Downregulation Of Mitochondrial Membrane Potential Research Articles

Synthesis, Characterization and Biological Evaluation of New Enamide Fluorinated‐Schiff Base Derivatives as Potential Cytotoxic and Apoptosis‐Inducing Agents

AbstractIn search for new anticancer candidates, a new series of enamide fluorinated‐Schiff base derivatives were synthesized and confirmed by spectroscopic techniques as well as elemental microanalyses. The newly prepared compounds were evaluated for their cytotoxic activity on MCF‐7 breast adenocarcinoma cell line. The benzaldehyde Schiff base and furfural Schiff base derivatives (IC50=3.67±0.15 and 2.06±0.08 μM, respectively) were found to be the most potent molecules of all the tested derivatives against the MCF‐7 cancer cells. Additionally, the furfural Schiff base compound was more potent than colchicine by 1.74‐fold, while benzaldehyde Schiff base compound was nearly equipotent with colchicine as standard drug. In addition to tubulin inhibition activity, enamide‐furfural Schiff base molecule caused cell cycle arrest in MCF‐7 cells at the G2/M phase (2.62‐fold more than control MCF‐7) and induced apoptotic death (48.97‐fold compared to untreated MCF‐7 control) as indicated by FACS analysis. Furthermore, apoptotic studies conducted by enamide‐furfural Schiff base derivative were accomplished by downregulation of mitochondrial membrane potential by 1.56‐fold relative to control untreated cells.

Selective pks+ Escherichia coli strains induce cell cycle arrest and apoptosis in colon cancer cell line.

pks+ Escherichia coli (E. coli) triggers genomic instability in normal colon cells which leads to colorectal cancer (CRC) tumorigenesis. Previously, we reported a significant presentation of pks+ E. coli strains in CRC patients' biopsies as compared to healthy cohorts. In this work, using anin vitro infection model, we further explored the ability of these strains in modulating cell cycle arrest and activation of apoptotic mediators in both primary colon epithelial cells (PCE) and CRC cells (HCT-116). Sixteen strains, of which eight tumours and the matching non-malignant tissues, respectively, from eight pks+ E. coli CRC patients were subjected to BrDU staining and cell cycle analysis via flow cytometry, while a subset of these strains underwent analysis of apoptotic mediators including caspase proteins, cellular reactive oxygen species (cROS) and mitochondrial membrane potential (MMP) via spectrophotometry as well as proinflammatory cytokines via flow cytometry. Data revealed that all strains exerted S-phase cell cycle blockade in both cells and G2/M phase in PCE cells only. Moreover, more significant upregulation of Caspase 9, cROS, proinflammatory cytokines and prominent downregulation of MMP were detected in HCT-116 cells indicating the potential role of pks related bacterial toxin as anticancer agent as compared to PCE cells which undergo cellular senescence leading to cell death without apparent upregulation of apoptotic mediators. These findings suggest the existence of discrepancies underlying the mechanism of action of pks+ E. coli on both cancer and normal cell lines. This work propounds the rationale to further understand the mechanism underlying pks+ E. coli-mediated CRC tumorigenesis and cancer killing.

Polyphyllin I Inhibits Cisplatin Resistance of Cervical Cancer Cells by Inducing Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

Resistance of cervical cancer to cisplatin chemotherapy compromises the therapeutic efficacy of the treatment. Polyphyllin I, a bioactive compound extracted from rhizoma of Paris polyphyllin, displays a selective inhibitory effect on a variety of tumor cells. Furthermore, it potentiates the chemosensitivity of cisplatin against cisplatin resistant gastric cancer cells and lung adenocarcinoma. However, its role in modulating chemosensitivity of cisplatin resistant cervical cancer cells remains unexplored. To this end, the repressive effect of polyphyllin I on proliferation, invasion and migration of cisplatin resistant cervical cancer cells compared to the parental cervical cancer cells as well as downregulation of mitochondrial membrane potential of cisplatin resistant cervical cancer cells, as well as upregulation of protein kinase R-like ER kinase/translation initiation factor 2-alpha kinase 3, protein-kinase and site-specific endoribonuclease/inositol-requiring enzyme 1, binding immunoglobulin protein, and C/EBP homologous transcription factor proteins. In conclusion, polyphyllin I stimulate mitochondrial dysfunction and endoplasmic reticulum stress to attenuate cisplatin resistance of cervical cancer.

