Increase in intracellular PGE<Subscript>2</Subscript> induces apoptosis in Bax-expressing colon cancer cell

Background  

NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE₂ is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE₂ known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE₂ in tumour progression thus appears complex and multipurpose.     

A miR-21 inhibitor enhances apoptosis and reduces G<Subscript>2</Subscript>-M accumulation induced by ionizing radiation in human glioblastoma U251 cells

MicroRNAs (miRNAs) are small noncoding RNAs that take part in diverse biological processes by suppressing target gene expression. Elevated expression of miR-21 has been reported in many types of human cancers. Radiotherapy is a standard adjuvant treatment for patients with glioblastoma. However, the resistance of glioblastoma cells to radiation limits the success of this treatment. In this study, we found that miR-21 expression was upregulated in response to ionizing radiation (IR) in U251 cells, which suggested that miR-21 could be involved in the response of U251 cells to radiation. We showed that a miR-21 inhibitor enhanced IR-induced glioblastoma cell growth arrest and increased the level of apoptosis, which was probably caused by abrogation of the G₂-M arrest induced by IR. Further research demonstrated that the miR-21 inhibitor induced the upregulation of Cdc25A. Taken together, these findings suggest that miR-21 inhibitor can increase IR-induced growth arrest and apoptosis in U251 glioblastoma cells, at least in part by abrogating G₂-M arrest, and that Cdc25A is a potential target of miR-21.     

Dichloroacetate induces apoptosis and cell-cycle arrest in colorectal cancer cells

Background:

Cancer cells are highly dependent on glycolysis. Our aim was to determine if switching metabolism from glycolysis towards mitochondrial respiration would reduce growth preferentially in colorectal cancer cells over normal cells, and to examine the underlying mechanisms.

Methods:

Representative colorectal cancer and non-cancerous cell lines were treated with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase.

Results:

Dichloroacetate (20 mM) did not reduce growth of non-cancerous cells but caused significant decrease in cancer cell proliferation (P=0.009), which was associated with apoptosis and G₂ phase cell-cycle arrest. The largest apoptotic effect was evident in metastatic LoVo cells, in which DCA induced up to a ten-fold increase in apoptotic cell counts after 48 h. The most striking G₂ arrest was evident in well-differentiated HT29 cells, in which DCA caused an eight-fold increase in cells in G₂ phase after 48 h. Dichloroacetate reduced lactate levels in growth media and induced dephosphorylation of E1α subunit of pyruvate dehydrogenase complex in all cell lines, but the intrinsic mitochondrial membrane potential was reduced in only cancer cells (P=0.04).

Conclusions:

Pyruvate dehydrogenase kinase inhibition attenuates glycolysis and facilitates mitochondrial oxidative phosphorylation, leading to reduced growth of colorectal cancer cells but not of non-cancerous cells.     

Intracellular clusterin induces G2-M phase arrest and cell death in PC-3 prostate cancer cells1

Enhanced clusterin gene expression has been related frequently to organ remodeling, tissue involution, and cell death. Whether clusterin represents a leading cause or a consequence of apoptosis induction is still a matter of debate. Clusterin is known as an extracellular secreted glycoprotein in the mature form. However, truncated isoforms of the protein and nuclear localization of clusterin have been described recently in association to cell death. Here, we show the biological effects triggered in PC-3 androgen-independent prostate cancer cells by overexpression of an intracellular, not secreted form of clusterin (intracellular-clusterin). Transient transfection of PC-3 cells with intracellular-clusterin resulted in nuclear localization signal-independent massive nuclear localization of the protein leading to G2-M phase blockade followed by caspase-dependent apoptosis. Constitutive expression of intracellular-clusterin (pFLAG- intracellular-clusterin) in recombinant PC-3 cells caused clonogenic toxicity. The rare pFLAG-intracellular clusterin surviving clones showed inhibition of the proliferation rate and altered phenotype with impaired mitosis and endoreduplication. In these cells, caspase-independent cell death was induced. Impaired cell cycle progression in pFLAG-intracellular-clusterin clones was associated to arrest at the G2-M checkpoint by down-regulation of the mitotic complex cyclin B1/cyclin-dependent kinase 1. Intriguingly, intracellular-clusterin was localized exclusively in the cytoplasm in stably transfected cells, suggesting a negative correlation between nuclear clusterin accumulation and cell survival. These findings may possibly explain the conflicting results obtained in different laboratories, suggesting that clusterin might be a proapoptotic or a survival gene, also opening new perspectives for the characterization of androgen-independent and apoptosis-resistant prostate cancer cells.     

ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH<Subscript>2</Subscript>-terminal kinase activation

Introduction  

The present study was designed to determine the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N) as a novel therapeutic agent in human breast cancers.     

Estrogen receptor beta decreases survival of p53-defective cancer cells after DNA damage by impairing G<Subscript>2</Subscript>/M checkpoint signaling

Estrogen receptor beta (ERβ) inhibits proliferation in different cellular systems by regulating components of the cell cycle machinery. Eukaryotic cells respond to DNA damage by arresting in G₁, S, or G₂ phases of the cell cycle to initiate DNA repair. Most tumor cells due to disruptions in the p53-dependent G₁ pathway are dependent on S-phase and G₂/M checkpoints to maintain genomic integrity in response to DNA damage. We report that induction of ERβ expression causes abrogation of the S-phase, and the Chk1/Cdc25C-mediated G₂/M checkpoints after cisplatin and doxorubicin exposure in p53-defective breast cancer cells but not in p53 wild-type mammary cells. This impairment of DNA damage response that involves BRCA1 downregulation and caspase-2 activation results in mitotic catastrophe and decreased cancer cell survival. These results indicate that in cancers where p53 is defective, assessment of the presence of ERβ may be of predictive value for the successful response to chemotherapy.     

Statin induces apoptosis and cell growth arrest in prostate cancer cells.

Statins are a class of low molecular weight drugs that inhibit the rate-limiting enzyme of the mevalonate pathway 3-hydroxy-3-methylglutaryl-CoA reductase. Statins have been approved and effectively used to control hypercholesterolemia in clinical setting. Recent study showed statin's antitumor activity and suggested a potential role for prevention of human cancers. In this study, we did cell viability, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays to evaluate the action of statins on prostate cancer cells and used Western blotting and RhoA activation assay to investigate the underlying molecular mechanism of action. Our data showed that lovastatin and simvastatin effectively decreased cell viability in three prostate cancer cell lines (PC3, DU145, and LnCap) by inducing apoptosis and cell growth arrest at G(1) phase. Both lovastatin and simvastatin induced activation of caspase-8, caspase-3, and, to a lesser extent, caspase-9. Both statins suppressed expression of Rb, phosphorylated Rb, cyclin D1, cyclin D3, CDK4, and CDK6, but induced p21 and p27 expression in prostate cancer cells. Furthermore, lovastatin and simvastatin suppressed RhoA activation and c-JUN expression, but not cyclooxygenase-2 expression. Our data showed that the antitumor activity of statins is due to induction of apoptosis and cell growth arrest. The underlying molecular mechanism of statin's action is mediated through inactivation of RhoA, which in turn induces caspase enzymatic activity and/or G(1) cell cycle. Future studies should focus on examining statins and other apoptosis-inducing drugs (e.g., cyclooxygenase-2 inhibitors or curcumin) together to assess their efficacy in prevention of prostate cancer.     

A2B adenosine receptor agonist induces cell cycle arrest and apoptosis in breast cancer stem cells via ERK1/2 phosphorylation

Purpose

It has been reported that cancer stem cells (CSCs) may play a crucial role in the development, recurrence and metastasis of breast cancer. Targeting signaling pathways in CSCs is considered to be a promising strategy for the treatment of cancer. Here, we investigated the role of the A2B adenosine receptor (A2BAR) and its associated signaling pathways in governing the proliferation and viability of breast cancer cell line derived CSCs.

Methods

CSCs were isolated from the breast cancer cell lines MCF-7 and MDA-MB-231 using a mammosphere assay. The effect of the A2BAR agonist BAY606583 on cell proliferation was evaluated using XTT and mammosphere formation assays, respectively. Apoptosis was assessed using Annexin-V staining and cell cycle analyses were performed using flow cytometry. The expression levels of Bax, Bcl-2, cyclin-D1, CDK-4 and (phosphorylated) ERK1/2 were assessed using Western blotting.

