Apoptosis In Human Tumors Research Articles

C-Jun N-terminal kinase signalling pathway in response to cisplatin.

Cisplatin (cis diamminedichloroplatinum II, cDDP) is one of the most effective cancer chemotherapeutic agents and is used in the treatment of many types of human malignancies. However, inherent tumour resistance is a major barrier to effective cisplatin therapy. So far, the mechanism of cDDP resistance has not been well defined. In general, cisplatin is considered to be a cytotoxic drug, for damaging DNA and inhibiting DNA synthesis, resulting in apoptosis via the mitochondrial death pathway or plasma membrane disruption. cDDP−induced DNA damage triggers signalling pathways that will eventually decide between cell life and death. As a member of the mitogen‐activated protein kinases family, c‐Jun N‐terminal kinase (JNK) is a signalling pathway in response to extracellular stimuli, especially drug treatment, to modify the activity of numerous proteins locating in the mitochondria or the nucleus. Recent studies suggest that JNK signalling pathway plays a major role in deciding the fate of the cell and inducing resistance to cDDP‐induced apoptosis in human tumours. c‐Jun N‐terminal kinase regulates several important cellular functions including cell proliferation, differentiation, survival and apoptosis while activating and inhibiting substrates for phosphorylation transcription factors (c‐Jun, ATF2: Activating transcription factor 2, p53 and so on), which subsequently induce pro‐apoptosis and pro‐survival factors expression. Therefore, it is suggested that JNK signal pathway is a double‐edged sword in cDDP treatment, simultaneously being a significant pro‐apoptosis factor but also being associated with increased resistance to cisplatin‐based chemotherapy. This review focuses on current knowledge concerning the role of JNK in cell response to cDDP, as well as their role in cisplatin resistance.

Effect of a Smac Mimetic (TL32711, Birinapant) on the Apoptotic Program and Apoptosis Biomarkers Examined with Validated Multiplex Immunoassays Fit for Clinical Use.

To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.

Apoptin Induced Tumour Specific Killing Via Protein Kinase C Isoforms: A Potential Therapeutic Strategy In Plasma Cell Dyscrasias

AbstractAbstract 5039There is mounting evidence that malignant cells have an intrinsic ability to prevent apoptosis. In the present study we provide evidence that the ectopic expression of Apoptin can restore the failing apoptosis program in myeloma cells via protein kinase C b (PKCb) and overcome intrinsic or acquired resistance to cell death. Apoptin (VP3), a chicken anemia virus (CAV)-derived protein has been shown to possess tumor specific cytotoxicity; its expression induces apoptosis in human tumor and transformed cells but there is little or no cytotoxic effect in normal human cells or cell lines derived from different tissues including peripheral blood mononuclear cells, fibroblast and epithelial cells. Several studies have shown that the tumor specific killing of Apoptin correlates with its phosphorylation and its subcellular localization. In cancer cells, Apoptin is localized in the nucleus and is phosphorylated on threonine108 by an as yet unknown kinase, whereas in normal cells Apoptin is detected in the cytoplasm and is essentially unphosphorylated. We developed a lentiviral vector encoding a GFP-Apoptin fusion gene (LV-GFP-AP), which delivers the Apoptin gene efficiently to haematopoietic cells. Apoptin significantly and selectively killed a number of leukemia cell lines including K562, HL60, U937, KG1 and NB4. In particular, the dexamethasone resistant multiple myeloma cell line MM1.R and the dexamethasone sensitive cell line MM1.S were efficiently killed by Apoptin. In contrast normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays.In addition, we showed that the dexamethasone resistant MM1.R cells were considerably more susceptible to Apoptin induced cell death than the parental matched MM1.S cells. This correlated with increased phosphorylation and activation of the Apoptin protein in MM1.R cells. Expression profiling of MM1.R and MM1.S cells identified a number of differentially expressed kinases. PKCb was over-expressed 9 fold in MM1.R cells and we showed, by immunoprecipitation and in vivo kinase studies, that this kinase was responsible for Apoptin phosphorylation. Analysis of the Apoptin amino acid sequence for potential phosphorylation sites indicated seven putative phosphorylation sites corresponding to the PKC kinase consensus motifs (S/TXK/R or S/TXXK/R). These sites included Thr-108, which has been previously shown to be phosphorylated in tumor cells, but not in normal cells. In vitro studies showed that recombinant Apoptin protein was phosphorylated by recombinant GST-PKCb protein at the Thr-108 site. Addition of a PKCb specific inhibitor resulted in diminished Apoptin phosphorylation whilst an unrelated inhibitor had no such effect. Furthermore, shRNA knockdown or drug mediated inhibition of PKCb in vivo significantly reduced Apoptin phosphorylation. Finally, we found that Apoptin mediated cell death proceeded via the up-regulation of PKCb, activation of caspase-9/3, cleavage of the PKCd catalytic domain and down-regulation of MERTK and AKT protein kinases. Collectively these results demonstrate a novel pathway for Apoptin activation involving PKCb and PKCd.Our results show that Apoptin is able to effectively eliminate multiple myeloma cells which have become resistant to dexamethasone. In addition, this study has led to the identification of tumor specific cellular targets such as PKCb, whose modulation by shRNAs and small molecule drugs can induce strong anti-myeloma effects. Importantly, the evidence from our data suggests that protein kinase C inhibitors may have an important therapeutic role in plasma cell neoplasia. Disclosures:No relevant conflicts of interest to declare.

