Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Antiproliferative activity of rosamultic acid is associated with induction of apoptosis,cell cycle arrest,inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells

BackgroundGastric cancer is the second leading cause of cancer related deaths after lung cancer globally. Among natural products, natural triterpenes represent a structurally diverse group of organic compounds with potent antitumor activity.PurposeThe objective of the present research work demonstrated the antiproliferative and apoptotic activity of rosamultic acid, a natural triterpenoid, in human gastric cancer (SGC-7901) cells. Its effect on cellular morphology, cell cycle arrest, DNA fragmentation and expression levels of caspase-3, caspase-8 and caspase-9 were also determined.MethodsAntiproliferative activity of rosamultic acid was evaluated by MTT assay. Phase contrast, fluorescence microscopy as well as flow cytometry using Hoechst 33342, acridine orange/ethidium bromide and Annexin V-FITC as cellular probes were used to evaluate the induction of apoptosis by rosamultic acid. Protein level expressions were analyzed by western blot analysis.ResultsThe results revealed that rosamultic acid induced dose-dependent as well as time dependent cytotoxic effects in SGC-7901 gastric cancer cells. It also led to a reduction in clonogenic activity along with inhibiting the cell migration. Characteristic features of apoptosis induced by rosamultic acid were observed and quantified. Cell cycle arrest at sub-G1 phase was induced by rosamultic acid along with downregulation of expression levels of CDK4, CDK6 and cyclin D1. Rosamultic acid also significantly led to the activation of caspase-3, -8 and -9 during the 48 h treatment along with cleaving PARP in a dose-dependent manner. DNA fragmentation following rosamultic acid treatment was also observed in these cells.ConclusionThe current study strongly reveals that rosamultic acid inhibits gastric cancer proliferation by inducing apoptosis mediated through cell cycle arrest, downregulation of cell cycle related protein expressions, inhibition of cell migration, DNA damage, and activation of caspases.     

Kin3 protein,a NIMA-related kinase of Saccharomyces cerevisiae,is involved in DNA adduct damage response

Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.     

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3′-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.     

Comparison of mutagenic and recombinogenic effects of some adenine analogues in Saccharomyces cerevisiae D7

2-Aminopurine, 2-amino-N⁶-hydroxyadenine and N⁶-hydroxyaminopurine were compared in suspension test with growing and non-growing cells for their mutagenic and recombinogenic (reciprocal and nonreciprocal) activities in Saccharomyces cerevisiae strain D7. Ethyl methanesulfonate was used as a positive control. No increases above spontaneous frequencies were observed when non- growing cells were treated with the base analogues although EMS induced concentration- dependent responses at all 3 genetic end-points. When growing cells were treated, HAP was recombinogenic and mutagenic and AHA was mutagenic, but only weakly recombinogenic. HAP induced comparable numbers of revertants at much lower concentrations than AHA. 2AP failed to induce any detectable response even at concentrations as high as 2400 μg/ml.     

Phosphatases,DNA Damage Checkpoints and Checkpoint Deactivation

Cells have evolved intricate and specialized responses to DNA damage, central to which are the DNA damage checkpoints that arrest cell cycle progression and facilitate the repair process. Activation of these damage checkpoints relies heavily on the activity of Ser/Thr kinases, such as Chk1 and Chk2 (Saccharomyces cerevisiae Rad53), which are themselves activated by phosphorylation. Only more recently have we begun to understand how cells disengage the checkpoints to reenter the cell cycle. Here, we review progress toward understanding the functions of phosphatases in checkpoint deactivation in S. cerevisiae, focusing on the non-redundant roles of the type 2A phosphatase Pph3 and the PP2C phosphatases Ptc2 and Ptc3 in the deactivation of Rad53. We discuss how these phosphatases may specifically recognize different phosphorylated forms of Rad53 and how each may independently regulate different facets of the checkpoint response. In conjunction with the independent dephosphorylation of other checkpoint proteins, such regulation may allow a more tailored response to DNA damage that is coordinated with the repair process, ultimately resulting in the resumption of growth.     

Inhibition of root growth by narciclasine is caused by DNA damage-induced cell cycle arrest in lettuce seedlings

Narciclasine (NCS) is an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs. Its phytotoxic effects on plant growth were examined in lettuce (Lactuca sativa L.) seedlings. Results showed that high concentrations (0.5–5 μM) of NCS restricted the growth of lettuce roots in a dose-dependent manner. In NCS-treated lettuce seedlings, the following changes were detected: reduction of mitotic cells and cell elongation in the mature region, inhibition of proliferation of meristematic cells, and cell cycle. Moreover, comet assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay indicated that higher levels NCS (0.5–5 μM) induced DNA damage in root cells of lettuce. The decrease in meristematic cells and increase in DNA damage signals in lettuce roots in responses to NCS are in a dose-dependent manner. NCS-induced reactive oxygen species accumulation may explain an increase in DNA damage in lettuce roots. Thus, the restraint of root growth is due to cell cycle arrest which is caused by NCS-induced DNA damage. In addition, it was also found that NCS (0.5–5 μM) inhibited the root hair development of lettuce seedlings. Further investigations on the underlying mechanism revealed that both auxin and ethylene signaling pathways are involved in the response of root hairs to NCS.     

Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior

Cell death induced by oxidative insult targeted to mitochondrial interior of A431 cells was investigated. For stimulated production of ROS in the inner space of mitochondria, safranin-mediated photodynamic treatment (PDT) was employed. Another photosensitizer, mTHPC, which diffusely localizes to cellular membranes, was used for comparison. Cell response to the oxidative insult in mitochondrial interior was different from the response to the photodamage produced in cellular membranes. Autophagy and apoptotic features of cell death in response to mTHPC-PDT was observed in a wide range of PDT doses. Cell response to the oxidative stress in mitochondrial interior was dose-dependent. Damage up to CD50 did not reveal hallmarks of dead cells. At intermediate damage (CD50), cells manifested enhanced autophagy and reduced population of S-phase, but not apoptosis. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation, in addition to autophagy and cell cycle arrest.     

Inhibition of the formation of autophagosome but not autolysosome augments ABT-751-induced apoptosis in TP53-deficient Hep-3B cells

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel antimicrotubule drug, ABT-751, in a tumor protein p53 ( TP53)-deficient hepatocellular carcinoma-derived Hep-3B cells. A series of in vitro assays indicated that ABT-751 caused the disruption of the mitotic spindle structure, collapse of mitochondrial membrane potential, generation of reactive oxygen species, DNA damage, G ₂/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Hep-3B cells accompanied by alteration of the expression levels of several DNA damage checkpoint proteins and cell cycle regulators. Subsequently, ABT-751 triggered apoptosis along with markedly upregulated several proapoptotic proteins involving in extrinsic, intrinsic, and caspase-mediated apoptotic pathways. A pan-caspase inhibitor suppressed ABT-751-induced apoptosis. ABT-751 also induced autophagy soon after the occurrence of apoptosis through the suppression of AKT serine/threonine kinase/mechanistic target of rapamycin signaling pathway. Exogenous expression of the TP53 gene significantly incurred both apoptosis and autophagy in Hep-3B cells. Pharmacological inhibition of autophagosome (early autophagy) but not autolysosome (late autophagy) enhanced ABT-751-induced apoptosis in TP53-deficient Hep-3B cells. Our study provided a new strategy to augment ABT-751-induced apoptosis in TP53-deficient cells.     

Effects of dihydroxy gymnemic triacetate (DGT) on expression of apoptosis associated proteins in human prostate cancer cell lines (PC3)

Abstract

Prostate cancer is the most common malignancies among men. The present study is aimed at the investigation of dihydroxy gymnemic triacetate (DGT) from Gymnema sylvestre on mitochondrial apoptotic pathway and cell cycle arrest. Treatment of DGT resulted in a dose-dependent inhibition of growth of PC-3 cells. The cell cycle arrest was observed at the G2/M phase and accumulation of apoptotic cells was observed in DGT-treated prostate cancer cell lines. The occurrence of apoptosis in these cells was observed by DNA fragmentation. These events were associated with increased levels of pro-apoptotic proteins Bax, Bad and reduced levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. DGT also induces the activation of caspase-9 and caspase-3. The above results, clearly, suggest that DGT induces apoptosis by the intrinsic pathways which could be very useful for the treatment of prostate cancer.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells

Background: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet.Material and methods: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis.Results: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways.Conclusion: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.     

Apoptosis induced by ardipusilloside III through BAD dephosphorylation and cleavage in human glioblastoma U251MG cells

Ardipusilloside III is a saponin newly isolated from Ardisia pusilla A.DC. Since saponins have exhibited broad anti-cancer and pro-apoptotic activity, we investigated the ability of ardipusilloside III to induce apoptosis in human glioblastoma U251MG cells, as well as the involvement of apoptotic signaling pathways. Ardipusilloside III markedly suppressed proliferation of U251MG cells in a time- and dose-dependent manner (P < 0.05, IC₅₀ = 8.2 μg/ml), but did not affect the growth of primary cultures of human astrocytes. Ardipusilloside III-treated U251MG cells underwent typical apoptotic changes. Exposure to a low dose of ardipusilloside III provoked G₂/M-phase cell cycle arrest, which preceded apoptosis characterized by the appearance of cells with sub-G₁ DNA content. However, a higher dose of ardipusilloside III induced apoptosis without first causing cell cycle arrest. In addition, ardipusilloside III exposure resulted in time-dependent BAD dephosphorylation and cleavage as well as activation of caspase-8 and caspase-3. Therefore, both the intrinsic pathway of apoptosis, mediated by BAD dephosphorylation and cleavage, and the extrinisic pathway of apoptosis, mediated by caspase-8 and caspase-3 activation, were involved in ardipusilloside III-induced apoptosis. These data suggest that ardipusilloside III is a reliable candidate for chemotherapeutic treatment of human glioblastomas, and should be investigated further. Hong Lin, Xiang Zhang, Guang Cheng, and Hai-Feng Tang contributed equally to the work.     

Cell cycle and morphological alterations as indicative of apoptosis promoted by UV irradiation in S. cerevisiae

An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy. In this work, we analyzed DNA content and cellular morphology of S. cerevisiae after H₂O₂ or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry. A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G₁ population was pointed out by flow cytometry. Sub-G₁ cells were isolated by flow sorting and analyzed by electron microscopy. This population showed condensed chromatin in the nucleus and cell shrinking. This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.     

Hyperthermia and paraquat-induced G1 arrest in the yeastSaccharomyces cerevisiae is independent of the RAD9 gene

Mutants of the RAD9 gene ofSaccharomyces cerevisiae are defective in cell cycle checkpoint arrest in G1 and G2 after treatment with DNA-damaging agents. It is demonstrated that the pronounced G1 arrest observed in yeast after hyperthermia treatment or exposure to paraquat-generated superoxide radicals does not depend on a functional RAD9 gene. For both types of treatments, the mechanism of cell cycle arrest must be considered different from the activation of the DNA damage checkpoint response.