Results

Our data revealed that the breast cancer cell line derived mammospheres were enriched for CSCs. We also found that A2BAR stimulation with its agonist BAY606583 inhibited mammosphere formation and CSC viability. In addition, we found that the application of BAY606583 led to CSC cell cycle arrest and apoptosis through the cyclin-D1/Cdk-4 and Bax/Bcl-2 pathways, respectively. Notably, we found that BAY606583 significantly down-regulated ERK1/2 phosphorylation in the breast cancer cell line derived CSCs.

Conclusions

From our results we conclude that A2BAR induces breast CSC cell cycle arrest and apoptosis through downregulation of the ERK1/2 cascade. As such, A2BAR may be considered as a novel target for the treatment of breast cancer.

     

Ascorbate modulation of H<Subscript>2</Subscript>O<Subscript>2</Subscript> and camptothecin-induced cell death in Jurkat cells

The effect of ascorbate on cell death was examined in Jurkat cells (human T-cell leukemia) by incubation with dehydroascorbate (DHA), which is rapidly taken up by cells and efficiently reduced to ascorbate. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of annexin V-labeled cells. In parallel, necrosis was estimated by the release of lactate dehydrogenase. Minor effects on cell death were observed when Jurkat cells were incubated with either DHA alone (100–1,000 M) or a single dose of 10 M H₂O₂. However, pre-incubation with DHA followed by exposure to H₂O₂ clearly stimulated both apoptosis and necrosis. In complete contrast, pre-incubation of cells with DHA significantly inhibited apoptosis, but did not affect necrosis, induced by the topoisomerase I inhibitor camptothecin. Our results indicate that intracellular ascorbate can modulate cell death in a manner which depends upon the nature of the apoptotic stimulus, which in turn has critical implications regarding the mechanism and potential application of ascorbate in cancer therapy.Abbreviations CPT 20-S-camptothecin lactone - DHA Dehydroascorbate - LDH Lactate dehydrogenase - Ac-DEVD-AMC Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin - PS Phosphatidylserine - GSH Glutathione - DTT Dithiothreitol     

Thimerosal induces apoptosis and G2/M phase arrest in human leukemia cells

Thimerosal is an organomercury compound with sulfhydryl-reactive properties. The ability of thimerosal to act as a sulfhydryl group is related to the presence of mercury. Due to its antibacterial effect, thimerosal is widely used as preservatives and has been reported to cause chemically mediated side effects. In the present study, we showed that the molecular mechanism of thimerosal induced apoptosis in U937 cells. Thimerosal was shown to be responsible for the inhibition of U937 cells growth by inducing apoptosis. Treatment with 2.5-5 microM thimerosal but not thiosalicylic acid (structural analog of thimerosal devoid of mercury) for 12 h produced apoptosis, G(2)/M phase arrest, and DNA fragmentation in a dose-dependent manner. Treatment with caspase inhibitor significantly reduced thimerosal-induced caspase 3 activation. In addition, thimerosal-induced apoptosis was attenuated by antioxidant Mn (III) meso-tetrakis (4-benzoic acid) porphyrin (Mn-TBAP). These data indicate that the cytotoxic effect of thimerosal on U937 cells is attributable to the induced apoptosis and that thimerosal-induced apoptosis is mediated by reactive oxygen species generation and caspase-3 activation.     

Down-regulation of Jagged-1 induces cell growth inhibition and S phase arrest in prostate cancer cells

Notch is an ancient cell signaling system that regulates cell fate specification, stem cell maintenance and initiation of differentiation in many tissues. It has been reported that Jagged-1, a Notch ligand, is significantly over expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues. Therefore, deregulation of Jagged-1 protein levels may play a role in prostate cancer cell growth and progression. Hence, the aim of our current study was to investigate the mechanistic role of Jagged-1 in prostate cancer cell growth and cell cycle progression. Our results show, for the first time, that down-regulation of Jagged-1 induces cell growth inhibition and S phase cell cycle arrest in prostate cancer cells, with reduced CDK2 kinase activity and increased p27 expression. These results suggest that Jagged-1 could be a potential therapeutic target for the treatment of prostate cancer.     

