Targeting of nav1.5 channel in metastatic breast cancer models in vitro and in vivo mice as A novel therapy

Development and examination of the effectiveness of new therapeutic interventions which the potential strength and popularity are growing for therapeutic purposes in the fight against cancer have great importance for both our country and the world. Ion channels are associated with proliferation, apoptosis, migration, angiogenesis and metastasis in cancer cell. It has been found that especially Nav1.5 is associated with high metastatic potential in vitro and the development of breast cancer in vivo. The importance of an increase in Nav1.5 channel especially the neonatal isoform expression and activity for influencing the intracellular events that are associated with breast cancer development and metastatic pathways was mentioned. Nevertheless, there is not a detailed study that evaluates intracellular protein changes and elaborative mechanism about the effects of the channel protein on breast cancer. Possible roles and effects of neonatal and adult subtypes of the Nav1.5 channel on breast cancer development and metastasis were evaluated in our study. In this study, gene silencing method by channel-specific siRNA was used. As in vitro experiments, cell proliferation test, colony formation capacity, tests associated with invasion and migration ability, apoptosis and cell cycle analysis by flow cytometry, the effects on chemotherapeutic resistance, western blot analysis for the evaluation of protein pathways that are associated with all these effects and Real-Time/RT-PCR experiments for mRNA expression of channel types were performed. Effects of Nav1.5 siRNA treatments in nanolipozomal carriers on both tumor growth and metastasis capacity of breast cancer were evaluated by performing xenograft orthotopic breast cancer model and xenograft lung metastasis model in vivo. In our study, expression of Adult and Neonatal Nav1.5 mRNAs are higher levels in MDA-MB-231 cell line compared to MCF7 and siRNAs that are specific to these two types downregulates the channel effectively were detected by PCR and Western blot methods. Specific Nav1.5 siRNA treatments realize a significant reduction in proliferation levels and colony formation abilities of breast cancer cells in MDA-MB-231, MDA-MB-468 ve MCF7 breast cancer cell lines were determined. However Nav1.5 siRNAs have not effects that are reducing cell proliferation and cytotoxic on normal breast epithelium cell line MCF10A were identified. Findings in relation with combinations of specific Nav1.5 siRNA treatments with a standard chemotherapeutic drug Paclitaxel increase the efficacy of the drug and reduce the drug resistance in MDA-MB-231 cell line were found. In addition to these results, it was observed that specific Nav1.5 siRNAs reduce significantly invasion, migration and wound-healing capacities of breast cancer cells in metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-468. In MDA-MB-231 cell line, these siRNAs increase the level of apoptosis and cause G1 arrest in cell cycle on cells were determined by flow cytometry method. İn vitro in MDA-MB-231 and MDA-MB-468 cell lines, significant reductions in the level of expression of the proteins that are involved in intracellular mechanisms regarding to all these effects of Nav1.5 siRNAs were observed. As in vivo experiments in MDA-MB-231 and MDA-MB-468 xenograft orthotopic breast cancer models, significant decrease in tumor size and tumor weight was seen in specific Nav1.5 treatment groups. On the other hand also in MDA-MB-231 xenograft lung metastasis model, significant decrease in size of tumor metastasis and photon counts per area in the lungs was found in same treatment groups. It was revealed with all these results that Nav1.5 channel has an important role in the metastasis, development and progression of breast cancer. Our study is of great importance in terms of being the first in the literature with the data that has revealed on many issues. Thus, with the appearance of effectiveness and details of this new treatment approach in the battle against cancer, it will be possible to transform it into a chronic disease by cutting off propagation and communication of breast cancer in organism, to increase the effectiveness of routine treatments and to completely defeat breast cancer by new studies to be made. Key Words: Breast cancer, Nanoliposome, Nav1.5, Metastasis/Invasion, siRNA.