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Inhibition of the nicotinic acetylcholine receptors by cobra venom α-neurotoxins: is there a perspective in lung cancer treatment?

Alama A, Bruzzo C, Cavalieri Z, Forlani A, Utkin Y, Casciano I, Romani M - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Lung Cancer Unit, Istituto Nazionale per la Ricerca sul Cancro, IST, Genova, Italy.

ABSTRACT
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.

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Presence of the α7-nACh receptor on NSCLC cell lines.A) Semiquantitative Alpha7 RT-PCR analysis on Human NSCLC adenocarcinoma e squamous cell carcinoma cell lines. GAPDH expression was utilized as internal control for mRNA integrity and cDNA quantification. NCI-H1975 and Calu1 are negative. C: No template control. B) qPCR alpha 7 expression in the same cell lines. On y-axis natural logaritm (ln) of fold change. NCI-H1650 was used as calibrator, GAPDH as reference gene. One ugr of Total RNA was retrotrascibed and the same amount of cDNA per sample (2 ul) was used (Ct GAPDH comprised between 15.65 and 17.65). NCI-H1975 and Calu1 resulted negative or with extremely low expression due to Ct>40. C) Western blot analysis for alpha 7. PC12 was loaded as positive control. Actin expression was utilized as internal control.
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pone-0020695-g001: Presence of the α7-nACh receptor on NSCLC cell lines.A) Semiquantitative Alpha7 RT-PCR analysis on Human NSCLC adenocarcinoma e squamous cell carcinoma cell lines. GAPDH expression was utilized as internal control for mRNA integrity and cDNA quantification. NCI-H1975 and Calu1 are negative. C: No template control. B) qPCR alpha 7 expression in the same cell lines. On y-axis natural logaritm (ln) of fold change. NCI-H1650 was used as calibrator, GAPDH as reference gene. One ugr of Total RNA was retrotrascibed and the same amount of cDNA per sample (2 ul) was used (Ct GAPDH comprised between 15.65 and 17.65). NCI-H1975 and Calu1 resulted negative or with extremely low expression due to Ct>40. C) Western blot analysis for alpha 7. PC12 was loaded as positive control. Actin expression was utilized as internal control.

Mentions: The interaction between α-cobratoxin and the α7 nicotinic receptor [32] was one of the experimental evidences behind the rationale of utilizing cobra venom toxins as anticancer agents [22]. Although this receptor is expressed on a wide spectrum of tissues and cell lines, a recent survey of the literature [22], [31] reported conflicting data on the expression of this receptor in A549, the cell line utilized for the in vivo and most of the in vitro anticancer assays on α-cobratoxin. Therefore, as an initial step to verify the activity of α-cobratoxin in NSCLC, we performed a semiquantitative RT-PCR and qPCR survey to demonstrate the expression of α7 nAChR in 5 lung cancer cell lines. Three of the cell lines utilized in the present study (A549, H1650 and SK-MES 1) were the same of the original set of experiments [18], [19], [20], [27]. As shown in Figure 1, Panel A, the α7 nicotinic receptor was readily detectable in A549, H1650 and SK-MES 1 but not in H1975 and CALU 1. The qPCR analysis confirmed the presence of different amount of the α7 nAChR mRNA in A549, H1650 and SK-MES 1 (Figure 1, Panel B). In agreement with the RT-PCR results, in H1975 and CALU 1 the α7 nAChR transcript, using the same amount of cDNA used for all the cell lines, appeared after cycle 40, a result that could be attributed either to an extremely low level of expression or to background noise


Inhibition of the nicotinic acetylcholine receptors by cobra venom α-neurotoxins: is there a perspective in lung cancer treatment?

Alama A, Bruzzo C, Cavalieri Z, Forlani A, Utkin Y, Casciano I, Romani M - PLoS ONE (2011)

Presence of the α7-nACh receptor on NSCLC cell lines.A) Semiquantitative Alpha7 RT-PCR analysis on Human NSCLC adenocarcinoma e squamous cell carcinoma cell lines. GAPDH expression was utilized as internal control for mRNA integrity and cDNA quantification. NCI-H1975 and Calu1 are negative. C: No template control. B) qPCR alpha 7 expression in the same cell lines. On y-axis natural logaritm (ln) of fold change. NCI-H1650 was used as calibrator, GAPDH as reference gene. One ugr of Total RNA was retrotrascibed and the same amount of cDNA per sample (2 ul) was used (Ct GAPDH comprised between 15.65 and 17.65). NCI-H1975 and Calu1 resulted negative or with extremely low expression due to Ct>40. C) Western blot analysis for alpha 7. PC12 was loaded as positive control. Actin expression was utilized as internal control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113800&req=5

pone-0020695-g001: Presence of the α7-nACh receptor on NSCLC cell lines.A) Semiquantitative Alpha7 RT-PCR analysis on Human NSCLC adenocarcinoma e squamous cell carcinoma cell lines. GAPDH expression was utilized as internal control for mRNA integrity and cDNA quantification. NCI-H1975 and Calu1 are negative. C: No template control. B) qPCR alpha 7 expression in the same cell lines. On y-axis natural logaritm (ln) of fold change. NCI-H1650 was used as calibrator, GAPDH as reference gene. One ugr of Total RNA was retrotrascibed and the same amount of cDNA per sample (2 ul) was used (Ct GAPDH comprised between 15.65 and 17.65). NCI-H1975 and Calu1 resulted negative or with extremely low expression due to Ct>40. C) Western blot analysis for alpha 7. PC12 was loaded as positive control. Actin expression was utilized as internal control.
Mentions: The interaction between α-cobratoxin and the α7 nicotinic receptor [32] was one of the experimental evidences behind the rationale of utilizing cobra venom toxins as anticancer agents [22]. Although this receptor is expressed on a wide spectrum of tissues and cell lines, a recent survey of the literature [22], [31] reported conflicting data on the expression of this receptor in A549, the cell line utilized for the in vivo and most of the in vitro anticancer assays on α-cobratoxin. Therefore, as an initial step to verify the activity of α-cobratoxin in NSCLC, we performed a semiquantitative RT-PCR and qPCR survey to demonstrate the expression of α7 nAChR in 5 lung cancer cell lines. Three of the cell lines utilized in the present study (A549, H1650 and SK-MES 1) were the same of the original set of experiments [18], [19], [20], [27]. As shown in Figure 1, Panel A, the α7 nicotinic receptor was readily detectable in A549, H1650 and SK-MES 1 but not in H1975 and CALU 1. The qPCR analysis confirmed the presence of different amount of the α7 nAChR mRNA in A549, H1650 and SK-MES 1 (Figure 1, Panel B). In agreement with the RT-PCR results, in H1975 and CALU 1 the α7 nAChR transcript, using the same amount of cDNA used for all the cell lines, appeared after cycle 40, a result that could be attributed either to an extremely low level of expression or to background noise

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Lung Cancer Unit, Istituto Nazionale per la Ricerca sul Cancro, IST, Genova, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus