Propidium Iodide Dual Staining Research Articles

Doxorubicin-induced chemoresistance in Duke's type B colon adenocarcinoma cell line is aggravated in the presence of TGF-β2 through non-apoptotic cell death.

The current challenge in clinical cancer treatment is chemoresistance. Colon cells have inherently higher xenobiotic transporters expression and hence can attain resistance rapidly. Increased levels of TGF-β2 expression in patients have been attributed to cancer progression, aggressiveness, and resistance. To investigate resistance progression, we treated doxorubicin (dox) to HT-29 colon adenocarcinoma cells in the presence or absence of TGF-β2 ligand. After 1, 3-, and 7-day treatment, we investigated cell proliferation, viability, and cytotoxicity by MTT, trypan blue staining, and lactate dehydrogenase enzyme release. The mechanism of cell death was elucidated by hoechst33342 and propidium iodide dual staining and apoptosis assay. The development of resistance was detected by rhodamine123 efflux and P-glycoprotein (P-gp)/MDR1 antibody staining through fluorimetry and flow cytometry. The colony formation ability of the cells was also elucidated. Inhibition of cell proliferation was noted after day 1, while a significant reduction in viability and a significant increase in lactate dehydrogenase release was detected after day 3. Reduction of intracellular rhodamine123 levels was detected after day 3 and was significantly lower in dox with TGF-β2 treatment compared to dox alone. Increased surface P-gp levels after days 3 and 7 were observed in the treated groups. Hoechst33342/propidium iodide staining and apoptosis assay indicated non-apoptotic cell death. The cells treated with TGF-β2 had higher colony formation ability. TGF-β2 expression might play a significant role in the development of chemoresistance to doxorubicin in Duke's type B colon adenocarcinoma cell line, HT-29.

Proteomic analysis of bovine mammary epithelial cells after in vitro incubation with S. agalactiae: potential biomarkers

Streptococcus agalactiae is one of the causative agents of subclinical mastitis, a common disease of dairy cows that causes great economic losses in the industry worldwide. It is thought that pathology is mainly due to inflammatory damage of bovine mammary epithelial cells (bMECs); however, the mechanism by which S. agalactiae damages the bMECs is not clear. The aim of this study was to evaluate the inflammatory effects of S. agalactiae on bMECs and the resulting changes in protein profiles. The bMECs were incubated with S. agalactiae for different times and assayed for cell viability by MTT assay, apoptosis by annexin V and propidium iodide dual staining, and morphological and ultrastructural changes by scanning and transmission electron microscopy. Quantitative real-time PCR was used to determine the effect of S. agalactiae on expression of mRNA of inflammatory factors in bMECs and protein levels were quantitated by liquid chromatography/mass spectrometry. Exposure to S. agalactiae significantly decreased the cell viability and triggered apoptosis, as well as up-regulating TNF-α, IL-1β and IL-6 mRNA, and inhibiting IL-8 expression. S. agalactiae also induced morphological and ultrastructural changes. Furthermore, we identified 325 up-regulated and 704 down-regulated proteins in the treated vs control group. All significant differentially expressed proteins (DSEPs) were classified into three major areas by function: biological processes, cellular components and molecular functions. These differentially expressed proteins included enzymes and proteins associated with various metabolic processes and cellular immunity. Pathway enrichment analysis showed that eight down-regulated signaling pathways were significantly enriched. Exposure to even subclinical levels of S. agalactiae can lead to inflammation and bMEC damage. Our data suggest some possible molecular mechanisms for the harmful effects of subclinical mastitis in dairy cows.

561 Time-dependence of UVB induced cellular mechanisms in cultured human keratinocytes

UVB-induced cyclobutane pyrimidine dimers (CPDs) are considered to be the main cause of acute sunburn and epidermal carcinogenesis. In humans, these lesions are repaired by nucleotide excision repair, but marsupials and lower organisms present photolyase enzyme which rapidly removes CPDs in a visible light-dependent process (photoreactivation). Previously we established an in vitro pseudouridin-modified mRNA encoding CPD-specific photolyase transfection on human keratinocyte cell lines, which was proved to be a proper method to avoid UVB-induced apoptosis. According to clinical experiences, there is a need for a treatment to diminish the deleterious effects of sunburn when UVB injury has already occurred and the first symptoms appear. Our aim was to determine the time interval after UVB exposure when keratinocyte apoptosis is still preventable. Normal human epidermal keratinocytes were transfected with lipofectamine-complexed, in vitro transcribed mRNA encoding CPD-specific photolyase. Cells were irradiated with 60 mJ/cm2 UVB. At 0, 6, 8 or 12 hours after UVB cells were either exposed to visible light (photoreactivation) or kept in the dark. Viability was measured by Annexin V and propidium iodide dual staining followed by flow cytometry, the relative amount of intracellular CPDs was analyzed by CPD-specific ELISA. Photolyase-mediated CPD removal restored cell viability close to the baseline conditions 0 to 6 hours after UVB treatment. The effect of CPD removal on keratinocyte survival began to decrease 8 hours after the UVB damage. In mRNA-transfected and photoreactivated cells 80% of CPDs were removed at 6 hours post-irradiation. 8 to 12 hours after UVB 40-60% of the CPDs were eliminated. Our results suggest that UV-induced keratinocyte apoptosis can be prevented within 6-8 hours after the UVB injury by the elimination of CPD photolesions. After that point the effect of CPD removal on cell viability is less feasible.

