The Zn(AP44eT)(NO3)2 coordination ratio of metal to ligand was 1:1, which was remarkably superior to 2-((3-aminopyridin-2-yl) methylene)-N, N-diethylhydrazinecarbothioamide (AP44eT·HCl) in many aspects, such as fluorescence and anti-tumour activity. Confocal fluorescence imaging showed that the Zn(AP44eT)(NO3)2 was aggregated in mitochondria. Moreover, Zn(AP44eT)(NO3)2 was more effective than the metal-free AP44eT·HCl in shortening the G2 phase in the MCF-7 cell cycle and promoting apoptosis of cancer cells. Supposedly, the effects of these complexes might be located mainly in the mitochondria and activated caspase-3 and 9 proteins.

Mitochondria-targeted delocalized lipophilic cation complexed with human serum albumin for tumor cell imaging and treatment.

Small molecule 5BMF is a novel mitochondria-targeted delocalized lipophilic cation (DLC) with good anti-tumor activity and fluorescence emission suitable for bioimaging. In this study, human serum albumin (HSA) complexed with 5BMF (5BMF@HSA) has been developed to further improve its solubility (from 1.61 to 5.41 mg/mL), and the fluorescent intensity of 5BMF@HSA was improved over 2 times. Nearly 10-fold 5BMF was released from 5BMF@HSA complex in acidic condition when compared with neutral/basic environment. Intracellular distribution of 5BMF was altered by HSA as its signals were observed in lysosomes where free 5BMF barely localized. Both 5BMF@HSA and 5BMF showed selective toxicity toward tumor cells in µM and nM range and effectively inhibited tumor growth in mice model. In summary, 5BMF@HSA shows improved solubility in aqueous buffer and enhanced fluorescence emission, and maintains tumor inhibition capability. It is a promising protein complex for tumor cell imaging and tumor treatment.

Developing an Anticancer Copper(II) Multitarget Pro-Drug Based on the His146 Residue in the IB Subdomain of Modified Human Serum Albumin.

Designing a multitarget anticancer drug with improved delivery and therapeutic efficiency in vivo presents a great challenge. Thus, we proposed to design an anticancer multitarget metal pro-drug derived from thiosemicarbazone based on the His146 residue in the IB subdomain of palmitic acid (PA)-modified human serum albumin (HSA-PA). The structure-activity relationship of six Cu(II) compounds with 6-methyl-2-formylpyridine-4N-substituted thiosemicarbazones were investigated, and then the multitarget capability of 4b was confirmed in cancer cell DNA and proteins. The structure of the HSA-PA-4b complex (HSA-PA-4b) revealed that 4b is bound to the IB subdomain of modified HSA, and that His146 replaces the nitrate ligand in 4b, coordinating with Cu2+, whereas PA is complexed with the IIA subdomain by its carboxyl forming hydrogen bonds with Lys199 and His242. In vivo data showed that 4b and the HSA-PA-4b complex inhibit lung tumor growth, and the targeting ability and therapeutic efficacy of the PA-modified HSA complex was stronger than 4b alone.

Evaluation of the antibacterial activity of a cationic polymer in aqueous solution with a convenient electrochemical method.

Quaternized chitosan is a cationic biopolymer with good antibacterial activity, biocompatibility, and biodegradability, and it has been widely applied in many fields. We have developed a convenient method to evaluate the antibacterial activity of hydroxypropyltrimethylammonium chloride chitosan (HACC) with a nonionic surfactant poloxamer in aqueous solution by monitoring the change of the oxidation peak current in cyclic voltammetry. Increasing values of the oxidation peak current were positively correlated with the antibacterial activity of HACC-poloxamer solutions. Optical microscope images, the zeta potential, and fluorescence spectroscopy showed that the aggregation state of HACC-poloxamer was related to the ratio of the two polymers and also to the antibacterial activity and oxidation peak current. At an HACC-to-poloxamer ratio of 1:0.75, the maximum surface charge density and the smooth edge of HACC-poloxamer aggregates can accelerate diffusion in aqueous solution. It is expected that this convenient method can be applied for a quick evaluation of the antibacterial activity of cationic biopolymers in aqueous solution. Graphical Abstract The cyclic voltammograms of MB in HACC/poloxamer solution, and the antibacterial efficiency against S. aureus after incubated with HACC (a) and 1/0.75 of HACC/poloxamer (b).

