- Email: [email protected]
Bcl-2 as a Target for Overcoming Chemoresistance in Small-Cell Lung Cancer
translational medicin e Bcl-2 as a Target for Overcoming Chemoresistance in Small-Cell Lung Cancer Dean A. Fennell Abstract Small-cell lung cancer (SCLC) is an aggressive malignancy that is frequently metastatic at presentation and has a poor prognosis. Although initially sensitive to primary therapy, acquisition of apoptosis resistance is typical, resulting in failure of secondary chemotherapy following relapse. Expression of the antiapoptosis protein Bcl-2 is prevalent in SCLC. The understanding of this oncoprotein’s function has increased dramatically over the past decade. In vitro and in vivo evidence supports a role for overexpression of Bcl-2 in SCLC and supports the notion that it is a major factor contributing to apoptosis resistance. Targeting Bcl-2 may provide a novel therapeutic approach to overcoming chemoresistance in SCLC. This article discusses the relevance of Bcl-2 to apoptosis susceptibility in SCLC and its exploitation using gene silencing to improve the clinical outcome in this disease. Clinical Lung Cancer, Vol. 4, No. 5, 307-313, 2003
Key words: Antisense oligonucleotides, Apoptosis, Caspases, Voltage-dependent anion channel
Introduction
chemotherapeutic combinations, little impact has been made in improving long-term survival over the past two decades.8 There is, therefore, a major need to address the therapeutic problem of emerging chemoresistance in SCLC in order to improve outcome.
Small-cell lung cancer (SCLC) affects approximately 20% of patients with pulmonary malignancy and is the most aggressive form of lung cancer, associated with a median survival from diagnosis of 2-4 months if untreated.1 Small-cell lung cancer displays exquisite sensitivity to induction chemotherapy, with objective response rates of 70%-80% in extensive-stage disease and complete remission rates of 20%-30%.2 In limited-stage disease, objective response rates are as high as 65%-90%, with complete remission rates of 45%-75%.3 Combination chemotherapy is the cornerstone of treatment due to the high probability of occult metastases; there is a 60% actuarial risk of developing central nervous system metastases within 2-3 years with prophylactic cranial irradiation.4 Despite the initial high response rate to chemotherapy, recurrent SCLC displays a transformation in phenotype with marked chemoresistance, resulting in an untreated median survival at recurrence of only 2-3 months.5 The efficacy of chemotherapy in this setting is poor; although this can be increased in patients receiving second-line therapy,6 this is highly dependent on the interval preceding relapse, with heavily pretreated patients rarely responding to further chemotherapy.7 Despite advances in diagnosis and emergence of new Lung Cancer Section, Department of Medical Oncology St Bartholomew’s Hospital, London, United Kingdom Submitted: Oct 25, 2002; Revised: Mar 7, 2003; Accepted: Mar 14, 2003 Address for correspondence: Dean A. Fennell, MD, PhD, Lung Cancer Section, Department of Medical Oncology, St Bartholomew’s Hospital, West Smithfield, London EC1A 7BE, United Kingdom Fax: 44-020-601-7577; e-mail: [email protected]
Apoptosis and Chemotherapy Over the past 2 decades, it has emerged that the efficacy of chemotherapy relies on the efficient induction of programmed cell death or apoptosis.9,10 This is a biochemically stereotyped and phylogenetically conserved process11,12 that plays a critical role in development,13,14 resulting in noninflammatory cell death and removal of cells. Resistance to chemotherapy-induced apoptosis results in the failure of therapy and is commonly associated with a defect in the intrinsic cellular apoptotic machinery (CAM).15,16 Recent advances have led to greater understanding of the underlying molecular detail associated with induction of apoptosis by chemotherapy coupled with an increased understanding of the functional genomics of many cancers, including SCLC.17,18 A growing list of proteins with antiapoptotic activity that are overexpressed in cancer have been discovered, with implications for susceptibility to chemotherapy-induced cell death and new drug development.19 Bcl-2 is a prototypical antiapoptosis protein that was discovered as the result of cloning of the translocation in B-cell non-Hodgkin’s (follicular) lymphoma, associated with hyperexpression of Bcl-2 due to juxtaposition with the immunoglobulin H chain promoter.20 Bcl-2 was the first member of an expanding family of proapoptotic and antiapoptotic proteins to be discovered. Bcl-2 prevents apoptosis and is high-
Clinical Lung Cancer March 2003
307
Bcl-2 in Small-Cell Lung Cancer ly expressed in SCLC.21-24 Evidence arising from studies of Bcl2 in SCLC suggest that this antiapoptotic protein is an attractive target for novel therapy.
Activation of the Cellular Apoptotic Machinery by Cancer Chemotherapy Despite a wide variety of primary molecular targets underlying tumor cell death induction by chemotherapeutic agents, their pharmacodynamics converge in the activation of the CAM. In the early 1990s, Jacobson and colleagues demonstrated that the nucleus is neither required for apoptotic cell death nor for the protection from cell death by Bcl-2.25 Mitochondria have emerged as an integrating center of cellular death signals following exposure to chemotherapy, irrespective of initial molecular interaction with the cell.16,26 These organelles, previously thought of as critical to life through their involvement in aerobic respiration, have now been shown to be intimately involved in the control of cell death.27,28
Proapoptotic Proteins Nucleated cells constitutively express Bcl-2 homologous proapoptotic proteins (PAPs), termed Bax and Bak in their cytosol.29-32 These proteins are folded into inactive conformations. However, upon induction of cell death by cytotoxic drugs, Bax and Bak unfold, resulting in their translocation to the outer mitochondrial membrane (OMM).33-36 These proteins are an essential component of the CAM that enable chemotherapy-induced apoptosis.37 Mitochondria are double-membraned organelles. The OMM and inner mitochondrial membrane (IMM) meet at regions characterized by junctional complexes; these play a critical role in cytosol-to-mitochondria signaling by directing translocation of PAPs38 and transducing signals from the mitochondria back to the cytosol, which result in the destruction of the cell.39
Mitochondrial Junctional Complexes Mitochondrial junctional complexes (also known as permeability transition pore complexes, PTPCs) consist of several proteins in the OMM, IMM, and innermembrane space (IMS); these include OMM-localized voltage-dependent anion channel (VDAC),40 peripheral benzodiazepine receptor,41 hexokinase II,42 IMS-localized creatine kinase,43 as well as IMM-localized adenine nucleotide translocator44 and cyclophilin D.39
Cytochrome C On docking with the OMM, PAPs initiate permeabilization of both the OMM and IMM, resulting in an irreversible commitment to cell death. The OMM permeabilization results in the release of apoptosis, inducing factors from the IMS that include cytochrome C,45-47 apoptosis-inducing factor,48 and second mitochondria-derived activator of caspases (SMAC).49,50 Some evidence suggests that this release of proteins is nonspecific and involves up to 100 proteins, implicating OMM rupture.51,52 However, in some experimental systems, cytochrome C appears to be released before other proteins.53 Shimizu and colleagues have proposed a model of the action of Bax and Bak,
308
Clinical Lung Cancer March 2003
