Cytotoxicity of PEGylated liposomes co-loaded with novel pro-apoptotic drug NCL-240 and the MEK inhibitor cobimetinib against colon carcinoma in vitro

Abstract

The overactivation of signaling pathways, such as the PI3K and MAPK, which are crucial to cell growth and survival, is a common feature in many cancer types. Though a number of advances have been made in the development of molecular agents targeting these pathways, their application as monotherapies has not significantly improved clinical outcome. A novel liposomal preparation was developed, co-loaded with NCL-240, a small-molecule inhibitor of the PI3K/mTOR pathway, along with cobimetinib, a MEK/ERK pathway inhibitor. This combination drug-loaded nanocarrier, (N+C)-LP, was able to significantly enhance the cytotoxicity of these drugs against colon carcinoma cells in vitro demonstrating a clear synergistic effect (combination index of 0.79). The (N+C)-LP was also able to induce cell cycle arrest of the cells, specifically in the G1 phase thereby preventing their progression to the S-phase, typical of the action of MEK inhibitors. Analyzing the apoptotic events, it was found that this effect on cell cycle regulation is followed by the induction of apoptosis. The quantified distribution of apoptotic events showed that the (N+C)-LP induced apoptosis significantly by over 3-4 fold (P<0.001) compared to other treatment groups. The co-loaded liposomal preparation was also targeted to the transferrin receptor of cancer cells by modifying the surface of the liposome with transferrin. FACS analysis showed that transferrin-mediated targeting enhanced the association of liposomes to HCT 116 cells by almost 5-fold. This could potentially allow for cancer cell-specific effects in vivo thereby minimizing any non-specific interactions of the liposomes with non-cancerous cells. Taken together, this study clearly shows that the combined inhibition of the PI3K and MEK pathways correlates with a significant anti-proliferative effect, due to cell-cycle regulation leading to the induction of apoptosis.

Keywords: Cobimetinib; Colon carcinoma; Liposomes; MEK; NCL-240; PI3K/AKT.

Figure 1

Transmission electron microscopy images of (A) Blank liposomes, (B) NCL liposomes, (C) Cobi liposomes and (D) NCL+Cobi liposomes by negative staining technique. (scale bar = 500 nm)

Figure 2

In vitro cytotoxicity analysis on HCT 116 cells showing enhanced cancer cell cytotoxicity of NCL and cobimetinib combinations

Figure 3

Analysis of cell cycle distribution showing cobimetinib-mediated cell cycle arrest in G1. (A) Histograms show the KS test which calculates difference in cell cycle distribution of the treatment groups compared to the control (the numerical value is an unsigned integer) followed by (B) the quantification of their distribution in the G1, S and G2 phases.

Figure 4

Analysis of NCL-240- and cobimetinib-mediated apoptosis. Images showing increased apoptosis of NCL+Cobi liposomes followed by the quantification of apoptotic events by random segmentation. (Apoptotic bodies shown with arrows) (Scale bar= 50 μm)

Figure 5

(A) Transferrin receptor expression and (B) cell association of transferrin-targeted liposomes.

Figure 6

In vitro cytotoxicity analysis of transferrin-targeted liposomes on HCT 116 cells.