Human Small Cell Lung Cancer Tumors Research Articles

Abstract C128: TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin.

Abstract Background: The molecular chaperone HSP90 has been considered a promising target for cancer therapy. We have previously reported the discovery of an orally available HSP90α/β selective inhibitor, TAS-116. TAS-116 led to tumor shrinkage in various human tumor xenograft models, while not inducing retinal toxicity in rats including at doses given above the maximum tolerated dose. Extensive small cell lung cancer (SCLC) is one of the most aggressive types of cancer, and has an unmet medical need for more effective treatments. An anthracycline, amrubicin (AMR), has been used to treat platinum resistant SCLC in Japan. Here we report on the possible application of TAS-116 to treat SCLC both as a single agent and in combination with AMR. Materials and Methods: In vitro drug combination effects were evaluated with calculation of the combination index and cell cycle analysis by fluorescence-activated cell sorting. To clarify the mechanism involved in TAS-116’s enhancement of anthracycline-induced apoptosis the extent of apoptosis was measured in combination with several kinase inhibitors including SB 218078 (CHK1) and MK-2206 (AKT). Antitumor activities of TAS-116 alone and in combination with AMR were evaluated in human SCLC xenograft models. Results: TAS-116 induced cell growth arrest and apoptosis in SCLC cell lines by inhibiting both PI3K/AKT and MAPK signaling through downregulation of HSP90 clients such as AKT and RAF1. TAS-116 also showed antitumor activity against human SCLC tumor xenograft models. In in vitro explorative combination studies in SCLC cell lines, structurally-related anthracyclines, AMR and doxorubicin (DOX), both showed synergistic effect upon apoptosis induction in combination with TAS-116. Moreover, TAS-116 enhanced the antitumor activity of AMR in human SCLC tumor xenograft models. Since anthracyclines exert antitumor activity based on their DNA damaging potential through topoisomerase II inhibition, we evaluated whether DNA damage signaling proteins and apoptosis-related proteins were affected by TAS-116 when given in combination with DOX. TAS-116 significantly reduced phosphorylation of CHK1, AKT and ERK1/2. In combination with these kinase inhibitors, SB 218078 significantly and MK-2206 moderately enhanced DOX-mediated apoptosis. Comparatively, TAS-116 enhanced apoptosis to a greater extent than SB 218078. Conclusion: TAS-116 showed antitumor activity against human SCLC tumor xenograft models alone and in combination with AMR. Our data suggest that TAS-116 enhanced the antitumor activity of anthracyclines through the combined actions of abrogation of the DNA damage-induced G2/M checkpoint and the inhibition of a survival signal by CHK1 and AKT downregulation, respectively. These results suggest that TAS-116 could be a promising agent to treat extensive SCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C128. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Akihiro Hashimoto, Kenjiro Ito, Makoto Kitade, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C128.

Anti‐angiogenic activity of alpha7‐nicotinic receptor antagonists in human small cell lung cancer

Small cell lung cancer (SCLC), a highly angiogenic tumor displays a 90% association with smoking. Nicotine promotes angiogenesis in lung cancer via alpha7‐nicotinic acetylcholine receptors (α7‐nAChRs). We conjectured that α7‐nAChR antagonists should suppress nicotine‐induced angiogenesis and may have potential applications for the therapy of SCLC. Here we show that the synthetic α7‐nAChR antagonist MG624 suppresses nicotine‐induced proliferation of human microvascular endothelial cells from the lung (HMEC‐L). MG624 displayed potent anti‐angiogenic activity in the Matrigel, rat aortic ring, and chicken chorioallantoic membrane models and in human SCLC tumors xenografted in athymic mice. The administration of MG624 did not cause any discomfort, gross toxicity or lethargy in mice. Chromatin immunprecipitation assays show that MG624 decreases the recruitment of Egr‐1 on FGF2 promoter thereby decreasing its transcription and suppressing nicotine‐induced angiogenesis. Currently, our laboratory is testing the anti‐angiogenic activity of other well characterized α7‐nAChR antagonists like MLA and COG. Our results suggest that α7‐nAChR based anti‐angiogenic agents may be useful for the therapy of human SCLC.

