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      • Record: found
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      Down-regulation of GRP78 is associated with the sensitivity of chemotherapy to VP-16 in small cell lung cancer NCI-H446 cells

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          Abstract

          Background

          Chemotherapy resistance remains a major obstacle for the treatment of small cell lung cancer (SCLC). Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum chaperone, plays a critical role in chemotherapy resistance in some cancers. However, whether the suppression of the chaperone can enhance the sensitivity of chemotherapy in SCLC is still unclear.

          Methods

          The SCLC NCI-H446 cells were divided into three groups: BAPTA-AM→A23187-treated group, A23187-treated group and control-group. Immunofluorescence, western blot and RT-PCR were used to assess the expression of GRP78 at both protein and mRNA levels. Cell apoptosis and the cell cycle distributions of the cells were analyzed by flow cytometry in order to evaluate the therapeutic sensitivity to VP-16.

          Results

          The expression of GRP78 at both protein and mRNA levels in the BAPTA-AM→A23187-treated cells dramatically decreased as compared to that in both A23187-treated and control groups. After treatment by VP-16, the percentage of apoptotic cells in BAPTA-AM→A23187-treated cells were: 33.4 ± 1.01%, 48.2 ± 1.77%, 53.0 ± 1.43%, 56.5 ± 2.13%, respectively, corresponding to the concentrations of BAPTA-AM 10, 15, 25, 40 μM, which was statistically significant high in comparison with the A23187-treated group and untreated-group (7.18 ± 1.03% and 27.8 ± 1.45%, respectively, p < 0.05). The results from analysis of cell cycle distribution showed that there was a significantly decreased in G 1 phase and a dramatically increased in S phase for the BAPTA-AM→A23187-treated cells as compared with the untreated cells.

          Conclusion

          BAPTA-AM is a strong inhibitor of GRP78 in the NCI-H446 cell line, the down-regulation of GRP78 can significantly increase the sensitivity to VP-16. The suppression of GRP78 may offer a new surrogated therapeutic approach to the clinical management of lung cancer.

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          Most cited references23

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          Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.

          R Rizzuto, M Brini, M. Murgia … (1993)
          Microdomains of high intracellular calcium ion concentration, [Ca2+]i, have been hypothesized to occur in living cells exposed to stimuli that generate inositol 1,4,5-trisphosphate (IP3). Mitochondrially targeted recombinant aequorin was used to show that IP3-induced Ca2+ mobilization from intracellular stores caused increases of mitochondrial Ca2+ concentration, [Ca2+]m, the speed and amplitude of which are not accounted for by the relatively small increases in mean [Ca2+]i. A similar response was obtained by the addition of IP3 to permeabilized cells but not by perfusion of cells with Ca2+ at concentrations similar to those measured in intact cells. It is concluded that in vivo, domains of high [Ca2+]i are transiently generated close to IP3-gated channels and sensed by nearby mitochondria; this may provide an efficient mechanism for optimizing mitochondrial activity upon cell stimulation.
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            Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation.

            Ramachandra Reddy, Changhui Mao, Peter Baumeister … (2003)
            A large number of correlative studies have established that the activation of the unfolded protein response (UPR) alters the cell's sensitivity to chemotherapeutic agents. Although the induction of the glucose-regulated proteins (GRPs) is commonly used as an indicator for the UPR, the direct role of the GRPs in conferring resistance to DNA damaging agents has not been proven. We report here that without the use of endoplasmic reticulum (ER) stress inducers, specific overexpression of GRP78 results in reduced apoptosis and higher colony survival when challenged with topoisomerase II inhibitors, etoposide and doxorubicin, and topoisomerase I inhibitor, camptothecin. While investigating the mechanism for the GRP78 protective effect against etoposide-induced cell death, we discovered that in contrast to the UPR, GRP78 overexpression does not result in G1 arrest or depletion of topoisomerase II. Caspase-7, an executor caspase that is associated with the ER, is activated by etoposide. We show here that specific expression of GRP78 blocks caspase-7 activation by etoposide both in vivo and in vitro, and this effect can be reversed by addition of dATP in a cell-free system. Recently, it was reported that ectopically expressed GRP78 and caspases-7 and -12 form a complex, thus coupling ER stress to the cell death program. However, the mechanism of how GRP78, a presumably ER lumen protein, can regulate cytosolic effectors of apoptosis is not known. Here we provide evidence that a subpopulation of GRP78 can exist as an ER transmembrane protein, as well as co-localize with caspase-7, as confirmed by fluorescence microscopy. Co-immunoprecipitation studies further reveal endogenous GRP78 constitutively associates with procaspase-7 but not with procaspase-3. Lastly, a GRP78 mutant deleted of its ATP binding domain fails to bind procaspase-7 and loses its protective effect against etoposide-induced apoptosis.
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              An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein.

              S. MUNRO, H Pelham (1986)
              We have characterized a cDNA clone that encodes a protein related to the 70 kd heat shock protein, but is expressed in normal rat liver. This protein has a hydrophobic leader and is secreted into the endoplasmic reticulum. We show that it is identical with two previously described proteins: GRP78, whose synthesis is induced by glucose starvation, and BiP, which is found bound to immunoglobulin heavy chains in pre-B cells. This protein, which is abundant in antibody-secreting cells, can be released from heavy chains by ATP, a reaction analogous to the release of hsp70 from heat shocked nuclear structures. We propose a specific role for this protein in the assembly of secreted and membrane-bound proteins.
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                Author and article information

                Journal
                Journal ID (nlm-ta): BMC Cancer
                Title: BMC Cancer
                Publisher: BioMed Central
                ISSN (Electronic): 1471-2407
                Publication date Collection: 2008
                Publication date (Electronic): 17 December 2008
                Volume: 8
                Page: 372
                Affiliations
                [1 ]Diagnostics Laboratory Center, Dalian Medical University, Dalian, Liaoning, PR China
                [2 ]Department of Respiratory Medicine, The Third People's Hospital of Dalian, Dalian, Liaoning, PR China
                [3 ]Department of Emergency Medicine, The Second Hospital Affiliated to Dalian Medical University, Dalian, Liaoning, PR China
                [4 ]Postgraduate School, Dalian Medical University, Dalian, Liaoning, 116044, PR China
                [5 ]Department of Histology and Embryology, Dalian Medical University, Dalian, Liaoning, PR China
                [6 ]Department of Respiratory Medicine, The Second Hospital Affiliated to Dalian Medical University, Dalian, Liaoning, PR China
                Article
                Publisher ID: 1471-2407-8-372
                DOI: 10.1186/1471-2407-8-372
                PMC ID: 2628931
                PubMed ID: 19087354
                SO-VID: 938b7113-e3c1-4ae2-a4fc-d25bc35f09b2
                Copyright © Copyright © 2008 Wang et al; licensee BioMed Central Ltd.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 14 July 2008
                Date accepted : 17 December 2008
                Categories
                Subject: Research Article

                ScienceOpen disciplines: Oncology & Radiotherapy
                Data availability:
                ScienceOpen disciplines: Oncology & Radiotherapy

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