Nuclear factor erythroid 2-related factor 2 protects bovine mammary epithelial cells against free fatty acid-induced mitochondrial dysfunction in vitro

Bovine mammary epithelial cells undergo an increase in metabolic rate, mitochondrial dysfunction, and oxidative stress after calving. Nuclear factor erythroid 2-related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays crucial roles in the regulation of mitochondrial function. The objective of this study was to investigate the role of NFE2L2 on mitochondrial function in bovine mammary epithelial cells under hyperlipidemic conditions. Three experiments were conducted as follows: (1) the immortalized bovine mammary epithelial cell line MAC-T was treated with various concentrations of free fatty acids (FFA; 0, 0.6, 1.2, or 2.4 mM) for 24 h to induce stress; (2) MAC-T cells were transfected with small interfering RNA targeting NFE2L2 (si-NFE2L2) and scrambled nontarget negative control (si-Control) for 48 h; and (3) MAC-T cells were pretreated with 10 μM sulforaphane (SFN), an activator of NFE2L2, for 24 h followed by treatment with 1.2 mM FFA for an additional 24 h. Results indicated that exogenous FFA challenge induced linear and quadratic increases in concentrations of mitochondrial reactive oxygen species (ROS). Compared with 0 mM FFA, mitochondrial membrane potential, mRNA abundance of oxidative phosphorylation complexes (CO I-V), protein abundance of nuclear respiratory factor 1 (NRF1), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), mitochondrial transcription factor A (TFAM), and NFE2L2 along with the contents of ATP, mitochondrial DNA (mtDNA), and total mitochondria were greater in the MAC-T challenged with 0.6 mM FFA group, but lower in the 1.2 and 2.4 mM FFA cultures. Knockdown of NFE2L2 via small interfering RNA led to greater mitochondrial ROS content and lower mitochondrial membrane potential along with contents of ATP, mtDNA, and total mitochondria. The SFN pretreatment upregulated protein abundance of NFE2L2 and attenuated the downregulation of NFE2L2 induced by FFA. Pretreatment with SFN attenuated the downregulation induced by FFA of PGC-1α, NRF1, and TFAM protein abundance along with contents of mtDNA and total mitochondria. Furthermore, SFN pretreatment attenuated the upregulation of mitochondrial ROS content, the downregulation of mitochondrial membrane potential, and the decreases in ATP, mtDNA, and mitochondrial content induced by FFA. Overall, data indicated that FFA inhibit NFE2L2, resulting in mitochondrial dysfunction and ROS production in bovine mammary epithelial cells. Thus, NFE2L2 may be a promising therapeutic target against metabolic challenge-driven mitochondrial dysfunction and oxidative stress in bovine mammary epithelial cells.

Selenite induced breast cancer MCF7 cells apoptosis through endoplasmic reticulum stress and oxidative stress pathway

