Pennogenyl Saponins from Paris quadrifolia L. Induce Extrinsic and Intrinsic Pathway of Apoptosis in Human Cervical Cancer HeLa Cells

Pennogenyl saponins are the active compounds of large number of plant species and consequently many polyherbal formulations. Hence, great interest has been shown in their characterization and in the investigation of their pharmacological and biological properties, especially anticancer. This present study reports on the evaluation of cytotoxic effects and explanation of the molecular mechanisms of action of the two pennogenyl saponins (PS 1 and PS 2) isolated from Paris quadrifolia L. rhizomes on human cervical adenocarcinoma cell line HeLa. To determine the viability of the cells treated with the compounds we used real-time cell proliferation analysis and found that the pennogenyl saponins PS 1 and PS 2 strongly inhibited the tumor cells growth with IC50 values of 1.11 ± 0.04 μg/ml and 0.87 ± 0.05 μg/ml, respectively. The flow cytometry analysis indicated that the two compounds induced apoptosis in a dose-dependent manner and decreased mitochondrial membrane potential in HeLa cells in the early stage of apoptosis. Quantitative PCR and Western Blot analysis showed that the two saponins significantly increased mRNA expression of FADD and BID as well as induced caspase-8 via increased of procaspase-8 processing in the treated cells. The results of this study suggest that both the extrinsic death receptor and intrinsic mitochondrial pathways are involved in the programmed cell death.     

Biochemical characterization of recombinant methionine aminopeptidases (MAPs) from Mycobacterium tuberculosis H37Rv

The aim of this study was to investigate the effect of dihydrotanshinone I (DI) in inhibiting the growth of human cervical cancer cells both in vitro and in vivo, and molecular targets in HeLa cells when treated by DI or irradiation with or without being combined. In this study, MTT, clonogenic assay, flow cytometry, and Western blotting were performed to assess the effect of treatment on cells. After treatment with IR, DI, and DI + IR, the apoptosis was 5.8, 13.3 and 22.5% (P < 0.05 vs. control), respectively. Clonogenic assay revealed that the survival of irradiated HeLa cell was significantly reduced by DI treatment. Combination treatment with IR and DI could down-regulate HPV E6 gene expression. Effect of DI on up-regulation of p21 expression and down-regulation of cyclin B1, p34(cdc2) expression in irradiated HeLa cell was concomitant with cell cycle arrest in G(2) phase. The significant increase in caspase-3 activity was also observed in the combination treatment. When HeLa cells were grown as xenografts in nude mice, combination treatment with DI and IR induced a significant decrease in tumor growth, and without signs of general or organ toxicity. These data suggest DI should be tested as the radiosensitizer in vitro and in vivo, which has potential in the treatment of human cervical cancer.     

The influence of saponins on cell membrane cholesterol

We studied the influence of structurally different saponins on the cholesterol content of cellular membranes. Therefore a cell culture model using ECV-304 urinary bladder carcinoma cells was developed. To measure the cholesterol content we used radiolabeled ³H-cholesterol which is chemically and physiologically identical to natural cholesterol. The cells were pre-incubated with ³H-cholesterol and after a medium change, they were treated with saponins to assess a saponin-induced cholesterol liberation from the cell membrane. In another experiment the cells were pre-incubated with saponins and after a medium change, they were treated with ³H-cholesterol to assess a saponin-induced inhibition of cholesterol uptake into the cell membrane. Furthermore, the membrane toxicity of all applied saponins was analyzed using extracellular LDH quantification and the general cytotoxicity was analyzed using a colorimetric MTT-assay and DNA quantification. Our results revealed a correlation between membrane toxicity and general cytotoxicity. We also compared the results from the experiments on the saponin-induced cholesterol liberation as well as the saponin-induced inhibition of cholesterol uptake with the membrane toxicity. A significant reduction in the cell membrane cholesterol content was noted for those saponins who showed membrane toxicity (IC₅₀ <60 μM). These potent membrane toxic saponins either liberated ³H-cholesterol from intact cell membranes or blocked the integration of supplemented ³H-cholesterol into the cell membrane. Saponins with little influence on the cell membrane (IC₅₀ >100 μM) insignificantly altered the cell membrane cholesterol content. The results suggested that the general cytotoxicity of saponins is mainly dependent on their membrane toxicity and that the membrane toxicity might be caused by the loss of cholesterol from the cell membrane.We also analyzed the influence of a significantly membrane toxic saponin on the cholesterol content of intracellular membranes such as those of endosomes and lysosomes. In these experiments ECV-304 cells were either incubated with ³H-cholesterol or with ³H-cholesterol and 5 μM saponin. After isolation of the endosomes/lysosomes their ³H-cholesterol content was measured. A significant influence of the saponins on the cholesterol content of endosomal/lysosomal membranes was not detected.     

