Ira Skvortsova

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation‐resistant prostate cancer cell lines, LNCaP‐IRR, PC3‐IRR, and Du145‐IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP‐IRR, PC3‐IRR, and Du145‐IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras‐mitogen‐activated protein kinase (MAPK) and phosphatidyl inositol 3‐kinase (PI3K)‐Akt and Jak‐STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2‐D DIGE followed by computational image analysis and MS. Twenty‐seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

MPC-6827: A Small-Molecule Inhibitor of Microtubule Formation That Is Not a Substrate for Multidrug Resistance Pumps

A novel series of 4-arylaminoquinazolines were identified from a cell-based screening assay as potent apoptosis inducers. Through structure-activity relationship studies, MPC-6827 and its close structural analogue, MPI-0441138, were discovered as proapoptotic molecules and mitotic inhibitors with potencies at low nanomolar concentrations in multiple tumor cell lines. Photoaffinity and radiolabeled analogues of MPC-6827 were found to bind a 55-kDa protein, and this binding was competed by MPC-6827, paclitaxel, and colchicine, but not vinblastine. MPC-6827 effectively inhibited the polymerization of tubulin in vitro, competed with colchicine binding, and disrupted the formation of microtubules in a variety of tumor cell lines, which together showed the molecular target as tubulin. Treatment of MCF-7 breast carcinoma or Jurkat leukemia cells with MPC-6827 led to pronounced G2-M cell cycle arrest followed by apoptosis. Apoptosis, as determined by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay, was preceded by loss of mitochondrial membrane potential, cytochrome c translocation from mitochondria to nuclei, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. MPC-6827 was equipotent in an in vitro growth inhibition assay in several cancer cell lines regardless of the expression levels of the multidrug resistance ABC transporters MDR-1 (Pgp-1), MRP-1, and BCRP-1. In B16-F1 allografts and in OVCAR-3, MIAPaCa-2, MCF-7, HT-29, MDA-MB-435, and MX-1 xenografts, statistically significant tumor growth inhibition was observed with MPC-6827. These studies show that MPC-6827 is a microtubule-disrupting agent with potent and broad-spectrum in vitro and in vivo cytotoxic activities and, therefore, MPC-6827 is a promising candidate for development as a novel therapeutic for multiple cancer types.

Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse

PURPOSE Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. METHODS AND MATERIALS Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. RESULTS In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. CONCLUSIONS The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E-cadherin, vimentin) may be considered as potential biomarkers to predict squamous cell carcinoma response after treatment with cetuximab in combination with radiation.

Crosstalk between DNA repair and cancer stem cell (CSC) associated intracellular pathways

DNA damaging agents (ionizing radiation and chemotherapeutics) are considered as most effective in cancer treatment. However, there is a subpopulation of carcinoma cells within the tumour demonstrating resistance to DNA damaging treatment approaches. It is suggested that limited tumour response to this kind of therapy can be associated with specific molecular properties of carcinoma stem cells (CSCs) representing the most refractory cell subpopulation. This review article presents novel data about molecular features of CSCs underlying DNA damage response and related intracellular signalling.

99mTc-labelled HYNIC-minigastrin with reduced kidney uptake for targeting of CCK-2 receptor-positive tumours

PurposeDifferent attempts have been made to develop a suitable radioligand for targeting CCK-2 receptors in vivo, for staging of medullary thyroid carcinoma (MTC) and other receptor-expressing tumours. After initial successful clinical studies with [DTPA0,DGlu1]minigastrin (DTPA-MG0) radiolabelled with 111In and 90Y, our group developed a 99mTc-labelled radioligand, based on HYNIC-MG0. A major drawback observed with these derivatives is their high uptake by the kidneys. In this study we describe the preclinical evaluation of the optimised shortened peptide analogue, [HYNIC0,DGlu1,desGlu2–6]minigastrin (HYNIC-MG11).Methods99mTc labelling of HYNIC-MG11 was performed using tricine and EDDA as coligands. Stability experiments were carried out by reversed phase HPLC analysis in PBS, PBS/cysteine and plasma as well as rat liver and kidney homogenates. Receptor binding and cell uptake experiments were performed using AR4-2J rat pancreatic tumour cells. Animal biodistribution was studied in AR4-2J tumour-bearing nude mice.ResultsRadiolabelling was performed at high specific activities and radiochemical purity was >90%. 99mTc-EDDA-HYNIC-MG11 showed high affinity for the CCK-2 receptor and cell internalisation comparable to that of 99mTc-EDDA-HYNIC-MG0. Despite high stability in solution, a low metabolic stability in rat tissue homogenates was found. In a nude mouse tumour model, very low unspecific retention in most organs, rapid renal excretion with reduced renal retention and high tumour uptake were observed.Conclusion99mTc-EDDA-HYNIC-MG11 shows advantages over 99mTc-EDDA-HYNIC-MG0 in terms of lower kidney retention with unchanged uptake in tumours and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labelling process still leave room for further improvement.

Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib

Targeting the ubiquitin-proteasome pathway with the proteasome inhibitor bortezomib has emerged as a promising approach for the treatment of several malignancies. The cellular and molecular effects of this agent on colorectal cancer cells are poorly characterized. This study investigated the antiproliferative effect of bortezomib on colorectal cancer cell lines (Caco-2 and HRT-18). In order to define the proteins potentially involved in the mechanisms of action, proteome profiling was applied to detect the proteins altered by bortezomib. The in vitro efficacy of bortezomib as a single agent in colorectal cancer cell lines was confirmed. Proteome profiling with two-dimensional PAGE followed by mass spectrometry revealed the up-regulation of the major inducible isoform of heat shock protein 70 (hsp72) and lactate dehydrogenase B in both cell lines, as well as the induction of aldo-keto reductase family 1 member B10 (AKR1B10) in HRT-18 cells. Both AKR1B10 and hsp72 exert cell-protective functions. This study shows for the first time a bortezomib-induced up-regulation of AKR1B10. Small interfering RNA–mediated inhibition of this enzyme with known intracellular detoxification function sensitized HRT-18 cells to therapy with the proteasome inhibitor. To further characterize the relevance of AKR1B10 for colorectal tumors, immunohistochemical expression was shown in 23.2% of 125 tumor specimens. These findings indicate that AKR1B10 might be a target for combination therapy with bortezomib. [Mol Cancer Ther 2009;8(7):1995–2006]

Cetuximab inhibits thymidylate synthase in colorectal cells expressing epidermal growth factor receptor

The monoclonal antibody cetuximab directed against the epidermal growth factor receptor (EGFR) is an attractive agent for targeted therapy in advanced colorectal cancer (CRC), especially when combined with 5‐fluorouracil (5‐FU)‐based chemotherapy. However, the mechanisms of cetuximab activity as chemosensitizer remain poorly understood. Using proteome‐fluorescence‐based technology, we found that cetuximab is able to suppress the expression of thymidylate synthase (TS), which is involved in the mechanism of 5‐FU action. Caco‐2, HRT‐18, HT‐29, WiDr and SW‐480 CRC cells were found to express EGFR. SW‐620 was used as EGFR‐negative cell line. Only in EGFR‐expressing cells cetuximab is able to inhibit TS expression. Combined treatment with cetuximab and 5‐FU revealed a synergistic anti‐tumor response that is closely correlated with functional activity of EGFR/mitogen‐activated protein kinase (MAPK) pathway. Moreover, no correlation was seen between constitutive TS protein expression, level of cetuximab‐induced TS down‐regulation and response either to 5‐FU alone or in combination with cetuximab. We demonstrated that only EGFR expression with high functional activity of EGFR/MAPK pathway is important for the synergistic effects between cetuximab and 5‐FU in the investigated cell lines.

Irreversible pan-ErbB tyrosine kinase inhibitor CI-1033 induces caspase-independent apoptosis in colorectal cancer DiFi cell line

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of colorectal carcinomas and represents a target for therapeutic interventions with signal transduction inhibitors. We investigated the ability of CI-1033 to induce apoptosis and inhibition of proliferation in the colorectal cancer cell lines DiFi and Caco-2, which both express high levels of EGFR. While in Caco-2 cells CI-1033 treatment at a concentration 0.1 μ M for 72 hours demonstrated only antiproliferative (53.7 ± 4.3%) but no pro-apoptotic effects, treatment of DiFi cells resulted in a reduced proliferation rate (31.4 ± 3.1%) and in apoptosis (44.2 ± 8.9%). In order to define proteins involved in the regulation of apoptosis, we aimed to determine differences in the proteome profile of both cell lines before and after treatment with CI-1033. Cellular proteins were analyzed by 2-D gel electrophoresis followed by computational image analysis and mass spectrometry. Our data show that DiFi cells differ from Caco-2 cells in nine significantly upregulated proteins, and their potential role in apoptosis is described. We demonstrate that induction of apoptosis was triggered via caspase-independent pathways. Overexpression of leukocyte elastase inhibitor (LEI) and translocation of cathepsin D to the cytosol accompanied by upregulation of other defined proteins resulted in Bax-independent AIF translocation from mitochondria into the nucleus and apoptosis. Definition of these proteins can pave the way for functional studies and contribute to a better understanding of the effects of CI-1033 and the pathways of caspase-independent cell death.

Proteomics of cancer stem cells

Abstract Purpose: New understanding of cancer stem cell (CSC) biology continues to emerge due to development of novel methods in genomics and proteomics. Analysis of nucleic acids (RNA, DNA) is widely used to elucidate molecular perturbations in malignant tumors and carcinoma cells, however genome data do not reflect the functional activities of encoded proteins. Therefore proteome-based methods could enhance knowledge about deregulation of pathways as a result of altered expression and activities of proteins in CSC. Methods and results: A sufficient number of CSC for proteomic analyses can be obtained in a variety of ways: Fluorescence (FACS) and magnetic (MACS) activated cell sorting, laser cell capture microdissection, and three-dimensional spheroid/organoid cell culture. These methods to enrich and isolate CSC can be performed either with or without staining using antibodies against currently known CSC-specific cell surface molecules, such as clusters of differentiation 44, 24, 133 (CD44, CD24, CD133), epithelial cell adhesion molecule (EpCAM), aldehyde-dehydrogenase-1 (ALDH1), etc. The most important limitation on using antibody-based staining of CSC is that we still do not possess definitive CSC surface markers. This review article discusses methods that could be used to study protein profiling of CSC and to identify novel CSC-specific biomarkers and therapeutic targets. Conclusion: Despite an opinion that the proteomic approach is time-consuming, laborious and difficult, this method can be used effectively to clarify which pathways are involved in regulating various intratumoral processes, including activation of CSC. Based on this point of view, searching and identification of single molecules as biomarkers or therapeutic targets could become possible when CSC-associated pathways are well described and clearly understood due to detailed investigation of the protein patterns in pre-clinical models and clinical samples.

Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-κB expression and proteasome activation in head and neck squamous carcinoma cell lines

This study aimed to characterize the antitumor activity of 5-Chloro-N-{2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl}-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 μmol/L with 17.16 ± 2.08%, 10.92 ± 1.22%, and 27.03 ± 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G0-G1 phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-κB (NF-κB) and NF-κB p65 phosphorylated at Ser536. The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-κB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer. [Mol Cancer Ther 2007;6(6):1898–908]