Abstract 4854: Inhibition of high-mobility group box 1 protein (HMGB1) release sensitizes cancer cells to chemotherapy

Abstract Metabolic and therapeutic stress can simultaneously provoke both adaptive and apoptotic responses in cancer cells. The sum of these programmed signals determines the fate of the cell: cell death or cell survival. As the catalogue of defects in pathways that control apoptosis in human tumors is expanding, understanding the significance of the alternative stress fate, autophagy, has become increasingly important. High-mobility group box 1 protein (HMGB1) is a highly conserved nuclear protein, acting as an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. In addition to its intranuclear roles, HMGB1 was also discovered recently to function as an extracellular signaling molecule during inflammation, cell differentiation, cell migration, and tumor metastasis. Ethyl pyruvate was first identified as an experimental agent that ameliorated inflammation, significantly reducing circulating levels of HMGB1 in animals with sepsis. Similarly, glycyrrhizin was identified as a direct small-molecule inhibitor of HMGB1 release and activity. To determine whether inhibition of HMGB1 release sensitizes cancer cells to chemotherapy, we pretreated cell with ethyl pyruvate or glycyrrhizin. These inhibitors attenuated melphalan-induced HMGB1 release. Moreover, inhibition of HMGB1 decreased autophagy by microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II/p62 levels assay, and increased apoptosis by flow cytometry and cleaved-poly (ADP-ribose) polymerase (PARP) assays in Panc2.03 and HCT116 tumor cells when treated with chemotherapy. To further characterize the role of HMGB1 release in cancer cells following chemotherapy, a target-specific short hairpin RNA (shRNA) to HMGB1 was transferred into Panc2.03 and HCT116 tumor cells. Transfection of HMGB1-shRNA led to a significant decrease in HMGB1 protein as well as release in these cells. Depletion of HMGB1 by shRNA in these cells decreased autophagy and rendered them significantly more sensitive to melphalan-induced apoptotic cell death. Thust HMGB1 release links autophagy and apoptosis, and serves as a potential drug target for therapeutic intervention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4854.

Celecoxib Toxicity Is Cell Cycle Phase Specific

Celecoxib inhibits proliferation and induces apoptosis in human tumors, but the molecular mechanisms for these processes are poorly understood. In this study, we evaluated the ability of celecoxib to induce toxicity in head and neck squamous cell carcinomas (HNSCC) and explored the relationships between celecoxib-induced cell cycle inhibition and toxicity in HNSCC. Celecoxib inhibited the proliferation of UM-SCC-1 and UM-SCC-17B cells both in vitro and in vivo, accompanied by G(1) phase cell cycle arrest and apoptosis. Celecoxib induced p21(waf1/cip1) at the transcriptional level independent of wild-type p53 function, leading to decreased expression of cyclin D1 and hypophosphorylation of Rb, with subsequent marked downstream decreases in nuclear E2F-1 protein expression and E2F transactivating activity by luciferase reporter assay. Cell cycle phase-specific cytometric sorting showed that celecoxib induced clonogenic toxicity preferentially to cells within the S phase greater than G(1) and G(2) phases. Levels of p21(waf1/cip1) and cyclin D1 protein were reduced in the S phase compared with the G(1) and G(2) phases, suggesting a possible protective role for p21(waf1/cip1) expression in celecoxib toxicity. In conclusion, we show that celecoxib has marked antiproliferative activity against head and neck cancer cells through transcriptional induction of p21(waf1/cip1) and G(1) phase accumulation leading to S phase-specific clonogenic toxicity. We additionally show that a profound inhibition of nuclear E2F function provides a possible mechanism for this S phase-specific toxicity.