which involves direct interaction with VDAC, resulting in a conformational change that leads to conduction of cytochrome into the cytosol.54-56 These proteins have been shown to form a large novel conductance with VDAC capable of conducting cytochrome C54; the existence of a novel, high conductance channel has also been demonstrated by Pavlov and colleagues, which correlates with the presence of Bax in the OMM,57 the so-called mitochondrial apoptosis channel. Cytochrome C directly activates a cascade of executioner serine proteases termed caspases. This occurs via the deoxyadenosine triphosphate–dependent formation of a heterotrimeric cytochrome C/Apaf-1/procaspase9 apoptosome, resulting in activation of caspase-945 followed by further downstream activation of other caspases including caspase-3.58 Caspases target several substrates within the cell, affecting the typical morphological features of apoptosis, including cell shrinkage, blebbing, chromatin condensation, and expression of outer plasma membrane phosphotidylserine (Figure 1). SMAC interacts with inhibitors of the apoptosis protein, XIAP, which inhibits caspase 3, caspase 7, and caspase 9,59-61 through an interaction with its BIR3 domain.62 Apoptosis-inducing factor (AIF) is a heat shock protein–70 inhibitable reduced nicotinamide adenine dinucleotide oxidoreductase that induces caspase independent cell death via translocation to the nucleus.63-65
Mitochondrial Depolarization At or around the time of docking of PAPs with mitochondria and OMM permeabilization, the IMM undergoes permeabilization via formation of a nonselective pore. This event, termed permeability transition, involves depolarization of the IMM potential established during respiratory electron transport.66,67 This process is directly induced by Bax68 through a mechanism that is not yet fully understood but is likely to directly or indirectly involve VDAC69 and adenine neucleotide translocation.70,71 The immediate consequence of mitochondrial depolarization is the failure of adenosine triphosphate (ATP) synthesis and death. Following permeability transition, the F1F0ATPase may function in reverse, resulting in hydrolysis of existing ATP stores in an attempt to stabilize the IMM potential.72
Bcl-2 Antiapoptosis Bcl-2 constitutively localizes to the OMM, the endoplasmic reticulum, and the outer nuclear membrane.73-77 In mitochondria, Bcl-2 is localized to contact sites and physically associates with PTPCs.71,78 Proapoptotic protein–induced permeability of the OMM is blocked by Bcl-2.79,80 The mechanism of this release is controversial. In the model proposed by Shimizu et al,56,81 VDAC is a target for regulation by Bcl-2, interacting stoichiometrically with Bax to antagonize induction of conformational change and so prevent passage of cytochrome C (Figure 2). Bcl-2 also prevents formation of the novel high conductance in the OMM.56,57 The consequence of failure of cytochrome C translocation is inhibition of caspase activation.82 The proapoptotic activity of AIF and SMAC are inhibited by Bcl-2 by blocking their release, suggesting regulation by Bcl-2 of nonspecific OMM permeability.83,84
Dean A. Fennell Figure 1 Caspase and Apoptosis CED3 Subfamily
ICE Subfamily
Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase 9 2 10 8 6 3 7 1 4 5 13 11 12 Caspase 6
Caspase 8
Caspase 3 Caspase 10
Caspase 2 CYT C APAF 1
dATP Pro
Caspase 9
Caspase 9
PARP, PAK2 (Blebbing), CAD (DNA fragmentation), Laminin (Condensation)
Caspase 7 Several caspases have been identified. Permeabilization of the outer mitochondrial membrane activates procaspase 9, which then activates effector caspases 3 and 7. Downstream caspase activation results in the manifestations of apoptosis via specific substrate cleavage. Abbreviations: APAF = apoptosis protease-activating protein-1; CAD = caspase-activated deoxyribonuclease; CED3 = Caenorhabditis elegans cell death 3; CYT C = cytochrome C; ICE = interleukin-1β–converting enzyme; PAK2 = p21-activated kinase 2; PARP = poly (ADP-ribose) polymerase
This Bcl-2 activity during cell death contrasts with the constitutive effects of Bcl-2 during normal respiration, which appears to interact with VDAC so as to increase conductance to pyridine nucleotides (adenosine diphosphate and ATP) required for oxidative phosphorylation.85-87 Bcl-2 also prevents permeability transition through an allosteric effect on the adenine nucleotide translocator although the mechanism of this regulation has not been elucidated.71
Expression of Bcl-2 in Small-Cell Lung Cancer Bcl-2 has been shown to be expressed in SCLC cell lines by immunocytochemistry as well as by Northern blot analysis to demonstrate mRNA expression21 and is prevalent in biopsy-derived SCLC tissue as shown in Table 1.22,23,88-94 Bcl-2 expression correlates with apoptosis sensitivity in vitro.95 Some studies have demonstrated adverse prognostic effects of high Bcl-2 expression in patients with SCLC by multivariate analysis.92,96 Owing to the high prevalence of Bcl-2 in SCLC cohorts, however, prognostic stratification has not been demonstrated in some reports.94,97 They argue that the prevalence of Bcl-2 expression may play an important role in antiapoptosis during tumorigenesis. Expression of Bcl-2 is higher in metastatic SCLC compared with localized disease98 and is regulated at the translational level by fibroblast growth factor-2.99 A very high proliferation index is observed in SCLC cells as measured by ki67 antigen expression; this may account for chemosensitivity despite Bcl-2 expression.100 The level of Bcl-2 expression in SCLC has a significant impact on apoptosis sensitivity; transfection-associated overexpres-
sion of Bcl-2 in SCLC can modulate sensitivity to chemotherapeutic agents in vitro.101 Sartorius and Krammer have recently examined the molecular changes associated with selection for resistance to cisplatin, etoposide, and doxorubin in 9 SCLC cell lines.102 Multidrug resistance was generated by subculture in subtherapeutic concentrations of etoposide. Drug resistance was associated with a substantial increase in the expression of Bcl-2. To date, no studies have been conducted to examine the molecular changes that accompany acquired multidrug resistance in SCLC in vivo.
Targeting Bcl-2 in Small-Cell Lung Cancer to Reverse Chemoresistance Antisense oligonucleotides (ASOs) enable transient and selective inhibition of polypeptide synthesis and provide a tool for investigating the function of proteins in living cells.103,104 Antisense oligonucleotides are short strands of modified DNA (usually 18-20 mer) that hybridize to their target mRNA to form a heteroduplex that triggers active degradation of the bound mRNA via RNaseH. By scanning the bcl-2 mRNA for effective candidate ASOs capable of downregulating bcl-2 mRNA and protein expression, Ziegler and colleagues identified a potent phosphorothioate ASO, termed 2009.105 Antisense oligonucleotide 2009 induced sequence-specific apoptosis in SCLC cell lines NCI-H69, NCI-H82, and NCI-417 in association with sequence-specific downregulation of Bcl-2. Control oligonucleotides did mediate toxicity, consistent with a role for Bcl-2 in controlling apoptosis threshold. Interestingly, the toxicity of ASO 2009 correlated with the basal level of
Clinical Lung Cancer March 2003
309
Bcl-2 in Small-Cell Lung Cancer lines.106 Potent combination indices were demonstrated for etoposide/ASO 2009 combinations in high Bcl-2–expressing NCI-H69 cells. However, no synergy was observed for combinations in the SCLC cell line NCI-H82, which exhibited the lowest level of Bcl-2 expression. A novel bispecific ASO targeting Bcl-2 and its antiapoptotic homologue Bcl-XL has been shown to exhibit sequence-specific killing of SCLC cells in vitro.107
Figure 2 Bcl-2 and Permeabilization
HKII Bcl-2
Bax PBR
Bcl-2 Antisense Therapy in Small-Cell Lung Cancer
VDAC
Oblimersen (G3139, Genasense™) is a phosphorothioatebased ASO that has shown activity in both solid tumors (nude mouse models of lymphoma, gastric cancer, and melanoma108111) and clinical trials (non-Hodgkin’s lymphoma and melanoma).109,112,113 Several studies in a variety of malignancies are ongoing (Table 2).109,112-114 Recently, the first phase I study in SCLC employing oblimersen was reported in patients with chemorefractory disease, which was defined as those patients experiencing relapse ≤ 3 months after the completion of primary therapy.114 A total of 12 patients were treated with paclitaxel 150 mg/m2 combined with oblimersen and administered as a continuous intravenous infusion over 7 days to allow for Bcl-2 protein downregulation. No objective responses were observed, although 2 patients developed stable disease; 1 patient who had the highest serum levels of oblimersen remained stable for 30 weeks.
