Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth

Abstract

In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.

Keywords: NF-κB; autophagy; glioblastoma (GBM); lactucopicrin (LCTP); oxidative stress; p62/SQSM1; temozolomide (TMZ).

Figure 1

(A) Chemical structure of LCTP. (B) IC50-values of U87Mg cells after 24, 48 and 72 h of incubation with LCTP. (C) Morphological changes of U87Mg glioblastoma cells treated every 24 h with LCTP 7.5 and 10 µM for 24, 48 and 72 h. Magnification 20×. (D) Time and dose-dependent growth inhibition of LCTP-treated U87Mg cells. (E) Cytotoxic effect of various concentrations of LCTP (7.5 and 10 µM) at 24, 48 and 72 h post-treatment on U87Mg cell line. (F) Quantification of the cell-free area in wound healing assay at T0 and 24 h post-scratch in control and LCTP-treated U87Mg cells. (G) Colony-forming assay of U87Mg treated 24 h with LCTP 7.5. µM. For all the experiments, values are the means ± SEM of 3 individual determinations. Unpaired t-test, p-value < 0.05. According to GraphPad Prism, * p-value 0.01 to 0.05 (significant), ** p-value 0.001 to 0.01 (very significant), *** p-value 0.0001 to 0.001 (Extremely significant), **** p-value < 0.0001 (Extremely significant).

Figure 2

(A) Western blot analysis of autophagy-associated proteins p62 and LC3BII in short-termtreated-U87Mg cells with LCTP 10 µM. Densitometric analysis of protein levels represent the means ± SEM of 3 individual determinations. Data were normalized to GAPDH and are expressed as fold change over control-treated cells. * Unpaired t-test, p-value < 0.05. (B) Western blot analysis of proliferative signals pERK/ERK/GAPDH and pAKT/AKT/GAPDH in short-termtreated-U87Mg cells with LCTP 10 µM. Densitometric analysis of protein levels represent the means ± SEM of 3 individual determinations and are expressed as fold change over control-treated cells, normalized to GAPDH. * Unpaired t-test, p-value < 0.05. According to GraphPad Prism, * p-value 0.01 to 0.05 (significant), ** p-value 0.001 to 0.01 (very significant), *** p-value 0.0001 to 0.001 (Extremely significant), **** p-value < 0.0001 (Extremely significant). (C) Immunofluorescence analysis of cytoskeleton proteins α-tubulin and Vimentin in U87Mgcells treated with LCTP 10 µM and vehicle control (DMSO 0.08%) for 20 min. Magnification 20× and 60×.

Figure 3

(A) Representative flow cytometry analysis of cell cycle arrest in G2/M and (B) plot of cell cycle distribution of propidium iodide (PI) staining in U87Mg, vehicle (left panel) and LCTP (7.5 µM) treated cells (right panel) for 24, 48 and 72 h. Values are the means ± SEM of 3 individual determinations. * Unpaired t-test, p-value < 0.05. (C) Western blot analysis of p53 and p21 in long-term LCTP--treated U87Mg cells. Data were normalized toβ-actin and are expressed as fold change over control-treated cells of 3 individual determinations. * Unpaired t-test, p-value < 0.05. (D) Western blot analysis of CDK2: the figure shows CDK2 analysis of U87Mg cells treated with LCTP 7.5 µM at different time (24, 48, and 72 h). Data were normalized to β-actin and are expressed as fold change over control-treated cells of 3 individual determinations. * Unpaired t-test, p-value < 0.05. (E) Western blot and densitometric analysis of procaspase 6 of LCTP-treated U87Mg cells at different time (24, 48 and 72 h). Values are the means ± SEM of 3 individual determinations and protein expression levels, normalized to β-actin, are expressed as fold change over control-treated cells. * Unpaired t-test, p-value < 0.05. (F) Western blot analysis of PARP in LCTP-treated U87Mg cells at different time (24, 48 and 72 h). Values as ratio of cleaved PARP to full-length PARP represent the means ± SEM of 3 individual determinations and are expressed as fold change over control-treated cells. * Unpaired t-test, p-value < 0.05. According to GraphPad Prism, * p-value 0.01 to 0.05 (significant), ** p-value 0.001 to 0.01 (very significant), *** p-value 0.0001 to 0.001 (Extremely significant), **** p-value < 0.0001 (Extremely significant). (G) DNA laddering of long-term treatment of U87Mg cells with LCTP 7.5 with respect to untreated cells.

Figure 4

(A) Effect of pre-treatment with ROS scavenger NAC 3 mM for 4 h on morphological changes and (B) cell viability (%) induced by different concentrations of LCTP (7.5, 10, 15 and 30 µM) on U87Mg cells at 24 h from treatment. Magnification 20×. (C) Western blot analysis of NF-κB p65 subunit of short-term treated U87Mg cells with LCTP 7.5 µM (30 min, 1 h, 2 h and 4 h). Data were normalized to GAPDH and are expressed as fold change over control-treated cells of 3 individual determinations. According to GraphPad Prism, ** p-value 0.001 to 0.01 (very significant), *** p-value 0.0001 to 0.001 (Extremely significant), **** p-value < 0.0001 (Extremely significant).

Figure 5

(A) IC50-values of U87Mg cells after 24, 48 and 72 h of incubation with TMZ. (B) IC50-values of TMZ at 24 h of U87Mg cells pre-treated with LCTP 7.5 and 10 µM for 24 h. (C) Synergistic effect of LCTP and conventional chemotherapy TMZ ongrowth of U87Mg cells. Values are the means ± SEM of 3 individual determinations. # p-value compared to cells treated with TMZ alone. (D) Synergistic effect of LCTP and conventional chemotherapy TMZ oncell viability of U87Mg cells. Values are the means ± SEM of 3 individual determinations. # p-value < 0.05 compared to cells treated with TMZ alone. According to GraphPad Prism, # p-value 0.01 to 0.05 (significant), ## p-value 0.001 to 0.01 (very significant), *** or ### p-value 0.0001 to 0.001 (Extremely significant), **** or #### p-value < 0.0001 (Extremely significant).