Small Cell Lung Cancer NCI-H446 Research Articles

The tyrosine kinase inhibitor nintedanib enhances the efficacy of 90Y-labeled B5209B radioimmunotherapy targeting ROBO1 without increased toxicity in small-cell lung cancer xenograft mice.

Small cell lung cancer (SCLC) has a poor prognosis, and Roundabout homolog 1 (ROBO1) is frequently expressed in SCLC. ROBO1-targeted radioimmunotherapy (RIT) previously showed tumor shrinkage, but regrowth with fibroblast infiltration was observed. The fibroblasts would support tumor survival by secreting growth factors and cytokines. Inhibition of fibroblasts offers a candidate strategy for increasing RIT efficacy. Here, we evaluated the efficacy of combination therapy with 90Y-labeled anti-ROBO1 antibody B5209B (90Y-B5209B) and the tyrosine kinase inhibitor nintedanib in SCLC xenograft mice. Subcutaneous NCI-H69 SCLC xenograft mice were divided into four groups: saline, nintedanib alone, RIT alone, and a combination of RIT with nintedanib (combination). A single dose of 7.4 MBq of 90Y-B5209B was injected intravenously. Nintedanib was orally administered at a dose of 400 µg five times a week for 4 weeks. Tumor volumes and body weights were measured regularly. Tumor sections were stained with hematoxylin and eosin or Masson trichrome. All six tumors in the combination therapy group disappeared, and four tumors showed no regrowth. Although RIT alone induced similar tumor shrinkage, regrowth was observed. Prolonged survival in the combination therapy group was found compared with the other groups. Temporary body weight loss was observed in RIT and combination therapy. There is no difference in fibroblast infiltration between RIT alone and the combination. Nintedanib significantly enhanced the anti-tumor effects of RIT with the 90Y-B5209B without an increase in toxicity. These findings encourage further research into the potential clinical application of combining RIT with nintedanib.

Single-Dose Cisplatin Pre-Treatment Enhances Efficacy of ROBO1-Targeted Radioimmunotherapy.

We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.

Preclinical evaluation of SC0191, a small molecule inhibitor of Wee1 kinase.

e15637 Background: TP53 mutation is common in cancer cells, especially in small cell lung cancer (SCLC) and pancreatic cancer (PC). The G1 checkpoint function is lost in these cancer cells, which more rely on G2 checkpoint to repair damaged DNA. Wee1 is a serine/threonine kinase, which phosphorylates the Tyr15 locus of CDC2, inhibits the activity of CDC2, and stays in the G2 phase. Wee1 inhibition can promote the cells with DNA damage into the M phase, thus driving the TP53 mutant cancer cells to apoptosis. The purpose of this study was to investigate the in vitro and in vivo antitumor activity of SC0191, a small molecule inhibitor of Wee1 kinase, in preclinical models of SCLC and PC with TP53 mutation. Methods: The kinase inhibiting activity of SC0191 was determined using the Wee1 kinase assays. The cellular anti-proliferative activity was evaluated with TP53 mutant NCI-H446 SCLC and BxPC-3 PC cell lines. The in vivo antitumor activity of SC0191 was evaluated in the NCI-H446 and BxPC-3 cells-derived xenograft (CDX) mouse models, respectively. Results: SC0191 displayed potent kinase inhibiting activity for Wee1 with IC50 22.3 nM. SC0191 significantly inhibited tumor cell proliferation in TP53 mutant NCI-H446 cells with IC50 231.1 nM and BxPC-3 cells with IC50 223.8 nM. Moreover, SC0191 oral administration showed significant antitumor efficacy which was better than AZD1775, in the NCI-H446 and BxPC-3 CDX mouse models, respectively. Conclusions: A novel Wee1 inhibitor, SC0191, has demonstrated excellent antitumor efficacy in TP53 mutant solid cancer preclinical studies, and represents a promising clinical candidate for treating solid tumors, such as SCLC and PC.

