Abstract:
Chemotherapy remains as the option for treating cancer in increasing the lifetime of the patient. The severe effects of chemotherapy drugs contribute to the developments of approaches based on silencing of cancer-causing genes using siRNA. Nonetheless, siRNA delivery vehicle is needed as they cannot diffuse across cellular membranes; thus leading to the development of carbonate apatite nanoparticle. By employing carbonate apatite nanoparticles, we showed that codelivery of siRNAs and drugs managed to block signalling pathways. Simultaneous delivery of ROS1 siRNA and 40nM Pax by carbonate apatite exhibited better effects in MCF7 cells where the cell viability was significantly reduced to 26% with 42% more sensitive towards the drug. Further analysis showed the treatment caused the disruption of phosphorylation in ERK1/2 pathway. Besides, silencing of RAF1 gene concurrently with 40nM Dox in MCF7 cells also resulted in low cell viability and relatively high enhancement of chemosensitivity, but this co-treatment did not affect the phosphorylation of ERK1/2 pathway. GRB2 gene seemed to be vital in MCF7 as its knockdown showed prominent cell death and chemosensitivity towards 8nM Pax, 40nM Pax and 40nM Cis. Nevertheless, all these three combinations failed to block the phosphorylation of ERK1/2 pathway. In 4T1 cells, co-delivery of SHC1 siRNA and 8nM or 40nM of Pax greatly decreased the cell viability to 29% and 17% respectively owing to the high sensitivity towards drug. Moreover, co-treatment of ROS1 siRNA and 40nM Dox reduced cell viability to 30% with the sensitivity for the drugs increased by 51%. Western blot results proved that this treatment had successfully blocked the phosphorylation of AKT1 (Ser473) pathway while partially blocked the phosphorylation of ERK1/2. From the in vitro results, treatment of ROS1 siRNA and Dox demonstrated effective cytotoxicity as well as interruption in AKT1 (Ser473) and ERK1/2 pathways. Thus, this combination was then used in in vivo study. Intratumoural injection of carbonate apatite conjugated with ROS1 siRNA and Dox also blocked the phosphorylation of AKT1 (Ser473) pathway. This concluded that carbonate apatite can be a promising tool for both in vitro and in vivo delivery of siRNAs targeting important cancer-related genes.
