Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.     

Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells

Polyclonal antibodies against plasminogen activator inhibitor type-I (PAI-1) caused rapid retraction and rounding of substrate-attached HT- 1080 cells. The kinetics and extent of antibody-mediated cell rounding were not dependent on either urokinase or plasmin activity. Cells adherent to vitronectin-coated substrates detached within 2 h of antibody addition. Cells adherent to fibronectin were unaffected by the antibodies. Immunoblotting of substrate-attached material indicated that HT-1080 cells deposited PAI-1 into vitronectin, but not fibronectin, dependent contacts. These data suggest that the antibody- mediated cell rounding resulted from a steric disruption of vitronectin- dependent adhesions, indicating that the binding site on vitronectin for PAI-1 is near, but does not overlap, the binding site for vitronectin receptor. The accumulation of PAI-1 into vitronectin- dependent adhesion sites correlated temporally with the preferential degradation of fibronectin from the substrate. HT-1080 cells adherent to either fibronectin or vitronectin were able to activate exogenous plasminogen to plasmin. Plasmin levels were increased 200% on cells adherent to fibronectin and 100% on cells adherent to vitronectin. In the presence of a neutralizing antibody against PAI-1, vitronectin adherent cells activated plasminogen to the same extent as fibronectin adherent cells. Plasmin levels of 200% above baseline were associated with retraction of cells from the substrate. The ability of vitronectin adherent cells to activate exogenous plasmin was completely blocked in the presence of neutralizing antibodies against urokinase. These data represent the first demonstration that vitronectin-associated PAI-1 regulates urokinase in focal contact areas.     

Changes in cAMP and fibronectin concentrations in adherent and suspension forms of a hybrid cell line

The coexistence of adherent (PCM3-M) and suspension (PCM3-S) cells in a hybrid cell line led to the investigation of changes in cellular adhesion. The clonal origin, stable karyotype and subculturing properties of this hybrid indicate that these cells represent different forms of an identical population. Time-lapse photography and mitotic studies signify that the PCM3-S cells were found in the mitotic phase of the cell cycle, while incorporation of [³H]thymidine into DNA by both PCM3-M and PCM3-S cells suggested that the adherent cells detach during the S phase. This was substantiated by inducing quiescence of the culture during the G2 phase, since a preferential increase in the suspension was subsequently observed. Thus the cell cycle studies are consistent with the proposal that floating cells are discernable in a culture of adherent cells because the point at which they round up for division occurs earlier in the cell cycle. Prostaglandin E₁ (2.8 μM) and 3-isobutyl-1-methylxanthine (0.1 mM) were shown to maximally stimulate cAMP production and incubating the hybrid in these agents dramatically decreased the percentage of floating cells, slowed the growth rate and increased the adhesion of the PCM3-M cells. However, determination of intracellular concentrations of cAMP indicated that the floating cells were not a manifestation of decreased cAMP concentrations. In addition, the cell surface glycoprotein, fibronectin, was found to be present on the PCM3-M cells but lost from the PCM3-S cells. Addition of extracted fibronectin to these floating cells resulted in a culture consisting exclusively of adherent PCM3-M cells, suggesting that an aberration in fibronectin-associated cell-substratum adhesion is responsible for the unusual growth of the hybrid.     

Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity

It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported here demonstrate that cell death can be prevented if 50 micro M CdSO(4) is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment. The post-cadmium recovery phase was characterized by DNA repair at the level of 50-200 kb and increased telomerase activity. Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal. Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis. Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress.     

Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content

BACKGROUND: Induction of apoptosis in adherent cell lines is associated with cell loss from the substratum. In this study the adenocarcinoma cell line, HT29, treated with indomethacin (400microM) has been employed as a model system to demonstrate how flow cytometric analysis can be used to quantify the changes that occur during this process. METHODS: Adherent and floating cell populations have been analyzed independently for effects on cell number, cell cycle characteristics and apoptosis using TUNEL assay and Annexin V binding. In addition apoptosis has been assessed using DNA laddering and morphology. RESULTS: Apoptosis was detected in adherent cells treated with indomethacin using Annexin V binding but not by other techniques employed in this study. In contrast, analysis of "floating" cells revealed the presence of apoptotic cells both in control and indomethacin treated cells using all the techniques employed. However quantification by flow cytometry showed that a significantly higher proportion of control "floaters" were late apoptotic/necrotic rather than apoptotic. DISCUSSION: The data here illustrate the need to interpret measures of apoptosis in adherent cell lines with care and the value of using flow cytometric techniques in the quantitative evaluation of the process.     