Selenium is an essential trace element for human, and has anti-tumor effects. In this study, we investigated the anti-tumor activity of sodium selenite (Na2SeO3) and explored its possible mechanisms involved in a breast cancer cell line. We found that Na2SeO3 could inhibit the cell viability of MCF7 cells, yet with minimal damage to human umbilical vein endothelial cells (HUVECs). The results of Hoechst staining and Western Blot showed that Na2SeO3 induced apoptosis of MCF7 cells. Na2SeO3 activated endoplasmic reticulum stress (ERS), as evidenced by the up-regulation of ERS-related proteins, including ATF6, p-eIF2α, ATF4, and CHOP, and the down-regulation of PERK. ATF6, p-eIF2α and apoptosis were decreased by pre-treatment with an ERS inhibitor (4-PBA). Na2SeO3 activated oxidative stress (OS) through increasing ROS generation and decreasing mitochondrial membrane potential (MMP) which induced apoptosis. Pre-treatment with an antioxidant (NAC) attenuated Na2SeO3-induced OS and cell apoptosis. Furthermore, ERS and OS had mutual effects. Pre-treatment with 4-PBA could act against the up-regulation of ROS and the down-regulation of MMP. Pre-treatment with NAC attenuated the expression of ATF6. At the same time, we found that treatment with Na2SeO3 promoted the phosphorylation of p38 and JNK, while inhibiting the phosphorylation of ERK. However, the up-regulation was inhibited after pre-treatment of NAC, and pre-treatment with 4-PBA inhibited the increase only of p38. Based on these results, our study provides a mechanistic understanding of how Na2SeO3 has antitumor effects against MCF7 cells through the OS and ERS pathway. OS and ERS interact with each other, and p38 is regulated by them.

Biological evaluation of mitochondria targeting small molecules as potent anticancer drugs

Cancer therapy targets specific metabolic pathways or a single gene. This may result in low therapeutic effects due to drug selectivity and drug resistance. Recent studies revealed that the mitochondrial membrane potential and transmembrane permeability of cancerous mitochondria are differed from normal mitochondria. Thus, chemotherapy targeting cancerous mitochondria could be an innovative and competent strategy for cancer therapy. Previously, our work with a novel group of mitochondria targeting small molecules presented promising inhibitory capability toward various cancer cell lines and suppressed adenosine triphosphate (ATP) generation. Therefore, it is critical to understand the anticancer effect and targeting mechanism of these small molecules. This study investigated the inhibitory activity of mitochondria targeting small molecules with human cervical cancer cells – HeLa to further explore their therapeutic potential. HeLa cells were exposed to 10 µM of synthesized compounds and presented elevation in intracellular reactive oxygen species (ROS) level, impaired mitochondrial membrane potential and upregulation of apoptosis as well as necrosis. In vivo, HeLa cell tumor-bearing BALB/c nude mice were treated with mitochondria targeting small molecules for 12 days consecutively. Throughout this chemotherapy study, no deleterious side effects nor the appearance of toxicity was observed. Furthermore, mitochondria targeting small molecules treated groups exhibited significant down-regulation with both tumor volume and tumor weight compared to the Doxorubicin (DOX) treated group. Thus, inhibition of mitochondrial ATP synthesis, activation of intracellular ROS production, down-regulation of mitochondrial membrane potential and upregulation of apoptosis and necrosis rates are the indications of cancer therapy. In this work, we examined the anticancer capability of four mitochondria targeting small molecules in vitro and in vivo, and demonstrated a novel therapeutic approach in cancer therapy with tremendous potential.

Inhibitory effects of 5-heptadecylresorcinol on the proliferation of human MCF-7 breast cancer cells through modulating PI3K/Akt/mTOR pathway

Abstract The effect of 5-heptadecylresorcinol (AR-C17) on the proliferation of human MCF-7 breast cancer cells was investigated in the present study. The results showed that AR-C17 treatment significantly suppressed MCF-7 cells proliferation, trigged intracellular ROS generation and decreased SOD2 expression. Furthermore, AR-C17 treatment could lead to MCF-7 cells apoptosis as evidenced by the activation of Bax/Bcl-2 expression ratio, active caspase activity and downregulation of mitochondrial membrane potential. Meanwhile, AR-C17 could activate MCF-7 cells autophagy by upregulating the protein levels of Atg7, Beclin-1 and LC3-Ⅱ as well as decreasing SQSTM1/p62 and increasing autophagosomes formation in cytoplasm. Inhibition of autophagy with chloroquine (CQ) enhanced AR-C17-induced cell apoptosis. In addition, AR-C17 exerted anti-cancer activity through modulating PI3K/Akt/mTOR signaling pathway which was evidenced by decreasing the protein expression of PI3K, p-Akt and p-mTOR. Co-treatment with PI3K agonist IGF-1 could alleviate the inhibitory effect of AR-C17. Taken together, AR-C17 could be used as potential nutraceutical for breast cancer management.

