Traditional Plant-Derived Compounds Inhibit Cell Migration and Induce Novel Cytoskeletal Effects in Glioblastoma Cells

Abstract

Glioblastomas (GBMs) are aggressive and invasive cancers of the brain, associated with high rates of tumour recurrence and poor patient outcomes despite initial treatment. Targeting cell migration is therefore of interest in highly invasive cancers such as GBMs, to prevent tumour dissemination and regrowth. One current aim of GBM research focuses on assessing the anti-migratory properties of novel or repurposed inhibitors, including plant-based drugs which display anti-cancer properties. We investigated the potential anti-migratory activity of plant-based products with known cytotoxic effects in cancers, using a range of two-dimensional (2D) and three-dimensional (3D) migration and invasion assays as well as immunofluorescence microscopy to determine the specific anti-migratory and phenotypic effects of three plant-derived compounds, Turmeric, Indigo and Magnolia bark, on established glioma cell lines. Migrastatic activity was observed in all three drugs, with Turmeric exerting the most inhibitory effect on GBM cell migration into scratches and from the spheroid edge at all the timepoints investigated (p < 0.001). We also observed novel cytoskeletal phenotypes affecting actin and the focal adhesion dynamics. As our in vitro results determined that Turmeric, Indigo and Magnolia are promising migrastatic drugs, we suggest additional experimentation at the whole organism level to further validate these novel findings.

Keywords: 2D/3D assays; anti-migratory; glioblastoma; indigo; invasion; magnolia; migration; turmeric.

Figure 1

The structure of three plant-derived compounds. (a) Magnolia possesses two APIs, Magnolol and Honokiol. (b) Indigo is derived from the Indigofera tinctoria plant. (c) Curcumin is the API found in Turmeric.

Figure 2

Actin localization categories identified via confocal microscopy. (a) Actin was localized cortically, around the perimeter of the cell, following treatment with Indigo (27.02 µg/mL). (b) Stress fiber formation across the cell body was observed in control, mock-treated cells. (c) Fragmented actin was located peripherally, but appeared to be formed from short, random actin filaments in Indigo (27.02 µg/mL) treated cells. (d) Magnified cell from (c) highlighting the fragmented actin phenotype. Arrows highlight examples for each actin category identified in cells. Red = actin; for (a–c) scale bar = 10 µm, for (d) scale bar = 100 µm.

Figure 3

Immunofluorescence images and actin localization analysis of both mock-treated and treated U87 cells. (a) Images of control, Magnolia (116.07 µg/mL), Indigo (27.02 µg/mL) and Turmeric (31.94 µg/mL) treated cells were imaged, with Alexa fluor 488 (secondary to mouse anti-vinculin) excited at a wavelength of 488nm, TRITC-conjugated phalloidin at 561 nm and DAPI at 405 nm. The mock-treated control group is characterized by actin stress fibers, whereas Turmeric appears to induce a shift towards cortical actin localization concomitant with a rounded, amoeboid morphology. Scale bar = 10 µm. (b) Mean percentage of actin localizations (mean ± SEM). Indigo appeared to exert the most significant changes in actin dynamics. Red = actin, green = vinculin and blue = DAPI. n ≥ 7 for each drug treatment, based on two repeats. Kruskal-Wallis: * = p ≤ 0.05 and ** = p ≤ 0.01.

Figure 4

Plant-derived drugs reduce FA numbers in U87 cells. (a) Images generated via confocal microscopy show vinculin expression and highlight a decrease in FA numbers in all three plant-based compounds in comparison to the mock-treated control. Arrows show example FAs for each treatment group. Scale bar = 10 µm. (b) Mean number of FAs per cell for each drug treatment (mean ± SEM). Magnolia, Indigo and Turmeric all decreased the number of FAs per cell, with Turmeric exhibiting the most inhibitory effect on FA generation; n ≥ 4 for each drug treatment. Kruskal-Wallis: * = p ≤ 0.05, ** = p ≤ 0.01 and *** = p ≤ 0.001.

Figure 5

Two-dimensional migration into scratches is significantly reduced by plant-derived compounds with established anti-cancer activity at low concentrations. (a) EVOS XL Core generated images demonstrated the extent of migration into scratches after 24 h incubation. Dashed red lines are used to outline the wound edges. Scale bar = 500 µm. (b) Mean SIR for each of the lower concentration treatment groups (mean ± SEM). All three compounds reduced the extent of 2D migration, but Turmeric (0.032 µg/mL) exerted the most statistically significant effect; n ≥ 11 for each drug treatment. ANOVA: * = p ≤ 0.05, and *** = p ≤ 0.001.