Bortezomib induces G2-M arrest in human colon cancer cells through ROS-inducible phosphorylation of ATM-CHK1

Colorectal cancer (CRC) is one of the most common cancers; however, the development of drugs to treat the condition has reached a plateau. Bortezomib (PS-341, Velcade?) is a proteasome inhibitor approved for the treatment of hematological malignancies, including multiple myeloma. A few trials of bortezomib, alone or in combination chemotherapy, for CRC patients have been reported; however, the results were largely inconclusive. This may be related to a lack of understanding of the drug's mechanism of action. Although bortezomib is reported to induce apoptosis and cell cycle arrest in various human cancer cells, the inhibitory mechanism involved is not clear. In this study, the effect of bortezomib as a treatment for human CRC was examined in vitro using three CRC cell lines. Bortezomib induced G2-M arrest in CRC cells. Investigation of G2-M phase-related cell cycle proteins involved in the response to bortezomib revealed that the ataxia telangiectasia mutated (ATM)-cell cycle checkpoint kinase 1 (CHK1) pathway, but not ATM and Rad3-related (ATR), was activated, resulting in the inactivation of cdc2. Bortezomib caused an increase in intracellular reactive oxygen species (ROS) and treatment with the ROS scavenger NAC inhibited phosphorylation of ATM leading to a decrease in the number of cells in G2-M phase. Thus, increased ROS levels after exposure to bortezomib resulted in ATM phosphorylation. In addition, knockdown of endogenous ATM by RNA interference resulted in decreased sensitivity to bortezomib. These results suggest that bortezomib induces G2-M arrest through ROS-inducible ATM phosphorylation and demonstrate that bortezomib is a potential candidate for further investigations in the treatment for CRC patients.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection in association with cell proliferation,apoptosis and prostaglandin E<Subscript>2</Subscript> levels

Objective:To evaluate the relationship between H.pylori infection with cell proliferation,apoptosis and PGE2 levels.Methods:A population-based study was conducted in Linqu,a high-risk area of gastric cancer in China.A total of 1523 subjects,aged 35-64,participating in a gastric cancer screening survey were investigated.H.pylori status were determined by 13C-urea breath test,expressions of Ki-67 were assessed by immunohistochemistry,apoptotic cells were detected by terminal deoxynucleotide transferase mediated dUTP nick end-labeling(TUNEL)method,and PGE2 levels were measured by enzyme immunoassay.Results:H.pylori infection was positively associated with cell proliferation activity.The mean and median percentage of Ki-67 labeling index(LI)in subjects with H.pylori positive were 14.1±10.3 and 12.0,significantly higher than those with H.pylori negative(?x±s:8.4±7.0;median:5.8;P<0.0001).Moreover,the prevalence rates of H.pylori infection showed a tendency to increase according to severity score of cell apoptosis(Ptrend <0.0001),from score 0 to 3,the percentage of H.pylori positivity increased from 67.5% to 96.7%.Furthermore,The mean and median of PGE2 concentration were 628.84±726.40 pg/mL and 411.33 pg/mL among subjects with H.pylori positive compared with 658.19±575.91pg/mL and 455.97 pg/mL among those with H.pylori negative(P=0.209).Conclusion:H.pylori infection was positively associated with increased cell proliferation and apoptosis activity,suggesting that H.pylori infection plays an important role in the gastric epithelial cell malignant transformation.     

Epigallocatechin-3-gallate induces apoptosis and cell cycle arrest in HTLV-1-positive and -negative leukemia cells

The objective of this study is to evaluate the efficacy of epigallocatechin gallate against ATL cells. The anti-proliferative and pro-apoptotic effects of EGCG were evaluated in HTLV-1-positive and -negative cells. EGCG exhibited a marked decrease in proliferation of ATL cells at 96 h of treatment. The results indicated that TGF-α was down-regulated whereas levels of TGF-β2 increased. Cell cycle distribution analysis revealed an increase in cells in the pre-G₁ phase which was confirmed by ELISA. The results on proteins showed an up-regulation of p53, Bax and p21 protein levels while the levels of Bcl-2α were down-regulated.     

