Dual Modulation of Autophagy and Apoptosis as Anticancer Mechanism of Action of Khaya grandiofoliola in Colon Carcinoma Cells

Abstract

Khaya grandiofoliola (Kh) is a medicinal plant with therapeutic properties. Studies have reported on the general bioactivity and anticancer potentials of the plant, but no investigations have yet investigated its anticancer mechanism of action. This study presents the first examination of the anticancer mechanism of action of the methanolic extract of Kh, alongside phytochemical profiling of its anticancer constituents. We conducted in vitro investigations into the mechanism of action of Kh and performed bioactivity-guided fractionation, with subsequent identification of its anticancer phytochemicals using HPLC and GC-MS, respectively. Kh posed a potent antiproliferative effect against colon carcinoma cells and an antioxidant property at low microgram levels. Furthermore, the treatment of Kh in Caco-2 cells led to the accumulation of p62 puncta, indicating inhibition of autophagic flux degradation. Kh impacts microtubule, induced G1 arrest, and late apoptosis induction in Caco-2 cells. Phytochemicals belonging to sesquiterpene alcohols were found most abundant in the Kh bioactive fractions. The identified phytochemicals are potential inducers of apoptosis, autophagy flux inhibition, and G1-phase arrest. Our findings suggest that the anticancer property of Kh is mediated through the dual modulation of autophagy and apoptosis. Further studies are needed to isolate the active compounds responsible for these effects and further elucidate the underlying molecular mechanisms.

Keywords: Khaya grandiofoliola; anticancer phytochemicals; apoptosis; autophagy; natural products.

Figure 1

Bioactivity potentials of the crude methanolic extract of Kh against Caco-2 cells. (A) represents the %viability curve, showing the IC50 value obtained from a triplicate of MTT tests. The %viability data are presented as mean ± standard deviation. ^a,b,c,d are the means within the same column, distinguished by different superscripts and exhibiting significant differences (p < 0.05). (B) represents the antioxidant potential of Kh via DPPH.

Figure 2

Cell cycle fraction analysis of the effect of Kh against Caco-2 cells. (A) = vehicular control; (B) = treatment at 50 µg/mL and (C) = treatment at 100 µg/mL.

Figure 3

Photomicrographs of Caco-2 cells treated with Kh at 50 and 100 µg/mL for 24 h. (A) The nuclei of treated cells at both concentration levels show nuclear fragmentation compared to the control. The immunostaining with β-tubulin shows a concentration-dependent degradation effect, while a concentration-dependent increase in the p62 puncta was observed compared with the control. All images were taken at 40× magnification. (B) Box plot displaying the concentration-dependent increment in the p62 intensity. The intensity data are presented as mean ± standard deviation. ^a,b,c are the means within the same column, distinguished by different superscripts and exhibiting significant differences (p < 0.05).

Figure 4

Apoptosis induction of crude methanolic extract of Kh against Caco-2 cells. (A) Vehicle control; (B) treated with 50 µg/mL and (C) treated with 100 µg/mL.

Figure 5

Bioactivity-guided fractionation of Kh executed via a gradient HPLC fractionation and MTT assay. (A) HPLC chromatograph; (B) bioactivity potential of all fractions using MTT assay.

Figure 6

GCMS identification of anticancer phytochemicals present in Kh. (A) Chromatograph showing the peaks of the phytochemicals. (B) Summary of the number of phytochemicals grouped according to their respective chemical classes and their percentage abundance. Heatmap generated via R programming software (RStudio 2024.12.1 +563).