Sodium butyrate induces apoptosis and accumulation of ubiquitinated proteins in human breast carcinoma cells

To investigate the possible relationship between apoptosis and the ubiquitin pathway we examined the patterns of ubiquitinated proteins in the human breast carcinoma MCF-7 cell line following induction of apoptotic death by sodium butyrate. Apoptosis in these cells was associated with internucleosomal DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase. By dual in situ antiubiquitin immunofluorescence and chromatin DNA staining, we demonstrated that ubiquitin fluorescence was increased specifically in cells that underwent sodium butyrate-mediated apoptosis. The extent of ubiquitin incorporation into protein conjugates was examined in both adherent (not yet apoptotic) and floating (apoptotic) cell populations. We found that apoptotic cells exhibited enhanced intensity of ubiquitin-immunoreactive conjugates, whereas adherent cells did not. In addition, two-dimensional immunoblot analysis of proteins from apoptotic cells identified a set of isomeric ubiquitinated conjugates located at a pI range of 4. 2 - 4.6 and a Mr approximately of 30 kDa. These data indicate that the ubiquitin pathway may play a role in the sodium butyrate-induced apoptotic program in breast carcinoma cells.     

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.     

Chemoprevention of Skin Cancer with 1,1-Bis (3′-Indolyl)-1-(Aromatic) Methane Analog through Induction of the Orphan Nuclear Receptor,NR4A2 (Nurr1)

Background

The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.

Methods

In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.

Results

The IC50 values of DIM-D were 68.7±7.3, 48.3±10.1 and 11.5±3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1±8.3, 186.1±5.2 and 56.7±3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.

Conclusion

Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.     

Taxol induced Bcl-2 protein phosphorylation in human hepatocellular carcinoma QGY-7703 cell line

Bcl-2 family proteins play a critical role in the regulation of apoptosis. Treatment of a human hepatocellular carcinoma cell line, QGY-7703, with Taxol induced apoptosis and Bcl-2 protein phosphorylation. Microscopic observation indicated that apoptotic bodies (0-15%) of Taxol-treated QGY cells appeared after 12 h of treatment, and apoptotic QGY cells gradually increased to 40% after 24 h and 70% after 48 h. A DNA fragmentation assay showed that Taxol induced genomic DNA cleavage into 200 bp DNA fragments. Bcl-2 protein was phosphorylated in Taxol-treated QGY cells within 3 h of treatment, and continued gradually up to 24 h. By 48 h, the protein was unphosphorylated. Other Bcl-2 family proteins, including Bax (a heterodimerization partner of Bcl-2), Bcl-XL, Bak and Bad, were expressed, but at constant levels. The results show a close correlation between Bcl-2 phosphorylation and apoptosis in QGY cells. The inactivation of Bcl-2 protein phosphorylation could be one of the key mechanisms needed for the induction of apoptosis in Taxol-treated QGY cells.     

Simple and effective method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells

We have established a simple and efficient method of electroporation applicable to gene transfer in mammalian cells. It uses a single decaying pulse of around 1 ms at room temperature in the medium such as Saline G appropriate for repair of pulse-induced pores in the plasma membrane. Many types of cells (both floating and adherent) could be transformed efficiently by the electric field strengths between 1-2 kV/cm. For instance P3U1, mouse myeloma cell, could be transformed by a pulse at 1.2 kV/cm with the frequency of 10(-2) per viable cells and with survivals of 90%. We have applied these conditions to transform tsBN2 cell line of BHK21/13 by a cosmid clone (approximately 45 kb) carrying the human gene complementing to tsBN2 mutation. Significant levels of transformation were observed for this gene. Since this gene can only work as a whole size (approximately 30 kb), the results show that electroporation is useful to introduce cosmid or possibly genomic DNA to mammalian cells.     

Analysis of aclarubicin-induced cell death in human fibroblasts

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.     

Jararhagin,a snake venom metalloproteinase,induces a specialized form of apoptosis (anoikis) selective to endothelial cells

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40μg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-x_L. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.     

Anti‐adhesive property of P‐selectin glycoprotein ligand‐1 (PSGL‐1) due to steric hindrance effect

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is an adhesive molecule that is known to be a ligand for P‐selectin. An anti‐adhesive property of PSGL‐1 has not been previously reported. In this study, we show that PSGL‐1 expression is anti‐adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL‐1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL‐1. PSGL‐1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL‐1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL‐1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL‐1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O‐glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL‐1 has Janus‐faced functions, being both adhesive and anti‐adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.     

A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells

Background: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness).Methods: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor- /cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation.Results: TNF- treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed.Conclusions: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.     

