scholarly journals Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds ofBorreria hispidaon Lung Cancer (A549) and Cervical Cancer (HeLa) Cell Lines

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
S. Rupachandra ◽  
D. V. L. Sarada
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Protein Fraction ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Peptide Mass Fingerprinting ◽  
Dependent Manner ◽  
Peptide Mass ◽  
Hela Cell Lines ◽  
Apoptotic Activity

A 35 KDa protein referred to as F3 was purified from the seeds ofBorreria hispidaby precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase ofPyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds ofBorreria hispidawith antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells.

scholarly journals Anticancer Potential of Biogenic Silver Nanoparticles: A Mechanistic Study

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 707
Author(s):  
Mohd Shahnawaz Khan ◽  
Alya Alomari ◽  
Shams Tabrez ◽  
Iftekhar Hassan ◽  
Rizwan Wahab ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Cancer Cells ◽  
Cell Lines ◽  
Human Life ◽  
Mechanistic Study ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Cell Viability Assay ◽  
Hct 116 ◽  
Mcf 7

The continuous loss of human life due to the paucity of effective drugs against different forms of cancer demands a better/noble therapeutic approach. One possible way could be the use of nanostructures-based treatment methods. In the current piece of work, we have synthesized silver nanoparticles (AgNPs) using plant (Heliotropiumbacciferum) extract using AgNO3 as starting materials. The size, shape, and structure of synthesized AgNPs were confirmed by various spectroscopy and microscopic techniques. The average size of biosynthesized AgNPs was found to be in the range of 15 nm. The anticancer potential of these AgNPs was evaluated by a battery of tests such as MTT, scratch, and comet assays in breast (MCF-7) and colorectal (HCT-116) cancer models. The toxicity of AgNPs towards cancer cells was confirmed by the expression pattern of apoptotic (p53, Bax, caspase-3) and antiapoptotic (BCl-2) genes by RT-PCR. The cell viability assay showed an IC50 value of 5.44 and 9.54 µg/mL for AgNPs in MCF-7 and HCT-116 cell lines respectively. We also observed cell migration inhibiting potential of AgNPs in a concentration-dependent manner in MCF-7 cell lines. A tremendous rise (150–250%) in the production of ROS was observed as a result of AgNPs treatment compared with control. Moreover, the RT-PCR results indicated the difference in expression levels of pro/antiapoptotic proteins in both cancer cells. All these results indicate that cell death observed by us is mediated by ROS production, which might have altered the cellular redox status. Collectively, we report the antimetastasis potential of biogenic synthesized AgNPs against breast and colorectal cancers. The biogenic synthesis of AgNPs seems to be a promising anticancer therapy with greater efficacy against the studied cell lines.

scholarly journals Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 630
Author(s):  
Hawon Yoo ◽  
Seul-Ki Choi ◽  
Jaeok Lee ◽  
So Hyeon Park ◽  
You Na Park ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Anticancer Agents ◽  
Tumor Growth Inhibition ◽  
Dependent Manner ◽  
Hsp27 Expression ◽  
Cancer Aggressiveness

Relationships between heat shock protein 27 (HSP27) and cancer aggressiveness, metastasis, drug resistance, and poor patient outcomes in various cancer types including non-small cell lung cancer (NSCLC) were reported, and inhibition of HSP27 expression is suggested to be a possible strategy for cancer therapy. Unlike HSP90 or HSP70, HSP27 does not have an ATP-binding pocket, and no effective HSP27 inhibitors have been identified. Previously, NSCLC cancer cells were sensitized to radiation and chemotherapy when co-treated with small molecule HSP27 functional inhibitors such as zerumbone (ZER), SW15, and J2 that can induce abnormal cross-linked HSP27 dimer. In this study, cancer inhibition effects of NA49, a chromenone compound with better solubility, longer circulation time, and less toxicity than J2, were examined in combination with anticancer drugs such as cisplatin and gefitinib in NSCLC cell lines. When the cytotoxic drug cisplatin was treated in combination with NA49 in epidermal growth factor receptors (EGFRs) WT cell lines, sensitization was induced in an HSP27 expression-dependent manner. With gefitinib treatment, NA49 showed increased combination effects in both EGFR WT and Mut cell lines, also with HSP27 expression-dependent patterns. Moreover, NA49 induced sensitization in EGFR Mut cells with a secondary mutation of T790M when combined with gefitinib. Augmented tumor growth inhibition was shown with the combination of cisplatin or gefitinib and NA49 in nude mouse xenograft models. These results suggest the combination of HSP27 inhibitor NA49 and anticancer agents as a candidate for overcoming HSP27-mediated drug resistance in NSCLC patients.

