Combined Effect of Anticancer Agents and Cytochrome C Decorated Hybrid Nanoparticles for Liver Cancer Therapy

Abstract

Hepatocellular carcinoma is an aggressive form of liver cancer that displays minimal symptoms until its late stages. Unfortunately, patient prognosis still remains poor with only 10% of patients surviving more than five years after diagnosis. Current chemotherapies alone are not offering efficient treatment, hence alternative therapeutic approaches are urgently required. In this work, we highlight the potential of combination of treatment of hepatocellular carcinoma with existing chemotherapies in combination with pro-apoptotic factor cytochrome C. In order to allow cytochrome C to cross the cellular membrane and become internalized, it has been immobilised onto the surface of hybrid iron oxide-gold nanoparticles. This novel approach has been tested in vitro on HepG2, Huh-7D and SK-hep-1 cell lines in order to elucidate potential as a possible alternative therapy with greater efficacy. The data from our studies show consistently that combining treatment of clinically used anticancer agents (doxorubicin, paclitaxel, oxaliplatin, vinblastine and vincristine) significantly increases the levels of apoptosis within the cell lines, which leads to cellular death. Hence, this combined approach may hold promise for future treatment regimes.

Keywords: apoptosis; combination therapy; cytochrome C; hybrid nanoparticle; liver cancer.

Figure 1

Cellular internalisation of HNP-c (hybrid nanoparticles with cytochrome C conjugated onto the surface) imaged using TEM (transmission electron microscopy) after exposure to (A) HepG2, (B) Huh-7D and (C) Sk-hep-1 cells with (D) quantification using ICP-OES (inductively coupled plasma − optical emission spectroscopy, n = 3, ±SD). * denotes significant increase compared to cytochrome C alone (p < 0.05).

Figure 2

Cell viability measured using the MTT assay of (A) doxorubicin, (B) paclitaxel, (C) oxaliplatin, (D) vinblastine and (E) vincristine as a single therapy and in combination with HNP-c after 48 h incubation in HepG2, Huh-7D and SK-hep-1 cell lines (n = 3, ±SD). * denotes significant decrease in IC₅₀ compared to single drug treatment (p < 0.05).

Figure 3

Apoptosis detection via Caspase-3 measurement in (A) HepG2, (B) Huh-7D and (C) SK-hep-1 cells incubated with chemotherapies both with and without HNP-c (n = 3, ±SD). Control refers to cells with no treatment. * denotes significance compared to single drug treatment (p < 0.05).

Figure 4

Western blot detection of poly (ADP-ribose) polymerase (PARP) cleavage in (A) HepG2, (B) Huh-7D and (C) SK-hep-1 cell lines incubated with chemotherapy both with and without HNP-c. Control refers to cells with no treatment.

Figure 5

Apoptosis detection via TUNEL assay measurement in (A) HepG2, (B) Huh-7D and (C) SK-hep-1 cells incubated with chemotherapies both with and without HNP-c (n = 3, ±SD). * denotes significance compared to single drug treatment (p < 0.05).

Figure 6

Apoptosis detection cells with Annexin V staining probe. HepG2 cells treated with single drugs and in combination with HNP-c demonstrated as (A) images and (B) quantitatively (n = 3, ±SD). Control refers to cells with no treatment. * denotes significance compared to single drug treatment (p < 0.05).