In-vitro Morphological Assessment of Apoptosis Induced by Nimbolide; A Limonoid from Azadirachta Indica (Neem Tree).

The present study was designed to investigate the in-vitro morphological assessment of apoptotic effect caused by nimbolide on the selected cancer cell lines (DU-145, PC-3, A-549) and normal cell lines (NIH3T3, CCD-18Co). The cells were grown in 6 well tissue culture plates after treatment with different concentrations of nimbolide and untreated control cells. Apoptotic and necrotic cells were measured using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and also by staining with annexin V and propidium iodide through flow cytometric analysis. The activity of caspase 3, 8, and 9 was measured by caspases colorimetric assay kits. The number of apoptotic and necrotic cells were significantly higher in all selected cancer cell lines treated with nimbolide as compared with untreated control cells, whereas in normal cell lines no significant difference was observed between nimbolide treated and untreated cells. The activity of caspase 3, 8, and 9 was also significantly higher in all cancer cell lines treated with nimbolide as compared with untreated control cells while it did not change significantly in normal cell lines as compared with untreated control. The results of the present study suggested that nimbolide induced apoptosis only in cancer cells without affecting the normal cells and one of the apoptosis inducing mechanism is through the activation of caspases signaling pathways. Therefore, nimbolide may be a novel promising candidate as an anticancer drug in future.

In-silico identification of small molecules targeting H-Ras and in-vitro cytotoxicity with caspase-mediated apoptosis in carcinoma cells.

H-Ras oncogene plays a critical role in the transformation of normal cells to a malignant phenotype through constitutive activation of the GTP bound protein leading to uncontrolled cell proliferation in several human cancers. Thus, H-Ras oncoprotein serves as an excellent target for anticancer drug discovery. To identify novel H-Ras inhibitors, we performed structure-based virtual screening of the Maybridge HitFinder™ library using Schrodinger suite. Thirty ligands from the chemical library were identified as they showed preferential in silico binding initially to H-Ras proteins with Gly12Val, Gly13Asp, and Gly12Val-Gly13Asp mutations. Absorption, distribution, metabolism, excretion, and toxicity profile confirmed drug-like properties of the compounds. Three representative molecules were tested for antiproliferative effect on T24 urinary bladder carcinoma cell line, MCF-7 breast cancer cell line and HDF-7 normal dermal fibroblast cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Two compounds (Cmpds) showed antiproliferative activity exclusively in the cancer cell lines with minimal effect on the control HDF-7 cells. The effect of compound treatment on cell cycle progression, assessed by propidium iodide (PI) staining, depicted increased arrest of T24 cell line in the sub G1 phase. Further, Annexin-V PI dual staining and pan caspase inhibitor Z-VAD-fmk indicated caspase-dependent apoptotic activity of Cmpds 1 and 3. Our findings demonstrate caspase-dependent apoptotic activity of Cmpds 1 and 3 selectively against Gly12Val mutated T24 cancer cell line implicating a potential for treatment of bladder cancer. We envisage that these molecules may be promising candidates with potential therapeutic value in H-Ras mutation-associated cancers.

