Typical Apoptotic Cell Death Research Articles

Anticancer Activity of the Acetylenic Derivative of Betulin Phosphate Involves Induction of Necrotic-Like Death in Breast Cancer Cells In Vitro.

Betulin (BT) is a natural pentacyclic lupane-type triterpene exhibiting anticancer activity. Betulin derivatives bearing propynoyloxy and phosphate groups were prepared in an effort to improve the availability and efficacy of the drug. In this study, a comparative assessment of the in vitro anticancer activity of betulin and its four derivatives was carried out using two human breast cancer cell lines: SK-BR-3 and MCF-7. In both studied cell lines, 30-diethoxyphosphoryl-28-propynoylbetulin (compound 4) turned out to be the most powerful inhibitor of growth and inducer of cellular death. Detailed examination of that derivative pertained to the mechanisms underlying its anticancer action. Treatment with compound 4 decreased DNA synthesis and up-regulated p21WAF1/Cip1 mRNA and protein levels in both cell lines. On the other hand, that derivative caused a significant increase in cell death, as evidenced by increased lactate dehydrogenase (LDH) release and ethidium homodimer uptake. Shortly after the compound addition, an increased generation of reactive oxygen species and loss of mitochondrial membrane potential were detected. The activation of caspase-3 and fragmentation of genomic DNA suggested an apoptotic type of cell death. However, analysis of cellular morphology did not reveal any nuclear features typical of apoptosis. Despite necrosis-like morphology, dead cells exhibited activation of the cascade of caspases. These observations have led to the conclusion that compound 4 pushed cells to undergo a form of necrotic-like regulated cell demise.

Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma.

Changes in zinc content and dysregulated zinc homeostatic mechanisms have been recognized in several solid malignancies such as prostate cancer, breast cancer, or pancreatic cancer. Moreover, it has been shown that zinc serum and/or tissue levels are altered in melanoma with varying effects on melanoma development and biology. This study was conducted to explore the effects of acute increases of intracellular zinc in a set of melanoma tissue explants obtained from clinical samples. Measurements of their zinc content showed an extant heterogeneity in total and free intracellular zinc pools associated with varying biological behavior of individual cells, e.g., autophagy levels and propensity to cell death. Use of zinc pyrithione elevated intracellular zinc in a short time frame which resulted in marked changes in mitochondrial activity and lysosomes. These alterations were accompanied by significantly enhanced autophagy flux and subsequent cell demise in the absence of typical apoptotic cell death markers. The present results show for the first time that acutely increased intracellular zinc in melanoma cells specifically enhances their autophagic activity via mitochondria and lysosomes which leads to autophagic cell death. While biologically relevant, this discovery may contribute to our understanding and exploration of zinc in relation to autophagy as a means of controlling melanoma growth and survival.

Berberine in combination with cisplatin induces necroptosis and apoptosis in ovarian cancer cells

BackgroundBerberine (BBR), a compound extracted from a variety of medicinal herbs, has been shown multiple pharmacological effects against cancer cells of different origins. Cisplatin (DDP) is known as an effective chemotherapeutic agent against cancer by inducing DNA damage and cell apoptosis. However, the effect of the combined used of BBR and DDP on cell necroptosis in ovarian cancer has not been reported.MethodsOVCAR3 and three patient-derived primary ovarian cancer cell lines (POCCLs) were chosen as the experimental objects. To determine the potential anti-cancer activity of BBR and DDP in combination, we firstly treated OVCAR3 and POCCLs cells with BBR and/or DDP. The cell viability of OVCAR3 and POCCLs with treatment of BBR or DDP for different hours was measured by CCK-8 assay. Flow cytometry was used to analyze cell cycle distribution and changes in apoptotic cells after treatment with BBR and/or DDP. The morphological changes of OVCAR3 cells were observed by using Transmission electron microscopy (TEM) analysis. Proliferation, apoptosis and necroptosis related markers of OVCAR3 and POCCLs with treatment of BBR or DDP were measured by RT-qPCR, western blotting and immunofluorescence assay.ResultsOur results demonstrated that BBR significantly inhibited the proliferation of OVCAR3 and primary ovarian cancer cells in a dose- and time-dependent manner. The combination treatment of BBR and DDP had a prominent inhibitory effect on cancer cell growth and induced G0/G1 cell cycle arrest. TEM revealed that the majority of cells after BBR or DDP treatment had an increasing tendency of typical apoptotic and necrotic cell death morphology. Besides, BBR and DDP inhibited the expression of PCNA and Ki67 and enhanced the expression and activation of Caspase-3, Caspase-8, RIPK3 and MLKL.ConclusionThis study proposed that the combination therapy of BBR and DDP markedly enhanced more ovarian cancer cell death by inducing apoptosis and necroptosis, which may improve the anticancer effect of chemotherapy drugs. The apoptosis involved the caspase-dependent pathway, while the necroptosis involved the activation of the RIPK3–MLKL pathway. We hope our findings might provide a new insight for the potential of BBR as a therapeutic agent in the treatment of ovarian cancer.