Capsaicin: a novel dietary therapeutic agent in human small cell lung cancers

Capsaicin, spicy ingredient of chili peppers, is used to topically treat pain and inflammation associated with a variety of diseases. However, emerging evidence shows that it can induce apoptosis in different transformed cell types in vitro and in animal models. The present study examines the apoptotic activity of capsaicin in human small cell lung cancer (SCLC). Here we show that capsaicin induced apoptosis in a panel of human SCLC in a concentration‐dependent and time‐dependent manner. Most importantly, capsaicin caused robust apoptosis in a panel of human SCLC cell lines, but did not affect normal human lung epithelial cells. The dietary administration of capsaicin decreased the growth H69 and DMS53 human SCLC tumors xenotransplanted in nude mice. The apoptotic activity of capsaicin was reversed with the generalized TRPV antagonist ruthenium red, indicating that TRPV receptors are involved in capsaicin induced apoptosis. However, antagonists to TRPV1, namely capsazepine and SB‐366791, did not affect the apoptotic activity of capsaicin, suggesting the involvement of TRPV1‐independent mechanism. In contrast, depletion of TRPV6 by siRNA ablated the apoptotic activity of capsaicin. Our findings suggest that capsaicin may have potential applications as a novel agent for management and therapy of human SCLCs.

Evaluation of Novel Orthotopic Nude Mouse Models for Human Small-Cell Lung Cancer

Although subcutaneous xenograft models have been widely used to evaluate the antitumor activity of new compounds, these models present a major disadvantage because the tumors do not accurately represent the cancer biology, especially with regard to metastasis and drug sensitivity. Effective murine models of small-cell lung cancer (SCLC) are needed. To provide strategies for studying new therapies and tumor biology, we developed three orthotopic models of human SCLC (H69A, a variant of the National Cancer Institute [NCI]-H69 cell line selected for invasiveness in vitro, NCI-H187, and NCI-N417) in nude mice. Tumor cells were injected into their lungs and new cell lines were established from these tumors (H69ALu, H187Lu, and N417Lu) to select for a reproducible tumor growth pattern and minimize variations in tumor size. In all three models tumors started as a solitary mass in the left lung and spread to mediastinal and axillary lymph nodes and to the right lung in a pattern similar to that observed in human SCLC. To test the accuracy of this model in representing SCLC as seen in the clinic, we compared the efficacy of chemotherapeutic agents in each model. Irinotecan significantly inhibited the growth and progression of all three human SCLC tumors, and cisplatin, paclitaxel, and etoposide significantly inhibited the growth and progression of H69ALu tumors over the control agent. We have established three orthotopic murine models of human SCLC closely resembling the course of human SCLC seen in the clinic including metastasis to lymph nodes and distant organs. They provide a means for better understanding the biology of this disease and will enable evaluation of novel therapeutic strategies.

MG624, an α7‐nicotinic receptor antagonist, inhibits angiogenesis in human small cell lung cancer

Small cell lung cancer (SCLC), a highly angiogenic tumor, displays a 90% association with smoking. Nicotine promotes angiogenesis in lung cancer via alpha‐7‐nicotinic acetylcholine receptors (α7nAChRs). Therefore, α7nAChR antagonists should attenuate nicotine‐induced angiogenesis and be useful for the therapy of SCLC. Here we show that the α7nAChR antagonist MG624 suppresses proliferation of human microvascular endothelial cells from the lung (HMEC‐L) in response to nicotine. MG624 displays potent anti‐angiogenic activity in the Matrigel, rat aortic ring and chicken chorioallantoic membrane models. MG624 also inhibited nicotine‐induced angiogenesis in human SCLC tumors xenografted in athymic mice. The administration of MG624 did not cause any discomfort or lethargy in mice. Chromatin immunprecipitation assays show that MG624 decreases the recruitment of Egr‐1 on FGF2 promoter thereby decreasing its transcription and suppressing nicotine‐induced angiogenesis. Our results suggest that anti‐angiogenic agents like MG624 may be useful for the therapy of human SCLC.