Volatile organic compound specific detection by electrochemical signals using a cell-based sensor

A cell-based in vitro exposure system was developed to determine whether oxidative stress plays a role in the cytotoxic effects of volatile organic compounds (VOCs) such as benzene, toluene, xylene, and chlorobenzene, using human epithelial HeLa cells. Thin films based on cysteine-terminated synthetic oligopeptides were fabricated for immobilization of the HeLa cells on a gold (Au) substrate. In addition, an immobilized cell-based sensor was applied to the electrochemical detection of the VOCs. Layer formation and immobilization of the cells were investigated with surface plasmon resonance (SPR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The adhered living cells were exposed to VOCs; this caused a change in the SPR angle and the VOC-specific electrochemical signal. In addition, VOC toxicity was found to correlate with the degree of nitric oxide (NO) generation and EIS. The primary reason for the marked increase in impedance was the change of aqueous electrolyte composition as a result of cell responses. The p53 and NF-kappaB downregulation were closely related to the magnitude of growth inhibition associated with increasing concentrations of each VOC. Therefore, the proposed cell immobilization method, using a self-assembly technique and VOC-specific electrochemical signals, can be applied to construct a cell microarray for onsite VOC monitoring.     

Binding and cytotoxicity of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and erythrocytes

The binding of Ricinus communis lectins to HeLa cells, Sarcoma 180 ascites tumor cells and human erythrocytes was studied in detail. Scatchard plots of binding of 125I-lectins to these cells gave biphasic lines except for HeLa cells at 0 degree C. The association constants of lectins for the three cell types at 37 degrees C were lower than those at 0 degree C. The numbers of total binding sites were estimated to be 7 to 16 X 10(7) per HeLa cell, 3 to 4 X 10(7) per Sarcoma 180 ascites tumor cell and 0.4 to 1 X 10(6) per erythrocyte. A fraction, 16 to 27% of the total amount of cell-bound lectin at 37 degrees C, appeared to be bound irreversibly as judged by non-removal on washing with 0.1 M lactose, whereas no lectin was irreversibly bound at 0 degree C. In the case of erythrocytes, no lectin became irreversibly bound even at 37 degrees C. The toxicity of lectins on HeLa cells and Sarcoma 180 ascites tumor cells was investigated. The toxicity of ricin D was 50 times for Sarcoma 180 ascites tumor cells and 140 times for HeLa cells as much as that for castor bean hemagglutinin. As to the sensitivities of both cell types to these lectins, it became apparent that Sarcoma 180 ascites tumor cells were more susceptible than HeLa cells.     

