Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

Differential cytotoxic effects ofAnnona squamosa seed extracts on human tumour cell lines: Role of reactive oxygen species and glutathione

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds ofAnnona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis byA. squamosa extracts can be selective for certain types of cancerous cells     

Modulation of genotoxic and cytotoxic effects of aromatic amines in monolayers of rat hepatocytes

Cultured rat hepatocytes exposed to 2-acetylaminofl uorene (AAF), 2-aminofl uorene (AF) or N-hydroxy-2-acetylaminofluorene (N-OH-AFF) for 3 hrs resulted in an increase in DNA repair measured as unscheduled DNA synthesis, with N-OH-AAF > AAF > AF. Cytotoxic effects were only seen with N-OH-AAF above 10^–6 M. -Naphthof avone increased the unscheduled DNA synthesis and cytotoxic effects of N-OH-AAF, whereas it decreased DNA repair and the covalent binding of AAF to cellular proteins. In contrast, very little effects of paraoxon were seen on the repair synthesis elicited by AAF, AF or N-OH-AAF. The addition of ascorbate reduced the covalent binding of AAF, the DNA repair synthesis caused by AAF and N-OH-AAF, and the cytotoxic effects of N-OH-AAF. The addition of pentachlorophenol or salicylamide all resulted in similar effects as ascorbate, through reduction of sulfation. Galactosamine, an inhibitor of glucuronidation, and the nucleophile GSH caused no or only minor effects of the activation of AAF, AF or N-OH-AAF as judged from the endpoints tested. These results are consistent with an arylnitrenium ion, a sulfate ester or a free radical as the arylamine metabolite causing cellular DNA damage, whereas the sulfate ester or a radical intermediate may be responsible for the cytotoxic effects of N-OH-AAF.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate monooxygenase - DMSO dimethyl sulfoxide - HU hydroxyurea - S-9 9000 g supernatants - LDH lactate dehydrogenase - UDS unscheduled DNA synthesis - ANF -naphthoflavone - GSH glutathione - PCP pentachlorophenol - MET metyrapone - PAR paraoxon - DEM dimethylmaleate     

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.     

Pyrimethanil: Between efficient fungicide against Aspergillus rot on cherry tomato and cytotoxic agent on human cell lines

Pyrimethanil, a synthetic fungicide widely used for the treatment of pre‐ and postharvest fungal diseases on different agricultural crops, was explored for its antifungal activity on different fungal strains. The effect of pyrimethanil on fungal ergosterol was tested by using Aspergillus niger as a model organism. Furthermore, it was investigated, if pyrimethanil can effectively reduce the appearance of Aspergillus rot in wounded cherry tomato fruits. The fungicide cytotoxic effect on different human cell lines was evaluated. In addition, its influence on cell proliferation was studied. A. niger was the most resistant fungal strain (MFC 1.88 μg μL^?1) to the effect of pyrimethanil. Addition of ergosterol doubled the MFC on A. niger, indicating that the compound might interfere with ergosterol, main sterol of fungal cell membrane. Disease incidence of A. niger in wounded cherry tomato fruits was not detected with pyrimethanil treatment of 0.75 mg/wound. Some cytotoxic effects of pyrimethanil on human cell lines were recorded already at 50 ng μL^?1, while the expression of Ki67 marker of proliferation was decreased with 150 ng μL^?1. These results altogether indicate that pyrimethanil is effective in reducing various fungal pathogens, but further use of this fungicide should be reevaluated because of its cytotoxicity.     

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N ¹,N ⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N ¹-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N ¹-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines.

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.     

Down-regulation of protein kinase C activity by sorbitol rapidly induces apoptosis in human gastric cancer cell lines

Although apoptosis-dependent involution of malignant tumors is associated with a number of non-surgical treatments including chemotherapy, most solid tumors, including gastric cancers, respond poorly to these therapies. In the hope of overcoming the resistance mechanism against non-surgical therapies, we studied the apoptosis-resistance mechanism in cells of gastric cancers as a model system. During the course of our study on apoptotic machinery in human gastric cancer cell lines, we previously found a rapid and efficient induction of apoptosis by sorbitol. In the present study, we demonstrated that the down-regulation of PKC activity in response to sorbitol is a major factor in the induction of apoptotic cell death in gastric cancer cells.     

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.     

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L -cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G ₀ and G ₂ phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.     

Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.     

Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion

This study evaluated whether cytoskeletal alterations during the ischemic conditions associated with kidney preservation could determine apoptosis. Cytoskeletal alterations are among the main effects of ischemia and may induce apoptosis. Rat kidneys were preserved in University of Wisconsin (UW) solution for 24 h. Some groups of animals underwent 45 min of warm ischemia (WI) to evaluate its effect on both the actin cytoskeleton and apoptosis (assessed by caspase-3 activity and TUNEL staining). Swinholide A (SwinA) and Latrunculin B (LB), two actin cytoskeleton-targeted agents, were administered to assess the effect of direct actin disruption on apoptosis. Jasplakinolide (JP), a compound that stabilizes actin filaments, was administered to evaluate the effect of actin stabilization. Apoptosis was evaluated at 3 h of ex vivo reperfusion using the isolated perfused rat kidney (IPK) model. Results: Apoptosis increased during reperfusion with WI or administration of actin disruptor agents. Administration of stabilizing agents reversed apoptosis in kidneys that had previously undergone WI or had received an actin disruptor agent. Conclusion: The disruption of the actin cytoskeleton during ischemic conditions associated with kidney preservation induces apoptosis upon reperfusion through caspase-3 activation.     