Apatinib Combined with CCI-779 Inhibits the Proliferation and Migration of Small Cell Lung Cancer NCI-H446 Cells In Vitro

背景与目的肺癌是世界上最常见的恶性肿瘤，其中小细胞肺癌是恶性程度最高的亚型，具有生长迅速、早期转移和高度血管化等特点。阿帕替尼（Apatinib）是我国自主研发的血管内皮生长因子受体2抑制剂，在多种实体瘤中疗效显著。本研究旨在探讨Apatinib对小细胞肺癌细胞株NCI-H446的体外作用以及联合哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）抑制剂CCI-779对小细胞肺癌的体外作用。方法体外培养小细胞肺癌细胞株NCI-H446，CCK8法、细胞凋亡实验、细胞周期实验及Transwell实验检测Apatinib及联合mTOR抑制剂CCI-779对NCI-H446细胞增殖、凋亡、周期及迁移的影响；Western blot实验检测血管内皮生长因子受体和细胞周期相关蛋白的表达。结果CCK8实验结果显示高浓度Apatinib能抑制NCI-H446细胞增殖；细胞凋亡实验结果显示高浓度Apatinib诱导NCI-H446细胞凋亡；Transwell实验结果显示高浓度Apatinib抑制NCI-H446细胞迁移；联合mTOR抑制剂CCI-779后，低浓度Apatinib便能抑制NCI-H446细胞增殖和迁移，诱导细胞凋亡。结论Apatinib对小细胞肺癌细胞株NCI-H446的作用具有浓度依赖性特征，高浓度Apatinib能够抑制NCI-H446细胞增殖和迁移，诱导细胞凋亡，与mTOR抑制剂CCI-779联用能增加NCI-H446细胞对Apatinib的敏感性。

Multimodality imaging of naturally active melanin nanoparticles targeting somatostatin receptor subtype 2 in human small-cell lung cancer.

Somatostatin receptor subtype 2 (SSTR2) is highly expressed in pulmonary neuroendocrine tumors, which account for approximately 25% of all lung cancers including small-cell lung cancer (SCLC). It is possible to establish SCLC-specific imaging agents for multimodal imaging to obtain tumor integrity information. Herein, we constructed novel multifunctional organic melanin nanoparticles (MNPs) as a carrier and surface-loaded somatostatin analog octreotide to produce a human small-cell lung cancer-targeted nanoprobe OCT-PEG-MNPs. MNPs have an excellent photoacoustic imaging (PAI) function and can be directly chelated with the magnetic resonance contrast agent Mn2+, and N-bromo succinimide (NBS) can be used as an oxidant to label the nanoparticles with the long half-life radionuclide 124I by an electrophilic substitution reaction. Therefore, (124I, Mn) OCT-PEG-MNPs can not only be used for PAI but also be used for positron emission tomography (PET) and magnetic resonance imaging (MRI). The NCI-H69 SCLC tumor xenograft model with high SSTR2 expression was constructed to evaluate the multimodal imaging ability of (124I, Mn) OCT-PEG-MNPs. This nanoprobe showed good imaging abilities in PAI, MRI and PET. The PA images showed that the photoacoustic signal in the NCI-H69 tumor site gradually increased with time, and the NCI-H69 xenograft showed a clear increase in the T1-weighted signal intensity after injection of Mn-OCT-PEG-MNPs at 24 h compared to that in the prescan. MicroPET and biodistribution studies showed that the uptake of NCI-H69 tumors (8.03 ± 0.37% ID g-1) was significantly higher than that in the control A549 model (3.35 ± 0.54% ID g-1) after injection of (124I, Mn) OCT-PEG-MNPs at 24 h. The (124I, Mn) OCT-PEG-MNPs were successfully applied to multimodal imaging in a small-cell lung cancer model with high SSTR2 expression. This nanoprobe may be considered for clinical trials since it combines the numerous advantages of organic nanoparticles.