Capsaicin induces apoptosis in human small cell lung cancer via the TRPV pathway

Capsaicin, the spicy ingredient of chili peppers, is used to topically treat pain and inflammation associated with a variety of diseases. However, emerging evidence shows that it can induce apoptosis in different transformed cell types in vitro and in animal models. The present study examines the apoptotic activity of capsaicin in human small cell lung cancer (SCLC). Here we show that capsaicin induced apoptosis in a panel of human SCLC in a concentration‐dependent and time‐dependent manner. Most importantly, capsaicin caused 5–6 fold apoptosis in a panel of human SCLC cell lines, but did not affect normal human lung epithelial cells. The dietary administration of capsaicin decreased the growth of H69 and DMS53 human SCLC tumors xenotransplanted in nude mice. The apoptotic activity of capsaicin was independent of TRPV1, the known receptor for capsaicin. In contrast, depletion of TRPV6 by siRNA ablated the apoptotic activity of capsaicin. Our findings suggest that capsaicin may have potential applications as a novel agent for management and therapy of human SCLCs.

MG624, an α7-nAChR antagonist, inhibits angiogenesis via the Egr-1/FGF2 pathway

Small cell lung cancer (SCLC) demonstrates a strong etiological association with smoking. Although cigarette smoke is a mixture of about 4,000 compounds, nicotine is the addictive component of cigarette smoke. Several convergent studies have shown that nicotine promotes angiogenesis in lung cancers via the α7-nicotinic acetylcholine receptor (α7-nAChR) on endothelial cells. Therefore, we conjectured that α7-nAChR antagonists may attenuate nicotine-induced angiogenesis and be useful for the treatment of human SCLC. For the first time, our study explores the anti-angiogenic activity of MG624, a small-molecule α7-nAChR antagonist, in several experimental models of angiogenesis. We observed that MG624 potently suppressed the proliferation of primary human microvascular endothelial cells of the lung (HMEC-Ls). Furthermore, MG624 displayed robust anti-angiogenic activity in the Matrigel, rat aortic ring and rat retinal explant assays. The anti-angiogenic activity of MG624 was assessed by two in vivo models, namely the chicken chorioallantoic membrane model and the nude mice model. In both of these experimental models, MG624 inhibited angiogenesis of human SCLC tumors. Most importantly, the administration of MG624 was not associated with any toxic side effects, lethargy or discomfort in the mice. The anti-angiogenic activity of MG624 was mediated via the suppression of nicotine-induced FGF2 levels in HMEC-Ls. MG624 decreased nicotine-induced early growth response gene 1 (Egr-1) levels in HMEC-Ls, and reduced the levels of Egr-1 on the FGF2 promoter. Consequently, this process decreased FGF2 levels and angiogenesis. Our findings suggest that the anti-angiogenic effects of MG624 could be useful in anti-angiogenic therapy of human SCLCs.

Capsaicin Displays Anti-Proliferative Activity against Human Small Cell Lung Cancer in Cell Culture and Nude Mice Models via the E2F Pathway

BackgroundSmall cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo.Methodology/Principal FindingsBrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation.Conclusions/SignificanceOur findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs.

NMDA receptors are expressed by small-cell lung cancer and are potential targets for effective treatment

We previously showed that functional N-methyl-D-aspartate (NMDA) receptors are expressed by human neuroblastoma cells. In this study we demonstrate functional NMDAR1 and NMDAR2 receptors are expressed by small-cell lung cancer (SCLC) classical cell lines NCI H146, NCI H345, and DMS 53, by variant cell line NCI H82, and by most SCLC tumors, and that these receptors are important for the growth of human SCLC tumor xenografts in mice. Reverse transcription-polymerase chain reaction demonstrated mRNA for both receptors, with sequences identical to those for human mRNAs, are expressed in all four cell lines, and these generated proteins of the expected sizes 120 and 170 kDa. Cell viability tests showed cell growth was significantly (P < 0.0001) impaired by NMDAR1 antagonists MK-801 and memantine. Ifenprodil and Ro25-6981, NMDAR2B antagonists at the polyamine site, also significantly (P < 0.001) inhibited the growth/survival of these cells. Alternatively, the glycine-binding antagonist, L701, 324, increased viability to 140% and 120% in NCI H345 and NCI H82 cells after 48 hours of incubation. Immunohistochemistry of SCLC tumors with our polyclonal antibodies gave specific positive staining for the NMDAR1 receptor in 8 of 10 tissues examined. Small amounts of these same antibodies significantly reduced the growth of NCI-H345 cells up to 25% (P < 0.001). When NCI H345 cells were grown as tumor xenografts in mice, the growth of these tumors was reduced by 60% (P < 0.001) by treatments with MK-801 over five days. All of these data point to active NMDAR receptors possibly having an important influence on SCLC growth and survival.

