The anticancer potential of tetrahydrocurcumin-phytosomes against oral carcinoma progression

Abstract

Background: Herbal medicine combined with nanotechnology offers an alternative to the increasing burden of surgery and/or chemotherapy, the main therapeutics of oral carcinoma. Phytosomes are nano-vesicular systems formed by the interaction between phospholipids and phyto-active components via hydrogen bonding, exhibiting superior efficacy over pure phytocomponents in drug delivery.

Methods: Tetrahydrocurcumin (THC)-phytosomes were prepared by thin film hydration method. After characterization, in vitro cytotoxicity, antiproliferative capacity, antioxidant potential and full apoptotic workup were paneled on oral squamous cell carcinoma (SCC4) in comparison with native THC-solution and cisplatin (3.58 µg/mL intravenous injection), as positive controls. In addition, we tested the three medications on normal oral keratinocytes and gingival fibroblasts to attest to their tissue-selectivity.

Results: Successful preparation of THC-phytosomes using 1:1 molar ratio of THC to phospholipid exhibited significantly increased aqueous solubility, good colloidal properties, and complete drug release after one hour. On SCC4 cells, THC-phytosomes, at their dose-/time-dependency at ~ 60.06 µg/mL escalated cell percentages in the S-phase with 32.5 ± 6.22% increase, as well as a startling 29.69 ± 2.3% increase in apoptotic population. Depletion of the cell colonies survival to 0.29 ± 0.1% together with restraining the migratory rate by -6.4 ± 6.8% validated THC-phytosomes' antiproliferative capacity. Comparatively, the corresponding results of THC-solution and cisplatin revealed 12.9 ± 0.9% and 25.8 ± 1.1% for apoptosis and 0.9 ± 0.1% and 0.7 ± 0.08% for colony survival fraction, respectively. Furthermore, the nanoformulation exhibited the strongest immuno-positivity to caspase-3, which positively correlated with intense mitochondrial fluorescence by Mitotracker Red, suggesting its implication in the mitochondrial pathway of apoptosis, a finding further explained by the enormously high Bax and caspase-8 expression by RT-qPCR. Finally, the THC groups showed the lowest oxidative stress index, marking their highest free radical-scavenging potential among the test groups.

Conclusions: THC-phytosomes are depicted to be an efficient nanoformulation that enhanced the anticancer efficacy over the free drug counterpart and the conventional chemotherapeutic. Additionally, being selective to cancer cells and less cytotoxic to normal cells makes THC-phytosomes a potential candidate for tissue-targeted therapy.

Keywords: Bax; Caspase; Mitotracker Red; S-phase; Squamous cell carcinoma.

Fig. 1

Characterization parameters of THC-phytosomes. (a) Release profiles of THC-phytosomes (1:1, 1:2, and 1:3) and THC suspension in 50% v/v ethanol at 37° C. (b) TEM of THC-phytosomes (P1). (c) FTIR spectrum of THC-phytosomes(P1) in comparison with THC and SPC. (d) DSC thermogram of THC-phytosomes (P1) in comparison with THC and SPC

Fig. 2

The dose-response curve of THC-phytosomes cytotoxicity on SCC4 cell line and normal oral epithelial cells. (a) The cytotoxic effects on cancerous SCC4 cells treated with the nanoformulation show a significant decrease in cell viability in a dose and time-dependent manner similar to the DMSO-dissolved THC-solution and the conventional chemotherapeutic agent. (b) The effect of the three drugs on the normal keratinocytes also shows an equivalent dose and time-dependent response, with THC-phytosomes having the highest calculated selectivity indices to oral cancer cells following 48 h of treatment. The bar charts are the mean ± SD of viability % from three independent experiments, each of triplicates in 96-well plates

Fig. 3

Wound closure rate evaluation of the different proposed treatments. (a) Photomicrographs of the scratch lines exposed to THC-phytosomes, THC-solution, and cisplatin at different timelines (24 h, 48 h, and 72 h) taking untreated oral cancerous cells as the negative control. The scratch line is the widest in the phytosomes-treated group at all time points, showing a significantly decreased migration rate percentage compared to the other proposed treatments. (b) A scatter plot with a bar graph representation of the migration percentage of SCC4 cells, where two-way ANOVA followed by Tukey’s multiple comparisons test reveals * of p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001, while ns (non-significant) = p > 0.05. The data is mean ± SD of triplicates (each is a well of a 6-well plate seeded with 25 × 10⁴ SCC4 cells)

