Enhancing human breast cancer cells destruction using combination of adenovirus expressing P53 and hyperthermia treatment

Abstract

In Malaysia, breast cancer is the most common cancer where 1 in 19 Malaysian women will be diagnosed with breast cancer by the age of 85. Moreover, lack of specific symptoms in the early stage of disease leading to delay in diagnosis. Unfortunately, current treatments by chemotherapeutic agents, surgery and radiation are not fully effective for the treatment of breast cancer. Thus, there is an urgency in developing new approaches for the treatment of breast cancer patients. In this study, a novel therapeutic regimen, combining the effects of recombinant adenovirus and hyperthermia was investigated. Firstly, Adenovirus serotype 5 was constructed by cloning of p53 gene into a defective recombinant adenovirus vector, Ad5-p53-DsRed Monomer N1. The Ad5-p53-DsRed Monomer N1 (MOI of 100) was then used to infect breast cancer cells (MDA-MB 231 and MCF-7) with or without combination of hyperthermia treatment (42ºC for 2 hours). The cell killing and viral concentration were then determined by MTT assay and viral plaque formation assay respectively. After that, the heat shock protein (Hsp70) and p53 protein expression in transfected cells were quantitated using ELISA assay. Activated-Caspase 3/7, 8 and 9 were also evaluated to study the apoptotic pathway of cancer cells. Furthermore, the novel protein interaction between nucleotide binding domain (NBD) Hsp70 and human Ad5 E1A 32 kDa motif (PNLVP); and NBD and p53 motif (SCMGGMNR) were investigated through bioinformatics tools such as Gromacs and Autodock softwares. It was found that MDA-MB 231 and MCF-7 cells infected with virus Ad5-p53- DsRed Monomer N1 alone resulted in 46.77±2.74% and 42.26±1.78% cell killing respectively while hyperthermia in combination with virus were 84.82±1.64% and 80.13±3.30% respectively. The Hsp70 expression of both cancer cells was also increased to 170.57% (MDA-MB 231) and 169.83% (MCF-7). Moreover, p53 expression in MDA-MB 231 and MCF-7 cells by virus combined with heat treatment (85.72 ng/L and 79.05 ng/L respectively) could lead to enhanced oncolytic property compared to virus treatment alone (47.82 ng/L and 40.54 ng/L respectively). In addition, caspase activity was first time reported that apoptosis process started at very early stage of infection in breast cancer cells with hyperthermia compared to virus alone. This was due to the evident that the highest kinetic energy was found in caspase 3 whereas virus alone the highest in caspase 8. In conclusion, Hsp70 induction by hyperthermia treatment enhanced Ad5-p53-DsRed Monomer N1 replication and oncolysis in MDA-MB 231 and MCF-7 cells through apoptotic pathway. Besides that, NBD of Hsp70 had the best interaction with PNLVP motif at 42°C. Thus, combining Ad5-p53 with hyperthermia treatment could be a potential approach for breast cancer treatment.