Cytotoxic Withanolides from the Whole Herb of Physalis angulata L.

Physalis angulata L. is a medicinal plant of the Solanaceae family, which is used to produce a variety of steroids. The present study reports on the cytotoxic withanolides of this plant. The species of Physalis angulata L. was identified by DNA barcoding techniques. Two new withanolides (1–2), together with six known analogues (3–8), were isolated from the whole plant of Physalis angulata L. The structures of these new compounds were determined on the basis of extensive spectroscopic data analyses and electronic circular dichroism (ECD) calculations. The withanolides exhibited strong cytotoxic activities against A549, Hela and p388 cell lines. Furthermore, compounds 1 and 2 induced typical apoptotic cell death in A549 cell line according to the evaluation of the apoptosis-inducing activity by flow cytometric analysis.

SPOT-008 Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukaemia cells from apoptosis

Introduction B-cell chronic lymphocytic leukaemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Material and methods B-cells were isolated from the peripheral blood of healthy donors and CLL patients (n=34) using the MACSxpress human B-CLL Cell Isolation Kit. Gene expression was assessed by RT-qPCR, protein expression and signalling pathway activation was assessed by Western Blot. Protein-protein interaction was confirmed by both Indirect Immuno-Fluorescence and co-immunoprecipitations. Cell death was assessed by Elisa Cell Death as well as Flow Cytometry. Studied receptors were inhibited either by RNA interference or pharmacological inhibitors. Results and discussions In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterisation revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand BDNF, which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signalling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death. Conclusion Together, our findings demonstrate that the NTSR2–TrkB interaction and the sustained activation of the signalling pathways under the control of these two actors plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.

Phytochemical analysis, antioxidant, antibacterial and cytotoxic activity of different plant organs of Eryngium serbicum L.

Plants from genus Eryngium are widely used in traditional medicine for treatment of various disorders. In this study, in vitro antioxidant, antibacterial and cytotoxic activity, as well as composition of bioactive constituents of the methanol extracts (flowers, leaves, stems and roots) of Eryngium serbicum are reported for the first time. By using LC-ESI–MS/MS, 22 bioactive compounds present in extracts were quantified. The most abundant compound observed in flower and stem extracts was chlorogenic acid (2704.23 μg/g and 1813.13 μg/g, respectively), while the most dominant compound in leaf and root extracts was rosmarinic acid (869.65 μg/g and 6467.69 μg/g, respectively). All examined extracts showed high contents of total phenolic compounds (expressed as mg GAE/g), flavonoids (expressed as mg RUE/g), flavonols (expressed as mg RUE/g) and total phenolic acids (expressed as mg CAE/g). Also, a notable antioxidant power of extracts was observed from the results of DPPH free-radical scavenging activity (as IC50, μg/mL), ABTS radical-cation scavenging activity (as IC50, μg/mL), reducing power activity (as RP50, μg/mL), as well as results of inhibitory activity toward lipid peroxidation (as IC50, μg/mL). With the exceptions of low activity of stem extract toward Enterococcus faecalis and Staphylococcus epidermidis, as well as leaf and stem extracts against Escherichia coli, all other examined extracts showed a notable activity toward test bacteria (MIC range: 15.6 μg/mL to 125 μg/mL). Crude E. serbicum extracts from flowers, leaves and stems showed selective cytotoxic activity towards tested cell lines, without effects on normal MRC-5 cells. After 72 h of exposure, all examined extracts were exerting a strong effect against colon (HCT-116, SW-480) and breast (MDA-MB-231) cancer cell lines. Flow cytometric analysis showed that examined extracts induced primary apoptotic type of cell death and cell cycle arrest. Due to strong activity, further in vivo studies and exact mechanism of action could be useful in designing new antibacterial and anticancer therapeutic agents.