Elimination of cells with abnormal nuclei in human epidermoid carcinoma cell line A431 by α-lipoic acid

Skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and malignant transformation. Antioxidants are able to affect these processes and eliminate transformed cells. The purpose of this work was to investigate the effect of α-lipoic acid (ALA) on human epidermoid carcinoma cell line A431. It was found that 100, 200, 300, 500 μM ALA added for 24, 48, 72 h inhibited cell proliferation and stimulated apoptosis. Most dying cells have abnormal nuclei (micronuclei, giant nuclei, nuclei with buds). Electron microscopy showed that cells with normal nuclear phenotypes after treatment with 200 μM ALA for 48 h had ultrastructural organizations typical for control cells. Thus, α-ALA not only triggers the apoptosis of carcinoma cells, but it may also activate the mechanism for eliminating cells with abnormal numbers of chromosomes.     

Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.     

14-3-3ε Mediates the Cell Fate Decision-Making Pathways in Response of Hepatocellular Carcinoma to Bleomycin-Induced DNA Damage

Background

Lack of understanding of the response of hepatocellular carcinoma (HCC) to anticancer drugs causes the high mortality of HCC patients. Bleomycin (BLM) that induces DNA damage is clinically used for cancer therapy, while the mechanism underlying BLM-induced DNA damage response (DDR) in HCC cells remains ambiguous. Given that 14-3-3 proteins are broadly involved in regulation of diverse biological processes (BPs)/pathways, we investigate how a 14-3-3 isoform coordinates particular BPs/pathways in BLM-induced DDR in HCC.

Methodology/Principal Findings

Using dual-tagging quantitative proteomic approach, we dissected the 14-3-3ε interactome formed during BLM-induced DDR, which revealed that 14-3-3ε via its associations with multiple pathway-specific proteins coordinates multiple pathways including chromosome remodeling, DNA/RNA binding/processing, DNA repair, protein ubiquitination/degradation, cell cycle arrest, signal transduction and apoptosis. Further, “zoom-in” investigation of the 14-3-3ε interacting network indicated that the BLM-induced interaction between 14-3-3ε and a MAP kinase TAK1 plays a critical role in determining cell propensity of apoptosis. Functional characterization of this interaction further revealed that BLM triggers site-specific phosphorylations in the kinase domain of TAK1. These BLM-induced changes of phosphorylations directly correlate to the strength of the TAK1 binding to 14-3-3ε, which govern the phosphorylation-dependent TAK1 activation. The enhanced 14-3-3ε-TAK1 association then inhibits the anti-apoptotic activity of TAK1, which ultimately promotes BLM-induced apoptosis in HCC cells. In a data-dependent manner, we then derived a mechanistic model where 14-3-3ε plays the pivotal role in integrating diverse biological pathways for cellular DDR to BLM in HCC.

Conclusions

Our data demonstrated on a systems view that 14-3-3ε coordinates multiple biological pathways involved in BLM-induced DDR in HCC cells. Specifically, 14-3-3ε associates with TAK1 in a phosphorylation-dependent manner to determine the cell fate of BLM-treated HCC cells. Not only individual proteins but also those critical links in the network could be the potential targets for BLM-mediated therapeutic intervention of HCC.     

All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells

The effect of all-trans retinoic acid (ATRA) on leukaemia cell differentiation, proliferation and induction of apoptosis was studied by using autonomously growing blast cells isolated from eight patients with acute myeloblastic leukaemia (AML) either at diagnosis ( n=4) or at relapse (n=4). No morphological or functional differentiation induced by ATRA was observed in any of the cases studied. In cell cultures, inhibition of leukaemia cell growth by ATRA was obvious, especially in the case of clonogenic cells, and it was both time- and concentration-dependent. Induction of apoptosis was more difficult to achieve. The cells retained over 90% viability in suspension when the ATRA exposure at any of the concentrations studied was 48 h or less. When the time of exposure to ATRA was longer than 48 h, the viability of the cells decreased in a concentration-dependent manner. Apoptosis was observed morphologically in each of the AML cases with 10-5 to 10-8 M ATRA, if the incubation time of cells in ATRA was 72 h. The percentage of apoptotic cells increased with increasing ATRA concentrations from 12± 9% of 10-8 M ATRA to 78±12% of 10-5 M ATRA. The DNA electrophoretic method was able to detect apoptosis in all the AML samples exposed to 10-7 and 10-6 ATRA for 48 h and occasionally in cases where lower concentrations and longer exposure time were used. In conclusion, the present study shows that it is possible to induce apoptotic leukaemia cell death in vitro with ATRA in AML, and this effect is dependent on both concentration and exposure time.     

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

Background

Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10–40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.

Methods

The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting.

Results and conclusion

In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1–2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01–16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0025-8) contains supplementary material, which is available to authorized users.