Pinellia pedatisecta agglutinin-based lectin blot analysis distinguishes between glycosylation patterns in various cancer cell lines

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer

International Journal of Gynecological Cancer ◽

10.1136/ijgc-00009577-200605000-00055 ◽

2006 ◽

Vol 16 (3) ◽

pp. 1314-1320 ◽

Cited By ~ 4

Author(s):

C. Leo ◽

L. C. Horn ◽

M. HÖCKEL

Keyword(s):

Cervical Cancer ◽

Cell Lines ◽

Cancer Cell ◽

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Cancer Cell Lines ◽

Cervical Cancer Cell ◽

Clinical Samples ◽

Cancer Tissue

Hypoxia plays a major role in the malignant progression of tumors. Here, we investigate the expression of Bcl-2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), a proapoptotic Bcl-2 family member, and its relationship to hypoxia in cervical cancer cell lines and clinical samples of cervical cancer. Cervical cancer cell lines were grown under hypoxia or normoxia, and BNIP3 mRNA expression was examined by Northern blot analysis. In 50 patients with cervical cancer, intratumoral oxygen measurement with the Eppendorf electrode and needle biopsies of the tumor were performed. The obtained tissue was subsequently analyzed by immunohistochemistry with an anti-BNIP3 antibody. Cervical cancer tissue collected upon surgery was used for Northern blot analysis of in vivo BNIP3 mRNA expression. BNIP3 mRNA is strongly induced under hypoxic conditions in all cervical cancer cell lines investigated. Furthermore, Northern blot analysis revealed that BNIP3 mRNA is expressed in cervical cancer tissue. Using immunohistochemistry, we demonstrated that BNIP3 protein is expressed in 82% of the investigated cervical cancers and that more advanced tumor stages showed significantly stronger BNIP3 expression. However, we observed no correlation between BNIP3 expression and intratumoral hypoxia. In conclusion, BNIP3 is expressed in different cervical cancer cell lines as well as in clinical samples of cervical cancer. Although BNIP3 is clearly hypoxia-inducible in vitro, our results suggest additional mechanisms of BNIP3 regulation in vivo. Our findings therefore highlight a discrepancy between in vitro models of tumor hypoxia and the complexity of human cancer.

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Loss of Beta-Catenin Expression in Metastatic Gastric Cancer

Journal of Clinical Oncology ◽

10.1200/jco.2003.10.017 ◽

2003 ◽

Vol 21 (9) ◽

pp. 1708-1714 ◽

Cited By ~ 53

Author(s):

Matthias P.A. Ebert ◽

Jun Yu ◽

Juliane Hoffmann ◽

Alba Rocco ◽

Christoph Röcken ◽

...

Keyword(s):

Gastric Cancer ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Blot Analysis ◽

Western Blot Analysis ◽

Gene Mutations ◽

Cancer Cell Lines ◽

Beta Catenin ◽

Gastric Cancers

Purpose: Beta-catenin (β-catenin) participates in intercellular adhesion and is an integral part of the Wnt signaling pathway. The role of β-catenin in the pathogenesis of gastric cancer and its metastasis is largely unknown. Patients and Methods: Immunohistochemistry and Western blot analysis were used to analyze the expression of β-catenin in 87 human gastric cancers, in metastasis and cancer cell lines. The β-catenin and the adenomatous polyposis coli (APC) genes were analyzed for gene mutations. Furthermore, methylation of the β-catenin promoter in cell lines was assessed by treatment with 5′-azadeoxycytidine and sodium bisulfite genomic sequencing. Results: β-Catenin expression was present at either the cell membrane or the cytoplasm in 34 of 75 primary gastric cancers. Expression of β-catenin was significantly more frequent in intestinal-type (P = .0049) and well-differentiated gastric cancers (P < .001). There were no quantitative differences between gastric cancers and the nonmalignant gastric tissues, as determined by Western blot analysis. One of 18 metastatic cancer lesions and four of five gastric cancer cell lines expressed β-catenin protein. N87 cells, derived from the liver metastasis of a gastric cancer, did not express β-catenin. Treatment with 5′-azadeoxycytidine restored β-catenin protein levels in this cell line, which exhibited significantly more 5-methylcytosines in the β-catenin promoter compared with the other cell lines. Conclusion: β-Catenin expression is lost in a subgroup of primary gastric cancers, is frequently absent in metastases, and exhibits nuclear localization in cancers with either β-catenin or APC gene mutations. Interestingly, the loss of β-catenin expression in metastatic gastric cancers may result from hypermethylation of the β-catenin promoter.