CK
Outer mitochondrial membrane
ANT
CyPD Inner mitochondrial membrane
Bcl-2 blocks permeabilization of both the outer mitochondrial membrane and the inner mitochondrial membrane via a direct interaction with the permeability transition pore complexes. Abbreviations: ANT = adenine neucleotide translocation; CK = creatine kinase; CyPD = cyclophilin D; HKII = hexokinase II; PBR = peripheral benzodiazepine receptor; VDAC = voltage-dependent anion channel
Conclusion
Bcl-2 expression. Studies to examine the effects of ASO 2009 in combination with chemotherapy have demonstrated synergy in SCLC cell Table 1
There is a paucity of research examining the molecular basis of chemoresistance that characterizes SCLC at relapse, a factor that underlies the poor survival rates associated with this disease. The antiapoptotic protein Bcl-2 is expressed in SCLC, is associated with acquired multidrug resistance, and represents a valid target for therapeutic intervention. Downregulation of antiapoptotic protein ex-
Bcl-2 Expression in Small-Cell Lung Cancer Number of Patients
% Bcl-2 Expression
Objective Response Rate (% Bcl-2+ vs. Bcl-2–)
Relative Median Survival (% Bcl-2+ vs. Bcl-2–)
Comments
Ben-Ezra et al,22 1994
23
65%
NR
NR
–
Higashiyama et al,23 1995
13
69%
NR
NR
–
24
77%
NR
NR
–
Jiang et al,89 1996
111
94%
NR
NR
–
Kaiser et al,90 1996
125
76%
40% vs. 27%
12% vs. 9.5% (P = 0.17)
No significant difference in survival detected
Yan et al,91 1996
29
93%
NR
NR
Bcl-2 does not correlate with clinical stage
Takayama et al,92 1996
38
55%
62% vs. 76%
NR
–
Stefanaki et al,93 1998
31
77%
NR
NR
–
Maitra et al,94 1999
42
57%
NR
11% vs. 13% (P = 0.2256)
Survival was independent of age, stage, therapy, and Bcl-2
Trial, Year
Higashiyama et
al,88
1996
In the minority of studies that analyzed survival data using Cox regression, Bcl-2 was not shown to be an independent prognostic factor. Abbreviation: NR = not reported
310
Clinical Lung Cancer March 2003
Dean A. Fennell Table 2
Ongoing Studies of Bcl-2 Antisense Oblimersen with and Without Standard Chemotherapy
Trial Phase Phase I114
Neoplasm
Synergistic Combination
Small-cell lung cancer
Paclitaxel
Phase II109,112 Non-Hodgkin’s lymphoma
–
Phase II
Mantle cell lymphoma
Rituximab, Cyclophosphamide, Vincristine, Doxorubicin, Prednisnone
Phase II
Prostate cancer
Docetaxel
Melanoma
Dacarbazine
Phase III
Non–small-cell lung cancer
Docetaxel
Phase III
Acute lymphocytic leukemia
Gemtuzumab Ozogamicin
Phase III
Multiple myeloma
High-dose Dexamethasone
Phase III
Chronic lymphocytic leukemia
Cyclophosphamide and Fludarabine
Phase
III113
pression by Bcl-2–targetting ASOs is an emerging therapeutic strategy that could lead to improvements in disease-free survival in SCLC. Other antisense strategies are currently being investigated in lung cancer and include targeting of c-raf by the oligonucleotide ISIS 5132 and protein kinase C by LY900003 (ISIS 3521, Affinitak™). However, further studies are required to determine the relative importance of these proteins as well as Bcl2 in relapsed SCLC.
Acknowledgements The author thanks Robin M. Rudd, MD, for reading the manuscript, and Finbarr E. Cotter, MD, PhD, for helpful discussions.
References 1. Okuno SH, Jett JR. Small cell lung cancer: current therapy and promising new regimens. Oncologist 2002; 7:234-238. 2. Simon GR, Wagner H. Small cell lung cancer. Chest 2003; 123:259S-271S. 3. Prasad US, Naylor AR, Walker WS, et al. Long term survival after pulmonary resection for small cell carcinoma of the lung. Thorax 1989; 44:784-787. 4. Nugent JL, Bunn PA Jr, Matthews MJ, et al. CNS metastases in small cell bronchogenic carcinoma: increasing frequency and changing pattern with lengthening survival. Cancer 1979; 44:1885-1893. 5. Albain KS, Crowley JJ, Hutchins L, et al. Predictors of survival following relapse or progression of small cell lung cancer. Southwest Oncology Group Study 8605 report and analysis of recurrent disease data base. Cancer 1993;72:1184-1191. 6. Chute JP, Kelley MJ, Venzon D, et al. Retreatment of patients surviving cancer-free 2 or more years after initial treatment of small cell lung cancer. Chest 1996; 110:165-171. 7. Sekine I, Nishiwaki Y, Kakinuma R, et al. Late recurrence of small-cell lung cancer: treatment and outcome. Oncology 1996; 53:318-321. 8. Chute JP, Venzon DJ, Hankins L, et al. Outcome of patients with small-cell lung cancer during 20 years of clinical research at the US National Cancer Institute. Mayo Clin Proc 1997; 72:901-912. 9. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26:239-257. 10. Kaufmann SH. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res 1989; 49:5870-5878. 11. Hengartner MO, Horvitz HR. Programmed cell death in Caenorhabditis elegans. Curr Opin Genet Dev 1994; 4:581-586. 12. Abrams JM. Competition and compensation: coupled to death in development and
cancer. Cell 2002; 110:403-406. 13. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell 1997; 88:347-354. 14. Meier P, Finch A, Evan G. Apoptosis in development. Nature 2000; 407:796-801. 15. Makin G, Dive C. Apoptosis and cancer chemotherapy. Trends Cell Biol 2001; 11:S22-S26. 16. Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000; 256:42-49. 17. Bangur CS, Switzer A, Fan L, et al. Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis. Oncogene 2002; 21:3814-3825. 18. Oh JM, Brichory F, Puravs E, et al. A database of protein expression in lung cancer. Proteomics 2001; 1:1303-1319. 19. Reed JC. Apoptosis-based therapies. Nat Rev Drug Discov 2002; 1:111-121. 20. Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 1984; 226:1097-1099. 21. Ikegaki N, Katsumata M, Minna J, et al. Expression of bcl-2 in small cell lung carcinoma cells. Cancer Res 1994; 54:6-8. 22. Ben-Ezra JM, Kornstein MJ, Grimes MM, et al. Small cell carcinomas of the lung express the Bcl-2 protein. Am J Pathol 1994; 145:1036-1040. 23. Higashiyama M, Doi O, Kodama K, et al. High prevalence of bcl-2 oncoprotein expression in small cell lung cancer. Anticancer Res 1995; 15:503-505. 24. Jiang SX, Sato Y, Kuwao S, et al. Expression of bcl-2 oncogene protein is prevalent in small cell lung carcinomas. J Pathol 1995; 177:135-138. 25. Jacobson MD, Burne JF, Raff MC. Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J 1994; 13:1899-1910. 26. Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1998; 1366:151-165. 27. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309-1312. 28. Van Loo G, Saelens X, Van Gurp M, et al. The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ 2002; 9:1031-1042. 29. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74:609-619. 30. Farrow SN, White JH, Martinou I, et al. Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K. Nature 1995; 374:731-733. 31. Chittenden T, Harrington EA, O'Connor R, et al. Induction of apoptosis by the Bcl-2 homologue Bak. Nature 1995; 374:733-736. 32. Kiefer MC, Brauer MJ, Powers VC, et al. Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature 1995; 374:736-739. 33. Wolter KG, Hsu YT, Smith CL, et al. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997; 139:1281-1292. 34. Nechushtan A, Smith CL, Hsu YT, et al. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J 1999; 18:2330-2341. 35. Nechushtan A, Smith CL, Lamensdorf I, et al. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 2001; 153:12651276. 36. Griffiths GJ, Corfe BM, Savory P, et al. Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak. Oncogene 2001; 20:7668-7676. 37. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001; 292:727-730. 38. De Giorgi F, Lartigue L, Bauer MK, et al. The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J 2002; 16:607-609. 39. Crompton M, Barksby E, Johnson N, et al. Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie 2002; 84:143152. 40. Szabo I, De Pinto V, Zoratti M. The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel. FEBS Lett 1993; 330:206-210. 41. McEnery MW. The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel (VDAC). J Bioenerg Biomembr 1992; 24:63-69. 42. Nakashima RA. Hexokinase-binding properties of the mitochondrial VDAC protein: inhibition by DCCD and location of putative DCCD-binding sites. J Bioenerg Biomembr 1989; 21:461-470. 43. O’Gorman E, Beutner G, Dolder M, et al. The role of creatine kinase in inhibition of mitochondrial permeability transition. FEBS Lett 1997; 414:253-257. 44. Belzacq AS, Vieira HL, Kroemer G, et al. The adenine nucleotide translocator in apoptosis. Biochimie 2002; 84:167-176. 45. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91:479-489. 46. Jurgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci U S A 1998; 95:49975002. 47. Narita M, Shimizu S, Ito T, et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A 1998; 95:14681-14686.
Clinical Lung Cancer March 2003
311
Bcl-2 in Small-Cell Lung Cancer 48. Lorenzo HK, Susin SA, Penninger J, et al. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ 1999; 6:516-524. 49. Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000; 102:33-42. 50. Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000; 102:43-53. 51. Single B, Leist M, Nicotera P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ 1998; 5:1001-1003. 52. Patterson SD, Spahr CS, Daugas E, et al. Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition. Cell Death Differ 2000; 7:137-144. 53. Doran E, Halestrap AP. Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites. Biochem J 2000; 348 Pt 2:343-350. 54. Shimizu S, Ide T, Yanagida T, et al. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J Biol Chem 2000; 275:12321-12325. 55. Shimizu S, Matsuoka Y, Shinohara Y, et al. Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J Cell Biol 2001; 152:237-250. 56. Shimizu S, Shinohara Y, Tsujimoto Y. Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator. Oncogene 2000; 19:4309-4318. 57. Pavlov EV, Priault M, Pietkiewicz D, et al. A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J Cell Biol 2001; 155:725-731. 58. Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376:37-43. 59. Chai J, Shiozaki E, Srinivasula SM, et al. Structural basis of caspase-7 inhibition by XIAP. Cell 2001; 104:769-780. 60. Riedl SJ, Renatus M, Schwarzenbacher R, et al. Structural basis for the inhibition of caspase-3 by XIAP. Cell 2001; 104:791-800. 61. Ekert PG, Silke J, Hawkins CJ, et al. DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9. J Cell Biol 2001; 152:483-490. 62. Liu Z, Sun C, Olejniczak ET, et al. Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 2000; 408:1004-1008. 63. Daugas E, Susin SA, Zamzami N, et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 2000; 14:729-739. 64. Loeffler M, Daugas E, Susin SA, et al. Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 2001; 15:758767. 65. Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397:441-446. 66. Hunter DR, Haworth RA, Southard JH. Relationship between configuration, function, and permeability in calcium-treated mitochondria. J Biol Chem 1976; 251:5069-5077. 67. Haworth RA, Hunter DR. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys 1979; 195:460-467. 68. Pastorino JG, Chen ST, Tafani M, et al. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J Biol Chem 1998; 273:7770-7775. 69. Tsujimoto Y, Shimizu S. The voltage-dependent anion channel: an essential player in apoptosis. Biochimie 2002; 84:187-193. 70. Marzo I, Brenner C, Kroemer G. The central role of the mitochondrial megachannel in apoptosis: evidence obtained with intact cells, isolated mitochondria, and purified protein complexes. Biomed Pharmacother 1998; 52:248251. 71. Brenner C, Cadiou H, Vieira HL, et al. Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene 2000; 19:329-336. 72. Rego AC, Vesce S, Nicholls DG. The mechanism of mitochondrial membrane potential retention following release of cytochrome c in apoptotic GT1-7 neural cells. Cell Death Differ 2001; 8:995-1003. 73. Monaghan P, Robertson D, Amos TA, et al. Ultrastructural localization of bcl-2 protein. J Histochem Cytochem 1992; 40:1819-1825. 74. Nguyen M, Millar DG, Yong VW, et al. Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH- terminal signal anchor sequence. J Biol Chem 1993; 268:25265-25268. 75. Nakai M, Takeda A, Cleary ML, et al. The bcl-2 protein is inserted into the outer membrane but not into the inner membrane of rat liver mitochondria in vitro. Biochem Biophys Res Commun 1993; 196:233-239. 76. Krajewski S, Tanaka S, Takayama S, et al. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 1993; 53:4701-4714. 77. Akao Y, Otsuki Y, Kataoka S, et al. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res 1994; 54:2468-2471. 78. Marzo I, Brenner C, Zamzami N, et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med
312
Clinical Lung Cancer March 2003
1998; 187:1261-1271. 79. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275:1129-1132. 80. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 1997; 275:1132-1136. 81. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 1999; 399:483-487. 82. Monney L, Otter I, Olivier R, et al. Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain. Biochem Biophys Res Commun 1996; 221:340-345. 83. Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBO J 2001; 20:6627-6636. 84. Susin SA, Zamzami N, Castedo M, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 1996; 184:1331-1341. 85. Vander Heiden MG, Chandel NS, Li XX, et al. Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc Natl Acad Sci U S A 2000; 97:4666-4671. 86. Vander Heiden MG, Li XX, Gottleib E, et al. Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J Biol Chem 2001; 276:19414-19419. 87. Vander Heiden MG, Chandel NS, Schumacker PT, et al. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Mol Cell 1999; 3:159-167. 88. Higashiyama M, Doi O, Kodama K, et al. Bcl-2 oncoprotein expression is increased especially in the portion of small cell carcinoma within the combined type of small cell lung cancer. Tumour Biol 1996; 17:341-344. 89. Jiang SX, Kameya T, Sato Y, et al. Bcl-2 protein expression in lung cancer and close correlation with neuroendocrine differentiation. Am J Pathol 1996; 148:837-846. 90. Kaiser U, Schilli M, Haag U, et al. Expression of bcl-2–protein in small cell lung cancer. Lung Cancer 1996; 15:31-40. 91. Yan JJ, Chen FF, Tsai YC, et al. Immunohistochemical detection of Bcl-2 protein in small cell carcinomas. Oncology 1996; 53:6-11. 92. Takayama K, Ogata K, Nakanishi Y, et al. Bcl-2 expression as a predictor of chemosensitivities and survival in small cell lung cancer. Cancer J Sci Am 1996; 2:212. 93. Stefanaki K, Rontogiannis D, Vamvouka C, et al. Immunohistochemical detection of bcl2, p53, mdm2 and p21/waf1 proteins in small-cell lung carcinomas. Anticancer Res 1998; 18:1689-1695. 94. Maitra A, Amirkhan RH, Saboorian MH, et al. Survival in small cell lung carcinoma is independent of Bcl-2 expression. Hum Pathol 1999; 30:712-717. 95. Breton C, Story MD, Meyn RE. Bcl-2 expression correlates with apoptosis induction but not loss of clonogenic survival in small cell lung cancer cell lines treated with etoposide. Anticancer Drugs 1998; 9:751-757. 96. Dingemans AM, Witlox MA, Stallaert RA, et al. Expression of DNA topoisomerase IIalpha and topoisomerase IIbeta genes predicts survival and response to chemotherapy in patients with small cell lung cancer. Clin Cancer Res 1999; 5:20482058. 97. Sloman A, D’Amico F, Yousem SA. Immunohistochemical markers of prolonged survival in small cell carcinoma of the lung. An immunohistochemical study. Arch Pathol Lab Med 1996; 120:465-472. 98. Pal’tsev MA, Demura SA, Kogan EA, et al. [Small cell carcinoma and carcinoids of the lung: morphology of apoptosis and expression of biomolecular markers of tumor growth]. Arkh Patol 2000; 62:11-17. 99. Pardo OE, Arcaro A, Salerno G, et al. Fibroblast growth factor-2 induces translational regulation of Bcl-XL and Bcl-2 via a MEK-dependent pathway: correlation with resistance to etoposide-induced apoptosis. J Biol Chem 2002; 277:12040-12046. 100. Eerola AK, Tormanen U, Rainio P, et al. Apoptosis in operated small cell lung carcinoma is inversely related to tumour necrosis and p53 immunoreactivity. J Pathol 1997; 181:172-177. 101. Ohmori T, Podack ER, Nishio K, et al. Apoptosis of lung cancer cells caused by some anti-cancer agents (MMC, CPT-11, ADM) is inhibited by bcl-2. Biochem Biophys Res Commun 1993; 192:30-36. 102. Sartorius UA, Krammer PH. Upregulation of Bcl-2 is involved in the mediation of chemotherapy resistance in human small cell lung cancer cell lines. Int J Cancer 2002; 97:584-592. 103. Bennett CF, Cowsert LM. Application of antisense oligonucleotides for gene functionalization and target validation. Curr Opin Mol Ther 1999; 1:359-371. 104. Dean NM. Functional genomics and target validation approaches using antisense oligonucleotide technology. Curr Opin Biotechnol 2001; 12:622-625. 105. Ziegler A, Luedke GH, Fabbro D, et al. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence. J Natl Cancer Inst 1997; 89:1027-1036. 106. Zangemeister-Wittke U, Schenker T, Luedke GH, et al. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines. Br J Cancer 1998; 78:1035-1042. 107. Zangemeister-Wittke U, Leech SH, Olie RA, et al. A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells. Clin Cancer Res 2000; 6:2547-2555. 108. Cotter FE, Johnson P, Hall P, et al. Antisense oligonucleotides suppress B-cell
Dean A. Fennell lymphoma growth in a SCID-hu mouse model. Oncogene 1994; 9:3049-3055. 109. Waters JS, Webb A, Cunningham D, et al. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma. J Clin Oncol 2000; 18:1812-1823. 110. Wacheck V, Heere-Ress E, Halaschek-Wiener J, et al. Bcl-2 antisense oligonucleotides chemosensitize human gastric cancer in a SCID mouse xenotransplantation model. J Mol Med 2001; 79:587-593. 111. Lopes de Menezes DE, Hudon N, McIntosh N, et al. Molecular and pharmacokinetic properties associated with the therapeutics of bcl-2 antisense
oligonucleotide G3139 combined with free and liposomal doxorubicin. Clin Cancer Res 2000; 6:2891-2902. 112. Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 1997; 349:1137-1141. 113. Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 2000; 356:1728-1733. 114. Rudin CM, Otterson GA, Mauer AM, et al. A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer. Ann Oncol 2002; 13:539-545.
