NCAM: a surface marker for human small cell lung cancer cells

Immunocytochemical and immunochemical techniques were used to study the expression of the neural cell adhesion molecule (NCAM) by human lung cancer cell lines. Intense surface staining for NCAM was found at light and electron microscopic levels on small cell lung cancer cells. The NCAM polypeptide of Mr 140,000 (NCAM 140) was detected by immunoblotting in all of 7 small cell lung cancer cell lines examined and in one out of two of the closely related large cell cancer cell lines: it was not detected in cell lines obtained from one patient with a mesothelioma, in two cases of adenocarcinoma, nor in two cases of squamous cell cancer. In contrast, neuron-specific enolase was found by immunoblotting in all the lung cancer cell lines tested and synaptophysin in all but the adenocarcinoma cell lines. These antigens were localized intracellularly. The specific expression of NCAM 140 by human small and large cell lung carcinomas suggests its potential as a diagnostic marker.     

Intratumoral anti-HuD immunotoxin therapy for small cell lung cancer and neuroblastoma

Background

Most patients with small cell lung cancer (SCLC) or neuroblastoma (NB) already show clinically detectable metastases at diagnosis and have an extremely poor prognosis even when treated with combined modalities. The HuD-antigen is a neuronal RNA-binding protein that is expressed in 100% of SCLC tumor cells and over 50% of neuroblastoma cells. The correlation between high titers of circulating anti-HuD antibodies in patients and spontaneous tumor remission suggests that the HuD-antigen might be a potential molecular target for immunotherapy.

Methods

We have constructed a new antibody-toxin compound (called BW-2) by assembling a mouse anti-human-HuD monoclonal antibody onto streptavidin/saporin complexes.

Results

We found that the immunotoxin BW-2 specifically killed HuD-positive human SCLC and NB cancer cells at very low concentrations in vitro. Moreover, intratumoral immunotoxin therapy in a nude mouse model of human SCLC (n?=?6) significantly reduced local tumor progression without causing toxicity. When the same intratumoral immunotoxin protocol was applied to an immunocompetent A/J mouse model of NB, significant inhibition of local tumor growth was also observed. In neuroblastoma allografted A/J mice (n?=?5) treated twice with intratumoral immunotoxin, significant tumor regression occurred in over 80% of the animals and their duration of tumor response was significantly prolonged.

Conclusions

Our study suggests that anti-HuD based immunotoxin therapy may prove to be an effective alternative treatment for patients with SCLC and NB.

     

Serum pleiotrophin as a diagnostic and prognostic marker for small cell lung cancer

Pleiotrophin (PTN) is involved in tumour progression, angiogenesis and metastasis. The purpose of this study was to investigate the expression level of PTN in the serum of patients with small cell lung cancer (SCLC) and to explore the clinical significance of PTN. Serum samples from 128 patients with SCLC, 120 healthy volunteers (HV) and 60 patients with benign lung disease (BLD) were collected. The levels of serum PTN were determined with ELISA and its correlation with the clinical data was examined. The serum PTN levels in SCLC patients were significantly higher than that in BLD patients (P < 0.05) or HV (P < 0.05). With a cutoff value of 258.18 ng/mL, the sensitivity and specificity of PTN to SCLC patients and BLD patients, SCLC patients and HV were 79.2% and 91.7%, 86.7% and 95.8% respectively. An area under the curve for all stages of SCLC resulting from PTN, which was significantly better than the other tumour markers tested including progastrin‐releasing peptide and neuron‐specific enolase. High serum PTN levels appear to correlate with poor survival in patients with SCLC. These results suggest that PTN levels in the serum could be a new effective biomarker for the diagnosis and prognosis of SCLC.     

Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells

The ability of cholecystokinin (CCK) to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. CCK-8 elevated the cytosolic Ca2+ levels in cell line NCI-H345 in a dose dependent manner. Nanomolar concentration of CCK-8 elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent CCK agonists such as gastrin-1 and nonsulfated CCK-8 but not inactive compounds such as CCK-27-32-NH2 elevated cytosolic Ca2+ levels. These data suggest that CCK receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.     

Neurotensin elevates cytosolic calcium in small cell lung cancer cells

The ability of neurotensin (NT) to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated using the fluorescent Ca²⁺ indicator Fura 2-AM. Using SCLC cell line NCI-H345, NT elevated cytosolic Ca²⁺ levels in a concentration-dependent manner. Using a 10 nM dose, NT and C-terminal fragments such as NT(8–13) but not N-terminal fragments such as NT(1–8) elevated the cytosolic Ca²⁺ levels. Because EGTA (5 mM) did not affect the NT response, NT may cause release of Ca²⁺ from intracellular stores. These data indicate that SCLC NT receptors may use Ca²⁺ as a second messenger.     