ODN 491, a novel antisense oligodeoxynucleotide that targets thymidylate synthase, exerts cell-specific effects in human tumor cell lines

Thymidylate synthase (TS) is essential for DNA replication and is a target for cancer chemotherapy. However, toxicity to normal cells and tumor cell drug resistance necessitate development of new therapeutic strategies. One such strategy is to use antisense (AS) technology to reduce TS mRNA and protein levels in treated cells. We have developed oligodeoxynucleotides (ODNs) that target different regions of TS mRNA, inhibit human tumor cell proliferation as single agents, and enhance cytotoxicity of clinically useful TS protein-targeting drugs. Here we describe ODN 491, a novel 20mer AS ODN complementary to a previously untargeted portion of the TS mRNA coding region. AS ODN 491 decreased TS mRNA levels to different degrees in a panel of human tumor-derived cell lines, and induced different physiological effects in a tumor cell line-dependent manner. ODN 491 (like AS TS ODN 83, previously shown to be effective) decreased TS protein levels in HeLa cells with a concomitant increase in sensitivity to TS-targeting chemotherapeutics. However (and contrary to HeLa cell response to an AS ODN 83), it did not, as a single agent, inhibit HeLa cell proliferation. In MCF-7 cells, ODN 491 treatment was less effective at reducing TS mRNA and did not reduce TS protein, nor did it enhance sensitivity to TS-targeting or other chemotherapeutics. Moreover, specifically in MCF-7 cells but not HeLa cells, ODN 491 as a single agent induced apoptosis. These data indicate that AS TS ODN 491 is an effective AS reagent targeting a novel TS mRNA region. However, treatment of tumor cell lines with AS TS ODNs targeting different TS mRNA regions results in a pattern of physiological effects that varies in a tumor cell line-specific fashion. In addition, the capacity of different AS TS ODNs to induce physiological effects does not correlate well with their capacity to reduce TS mRNA and/or protein and, further, depends on the region of TS mRNA selected for targeting. Recognition of tumor cell-specific and mRNA region-specific variability in response to AS TS ODNs will be important in designing AS TS ODNs for potential clinical use.     

Antitumor effect of F-PBF(beta-TrCP)-induced targeted PTTG1 degradation in HeLa cells

Pituitary tumor-transforming gene 1 (PTTG1), a proto-oncogene, is associated with tumor formation, proliferation and invasiveness. F-PBF(beta-TrCP), a fusion protein, was produced by replacing the WD40-repeat of F-box protein beta-TrCP with the PTTG1-binding factor (PBF) for targeted degradation of PTTG1. To evaluate the function of F-PBF(beta-TrCP), PTTG1-EGFP fusion protein was constructed. Our results showed that F-PBF(beta-TrCP) can both degrade exogenous PTTG1-EGFP fusion protein in COS-7 cells and endogenous PTTG1 protein in HeLa cells and the targeted PTTG1 knock down resulted in bFGF mRNA level down-regulation and inhibition of proliferation and clonogenicity in HeLa cells. In conclusion, targeted degradation of PTTG1 by F-PBF(beta-TrCP) has antitumor activity in vitro in HeLa cells. These results suggest that F-PBF(beta-TrCP) could be used for cancer treatment by targeted degradation of PTTG1.     

Real-time monitoring of cisplatin-induced cell death

Since the discovery of cisplatin more than 40 years ago and its clinical introduction in the 1970s an enormous amount of research has gone into elucidating the mechanism of action of cisplatin on tumor cells. With a novel cell biosensor chip system allowing continuous monitoring of respiration, glycolysis, and impedance we followed cisplatin treatment of different cancer cell lines in real-time. Our measurements reveal a first effect on respiration, in all cisplatin treated cell lines, followed with a significant delay by interference with glycolysis in HT-29, HCT-116, HepG2, and MCF-7 cells but not in the cisplatin-resistant cell line MDA-MB-231. Most strikingly, cell death started in all cisplatin-sensitive cell lines within 8 to 11 h of treatment, indicating a clear time frame from exposure, first response to cisplatin lesions, to cell fate decision. The time points of most significant changes were selected for more detailed analysis of cisplatin response in the breast cancer cell line MCF-7. Phosphorylation of selected signal transduction mediators connected with cellular proliferation, as well as changes in gene expression, were analyzed in samples obtained directly from sensor chips at the time points when changes in glycolysis and impedance occurred. Our online cell biosensor measurements reveal for the first time the time scale of metabolic response until onset of cell death under cisplatin treatment, which is in good agreement with models of p53-mediated cell fate decision.     