Quercetin suppresses DNA double-strand break repair and enhances the radiosensitivity of human ovarian cancer cells via p53-dependent endoplasmic reticulum stress pathway.

Quercetin is proven to have anticancer effects for many cancers. However, the role of tumor suppressor p53 on quercetin’s radiosensitization and regulation of endoplasmic reticulum (ER) stress response in this process remains obscure. Here, quercetin exposure resulted in ER stress, prolonged DNA repair, and the expression of p53 protein; phosphorylation on serine 15 and 20 increased in combination with X-irradiation. Quercetin pretreatment could potentiate radiation-induced cell death. The combination of irradiation and quercetin treatment aggravated DNA damages and caused typical apoptotic cell death; as well the expression of Bax and p21 elevated and the expression of Bcl-2 decreased. Knocking down of p53 could reverse all the above effects under quercetin in combination with radiation. In addition, quercetin-induced radiosensitization was through stimulation of ATM phosphorylation. In human ovarian cancer xenograft model, combined treatment of quercetin and radiation significantly restrained the growth of tumors, accompanied with the activation of p53, CCAAT/enhancer-binding protein homologous protein, and γ-H2AX. Overall, these results indicated that quercetin acted as a promising radiosensitizer through p53-dependent ER stress signals.

Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukemia cells from apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors drives cell transformation, and thus plays a critical role in malignancies. In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterization revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand brain-derived neurotrophic factor (BDNF), which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death, reflected by DNA fragmentation and Annexin V presentation. Together, our findings demonstrate that the NTSR2–TrkB interaction plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent-kaurane diterpenoid, 11β,16α-dihydroxy-ent-kauran-19-oic acid (1), together with eight known analogues 2 - 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid (3), was converted to seven derivatives, 10 - 16. All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG-2), and stomach (SGC-7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 - 16, with 15-keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC50 value ranging from 0.05 to 3.71 μm. Compounds 10 - 12, with oxime on C(15) showed moderate inhibitory effects and compounds 1 - 9 showed no cytotoxicities on them. Structure-activity relationships were also discussed based on the experimental data obtained. The known derivative, 15-oxokaurenoic acid 4-piperdin-1-ylbutyl ester (17), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis-inducing activity by flow-cytometric analysis.

Neuronal death signaling pathways triggered by mutant LRRK2

Autosomal dominantly inherited mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. While considerable progress has been made in understanding its function and the many different cellular activities in which it participates, a clear understanding of the mechanism(s) of the induction of neuronal death by mutant forms of LRRK2 remains elusive. Although several in vivo models have documented the progressive loss of dopaminergic neurons of the substantia nigra, more complete interrogations of the modality of neuronal death have been gained from cellular models. Overexpression of mutant LRRK2 in neuronal-like cell lines or in primary neurons induces an apoptotic type of cell death involving components of the extrinsic as well as intrinsic death pathways. While informative, these studies are limited by their reliance upon isolated neuronal cells; and the pathways triggered by mutant LRRK2 in neurons may be further refined or modulated by extracellular signals. Nevertheless, the identification of specific cell death-associated signaling events set in motion by the dominant action of mutant LRRK2, the loss of an inhibitory function of wild-type LRRK2, or a combination of the two, expands the landscape of potential therapeutic targets for future intervention in the clinic.