Vasopressin elevates cytosolic calcium in small cell lung cancer cells

The ability of vasopressin to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca²⁺ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca²⁺ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca²⁺ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca²⁺. The response to vasopressin was strongly blocked by [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,O-MeTyr²,Arg⁸]vasopressin and weakly blocked by [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,O-MeTyr²,Orn⁸]vasotocin. These data suggest that V₁ vasopressin receptors are present on SCLC cells.     

Chemotherapy of small cell lung cancer

The author summarizes the most recent information on small cell lung cancer. Reviews the epidemiology, prognostic markers and stages of small cell lung cancer Details the more frequently used combined therapeutic modalities, the criteria of the optimal therapeutic approaches and the achieved remission rates. Based on the most recent data, summarizes the new chemotherapeutic agents and their most effective combinations, the second line treatment and immunotherapy. Finally tries to answer the most frequently asked questions.     

Markers of small cell lung cancer

Lung cancer is the number one cause of cancer death; however, no specific serum biomarker is available till date for detection of early lung cancer. Despite good initial response to chemotherapy, small-cell lung cancer (SCLC) has a poor prognosis. Therefore, it is important to identify molecular markers that might influence survival and may serve as potential therapeutic targets. The review aims to summarize the current knowledge of serum biomarkers in SCLC to improve diagnostic efficiency in the detection of tumor progression in lung cancer. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC is emphasized. Recent findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomic technology for detecting lung cancer are also described. It is believed that implementing these new research techniques will facilitate and improve early detection, prognostication and better treatment of SCLC.     

Camptothecin-somatostatin conjugates inhibit the growth of small cell lung cancer cells

The effects of camptothecin-somatostatin (CPT-SS) conjugates were investigated on small cell lung cancer (SCLC) cells. CPT was coupled to a SS agonist (SSA), c(Cys-Phe-DTrp-Lys-Thr-Cys)Thr-NH2 using the built in nucleophile assisted-releasing group (L1) N-methyl-aminoethyl-Gly-Dser-Nle-Dtyr-Dser or (L2) aminoethyl-Gly-Dser-Nle-Dtyr-Dser. The resulting CPT-L1-SSA and CPT-L2-SSA inhibited the specific binding of [125I-Tyr11]SS to NCI-H69 cell membranes with IC50 values of 0.2 and 2.1 nM, respectively. [125I]CPT-L1-SSA was internalized by SCLC cells at 37 degrees C but not at 4 degrees C. CPT-L1-SSA and CPT-L2-SSA inhibited in a dose-dependent manner the increase in adenylylcyclase activity caused by 25 microM forskolin. In vitro, 0.3 microM CPT-L1-SSA half-maximally inhibited the clonal growth of SCLC cells and 1 microM CPT-L1-SSA strongly inhibited 3H-thymidine incorporation into DNA and trypan-blue exclusion. These results suggest that CPT conjugated peptides such as CPT-L1-SSA may prove useful for exploring the efficacy of receptor-directed cytotoxicity to inhibit the proliferation of SCLC cells.     

High affinity binding of cholecystokinin to small cell lung cancer cells

The binding of 125I-Bolton Hunter-cholecystokinin octapeptide (125I-BH-CCK-8) to small cell lung cancer cell lines was investigated. 125I-BH-CCK-8 bound with high affinity (Kd = 2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific 125I-BH-CCK-8 binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease.     

Sigma ligands inhibit the growth of small cell lung cancer cells

The effects of sigma ligands on small cell lung cancer (SCLC) cells were investigated. 125I-N-(2-(piperidino)ethyl)-2-iodobenazmide (2-IBP) bound with high affinity to SCLC cell line NCI-H209 and NCI-N417. Specific 125I-2-IBP binding was inhibited with high affinity by ifendipine, haloperidol, (2-piperidinyl-aminoethyl)-4-iodobenzamide (IPAB) and 1,3-ditolylguanidine (DTG) with IC50 values of 3, 10, 15 and 90 nM respectively. In vitro, 10 microM 2-IBP, haloperidol or IPAB inhibited NCI-N417 proliferation using a MTT or clonogenic assay. In vivo, 4 mg/kg IPAB or 2-IBP inhibited NCI-N417 xenograft proliferation. 125I-2-IBP localized to the SCLC tumors after subcutaneous injection. These results suggest that sigma ligands may be utilized to localize and inhibit the proliferation of SCLC tumors.     

TSA对非小细胞肺癌A549细胞放疗增敏性的研究

目的:探讨组蛋白去乙酰化酶抑制剂(HDIs)trichostatinA(TSA)对非小细胞肺癌(NSCLC)A549细胞放疗敏感性的影响.方法:以5Gy γ-射线照射细胞,或同时以TSA处理细胞.采用MTT法检测细胞存活率,Armexin V-PI染色检测细胞凋亡,流式细胞仪检测caspase-3活性.结果:SGy γ-射线可轻度降低细胞存活率,仅有少量细胞发生凋亡,但同时以TSA和γ-射线处理细胞,细胞存活率明显下降,凋亡细胞显著增多,且明显提高活化的caspase-3水平.结论:TSA通过促进caspase-3激活增强A549细胞对γ-射线的敏感性.     

Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells

The ability of bombesin-like peptides to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. Nanomolar concentrations of bombesin elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent bombesin receptor agonists, such as gastrin releasing peptide (GRP) or (GRP)14-27 elevated cytosolic Ca2+ levels whereas inactive compounds such as (D-Trp8)bombesin or (GRP)1-16 did not. Furthermore, the bombesin receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P (30 microM) had no effect on the Ca2+ levels by itself but antagonized the increase in Ca2+ caused by 10 nM or 100 nM bombesin. These data suggest that bombesin receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.     

Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells

Small cell lung cancer (SCLC) cells migration across human brain microvascular endothelial cells (HBMECs) is an essential step of brain metastases. Here we investigated signalling pathways in HBMECs contributing to the process. Inhibition of endothelial Rho kinase (ROCK) with Y27632 and overexpression of ROCK dominant-negative mutant prevented SCLC cells, NCI-H209, transendothelial migration and the concomitant changes of tight junction. Conversely, inhibition of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) had no effects. Furthermore, endothelial RhoA protein was activated during NCI-H209 cells transendothelial migration. Rho/ROCK participated in NCI-H209 cells transendothelial migration through regulating actin cytoskeleton reorganization. These results suggested that Rho/ROCK was required for SCLC cells transendothelial migration.     

The possibility of surgery for small cell lung cancer (state of art)

The author provides information about epidemiology as well as surgical practice for small cell lung cancer (SCLC) in Hungary. It is emphasized that, based on the author's experience and on international consensus, TNM system is the basis of accurate oncological treatment. The oncological management of SCLC is summarized, which is based on surgery. SCLC, especially cases undergoing surgery, are often detected as a peripheral nodule which should be examined preoperatively by special algorithm. Despite the published favorable results, surgical treatment alone is not accepted nowadays as a correct oncological point of view. The aim of adjuvant therapy is to improve survival and to decrease local recurrence. The 5-year survival of SCLC according to stages is between 4 and 60%. After the late 80's, the complex multimodality oncotherapy has started, with neoadjuvant treatment. SCLC is chemosensitive, even at N2 stage after down-staging surgery might be available. The late results show 20-46% cumulative 5-year survival rate. However, in the case of N2 disease, only 15-30% 5-year survival is achieved. This is a critical question for surgery of SCLC, because the dominant opinion is that for N2 disease and especially residual N2 surgery does not prolong survival. SCLC often occurs in combination with NSCLC. Therefore, salvage operation is a possible choice to remove the residual chemo/radiation resistant SCLC and NSCLC components. In conclusion, surgery has an advantage for SCLC therapy especially in patients with stage I-II disease. Value of surgery for stage III/a disease is under discussion and it is not recommended in Hungarian practice. To use a complex neoadjuvant protocol is advised which provides from 20 up to 40% five-year survival rate.     

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.     

miR-126 inhibits proliferation of small cell lung cancer cells by targeting SLC7A5

Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median survival of 18 months. Since miR-126 is under-expressed in the majority of SCLC tumors, we investigated the effect of miR-126 overexpression on the proliferation and cell cycle distribution of H69 cells. Our results demonstrate that miR-126 inhibits proliferation of H69 cells, by delaying the cells in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct target of miR-126.     

Monitoring tumour cells in the peripheral blood of small cell lung cancer patients

BACKGROUND: Flow cytometry was used to enumerate tumour cells in longitudinal studies of peripheral blood from small cell lung cancer (SCLC) patients, together with magnetic bead selection to isolate and identify these cells. As part of a trial, 11 patients received either standard (four weekly) chemotherapy with ifosfamide, carboplatin, and etoposide (ICE) or accelerated (two weekly) ICE with filgrastim (granulocyte colony-stimulating factor [G-CSF]) and autologous stem cell support. METHODS: Fresh venous blood was taken throughout treatment and follow-up. Aliquots were stained with a "tumour-specific" antibody against epithelial tissue (Ber EP4), verified as a good marker of SCLC cells by immunohistochemistry. Matched samples labelled with Ber EP4 were separated magnetically by adding a secondary bead-antibody conjugate for confirmation of tumour cell identity. RESULTS: Circulating tumour cells were detected and monitored throughout treatment periods. An initial rise in circulating cells after the first cycle was followed by a fall in both treatment arms to baseline levels set by normal controls. This was achieved by week 12 in the accelerated treatment arm and by week 24 in the standard arm. CONCLUSIONS: Flow cytometry and magnetic bead isolation can be used to identify changes in numbers of circulating tumour cells in patients undergoing chemotherapy for SCLC and thereafter during follow-up periods. Absence of tumour cells may indicate a more favourable patient group who would benefit from a more intense course of treatment.