In vitro α-amylase, α-glucosidase, lipase inhibitory and cytotoxic activities of tuber extracts of Kedrostis africana (L.) Cogn

Kedrostis africana, is a tuberous plant commonly used by traditional healers in the Eastern Cape Province of South Africa for the management of obesity. The aim of this study was to investigate the antiobesity and cytotoxic effects of Kedrostis africana extracts in vitro The α-amylase, α-glucosidase and lipase inhibitory activities of aqueous and ethanol extracts of Kedrostis africana tuber were investigated while the cytotoxic effects of these extracts were analyzed using Hoechst 33342 and propidium iodide (PI) dual staining in combination with Molecular Devices ImageXpress Micro XLS Widefield microscope for high content analysis on human cervical (HeLa) cell line. The ethanol extract exhibited the strongest inhibitory effect on pancreatic lipase (IC50 = 381.86 μg/ml) and on α-glucosidase (IC50 = 157.99 μg/mL) while the aqueous extract has strongest α-amylase (IC50 = 439.45 μg/ml). Both tuber extracts were found nontoxic at tested concentrations on HeLa cell lines as confirmed by the Hoechst 33342 and propidium iodide dual staining respectively. This study revealed that both the aqueous and ethanol tuber extract of K. africana exerts a certain degree of inhibitory effect on α-amylase, α-glucosidase and lipase and were also nontoxic to HeLa cell line at tested concentrations.

Antitumor activity of chitosan from mayfly with comparison to commercially available low, medium and high molecular weight chitosans.

Insects' cuticles have a potential to be evaluated as a chitin source. Especially adults of aquatic insects like mayflies (order Ephemeroptera) swarm in enormous numbers in artificially lit areas while mating in spring and then die by leaving huge amounts of dead insects' bodies. Here in this study, mayfly corpses were harvested and used for production of low MW chitosan. Dried mayfly bodies had 10.21% chitin content; mayfly chitin was converted into chitosan with efficiency rate of 78.43% (deacetylation degree, 84.3%; MW, 3.69kDa). Cytotoxicity and anti-proliferative activity of mayfly and commercially available shrimp chitosans (low, medium, and high MW) were determined on L929 fibroblast and three different cancer types including HeLa, A549, and WiDr. Apoptosis and necrosis stimulating potential of mayfly and commercial chitosans were also evaluated on A549 and WiDr cells using acridine orange and propidium iodide dual staining to observe morphological changes in nuclei and thus to reveal the predominant cell death mechanism. The effects of chitosans have varied depending on cell types, concentration, and chitosan derivatives. Mayfly and low MW chitosans had a cytotoxic effect at a concentration of 500μgmL-1 on non-cancer cells. At concentrations below this value (250μgmL-1), mayfly and commercial chitosans except high MW one exhibited strong inhibitory activity on cancer cells especially A549 and WiDr cells. Mayfly chitosan induced early and late apoptosis in A549 cells, but late apoptosis and necrosis in WiDr cells. This study suggests that dead bodies of mayflies can be used for production of low MW chitosan with anti-proliferative activity.

Induction of apoptosis and cell cycle arrest by ethyl acetate fraction of Phoenix dactylifera L. (Ajwa dates) in prostate cancer cells

Ethnopharmacological relevancePhoenix dactylifera L. (Ajwa date) has high nutritive value and are consumed in Arabian Peninsula as an essential diet. Phoenix dactylifera L. have been mentioned in folk remedies of traditional Egyptian medicine and alternative medicine, for numerous health benefits including cancer treatment. The aim of the study is to evaluate the anticancer effects of the extract of Ajwa Date on human Prostate cancer cell line (PC3). Materials and methodsAntiproliferative effect was measured using MTT assay. The long-term effect of EAFAD was determined using colony assay. Different stains like Giemsa and fluorescent stains (DAPI and acridine orange / Ethidium bromide) measured morphological changes. Loss of mitochondrial membrane potential and increased oxidative stress were measured using JC-1 and DCFH-DA dyes. DNA degradation was analyzed by comet assay. Cell cycle distribution was measured by flow cytometer. The apoptotic cell was quantified by annexin V-FITC and Propidium iodide dual staining using flow cytometer. ResultsPC3 cell line was treated with ethyl acetate fractions of Ajwa dates (EAFAD) to study their morphological and cellular changes and induction of apoptosis. MTT assay showed the strong inhibitory effect of EAFAD on PC3 cells. Loss of mitochondrial membrane potential and increased oxidative stress were observed in EAFAD treated cells, which suggested mitochondrial involvement in apoptosis. Comet assay proved DNA fragmentation induced by EAFAD. Flow Cytometer results demonstrated that Annexin V-FITC and propidium iodide staining showed that EAFAD induced apoptosis and arrest the cell cycle in S phase. ConclusionOur results suggested EAFAD has potential therapeutics properties for prostate cancer.