619 POSTER Combined inhibition of P13K/AKT and MAPK esignaling is required to inhibit translational initiation and to induce apoptosis in human tumors

619 POSTER Combined inhibition of P13K/AKT and MAPK esignaling is required to inhibit translational initiation and to induce apoptosis in human tumors

A Novel Transcriptional Inhibitor Induces Apoptosis in Tumor Cells and Exhibits Antiangiogenic Activity

Using a high-throughput cell-based assay, we identified a nucleoside analogue 4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo(2,3-d)-pyrimidine-5-carboxamide (ARC), which has the properties of a general transcriptional inhibitor. Specifically, ARC inhibits the phosphorylation of RNA polymerase II by positive transcription elongation factor-b, leading to a block in transcriptional elongation. ARC was able to potently repress p53 targets p21 and hdm2 (human homologue of mdm2) protein levels, but dramatically increased p53 levels similar to other transcriptional inhibitors, including flavopiridol. This increase in p53 corresponded to the down-regulation of short-lived protein hdm2, which is a well-established negative regulator of p53. Remarkably, ARC induced potent apoptosis in human tumor and transformed, but not in normal cells, and possessed strong antiangiogenic activity in vitro. Although ARC promoted the accumulation of p53, ARC-induced apoptosis in tumor cells was p53-independent, suggesting that it may be useful for the treatment of tumors with functionally inactive p53. Furthermore, cell death induced by ARC had a strong correlation with down-regulation of the antiapoptotic gene survivin, which is often overexpressed in human tumors. Taken together, our data suggests that ARC may be an attractive candidate for anticancer drug development.

Novel N-thiolated β-lactam antibiotics selectively induce apoptosis in human tumor and transformed, but not normal or nontransformed, cells

Historically, it has been shown that the β-lactam antibiotics play an essential role in treating bacterial infections while demonstrating selectivity for prokaryotic cells. We recently reported that certain N-methylthio-substituted β-lactam antibiotics had DNA-damaging and apoptosis-inducing activities in various tumor cells. However, whether these compounds affect human normal or nontransformed cells was unknown. In the current study, we first show that a lead compound (lactam 1) selectively induces apoptosis in human leukemic Jurkat T, but not in the nontransformed, immortalized human natural killer (NK) cells. Additionally, we screened a library of other N-methylthiolated β-lactams to determine their structure–activity relationships (SARs), and found lactam 12 to have the highest apoptosis-inducing activity against human leukemic Jurkat T cells, associated with increased DNA-damaging potency. Furthermore, we demonstrate that lactam 12, as well as lactam 1, potently inhibits colony formation of human prostate cancer cells. We also show that lactam 12 induces apoptosis in human breast, prostate, and head-and-neck cancer cells. Finally, lactam 12 induces apoptosis selectively in Jurkat T and simian virus 40-transformed, but not in nontransformed NK and parental normal fibroblast, cells. Our results suggest that there is potential for developing this class of β-lactams into novel anticancer agents.

Perspectives for cancer therapies with cdk2 inhibitors.

Modern anticancer strategies are designed against specific molecular targets with the goal of sparing normal, non-neoplastic tissues. Choosing specific molecular targets, however, is problematic. Cdk2 (Cyclin dependent kinase 2, cell division kinase 2, p33) is an important candidate target for therapeutic intervention. Phosphorylation of retinoblastoma protein (pRb) by Cdk2 is the penultimate step in the transition from G1 to S phase. Inhibition of this step could potentially result in inhibition of proliferation, cytostasis and possibly apoptosis in human tumors. Cdk2 also plays a critical role in the transition through S phase and the S to G2 transition as well. Inhibitors of the cyclin dependent kinases, such as flavopiridol and UCN-01, are currently in clinical trials. While demonstrating clinical activity, neither acts specifically against Cdk2. Other more specific Cdk2 inhibitors are currently in preclinical development. Further studies to explore the therapeutic worth of such agents are warranted.

Inactivation of Myc-induced p53-dependent apoptosis in human tumors.

The Myc family of oncoproteins promote cell growth and are frequently overexpressed in human tumors. However, Myc can also trigger cell death by apoptosis. This is at least in part mediated via the ARF-p53 pathway. Myc activation leads to a selection for inactivation of ARF or p53, allowing cell survival and tumor progression. Restoration of p53-dependent apoptosis by various means is an attractive approach for new cancer therapy.

P53 regulates apoptosis in human retinoblastoma.

