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Ann Thorac Surg 2004;78:1207-1214
© 2004 The Society of Thoracic Surgeons
a Department of Thoracic and Cardiovascular Surgery, University of Virginia, Charlottesville, Virginia, USA
Accepted for publication April 5, 2004.
* Address reprint requests to Dr Jones, Department of Surgery, Box 800679, University of Virginia, Charlottesville, VA, USA 22908-0679
djones{at}virginia.edu
Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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Abstract |
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B in non–small-cell lung cancer (NSCLC). We hypothesized that inhibition of NF-B by using the proteasome inhibitor bortezomib (Velcade) would sensitize NSCLC to gemcitabine-induced apoptosis.
METHODS: Tumorigenic NSCLC cell lines (H157 and A549) were treated with nothing, gemcitabine, bortezomib, or both compounds. NF-B activity was determined by nuclear p65 protein levels, electrophoretic mobility shift assays, and reverse transcription-polymerase chain reaction of the NF-B–regulated genes interleukin-8, c-IAP2, and Bcl-xL. The p21 and p53 protein levels were determined in similarly treated cells. Cell-cycle dysregulation was assessed by fluorescence-activated cell sorting analysis. Cell death and apoptosis were quantified by clonogenic assays, caspase-3 activation, and DNA fragmentation. NSCLC A549 xenografts were generated and treated as noted previously. Tumor growth was assessed over a 4-week treatment period. Statistical analysis was performed with analysis of variance.
RESULTS: Gemcitabine enhanced nuclear p65 levels, NF-B binding to DNA, and transcription of all NF-B–regulated genes. Bortezomib inhibited each of these effects. Combined gemcitabine and bortezomib enhanced p21 and p53 expression and induced S-phase and G2/M cell-cycle arrests, respectively. Combined treatment killed 80% of the NSCLC cells and induced apoptosis, as determined by caspase-3 activation (p = 0.05) and DNA fragmentation (p = 0.02). NSCLC xenografts treated with combination therapy grew significantly slower than xenografts treated with gemcitabine alone (p = 0.02).
CONCLUSIONS: Bortezomib inhibits gemcitabine-induced activation of NF-B and sensitizes NSCLC to death in vitro and in vivo. This combined treatment strategy warrants further investigation and may represent a reasonable treatment strategy for select patients with NSCLC given the current clinical availability of both drugs.
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Introduction |
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| Dr Jones discloses that he has a financial relationship with Millennium Pharmaceuticals.
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One strategy to improve the response of malignant cells to molecularly targeted therapy is to inhibit central mediators of resistance to death by using small-molecule inhibitors of these antiapoptotic signaling pathways. The resistance of tumor cells to undergo apoptosis is a natural and vital process for all malignancies [7]. A primary mechanism of apoptotic resistance in NSCLC and other cancers is through activation of the antiapoptotic transcription factor nuclear factor (NF)-B [8]. In addition to the basal overexpression of NF-B subunits in NSCLC, exposure to chemotherapy further enhances NF-B transcriptional activity, thus attenuating the efficacy of the chemotherapy agents [9–11]. NF-B is normally sequestered in the cytoplasm of nonstimulated cells by inhibitory IB proteins [12]. Stress signals emanated from the nucleus in response to DNA damage induce IB degradation by the 26S proteasomes, thus liberating NF-B for nuclear translocation, where it drives transcription of selective antiapoptotic genes, including c-IAP1, c-IAP2, Bcl-xL, MnSOD, and others [13, 14].
Previous work from our laboratory and others has shown that direct inhibition of NF-B by using an adenoviral delivery of a dominant-negative IB (SR-IB) abrogates NF-B activity after treatment of NSCLC with chemotherapy and sensitizes these cells to chemotherapy-induced apoptosis [9, 10, 13]. Unfortunately, adenoviral-dependent delivery of the SR-IB transgene has precluded clinical use of this therapy [15]. The recent development and Food and Drug Administration approval of the pharmacologic proteasome inhibitor bortezomib (Velcade; Millennium Pharmaceuticals, Cambridge, MA) may provide a clinically relevant alternative strategy for NF-B inhibition.
Bortezomib, formerly known as PS-341, is a novel 26S proteasome inhibitor that stabilizes phosphorylated IB, inhibits the nuclear translocation and transcriptional activity of NF-B, and sensitizes malignant cells to chemotherapy-induced apoptosis [16, 17]. Bortezomib has also been shown to induce a G2/M cell-cycle arrest [18, 19]. It is important to note that bortezomib has been well tolerated in patients when it is administered as a single agent, thus indicating that it may have potential for use as part of a combination therapy [20, 21].