In vitro antiproliferative and apoptosis inducing effect of a methanolic extract of Azadirachta indica oil on selected cancerous and noncancerous cell lines

Objective: To find the cytotoxic and apoptotic effects of neem oil extract on the selected cancerous (A-549, PC-3 and DU-145) and noncancerous (NIH3T3 and CCD-18Co) cell lines. Methods: Viability and cytotoxic effect induced by the extract was measured by using MTT assay and apoptotic effect of the extract was evaluated by using Hoechst 33342 and propidium iodide dual staining through a fluorescent microscope and activity of caspases 3, 8 and 9 through colorimetric assay kits. Results: The results showed that neem oil extract significantly reduced the viability in all selected cancer cells treated with varying concentrations of extract as compared with untreated cells and had less effect on noncancerous cell lines. It significantly increased the percentage of necrotic and apoptotic cells, and caspases 3, 8 and 9 activities in all cancer cells treated with extract as compared with untreated cells whereas no effect on noncancerous cell lines. It suggested that neem oil extract exerted a higher cytotoxic effect on cancer cells than normal cells and lower concentration induced apoptosis only in cancer cells. One of the apoptosis-inducing mechanism was through the activation of caspases signaling pathways. Conclusion: Conclusively, it implies that neem oil extract may contain one or more potential agents that can be used as a safe and effective anticancer therapy.

Effect of sucrose on cryopreservation of pig spermatogonial stem cells

Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells (pSSCs) has not been tested. The aim of this work was to study the effect of sucrose during pSSC cryopreservation and to find the most effective concentration in freezing medium. pSSCs were cryopreserved with freezing media containing different concentrations of sucrose (70, 140, 210, and 280 mmol L−1) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue (TB) staining, SYBR-14/propidium iodide (PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L−1 sucrose. Moreover, the 210 mmol L−1 sucrose group yielded the highest survival rate among all the groups (P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR (qRT-PCR) indicated that the mRNA levels of three apoptosis-promoting genes (BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing (P<0.05). Moreover, the mRNA level of one anti-apoptotic gene (XIAP) was significantly lower in thawed cells than in cells before freezing (P<0.05). When comparing the mRNA expression of apoptosis-related genes in thawed cells, the mRNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups (P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L−1 sucrose group. Both qRT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted pSSCs survival after freezing and thawing, especially at a concentration of 210 mmol L−1, which possibly assisted pSSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of pSSCs.

A role of ghrelin in canine mammary carcinoma cells proliferation, apoptosis and migration

BackgroundGhrelin is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/GHS-R axis may have an important role in tumor growth and progression. However, a role of ghrelin in canine tumors remains unknown. Thus, the aim of our study was two-fold: (1) to assess expression of ghrelin and its receptor in canine mammary cancer and (2) to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. The expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines (isolated from primary tumors and their metastases) was examined using Real-time qPCR and immunohistochemistry. For apoptosis analysis the Annexin V and propidium iodide dual staining was applied whereas cell proliferation was evaluated by MTT assay and BrdU incorporation test. The influence of ghrelin on cancer cells migration and invasion was assessed using Boyden chamber assays and wound healing assay.ResultsThe highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3rd grade of malignancy. Higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. In vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. Growth hormone secretagogue receptor inhibitor ([D-Lys3]-GHRP6) as well as RNA interference enhances early apoptosis.ConclusionThe presence of ghrelin and GHS-R in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. Our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis.

The venom of the spider Macrothele raveni induces apoptosis in the myelogenous leukemia K562 cell line

Spider venoms are a rich source of bioactive compounds with therapeutic potential. In traditional Chinese medicine, spiders and spider venoms have been used in the treatment of various ailments. In the present study, the venom of the spider Macrothele raveni potently suppressed cell growth in the myelogenous leukemia K562 cell line in a dose and time-dependent manner with an IC50 of 5.1μg/mL. The venom also had a low inhibitory effect on human lymphocytes with an IC50 of approximately 36.4μg/mL, indicating that the venom is relatively selective for leukemic cells. Venom treated K562 cells showed typical morphological indicators of apoptosis including condensation of nuclei and fragmentation of DNA. Annexin V-FITC and propidium iodide dual staining further demonstrated that the venom had potent apoptogenic activity. Venom treatment induced caspase 3 and caspase 8 activation in K562 cells and promoted PARP cleavage. The present results indicate that the venom of the spider M. raveni potently and selectively suppresses the growth of K562 cells by inducing apoptosis via caspase 3 and caspase 8 mediated signaling pathways.