Apoptosis induction in K562 human myelogenous leukaemia cells is connected to the modulation of Wnt/β-catenin signalling by BHX, a novel pyrazoline derivative.

The goal of this study was to explore the effects of BHX on human chronic myeloid leukaemia (CML) cells and to elucidate the underlying molecular mechanism. CML cell line K562 cells were treated with BHX. The effects of BHX on cell proliferation, apoptosis and cell cycle were detected. Subsequently, the caspase, ATP activity, Ca2+ , ROS and mitochondrial membrane potential (MMP) levels treated with various concentrations of BHX were analysed. The variation of relevant proteins and genes was detected. Further, toxicity of BHX on peripheral blood cells, bone marrow-nucleated cells (BMNC) and organ index were investigated on mice. Results showed that BHX suppressed K562 cell proliferation in a dose-dependent manner and induced apoptosis and G0/G1 phase arrest. BHX induced mitochondria-mediated apoptosis, which was associated with downregulation of MMP, activation of caspase-3 and caspase-9, generation of intracellular ROS and elevation of Ca2+ in K562 cells. In treated cells, ATP levels were decreased, expression of total β-catenin, phosphorylated β-catenin and β-catenin in the nucleus was decreased, and expression of cell cycle-related proteins was decreased. Further analysis revealed that BHX lowered the transcriptional level of β-catenin. Lastly, BHX treatment significantly reduced the number of white blood cells, but had no effect on BMNC and organ index. These findings provide further insight into the potential use of BHX as an anti-cancer agent against human leukaemia.

Typhonium giganteum Lectin Exerts A Pro-Inflammatory Effect on RAW 264.7 via ROS and The NF-κB Signaling Pathway.

Typhonii rhizoma, a widely used herb in traditional Chinese medicine, has acute irritating toxicity related to Typhonium giganteum lectin (TGL). TGL exhibits acute inflammatory effects, but the underlying molecular mechanisms are largely unknown. This paper is designed to assess the pro-inflammatory response of TGL on RAW 264.7 cells. RAW 264.7 treated with 6.25, 12.5, 25, and 50 µg/mL TGL showed elevated levels of inflammatory factors (TNF-α, IL-1β) and of p-IκB and p-p65, all dose-dependent, indicating that TGL had a substantial inflammatory effect and mobilized the nuclear factor-κB (NF-κB) pathway. All four TGL treatments also induced the up-regulation of reactive oxygen species (ROS) and cytosolic free Ca2+ and down-regulation of mitochondrial membrane potential (MMP). The production of cytokines and p-IκB, p-p65 were reduced by N-acetylcysteine (NAC), an ROS scavenger, which somewhat abrogated ROS production. The results showed the TGL-activated inflammatory signaling pathway NF-κB to be associated with the overproduction of ROS. Moreover, 50 μg/mL treatment with TGL led to cell apoptosis after 1 h and increased necrosis over time. These results provided potential molecular mechanisms for the observed inflammatory response to TGL including up-regulation of ROS and cytosolic free Ca2+, down-regulation of MMP, the mobilization of the NF-κB pathway, and the subsequent overproduction of pro-inflammatory factors resulting in apoptosis. Long-term stimulation with TGL resulted in strong toxic effects related to inflammation that induced necrosis in macrophages.