麻疹病毒沪191对HeLa肿瘤细胞抑制作用的体内外实验研究

研究麻疹病毒减毒疫苗沪191株(MV沪191)在组织培养中和裸鼠体内对HeLa肿瘤细胞的抑制作用.用空斑实验测定MV沪191感染HeLa细胞后细胞裂解液中病毒量;用MTF试验测定MV沪191感染对细胞活性的影响;用流式细胞仪分析测定MV沪191感染引起的细胞凋亡和对细胞周期的影响;HeLa肿瘤细胞背部皮下接种BALB/C裸鼠引起的肿瘤,评估MV沪191体内抑瘤作用.MV沪191感染HeLa细胞后可引起广泛的CPE,感染的HeLa细胞与对照组相比细胞活性明显降低.MV沪191感染HeLa细胞后随着时间延长,G1/G0细胞率明显增多,S期率明显减少,细胞凋亡率明显增加(P＜0.01).给药第60天时瘤内治疗组、静脉治疗组和对照组肿瘤体积平均分别为15.5、64.6、156.4 mm3.瘤内治疗组与对照组相比有显著差异(P＜0.01);静脉治疗组与对照组相比有明显差异(P＜0.05).MV沪191减毒株在组织培养中和裸鼠体内对HeLa肿瘤有明显的杀伤作用.     

Microelectronic-sensing assay to detect presence of Verotoxins in human faecal samples

Aims: To develop a novel Vero cell assay that implements a real‐time cell electronic sensing (RT‐CES) system for the determination of the presence of verotoxin‐producing Escherichia coli (VTEC). The assay overcomes the major drawbacks in conventional Vero cell assay, for example, labour‐intensive and time‐consuming. Methods and Results: Cells were grown onto the surfaces of microelectronic sensors that are integrated into the bottom surfaces of the microtiter plate. Cellular viability was monitored in real‐time and quantified based on changes in the sensor’s electrical impedance. For cell viability measurement, the data generated on the RT‐CES system correlated well with those obtained by the Vero cell assay for Verotoxins. To assess cytotoxicity, test cells growing on microelectronic sensors were treated with either supernatant from pure cultures, or stool samples. The specific neutralizing antibodies of VT1 and VT2 were used to identify specific toxins in the samples. Conclusions: The RT‐CES assay provides a sensitive measurement comparable to conventional crystal violet assay. The assay has been successfully and specifically used to identify VTEC in human faecal samples. Significance and Impact of the Study: The RT‐CES assay significantly shortens the testing time from 48 to 72 h required by the crystal violet assay to only 15 h with automated operation.     

Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells

We found that benomyl, a benzimidazole fungicide, strongly suppressed the reassembly of cold-depolymerized spindle microtubules in HeLa cells. Benomyl perturbed microtubule-kinetochore attachment and chromosome alignment at the metaphase plate. Benomyl also significantly decreased the distance between the sister kinetochore pairs in metaphase cells and increased the level of the checkpoint protein BubR1 at the kinetochore region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed the effects of benomyl on the signal transduction pathways in relation to mitotic block, bcl2 phosphorylation and induction of apoptosis. The results suggest that benomyl causes loss of tension across the kinetochores, blocks the cell cycle progression at mitosis and subsequently, induces apoptosis through the bcl2-bax pathway in a manner qualitatively similar to the powerful microtubule targeted anticancer drugs like the vinca alkaloids and paclitaxel. Considering the very high toxicity of the potent anticancer drugs and the low toxicity of benomyl in humans, we suggest that benomyl could be useful as an adjuvant in combination with the powerful anticancer drugs in cancer therapy.     

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.     

