Apoptosis Of Small Cell Lung Cancer Cells Research Articles

Targeting Gastrin-Releasing Peptide Receptors on Small Cell Lung Cancer Cells with a Bispecific Molecule that Activates Polyclonal T Lymphocytes

Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNgamma production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule-armed human T cells significantly reduced SCLC tumor burdens in a mouse model. The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor-positive tumors, such as SCLC, are warranted.

Inhibition of Akt/Protein Kinase B Signaling by Naltrindole in Small Cell Lung Cancer Cells

The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic delta opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3beta and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.

Reduced expression of the neuron restrictive silencer factor permits transcription of glycine receptor alpha1 subunit in small-cell lung cancer cells.

Small-cell lung cancer (SCLC) cells express various markers of neuronal differentiation associated with deficient activity of the neuron-restricted silencer factor (NRSF). Here, we characterize mechanisms by which NRSF target genes are upregulated in SCLC and their functional consequences for cell survival. Since the glycine receptor (GlyR) alpha1 subunit gene, GLRA1, contains a sequence motif for NRSF binding (NRSE) within its 5' UTR, it served as a cellular surrogate marker for NRSF activity. Expression of GLRA1 in nontransformed cells is largely restricted to cells in the spinal cord, retina and brain stem. In experiments described here, we detected GLRA1 transcripts in three out of four SCLC-derived cell lines and in three of five biopsy samples obtained from SCLCs. In contrast, no GLRA1 transcripts were found in 10 nonmalignant nor 15 non-small-cell lung cancer biopsies. Consistent with this observation, NRSF-mediated suppression of an expression construct harbouring the NRSE of the GLRA1 (GLRA1 NRSE) gene was impaired in three of four 'classic' SCLC cell lines, whereas exogenous overexpression of NRSF in NRSF-deficient SCLC cell lines reconstituted silencing of the reporter plasmid. The level of NRSF transcripts as well as the level of specifically bound NRSF to the NRSE correlated with the level of GLRA1 transcripts in SCLC cell lines. Splice variants encoding truncated NRSF proteins and expressed in some SCLC did not antagonize the repression of NRSE-containing genes. Most interestingly, reconstitution of NRSF expression induced apoptosis in SCLC cells, suggesting that inhibition of NRSF activity is a crucial step in the carcinogenesis of a subgroup of SCLC.

In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways.

Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.

An anti-GD2 monoclonal antibody enhances apoptotic effects of anti-cancer drugs against small cell lung cancer cells via JNK (c-Jun terminal kinase) activation.

Small cell lung cancer (SCLC) cell lines specifically express ganglioside GD2, and anti‐GD2 monoclonal antibodies (mAbs) caused suppression of cell growth and induced apoptosis of SCLC cells with single use. Here, enhancement of the cytotoxic effects of various anti‐cancer drugs with an anti‐GD2 mAb was demonstrated. The cytotoxicity of all six drugs examined was markedly enhanced, i.e. 2.4–7.8–fold increase of cell sensitivity in terms of IC50. In particular, the combination of cisplatin (CDDP) with an anti‐GD2 mAb resulted in prominent enhancement of cytotoxicity even in low‐moderate GD2–expressing lines. The anti‐GD2 mAb induced weak activation of c‐Jun terminal kinase (JNK) in SCLC cells, and all anti‐cancer drugs also induced its activation to various degrees. When CDDP and an anti‐GD2 mAb were used together, significantly stronger JNK activation was observed corresponding to the cytotoxic effects, suggesting that synergistic phosphorylation of JNK with two reagents induced prominent apoptosis. The essential role of JNK in the induction of SCLC apoptosis with CDDP and anti‐GD2 mAb was confirmed by experiments with a JNK inhibitor, curcumin. These results suggest that anti‐GD2 mAbs would be very efficient in combination with anti‐cancer drugs, both to achieve SCLC‐specific cytotoxicity and to enhance its magnitude.

A study on apoptosis induced by As₂O₃ for small cell lung cancer

To study apoptosis of small cell lung cancer (SCLC) cell induced by As₂O₃ and enhancement effect of interferon (IFN) on As₂O₃. DNA gel electrophoresis, terminal deoxynucleotidyl transferase nick end labeling (TUNEL) and immunocytochemistry were used to detect the apoptosis induced by As₂O₃ with different doses and time and the expression of Bcl-2 or PCNA. Within certain extent of concentration, As₂O₃ could induce apoptosis of SCLC cells. And apoptotic cells increased and expression of Bcl 2 and PCNA decreased with time prolonged or dose increased. The apoptosis inducing effect of As₂O₃ combined with IFN was much higher than that of As₂O₃ alone. As₂O₃ may induce apoptosis of SCLC cells and the efficiency of apoptosis is related to dose and time. IFNα-2b might enhance the effect of apoptosis on SCLC induced by As₂O₃.

Arg(6), D-Trp(7,9), N(me)Phe(8)]-substance P (6-11) (antagonist G) induces AP-1 transcription and sensitizes cells to chemotherapy.

[Arg6, D-Trp7,9, NmePhe8]-substance P (6–11) (antagonist G) inhibits small cell lung cancer (SCLC) growth and is entering Phase II clinical investigation for the treatment of SCLC. As well as acting as a neuropeptide receptor antagonist, antagonist G stimulates c-jun-N-terminal kinase (JNK) activity and apoptosis in SCLC cells. We extend these findings and show that the stimulation of JNK and apoptosis by antagonist G is dependent upon the generation of reactive oxygen species (ROS) being inhibited either by anoxia or the presence of N-acetyl cysteine (n-AC). Antagonist G is not intrinsically a free radical oxygen donor but stimulates free radical generation specifically within SCLC cells (6.2-fold) and increases the activity of the redox-sensitive transcription factor AP-1 by 61%. In keeping with this, antagonist G reduces cellular glutathione (GSH) levels (38% reduction) and stimulates ceramide production and lipid peroxidation (112% increase). At plasma concentrations achieved clinically in the phase I studies, antagonist G augments, more than additively, growth inhibition induced by etoposide. Our results suggest that antagonist G may be particularly effective as an additional treatment with standard chemotherapy in SCLC. These novel findings will be important for the clinical application of this new and exciting compound and for the future drug development of new agents to treat this aggressive cancer. © 2000 Cancer Research Campaign