Antiproliferative effect of sweet orange peel and its bioactive compounds against human hepatoma cells, in vitro and in vivo

The ethyl acetate extracts from sweet orange peel (EESP) and its bioactive compounds against human hepatoma cells, in vitro and in vivo, is investigated. EESP (50–500µg/ml) exhibits significant cytotoxicity on HepG2 cells. This antiproliferative action is partly attributable to cell cycle arrest in the G0/G1 phase and apoptosis induction. EESP induces apoptosis through an increase in the Bax/Bcl-2 ratio, the down-regulation of mitochondrial membrane potential and the up-regulation of caspase-3. Excessive ROS generated by EESP contributes to apoptosis of HepG2 cells. According to the HPLC-DAD and HPLC-MS/MS analysis, nobiletin, tangeretin and another twelve compounds were present in EESP, which may account for its antiproliferative effect. When HepG2 cells are grown as xenografts in nude mice, the oral administration of EESP induces a significant decrease in tumor growth. Collectively, these results suggest that EESP could be a potential useful source for the future application of EESP in liver cancer therapy.

Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450.

In current study, we investigated the anti-tumor effect of luteolin in human ESCC cell lines in vitro and in vivo and tried to explore the potential mechanisms. Results from flow cytometry showed that luteolin could induce apoptosis and caspase-3 activation and induce cell cycle arrest at G2/M phase in a dose- and time-dependent manner in EC1 and KYSE450 cells. JC-1 test results showed that membrane potential of mitochondria after luteolin treatment was down-regulated and this was an indicator for intrinsic apoptosis. Western Blot results showed the expression of cell cycle regulatory protein p21 and p53 increased and three apoptosis related proteins that participate in mitochondrial apoptotic pathway, namely, Bim, CYT-c and cPARP, also increased in luteolin treated cells compared with control groups. We further confirmed that luteolin could significantly inhibit the growth of ESCC tumors in xenograft mouse models and no evidence of systemic toxicity was observed. Our results suggest that luteolin can induce cell apoptosis and cell cycle arrest in G2/M phase through mitochondrial pathway in EC1 and KYSE450 cell lines and proper utilization of luteolin might be a practical approach in ESCC chemotherapy.

Bacterial-excreted small volatile molecule 2-aminoacetophenone induces oxidative stress and apoptosis in murine skeletal muscle.

Oxidative stress induces mitochondrial dysfunction and facilitates apoptosis, tissue damage or metabolic alterations following infection. We have previously discovered that the Pseudomonas aeruginosa (PA) quorum sensing (QS)-excreted small volatile molecule, 2-aminoacetophenone (2-AA), which is produced in infected human tissue, promotes bacterial phenotypes that favor chronic infection, while also compromising muscle function and dampens the pathogen-induced innate immune response, promoting host tolerance to infection. In this study, murine whole-genome expression data have demonstrated that 2-AA affects the expression of genes involved in reactive oxygen species (ROS) homeostasis, thus producing an oxidative stress signature in skeletal muscle. The results of the present study demonstrated that the expression levels of genes involved in apoptosis signaling pathways were upregulated in the skeletal muscle of 2-AA-treated mice. To confirm the results of our transcriptome analysis, we used a novel high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) method and observed increased levels of bisallylic methylene fatty acyl protons and vinyl protons, suggesting that 2-AA induces skeletal muscle cell apoptosis. This effect was corroborated by our results demonstrating the downregulation of mitochondrial membrane potential in vivo in response to 2-AA. The findings of the present study indicate that the bacterial infochemical, 2-AA, disrupts mitochondrial functions by inducing oxidative stress and apoptosis signaling and likely promotes skeletal muscle dysfunction, which may favor chronic/persistent infection.