Therefore, the purpose of this study was to determine whether treatment with the proteasome inhibitor bortezomib would abrogate the gemcitabine-induced activation of NF-B and sensitize NSCLC to gemcitabine-induced apoptosis in vitro and in an NSCLC xenograft model.
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Material and Methods |
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Cell-Survival Assays
H157 and A549 cells were grown to 60% confluency on 12-well culture plates. Cells were treated with gemcitabine (1 to 200 µmol/L) for 24 hours, and cell survival was determined by sequentially incubating the cells in 1% glutaraldehyde, 0.5% crystal violet dye, and distilled water at room temperature for 15 minutes each. Cells were dried, and crystal violet dye was eluted by incubating the stained cells in Sorenson's solution (30 mmol/L sodium citrate, 0.02 mol/L HCl, and 50% ethanol) at room temperature for 15 minutes. Optical density of the eluent was measured at a wavelength of 570 nm.
Western Blotting
NSCLC cells that were 40% to 50% confluent on 100-mm plates were treated with nothing, gemcitabine (10 µmol/L), bortezomib (50 nmol/L), or both agents for 12 hours. Tumor necrosis factor (10 ng/mL; Sigma-Aldrich) treatment for 15 minutes was used as a positive control. Nuclear extracts were prepared by incubating pelleted cells in cytoplasmic extract buffer [0.075% NP-40, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 50 mmol/L KCl, 1 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L dithiothreitol] containing protease inhibitors at 4°C for 6 minutes followed by centrifugation at 1,800g. Pelleted nuclei were washed with cytoplasmic extract buffer without NP-40 and subsequently lysed in nuclear extract buffer (20 mmol/L Tris, 420 mmol/L NaCl, and 1.5 mmol/L MgCl) containing protease inhibitors at 4°C for 10 minutes. Nuclear extracts were cleared by centrifugation at 13,000g, and protein concentrations were determined by using the Pierce (Rockford, IL) BCA protein assay kit. Nuclear extracts (30 µg per lane) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies against p65 and RNA pol II were used for immunoblotting. Whole cell lysates were prepared from similarly treated cells by using radioimmunoprecipitation assay lysis buffer. Proteins (50 µg per lane) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were immunoblotted with antibodies to p21, p53, and ß-tubulin.
Electrophoretic Mobility Shift Assay
NSCLC cells were treated and nuclear extracts were prepared as described previously. Nuclear extracts (5 µg per treatment condition) were incubated in binding buffer (10 mmol/L Tris, 50 mmol/L NaCl, 0.5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L dithiothreitol, 10% glycerol, and 1 µg of poly dI-dC) for 20 minutes at 20°C with a 32P-labeled double-stranded probe containing the class I major histocompatibility complex promoter (5'-CAGGGCTGGGGATTCCCCATCTCCACAGTTTCACTTC-3'). DNA-protein complexes were resolved on a 5% acrylamide gel and dried before exposure for autoradiography.
Reverse Transcription-Polymerase Chain Reaction
NCSLC cells that were 40% to 50% confluent on 60-mm plates were treated for 24 hours under previously described experimental conditions. Cells were lysed with TRIzol (Invitrogen, Carlsbad, CA), and proteins were extracted with chloroform. RNAs were precipitated with isopropanol and washed with 70% ethanol. Complementary DNAs (cDNAs) were created by using the Advantage RT for polymerase chain reaction (PCR) enzyme (Clontech, Palo Alto, CA) according to the manufacturer's instructions, and cDNAs were amplified by PCR by using Platinum Taq (Invitrogen) and primers for interleukin-8 (5'-CTTCCAAGCTGGCCGTGG-3' and 5'-TGAATTCTCAGCCTTCTT-3'), Bcl-xL (5'-TCTCAG-AGCAACCGGGAG-3' and 5'-TGGCCCTTTCGGCTCTCG-3'), and c-IAP2 (5'-AACTTTCTC-CAGGTCCAAAATGAATAA-3' and 5'-TCTACATATTCAACTTTCCCCGCCGGG-3'). As a control, glyceraldehyde phosphate dehydrogenase cDNA was amplified by using the primers 5'-GTGAGGAGGGGAGATTCAG-3' and 5'-GCATCCTGGGCTACACTG-3'. PCR products were resolved on a 0.8% agarose gel.