State of nucleolar proteins B23/nucleophosmin and UBF in HeLa cells during apoptosis induced by tumor necrosis factor

The structural state of two major nucleolar proteins, UBF and B23/nucleophosmin (both monomeric and oligomeric forms), was for the first time established in HeLa cells treated with apoptosis inducers: tumor necrosis factor (TNF-alpha), emetine, and their combination. The treatment of the cells with either TNF-alpha or emetine did not induce apoptosis and affect the state of UBF and nucleophosmin (both monomers and oligomers). Apoptosis was rather pronounced only if HeLa cells were treated with a mixture of TNF-alpha and emetine. States of the UBF and B23 proteins were analyzed in samples containing 25, 45, and 100% of cells with apoptotic nuclei. It was shown by immunoblotting that TNF-alpha-induced apoptosis of HeLa cells was associated with proteolysis of UBF and production of a 76-kD fragment, the content of which increased in correlation with the fraction of apoptotically changed cells. The N- and C-terminal amino acid sequences of UBF and its 76-kD fragment were characterized, and the site of the apoptosis-induced specific proteolysis was identified. As differentiated from UBF, protein B23 did not undergo proteolytic degradation during the TNF-alpha-induced apoptosis of HeLa cells and its content was unchanged even in the cell fraction with fragmentation of virtually all nuclei. However, the ratio between the monomeric and oligomeric states of B23 protein was changed in apoptotic cells, and apoptosis-specific forms of nucleophosmin were detected.     

High spatial resolution impedance measurement of EIS sensors for light addressable cell adhesion monitoring

In this paper, impedance measurement of electrolyte-insulator-semiconductor (EIS) structure with high spatial resolution was proposed to monitor cell adhesion. The light addressing ability of this work overcomes the geometrical restrict of cell culture on conventional impedance detection devices such as interdigitated electrode (IDE) and electric cell-substrate impedance sensing (ECIS). Instead of studying cells on predetermined sites of IDE and ECIS, cells cultured anywhere on EIS sensor surface can be addressed and selected as target cells. Principle and primary models for high resolution impedance detection were described and tested by experiments. The EIS sensor was investigated in terms of its intrinsic characteristics, like impedance behavior, voltage characteristic, frequency dependency and photovoltaic effect. Optimized working condition was studied for cell experiments. Cell adhesion under treatment of 0.1% Triton X-100 was monitored using rat kidney cells as the source. Results showed good sensitivity (10% change of impedance) and resolution (40 μm) for cell adhesion impedance detection and suggested this work should be suitable for monitoring cell impedance. Further improvements on sensitivity, spatial resolution were discussed as well as the further applications for single cell monitoring and cell adhesion imaging.     

Activation of AMPK is necessary for killing cancer cells and sparing cardiac cells

ErbB2 targeted therapies represent an attractive strategy in breast cancer. Herceptin, an anti-ErbB2 monoclonal antibody, is an approved treatment for patients with ErbB2-overexpressing breast cancers. ErbB2 signaling can also be blocked using small molecule tyrosine kinase inhibitors, like Lapatinib, that compete with ATP for binding at the ErbB2 catalytic kinase domain. The principal adverse event attributable to Herceptin is cardiac toxicity. Data from clinical trials show that, unlike Herceptin, Lapatinib may have reduced cardiac toxicity. This study was conducted to elucidate pathways which may contribute to cardiac toxicity or survival using Lapatinib and Herceptin. Our results show that treatments directed to ErbB1/2 receptors using GW-2974 (a generic ErbB1/2 inhibitor) activated AMPK, a key regulator in mitochondrial energy production pathways in human cardiac cells and cancer cells. Although Herceptin down-regulates tumor survival pathways, AMPK fails to be activated in tumor and cardiac cells. When treated in combination with TNF-α, a known cytokine associated with cardiac toxicity, GW-2974 protected cardiac cells from cell death whereas Herceptin contributed to TNF-α-induced cellular killing. Since activity of AMPK in cardiac cells is associated with stress induced survival in response to cytokines or energy depletion, cardiac toxicity by Herceptin may be a consequence of failure to induce stress-related survival mechanisms. Thus, the ability to activate AMPK after treatment with tyrosine kinase inhibitors may be a crucial factor for increased efficacy against the tumor and decreased risk of cardiomyopathy.     