Abstract 2808: Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450

Abstract Esophageal squamous carcinoma (ESCC) may be developed through a progressive sequence from mild to severe dysplasia, carcinoma in situ, and finally, invasive carcinoma. Chemoprevention can block or weaken the influence of development. Recent study has shown luteolin, a bioflavonoid, possesses anti-inflammatory, antioxidant, and anti-proliferative effects, and it might have a preventive effect in this progress. In this study, we focused on the effect of luteolin on cell cycle regulation in human Esophageal Squamous Carcinoma Cell Line EC1 and KYSE450 Cells in vitro and its potential mechanisms. Observations by flow cytometer showed that luteolin inhibited cell cycle progression at G2/M phase in a dose- and time-dependent manner. We also found that luteolin could induce cell apoptosis via decreasing activation of caspase-3 and down-regulation of mitochondrial membrane potential. Western blot results showed the protein expression of cycle related protein CyclinD1 and apoptosis related proteins caspase-3, caspase-9, and Bak were also significantly decreased in luteolin treated cells compared with the non-luteolin treated cells. Our results suggest that the proper use of luteolin might be a practical approach to the prevention of esophageal carcinoma via the inhibition of cell proliferation and other potential high risk regions for this disease. Citation Format: Ping Chen, Tao Hu, Yane Ma, Xiaoyu Chen, Liping Dai, Ningjing Lei, Ziming Dong, Pei Li. Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2015-2808

Inhibitory effects of 3-bromopyruvate in human nasopharyngeal carcinoma cells.

Tumor cells depend on aerobic glycolysis for adenosine triphosphate (ATP) production, which is therefore targeted by therapeutic agents. The compound 3-bromopyruvate (3-BrPA), a strong alkylating agent and hexokinase inhibitor, inhibits tumor cell glycolysis and the production of ATP, causing apoptosis. 3-BrPA induces apoptosis of nasopharyngeal carcinoma (NPC) cell lines HNE1 and CNE-2Z, which may be related to its molecular mechanisms. In the present study, we investigated the effects of 3-BrPA on the viability, reactive oxygen species (ROS), apoptosis and other types of programmed cell death in NPC cells in vitro and in vivo. PI staining showed significant apoptosis in NPC cells accompanied by the overproduction of ROS and downregulation of mitochondrial membrane potential (MMP, ΔΨm) by 3-BrPA. However, the ROS scavenger N-acetyl-L-cysteine (NAC) significantly reduced 3-BrPA-induced apoptosis by decreasing ROS and facilitating the recovery of MMP. We elucidated the molecular mechanisms underlying 3-BrPA activity and found that it caused mitochondrial dysfunction and ROS production, leading to necroptosis of NPC cells. We investigated the effects of the caspase inhibitor z-VAD-fmk, which inhibits apoptosis but promotes death domain receptor (DR)-induced NPC cell necrosis. Necrostatin-1 (Nec-1) inhibits necroptosis, apparently via a DR signaling pathway and thus abrogates the effects of z-VAD‑fmk. In addition, we demonstrated the effective attenuation of 3-BrPA-induced necrotic cell death by Nec-1. Finally, animal studies proved that 3-BrPA exhibited significant antitumor activity in nude mice. The present study is the first demonstration of 3-BrPA-induced non-apoptotic necroptosis and ROS generation in NPC cells and provides potential strategies for developing agents against apoptosis‑resistant cancers.

Protective Effect of Arctigenin against MPP+ and MPTP-induced Neurotoxicity

The potential protective effects of arctigenin on 1-methyl-4-phenylpyridinium ion and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride-induced neurotoxicity were examined, and the results indicated that arctigenin could improve the movement behaviors and upregulate dopamine and γ-aminobutyric acid levels in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyride-induced neurotoxicity mouse model. A further in vitro experiment showed that the pretreatment with arctigenin on cultured human neuroblastoma SH-SY5Y cells could obviously attenuate the decrease of cell survival rates caused by treatment with 1-methyl-4-phenylpyridinium ion by way of acting against cell apoptosis through the decrease of Bax/Bcl-2 and caspase-3, and by antioxidative action through reduction of the surplus reactive oxygen species production and downregulation of mitochondrial membrane potential. It is for the first time that a neuroprotective activity of arctigenin in both in vitro and in vivo experiments was reported, enlightening that arctigenin could be useful as a potential therapeutic agent for Parkinson's disease.