Clinical Lung Cancer March 2003
313
Key words: Antisense oligonucleotides, Apoptosis, Caspases, Voltage-dependent anion channel
Introduction
chemotherapeutic combinations, little impact has been made in improving long-term survival over the past two decades.8 There is, therefore, a major need to address the therapeutic problem of emerging chemoresistance in SCLC in order to improve outcome.
Small-cell lung cancer (SCLC) affects approximately 20% of patients with pulmonary malignancy and is the most aggressive form of lung cancer, associated with a median survival from diagnosis of 2-4 months if untreated.1 Small-cell lung cancer displays exquisite sensitivity to induction chemotherapy, with objective response rates of 70%-80% in extensive-stage disease and complete remission rates of 20%-30%.2 In limited-stage disease, objective response rates are as high as 65%-90%, with complete remission rates of 45%-75%.3 Combination chemotherapy is the cornerstone of treatment due to the high probability of occult metastases; there is a 60% actuarial risk of developing central nervous system metastases within 2-3 years with prophylactic cranial irradiation.4 Despite the initial high response rate to chemotherapy, recurrent SCLC displays a transformation in phenotype with marked chemoresistance, resulting in an untreated median survival at recurrence of only 2-3 months.5 The efficacy of chemotherapy in this setting is poor; although this can be increased in patients receiving second-line therapy,6 this is highly dependent on the interval preceding relapse, with heavily pretreated patients rarely responding to further chemotherapy.7 Despite advances in diagnosis and emergence of new Lung Cancer Section, Department of Medical Oncology St Bartholomew’s Hospital, London, United Kingdom Submitted: Oct 25, 2002; Revised: Mar 7, 2003; Accepted: Mar 14, 2003 Address for correspondence: Dean A. Fennell, MD, PhD, Lung Cancer Section, Department of Medical Oncology, St Bartholomew’s Hospital, West Smithfield, London EC1A 7BE, United Kingdom Fax: 44-020-601-7577; e-mail: [email protected]
Apoptosis and Chemotherapy Over the past 2 decades, it has emerged that the efficacy of chemotherapy relies on the efficient induction of programmed cell death or apoptosis.9,10 This is a biochemically stereotyped and phylogenetically conserved process11,12 that plays a critical role in development,13,14 resulting in noninflammatory cell death and removal of cells. Resistance to chemotherapy-induced apoptosis results in the failure of therapy and is commonly associated with a defect in the intrinsic cellular apoptotic machinery (CAM).15,16 Recent advances have led to greater understanding of the underlying molecular detail associated with induction of apoptosis by chemotherapy coupled with an increased understanding of the functional genomics of many cancers, including SCLC.17,18 A growing list of proteins with antiapoptotic activity that are overexpressed in cancer have been discovered, with implications for susceptibility to chemotherapy-induced cell death and new drug development.19 Bcl-2 is a prototypical antiapoptosis protein that was discovered as the result of cloning of the translocation in B-cell non-Hodgkin’s (follicular) lymphoma, associated with hyperexpression of Bcl-2 due to juxtaposition with the immunoglobulin H chain promoter.20 Bcl-2 was the first member of an expanding family of proapoptotic and antiapoptotic proteins to be discovered. Bcl-2 prevents apoptosis and is high-
Clinical Lung Cancer March 2003
307
Bcl-2 in Small-Cell Lung Cancer ly expressed in SCLC.21-24 Evidence arising from studies of Bcl2 in SCLC suggest that this antiapoptotic protein is an attractive target for novel therapy.
Activation of the Cellular Apoptotic Machinery by Cancer Chemotherapy Despite a wide variety of primary molecular targets underlying tumor cell death induction by chemotherapeutic agents, their pharmacodynamics converge in the activation of the CAM. In the early 1990s, Jacobson and colleagues demonstrated that the nucleus is neither required for apoptotic cell death nor for the protection from cell death by Bcl-2.25 Mitochondria have emerged as an integrating center of cellular death signals following exposure to chemotherapy, irrespective of initial molecular interaction with the cell.16,26 These organelles, previously thought of as critical to life through their involvement in aerobic respiration, have now been shown to be intimately involved in the control of cell death.27,28
Proapoptotic Proteins Nucleated cells constitutively express Bcl-2 homologous proapoptotic proteins (PAPs), termed Bax and Bak in their cytosol.29-32 These proteins are folded into inactive conformations. However, upon induction of cell death by cytotoxic drugs, Bax and Bak unfold, resulting in their translocation to the outer mitochondrial membrane (OMM).33-36 These proteins are an essential component of the CAM that enable chemotherapy-induced apoptosis.37 Mitochondria are double-membraned organelles. The OMM and inner mitochondrial membrane (IMM) meet at regions characterized by junctional complexes; these play a critical role in cytosol-to-mitochondria signaling by directing translocation of PAPs38 and transducing signals from the mitochondria back to the cytosol, which result in the destruction of the cell.39
Mitochondrial Junctional Complexes Mitochondrial junctional complexes (also known as permeability transition pore complexes, PTPCs) consist of several proteins in the OMM, IMM, and innermembrane space (IMS); these include OMM-localized voltage-dependent anion channel (VDAC),40 peripheral benzodiazepine receptor,41 hexokinase II,42 IMS-localized creatine kinase,43 as well as IMM-localized adenine nucleotide translocator44 and cyclophilin D.39
Cytochrome C On docking with the OMM, PAPs initiate permeabilization of both the OMM and IMM, resulting in an irreversible commitment to cell death. The OMM permeabilization results in the release of apoptosis, inducing factors from the IMS that include cytochrome C,45-47 apoptosis-inducing factor,48 and second mitochondria-derived activator of caspases (SMAC).49,50 Some evidence suggests that this release of proteins is nonspecific and involves up to 100 proteins, implicating OMM rupture.51,52 However, in some experimental systems, cytochrome C appears to be released before other proteins.53 Shimizu and colleagues have proposed a model of the action of Bax and Bak,
308
Clinical Lung Cancer March 2003
which involves direct interaction with VDAC, resulting in a conformational change that leads to conduction of cytochrome into the cytosol.54-56 These proteins have been shown to form a large novel conductance with VDAC capable of conducting cytochrome C54; the existence of a novel, high conductance channel has also been demonstrated by Pavlov and colleagues, which correlates with the presence of Bax in the OMM,57 the so-called mitochondrial apoptosis channel. Cytochrome C directly activates a cascade of executioner serine proteases termed caspases. This occurs via the deoxyadenosine triphosphate–dependent formation of a heterotrimeric cytochrome C/Apaf-1/procaspase9 apoptosome, resulting in activation of caspase-945 followed by further downstream activation of other caspases including caspase-3.58 Caspases target several substrates within the cell, affecting the typical morphological features of apoptosis, including cell shrinkage, blebbing, chromatin condensation, and expression of outer plasma membrane phosphotidylserine (Figure 1). SMAC interacts with inhibitors of the apoptosis protein, XIAP, which inhibits caspase 3, caspase 7, and caspase 9,59-61 through an interaction with its BIR3 domain.62 Apoptosis-inducing factor (AIF) is a heat shock protein–70 inhibitable reduced nicotinamide adenine dinucleotide oxidoreductase that induces caspase independent cell death via translocation to the nucleus.63-65
Mitochondrial Depolarization At or around the time of docking of PAPs with mitochondria and OMM permeabilization, the IMM undergoes permeabilization via formation of a nonselective pore. This event, termed permeability transition, involves depolarization of the IMM potential established during respiratory electron transport.66,67 This process is directly induced by Bax68 through a mechanism that is not yet fully understood but is likely to directly or indirectly involve VDAC69 and adenine neucleotide translocation.70,71 The immediate consequence of mitochondrial depolarization is the failure of adenosine triphosphate (ATP) synthesis and death. Following permeability transition, the F1F0ATPase may function in reverse, resulting in hydrolysis of existing ATP stores in an attempt to stabilize the IMM potential.72