Studies on the mechanism of arsenic trioxide-induced apoptosis in HepG<Subscript>2</Subscript> human hepatocellular carcinoma cells

OBJECTIVE To study the anti-tumor effect of arsenic trioxide on the HepG2 human hepatocellular carcinoma cell line, and to explore its mechanism of action. METHODS The MTT assay was used to determine the inhibitory effect of As2O3 on HepG2 cells at various As2O3 concentrations. The expression of p-JNK, caspase-3 and PARP was detected by Western blots. RESULTS As2O3 markedly inhibited the growth of the HepG2 cells and induced apoptosis. The results of Western blot analysis showed that the As2O3-induced apoptosis was accompanied by caspase-3 and PARP activation. p-JNK was detected at 10 min following As2O3 treatment, and preceded to peak at 20 min, and decreased by 30 min. The total protein content did not obviously change. The activation of JNK occurred prior to cell apoptosis. SP600125, a JNK inhibitor, suppressed the As2O3-induced activation of caspase-3 and PARP cleavage. CONCLUSION As2O3 inhibits the proliferation of human HepG2 hepatocellular carcinoma cells by inducing apoptosis in vitro. As2O3-induced apoptosis is accessed through the caspase-3 pathway. The JNK signal-transduction pathway and caspase-3 are involved upstream in the As2O3 induced HepG2 apoptotic response.     

EHMT1 knockdown induces apoptosis and cell cycle arrest in lung cancer cells by increasing CDKN1A expression

Dozens of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases such as cancer remain largely unclear. Here, we demonstrate the involvement of EHMT1, a histone lysine methyltransferase, in lung cancer. Immunohistochemical analysis indicated that the expression levels of EHMT1 are significantly elevated in human lung carcinomas compared with non‐neoplastic lung tissues. Through gene ontology analysis of RNA‐seq results, we showed that EHMT1 is clearly associated with apoptosis and the cell cycle process. Moreover, FACS analysis and cell growth assays showed that knockdown of EHMT1 induced apoptosis and G1 cell cycle arrest via upregulation of CDKN1A in A549 and H1299 cell lines. Finally, in 3D spheroid culture, compared to control cells, EHMT1 knockdown cells exhibited reduced aggregation of 3D spheroids and clear upregulation of CDKN1A and downregulation of E‐cadherin. Therefore, the results of the present study suggest that EHMT1 plays a critical role in the regulation of cancer cell apoptosis and the cell cycle by modulating CDKN1A expression. Further functional analyses of EHMT1 in the context of human tumorigenesis may aid in the development of novel therapeutic strategies for cancer.     

FR901228, a novel histone deacetylase inhibitor, induces cell cycle arrest and subsequent apoptosis in refractory human pancreatic cancer cells

Histone deacetylase (HDAC) inhibitors have antiproliferative activity against human cancer cells via cell cycle arrest, differentiation, and apoptosis. However, no report has focused on the apoptotic potential of HDAC inhibitors in refractory human pancreatic cancer. This study was designed to examine the apoptotic potential of FR901228, a novel HDAC inhibitor, in five human pancreatic cancer cell lines: Capan-1, BxPC-3, HPAF, Panc-1, and MIAPaCa-2. FR901228 markedly inhibited the proliferation of all five cell lines (IC50: 1-500 nM), with the greatest effect in MIAPaCa-2 cells. Treatment of each cell line with FR901228 (10-100 nM) caused cell cycle arrest at the G1 or G2/M phase and subsequent apoptosis. FR901228 induced expression of hyperacetylated histone H3 after 3 h of treatment and overexpression of p21Waf-1 after 6 h. In addition, FR901228 induced apoptosis by activating caspase-3, which led to cleavage of p21Waf-1 into a 15-kDa breakdown product and drove cancer cells from cell cycle arrest into apoptosis. FR901228 also decreased the protein level of survivin dramatically. Our results show that FR901228 markedly inhibits the growth of pancreatic cancer cells, not only through cell cycle arrest, but also through subsequent apoptosis; this was accompanied by caspase-3 activation, survivin degradation, and p21Waf-1 cleavage. FR901228 may prove clinically useful as an agent for refractory pancreatic cancers.