scholarly journals Nuclear upregulation of PI3K p110β correlates with increased rRNA transcription in endometrial cancer cells

2019 ◽  
Author(s):  
Fatemeh Mazloumi Gavgani ◽  
Thomas Karlsson ◽  
Ingvild L Tangen ◽  
Andrea Papdiné Morovicz ◽  
Victoria Smith Arnesen ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Pi3k Pathway ◽  
Clinical Samples ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cytoplasmic Staining ◽  
Ec Cells

AbstractGenes encoding for components of the phosphoinositide 3-kinase (PI3K) pathway are frequently mutated in cancer, including inactivating mutations of PTEN and activating mutations of PIK3CA, encoding the PI3K catalytic subunit p110α. PIK3CB, encoding p110β, is rarely mutated, but can contribute to tumourigenesis in some PTEN-deficient tumours. The underlying molecular mechanisms are however poorly understood. By analysing cell lines and annotated clinical samples, we have previously found that p110β is highly expressed in endometrial cancer (EC) cell lines and that PIK3CB mRNA levels increase early in primary tumours correlating with lower survival. Selective inhibition of p110α and p110β led to different effects on cell signalling and cell function, p110α activity being correlated to cell survival in PIK3CA mutant cells and p110β with cell proliferation in PTEN-deficient cells. To understand the mechanisms governing the differential roles of these isoforms, we assessed their sub-cellular localisation. p110α was cytoplasmic whereas p110β was both cytoplasmic and nuclear with increased levels in both compartments in cancer cells. Immunohistochemistry of p110β in clinically annotated patient tumour sections revealed high nuclear/cytoplasmic staining ratio, which correlated significantly with higher grades. Consistently, the presence of high levels of p110β in the nuclei of EC cells, correlated with high levels of its product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in the nucleus. Using immunofluorescence labelling, we observed both p110β and PtdIns(3,4,5)P3 in the nucleoli of EC cell lines. The production of nucleolar PtdIns(3,4,5)P3 was dependent upon p110β activity. EC cells with high levels of nuclear PtdIns(3,4,5)P3 and p110β showed elevated nucleolar activity as assessed by the increase in 47S pre-rRNA transcriptional levels in a p110β-dependent manner. Altogether, these results present a nucleolar role for the PI3K pathway that may contribute to tumour progression in endometrial cancer.

scholarly journals Hesperidin Induces Mitochondria Mediated Intrinsic Apoptosis in HPV-positive Cervical Cancer Cells via Regulation of E6/p53 Expression

2020 ◽  
Author(s):  
Fang Ren ◽  
Gong Zhang ◽  
Caiyu Li ◽  
Gailing Li ◽  
Yuan Cao ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Protein Level ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
P53 Expression ◽  
Cervical Cancer Cells ◽  
Apoptotic Proteins

Abstract Background: Hesperetin, an active compound found in citrus fruits, possesses antiproliferative effects toward several types of cancer cell lines, including cervical cancer. In this study, we explore the antitumor effects of Hesperetin on the human cervical cancer human papilloma virus (HPV)-positive (CaSki and HeLa) and HPV-negative (C-33A) cell lines and further elucidated the underlying mechanisms of this action. Methods: Cell viability and proliferation was measured by the MTT assay and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, respectively. dUTP-fluorescein nick end-labeling (TUNEL) staining, Annexin V‑fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining and flow cytometry was used to assess the degree of apoptosis. JC-1 staining assay was used to evaluate the change of mitochondrial membrane potential (ΔΨm) and Western blot assays were used to determine apoptosis-related factors at protein level. Results: Hesperetin (100, 200 and 400 μM) exhibited a significant exclusive inhibitory effect against the growth of HPV-infected CaSki and HeLa cancer cells via induction of apoptosis in a concentration-dependent manner, while it was almost not active in HPV-negative C-33A cancer cells and normal cervix epithelial H8 cells. Moreover, this antitumor effect executed by Hesperetin was associated with disruption of ΔΨm, the release of cytochrome c from mitochondria, activation of pro-apoptotic proteins (Bax, cleaved caspase-3 and caspase-9) and inhibition of anti-apoptotic proteins (Bcl-2). During this process, cleaved caspase-8 remained unchanged. In addition, Hesperetin led to a downregulation of E6 oncoprotein expression and upregulation of tumor suppressor protein p53 level. Conclusions: Collectively, these results implicated that Hesperetin can induce apoptosis of HPV‑positive cervical cancer cells via a mitochondria-mediated intrinsic signaling pathway, together with the repression of E6 and enhancement of p53 protein level, indicating Hesperetin may be considered as a potential candidate for the development of innovative anti-HPV cervical cancer agents.