Abstract B09: Evaluating transcription factor networks as targets for the treatment of small cell lung cancer

Abstract Achaete-scute homolog 1 (ASCL1) is a transcription factor that is highly expressed in most small cell lung cancer (SCLC) tumors and cell lines and is critical for their proliferation. Inhibiting ASCL1 transcriptional activity may thus be a valid therapeutic strategy for SCLC. However, transcription factors (TFs) have been historically difficult to target. Given that TFs belong to complex regulatory networks, we propose to identify and target multiple components within an ASCL1-transcriptional network rather than ASCL1 alone. We have previously detected ASCL1-bound genomic regions associated with the active chromatin epigenetic mark, H3K27Ac in SCLC cells. Here we report the identification of an ASCL1-containing transcriptional network in SCLC cells and evaluate its targeting by the transcriptional inhibitor, mithramycin. We conducted chromatin immunoprecipitation (ChIP) by immunoprecipitating the H3K27Ac chromatin mark from NCI-H2107 and NCI-H69 SCLCs (5X107 cells) nuclear lysates with10 μg H3K27Ac antibodies (Abcam-Ab4729), and sequenced ChIP libraries on an Illumina High-Seq2000. Further characterization using siRNA transfections, immunoblotting and MTS-cell proliferation assays were conducted following standard procedures. We found 82 overlapping TF genes, including lineage-specific TF genes for ASCL1, Forkhead box protein A2 (FOXA2), and nuclear factor 1 B-type (NFIB), associated with the active chromatin epigenetic marks H3K27Ac in SCLC cell lines. This suggests that ASCL1, FOXA2, and NFIB may belong to the same transcriptional network. siRNA-mediated knockdown of ASCL1 leads to decreased FOXA2 levels, but it has no effect on NFIB levels, implicating ASCL1 as a transcriptional regulator of FOXA2 but not NFIB. Mithramycin stops cell proliferation and reduces ASCL1, FOXA2 but not NFIB protein levels. In conclusion, our studies identify the beginning of a transcriptional network necessary for SCLC proliferation in which ASCL1 regulates FOXA2. Targeting multiple TFs within a network with mithramycin can stop cell proliferation and as such suggests a therapeutic strategy for SCLC. Because of the importance of NFIB in mediating metastatic behavior of SCLC, these data indicate that approaches other than targeting ASCL1 and use of mithramycin will be needed to target NFIB. Citation Format: Karine Pozo, Rahul K. Kollipara, John D. Minna, Adi F. Gazdar, Jane E. Johnson. Evaluating transcription factor networks as targets for the treatment of small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B09.

Effects of MnSOD silence on in vitro tumorigenicity in NCI-H446 cells

To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms. Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot. Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells. Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.

Effects of three IL-15 variants on NCI-H446 cell proliferation and expression of cell cycle regulatory molecules.

Interleukin 15 (IL-15) is a cytokine exhibiting antitumor characteristic similar to that of IL-2. However, in human tissues and cells, IL-15 expression and secretion is very limited, suggesting IL-15 functions mainly intracellularly. In the present study, we assessed the effects of transfecting NCI-H446 small cell lung cancer cells with genes encoding three IL-15 variants: prototypical IL-15, mature IL-15 peptide, and modified IL-15 in which the IL-2 signal peptide is substituted for the native signal peptide. NCI-H446 cells transfected with empty plasmid served as the control group. We found that IL-15 transfection effectively inhibited NCI-H446 cell proliferation and arrested cell cycle progression, with the modified IL-15 carrying the IL-2 signal peptide exerting the greatest effect. Consistent with those findings, expression each of the three IL-15 variants reduced growth of NCI-H446 xenograph tumors, and the modified IL-15 again showed the greatest effect. In addition, IL-15 expression led to down-regulation of the positive cell cycle regulators cyclin E and CDK2 and up-regulation of the negative cycle regulators p21 and Rb. These findings suggest IL-15 acts as a tumor suppressor that inhibits tumor cell proliferation by inducing cell cycle arrest.