Artesunate induces apoptosis and inhibits growth of Eca109 and Ec9706 human esophageal cancer cell lines in vitro and in vivo.

Esophageal cancer is a common malignant tumor worldwide with a high incidence rate in China and it is a great threat to human health. Combined modality therapy is used for chemotherapeutic treatment of esophageal cancer; however, drug resistance and side effects of the drugs is a major barrier to the success of chemotherapy. As chemotherapy with common drugs is far from providing satisfactory clinical outcomes for patients with esophageal cancer, more efficient drugs are urgently required. Artesunate (Art) is the first-line treatment option for malaria; however, it was recently revealed that Art has remarkable anti-tumor activity, making it a novel candidate for cancer chemotherapy. Although the anti-cancer effects of Art have been well documented, its potential against esophageal cancer has rarely been explored. The present study aimed to investigate the significance and mechanism of the anti-proliferative activity of Art on esophageal cancer cells in vitro and in vivo. In the in vitro experiments, Art inhibited the growth as well as induced cell apoptosis and cell cycle arrest of esophageal cancer cell lines (Eca109 and Ec9706) in a concentration-dependent manner. Furthermore, downregulation of mitochondrial membrane potential, B-cell lymphoma-2 (BCL-2) and CDC25A, as well as upregulation of BCL-2‑associated X protein (Bax) and caspase-3 expression in Art-treated cells were identified. In addition, an in vivo study showed that Art produced a dose-dependent tumor regression in nude mice, while side effects were low. The anti-tumor activity of 200 mg/kg Art was similar to that of 3 mg/kg cisplatin. In conclusion, Art exerted concentration-dependent inhibitory activity against esophageal cancer in vivo and in vitro by inducing cell apoptosis and cell cycle arrest through affecting mitochondrial membrane potential, BCL-2, Bax, caspase-3 and CDC25A.

Hydroxyl Radical (·OH) Played a Pivotal Role in Oridonin-Induced Apoptosis and Autophagy in Human Epidermoid Carcinoma A431 Cells

Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.

Molecular mechanism study of chemosensitization of doxorubicin-resistant human myelogenous leukemia cells induced by a composite polymer micelle

The present study was aimed to overcome the multidrug resistance (MDR) of tumor cells which accounts for the failure of clinical chemotherapy. A novel doxorubicin (DOX)-loaded composite micelle consisting of polyethylene glycol (PEG)–polycaprolactone (PCL)/Pluronic P105 has been developed and was proved to inhibit the drug resistance of human myelogenous leukemia (K562/ADR) cells. The modulation mechanism that DOX-loaded the composite micelle inhibited MDR was for the first time investigated at cell levels. Results indicated that the cytotoxicity in K562/ADR cells treated by DOX-loaded PEG–PCL/P105 composite micelle was about 4 times higher than DOX solution at 12 μg/mL of DOX. Confocal images showed that the DOX-loaded composite micelles gradually entered into cytoplasm and nucleus, and stayed in intracellular much longer than DOX solution. All the micelles (PEG–PCL micelle, P105 micelle and PEG–PCL/P105 composite micelle) did not change Pgp expression on the surface of K562/ADR cells. However, further study revealed that micelle containing of P105 (P105 or PEG–PCL/P105 composite micelle) significantly decreased ATP level, and consequently restricted the activity of Pgp by down-regulation of mitochondrial membrane potential. On the other hand, the PEG–PCL micelle had no effect on both mitochondrial membrane potential and ATP level of the K562/ADR cells, but its access to K562/ADR cells through endocytic pathway avoided the recognition of Pgp. The PEG–PCL/P105 composite micelle was designed based on the combination of P105-mediated down regulation of mitochondrial membrane potential the malignant cells and PEG–PCL-mediated internalization effect. Therefore, the novel composite micelle is a promising drug delivery system for anticancer drug to overcome MDR.