scholarly journals Autumn Royal and Ribier Grape Juice Extracts Reduced Viability and Metastatic Potential of Colon Cancer Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Manuel Valenzuela ◽  
Lorena Bastias ◽  
Iván Montenegro ◽  
Enrique Werner ◽  
Alejandro Madrid ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Grape Juice ◽  
Metastatic Potential ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Colon Cancer Cells ◽  
Gene Expressions ◽  
Anticancer Properties

Antioxidants are known to be beneficial to health. This paper evaluates the potential chemopreventive and anticancer properties of phenolic compounds present in grape juice extracts (GJE) from Autumn Royal and Ribier varieties. The effects of these GJE on viability (SRB day assay) and metastatic potential (migration and invasion parameters) of colon cancer cell lines HT-29 and SW-480 were evaluated. The effects of GJE on two matrix metalloproteinase gene expressions (MMP2 and MMP9) were also evaluated via qRT-PCR. In the former, GJE reduced cell viability in both cell lines in a dose-dependent manner. GJE treatment also reduced cell migration and invasion. Moreover, MMP-2 and MMP-9 gene expression diminished depending on extract and on cell type.Conclusions. These results provide novel information concerning anticancer properties of selected GJE by revealing selective cytotoxicity and the ability to reduce invasiveness of colon cancer cells.

scholarly journals Comparison of the effect of rhodium citrate-associated iron oxide nanoparticles on metastatic and non-metastatic breast cancer cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Natalia Lemos Chaves ◽  
Danilo Aquino Amorim ◽  
Cláudio Afonso Pinho Lopes ◽  
Irina Estrela-Lopis ◽  
Julia Böttner ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Breast Cancer Cells ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Cytotoxic Action ◽  
Dependent Manner ◽  
Metastatic Cells ◽  
Mcf 7

Abstract Background Nanocarriers have the potential to improve the therapeutic index of currently available drugs by increasing drug efficacy, lowering drug toxicity and achieving steady-state therapeutic levels of drugs over an extended period. The association of maghemite nanoparticles (NPs) with rhodium citrate (forming the complex hereafter referred to as MRC) has the potential to increase the specificity of the cytotoxic action of the latter compound, since this nanocomposite can be guided or transported to a target by the use of an external magnetic field. However, the behavior of these nanoparticles for an extended time of exposure to breast cancer cells has not yet been explored, and nor has MRC cytotoxicity comparison in different cell lines been performed until now. In this work, the effects of MRC NPs on these cells were analyzed for up to 72 h of exposure, and we focused on comparing NPs’ therapeutic effectiveness in different cell lines to elect the most responsive model, while elucidating the underlying action mechanism. Results MRC complexes exhibited broad cytotoxicity on human tumor cells, mainly in the first 24 h. However, while MRC induced cytotoxicity in MDA-MB-231 in a time-dependent manner, progressively decreasing the required dose for significant reduction in cell viability at 48 and 72 h, MCF-7 appears to recover its viability after 48 h of exposure. The recovery of MCF-7 is possibly explained by a resistance mechanism mediated by PGP (P-glycoprotein) proteins, which increase in these cells after MRC treatment. Remaining viable tumor metastatic cells had the migration capacity reduced after treatment with MRC (24 h). Moreover, MRC treatment induced S phase arrest of the cell cycle. Conclusion MRC act at the nucleus, inhibiting DNA synthesis and proliferation and inducing cell death. These effects were verified in both tumor lines, but MDA-MB-231 cells seem to be more responsive to the effects of NPs. In addition, NPs may also disrupt the metastatic activity of remaining cells, by reducing their migratory capacity. Our results suggest that MRC nanoparticles are a promising nanomaterial that can provide a convenient route for tumor targeting and treatment, mainly in metastatic cells.

scholarly journals Anticancer Potential of Fruit Extracts from Vatica diospyroides Symington Type SS and Their Effect on Program Cell Death of Cervical Cancer Cell Lines

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Atchara Chothiphirat ◽  
Kesara Nittayaboon ◽  
Kanyanatt Kanokwiroon ◽  
Theera Srisawat ◽  
Raphatphorn Navakanitworakul
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Morphological Changes ◽  
Annexin V ◽  
Siha Cell ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Cervical Cancer Cells ◽  
Dose Dependent