Wnt inhibitor XAV939 suppresses the viability of small cell lung cancer NCI-H446 cells and induces apoptosis.

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer due to a fast tumor doubling time and early hematogenous spread. Advances in the treatment of non-small cell lung cancer using targeted therapies having been made, but no targeted drugs for SCLC have been approved. The Wnt signaling pathway is associated with tumor progression and metastasis; therefore, the inhibition of Wnt/β-catenin signaling is a strategy for anticancer drugs. Tankyrase 1 (TNKS1) is overexpressed in a number of types of cancer and XAV939 is a small molecule inhibitor of TNKS1 which may inhibit tumor growth. The present study aimed to investigate the potential molecular mechanisms underlying XAV939-induced suppression of the viability of SCLC cells. MTT assays were used to determine the viability-inhibition rate of cells and to identify the drug concentration which optimally inhibited cell viability. Flow cytometry was used to determine whether XAV939 induced apoptosis of SCLC cells, and to analyze the effect of the drug on the cell cycle. The results of the present study identified that XAV939 inhibited the viability of NCI-H446 cells in a dose-dependent manner, but cisplatin inhibited NCI-H446 cell viability in a time- and dose-dependent manner. The combination of XAV939 and cisplatin exhibited a slightly more pronounced inhibition of cell viability at an increased dose of XAV939. In addition, XAV939 markedly induced cell apoptosis of the SCLC cell line H446 by increasing the proportion of cells in the G0/G1 phase, leading to inhibition of the cell cycle. The results of the present study indicated that XAV939 inhibited the viability of the NCI-H446 SCLC cell line by inducing cell apoptosis through the Wnt signaling pathway. Therefore, XAV939 may be useful for the treatment of SCLC.

Ouabain targets the Na+/K+-ATPase α3 isoform to inhibit cancer cell proliferation and induce apoptosis.

Ouabain has been used for the treatment of heart failure and atrial fibrillation. Its potential anticancer effect has also attracted great interest. The aim of the present study was to evaluate the anticancer effect of ouabain and investigate its molecular target. The effects of ouabain on the viability of and induction of cellular death on OS-RC-2 renal cancer cells were examined using the MTT assay and acridine orange/ethidium bromide staining. The levels of Ca2+ and reactive oxygen species were determined using Fura-3-acetoxymethyl ester and dichloro-dihydro-fluorescein diacetate probes, respectively. Apoptosis was examined using annexin V-fluorescein isothiocyanate/propidium iodide staining and western blotting. The expression profile of the different Na+/K+-ATPase (NKA) isoforms in NCI-H446 small cell lung cancer cells was determined using immunocytochemistry and reverse transcription polymerase chain reaction analysis. In the present study, it was demonstrated that ouabain inhibited cancer cell proliferation and induced apoptosis while no significant difference in the expression of NKA α1 and α3 isoforms was detected following 48 h of ouabain treatment. Furthermore, expression of NKA α3 but not the α1 isoform was associated with ouabain sensitivity. The results of the present study indicated that ouabain targets the NKA α3 isoform, inhibits cancer cell proliferation and induces apoptosis.

Cisplatin and photodynamic therapy exert synergistic inhibitory effects on small-cell lung cancer cell viability and xenograft tumor growth

Lung cancer is the leading cause of cancer death worldwide. Small-cell lung cancer (SCLC) is an aggressive type of lung cancer that shows an overall 5-year survival rate below 10%. Although chemotherapy using cisplatin has been proven effective in SCLC treatment, conventional dose of cisplatin causes adverse side effects. Photodynamic therapy, a form of non-ionizing radiation therapy, is increasingly used alone or in combination with other therapeutics in cancer treatment. Herein, we aimed to address whether low dose cisplatin combination with PDT can effectively induce SCLC cell death by using in vitro cultured human SCLC NCI-H446 cells and in vivo tumor xenograft model. We found that both cisplatin and PDT showed dose-dependent cytotoxic effects in NCI-H446 cells. Importantly, co-treatment with low dose cisplatin (1 μM) and PDT (1.25 J/cm2) synergistically inhibited cell viability and cell migration. We further showed that the combined therapy induced a higher level of intracellular ROS in cultured NCI-H446 cells. Moreover, the synergistic effect by cisplatin and PDT was recapitulated in tumor xenograft as revealed by a more robust increase in the staining of TUNEL (a marker of cell death) and decrease in tumor volume. Taken together, our findings suggest that low dose cisplatin combination with PDT can be an effective therapeutic modality in the treatment of SCLC patients.