Bcl-2 Antiapoptosis Bcl-2 constitutively localizes to the OMM, the endoplasmic reticulum, and the outer nuclear membrane.73-77 In mitochondria, Bcl-2 is localized to contact sites and physically associates with PTPCs.71,78 Proapoptotic protein–induced permeability of the OMM is blocked by Bcl-2.79,80 The mechanism of this release is controversial. In the model proposed by Shimizu et al,56,81 VDAC is a target for regulation by Bcl-2, interacting stoichiometrically with Bax to antagonize induction of conformational change and so prevent passage of cytochrome C (Figure 2). Bcl-2 also prevents formation of the novel high conductance in the OMM.56,57 The consequence of failure of cytochrome C translocation is inhibition of caspase activation.82 The proapoptotic activity of AIF and SMAC are inhibited by Bcl-2 by blocking their release, suggesting regulation by Bcl-2 of nonspecific OMM permeability.83,84
Dean A. Fennell Figure 1 Caspase and Apoptosis CED3 Subfamily
ICE Subfamily
Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase Caspase 9 2 10 8 6 3 7 1 4 5 13 11 12 Caspase 6
Caspase 8
Caspase 3 Caspase 10
Caspase 2 CYT C APAF 1
dATP Pro
Caspase 9
Caspase 9
PARP, PAK2 (Blebbing), CAD (DNA fragmentation), Laminin (Condensation)
Caspase 7 Several caspases have been identified. Permeabilization of the outer mitochondrial membrane activates procaspase 9, which then activates effector caspases 3 and 7. Downstream caspase activation results in the manifestations of apoptosis via specific substrate cleavage. Abbreviations: APAF = apoptosis protease-activating protein-1; CAD = caspase-activated deoxyribonuclease; CED3 = Caenorhabditis elegans cell death 3; CYT C = cytochrome C; ICE = interleukin-1β–converting enzyme; PAK2 = p21-activated kinase 2; PARP = poly (ADP-ribose) polymerase
This Bcl-2 activity during cell death contrasts with the constitutive effects of Bcl-2 during normal respiration, which appears to interact with VDAC so as to increase conductance to pyridine nucleotides (adenosine diphosphate and ATP) required for oxidative phosphorylation.85-87 Bcl-2 also prevents permeability transition through an allosteric effect on the adenine nucleotide translocator although the mechanism of this regulation has not been elucidated.71
Expression of Bcl-2 in Small-Cell Lung Cancer Bcl-2 has been shown to be expressed in SCLC cell lines by immunocytochemistry as well as by Northern blot analysis to demonstrate mRNA expression21 and is prevalent in biopsy-derived SCLC tissue as shown in Table 1.22,23,88-94 Bcl-2 expression correlates with apoptosis sensitivity in vitro.95 Some studies have demonstrated adverse prognostic effects of high Bcl-2 expression in patients with SCLC by multivariate analysis.92,96 Owing to the high prevalence of Bcl-2 in SCLC cohorts, however, prognostic stratification has not been demonstrated in some reports.94,97 They argue that the prevalence of Bcl-2 expression may play an important role in antiapoptosis during tumorigenesis. Expression of Bcl-2 is higher in metastatic SCLC compared with localized disease98 and is regulated at the translational level by fibroblast growth factor-2.99 A very high proliferation index is observed in SCLC cells as measured by ki67 antigen expression; this may account for chemosensitivity despite Bcl-2 expression.100 The level of Bcl-2 expression in SCLC has a significant impact on apoptosis sensitivity; transfection-associated overexpres-
sion of Bcl-2 in SCLC can modulate sensitivity to chemotherapeutic agents in vitro.101 Sartorius and Krammer have recently examined the molecular changes associated with selection for resistance to cisplatin, etoposide, and doxorubin in 9 SCLC cell lines.102 Multidrug resistance was generated by subculture in subtherapeutic concentrations of etoposide. Drug resistance was associated with a substantial increase in the expression of Bcl-2. To date, no studies have been conducted to examine the molecular changes that accompany acquired multidrug resistance in SCLC in vivo.
Targeting Bcl-2 in Small-Cell Lung Cancer to Reverse Chemoresistance Antisense oligonucleotides (ASOs) enable transient and selective inhibition of polypeptide synthesis and provide a tool for investigating the function of proteins in living cells.103,104 Antisense oligonucleotides are short strands of modified DNA (usually 18-20 mer) that hybridize to their target mRNA to form a heteroduplex that triggers active degradation of the bound mRNA via RNaseH. By scanning the bcl-2 mRNA for effective candidate ASOs capable of downregulating bcl-2 mRNA and protein expression, Ziegler and colleagues identified a potent phosphorothioate ASO, termed 2009.105 Antisense oligonucleotide 2009 induced sequence-specific apoptosis in SCLC cell lines NCI-H69, NCI-H82, and NCI-417 in association with sequence-specific downregulation of Bcl-2. Control oligonucleotides did mediate toxicity, consistent with a role for Bcl-2 in controlling apoptosis threshold. Interestingly, the toxicity of ASO 2009 correlated with the basal level of
Clinical Lung Cancer March 2003
309
Bcl-2 in Small-Cell Lung Cancer lines.106 Potent combination indices were demonstrated for etoposide/ASO 2009 combinations in high Bcl-2–expressing NCI-H69 cells. However, no synergy was observed for combinations in the SCLC cell line NCI-H82, which exhibited the lowest level of Bcl-2 expression. A novel bispecific ASO targeting Bcl-2 and its antiapoptotic homologue Bcl-XL has been shown to exhibit sequence-specific killing of SCLC cells in vitro.107
Figure 2 Bcl-2 and Permeabilization
HKII Bcl-2
Bax PBR
Bcl-2 Antisense Therapy in Small-Cell Lung Cancer
VDAC
Oblimersen (G3139, Genasense™) is a phosphorothioatebased ASO that has shown activity in both solid tumors (nude mouse models of lymphoma, gastric cancer, and melanoma108111) and clinical trials (non-Hodgkin’s lymphoma and melanoma).109,112,113 Several studies in a variety of malignancies are ongoing (Table 2).109,112-114 Recently, the first phase I study in SCLC employing oblimersen was reported in patients with chemorefractory disease, which was defined as those patients experiencing relapse ≤ 3 months after the completion of primary therapy.114 A total of 12 patients were treated with paclitaxel 150 mg/m2 combined with oblimersen and administered as a continuous intravenous infusion over 7 days to allow for Bcl-2 protein downregulation. No objective responses were observed, although 2 patients developed stable disease; 1 patient who had the highest serum levels of oblimersen remained stable for 30 weeks.
CK
Outer mitochondrial membrane
ANT
CyPD Inner mitochondrial membrane
Bcl-2 blocks permeabilization of both the outer mitochondrial membrane and the inner mitochondrial membrane via a direct interaction with the permeability transition pore complexes. Abbreviations: ANT = adenine neucleotide translocation; CK = creatine kinase; CyPD = cyclophilin D; HKII = hexokinase II; PBR = peripheral benzodiazepine receptor; VDAC = voltage-dependent anion channel
Conclusion
Bcl-2 expression. Studies to examine the effects of ASO 2009 in combination with chemotherapy have demonstrated synergy in SCLC cell Table 1
There is a paucity of research examining the molecular basis of chemoresistance that characterizes SCLC at relapse, a factor that underlies the poor survival rates associated with this disease. The antiapoptotic protein Bcl-2 is expressed in SCLC, is associated with acquired multidrug resistance, and represents a valid target for therapeutic intervention. Downregulation of antiapoptotic protein ex-
Bcl-2 Expression in Small-Cell Lung Cancer Number of Patients
% Bcl-2 Expression
Objective Response Rate (% Bcl-2+ vs. Bcl-2–)
Relative Median Survival (% Bcl-2+ vs. Bcl-2–)
Comments
Ben-Ezra et al,22 1994
23
65%
NR
NR
–
Higashiyama et al,23 1995
13
69%
NR
NR
–
24
77%
NR
NR
–
Jiang et al,89 1996
111
94%
NR
NR
–
Kaiser et al,90 1996
125
76%
40% vs. 27%
12% vs. 9.5% (P = 0.17)
No significant difference in survival detected
Yan et al,91 1996
29
93%
NR
NR
Bcl-2 does not correlate with clinical stage
Takayama et al,92 1996
38
55%
62% vs. 76%
NR
–
Stefanaki et al,93 1998
31
77%
NR
NR
–
Maitra et al,94 1999
42
57%
NR
11% vs. 13% (P = 0.2256)
Survival was independent of age, stage, therapy, and Bcl-2
Trial, Year
Higashiyama et
al,88
1996
In the minority of studies that analyzed survival data using Cox regression, Bcl-2 was not shown to be an independent prognostic factor. Abbreviation: NR = not reported
310
Clinical Lung Cancer March 2003
Dean A. Fennell Table 2
Ongoing Studies of Bcl-2 Antisense Oblimersen with and Without Standard Chemotherapy
Trial Phase Phase I114
Neoplasm
Synergistic Combination
Small-cell lung cancer
Paclitaxel
Phase II109,112 Non-Hodgkin’s lymphoma
–
Phase II
Mantle cell lymphoma
Rituximab, Cyclophosphamide, Vincristine, Doxorubicin, Prednisnone
Phase II
Prostate cancer
Docetaxel
Melanoma
Dacarbazine
Phase III
Non–small-cell lung cancer
Docetaxel
Phase III
Acute lymphocytic leukemia
Gemtuzumab Ozogamicin
Phase III
Multiple myeloma
High-dose Dexamethasone
Phase III
Chronic lymphocytic leukemia
Cyclophosphamide and Fludarabine
Phase
III113
pression by Bcl-2–targetting ASOs is an emerging therapeutic strategy that could lead to improvements in disease-free survival in SCLC. Other antisense strategies are currently being investigated in lung cancer and include targeting of c-raf by the oligonucleotide ISIS 5132 and protein kinase C by LY900003 (ISIS 3521, Affinitak™). However, further studies are required to determine the relative importance of these proteins as well as Bcl2 in relapsed SCLC.