Developing an Anticancer Copper(II) Pro-Drug Based on the His242 Residue of the Human Serum Albumin Carrier IIA Subdomain.

To increase delivery efficiency, anticancer activity, and selectivity of anticancer metal agents in vivo, we proposed to develop the anticancer metal pro-drug based on His242 residue of the human serum albumin (HSA) carrier IIA subdomain. To confirm our hypothesis, we prepared two Cu(II) compounds [Cu(P4 mT)Cl and Cu(Bp44 mT)Cl] by modifying Cu(II) compound ligand structure. Studies with two HSA complex structures revealed that Cu(P4 mT)Cl bound to the HSA subdomain IIA via hydrophobic interactions, but Cu(Bp44 mT)Cl bound to the HSA subdomain IIA via His242 replacement of a Cl atom of Cu(Bp44 mT)Cl, and a coordination to Cu(2+). Furthermore, Cu(II) compounds released from HSA could be regulated at different pHs. In vivo data revealed that the HSA-Cu(Bp44 mT) complex increased copper's selectivity and capacity of inhibiting tumor growth compared to Cu(Bp44 mT)Cl alone.

Multidrug Delivery Systems Based on Human Serum Albumin for Combination Therapy with Three Anticancer Agents.

When administering several anticancer drugs within a single carrier, it is important to regulate their spatial distribution so as to avoid possible mutual interference and to thus enhance the drugs' selectivity and efficiency. To achieve this, we proposed to develop human serum albumin (HSA)-based multidrug delivery systems for combination anticancer therapy. We used three anticancer agents (an organic drug [5-fluorouracil, or 5FU], a metallic agent [2-benzoylpyridine thiosemicarbazide copper II, or BpT], and a gene agent [AS1411]) to treat liver cancer and confirm our hypothesis. The structure of the HSA-palmitic acid (PA)-5FU-BpT complex revealed that 5FU and BpT, respectively, bind to the IB and IIA subdomains of HSA. Our MALDI-TOF-MS spectral data show that one AS1411 molecule is conjugated to Cys-34 of the HSA-5FU-BpT complex via a linker. Compared with unregulated three-drug combination therapy, the HSA-5FU-BpT-AS1411 complex enhances cytotoxicity in Bel-7402 cells approximately 7-fold in vitro; however, in normal cells it does not raise cytotoxicity levels. Importantly, our in vivo results demonstrate that the HSA-5FU-BpT-AS1411 complex is superior to the unregulated three-drug combination in enhancing targeting ability, inhibiting liver tumor growth, and causing fewer side effects.

Developing Anticancer Copper(II) Pro-drugs Based on the Nature of Cancer Cells and the Human Serum Albumin Carrier IIA Subdomain.

To synergistically enhance the selectivity and efficiency of anticancer copper drugs, we proposed and built a model to develop anticancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper(II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1-C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with the Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3-5-fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find that the HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1) and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

Recent progress in thiocarbazone metal complexes for cancer therapy via mitochondrial signalling pathway.

Mitochondria are the energy factories of cells and are important targets for the development of novel tumour treatment strategies owing to their involvement in processes such as apoptosis, oxidative stress, and metabolic programming. Thiosemicarbazone metal complexes target mitochondria and reduce mitochondrial membrane potential. The breakdown of mitochondrial membrane potential is a key event in the early stage of apoptosis, which releases cytochrome C and other pro-apoptotic factors, activates the intracellular apoptotic enzyme cascade, and eventually causes irreversible apoptosis of tumour cells. Thiosemicarbazone metal complexes targeting the mitochondria have recently emerged as potential antitumour agents; therefore, this review describes the structural diversity of thiosemicarbazone metal [Fe(III), Cu(II), Ni(II), Zn(II), Ga(III), Pb(II), Au(III), and Ir(III)] complexes and explores their anti-tumour mechanisms that target mitochondrial pathways.

Long noncoding RNA LINC00885 upregulates NCK1 to promote cell viability and migration of triple-negative breast cancer cells through sponging miR-654-3p.