A candidate Chinese medicine preparation-Fructus Viticis Total Flavonoids inhibits stem-like characteristics of lung cancer stem-like cells.

BackgroundCancer stem cells (CSCs) are considered as the origin of tumor relapse. Here, we investigated the effects of Fructus Viticis total flavonoids (FVTF) on the characteristics of lung cancer stem-like cells (LCSLCs) derived from human small cell lung cancer NCI-H446 cell line and its potential mechanism.MethodsHuman small cell lung cancer NCI-H446 cell line was cultured in vitro. The CD133+ cells were sorted from NCI-H446 cell line by magnetic separation.The suspended culture with stem cell-conditioned medium was used to amplify CD133+ sphere-forming cells (SFCs). The stem cell characteristics of CD133+ SFCs were evaluated using cell self-renewal capacity by tumor sphere formation assay, migration and invasion capacity by Transwell assay, tumorigenicity by xenograft model in nude mouse and cancer stem cell markers expression levels by western blot. The effects of FVTF on the properties of LCSLCs were examined by tumorsphere formation assay and transwell chamber assay. The expression level of p-Akt was determined by western blot analysis.ResultCD133+ SFCs derived from human small cell lung cancer NCI-H446 cells exhibited stemness properties of tumorsphere formation and tumorigenesis capacity comparing to the parental cells. FVTF relative selectively inhibited the proliferation of LCSLCs, suppressed tumor sphere forming capacity and migration and invasion of LCSLCs, and down-regulated the protein expression of stem cell markers (CD133, CD44 and ALDH1), self-renewal associated transcription factors (Bmi1, Nanog and OCT4) and invasion associated transcription factors (Twist1 and Snail1) in a dose-dependent manner. Moreover, we found that FVTF treatment could significantly decrease the phosphorylation level of Akt in LCSLCs. Meanwhile, LY294002 and FVTF synergistically inhibited the characteristics of LCSLCs.ConclusionFVTF inhibits the characteristics of LCSLCs through down-regulating expression of p-Akt.

Inactivated Tianjin strain, a novel genotype of Sendai virus, induces apoptosis in HeLa, NCI-H446 and Hep3B cells.

The Sendai virus strain Tianjin is a novel genotype of the Sendai virus. In previous studies, ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) demonstrated antitumor effects on human breast cancer cells. The aim of the present study was to investigate the in vitro antitumor effects of UV-Tianjin on the human cervical carcinoma HeLa, human small cell lung cancer NCI-H446 and human hepatocellular carcinoma Hep 3B cell lines, and the possible underlying mechanisms of these antitumor effects. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that UV-Tianjin treatment inhibited the proliferation of HeLa, NCI-H446 and Hep 3B cells in a dose- and time-dependent manner. Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide double staining indicated that UV-Tianjin induced dose-dependent apoptosis in all three cell lines with the most significant effect observed in the HeLa cell line. In the HeLa cell line, UV-Tianjin-induced apoptosis was further confirmed by the disruption of the mitochondria membrane potential and the activation of caspases, as demonstrated by fluorescent cationic dye and colorimetric assays, respectively. In addition, western blot analysis revealed that UV-Tianjin treatment resulted in significant upregulation of cytochrome c, apoptosis protease activating factor-1, Fas, Fas ligand and Fas-associated protein with death domain, and activated caspase-9, −8 and −3 in HeLa cells. Based on these results, it is hypothesized that UV-Tianjin exhibits anticancer activity in HeLa, NCI-H446 and Hep 3B cell lines via the induction of apoptosis. In conclusion, the results of the present study indicate that in the HeLa cell line, intrinsic and extrinsic apoptotic pathways may be involved in UV-Tianjin-induced apoptosis.