Fluorescence-Activated Cell Sorting Analysis
NSCLC cells were treated with the appropriate pharmacologic agents for 12 hours. Cells were washed once with 1x phosphate-buffered saline, trypsinized, and resuspended in ice-cold 70% ethanol for 24 hours. Fixed cells were incubated for 12 hours in a propidium iodide–staining solution (0.1% Triton X-100, 0.2 mg/mL ribonuclease, and 0.02 mg/mL propidium iodide). Cellular DNA contents were determined by flow cytometry.
Apoptosis Assays
NSCLC cells were plated at 5 x 105 cells per well in 12-well plates. Cells were either left alone or treated for 24 hours with gemcitabine (10 µmol/L), bortezomib (50 nmol/L), or both gemcitabine and bortezomib. Apoptosis was quantified by detection of caspase-3 activation and DNA fragmentation. Caspase-3 activity was determined by the addition of an APC-DEVD protein conjugate (Calbiochem, San Diego, CA) to cellular extracts containing 25 µg of protein. Fluorescence of caspase-3–cleaved protein conjugates was determined fluorometrically. DNA fragmentation was determined by evaluation of cellular nucleosome formation by using the Cell Death Detection ELISA Plus kit (Roche, Indianapolis, IN) according to the manufacturer's instructions.
Xenograft Development and Treatment
Experiments in this study were performed in accordance with a written protocol approved by our institutional animal care and use committee. Human NSCLC xenografts were generated in athymic nude mice (Taconic, Germantown, NY) by injecting 2 x 106 A549 cells suspended in serum-free RPMI (100 µL per injection site). Once the tumors achieved a volume of 0.5 cm3, the mice were randomized to receive no treatment, gemcitabine (intraperitoneally, 125 mg/kg twice weekly), bortezomib (intravenously, 0.6 mg/kg twice weekly), or both gemcitabine and bortezomib at the same doses (n = 8 per group). Tumors were measured and volumes calculated (volume = length x short axis2 every other day for 4 weeks. At the conclusion of the treatment period, mice were killed, and tumors were harvested.
Statistical Analysis
Where appropriate, statistical analysis was performed by analysis of variance. Additionally, post hoc analyses were performed with Tukey's honestly significant different test and Bonferroni's test to compare the combined treatment group with each of the other treatment conditions.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDiscussionAcknowledgmentsReferences |
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BB, NSCLC cells were treated with nothing, gemcitabine, bortezomib, or both gemcitabine and bortezomib for 12 hours. Nuclear extracts were prepared and were evaluated by Western blot analysis probing for p65, the transcriptionally active subunit of NF-B (Fig 2A). In each cell line, gemcitabine enhanced the nuclear p65 concentration, and this was blocked by the addition of bortezomib. To determine whether nuclear concentrations of p65 correlated with the enhanced DNA binding function of NF-B, an electrophoretic mobility shift assay was performed on the A549 cells after identical treatments as noted previously (Fig 2B). Similarly, gemcitabine enhanced the DNA binding of NF-B, and this was completely blocked by bortezomib.
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B–dependent transcriptional activity, H157 and A549 cells were treated as described previously, and purified messenger RNAs were evaluated by reverse transcription-PCR by using primers specific to the NF-B–dependent genes interleukin-8, c-IAP2, and Bcl-xL and the housekeeping gene glyceraldehyde phosphate dehydrogenase (Fig 2C). Gemcitabine upregulated the transcriptional activity of all NF-B–regulated genes, and bortezomib completely blocked these effects.
Gemcitabine and Bortezomib Disrupt Cell-Cycle Progression in NSCLC
Cell-cycle progression is critical to the sensitization of cells to the damaging effects of chemotherapy. Specifically, the toxicity of the nucleotide analogue gemcitabine is proportional to the amount of drug incorporated into the DNA during S-phase [22]. To determine what effect gemcitabine and bortezomib have on progression through the cell cycle, the regulatory proteins p53 and p21 were evaluated in the H157 and A549 cells by Western blot analysis after a 12-hour treatment period (Fig 3A). Both gemcitabine and bortezomib enhanced expression of p53 in the A549 cells that express wild-type p53, but treatment with both agents did not seem to have an additive effect on p53 expression. Expression of p53 was not detected after any treatment condition in the H157 cells that did not express wild-type p53. Bortezomib also enhanced expression of p21 in both cell lines despite the lack of functional p53 in the H157 cells, but gemcitabine had no effect on p21 expression.