Ghrelin protects H9c2 cells from hydrogen peroxide-induced apoptosis through NF-κB and mitochondria-mediated signaling

Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. Herein we investigate the protective effects of ghrelin in H2O2-induced apoptosis of H9c2 cells, as well as the possible molecular mechanisms involved. To study apoptosis, the cells were assessed by morphologic examination, MTS assay, Annexin V–propidium iodide dual staining and TUNEL analysis. Intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential were also measured. To investigate the underlying molecular mechanisms, the expression of Bcl-2, Bax, active caspase-9 and NF-κB were assessed by Western blotting, and caspase-3 activity was determined by a colorimetric activity assay kit. After stimulation with H2O2 for 18h, H9c2 cells viability decreased significantly; a large fraction of cells underwent apoptosis. We observed a dose-dependent rescue of H9c2 cells from H2O2-induced apoptosis in the presence of different ghrelin concentrations. Preincubation with ghrelin also restored the ROS and mitochondrial membrane potential levels that had been altered by H2O2 treatment. Moreover, ghrelin decreased H2O2-induced Bax production and caspase-9 activation, and increased Bcl-2 levels. NF-κB phosphorylation was also significantly inhibited by ghrelin in H2O2-treated cells. Caspase-3 activation was suppressed by ghrelin in H2O2-treated H9c2 cells in a dose-dependent manner. In summary, ghrelin protects H9c2 cells from oxidative stress-induced apoptosis through downregulation of Bax expression, caspase-9 activation and NF-κB phosphorylation, and upregulation of Bcl-2 expression. Caspase-3 activation was also reduced in a dose-dependent manner. These data suggest that ghrelin might protect against cardiovascular disease by protecting the mitochondria.

Cyclooxygenase-2 Suppresses Polymorphonuclear Neutrophil Apoptosis After Acute Lung Injury

Polymorphonuclear neutrophil (PMN) apoptosis is suppressed after acute lung injury (ALI), and strategies aimed at inducing PMN apoptosis are thought to be promising therapies for ALI. However, the mechanisms underlying PMN apoptotic suppression are unknown. Cyclo-oxygenase-2 (COX-2) has been shown to regulate tumor cell apoptosis and is up-regulated by inflammatory mediators in PMN. Therefore, we set out to determine whether up-regulation of COX-2 expression contributes to PMN apoptosis after ALI. Experimental ALI was established in New Zealand rabbits by blunt chest trauma, and a correlation analysis of COX-2 immunohistochemical staining in lung tissue and PMN apoptosis in bronchoalveolar lavage fluid (BALF) was performed. Apoptosis was measured by flow cytometric analysis of annexin V and propidium iodide dual staining. As an in vitro correlate, normal PMNs were treated with BALF from injured lung (BALFALI) in the presence or absence of the COX-2 inhibitor, NS398. COX-2 mRNA levels and PMN apoptosis were then measured. PMN apoptosis was significantly decreased in BALF after injury. In contrast, COX-2 expression was significantly increased after injury. COX-2 protein expression and PMN apoptosis exhibited a strong inverse correlation (gamma = -0.75, p < 0.01). In vitro experiments revealed apoptosis of normal PMNs was significantly decreased by the addition of BALFALI. The addition of BALFALI was also associated with increased COX-2 mRNA levels. Treatment of cultures with NS398, 10 minutes before BALFALI addition, partially reversed all of these effects. Up-regulation of intrapulmonary COX-2 expression contributes to the suppression of PMN apoptosis after ALI.

Tumor necrosis factor- $$\alpha$$ induces caspase-independent cell death in human neutrophils via reactive oxidants and associated with calpain activity

Apoptosis mediated by caspase activation is important in the neutrophil homeostasis and resolution of tissue inflammation. Paradoxically, our previous study demonstrated that broad-spectrum caspase inhibition augmented tumor necrosis factor (TNF)-alpha-induced cell death in the human neutrophils. Therefore, we further explored the mechanisms related to the caspase-independent cell death in the neutrophils. The cell apoptosis/necrosis was determined by annexin V and propidium iodide dual staining in flow cytometry. Their morphological changes were observed under light microscopy. Fluorogenic substrates were used to measure the intracellular oxidative reactions and the activities of proteinases, calpains. Calpain inhibitors and antioxidants were used to elucidate the relationship of calpains and oxidants with the neutrophil cell death. Our results verified the caspase-independent cell death pathway in the zVAD-sensitized, TNF-alpha-stimulated neutrophils. Furthermore, the cell death was accompanied with increased calpain and oxidative activities in the cells. Calpain inhibitors, zLLY, as well as anti-oxidants, catalase and DMSO, were able to attenuate the cell death in the zVAD-sensitized, TNF-alpha-induced neutrophils. Pretreating the neutrophils with G-CSF or GM-CSF was not able to reduce the cell death. These results demonstrate that, in human neutrophils, TNF-alpha-induces a caspase-independent cell death signal, which is related to calpain and oxidative activities and cannot be rescued by the growth factor-related signaling mechanism.