Increased insulin-like growth factor 1 receptor (IGF1R) expression in small cell lung cancer and the effect of inhibition of IGF1R expression by RNAi on growth of human small cell lung cancer NCI-H446 cell

Insulin-like growth factor 1 receptor (IGF1R) is a tyrosine kinase receptor implicated in tumourigenesis that may be an attractive target for anti-cancer treatment. In this study, the expression and clinical significance of IGF1R were investigated in serum and lung cancer tissues from small cell lung cancinoma (SCLC). We also compared the effect of IGF1R up-regulation and IGF1R inhibition on viability and apoptosis of NCI-H446 cells. We found the concentration of IGF1R in blood serum was significantly increased and positive IGF1R protein in cancer tissue was more prevalent in SCLC. A statistically significant correlation among IGF1R-positve tumors, lymph node metastasis and local invasion was discussed. Furthermore, IGF1R overexpression lead to an increase of cell survival and suppressed cell apoptosis, IGF1R silencing mediated by RNAi abrogate this response of NCI-H446 cells. Our results further demonstrated that the effects of these treatments may be assigned to the effective inhibition of lung cancer cells from Akt/P27Kip1 pathway in IGF-1R signaling. These features may have important implications for future anti-IGF1R therapeutic approaches.

99mTc-octreotide receptor scintigraphy in NCI-H446 small cell lung cancer nude mice model

背景与目的小细胞肺癌恶性程度高，早期诊断对其预后有重要价值，目前的检查方法比较局限，传统影像学方法特异性差，而PET/CT价格昂贵，难于推广应用。小细胞肺癌属神经内分泌肿瘤，高表达生长抑素受体，是其早期进行分子影像诊断的理论基石。本实验旨在观察99mTc-octreotide在正常裸鼠体内的分布、代谢及荷人NCI-H446小细胞肺癌裸鼠模型体内显像变化，为临床小细胞肺癌早期诊断奠定基础。方法建立人小细胞肺癌的裸鼠肿瘤模型，正常裸鼠及荷瘤鼠静脉注射99mTc-octreotide显像剂后行动态及延迟显像。运用感兴趣区（region of interest, ROI）技术勾画各时相裸鼠各脏器、肿瘤（T）及肿瘤对侧对应部位（N）放射性计数，计算相应T/N比值，并建立30 min内各ROI的时间-放射性（A-T）曲线。结果① 正常裸鼠的肾脏、肝脏内99mTc-octreotide分布最多，肺部、心脏部位分布较低，头部放射性分布最少，99mTc-octreotide主要通过泌尿系统排泄；各脏器30 min内A-T曲线显示放射性分布随时间延迟呈逐渐下降趋势。②5例荷瘤裸鼠的肿瘤显像均呈阳性；静脉注射99mTc-octreotide后肿瘤部位在3 h显像最清楚，整个检查时间内肝脏放射性强度明显高于肿瘤组织，肺部放射性与肿瘤部位较相近。半定量分析结果显示，静脉注射99mTc-octreotide后肿瘤组织与对侧肢体肌肉的T/N比值在0.5 h、2 h、3 h、4 h分别为1.163±0.03、2.08±0.12、3.03±0.23、2.689±0.31；各时相T/N比值差异有统计学意义（F=51.69, P < 0.000, 1）；通过两两比较发现，静脉注射显像剂后3 h的T/N比值与其他各时相差异均有统计学意义（P < 0.05）；不同检查时间肝脏部位的放射性平均计数高于肿瘤部位，肺部的平均计数与肿瘤相近。肿瘤部位A-T曲线显示，注射99mTc-octreotide后2 min-3 min出现一过性放射性分布高峰。结论运用99mTc-octreotide作显像剂，人小细胞肺癌NCI-H446裸鼠模型具有极高的显像阳性率，且3 h肿瘤显像最清楚。

Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Small cell lung cancer (SCLC) accounts for nearly 15% of human lung cancers and is one of the most aggressive solid tumors. The SCLC cells are thought to derive from self-renewing pulmonary neuroendocrine cells by oncogenic transformation. However, whether the SCLC cells possess stemness and plasticity for differentiation as normal stem cells has not been well understood thus far. In this study, we investigated the expressions of multilineage stem cell markers in the cancer cells of SCLC cell line (NCI-H446) and analyzed their clonogenicity, tumorigenicity, and plasticity for inducing differentiation. It has been found that most cancer cells of the cell line expressed multilineage stem cell markers under the routine culture conditions and generated single-cell clones in anchorage-dependent or -independent conditions. These cancer cells could form subcutaneous xenograft tumors and orthotopic lung xenograft tumors in BALB/C-nude mice. Most cells in xenograft tumors expressed stem cell markers and proliferation cell nuclear antigen Ki67, suggesting that these cancer cells remained stemness and highly proliferative ability in vivo. Intriguingly, the cancer cells could be induced to differentiate into neurons, adipocytes, and osteocytes, respectively, in vitro. During the processes of cellular phenotype-conversions, autophagy and apoptosis were two main metabolic events. There is cross-talking between autophagy and apoptosis in the differentiated cancer cells. In addition, the effects of the inhibitor and agonist for Sirtuin1/2 on the inducing osteogenic differentiation indicated that Sirtuin1/2 had an important role in this process. Taken together, these results indicate that most cancer cells of NCI-H446 cell line possess stemness and plasticity for multilineage differentiation. These findings have potentially some translational applications in treatments of SCLC with inducing differentiation therapy.

Biodistribution and preparation of technetium-99m-labeled D-D3 monoclonal antibody against pro-gastrin-releasing peptide(31-98) in mice

Background We previously reported that iodine-131(131I)-labeled anti-progastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, 131I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72-96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m (99mTc) and to investigate the biodistribution of this antibody in healthy ICR mice. Methods D-D3 was labeled with 99mTc via the 2-mercaptoethanol reduction method. 99mTc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of 99mTc-D-D3 was determined with cell conjugation assays. 99mTc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g). Results The labeling rate and the radiochemical purity of 99mTc-D-D3 were (73.87±2.89)% and (94.13±4.49)%, respectively. The immunobinding rates of 99mTc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2±2.37)% and (24.3±1.46)%, respectively. The distribution data of normal ICR mice demonstrated that 99mTc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle. Conclusions 99mTc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. 99mTc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, 99mTc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Effect of sulforaphane on human small cell lung cancer NCI-H446 cells proliferation, invasion and the activity of MMP-9