Figure 6

Two-dimensional migration into scratches is significantly reduced by plant-derived compounds with established anti-cancer activity at their IC₂₅ concentrations. (a) EVOS XL Core generated images demonstrated the extent of migration into scratches after 24 h incubation. Dashed red lines are used to highlight the wound edges. Scale bar = 500 µm. (b) Mean SIR for each of the lower concentration treatment groups (mean ± SEM). All three compounds reduced 2D migration, but Indigo (27.02 µg/mL) produced the lowest SIR, although this was just as statistically significant as for Turmeric when compared to the control; n ≥ 11 for each drug treatment. ANOVA: *** = p ≤ 0.001.

Figure 7

Cell migration from spheroids was reduced following treatment with a low concentration of natural products. (a) Images obtained using an EVOS XL Core imaging system showed an increase in migration into the collagen matrix over a 48 h period. Spheroids in the treated groups appeared visually consistent with the control. Scale bar = 500 µm. (b) Mean MIs for each of the lower drug concentrations, at the migrating front and edge at all timepoints (mean ± SEM). Indigo (0.027 g/mL) treated spheroids had the most significantly reduced edge MI at 48 h. Magnolia (0.116 µg/mL) exerted the most anti-migratory effect for the migrating front after 24 h, but this was not statistically significant; n ≥ 7 for each drug treatment. ANOVA (front MI 24 h, edge MI 24 h and front MI 48 h) and Kruskal-Wallis (edge MI 48 h): * = p ≤ 0.05.

Figure 8

Decrease in 3D GBM cell migration after treatment with a high concentration of plant-based drugs. (a) Spheroid images taken over 48 h highlight a decrease in migration in the Turmeric (31.94 µg/mL) group. Scale bar = 500 µm. (b) The mean MI of both treated and mock-treated spheroids (mean ± SEM). At the 24 h migrating edge and both 48 h fronts, Turmeric (31.94 µg/mL) produced the lowest and most statistically significant MI; n ≥ 7 for each drug treatment. ANOVA (front MI 24 h, edge MI 24 h and front MI 48 h) and Kruskal-Wallis (edge MI 48 h): * = p ≤ 0.05, ** = p ≤ 0.01 and *** = p ≤ 0.001.

Figure 9

Immunofluorescence of both migrating U87 cells and spheroids within their respective collagen plugs. (a) Images of each treatment group were acquired, with Alexa fluor 488 (secondary to mouse anti-vinculin) excited at a wavelength of 488nm, TRITC-conjugated phalloidin at 561 nm and DAPI at 405 nm. Turmeric (31.94 µg/mL) cells are morphologically rounded in comparison to the control, and actin is less visible within migrating cells. Scale bar = 100 µm. (b) Similarly, spheroids treated with the lower concentrations of the plant-based compounds were visualized, but there were no obvious differences in cell morphology as a result of drug treatment. Red = actin, green = vinculin and blue = DAPI. n ≥ 2 for each drug treatment. Scale bar = 100 µm.

Figure 10

The potential effects of Curcumin on the PI3K-PKB-mTOR pathway. Receptor tyrosine kinases (RTKs) activate Ras and PI3K following the conversion of PIP2 into PIP3. PKB is then activated via phosphorylation, where it inhibits tuberous sclerosis protein (TSC), a GTPase responsible for converting Rheb-GTP into Rheb-GDP. Rheb (Ras homolog enriched in brain) controls mTORC1 expression, exerting effects on growth and proliferation. Curcumin, the active compound of Turmeric, has been shown to up-regulate miR-206, which reduces CREB protein expression, preventing PTEN down-regulation and therefore inhibiting mTORC1 activity by preventing PKB phosphorylation. The lack of mTORC1 activity induces changes in cytoskeletal dynamics, due to direct effects on migration. Created using Smart Servier Medical Art (https://smart.servier.com/ (accessed on 3 May 2024)).

Figure 11

Indirubin exerts anti-migratory effects via GSK-3 inhibition. Arf6 activation induced by GSK-3 promotes actin localization, whilst Prune increases FAK expression. Maintenance of FA turnover is controlled by GSK-3-mediated FAK inhibition. PTEN inhibition also depends on GSK-3 expression, promoting activation of the PI3K-PKB-mTORC1 pathway, therefore leading to growth, proliferation and migration. Actin localization and FA dynamics are altered via GSK-3 inhibition. Created using Smart Servier Medical Art (https://smart.servier.com/ (accessed on 3 May 2024)).

Figure 12

Magnolia bark reduces cell migration through the action of both APIs, Magnolol and Honokiol. FAK phosphorylation is inhibited by Magnolol, down-regulating N-cadherins, β-cadherins and integrins. N-cadherin and β-cadherin complexation is increased by Magnolol, resulting in reduced migration. AMPK and PI3K are inhibited by Honokiol, consequently decreasing mTORC1 activity and migration by altering actin localization in the cytoskeleton. Created using Smart Servier Medical Art (https://smart.servier.com/ (accessed on 3 May 2024)); Magnolia bark image created by siewlingc (https://www.freeimg.net/photo/1000145/traditionalchinesemedicine-chineseherb-bark (accessed on 3 May 2024)).