Vatica diospyroides Symington is locally known as Chan-Ka-Pho in Thailand. Ancient people have used it as therapeutic plant for cardiac and blood tonic cure. The purpose of this study was to investigate the potential cytotoxicity and selectivity of the extracts from V. diospyroides type SS fruit on cervical cancer HeLa and SiHa cell lines and to examine its underlying mechanism of action. MTT assay revealed that the extracts showed inhibition of cell survival in a dose-dependent manner and exhibited highly cytotoxic activity against both HeLa and SiHa cells with IC50 value less than 20 μg/mL along with less toxicity against L929 cells. Acetone cotyledon extract (ACE) showed the best selectivity index value of 4.47 (HeLa) and 3.51 (SiHa). Distinctive morphological changes were observed in ACE-treated cervical cancer cells contributing to apoptosis action. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that ACE induced apoptosis in HeLa and SiHa cell lines in a dose-dependent manner. Treatment of ACE with half IC50 caused DNA fragmentation and also activated increasing of bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. The results suggest that ACE has potent and selective cytotoxic effect against cervical cancer cells and the potential to induce bax and caspase-8-dependent apoptosis. Hence, the ACE could be further exploited as a potential lead in cancer treatment.

HIF-1 Supports the Survival of Multiple Myeloma Cells through the Induction of Survivin Gene.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 110-110 ◽  
Author(s):  
Keita Kirito ◽  
Hu Yongzhen ◽  
Kozue Yoshida ◽  
Toru Mitsumori ◽  
Kei Nakajima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cancer Cells ◽  
Cell Lines ◽  
Dependent Manner ◽  
Survivin Mrna ◽  
Myeloma Cells ◽  
Survivin Gene ◽  
Growth And Survival ◽  
Induced Apoptosis