Objective To investigate the effects of sulforaphane (SFN) on proliferation and invasion of human small cell lung cancer NCI-H446 and the activity of matrix metalloproteinase (MMP) -9.Methods NCI-H446 cells were cultured with 0,25,50,100 μmol/L SFN for 24 ～ 72 h,then MTT assay was employed to detect cell proliferation.Chamber invasion assay was used to study the cell invasion,and gelatin zymography assay was implied in MMP-9 enzyme activity.Results After treatment of 25,50,100μmol/L SFN,the growth of NCI-H446 cells were inhibited.When cells were incubated with 25,50,100μmol/L of SFN for 72h,the inhibition ratio was ( 11.1 ± 2.26 ) ％,( 25.2 ± 3.24 ) ％ and ( 44.6 ±4.2) ％,respectively,the difference was statistically significant compared with the solvent control group ( t =10.685,8.417,5.264,P ＜0.05 ).Chamber invasion assay showed that NCI-H446 cell invasion could be reduced.25,50,100 μmol/L of SFN could decrease the trans-membrane cells to (48.6 ± 1.84)％,(35.4 ± 2.22) ％ and (27.8 ± 1.36) ％,and it was statistically significant compared with the solvent control group ( t =6.341,5.562,4.925,P ＜0.05 ),respectively.In addition,MMP-9 activity was significantly inhibited by SFN.25,50,100 μmol/L of SFN could decrease the gray value of MMP-9 to 764 ±18.4,685 ± 14.74 and 638 ± 21.54 ( control group 822 ± 12.53,t =4.971,7.582,11.235,respectively,P ＜0.05).Conclusions SFN can inhibit NCI-H446 cells growth,invasion and the activity ofMMP-9. Key words: Isothiocyanates/pharmacology; Small cell lung carcinoma/metabolism/pathology; Cell line, tumor; Cell proliferation; Neoplasm invasiveness; Matrix metalloproteinase 9/metabolism

The Rho-kinase inhibitor inhibits proliferation and metastasis of small cell lung cancer

The purpose of this study was to investigate the effects of Rho-kinase inhibitor on the growth, proliferation, apoptosis, adhesion, invasion and migration of NCI-H446 small cell lung cancer cells and to explore the underlying molecular mechanisms involved in this process. After treatment to NCI-H446 small cell lung cancer cells with Fasudil, a Rho-kinase inhibitor, cell biological behaviors were observed. Matrix metalloproteinase activity and Western blot assay were used to evaluate underlying molecular mechanisms. The IC50 of Fasudil to NCI-H446 small cell lung cancer cells was approximately 0.86mg/ml (95% confidence limits: 0.65–1.17mg/ml). After treatment with 0.75mg/ml Fasudil, the ability of NCI-H446 small cell lung cancer cells, including growth, proliferation, adhesion, migration, and invasion were decreased, while their apoptosis was increased significantly. On the molecular level, the total amounts of active MMP2 and MMP9 were decreased about 20.5% (P<0.05) and 57.5% (P<0.01) respectively. Myosin phosphatase targeting subunit 1 phosphorylation (P-MYPT1) was reduced by 27.9% (P<0.05). The activation of caspase-3, and PARP cleavage in experimental group were significantly higher than those in normal control group (P<0.01). Meanwhile, treatment with Fasudil led to a remarkable reduction of the phosphorylated STAT3 (P-STAT3) (P<0.01). Taken together, our findings show that Fasudil prevents the growth, metastasis and induces apoptosis of NCI-H446 small cell lung cancer cells by inhibiting the Rho/Rho-kinase pathway. Changes in MMP2, MMP9, P-MYPT1, caspase-3, PARP cleavage and P-STAT3 may be one of its molecular mechanisms.

Curcumin Induces Small Cell Lung Cancer NCI-H446 Cell Apoptosis via the Reactive Oxygen Species-Mediated Mitochondrial Pathway and Not the Cell Death Receptor Pathway

Curcumin (diferuloylmethane), an active component of the spice turmeric, induces apoptosis in several types of malignancies. However, little is known about its anticancer activity in small cell lung cancer (SCLC). SCLC represents a highly malignant and particularly aggressive form of cancer, with early and widespread metastases and a poor prognosis. In this study, we found that curcumin does not activate caspase-8 cleavage or alter the expression of apoptotic receptors FAS and TRAIL in NCI-H446 cells, suggesting that curcumin-induced apoptosis is not associated with death receptor-mediated pathways in these cells. Instead, curcumin caused apoptosis by increasing Bax expression while decreasing the expression of Bcl-2 and Bcl-xL. Curcumin induced a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. In addition, curcumin-induced apoptosis was accompanied by an increase of intracellular reactive oxygen species (ROS) level. These results indicated that a ROS-mediated mitochondrial pathway played an important role in the process of curcumin-induced apoptosis of human SCLC NCI-H446 cells.