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Gemcitabine and Bortezomib Induce Apoptosis in NSCLC
NSCLC cells were treated with nothing, gemcitabine, bortezomib, or both compounds for 24 hours and were analyzed for evidence of apoptosis. Apoptosis was measured by quantifying caspase-3 activity and the degree of DNA fragmentation (Fig 4). These independent assays detected 2 separate hallmarks of apoptosis that become maximally activated at different phases of apoptosis, and this contributed to some variance between assays. In each cell line, combined treatment with both gemcitabine and bortezomib induced considerably more caspase-3 activity and caused considerably greater DNA fragmentation than any of the other treatment conditions, as determined by analysis of variance (p 
0.001) and post hoc analysis by Bonferroni's test (p 0.02). These results further support our understanding that the diminished crystal violet dye uptake in Figure 1 was the result of cell growth arrest rather than the induction of apoptosis, because only minimal levels of apoptosis were observed in this assay after treatment with gemcitabine alone. Additionally, bortezomib dose-escalation studies did not result in meaningful enhancement of cell death (data not shown).
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Comment |
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B plays as a central regulator of antiapoptotic signaling pathways involved in the resistance of NSCLC to chemotherapy-induced apoptosis [9, 10, 24–26].
Currently, no known selective small-molecule inhibitors of NF-B are available for clinical use. Therefore, my colleagues and I, and others, have focused on molecular targets that inhibit pathways critical for NF-B activation. Unfortunately, many of the compounds that inhibit NF-B activity have limited clinical applicability secondary to their solubility or toxicity profiles. In contrast, bortezomib is a water-soluble agent that has been well tolerated in clinical trials in cancer patients, including those with lung cancer [21, 27, 28]. Toxicity associated with bortezomib has been mild and typically includes thrombocytopenia, fatigue, and peripheral neuropathies [20].
As shown in Figures 1 and 4, treatment of NSCLC cells with gemcitabine alone did not induce considerable cell death or apoptosis despite marked dose escalation. This in vitro response parallels what is seen clinically, because the overall response rate of NSCLC to gemcitabine, as a single agent, is only 21% [29]. The lack of tumor responsiveness to gemcitabine in vitro correlated with the nuclear translocation of NF-B and subsequent activation of several NF-B–dependent antiapoptotic genes. It is important to note that bortezomib abrogated gemcitabine-induced NF-B activation, sensitized cells to apoptosis in vitro, and inhibited tumor growth in vivo. The absence of overt signs of toxicity in tumor-bearing mice further indicates that the clinical application of combined treatment of NSCLC with gemcitabine and bortezomib remains plausible.
Previous trials involving combination chemotherapy in patients with cancer indicate that the scheduling sequence influences both the therapeutic efficacy and toxicity profiles [30]. Similarly, a recent publication by Fahy and associates [16] shows that the cytotoxic effects of combined treatment with gemcitabine and bortezomib in vitro are markedly greater when gemcitabine is administered first followed by bortezomib, compared with simultaneous treatment or treatment in the reverse order, in a pancreatic cancer model. Preliminary studies by our laboratory have corroborated these findings by analysis of cell-survival assays performed in vitro as well as by further studies of NSCLC xenografts. In these preliminary studies we noted decreased cell survival in vitro and less xenograft growth when gemcitabine was administered before bortezomib.
Although the time course of critical NF-B–dependent gene activation has not been elucidated, the results of Fahy and associates [16] suggest that activation of NF-B may be critical as a delayed event in overcoming proapoptotic signals that result from genotoxic stressors. Additionally, drug interactions between gemcitabine and bortezomib that extend beyond NF-B activation or inhibition may have contributed to the results.
In addition to NF-B inhibition, bortezomib may sensitize cells to gemcitabine-induced apoptosis through mechanisms that involve cell-cycle dysregulation. Previous reports indicate that both gemcitabine and bortezomib disrupt progression through the cell cycle, and this is consistent with our current results (Fig 3) [18, 23]. Pretreatment with bortezomib disrupts cell-cycle progression at the G2/M checkpoint and would be expected to protect cells from entering the S-phase, where gemcitabine exerts its greatest cytotoxicity [22]. Thus, by arresting cell-cycle progression, pretreatment with bortezomib before gemcitabine may diminish the efficacy of gemcitabine. Conversely, the addition of bortezomib to cells previously treated with gemcitabine may enhance gemcitabine's cytotoxicity by influencing cell-cycle progression through the G1/S checkpoint. DNA damage caused by radiation or chemotherapy leads to dephosphorylation of the retinoblastoma protein and transcriptional downregulation of cyclin A, which is necessary for progression through the G1/S checkpoint [31, 32]. Cyclin A depletion protects cells from further DNA damage by preventing the entry of cells into the S-phase, where they are most sensitive to gemcitabine [22]. It is important to note that bortezomib has been shown to stabilize cyclin A expression and therefore would counteract the gemcitabine-induced cyclin A depletion and force cells to enter S-phase [19]. The conflicting cell cycle–deregulatory effects of gemcitabine and bortezomib may have contributed to the relative normalization of the cell-cycle profile after the combined treatment (Fig 3B) and may enhance gemcitabine-induced toxicities by forcing cell-cycle progression through S-phase.