Capsaicin: a potential therapeutic agent for human small cell lung cancer

Capsaicin the major active ingredient of chili peppers can induce apoptosis in multiple human cancer cell lines in cell culture and animal models. The present study examines the anti‐neoplastic activity of capsaicin in human small cell lung cancer (SCLC). SCLC is characterized by rapid progression, early metastasis and low survival rates. Our study showed that capsaicin induced 5–6 fold increase in apoptosis in multiple human SCLC cell lines. Most interestingly, the apoptotic activity of capsaicin was only displayed in SCLC cells and not in normal human lung epithelial cells. Capsaicin also suppressed the invasion of human SCLC cells as determined by Boyden Chamber assays. The dietary administration of capsaicin decreased the growth of human SCLC tumors xenotransplanted in nude mice. The growth‐inhibitory activity of capsaicin was independent of TRPV1, which is known to be the classical receptor for capsaicin. In contrast, the growth‐inhibitory activity of capsaicin requires the TRPV6 receptor, and depletion of TRPV6 by siRNA ablated the growth‐inhibitory activity of capsaicin. Our findings suggest that capsaicin may have potential applications as a novel agent for management and therapy of human SCLCs. Funding for our study was supported by a COBRE Pilot Grant, CDDC Pilot Grant, an MU‐ADVANCE Fellowship, and the Flight Attendant Medical Research Institute YCSA Grant.

Impact of STI571 (Imatinib) on the Pharmacokinetics and In Vivo Efficacy of Etoposide and/or Ifosfamide.

Using a human small cell lung cancer (SCLC) xenografted in nude mice, we previously reported an increase of tumor growth inhibition following chemotherapy in combination with STI571 (imatinib)(Int J Cancer. 2005; 113:849–856). This led us to study the in vivo impact of STI571 on the pharmacokinetics of chemotherapy. Plasma, urine, and fecal concentrations of etoposide were determined by a validated high performance liquid chromatography method, and plasma concentrations of ifosfamide were determined by a validated GAS chromatography assay with nitrogen-phosphorus detection. C-kit and MDR1/ABCB1 transcripts were quantified using real-time quantitative reverse transcription-PCR assays. We first observed an increase of the Cmax of both etoposide and ifosfamide on days 1 and 3 when mice were treated by chemotherapy combined with STI571 compared to chemotherapy alone. We then observed a high significant increase of the AUC 0–3h (Area Under the concentration-time Curve) of etoposide when mice were treated with etoposide + STI571, due to a decrease of its fecal excretion, without impact of concomitant administration of the CYT3A4 inhibitor fluconazole. As previously shown, this effect was observed despite the absence of c-kit receptor mRNA expression. Finally, we showed that STI571 enhanced the etoposide-induced tumor growth inhibition of a human xenografted lymphoma, but not that of an in vivo human SCLC tumor, and that this antitumor effect was correlated with the mRNA level of the MDR1/ABCB1 gene. This last observation suggests that imatinib-induced pharmacokinetic changes could be due to inhibition mediated via the MDR modulator of the P-glycoprotein transporter resulting in decreased elimination of etoposide. In conclusion, STI571 can reverse MDR1 drug resistance and potentiate the effects of etoposide in MDR1-expressing cancer cells such as lymphoma cell lines. These results show that further prospective phase I and II clinical trials with combinations of etoposide-based chemotherapy and imatinib are therefore warranted in some cancer patients, such as malignant lymphoma patients, with careful toxicologic monitoring.