Fig. 4

Clonogenic survival assay. (a) Representative photographs of cellular morphology of colonies formed after SCC4 cells treatment with phytosomes, solution, and cisplatin. (b) The survival fraction plot of cancer cells after administering the IC50 of the three medications, taking untreated cells as the control. A one-way ANOVA test followed by Tukey’s multiple comparisons test exhibits * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** p < 0.0001, and ns codes for p > 0.05. The data is mean ± SD of triplicates (each replica is one well of a 6-well plate seeded with 25 × 10⁴ SCC4 cells)

Fig. 5

Cell cycle analysis by flow cytometry. (a) Representative RNase PI-marked histograms display the THC-phytosomes-induced arrest in the S-phase at 24 and 48 h. Cisplatin causes an arrest at the S-phase at 24 h, then shifts to an M-phase cell cycle arrest at 48 h. (b) A bar plot represents the cell population percentages in different cell cycle phases. A two-way ANOVA followed by multiple comparisons test shows * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** p < 0.0001, while ns codes for p > 0.05. Data expressed as mean ± SD of triplicates (each replicate is one well of a 6-well plate seeded with 25 × 10⁴ cancer cells)

Fig. 6

The apoptotic panel of the THC-phytosomes. (a) Representative annexin V/PI flow cytometer dot plots reveal that THC-phytosomes successfully enhance apoptosis in SCC4 cells at 24 and 48 h. (b) An interleaved scatter with bars quantifying the apoptotic cell population percentages in different proposed treatments at both time points. Data expressed as mean ± SD of triplicates (each is one well of 6-well plate seeded with 25 × 10⁴ cancerous cells), where two-way ANOVA followed by multiple comparisons test displays * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** p < 0.0001, while ns codes for p > 0.05. (c) Hoechst nuclear-staining photomicrographs (scale bar = 25 μm) show nuclear condensation (red arrow), fragmentation (green arrow), and blebbing of the nuclear membrane (orange arrow). The apoptotic changes appear most evident in the phytosomes group. Cisplatin shows chromatin fading (yellow arrow), indicating necrosis. (d) H&E staining cytological images (scale bar = 20 μm) show the proliferating control cancer cells as well as multinucleated tumor giant cells, depicting the aggressiveness of the cancer behavior. Phytosomes-treated cells are undergoing apoptosis and show extreme shrinkage and apoptotic bodies (inset). With less abundance, the solution-treated group shows similar effects, while cisplatin shows sheets of ghosted cells undergoing cloudy degeneration and nuclear fading

Fig. 7

THC-phytosomes induce mitochondria-dependent apoptosis. (a) Mitotracker Red-staining micrographs (scale bar = 25 μm) show the most intense fluorescence in the phytosomes group, indicating mitochondrial swelling and fission, followed by the solution group. (b) A graphical representation of the fluorescence intensity emitted by the cells after Mitotracker Red staining. (c) IHC stained photomicrographs (scale bar = 20 μm) show the highest positivity of caspase-3 antibody in the phytosomes group with cytoplasmic homing of the stain and nuclei showing nuclear fragmentation, characteristic of apoptosis. (d) A quantitative graphical representation of the optical density displayed by the up taken stain in the different groups. (e) Heat map illustration of correlation matrix shows varying degrees of positive correlation between log₁₀ fluorescence intensity of Mitotracker Red and the optical density of caspase-3 protein expression by IHC among all treated groups. The green circles signal out the significant positive correlation present in both THC cohorts. A one-way ANOVA test followed by Tukey’s test shows * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** p < 0.0001, and ns codes for p > 0.05. The data is the mean ± SD of five random microscopical fields from triplicate wells of 6-well plate seeded with 25 × 10⁴ SCC4 cells. Correlation coefficient r was measured using Pearson’s method

Fig. 8

RTqPCR analysis and oxidative stress biomarkers assay. (a) Bar plots for RTqPCR analysis show the unparallel effect of THC-phytosomes compared with other groups in expression of caspase-8 and Bax, apoptotic genes. (b) Another bar graph shows both THC groups culminating the lowest MDA/TAC oxidative stress index of the different test groups compared with the control, highlighting their superior radical-scavenging potential. A one-way ANOVA test followed by Tukey’s test shows * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** p < 0.0001, and ns codes for p > 0.05. The data is the mean ± SD of triplicate wells of 6-well plate seeded with 25 × 10⁴ SCC4 cells