Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier

Abstract

Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37°C) but aggregates when the temperature is raised above 39°C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37°C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42°C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.

A thermally responsive biopolymer conjugated to an acid-sensitive derivative of paclitaxel stabilizes microtubules, arrests cell cycle, and induces apoptosis

“…It is likely that SynB1-mediated endocytosis that has slower kinetics than the free drug requires more time to get completely internalized and increase the intracellular concentration of paclitaxel to exert similar effects as free drug. Thus, the role of hyperthermia combined with SynB1 mediated cellular uptake is to increase the amount of polypeptide delivered inside the cell, which is consistent with other findings [9,14,15,24,29,34].…”

“…A 59.2 kDa ELP with a T t of~40°C (ELP1) is ideal for thermal targeting and has been used for thermally targeted delivery of doxorubicin and anti-cancer therapeutic peptides [13][14][15], and reviewed in [5]. Since hyperthermia is expected to enhance the vascular permeability of the tumor, the application of heat could also augment tumor vasculature penetration by the ELP macromolecule.…”

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

“…2A). Previously, neutral elastin-like polypeptides were employed as delivery vehicles for small therapeutic peptides [36] and proteins [37]. However, with those systems uptake into cancerous tissue and cells was mediated by exploiting their thermoresponsive properties and fused CCP motifs, respectively.…”

Nanotechnological Approaches to Therapeutic Delivery Using Elastin-Like Recombinamers

“…One of the first ELR-p21 recombinamers, which was combined with Bac CPP, showed internalization of ELR aggregates and further anti-proliferative effects by the inhibition of the cell cycle in SKOV-3 ovarian cancer cells and MCF-7 breast cancer ones. 52 An analogous fusion recombinamer was combined with gemcitabine, a chemotherapeutic agent used in the treatment of pancreatic cancer, to enhance the anti-tumour effect while lowering the drug dose to reduce its side effects. The results showed cytotoxicity in MIA PaCa-2 and Panc-1 pancreatic cancer cell lines and tumour growth inhibition in mice in vivo after hyperthermia.…”