Glycofullerenes as non-receptor tyrosine kinase inhibitors- towards better nanotherapeutics for pancreatic cancer treatment

Abstract

The water-soluble glycofullerenes GF1 and GF2 were synthesized using two-step modified Bingel-Hirsch methodology. Interestingly, we identified buckyballs as a novel class of non-receptor Src kinases inhibitors. The evaluated compounds were found to inhibit Fyn A and BTK proteins with IC₅₀ values in the low micromolar range, with the most active compound at 39 µM. Moreover, we have demonstrated that formation of protein corona on the surface of [60]fullerene derivatives is changing the landscape of their activity, tuning the selectivity of obtained carbon nanomaterials towards Fyn A and BTK kinases. The performed molecular biology studies revealed no cytotoxicity and no influence of engineered carbon nanomaterials on the cell cycle of PANC-1 and AsPC-1 cancer cell lines. Incubation with the tested compounds resulted in the cellular redox imbalance triggering the repair systems and influenced the changing of protein levels.

Figure 1

(A) Structures of GF1 and GF2 glycofullerenes; (B) Inhibitory activity of glycofullerenes on a panel of non-receptor tyrosine kinases.

Figure 2

qRT-PCR analysis of expression of FYN and LCK in PANC-1 (A) and AsPC-1 cells (B) after 24-hour and 72-hour incubation with glycofullerenes (1 mg/mL). The results are shown as the mean ± SD of four independent measurements, each in triplicate. Data were analyzed using one-way ANOVA with Bonferroni’s post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control and respective compounds.

Figure 3

The effect of glycofullerenes (1 mg/mL) on the expression of Fyn, Lck, p21, p53, and HO-1 in PANC-1 and AsPC-1 cells (A). Densitometric analyses of these proteins were normalized to GAPDH, vinculin, or β-actin. The results are the mean ± SD of three independent experiments. Statistical differences were analyzed using one-way ANOVA with a Bonferroni’s post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group and respective compounds (B).

Figure 4

Influence of glycofullerenes on the cell cycle progression in pancreatic cell lines. The representative histograms from flow cytometry show cell cycle distribution after 24-hour incubation with glycofullerenes (0.1 and 1 mg/mL) in PANC-1 (A) and AsPC-1 (B) cells. The graph shows the mean ± SD percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle from three independent experiments.

Figure 5

Influence of glycofullerenes (1 mg/mL) on autophagy induction in PANC-1 (A) and AsPC-1 (B) cells. The histograms show the autophagy induction ratio that was calculated by comparison of fluorescence from control cells (blue or gray peak) to the tested compounds (red peak). As a positive control, Imatinib (25 μM) was used. Data were analyzed using one-way ANOVA with Bonferroni’s post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control (untreated cells).