Abstract In spite of the recent development of therapeutic strategies, multiple myeloma (MM) still remains incurable. Several cytokines and chemokines contribute to progression of the disease and acquisition of resistance to chemotherapy. These humoral factors support the growth and survival of myeloma cells through the regulation of transcription factors including NF-κB, Stat3 and FOXO3a. Hypoxia inducible factor-1 (HIF-1) is an important transcription factor that is activated under low oxygen tension and controls dozens of genes involved in angiogenesis, energy production and resistance to apoptosis. Interestingly, HIF-1 is frequently activated in cancer cells even under normoxic condition and it is well established that HIF-1 expression and activation correlates with tumor progression and resistance to cancer treatments. In this study, we investigated whether HIF-1 is involved in the biology of multiple myeloma. To this end, we used three MM cell lines U266, RPMI8226 and KMM-1. After informed consent, we also prepared primary MM cells from bone marrow samples of patients (n=5) using anti-CD138 magnetic beads. Initially, we treated MM cells with insulin-like growth factor-1 (IGF-1) and IL-6, both of which are major growth and survival factors for myeloma cells. Treatment with IGF-1 and, to be a lesser degree, IL-6 clearly enhanced expression of HIF-1α, a subunit of HIF-1, in all three cell lines. Similar results were obtained from isolated primary MM cells. Based on several lines of evidence that survivin, a member of inhibitor of apoptosis (IAP) family protein, is transcriptionally regulated by HIF-1 in breast cancer cells, and that this anti-apoptotic factor is important for growth of MM cells, we examined whether HIF-1 supports the survival of MM cells through the induction of survivin. Quantitative RT-PCR assay revealed that IGF-1 increased survivin mRNA both in MM cell lines and in primary MM cells. In addition, IGF-1 activated survivin gene promoter containing a HIF-1-binding site. To confirm that IGF-1-induced activation of survivin gene is mediated by HIF-1, we treated MM cell lines with echinomycin, an inhibitor of DNA-binding activity of HIF-1. As expected, echinomycin inhibited IGF-1-induced survivin gene expression in a dose-dependent manner. The inhibitor also induced apoptosis of MM cells, and IGF-1 could not rescue the MM cells from echinomycin-induced apoptosis. Furthermore, echinomycin enhanced melphalan-induced apoptosis of MM cells. To further examine the involvement of HIF-1 in IGF-1-induced survivin gene expression, we generated three independent HIF-1α knockdown KMM-1 clones using siRNA system. Survivin mRNA was not detected in the HIF-1α knockdown cells, and these clones easily underwent apoptosis even in the presence of IGF-1, compared to the parental cells. Taken together, HIF-1 plays a pivotal role in survival of MM cells through the induction of survivin gene. In conclusion, HIF-1 might be an attractive therapeutic target for MM.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Identification of a Novel Auto-Antibody Highly Prevalent in Patients with Hepatitis-Associated and Idiopathic Aplastic Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3200-3200
Author(s):  
Hiroyuki Takamatsu ◽  
Zhirong Qi ◽  
Tomoyuki Sakurai ◽  
Luis Espinoza ◽  
Naomi Sugimori ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Peptide Mass Fingerprinting ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Healthy Individuals ◽  
Peptide Mass ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract Abstract 3200 Poster Board III-137 Hepatitis-associated aplastic anemia (HAA) is a subset of acquired AA that is highly responsive to immunosuppressive therapy. The target antigens of the immune system attack in HAA are thought to be a protein shared by both liver and hematopoietic stem cells, since it is usually associated with severe hepatitis of unknown etiology. Screening sera from patients with HAA for the presence of antibodies (Abs) recognizing liver cell-derived proteins may be useful in identifying novel auto-antigens in AA. To test this hypothesis, sera from HAA patients were examined using immunoblotting with a lysate of a hepatocellular carcinoma cell line Huh7 and subsequent peptide mass fingerprinting. Methods and Results The serum of a patient with typical HAA (a 23 year-old male) possessing a small population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells was used for Western blotting (WB) with the lysates of Huh7. A distinct band of 70 kDa protein was revealed. The same band was revealed when the culture supernatant of Huh7 cells was subjected to WB. The peptide mass fingerprinting of the 70 kDa band identified this protein to be heat shock protein (HSP) 72. HSP72 is a stress-inducible protein and extracellular HSP72 enhances the cytotoxicity of CD4+ T cells and NK cells. An examination of the sera from HAA patients, idiopathic acquired AA (IAA) patients and healthy individuals with WB revealed the anti-HSP72 Abs to be detected in 10 of 12 (83%) HAA patients and in 57 of 80 (71%) IAA patients while it was detected only in 8 of 59 (14%) healthy individuals. The prevalence of anti-HSP72 Abs in AA was markedly higher than that of anti-kinectin Abs (39%), anti-PMS1 Abs (10%), anti-DRS-1 Abs (38%) or anti-moesin Abs (37%) reported previously. Anti-HSP72 Abs were frequently detectable both in patients with IAA possessing PNH-type cells (63%) and in patients without PNH-type cells (86%), a finding contrasting to the higher prevalence of anti-DRS-1 Abs and anti-moesin Abs in patients with PNH-type cells than in those without PNH-type cells reported previously. Although anti-HSP72 Abs were detectable in the sera of patients with rheumatoid arthritis and systemic lupus erythematosus, the prevalence was 15% (4 of 27) and 20% (1 of 5), respectively. In contrast to a previous report that detected anti-HSP72 Abs in 24% of patients with chronic hepatitis C, WB failed to detect the Abs in the sera of 4 patients with autoimmune hepatitis and 5 with hepatitis B or C. Ten patients with HAA were treated with immunosuppressive therapy, and 7 of the 8 responders expressed anti-HSP72 Abs. The quantification of the gene expression level of HSP72 by blood cells using real-time PCR demonstrated that the HSP72 mRNA levels were markedly higher in myeloid leukemia cell lines as well as CD34+ cells isolated from 3 healthy individuals in comparison to that in lymphoid or monocytoid leukemia cell lines. HSP72/GAPDH ratios of PBMCs and CD34+ cells from 3 healthy individuals, K562, KH88, OUN-1 were 0.51, 1.31, 1.02, 0.07 and 0.09 respectively. Other leukemia cell lines such as Daudi, Molt-4 and THP-1 did not display detectable levels of HSP72 mRNA. The cell surface expression of HSP72 was examined in various kinds of leukemia cell lines and CD34+ bone marrow (BM) cells derived from 3 healthy individuals using Ab to HSP72 (Clone C92F3A-5) because previous studies demonstrated heat-inducible expression of HSP72 by K562. Flow cytometry detected cell surface HSP72 on immature CML cell lines such as K562 but not on CD34+ BM cells, acute promyelocytic leukemia cell lines such as NB-4 and HL-60, and lymphoid leukemia cell lines such as Molt-4 and Daudi. Exposure to 42°C for 2 h increased the HSP72 expression on K562 cells and Molt-4 cells but not on CD34+ cells. Conclusion Anti-HSP72 Ab is the most prevalent auto-Ab in AA among the auto-Abs previously detected. Given the increased expression of HSP72 by immature myeloid cells as well as stress-inducible cell surface expression of the molecule, immune responses to HSP72 may thus play an essential role in the pathogenesis of HAA and IAA. Disclosures No relevant conflicts of interest to declare.

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