The involvement of ROS overproduction and mitochondrial dysfunction in PBDE-47-induced apoptosis on Jurkat cells

2,2′,4,4′-Tetrabromodiphenyl ether (PBDE-47), as one of the congeners of polybrominated diphenyl ethers (PBDEs), is widely present and threatens the human health in many aspects. This study aims to investigate the toxic effects of PBDE-47 on cell viability, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of Jurkat cells in vitro. The results showed that PBDE-47 significantly inhibited the viability of Jurkat cells in a dose-dependent manner by alamar blue assay. Significant induction of apoptosis was detected in Jurkat cells at 25–100 μM by propidium iodide staining, accompanied with overproduction of ROS and downregulation of MMP. Furthermore, N-acetyl-L-cysteine (NAC), a widely used ROS scavenger, significantly reduced the PBDE-47-induced apoptosis by decreasing ROS level and mediating recovery of the MMP. In conclusion, the results of this study suggest that PBDE-47 could induce apoptosis in Jurkat cells and ROS and mitochondrial dysfunction play important roles in the apoptotic process.

Anti‐oxidants reverse uraemia‐induced down‐regulation of mitochondrial membrane potential and interleukin‐10 production

The anti-inflammatory cytokine, interleukin-10 (IL-10), has potent immunomodulatory effects. We hypothesized that previously reported defective synthesis of IL-10 by immunocompetent cells exposed to a uraemic milieu may be due to impaired mitochondrial membrane potential (MMP). The human promonocytic THP-1 cell line was differentiated to monocytes and incubated with pooled control or uraemic plasma with and without catalase or N-acetyl L-cysteine (NAC). Basal hydrogen peroxide (H(2)O(2)) production was measured by flow cytometry. To measure MMP, cells were stained with rhodamine 123 (Rh123) and the uptake of Rh123 assessed by flow cytometry. To assess the relative contribution of the NADPH oxidase and mitochondrial electron transport chain (ELT) to endotoxin (ET)-stimulated IL-10 production among monocytic cells, cells were incubated with and without a selective NADPH oxidase inhibitor, apocynin and mitochondrial ELT inhibitors, diphenyliodinium and rotenone, washed and ET-stimulated IL-10 production was measured. In other experiments, cells were incubated with pooled control or uraemic plasma in the presence or absence of antioxidants followed by overnight incubation with ET. IL-10 production by monocytes in the cell supernatant was then quantified. Basal H(2)O(2) production was significantly higher among differentiated THP-1 cells exposed to uraemic plasma compared with normal plasma (180.57 +/- 10.24 vs. 41.57 +/- 8.98 MCI; P = 0.02). Uraemic plasma also down regulated MMP (4.60 +/- 1.28 vs. 8.00 +/- 1.59 MCI with normal plasma; P = 0.03). Both diphenyliodinium and rotenone, selective inhibitors of the mitochondrial ELT, inhibited ET-stimulated IL-10 production. In contrast, apocynin, a selective NADPH oxidase inhibitor, did not inhibit ET-stimulated IL-10 production. Further, ET-stimulated IL-10 production by cells incubated with uraemic plasma was significantly lower when compared to cells exposed to normal plasma. Pre-incubation with catalase and NAC restored uraemia-induced down regulation of MMP. In addition, ET-stimulated IL-10 production by cells incubated with uraemic plasma was also restored by both catalase and NAC. Our observations suggest that ET-stimulated IL-10 synthesis by monocytic cells is mitochondrial ELT-dependent and NADPH oxidase-independent. Monocytic cells exposed to a uraemic environment exhibit higher basal ROS production, lower MMP, and impaired ET-stimulated IL-10 synthesis. Anti-oxidants restore MMP and up-regulate ET-stimulated IL-10 synthesis.