αO-Conotoxin GeXIVA Inhibits the Growth of Breast Cancer Cells via Interaction with α9 Nicotine Acetylcholine Receptors.

The α9-containing nicotinic acetylcholine receptor (nAChR) is increasingly emerging as a new tumor target owing to its high expression specificity in breast cancer. αO-Conotoxin GeXIVA is a potent antagonist of α9α10 nAChR. Nevertheless, the anti-tumor effect of GeXIVA on breast cancer cells remains unclear. Cell Counting Kit-8 assay was used to study the cell viability of breast cancer MDA-MD-157 cells and human normal breast epithelial cells, which were exposed to different doses of GeXIVA. Flow cytometry was adopted to detect the cell cycle arrest and apoptosis of GeXIVA in breast cancer cells. Migration ability was analyzed by wound healing assay. Western blot (WB), quantitative real-time PCR (QRT-PCR) and flow cytometry were used to determine expression of α9-nAChR. Stable MDA-MB-157 breast cancer cell line, with the α9-nAChR subunit knocked out (KO), was established using the CRISPR/Cas9 technique. GeXIVA was able to significantly inhibit the proliferation and promote apoptosis of breast cancer MDA-MB-157 cells. Furthermore, the proliferation of breast cancer MDA-MB-157 cells was inhibited by GeXIVA, which caused cell cycle arrest through downregulating α9-nAChR. GeXIVA could suppress MDA-MB-157 cell migration as well. This demonstrates that GeXIVA induced a downregulation of α9-nAChR expression, and the growth of MDA-MB-157 α9-nAChR KO cell line was inhibited as well, due to α9-nAChR deletion. GeXIVA inhibits the growth of breast cancer cell MDA-MB-157 cells in vitro and may occur in a mechanism abolishing α9-nAChR.

Differential Expression of Nicotine Acetylcholine Receptors Associates with Human Breast Cancer and Mediates Antitumor Activity of αO-Conotoxin GeXIVA.

Nicotinic acetylcholine receptors (nAChRs) are membrane receptors and play a major role in tumorigenesis and cancer progression. Here, we have investigated the differential expression of nAChR subunits in human breast cancer cell lines and breast epithelial cell lines at mRNA and protein levels and the effects of the αO-conotoxin GeXIVA, antagonist of α9α10 nAChR, on human breast cancer cells. Reverse transcription polymerase chain reaction (PCR) demonstrated that all nAChR subunits, except α6, were expressed in the 20 tested cell lines. Real time quantitative PCR (QRT-PCR) suggested that the mRNA of α5, α7, α9 and ß4 nAChR subunits were overexpressed in all the breast cancer cell lines compared with the normal epithelial cell line HS578BST. α9 nAChR was highly expressed in almost all the breast cancer cell lines in comparison to normal cells. The different expression is prominent (p < 0.001) as determined by flow cytometry and Western blotting, except for MDA-MB-453 and HCC1395 cell lines. αO-conotoxin GeXIVA that targeted α9α10 nAChR were able to significantly inhibit breast cancer cell proliferation in vitro and merits further investigation as potential agents for targeted therapy.

Engineered Conotoxin Differentially Blocks and Discriminates Rat and Human α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (nAChR) is present in the central nervous system and plays an important role in cognitive function and memory. α-Conotoxin LvIB, identified from genomic DNA of Conus lividus, its three isomers and four globular isomer analogues were synthesized and screened at a wide range of nAChR subtypes. One of the analogues, amidated [Q1G,ΔR14]LvIB, was found to be a potent blocker of rat α7 nAChRs. Importantly, it differentiates between α7 nAChRs of human (IC50: 1570 nM) and rat (IC50: 97 nM). Substitutions between rat and human α7 nAChRs at three key mutation sites revealed that no single mutant could completely change the activity profile of amidated [Q1G,ΔR14]LvIB. Rather, we found that the combined influence of Gln141, Asn184, and Lys186 determines the α7 nAChR species specificity of this peptide. This engineered α4/4 conotoxin has potential applications as a template for designing ligands to selectively block human α7 nAChRs.

Identification of Crucial Residues in α-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor.

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1ß1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1ß1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1ß1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1ß1δε nAChR, and showed loss of activity on α3ß2 and α3ß4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1ß1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1ß1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1ß1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

α-Conotoxin [S9A]TxID Potently Discriminates between α3ß4 and α6/α3ß4 Nicotinic Acetylcholine Receptors.

α3ß4 nAChRs have been implicated in various pathophysiological conditions. However, the expression profile of α3ß4 nAChRs and α6/α3ß4 nAChRs overlap in a variety of tissues. To distinguish between these two subtypes, we redesigned peptide 1 (α-conotoxin TxID), which inhibits α3ß4 and α6/α3ß4 nAChR subtypes. We systematically mutated 1 to evaluate analogue selectivity for α3ß4 vs α6/α3ß4 nAChRs expressed in Xenopus laevis oocytes. One analogue, peptide 7 ([S9A]TxID), had 46-fold greater potency for α3ß4 versus α6/α3ß4 nAChRs. Peptide 7 had IC50s > 10 µM for other nAChR subtypes. Molecular dynamics simulations suggested that Ser-9 of TxID was involved in a weak hydrogen bond with ß4 Lys-81 in the α6ß4 binding site but not in the α3ß4 binding site. When Ser-9 was substituted by an Ala, this hydrogen bond interaction was disrupted. These results provide further molecular insights into the selectivity of 7 and provide a guide for designing ligands that block α3ß4 nAChRs.