BACKGROUND: LINC00885 is a novel oncogenic long noncoding RNA (LncRNA) which is upregulated in various types of cancer, but its function in triple-negative breast cancer (TNBC) remains unknown. OBJECTIVE: This study aimed to determine the role of LINC00885 on TNBC development. METHODS: Clinical interrelation and survival analysis were determined using online database. The CCK-8 and Transwell assays were used to detect the proliferation and migration behaviors in TNBC cell lines. The interaction among genes was detected by RNA pull down assay. RESULTS: LncRNA LINC00885 was highly expressed in TNBC compared to normal breast like. Low levels of LINC00885 was related to good prognosis in TNBC patients compared to TNBC patients with high LINC00885. LINC00885-downregulation inhibited, whereas LINC00885-overexpression promoted the proliferation and migration capability of TNBC cell lines. In TNBC cell lines, noncatalytic region of tyrosine kinase 1 (NCK1) expression was positively associated with LINC00885 expression, and shRNA-mediated the depletion of NCK1 significantly abolished LINC00885 upregulation-mediated pro-tumor effects. Combined with online databases, miR-654-3p was screened as the direct target gene of LINC00885, which could directly bind to 3'-untranslated regions (3'-UTR) of NCK1, resulting in the decreased expression of NCK1 in TNBC cell lines. LINC00885 overexpression-mediated the upregulation of NCK1 was abrogated by miR-654-3p mimics. MiR-654-3p mimics significantly rescued the tumor promotive role caused by LINC00885-overexpression. However, exogenous NCK1 notably eliminated the anti-tumor effects caused by miR-654-3p mimics in LINC00885-overexpressed cells. CONCLUSIONS: LINC00885 is expressed at a high level in TNBC. LINC00885 promoted proliferation and migration by regulating the miR-654-3p/NCK1 axis in TNBC cell lines. Possibly, LINC00885 can be served as a potential therapeutic target for TNBC.

Advances in molecular agents targeting toll-like receptor 4 signaling pathways for potential treatment of sepsis.

Sepsis is a systemic inflammatory response syndrome caused by an infection. Toll-like receptor 4 (TLR4) is activated by endogenous molecules released by injured or necrotic tissues. Additionally, TLR4 is remarkably sensitive to infection of various bacteria and can rapidly stimulate host defense responses. The TLR4 signaling pathway plays an important role in sepsis by activating the inflammatory response. Accordingly, as part of efforts to improve the inflammatory response and survival rate of patients with sepsis, several drugs have been developed to regulate the inflammatory signaling pathways mediated by TLR4. Inhibition of TLR4 signal transduction can be directed toward either TLR4 directly or other proteins in the TLR4 signaling pathway. Here, we review the advances in the development of small-molecule agents and peptides targeting regulation of the TLR4 signaling pathway, which are characterized according to their structural characteristics as polyphenols, terpenoids, steroids, antibiotics, anthraquinones, inorganic compounds, and others. Therefore, regulating the expression of the TLR4 signaling pathway and modulating its effects has broad prospects as a target for the treatment of lung, liver, kidneys, and other important organs injury in sepsis.

Comparative evaluation of the structure and antitumor mechanism of mononuclear and trinucleated thiosemicarbazone Cu(II) complexes.

The ratio of ligand to Cu(II) ions has an essential effect on the geometrical configuration and anti-tumour activity of metal-based complexes. In this work, we synthesised two Cu(II) thiosemicarbazone complexes, namely, [Cu(L)(Cl)] (C1) and [Cu3(L)2(Cl)4] (C2), by controlling the ratio of Cu(II) ion to ligand, to evaluate their anti-tumour activity. The ability of C1 to catalyze hydrogen peroxide to produce reactive oxygen species (ROS) was significantly higher than that of Cu(II) ion. Moreover, the bridge of Cu(II) and two molecules generated a new complex (C2), which, in contrast to C1, enhanced the generation of Fenton-like-triggered ROS. Consequently, the produced ROS depleted reduced glutathione, caused oxidative cell stress and promoted apoptosis through mitochondrial apoptotic pathways. In addition, C2 exhibited better tumour suppression than C1 in a nude mouse tumour xenograft model.

Advances in thiosemicarbazone metal complexes as anti-lung cancer agents.

The great success of cisplatin as a chemotherapeutic agent considerably increased research efforts in inorganic biochemistry to identify more metallic drugs having the potential of treating lung cancer. Metal coordination centres, which exhibit a wide range of coordination numbers and geometries, various oxidised and reduced states and the inherent ligand properties offer pharmaceutical chemists a plethora of drug structures. Owing to the presence of C=N and C=S bonds in a thiosemicarbazone Schiff base, N and S atoms in its hybrid orbital has lone pair of electrons, which can generate metal complexes with different stabilities with most metal elements under certain conditions. Such ligands and complexes play key roles in the treatment of anti-lung cancer. Research regarding metallic anti-lung cancer has advanced considerably, but there remain several challenges. In this review, we discuss the potential of thiosemicarbazone Schiff base complexes as anti-lung cancer drugs, their anti-cancer activities and the most likely action mechanisms involving the recent families of copper, nickel, platinum, ruthenium and other complexes.