Radionuclide imaging of small-cell lung cancer (SCLC) using 99mTc-labeled neurotensin peptide 8–13

To prepare 99m technetium (99mTc)-labeled neurotensin (NT) peptide and to evaluate the feasibility of imaging oncogene NT receptors overexpressed in human small-cell lung cancer (SCLC) cells. The NT analogue (Nalpha-His)Ac-NT(8-13) was synthesized such that histidine was attached at the N-terminus. The analogue was labeled with [99mTc(H2O)3(CO)3] at pH 7. 99mTc-(Nalpha-His)Ac-NT(8-13) in vitro stability was determined by challenging it with 100 times the molar excess of DTPA, human serum albumin (HSA) and cysteine. The affinity, 99mTc-(Nalpha-His)Ac-NT(8-13) binding to SCLC cell line NCI-H446, was studied in vitro. Biodistribution and imaging with 99mTc-(Nalpha-His)Ac-NT(8-13) were performed at 4 and 12 h postinjection, and tissue distribution and imaging after receptor blocking were carried out at 4 h in nude mice bearing human SCLC tumor. Blood clearance was determined in normal mice. The affinity constant (Kd) of 99mTc-(Nalpha-His)Ac-NT(8-13) to SCLC cells was 0.56 nmol/L. When challenged with 100 times the molar excess of DTPA, HSA or cysteine, more than 97+/-1.8% radioactivity remained as 99mTc-(Nalpha-His)Ac-NT(8-13). Tumor-to-muscle ratio was 3.35+/-1.01 at 4 h and 4.20+/-1.35 at 12 h postinjection. The excretory route of 99mTc-(Nalpha-His)Ac-NT(8-13) was chiefly through the renal pathway. In the receptor-blocking group treated with unlabeled (Nalpha-His)Ac-NT(8-13), tumor-to-muscle ratio at 4 h was 1.25+/-0.55. The results suggest that 99mTc-(Nalpha-His)Ac-NT(8-13) specifically binds to the SCLC cells and made 99mTc-(Nalpha-His)Ac-NT(8-13) a desirable compound for further studies in planar or SPECT imaging of oncogene receptors overexpressed in SCLC cells.

Targeting small-cell lung cancer with novel fluorescent analogs of somatostatin

Early, accurate detection of small-cell lung cancer (SCLC), before it becomes systemic, is essential for successful treatment. Fluorescence-based imaging provides safe, sensitive detection of malignancies. Targeted delivery of fluorophores increases sensitivity of endoscopic imaging. We synthesized novel somatostatin analogs, based on backbone cyclic peptides, and conjugated them with fluorescent agents. Nineteen conjugates differing in core peptide, length of alkyl linker and fluorescence moiety (rhodamine and fluorescein) were tested in vitro, using a receptor binding assay, and nine of the more promising conjugates were tested in vivo by fiber-optic spectrofluorimetry and quantitative spectral imaging, on an H69 human SCLC tumor mouse xenograft model. The lead compound showed exceptional tumor/normal tissue ratios, ranging from 9 to 90, and has potential for targeting SCLC overexpressing somatostatin receptors.

In vivo efficacy of STI571 in xenografted human small cell lung cancer alone or combined with chemotherapy