Acknowledgements The author thanks Robin M. Rudd, MD, for reading the manuscript, and Finbarr E. Cotter, MD, PhD, for helpful discussions.
References 1. Okuno SH, Jett JR. Small cell lung cancer: current therapy and promising new regimens. Oncologist 2002; 7:234-238. 2. Simon GR, Wagner H. Small cell lung cancer. Chest 2003; 123:259S-271S. 3. Prasad US, Naylor AR, Walker WS, et al. Long term survival after pulmonary resection for small cell carcinoma of the lung. Thorax 1989; 44:784-787. 4. Nugent JL, Bunn PA Jr, Matthews MJ, et al. CNS metastases in small cell bronchogenic carcinoma: increasing frequency and changing pattern with lengthening survival. Cancer 1979; 44:1885-1893. 5. Albain KS, Crowley JJ, Hutchins L, et al. Predictors of survival following relapse or progression of small cell lung cancer. Southwest Oncology Group Study 8605 report and analysis of recurrent disease data base. Cancer 1993;72:1184-1191. 6. Chute JP, Kelley MJ, Venzon D, et al. Retreatment of patients surviving cancer-free 2 or more years after initial treatment of small cell lung cancer. Chest 1996; 110:165-171. 7. Sekine I, Nishiwaki Y, Kakinuma R, et al. Late recurrence of small-cell lung cancer: treatment and outcome. Oncology 1996; 53:318-321. 8. Chute JP, Venzon DJ, Hankins L, et al. Outcome of patients with small-cell lung cancer during 20 years of clinical research at the US National Cancer Institute. Mayo Clin Proc 1997; 72:901-912. 9. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26:239-257. 10. Kaufmann SH. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Res 1989; 49:5870-5878. 11. Hengartner MO, Horvitz HR. Programmed cell death in Caenorhabditis elegans. Curr Opin Genet Dev 1994; 4:581-586. 12. Abrams JM. Competition and compensation: coupled to death in development and
cancer. Cell 2002; 110:403-406. 13. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell 1997; 88:347-354. 14. Meier P, Finch A, Evan G. Apoptosis in development. Nature 2000; 407:796-801. 15. Makin G, Dive C. Apoptosis and cancer chemotherapy. Trends Cell Biol 2001; 11:S22-S26. 16. Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000; 256:42-49. 17. Bangur CS, Switzer A, Fan L, et al. Identification of genes over-expressed in small cell lung carcinoma using suppression subtractive hybridization and cDNA microarray expression analysis. Oncogene 2002; 21:3814-3825. 18. Oh JM, Brichory F, Puravs E, et al. A database of protein expression in lung cancer. Proteomics 2001; 1:1303-1319. 19. Reed JC. Apoptosis-based therapies. Nat Rev Drug Discov 2002; 1:111-121. 20. Tsujimoto Y, Finger LR, Yunis J, et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 1984; 226:1097-1099. 21. Ikegaki N, Katsumata M, Minna J, et al. Expression of bcl-2 in small cell lung carcinoma cells. Cancer Res 1994; 54:6-8. 22. Ben-Ezra JM, Kornstein MJ, Grimes MM, et al. Small cell carcinomas of the lung express the Bcl-2 protein. Am J Pathol 1994; 145:1036-1040. 23. Higashiyama M, Doi O, Kodama K, et al. High prevalence of bcl-2 oncoprotein expression in small cell lung cancer. Anticancer Res 1995; 15:503-505. 24. Jiang SX, Sato Y, Kuwao S, et al. Expression of bcl-2 oncogene protein is prevalent in small cell lung carcinomas. J Pathol 1995; 177:135-138. 25. Jacobson MD, Burne JF, Raff MC. Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J 1994; 13:1899-1910. 26. Susin SA, Zamzami N, Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1998; 1366:151-165. 27. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309-1312. 28. Van Loo G, Saelens X, Van Gurp M, et al. The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ 2002; 9:1031-1042. 29. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74:609-619. 30. Farrow SN, White JH, Martinou I, et al. Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K. Nature 1995; 374:731-733. 31. Chittenden T, Harrington EA, O'Connor R, et al. Induction of apoptosis by the Bcl-2 homologue Bak. Nature 1995; 374:733-736. 32. Kiefer MC, Brauer MJ, Powers VC, et al. Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature 1995; 374:736-739. 33. Wolter KG, Hsu YT, Smith CL, et al. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997; 139:1281-1292. 34. Nechushtan A, Smith CL, Hsu YT, et al. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J 1999; 18:2330-2341. 35. Nechushtan A, Smith CL, Lamensdorf I, et al. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 2001; 153:12651276. 36. Griffiths GJ, Corfe BM, Savory P, et al. Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak. Oncogene 2001; 20:7668-7676. 37. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001; 292:727-730. 38. De Giorgi F, Lartigue L, Bauer MK, et al. The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J 2002; 16:607-609. 39. Crompton M, Barksby E, Johnson N, et al. Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie 2002; 84:143152. 40. Szabo I, De Pinto V, Zoratti M. The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel. FEBS Lett 1993; 330:206-210. 41. McEnery MW. The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel (VDAC). J Bioenerg Biomembr 1992; 24:63-69. 42. Nakashima RA. Hexokinase-binding properties of the mitochondrial VDAC protein: inhibition by DCCD and location of putative DCCD-binding sites. J Bioenerg Biomembr 1989; 21:461-470. 43. O’Gorman E, Beutner G, Dolder M, et al. The role of creatine kinase in inhibition of mitochondrial permeability transition. FEBS Lett 1997; 414:253-257. 44. Belzacq AS, Vieira HL, Kroemer G, et al. The adenine nucleotide translocator in apoptosis. Biochimie 2002; 84:167-176. 45. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91:479-489. 46. Jurgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci U S A 1998; 95:49975002. 47. Narita M, Shimizu S, Ito T, et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A 1998; 95:14681-14686.