To determine whether apoptosis is a significant mode of cell death in human retinoblastoma (RB) and if it is regulated by the expression of p53. Apoptosis was analyzed using the criterion of internucleosomal DNA degradation as determined by agarose gel electrophoresis of DNA isolated from tumor specimens. Individual cells undergoing apoptosis were identified using terminal transferase-mediated biotin-dUTP nick end labeling (TUNEL) of fragmented DNA. The expression of p53 and WAF1 (a protein involved in p53-mediated cell cycle arrest) in human RB was determined by immunocytochemical analysis. The function of p53 in human RB cell lines was tested by transfecting them with a complementary DNA encoding a temperature-sensitive isoform of murine p53 under the control of a strong viral promoter. DNA from RB tumor specimens showed a strong nucleosomal ladder of DNA fragments typical of apoptosis. The TUNEL staining indicated that poorly and moderately differentiated cells in tumors were undergoing DNA fragmentation. Immunoreactivity for p53 was variable. Cells expressing low levels of p53 seemed viable and expressed WAF1. Cells expressing high levels of p53 were found immediately adjacent to cells undergoing apoptosis. Human RB cells in culture that were expressing a murine temperature-sensitive isoform of p53 died at temperatures that allow this protein to assume a wild-type conformation. Apoptotic cell death is prevalent in RB. The close association of p53-immunoreactive cells and cells undergoing apoptosis in human tumors, and the ability of exogenous p53 to stimulate cell death in cultured human RB cells, suggests that p53 plays a role in regulating cell death in RB.

Apoptosis and mitotic cell death: their relative contributions to normal-tissue and tumour radiation response

The target-cell theory of tissue responses is reviewed with reference to the radiosensitivity of proposed target cells in bone marrow, intestine, epidermis, and spermatogenesis. The difficulties in precisely identifying target cells using histological/cell marker criteria, and hence determining the role of their mode of death in tissue responses, are being circumvented to some extent by the recent use of mice deficient in gene products required for radiation-induced apoptosis. In this case cell death results from 'mitotic cell death' and e.g. in the case of p53, any remaining p53-independent apoptosis. In the p53 null mouse, cell survival levels are increased in bone marrow and intestine but decreased in the testis. Different interpretations, based on the lack of p53-dependent apoptosis or the lack of a permanently induced G1-arrest in the case of marrow fibroblasts, can be applied to the results for different cell types. Hence both apoptosis and mitotic cell death can variously be involved as contributing to target-cell sensitivity and hence to early reactions in these tissues after irradiation. It is still unclear whether, or how, the mode of cell death (apoptotic versus mitotic) determines the radiosensitivity and response of tumours. In experimental tumours, the levels of radiation-induced apoptosis have been shown to correlate both with the in vivo response to radiation and the degree of spontaneous apoptosis in the tumours. Measurements of spontaneous apoptosis in human tumours, however, have yielded conflicting data with high apoptotic levels significantly correlating with both good and poor prognosis in different studies. There is one report of a lack of relationship between intrinsic radiosensitivity and spontaneous apoptosis in cervical cancers. In contrast several studies have reported correlations between apoptosis levels and the degree of tumour cell proliferation. Tumour hypoxia has also been shown to increase apoptosis. These data suggest that tumour apoptosis may be a reflection of intrinsic radiosensitivity, tumour cell proliferation and tumour hypoxia. Its relative importance will probably be tumour type, size and stage related.

The extent of apoptosis in different types of high grade prostatic carcinoma.

Apoptosis is the genetically mediated mechanism by which individual cells are deleted from living tissues. The extent of apoptosis in human tumours has not been examined in detail. In this study, apoptotic indices were assessed semiquantitatively by light microscopy in routine haematoxylin and eosin (H & E) stained sections of 15 high-grade carcinomas of the prostate by light microscopy, using the 40 x objective, counting 2000-2500 tumour cells per case. The mean apoptotic index for nine solid undifferentiated carcinomas was 1.7% (range 0.2-2.4%), for three small cell undifferentiated carcinomas 25.2% (range 10-37%), and for three secondary transitional cell carcinomas of the prostate 5.5% (range 2.4-9.1%). In general, apoptosis was easily identified, using the 40 x objective. There were minor variations in apoptosis from field to field in each tumour, and greater variability in the apoptotic index was observed among the small cell undifferentiated carcinomas and transitional cell carcinomas. In conclusion, assessment of apoptosis in routine H & E sections of human tumours is feasible, but it remains to be determined whether the extent of apoptosis correlates with other determinants of biological behaviour.