STI571, or imatinib, selectively inhibits BCR/ABL, PDGFR and c-kit kinase activity. It has been reported that a large proportion of small cell lung cancer (SCLC) cell lines and tumors express c-kit and that STI571 inhibits tumor cell growth. We therefore investigated the therapeutic efficacy of STI571, alone or combined with chemotherapy, in human SCLC cells or tumors xenografted into nude mice. The level of c-kit mRNA expression was variable in SCLC tumors (positive for 2 of 4 xenografts), and c-kit protein was not detected by immunohistochemistry. On the 4 xenografted tumors, PDGFRalpha and PDGFRbeta were not detected by immunohistochemistry. STI571 induced inhibition of proliferation of the SCLC6 cell line without inducing apoptosis; in contrast, in combination with etoposide or topotecan, the growth inhibition of SCLC6 cells induced by STI571 was increased, with apoptotic DNA fragmentation. Four human SCLC xenografts (SCLC6, SCLC61, SCLC74 and SCLC108) were transplanted into mice. After intraperitoneal injection of STI571, we observed 80%, 40% and 78% growth inhibition of SCLC6, SCLC61 and SCLC108 tumors, respectively, without any significant inhibition of SCLC74 tumor growth. In mice bearing responsive SCLC tumors, we observed an increase of growth inhibition induced by chemotherapy (etoposide + ifosfamide or topotecan) by concomitant and continuous administration of STI571, associated with an increase of toxic deaths. In SCLC6-bearing mice receiving sequential treatments, we observed a reduction of toxic deaths but a decrease of synergistic antitumor efficacy. In conclusion, the efficacy of STI571 alone in SCLC xenografted tumors was variable and did not depend on c-kit expression. Moreover, a significant increase of chemotherapy-induced growth inhibition was obtained by concomitant administration of STI571 that should be carefully investigated in SCLC patients.

Imatinib mesylate efficiently achieves therapeutic intratumor concentrations in vivo but has limited activity in a xenograft model of small cell lung cancer.

Despite recent advances in cancer therapy, long-term survival in small cell lung cancer (SCLC) remains uncommon, underscoring the need for novel therapeutic approaches. Previous studies have identified constitutive expression of the receptor tyrosine kinase, c-Kit, and its ligand, stem cell factor, in a substantial proportion of SCLC specimens. The purpose of this study was to determine whether imatinib mesylate, an inhibitor of c-Kit, could achieve therapeutic concentrations in tumors and in brain (a frequent site of SCLC metastasis) and interfere with SCLC tumor growth in vivo. Human SCLC tumor cell lines with constitutive c-kit expression and tyrosine phosphorylation (NCI-H209, NCI-H526, and NCI-H1607) were used to establish SCLC tumor xenografts in NCr nude (nu/nu)-immunodeficient mice. SCLC tumor-bearing mice were randomly assigned to imatinib or control (water) administered twice a day by oral gavage. Imatinib concentrations in plasma, brain, and tumor were quantitated and correlated with tumor response. Therapeutic concentrations of imatinib were achieved in plasma and tumor xenografts but not in the brain. Imatinib blocked the constitutive activation of c-kit in SCLC tumor cell lines in vitro but had a negligible effect on SCLC xenograft growth in vivo. Orally administered imatinib rapidly reaches therapeutic concentrations in SCLC xenografts, suggesting the feasibility of combining imatinib with other novel or traditional chemotherapeutic agents in SCLC or other solid tumors. The c-Kit signaling pathway does not appear to play a critical role in SCLC proliferation and viability in vivo, however, suggesting that imatinib is unlikely to be effective as monotherapy for SCLC.

The role of RAD51 in etoposide (VP16) resistance in small cell lung cancer.

Etoposide (VP16) is a potent inducer of DNA double-strand breaks (DSBs) and is efficiently used in small cell lung cancer (SCLC) therapy. However, acquired VP16 resistance remains an important barrier to effective treatment. To understand the underlying mechanisms for VP16 resistance in SCLC, we investigated DSB repair and cellular VP16 sensitivity of SCLC cells. VP16 sensitivity and RAD51, DNA-PK(cs), topoisomerase IIalpha and P-glycoprotein protein levels were determined in 17 SCLC cell lines. In order to unravel the role of RAD51 in VP16 resistance, we cloned the human RAD51 gene, transfected SCLC cells with RAD51 sense or antisense constructs and measured the VP16 resistance. Finally, we measured VP16-induced DSBs in the 17 SCLC cell lines. Two cell lines exhibited a multidrug-resistant phenotype. In the other SCLC cell lines, the cellular VP16 resistance was positively correlated with the RAD51 protein level. In addition, downregulation or overexpression of the RAD51 gene altered the VP16 sensitivity. Furthermore, the levels of the RAD51 and DNA-PK(cs) proteins were related to VP16-induced DSBs. The results suggest that repair of VP16-induced DSBs is mediated through both RAD51-dependent homologous recombination and DNA-PK(cs)-dependent nonhomologous end-joining and may be a determinant of the variation in clinical treatment effect observed in human SCLC tumors of identical histologic subtype. Finally, we propose RAD51 as a potential target to improve VP16 efficacy and predict tumor resistance in the treatment of SCLC patients.