Platelet-endothelial cell adhesion molecule-1 (CD31) recycles and induces cell growth inhibition on human tumor cell lines

CD31 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, and most leukocytes. This report demonstrates by Western Blot and immunofluorescence that some human melanoma and adenocarcinoma cell lines express CD31 on the cell surface. The surface expression of CD31 was regulated by cell-cell contact, being higher on sparse and spontaneously detached cells. Indeed, fixing and permeabilizing tumor cells revealed a cytoplasmic pool, which was confirmed by confocal microscopy. Some of the plasma surface molecule is endocytosed following mAb binding. Engagement of CD31 on tumor cells via domain-3 inhibited proliferation by inducing cell apoptosis. On the other hand, apoptosis does not increase CD31 expression. Overall, these results indicate that there is an intracellular pool of CD31 on some tumor cells, which modulates CD31 surface expression and its role in cancer cell growth and metastasis. Thus, the expression of CD31 and its role in cell survival in some tumor cells appears to differ from endothelial cells.     

Plants from Northwestern Anatolia Display Selective Cytotoxicity and Induce Mitotic Catastrophe: A Study on Anticancer and Genotoxic Activities

Anatolia is rich in floristic diversity with a high rate of endemism. Eight plant species from northwestern Anatolia were evaluated for their anti-growth properties in two malignant (MCF-7 and MDA-MB-231) and a non-malignant (MCF-10A) breast cell lines. The two most active extracts, Achillea multifida (AME) and Astragalus sibthorpianus (ASE), induced apoptotic cell death in all cell lines. The major phenolic compounds in AME were identified as chlorogenic acid, and catechins in ASE. ASE displayed selective cytotoxicity against breast cancer cells, with DNA damage repair in non-malignant cells contributing to its selectivity. Conversely, AME induced DNA damage in a time-dependent manner and displayed a dual dose-dependent biological activity, resulting in mitotic catastrophe and apoptosis at different doses. Most plant species exhibited moderate to strong cytotoxicity, highlighting their medicinal and economic potential and the need for their protection.     

Interferon-gamma induces reactive oxygen species and endoplasmic reticulum stress at the hepatic apoptosis

Interferon-gamma (IFN-gamma) induces cell-cycle arrest and p53-independent apoptosis in primary cultured hepatocytes. However, the detailed mechanism, including regulating molecules, is still unclear. In this study, we found that IFN-gamma induced generation of reactive oxygen species (ROS) in primary hepatocytes and that pyrrolidinedithiocarbamate (PDTC), an anti-oxidant reagent, completely suppressed IFN-gamma-induced hepatic apoptosis. PDTC blocked apoptosis downstream from IRF-1 and upstream from caspase activation, suggesting that the generation of ROS occurred between these stages. However, IFN-gamma also induced the generation of ROS in IRF-1-deficient hepatocytes, cells insensitive to IFN-gamma-induced apoptosis. Moreover, a general cyclooxygenase (COX) inhibitor, indomethacin (but not the cyclooxygenase 2-specific inhibitor, NS-398) also inhibited the apoptosis without blocking the generation of ROS. Both PDTC and indomethacin also blocked IFN-gamma-induced release of cytochrome c from mitochondria. These results suggest that ROS are not the only or sufficient mediators of IFN-gamma-induced hepatic apoptosis. In contrast, we also found that IFN-gamma induced endoplasmic reticulum (ER) stress proteins, CHOP/GADD153 and caspase 12, in wild-type primary hepatocytes, but induced only caspase 12 and not CHOP/GADD153 protein in IRF-1-deficient hepatocytes. These results suggest that IFN-gamma induces ER stress in primary hepatocytes. Both the ROS and ER stress induced by IFN-gamma may be complementary mediators that induce apoptosis in primary hepatocytes.     

Antitumor and cytotoxic effects of Phyllanthus polyphyllus on Ehrlich ascites carcinoma and human cancer cell lines

To evaluate the antitumor and cytotoxic activity of methanol extract of Phyllanthus polyphyllus (MPP) in mice and human cancer cell lines, the antitumor activity of MPP was evaluated against an Ehrlich ascites carcinoma (EAC) tumor model. The activity was assessed using survival time, hematological studies, lipid peroxidation (LPO), antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione S-transferase (GST), solid tumor mass, and short-term in vitro cytotoxicity. The cytotoxic activity of MPP was evaluated using human breast cancer (MCF7), colon cancer (HT29), and liver cancer (HepG2) cell lines Oral administration of MPP (200 and 300 mg/kg) increased the survival time and significantly reduced the solid tumor volume in a dose-dependent manner. Hematological parameters, protein, and packed cellular volume (PCV), which were altered by tumor inoculation, were restored. MPP significantly decreased the levels of LPO, GPx, GST, and significantly increased the levels of SOD and CAT. In a cytotoxicity study against human cancer cell lines, MPP was found to have IC50 values of 27, 42 and 38 microg/ml on MCF-7, HT-29, and HepG2 cells respectively. MPP possessed significant antitumor and cytotoxic activity on EAC and human cancer cell lines.