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Neferine-induced apoptosis is dependent on the suppression of Bcl-2 expression via downregulation of p65 in renal cancer cells

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmz061 ◽

2019 ◽

Vol 51 (7) ◽

pp. 734-742 ◽

Cited By ~ 3

Author(s):

Eun-Ae Kim ◽

Eon-Gi Sung ◽

In-Hwan Song ◽

Joo-Young Kim ◽

Hwa-Jung Sung ◽

...

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Renal Cancer ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cancer Cell Lines ◽

Induced Apoptosis

Abstract Neferine is an alkaloid extracted from a seed embryo of Nelumbo nucifera and has recently been shown to have anticancer effects in various human cancer cell lines. However, the detailed molecular mechanism of neferine-induced apoptosis has not been elucidated in renal cancer cells. In the present study, we observed that neferine induced inhibition of cell proliferation and apoptosis in Caki-1 cells in a dose-dependent manner by using MT assay and flow cytometry and that neferine-mediated apoptosis was attenuated by pretreatment with N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone, a pan-caspase inhibitor. Treatments with neferine dose-dependently downregulated B cell lymphoma-2 (Bcl-2) expression at the transcriptional level determined by reverse transcriptase-polymerase chain reaction. The forced expression of Bcl-2 and p65 attenuated the neferine-mediated apoptosis in Caki-1 cells. In addition, neferine induced apoptosis by downregulating Bcl-2 and p65 expression in the other two kidney cancer cell lines determined by flow cytometry and western blot analysis. Finally, we observed that treatment with neferine induced apoptosis by inhibiting the NF-κB pathway through caspase-mediated cleavage of the p65 protein by western blot analysis. Collectively, this study demonstrated that neferine-induced apoptosis is mediated by the downregulation of Bcl-2 expression via repression of the NF-κB pathway in renal cancer cells.

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Curcumin inhibits constitutive STAT3 phosphorylation in human pancreatic cancer cell lines and down-regulates survivin/BIRC5 gene expression

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.15030 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 15030-15030

Author(s):

W. Glienke ◽

L. Maute ◽

E. Milz ◽

N. Bauer ◽

L. Bergmann

Keyword(s):

Pancreatic Cancer ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Blot Analysis ◽

Protein Level ◽

Western Blot Analysis ◽

Cellular Proliferation ◽

Pancreatic Cancer Cell ◽

Cancer Cell Lines

15030 The anti-apoptotic gene survivin/BIRC5 plays an important role in cellular proliferation and survival of cancer cells. Regulated by constitutively activated STAT3, Survivin/BIRC5 may be a target for inhibiting cellular proliferation in pancreatic cancer cell lines. The purpose of this study was (a) to determine the effect of curcumin (diferuloylmethane) on survivin/BIRC5 expression and (b) a possible role of STAT3 activation in Survivin/BIRC5 expression in pancreatic cancer cell lines. We have incubated four pancreatic cancer cell lines with different amounts of curcumin. The expression of Survivin/BIRC5 on mRNA and protein level was measured with RT-PCR and western blot analysis. The activation of STAT3 through phosphorylation, contributing to the regulation of survivin was analyzed with western blot analysis. The incubation of the pancreatic cancer cell lines with curcumin resulted in a down-regulation of cellular proliferation in all cell lines tested but with different rates of inhibition. The expression of survivin/BIRC5 on mRNA and protein level was significantly down-regulated after treatment with 30 μM. Curcumin blocked the phosphorylation of STAT3 in a concentration-dependent manner. Treatment of pancreatic cancer cell lines with curcumin resulted in an induction of apoptosis as measured with FacScan analysis. We conclude that curcumin inhibits several key factors in cancer cellular pathways and may have the potential to be investigated in pancreatic cancer. No significant financial relationships to disclose.

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Combination of fulvestrant and lapatinib in non-HER2-overexpressing and adriamycin-resistant breast cancer cell lines

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.14050 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 14050-14050 ◽

Cited By ~ 1

Author(s):

A. M. Emde ◽

K. Maslak ◽

H. Liu ◽

A. E. Reles ◽

K. Possinger ◽

...

