Selective apoptosis induction in MCF-7 cell line by truncated minimal functional region of Apoptin

Abstract

BackgroundChicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has a unique capability in inducing apoptosis in cancer cells but not in normal cells. Five truncated Apoptin proteins were analyzed to determine their selective ability to migrate into the nucleus of human breast adenocarcinoma MCF-7 cells for inducing apoptosis.MethodsFor identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N’ terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect.ResultsThree of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32–83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1–31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin’s signature targeting activity.ConclusionsTherefore, the critical stretch spanning amino acid 1–31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across cellular membrane, simultaneously promoting protein delivery for selective and effective breast cancer therapy.

New insights into the evolutionary features of viral overlapping genes by discriminant analysis

“…A truncated apoptin lacking residues 1-43 is a soluble, non-aggregating protein that maintains most of the properties of wildtype apoptin when transfected into human cancer cells (Rui-Martinez et al, 2017). Two other constructs, the first formed by PRS and NLSs and the other by LRS and NLSs, can induce selective apoptosis in a breast cancer cell line and in glioma cells, respectively (Shen Ni et al 2013;Zhang et al, 2017).…”

“…Another example is the X protein of Borna disease virus, which shows protective properties against neurodegeneration in vitro and in vivo (Szelechowski et al, 2014). To increase their therapeutic effectiveness, researchers have constructed N-terminal deleted forms or short peptides both for apoptin (Shen Ni et al, 2013;Ruiz-Martínez et al, 2017;Zhang et al, 2017) and X protein (Szelechowski et al, 2014;Ferré et al, 2017).…”

On Advances in Cancer Suicide-genes Therapy

“…This allows a specific induction of apoptosis in cancer cells. [47] This ability to kill only tumor cells has been reported in breast [48] , head and neck [49] , prostate [50,51] , lung cancer, osteosarcoma [50] , hepatocellular [52] and nasopharyngeal [53] carcinoma. A study analyzed the ability to induce apoptosis of five truncated apoptin proteins in human breast adenocarcinoma MCF-7 cells and human liver Chang cells as control.…”

“…Three out of the five truncated proteins targeted the nucleus of the MCF-7 cells initiating apoptosis and showing no effect on normal cells. [48] Dacarbazine is an agent used in the treatment against malignant melanoma. When combined with apoptin they have demonstrated a synergistic antitumor effect in human and mouse B16-F1 melanoma cells.…”

Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

“…SiRNA cocktail targeting different region of viral genome lowers the chances of viral mutant formation and prevent viral escape (24)(25)(26)(27). Nevertheless, besides virus infections, siRNAs can be utilized to silence multiple genes at a time which could benefit to treat brain tumorigenesis involving multiple genetic alterations and also targeting a suitable apoptotic gene as a treatment (28,29). In addition, the availability to maintains the wide array of cells including islets of Langerhans could provide a strong foundation to use siRNAs for diabetic studies (30,31).…”