DNA repair rate and etoposide (VP16) resistance of tumor cell subpopulations derived from a single human small cell lung cancer

Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated. The tumor was classified as intermediate-type SCLC. The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro. Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs). The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant. In order to explain the VP16 resistant phenotype several mechanisms where considered. The p53 status, P-glycoprotein, MRP, topoisomerase IIα, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance. In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PK cs) and RAD51 was observed. The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PK cs and RAD51 compared with the sensitive 54A subline. We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype.

Targeting Stealth liposomes in a murine model of human small cell lung cancer

Tumor accumulation and therapeutic activity of Stealth liposomes loaded with doxorubicin (DXR) were examined in Balb/c nude mice xenografts inoculated subcutaneously with the human small cell lung cancer (SCLC) cell line, H69. Mice were treated with non-targeted liposomes (SL) or liposomes targeted with antagonist G coupled to the liposome surface (SLG). SLG showed 30–44-fold higher binding to H69 cells harvested from H69 xenografts than SL. At 48 and 72 h post injection, tumor accumulation of [ 125I]tyraminylinulin-containing liposomes was shown to be dependent on liposome size but independent of the presence of the targeting ligand. Maximum tumor uptake of either SLG or SL ranged from 2 to 4% of injected dose/g of tissue. In therapeutic studies, mice received three weekly injections of 3 or 6 mg free DXR/kg or 3 or 10 mg liposomal DXR/kg at initial tumor volumes of either 7 or 33 mm 3. The therapeutic efficacy of DXR-containing SL or SLG was significantly improved over free DXR, but SLG did not improve anti-tumor efficacy relative to SL. Stealth liposomes containing DXR have potential as a therapy against human SCLC tumors.

Rhenium-188-labeled anti-neural cell adhesion molecule antibodies with 2-iminothiolane modification for targeting small-cell lung cancer.

We have evaluated the potential of 188Re-labeled monoclonal antibodies (MAbs) modified with 2-iminothiolane (2IT) for targeting small-cell lung cancer (SCLC). Radiolabeled MAbs NK1NBL1 and C218 recognizing neural cell adhesion molecule were injected i.v. into athymic mice inoculated with human SCLC tumors, and the biodistribution was examined. NK1NBL1 localized in the tumors better than C218. 188Re-labeled MAbs cleared from the blood faster than 125I-labeled counterparts, resulting in higher tumor-to-blood ratios. In conclusion, the 188Re-labeled MAbs are attractive candidates for imaging and therapy of SCLC.

Effect of radiation therapy on small-cell lung cancer is reduced by ubiquinone intake.

The effect of oral ubiquinone (Q10) intake on the in vivo response of tumors to single dose radiotherapy was examined. The human small-cell lung cancer (SCLC) line CPH 054A, which is sensitive to relatively low doses of X-radiation, was grown as subcutaneous transplants in the flanks of nude nu/nu mice. When macroscopical growth was established, groups of mice received either 10, 20 or 40 mg/kg Q10 in 30 mL soy oil intragastrically daily on 4 consecutive days. Controls received either 30 mL of pure soy oil or nothing. Three h after the last dose half of the tumors in each group received a single radiation dose of 5 Gy, using a 300 kV therapeutic unit. The macroscopic growth pre- and posttreatment was analyzed according to a transformed Gompertz algorithm using the software program GROWTH. Treatment with Q10 or soy oil alone had no effect on tumor growth compared with untreated controls. Groups of tumors that received Q10 and radiotherapy had a significantly lower specific growth delay (SGD) than the radiotherapy-only groups. This effect was significant at 40 mg/kg and borderline at 20 mg/kg, whereas at 10 mg/kg no radioprotection was seen. We conclude that systemic Q10 reduces the response to single dose tumor irradiation inxenotransplanted human SCLC tumors.