Pyridoxal hydrochloride thiosemicarbazones with copper ions inhibit cell division via Topo-I and Topo-IIɑ.

Topoisomerase (Topo) accelerates cell growth and division, and has been a theoretical target for anti-cancer drugs for decades. A series of pyridoxal thiosemicarbazone (PLT) ligands were designed and synthesized, and the dependence of their antiproliferative activity on copper was investigated. The insertion of N-cyclohexyl-2-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methylene)-N-methylhydrazinecarbothioamide hydrochloride (compound 9) and Chlorido(N-cyclohexyl-2-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methylene)-N-methylhydrazinecarbothioamide hydrochloride-O,N,S)­copper(II) nitrate (9-Cu complex) into Topo-I and Topo-II prevented uncoiling of DNA through hydrogen bonds and intermolecular forces. The combination of PLT derivatives and copper gluconate (CuGlu) improved their anti-tumour activity against a cell line with high expression of topoisomerase (SK-BR-3). The non-linear regression equations of the inhibitory activity and anti-tumour activity of Topo-I and Topo-IIɑ in SK-BR-3 cells had R2 values of 0.93 and 0.94, respectively. In addition to lipophilicity, inhibition of topoisomerase also affected the activity of PLT ligands by coordinating with copper ions. At the cellular level, PLTs and CuGlu penetrate the cell membrane to form metabolites in the cell, thus selectively inhibiting the activity of Topo-I and Topo-IIɑ, and ultimately inhibiting cell division. These findings will inform the design of future anti-cancer thiosemicarbazone drugs.

Mechanism of vitamin B6 benzoyl hydrazone platinum(II) complexes overcomes multidrug resistance in lung cancer.

To overcome the resistance of tumour cells to cis-diaminedichloroplatinum(II) (cisplatin, DDP), we designed and synthesised platinum(II) complexes with copper coordination active sites using vitamin B6 and benzohydrazide derivatives as raw materials.The 3D structures of the complexes were confirmed by X-ray single-crystal diffraction. The results of the biological activity assay showed that the Pt(II) complexes (VB6-Pt1 and VB6-Pt2) have higher anti-tumour activity on detected typical lung cancer cells than DDP. Among them, VB6-Pt1 (IC50 = 0.78 µM) efficiently reversed DDP resistance in A549/DDP cell line and increased selectivity index (26) against mortal MRC-5 fibroblasts. The study showed that VB6-Pt1 overcomes tumor drug resistance by significantly increasing the level of reactive oxyge species and inducing lysosomal membrane permeability, which leads to mitochondrial dysfunction and cell apoptosis. Furthermore, the inhibitory effect of VB6-Pt1 on A549 xenograft tumours was 81.5%, which was much higher than that of cisplatin (50.0%), without significantly increasing p-glycoprotein (P-gp) protein expression. The copper-coordinated active site in Pt(II) complexes may be a key factor in their ability to overcome DDP-resistant cancer cells.

Antioxidative and immunological effects of Cyclocarya paliurus polysaccharides on the spleen injury of diabetic rat.

OBJEVTIVE: To investigate the effects of Cyclocarya paliurus (C. paliurus) polysaccharides on the spleen injury of diabetic rats. METHODS: Animals were divided into 6 groups, including normal group, model group, control group, low-dose group of C. paliurus polysaccharides treatment, middle-dose group of C. paliurus polysaccharides treatment and high-dose group of C. paliurus polysaccharides treatment. Histological analysis of spleen was analyzed using hematoxilin and eosin. Levels of biological parameters and anti-oxidative enzymes were determined by spectrophotometry. Interleukin-7 (IL-7) and IL-10 were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with that of model group, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase level increased 78.63% (P < 0.05), 51.76% (P < 0.05), 2.95 times (P < 0.01) and 41.11% (P < 0.05) in the high-dose group of C. paliurus polysaccharides treatment, respectively. IL-7 and IL-10 increase 1.66 (P < 0.01) and 1.21 times (P < 0.01) in the high-dose group of C. paliurus polysaccharides treatment, respectively. CONCLUSION: It is suggested that C. paliurus polysaccharides may play a protecting role for spleen injury of diabetic rats by enhancing the antioxidative ability and evaluating the immunity.