Cytotoxic and Apoptosis-inducing Activities of Taraxastane-type Triterpenoid Derivatives in Human Cancer Cell Lines.

Twenty-eight taraxastane-type triterpenoid derivatives 4-31 were prepared from the naturally occurring triterpenoids faradiol (1) and heliantriol C (3). The cytotoxic activities of these compounds and arnidiol (2) were evaluated in leukemia (HL60), lung (A549), duodenal (AZ521), and breast (SK-BR-3) cancer cell lines. 21-Oxoarnidiol (18) and faradiol 3,16-di-O-l-alaninate (31) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5-2.7μm. In particular, flow cytometric analysis indicated that compound 31 induced typical apoptotic cell death in HL60 cells. These results suggested that taraxastane-type triterpenoid derivatives might provide useful antitumor agents with apoptosis-inducing activity.

Chemoresistance is associated with increased cytoprotective autophagy and diminished apoptosis in bladder cancer cells treated with the BH3 mimetic (-)-Gossypol (AT-101).

BackgroundAcquired resistance to standard chemotherapy causes treatment failure in patients with metastatic bladder cancer. Overexpression of pro-survival Bcl-2 family proteins has been associated with a poor chemotherapeutic response, suggesting that Bcl-2-targeted therapy may be a feasible strategy in patients with these tumors. The small-molecule pan-Bcl-2 inhibitor (−)-gossypol (AT-101) is known to induce apoptotic cell death, but can also induce autophagy through release of the pro-autophagic BH3 only protein Beclin-1 from Bcl-2. The potential therapeutic effects of (−)-gossypol in chemoresistant bladder cancer and the role of autophagy in this context are hitherto unknown.MethodsCisplatin (5637rCDDP1000, RT4rCDDP1000) and gemcitabine (5637rGEMCI20, RT4rGEMCI20) chemoresistant sub-lines of the chemo-sensitive bladder cancer cell lines 5637 and RT4 were established for the investigation of acquired resistance mechanisms. Cell lines carrying a stable lentiviral knockdown of the core autophagy regulator ATG5 were created from chemosensitive 5637 and chemoresistant 5637rGEMCI20 and 5637rCDDP1000 cell lines. Cell death and autophagy were quantified by FACS analysis of propidium iodide, Annexin and Lysotracker staining, as well as LC3 translocation.ResultsHere we demonstrate that (−)-gossypol induces an apoptotic type of cell death in 5637 and RT4 cells which is partially inhibited by the pan-caspase inhibitor z-VAD. Cisplatin- and gemcitabine-resistant bladder cancer cells exhibit enhanced basal and drug-induced autophagosome formation and lysosomal activity which is accompanied by an attenuated apoptotic cell death after treatment with both (−)-gossypol and ABT-737, a Bcl-2 inhibitor which spares Mcl-1, in comparison to parental cells. Knockdown of ATG5 and inhibition of autophagy by 3-MA had no discernible effect on apoptotic cell death induced by (−)-gossypol and ABT-737 in parental 5637 cells, but evoked a significant increase in early apoptosis and overall cell death in BH3 mimetic-treated 5637rGEMCI20 and 5637rCDDP1000 cells.ConclusionsOur findings show for the first time that (−)-gossypol concomitantly triggers apoptosis and a cytoprotective type of autophagy in bladder cancer and support the notion that enhanced autophagy may underlie the chemoresistant phenotype of these tumors. Simultaneous targeting of Bcl-2 proteins and the autophagy pathway may be an efficient new strategy to overcome their “autophagy addiction” and acquired resistance to current therapy.

Three new and other limonoids from the hexane extract of Melia azedarach fruits and their cytotoxic activities.

