alpha-cobratoxin and Lung-Neoplasms

Nicotine exerts its oncogenic effects through the binding to nicotinic acetylcholine receptors (nAChRs) and the activation of downstream pathways that block apoptosis and promote neo-angiogenesis. The nAChRs of the α7 subtype are present on a wide variety of cancer cells and their inhibition by cobra venom neurotoxins has been proposed in several articles and reviews as a potential innovative lung cancer therapy. However, since part of the published results was recently retracted, we believe that the antitumoral activity of cobra venom neurotoxins needs to be independently re-evaluated.We determined the activity of α-neurotoxins from Naja atra (short-chain neurotoxin, α-cobrotoxin) and Naja kaouthia (long-chain neurotoxin, α-cobratoxin) in vitro by cytotoxicity measurements in 5 lung cancer cell lines, by colony formation assay with α7nAChRs expressing and non-expressing cell lines and in vivo by assessing tumor growth in an orthotopic Non-Obese Diabetic/Severe Combined Immunodeficient (NOD/SCID) mouse model system utilizing different treatment schedules and dosages.No statistically significant reduction in tumor growth was observed in the treatment arms in comparison to the control for both toxins. Paradoxically α-cobrotoxin from Naja atra showed the tendency to enhance tumor growth although, even in this case, the statistical significance was not reached.In conclusion our results show that, in contrast with other reports, the nAChR inhibitors α-cobratoxin from N. kaouthia and α-cobrotoxin from N. atra neither suppressed tumor growth nor prolonged the survival of the treated animals.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Clone Cells; Cobra Neurotoxin Proteins; Drug Screening Assays, Antitumor; Elapid Venoms; Humans; Luminescent Measurements; Lung Neoplasms; Mice; Receptors, Nicotinic; Toxicity Tests

Studies strongly suggest that the nicotinic acetylcholine receptors for nicotine (nAChRs) play a significant role in lung cancer predisposition and natural history. The nAChR alpha7 subunit has been found to be pivotal in the control of nicotine-induced lung cancer development and in growth signal transduction induced by nicotine binding to nAChRs.. To investigate the anticancer effects of alpha7-nAChR antagonists.. (1) To check the correlation between alpha7-nAChR presence and alpha-cobratoxin (alpha-CbT) sensitivity, binding experiments were performed in various normal human cells, lung cancer cell lines, and primary tumoral cells; (2) to demonstrate that alpha-CbT might be an efficient adjuvant therapy for non-small cell lung cancer (NSCLC) we expanded our previous observations to a panel of NSCLCs of various subtypes orthotopically grafted on nonobese diabetic/severe combined immunodeficient mice; (3) to gain insight into the mechanism of alpha-CbT-induced tumor reduction, the cells obtained after enzymatic digestion of tumors were analyzed for procaspase-9, Bax, Bad, and Bcl-X(L) protein; and (4) Snail/E-cadherin expression was evaluated to acquire information about the chemoresistance of cancer cells to alpha-CbT.. We report herein the results of an experimental strategy aimed at investigating the antitumor effects of a powerful alpha7-nAChR antagonist, alpha-CbT, in an in vivo setting set to mimic the clinical setting of lung cancer; in addition, a possible explanation for alpha-CbT selectivity toward cancer cells is presented.. We report the prolonged survival of alpha-CbT-treated animals in our mouse model of NSCLC, which is most likely the result of multiple mechanisms, including various antiproliferative and antiangiogenic effects.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Apoptosis; Blotting, Western; Bungarotoxins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cobra Neurotoxin Proteins; Humans; Immunohistochemistry; Ki-67 Antigen; Lung Neoplasms; Mice; Mice, Inbred NOD; Neoplasm Transplantation; Neoplasms, Experimental; Nicotinic Antagonists; Platelet Endothelial Cell Adhesion Molecule-1; Receptors, Nicotinic