Characterization of Anoectochilus roxburghii polysaccharide and its therapeutic effect on type 2 diabetic mice.

Anoectochilus roxburghii is a traditional herb in China that can be potentially used to treat diabetes. A novel polysaccharide ARLP-W was isolated from Anoectochilus roxburghii by chromatography on DEAE-52 cellulose. Chemical analysis indicated that ARLP-W (8.1 × 104 Da) was mainly composed of mannose and glucose. The main linkages of glycosidic bonds of ARLP-W were ß-1, 4-Manp and α-1, 4-Glcp. The terminal Glcp was connected to Manp-via O-3. RT-qPCR and western blotting analysis showed that ARLP-W caused a significant reduction in the levels of the key gluconeogenesis enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver. The results of the insulin resistance tests indicated that ARLP-W increased glucose absorption. These results indicate that ARLP-W has a good therapeutic effect on type 2 diabetes and can assist with further development and application treatment of diabetes.

Synthesis, crystal structure and antiproliferative mechanisms of gallium(iii) complexes with benzoylpyridine thiosemicarbazones.

We have prepared six thiosemicarbazone ligands and synthesized the corresponding Ga(iii) complexes. The antitumor activity of the ligand increases with its lipophilicity, and the antitumor activity of the Ga(iii) complexes is affected by the ligands. Since C6 has the highest anticancer proliferative activity (0.14 ± 0.01 µM) against HepG-2 (Human hepatocarcinoma cell line), we characterized its structure by X-ray single crystal diffraction and explored its antiproliferation mechanism. Anti-tumor mechanism results show that Ga(iii) complex (C6) promoted HepG-2 cell cycle arrest in the G1 phase by regulating the expression of cell cycle-associated proteins (Cdk 2, cyclin A and cyclin E). Ga(iii) complex (C6) promotes apoptosis by consuming intracellular iron, enhancing intracellular reactive oxygen species (ROS), activating caspase-3/9, releasing cytochromes and apoptotic protease activating factor-1 (apaf-1).

Molecular characterization of AwSox2 from bivalve Anodonta woodiana: Elucidating its player in the immune response.

Sox2 is an embryonal stem cell Ag essential for early embryonic development, tissue homeostasis and immune regulation. In the current study, one complete Sox2 cDNA sequence was cloned from freshwater bivalve Anodonta woodiana and named AwSox2. Histological changes of testis derived from Bisphenol A (BPA) treatment were analyzed by hematoxylin and eosin staining. Expressions of AwSox2 derived from BPA, LPS and polyinosinic:polycytidylic (Poly I:C) challenge were measured by quantitative real-time PCR. The full-length cDNA of AwSox2 contained an open reading frame of 927 nucleotides bearing the typical structural features of Sox2 family. Obvious degeneration, irregular arrangement of spermatids, and clotted dead and intertwined spermatids were observed in BPA-treated groups. Administration of BPA could result in a dose-dependent up-regulation of AwSox2 expression in the male gonadal tissue of A. woodiana. In addition, expression of AwSox2 was significantly induced by LPS and Poly I:C treatment in the hepatopancreas, gill and hemocytes, compared with that of control group. These results indicated that up-regulations of AwSOx2 are closely related to apoptosis of spermatogonial stem cells derived from BPA treatment as well as enhancement of immune defense against LPS and Poly I:C challenge in A. woodiana.

Structure-activity relationships of 2­quinolinecarboxaldehyde thiosemicarbazone gallium(III) complexes with potent and selective anticancer activity.

Six gallium(III) complexes (Ga1-Ga6) with 2­quinolinecarboxaldehyde thiosemicarbazone analogues were synthesized and characterized. These gallium(III) complexes exhibited potent anticancer activity and exceeded that of the corresponding metal free ligands. Importantly, these gallium(III) complexes have a strong selectivity for tumor cells. Through the study of cellular mechanisms, we have found that the lipophilicity of ligands is closely linked to the antitumor activity of gallium(III) complexes. Additionally, we have chosen Ga6 with the best anti-tumor activity to study the mechanism of apoptosis. Caspase-3 and 9 activation and Annexin V-FITC/Propidium iodide (PI) dual-staining studies revealed that Ga6 promote apoptosis in A549 cells lines. Ga6 induces intracellular reactive oxygen species (ROS) and disrupts mitochondrial membrane potential.