Keyword(s):

Breast Cancer ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Mtt Assay ◽

Blot Analysis ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Mcf7 Cells

14050 Background: We evaluated whether combination of lapatinib, a dual tyrosine kinase inhibitor against EGFR and HER2, and fulvestrant, a full estrogen receptor antagonist, is superior in EGFR and HER2 overexpressing and non-overexpressing breast cancer cell lines regarding cell growth inhibition and effects on the protein expression level on special proteins such as PDK1 and ERK1/2. Methods: MTT assay and western blot analysis were performed in the different breast cancer cell lines BT474, T47D, MCF-7 and Adriamycin resistant MCF7 cells. Incubation time was 72h. Concentration of both agents in western blot: 1x10exp-7M, in MTT assay 10exp-11M to 10exp-5M. Results: MTT assay showed a significant stronger proliferation inhibition by lapatinib and fulvestrant in BT474 cells and non- overexpressing T47D cells at a concentration of 10E-7M compared to single agent treatment. By western blot analysis, we found a synergistic downregulation of PDK1 in the combination treatment both in BT474 and AR MCF7 cells, whereas not in T47D cells. In MCF7 cells a downregulation of p-PDK1 was observed after treatment with fulvestrant alone as well as lapatinib plus fulvestrant. Regarding ERK1/2, a synergistic downregulation could be observed in AR MCF7 cells. A downregulation of a p-ERK was detected in MCF-7 cells after treatment with lapatinib, fulvestrant or both. Conclusion: A synergistic action of lapatinib and fulvestrant was observed in all 4 cell lines despite their different receptor status regarding EGFR, HER2 and ER alpha. Shadeo et al. (2005) showed different copy number profiles in these breast cancer cell lines regarding the examined pathways. We could show by western blot analysis that the combination treatment had inhibitory effect on these cell lines according to their individual up-regulated pathways. Altogether, this suggests that the combination is a promising treatment not only in EGFR and HER2 over-expressing breast cancer and that treatment effect is also dependent on up-regulated pathways more likely than receptor status. No significant financial relationships to disclose.

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Molecular characterization of human tensin

Biochemical Journal ◽

10.1042/bj3510403 ◽

2000 ◽

Vol 351 (2) ◽

pp. 403-411 ◽

Cited By ~ 30

Author(s):

Huaiyang CHEN ◽

Akiko ISHII ◽

Wai-Keung WONG ◽

Lan Bo CHEN ◽

Su Hao LO

Keyword(s):

Amino Acid ◽

Cell Lines ◽

Cancer Cell ◽

Focal Adhesion ◽

Blot Analysis ◽

Cancer Cell Lines ◽

Cag Repeat ◽

Actin Binding ◽

Breast Cancer Cell Lines ◽

Human Cdna

Tensin is a focal-adhesion molecule that binds to actin filaments and interacts with phosphotyrosine-containing proteins. To analyse tensin's function in mammals, we have cloned tensin cDNAs from human and cow. The isolated approx. 7.7-kb human cDNA contains an open reading frame encoding 1735 amino acid residues. The amino acid sequence of human tensin shares 60% identity with chicken tensin, and contains all the structural features described previously in chicken tensin. This includes the actin-binding domains, the Src homology domain 2, and the region similar to a tumour suppressor, PTEN. Two major differences between human and chicken tensin are (i) the lack of the first 54 residues present in chicken tensin, and (ii) the addition of 34- and 38-residue inserts in human and bovine tensin. In addition, our interspecies sequencing data have uncovered the presence of a glutamine/CAG repeat that appears to have expanded in the course of evolution. Northern-blot analysis reveals a 10-kb message in most of the human tissues examined. An additional 9-kb message is detected in heart and skeletal muscles. The molecular mass predicted from the human cDNA is 185kDa, although both endogenous and recombinant human tensin migrate as 220-kDa proteins on SDS/PAGE. The discrepancy is due to the unusually low electrophoretic mobility of the central region of the tensin polypeptide (residues 306–981). A survey of human prostate and breast cancer cell lines by Western-blot analysis shows a lack of tensin expression in most cancer cell lines, whereas these lines express considerable amounts of focal-adhesion molecules such as talin and focal-adhesion kinase. Finally, tensin is rapidly cleaved by a focal-adhesion protease, calpain II. Incubation of cells with a calpain inhibitor, MDL, prevented tensin cleavage and induced morphological change in these cells, suggesting that cleavage of tensin and other focal-adhesion constituents by calpain disrupts maintenance of normal cell shape.

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