Limitations of this experimental design include the fact that only 2 NSCLC cell lines were evaluated. We selected the H157 and A549 cell lines because of their inherent resistance to gemcitabine-induced apoptosis, which is reflective of the clinical response [2]. Another limitation is that only gemcitabine was used in combination with bortezomib in this experiment: the combination of bortezomib with other currently available drugs may have had a different effect. Additionally, a dose-response curve with both compounds may need to be generated in our in vivo model. Studies performed in vivo used only the A549 cell line, which was selected because of its relatively slow growth rate compared with the H157 line and other NSCLC xenografts generated from other cell lines. The relatively slow growth of A549 xenografts allows for a more extensive treatment period before the tumors became excessively large. Our in vivo studies were further limited by the selection of a single dose for each drug evaluated rather than the establishment of dose-response curves. Because gemcitabine and bortezomib are both clinically available, the doses used in this study were chosen to reflect what is clinically relevant.
This investigation involved the combined treatment of NSCLC in vivo with gemcitabine and bortezomib. In a disease for which meaningful progress has been lacking, molecularly targeted therapy seems to hold promise for the future. Use of both in vitro and in vivo studies allowed us to increase our understanding of the molecular signaling events occurring within the cells while simultaneously permitting us to test our hypothesis in an animal model of lung cancer. This report suggests that combined gemcitabine and bortezomib treatment deserves further investigation for patients with NSCLC.
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Discussion |
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DR DENLINGER: That is an important question. Thus far we have not specifically looked at proliferating cells such as the bone marrow or gastrointestinal epithelial cells, for signs of toxicity in the animals following treatment. Importantly, we have not observed overt signs of toxicity in mice treated with Velcade, Gemcitabine or the combined therapy over the four-week treatment period. Moreover, mice in all treatment groups continued to gain weight and demonstrated normal activity patterns.
DR THOMAS A. D’AMICO (Durham, NC): How certain can we be that NF-B inhibition is an important step in human tumors and whether Velcade will cause proteasome inhibition in humans to do the same thing?
DR DENLINGER: The best way that I can directly address the importance of NF-B inhibition in non-small cell lung cancer is to refer to some of our previous studies performed in vitro using adenoviral-mediated NF-B inhibition. In this model, selective NF-B inhibition dramatically sensitized cells to chemotherapy-induced apoptosis. This suggests that NF-B does play an important role in protecting cells from the toxic effects of chemotherapy. In this study, Velcade did inhibit NF-B-dependent anti-apoptotic gene transcription, and Velcade did sensitize non-small cell lung cancer cells to apoptosis. In addition, Velcade also affected other cell signaling pathways such as those regulating cell-cycle progression. Although this study does not indicate that the interaction between gemcitabine and Velcade is mediated exclusively through NF-B inhibition, I think the most important point is that they appear to synergistically induce apoptosis in vitro and inhibit tumor growth in vivo.
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References |
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B. Semin Thorac Cardiovasc Surg 2004;16:28–39
B chemosensitizes non-small cell lung cancer through cytochrome c release and caspase activation. J Thorac Cardiovasc Surg. 2002;123:310–317B sensitizes non-small cell lung cancer cells to chemotherapy induced apoptosis. Ann Thorac Surg. 2000;70:930–937B transcription factor. Oncogene. 1999;18:6910–6924[Medline]
B. Science. 1996;274:784–787B as a therapeutic target in cancer. Trends Mol Med. 2002;8:385–389[Medline]
B transcription factors in apoptosis of human hepatocellular carcinoma cells. Cancer. 2002;95:1696–1705[Medline]
B through the Akt pathway. J Biol Chem. 2003;278:18980–18989B inhibition sensitizes gemcitabine-resistant NSCLC to apoptosis. J Am Coll Surg. 2003;197:S29
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p 
p 
= no treatment; 
= Velcade; = gemcitabine; 
= gem/Velcade. (RFU = relative fluorescence units; OD = optical density.)