Synthesis and cytotoxic activity of diosgenyl saponin analogues

Diosgenyl saponins are steroidal glycosides that are often found as major components in many traditional oriental medicines. Recently, a number of naturally occurring diosgenyl saponins have been shown to exert cytotoxic activity against several strains of human cancer cells. Use of these saponin compounds for cancer treatment is hampered due to the lack of understanding of their action mechanism as well as limited access to such structurally complicated molecules. In the present paper, we have prepared a group of diosgenyl saponin analogues which contain a beta-D-2-amino-2-deoxy-glucopyranose residue having different substituents at the amino group. Moderate cytotoxic activity is found for most analogues against neuroblastoma (SK-N-SH) cells, breast cancer (MCF-7) cells, and cervical cancer (HeLa) cells. The analogue 13 that contains an alpha-lipoic acid residue exhibits the highest potency against all three cancer cell lines with IC(50) ranging from 4.8 microM in SK-N-SH cells to 7.3 microM in HeLa cells. Preliminary mechanistic investigation with one saponin analogue (10) shows that the compound induces cell cycle arrest at G(1) phase in SK-N-SH cells, but the same compound induces cell cycle arrest at G(2) phase in MCF-7 cells. This result suggests that the cytotoxic activity of these saponin analogues may involve different action mechanisms in cell lines derived from different cancer sites.     

Bacterially-Derived Nanocells for Tumor-Targeted Delivery of Chemotherapeutics and Cell Cycle Inhibitors

Chemotherapeutic drug therapy in cancer is seriously hampered by severe toxicity primarily due to indiscriminate drug distribution and consequent collateral damage to normal cells. Molecularly targeted drugs such as cell cycle inhibitors are being developed to achieve a higher degree of tumor cell specificity and reduce toxic side effects. Unfortunately, relative to the cytotoxics, many of the molecularly targeted drugs are less potent and the target protein is expressed only at certain stages of the cell cycle thus necessitating regimens like continuous infusion therapy to arrest a significant number of tumor cells in a heterogeneous tumor mass. Here we discuss targeted drug delivery nanovectors and a recently reported bacterially-derived 400nm sized minicell that can be packaged with therapeutically significant concentrations of chemotherapeutic drugs, targeted to tumor cell surface receptors and effect intracellular drug delivery with highly significant anti-tumor effects in-vivo. We also report that molecularly targeted drugs can also be packaged in minicells and targeted to tumor cells with highly significant tumor growth-inhibition and regression in mouse xenografts despite administration of minute amounts of drug. This targeted intracellular drug delivery may overcome many of the hurdles associated with the delivery of cytotoxic and molecularly targeted drugs.     

Advanced electrochemical sensors for cell cancer monitoring

The possibility of using minimally invasive analytical instruments to monitor cancerous cells and their interactions with analytes provide great advances in cancer research and toxicology. The real success in the development of a reliable sensor for cell monitoring depends on the ability to design powerful instrumentation that will facilitate efficient signal transduction from the biological process that occurs in the cellular environment. The resulting sensor should not affect cell viability and must function as well as adapt the system to the specific conditions imposed by the cell culture. Due to their performance, electrochemical biosensors could be used as an effective instrument in cell cancer research for studying biochemical processes, cancer development and progression as well as toxicity monitoring. Current research in this direction is conducted through high-throughput, compact, portable, and easy to use sensors that enable measurement of cells' activity in their optimum environment. This paper discusses the potential of a high-throughput electrochemical multisensor system, so-called the DOX system for monitoring cancerous cells and their interaction with chemical toxins. We describe the methodology, experiments, and the operation principle of this device, and we focus on the challenges encountered in optimizing and adapting the system to the specific cell-culture conditions. The DOX system is also compared with conventional cell-culture techniques.