A defatted fraction obtained from the hexane extract of the fruits of Melia azedarach L. (chinaberry tree; Meliaceae) exhibited cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines with IC50 values in the range of 2.9-21.9 μg/ml. Three new limonoids, 3-deacetyl-4'-demethylsalannin (5), 3-deacetyl-28-oxosalannin (14), and 1-detigloylohchinolal (17), along with 16 known limonoids, 1-4, 6-13, 15, 16, 18, and 19, and one known triterpenoid, 20, were isolated from the fraction. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. These compounds were evaluated for their cytotoxic activities against the four cancer cell lines mentioned above. 3-Deacetyl-4'-demethyl-28-oxosalannin (16), which exhibited potent cytotoxicity against AZ521 (IC50 3.2 μM) cells, induced typical apoptotic cell death in AZ521 cells upon evaluation of the apoptosis-inducing activity by flow cytometry. This work provided, furthermore, valuable information on the structural features of limonoids of the fruits and/or seeds of Melia azedarach and related Meliaceae plants, M. toosendan and Azadirachta indica.

αNAC inhibition of the FADD-JNK axis plays anti-apoptotic role in multiple cancer cells.

Nascent polypeptide-associated complex α (αNAC) is reportedly overexpressed in several types of cancers and regulates cell apoptosis under hypoxic conditions in HeLa cells. The aim of our study was to investigate the apoptotic function of αNAC in cancer progression. First, we observed the cellular effects of αNAC depletion. Mouse αNAC was used to restore the protein level and verify the effect. An Annexin V assay, a caspase activity reporter assay, an apoptotic molecular marker, and a colony formation assay were used as markers to investigate the mechanisms of cell death caused by αNAC depletion. The Cancer 10-pathway reporter assay was used to screen downstream pathways. PCR site-directed deletion based on the functional domains of αNAC was used to construct deletion mutants. Those functional domain deletion mutants were used to recover the apoptotic phenotype caused by αNAC depletion. Finally, the role of αNAC in TNF-related apoptosis-inducing ligand (TRAIL) treatment was investigated in vitro. We found that depletion of αNAC in multiple types of cancer cells induce typical apoptotic cell death. This anti-apoptotic function is mediated by the FADD/c-Jun N-terminal kinase pathway. Intact αNAC is required for the direct binding of FADD as well as its anti-apoptosis function. Either αNAC depletion or the deletion of the ubiquitin-associated domain of αNAC sensitizes L929 cancer cells to mTRAIL treatment. Our study revealed a αNAC anti-apoptotic function in multiple types of cancer cells and suggested its potential in cancer therapy.

Nanosized Zinc Oxide Induces Toxicity in Human Lung Cells

Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, biosensors, food additives, pigments, rubber manufacture, and electronic materials. With the wide application of ZnO-NPs, concern has been raised about its unintentional health and environmental impacts. This study investigates the toxic effects of ZnO-NPs in human lung cells. In order to assess toxicity, human lung epithelial cells (L-132) were exposed to dispersion of 50 nm ZnO-NPs at concentrations of 5, 25, 50, and 100 μg/mL for 24 h. The toxicity was evaluated by observing changes in cell morphology, cell viability, oxidative stress parameters, DNA damage analysis, and gene expression. Exposure to 50 nm ZnO-NPs at concentrations between 5 and 100 μg/mL decreased cell viability in a concentration-dependent manner. Morphological examination revealed cell shrinkage, nuclear condensation, and formation of apoptotic bodies. The oxidative stress parameters revealed significant depletion of GSH level and increase in ROS levels suggesting generation of oxidative stress. ZnO-NPs exposure caused DNA fragmentation demonstrating apoptotic type of cell death. ZnO-NPs increased the expression of metallothionein gene, which is considered as a biomarker in metal-induced toxicity. To summarize, ZnO-NPs cause toxicity in human lung cells possibly through oxidative stress-induced apoptosis.