Clinical Lung Cancer March 2003
311
Bcl-2 in Small-Cell Lung Cancer 48. Lorenzo HK, Susin SA, Penninger J, et al. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ 1999; 6:516-524. 49. Du C, Fang M, Li Y, et al. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 2000; 102:33-42. 50. Verhagen AM, Ekert PG, Pakusch M, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 2000; 102:43-53. 51. Single B, Leist M, Nicotera P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ 1998; 5:1001-1003. 52. Patterson SD, Spahr CS, Daugas E, et al. Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition. Cell Death Differ 2000; 7:137-144. 53. Doran E, Halestrap AP. Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites. Biochem J 2000; 348 Pt 2:343-350. 54. Shimizu S, Ide T, Yanagida T, et al. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J Biol Chem 2000; 275:12321-12325. 55. Shimizu S, Matsuoka Y, Shinohara Y, et al. Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J Cell Biol 2001; 152:237-250. 56. Shimizu S, Shinohara Y, Tsujimoto Y. Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator. Oncogene 2000; 19:4309-4318. 57. Pavlov EV, Priault M, Pietkiewicz D, et al. A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J Cell Biol 2001; 155:725-731. 58. Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376:37-43. 59. Chai J, Shiozaki E, Srinivasula SM, et al. Structural basis of caspase-7 inhibition by XIAP. Cell 2001; 104:769-780. 60. Riedl SJ, Renatus M, Schwarzenbacher R, et al. Structural basis for the inhibition of caspase-3 by XIAP. Cell 2001; 104:791-800. 61. Ekert PG, Silke J, Hawkins CJ, et al. DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9. J Cell Biol 2001; 152:483-490. 62. Liu Z, Sun C, Olejniczak ET, et al. Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 2000; 408:1004-1008. 63. Daugas E, Susin SA, Zamzami N, et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 2000; 14:729-739. 64. Loeffler M, Daugas E, Susin SA, et al. Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J 2001; 15:758767. 65. Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397:441-446. 66. Hunter DR, Haworth RA, Southard JH. Relationship between configuration, function, and permeability in calcium-treated mitochondria. J Biol Chem 1976; 251:5069-5077. 67. Haworth RA, Hunter DR. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys 1979; 195:460-467. 68. Pastorino JG, Chen ST, Tafani M, et al. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J Biol Chem 1998; 273:7770-7775. 69. Tsujimoto Y, Shimizu S. The voltage-dependent anion channel: an essential player in apoptosis. Biochimie 2002; 84:187-193. 70. Marzo I, Brenner C, Kroemer G. The central role of the mitochondrial megachannel in apoptosis: evidence obtained with intact cells, isolated mitochondria, and purified protein complexes. Biomed Pharmacother 1998; 52:248251. 71. Brenner C, Cadiou H, Vieira HL, et al. Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene 2000; 19:329-336. 72. Rego AC, Vesce S, Nicholls DG. The mechanism of mitochondrial membrane potential retention following release of cytochrome c in apoptotic GT1-7 neural cells. Cell Death Differ 2001; 8:995-1003. 73. Monaghan P, Robertson D, Amos TA, et al. Ultrastructural localization of bcl-2 protein. J Histochem Cytochem 1992; 40:1819-1825. 74. Nguyen M, Millar DG, Yong VW, et al. Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH- terminal signal anchor sequence. J Biol Chem 1993; 268:25265-25268. 75. Nakai M, Takeda A, Cleary ML, et al. The bcl-2 protein is inserted into the outer membrane but not into the inner membrane of rat liver mitochondria in vitro. Biochem Biophys Res Commun 1993; 196:233-239. 76. Krajewski S, Tanaka S, Takayama S, et al. Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 1993; 53:4701-4714. 77. Akao Y, Otsuki Y, Kataoka S, et al. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res 1994; 54:2468-2471. 78. Marzo I, Brenner C, Zamzami N, et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med
312
Clinical Lung Cancer March 2003
1998; 187:1261-1271. 79. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275:1129-1132. 80. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 1997; 275:1132-1136. 81. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 1999; 399:483-487. 82. Monney L, Otter I, Olivier R, et al. Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain. Biochem Biophys Res Commun 1996; 221:340-345. 83. Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBO J 2001; 20:6627-6636. 84. Susin SA, Zamzami N, Castedo M, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 1996; 184:1331-1341. 85. Vander Heiden MG, Chandel NS, Li XX, et al. Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc Natl Acad Sci U S A 2000; 97:4666-4671. 86. Vander Heiden MG, Li XX, Gottleib E, et al. Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J Biol Chem 2001; 276:19414-19419. 87. Vander Heiden MG, Chandel NS, Schumacker PT, et al. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Mol Cell 1999; 3:159-167. 88. Higashiyama M, Doi O, Kodama K, et al. Bcl-2 oncoprotein expression is increased especially in the portion of small cell carcinoma within the combined type of small cell lung cancer. Tumour Biol 1996; 17:341-344. 89. Jiang SX, Kameya T, Sato Y, et al. Bcl-2 protein expression in lung cancer and close correlation with neuroendocrine differentiation. Am J Pathol 1996; 148:837-846. 90. Kaiser U, Schilli M, Haag U, et al. Expression of bcl-2–protein in small cell lung cancer. Lung Cancer 1996; 15:31-40. 91. Yan JJ, Chen FF, Tsai YC, et al. Immunohistochemical detection of Bcl-2 protein in small cell carcinomas. Oncology 1996; 53:6-11. 92. Takayama K, Ogata K, Nakanishi Y, et al. Bcl-2 expression as a predictor of chemosensitivities and survival in small cell lung cancer. Cancer J Sci Am 1996; 2:212. 93. Stefanaki K, Rontogiannis D, Vamvouka C, et al. Immunohistochemical detection of bcl2, p53, mdm2 and p21/waf1 proteins in small-cell lung carcinomas. Anticancer Res 1998; 18:1689-1695. 94. Maitra A, Amirkhan RH, Saboorian MH, et al. Survival in small cell lung carcinoma is independent of Bcl-2 expression. Hum Pathol 1999; 30:712-717. 95. Breton C, Story MD, Meyn RE. Bcl-2 expression correlates with apoptosis induction but not loss of clonogenic survival in small cell lung cancer cell lines treated with etoposide. Anticancer Drugs 1998; 9:751-757. 96. Dingemans AM, Witlox MA, Stallaert RA, et al. Expression of DNA topoisomerase IIalpha and topoisomerase IIbeta genes predicts survival and response to chemotherapy in patients with small cell lung cancer. Clin Cancer Res 1999; 5:20482058. 97. Sloman A, D’Amico F, Yousem SA. Immunohistochemical markers of prolonged survival in small cell carcinoma of the lung. An immunohistochemical study. Arch Pathol Lab Med 1996; 120:465-472. 98. Pal’tsev MA, Demura SA, Kogan EA, et al. [Small cell carcinoma and carcinoids of the lung: morphology of apoptosis and expression of biomolecular markers of tumor growth]. Arkh Patol 2000; 62:11-17. 99. Pardo OE, Arcaro A, Salerno G, et al. Fibroblast growth factor-2 induces translational regulation of Bcl-XL and Bcl-2 via a MEK-dependent pathway: correlation with resistance to etoposide-induced apoptosis. J Biol Chem 2002; 277:12040-12046. 100. Eerola AK, Tormanen U, Rainio P, et al. Apoptosis in operated small cell lung carcinoma is inversely related to tumour necrosis and p53 immunoreactivity. J Pathol 1997; 181:172-177. 101. Ohmori T, Podack ER, Nishio K, et al. Apoptosis of lung cancer cells caused by some anti-cancer agents (MMC, CPT-11, ADM) is inhibited by bcl-2. Biochem Biophys Res Commun 1993; 192:30-36. 102. Sartorius UA, Krammer PH. Upregulation of Bcl-2 is involved in the mediation of chemotherapy resistance in human small cell lung cancer cell lines. Int J Cancer 2002; 97:584-592. 103. Bennett CF, Cowsert LM. Application of antisense oligonucleotides for gene functionalization and target validation. Curr Opin Mol Ther 1999; 1:359-371. 104. Dean NM. Functional genomics and target validation approaches using antisense oligonucleotide technology. Curr Opin Biotechnol 2001; 12:622-625. 105. Ziegler A, Luedke GH, Fabbro D, et al. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence. J Natl Cancer Inst 1997; 89:1027-1036. 106. Zangemeister-Wittke U, Schenker T, Luedke GH, et al. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines. Br J Cancer 1998; 78:1035-1042. 107. Zangemeister-Wittke U, Leech SH, Olie RA, et al. A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells. Clin Cancer Res 2000; 6:2547-2555. 108. Cotter FE, Johnson P, Hall P, et al. Antisense oligonucleotides suppress B-cell
Dean A. Fennell lymphoma growth in a SCID-hu mouse model. Oncogene 1994; 9:3049-3055. 109. Waters JS, Webb A, Cunningham D, et al. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma. J Clin Oncol 2000; 18:1812-1823. 110. Wacheck V, Heere-Ress E, Halaschek-Wiener J, et al. Bcl-2 antisense oligonucleotides chemosensitize human gastric cancer in a SCID mouse xenotransplantation model. J Mol Med 2001; 79:587-593. 111. Lopes de Menezes DE, Hudon N, McIntosh N, et al. Molecular and pharmacokinetic properties associated with the therapeutics of bcl-2 antisense
oligonucleotide G3139 combined with free and liposomal doxorubicin. Clin Cancer Res 2000; 6:2891-2902. 112. Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 1997; 349:1137-1141. 113. Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 2000; 356:1728-1733. 114. Rudin CM, Otterson GA, Mauer AM, et al. A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer. Ann Oncol 2002; 13:539-545.
Clinical Lung Cancer March 2003
313