Nicotinic acetylcholine receptors (nAChR) are expressed on bronchial epithelial and non-small cell lung cancer cells and are involved in cell growth regulation. Nicotine (classical nAChR agonist) induced cell proliferation, whereas nAChR antagonists, d- tubocurarine or alpha-cobratoxin (alpha-CbT), induced cell death. In the current study, we further explored the antitumor potential mechanisms and activities of alpha-CbT. NOD/SCID mice were grafted intraperitoneally or orthotopically and treated with alpha-CbT. alpha-CbT treatment [0.04 ng/kg or 0.12 ng/kg] induced a strong reduction in tumor size ( approximately 90%) in comparison with mice treated with the vehicle alone. Tumor inhibition was related to severe induction of apoptosis. Moreover, neoangiogenesis was strongly inhibited (reduction of cells positive to vascular endothelial growth factor and CD31). Biochemical analyses of the cells, isolated by the primary lung tumor in alpha-CbT-treated mice, showed apoptosis features characterized by: (i) inhibition of BAD phosphorylation at Ser(112) and Ser(136); (ii) BAD dissociation from 14-3-3; (iii) BAD association with BCL-XL; and (iv) cleavage of caspase-9. Moreover, these cells were unable to grow in soft agar and develop tumor, when reinjected into mice. The small interfering RNA-mediated silencing of the alpha7-nAChR gene confirmed that alpha-CbT specifically inhibited the alpha7-nAChR-mediated survival pathway in A549 cells. Furthermore, the specificity of alpha-CbT is reinforced by the lack of effect of short chain toxin (Erabutoxin-a). Once more, the no effect of the low-affinity R33E-modified alpha-CbT strengthened the specificity of this inhibition. Although alpha7-nAChR antagonists, such as alpha-CbT, are unlikely to be a primary therapy, it may provide lead compounds for the design of clinically useful drugs.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Apoptosis; Bungarotoxins; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cobra Neurotoxin Proteins; Humans; Immunoprecipitation; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Nicotinic Antagonists; Receptors, Nicotinic; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous; Tumor Cells, Cultured

Lung cancer is the most common cause of cancer death in the world. Cigarette smoking represents the major risk factor. Nicotine, an active component of cigarettes, can induce cell proliferation, angiogenesis and apoptosis resistance. All these events are mediated through the nicotinic acetylcholine receptor (nAChR) expressed on lung cancer cells. We speculate that new insights into the pathophysiological roles of nAChR may lead to new therapeutic avenues to reduce non-small cell lung cancer (NSCLC) tumour growth.. Human samples of NSCLC, cell lines and mouse models were utilized in Western blotting, reverse transcriptase polymerase chain reaction and apoptosis studies.. Human NSCLC tissues expressed alpha7-nAChR. This expression was higher in smoking patients with squamous carcinomas than those with adenocarcinomas and in male smoking patients than in females. All the data support the hypothesis that major expression of alpha7-nAChR is related to major activation of the Rb-Raf-1/phospho-ERK/phospho-p90RSK pathway. alpha7-nAChR antagonists, via mitochondria associated apoptosis, inhibited proliferation of human NSCLC primary and established cells. Nicotine stimulates tumour growth in a murine model, A549 cells orthotopically grafted. The effects of nicotine were associated with increases in phospho-ERK in tumours. Proliferation effects of nicotine could be blocked by inhibition of alpha7-nAChR by the high affinity ligand alpha-cobratoxin.. These results showed that alpha7-nAChR plays an important role in NSCLC cell growth and tumour progression as well as in cell death.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Apoptosis; Bungarotoxins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cobra Neurotoxin Proteins; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lung Neoplasms; Male; Mice; Mice, SCID; Models, Biological; Nicotine; Proto-Oncogene Proteins c-raf; Receptors, Nicotinic; Ribosomal Protein S6 Kinases, 90-kDa; Tubocurarine; Xenograft Model Antitumor Assays

Nicotinic acetylcholine receptors (nAChR) are expressed on normal bronchial epithelial and nonsmall cell lung cancer (NSCLC) cells and are involved in cell growth regulation. Nicotine induced cell proliferation. The purpose of this study was to determine if interruption of autocrine nicotinic cholinergic signaling might inhibit A549 NSCLC cell growth. For this purpose alpha-Cobratoxin (alpha-CbT), a high affinity alpha7-nAChR antagonist was studied. Cell growth decrease was evaluated by Clonogenic and MTT assays. Evidence of apoptosis was identified staining cell with Annexin-V/PI. Characterization of the basal NF-kappaB activity was done using the Trans-AM NF-kappaB assay colorimetric kit. "In vivo" antitumour activity was evaluated in orthotopically transplanted nude mice monitored by In vivo Imaging System technology. alpha-CbT caused concentration-dependent cell growth decrease, mitochondrial apoptosis caspases-9 and 3-dependent, but caspase-2 and p53-independent and down-regulation of basal high levels of activated NF-kappaB. alpha-CbT treatment determines a significant reduction of tumor growth in nude mice orthotopically engrafted with A549-luciferase cells (4.6% of living cells vs. 31% in untreated mice). No sign of toxicity was reported related to treatment. These findings suggest that alpha7-nAChR antagonists namely alpha-CbT may be useful adjuvant for treatment of NSCLC and potentially other cancers.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cobra Neurotoxin Proteins; Down-Regulation; Elapid Venoms; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice; Mice, Nude; NF-kappa B; Receptors, Nicotinic; Signal Transduction