Ginseng Treatment Attenuates Chronic Cyclosporine Nephropathy via Reducing Oxidative Stress in an Experimental Mouse Model

Background: This study was performed to investigate whether ginseng extract has a protective effect in an experimental mouse model of chronic cyclosporine (CsA) nephropathy. Methods: Mice were treated with CsA (30 mg/kg/day, subcutaneously) with or without Korean red ginseng extract (KRG) (0.2, 0.4 g/kg/day, orally) on a 0.01% salt diet for 4 weeks. The effect of KRG on CsA-induced renal injury was evaluated by assessing renal function and pathology, mediators of inflammation, tubulointerstitial fibrosis and apoptotic cell death. Using an in vitro model, we also examined the effect of KRG on CsA-treated proximal tubular cells (HK-2). Oxidative stress was measured by assessing 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in 24-hour urine, tissue sections, and culture media. Results: Four weeks of CsA treatment caused renal dysfunction, typical pathologic lesions and apoptotic cell death. KRG treatment reduced serum creatinine and blood urea nitrogen and histopathology and increased creatinine clearance. Proinflammatory and profibrotic molecules such as induced nitric oxide synthase, cytokines, transforming growth factor (TGF)-β1 and TGF-β1-inducible gene h3 and apoptotic cell death, also decreased with KRG treatment. Consistent with these results, in vitro studies showed that addition of KRG protected against CsA-induced morphological changes, cytotoxicity, inflammation, and apoptotic cell death as demonstrated by annexin V binding. These changes were accompanied by decrease in the level of 8-OHdG in urine and culture supernatant after KRG treatment. Conclusion: The results of our in vivo and in vitro studies demonstrate that KRG has a protective effect in CsA-induced renal injury via reducing oxidative stress.

Cytotoxic and Apoptosis‐Inducing Activities of Steviol and Isosteviol Derivatives against Human Cancer Cell Lines

Seventeen steviol derivatives, i.e., 2-18, and 19 isosteviol derivatives, i.e., 19-37, were prepared from a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, nine steviol derivatives, i.e., 5-9 and 11-14, and five isosteviol derivatives, i.e., 28-32, exhibited activities with single-digit micromolar IC(50) values against one or more cell lines. All of these active compounds possess C(19)-O-acyl group, and among which, ent-kaur-16-ene-13,19-diol 19-O-4',4',4'-trifluorocrotonate (14) exhibited potent cytotoxicities against four cell lines with IC(50) values in the range of 1.2-4.1 μM. Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis-inducing activity by flow-cytometric analysis. These results suggested that acylation of the 19-OH group of kaurane- and beyerane-type diterpenoids might be useful for enhancement of their cytotoxicities with apoptosis-inducing activity.

Acriflavine enhances radiosensitivity of colon cancer cells through endoplasmic reticulum stress-mediated apoptosis

Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by γ-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), eIF-2α (eukaryotic initiation factor 2α), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1α by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer.

DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound.

Cytotoxic and Apoptosis-Inducing Activities of Triterpene Acids from Poria cocos

Six lanostane-type triterpene acids (1a-6a), isolated from Poria cocos , and their methyl ester (1b-6b) and hydroxy derivatives (1c-6c) were prepared. Upon evaluation of the cytotoxic activity of these compounds against leukemia (HL60), lung (A549), melanoma (CRL1579), ovary (NIH:OVCAR-3), breast (SK-BR-3), prostate (DU145), stomach (AZ521), and pancreas (PANC-1) cancer cell lines, 11 compounds (5a, 6a, 2b-5b, 1c, and 3c-6c) exhibited activity with single-digit micromolar IC(50) values against one or more cell lines. Poricotriol A (1c), a hydroxy derivative of poricoic acid A (1a), exhibited potent cytotoxicities against six cell lines with IC(50) values of 1.2-5.5 μM. Poricotriol A induced typical apoptotic cell death in HL60 and A549 cells on evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis in HL60 cells showed that poricotriol A activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that poricotriol A induced apoptosis via both mitochondrial and death receptor pathways in HL60. On the other hand, poricotriol A did not activate caspases-3, -8, and -9, but induced translocation of apoptosis-inducing factor (AIF) from mitochondria and increased the ratio of Bax/Bcl-2 in A549. This suggested that poricotriol A induced apoptosis via the mitochondrial pathway mostly by translocation of AIF, independent from the caspase pathway in A549. Furthermore, poricotriol A was shown to possess high selective toxicity in lung cancer cells since it exhibited only weak cytotoxicity against a normal lung cell line (WI-38).