The Leaf Extracts of Toona sinensis and Fermented Culture Broths of Antrodia camphorata Synergistically Cause Apoptotic Cell Death in Promyelocytic Leukemia Cells

Abstract

Toona sinensis is a common edible vegetable that is used in certain Chinese dishes and has importance in folk medicine. The leaf extracts of T sinensis possess and exhibit anticancer efficacy against various cancer cell types. In Taiwanese folklore, Antrodia camphorata, also known as "Niu-Cheng-Zi," is used in traditional medicine to treat various illnesses. Its fruit and mycelium possess various potent antiproliferative properties. Two studies from our group have reported that T sinensis or A camphorata has the ability to cause apoptosis in various cancer cells. Conversely, underlying molecular mechanisms and any beneficial effects remain unknown. This study shows anticancer efficacy for both T sinensis and A camphorata co-treatments that target HL-60 cells. The combination index values indicate that 40 µg/mL of T sinensis and 25 µg/mL of A camphorata as a combined treatment shows a synergetic effect, which reduces HL-60 cell proliferation. Alternately, this treatment exhibited no cytotoxic effects for human umbilical vein endothelial cells. Western blot data showed that T sinensis and A camphorata as a combined treatment result in augmented expression of apoptosis, cytochrome c release, Bcl-2 inhibition, expression of Bax, Fas, and FasL, as well as the cleavage of Bid in HL-60 cells. Moreover, this combined treatment overshadowed monotherapy in its ability to inhibit uPAR, MMP-9, MMP-2, COX-2 expression, and PGE₂ secretions. Our study strongly implies that this combined treatment offers more beneficial effects to suppress and treat leukemia due to apoptosis-mediated cell inhibition. Further in vivo studies related to the combined treatment could establish its future potential.

Keywords: Antrodia camphorate; COX-2; HL-60 cells; MMP; Toona sinensis; apoptosis; leukemia.

Figure 1.

Effect of Toona sinensis and Antrodia camphorata co-treatment on HL-60 cell viability. (A, B) HL-60 cells were treated with 25 µg/mL T sinensis or 40 µg/mL A camphorata or in combination for 24 hours. Morphological changes were observed under the phase-contrast microscope (200× magnification). Using the trypan blue exclusion assay, HL-60 cell viability was determined. (C) HUVECs (human umbilical vein endothelial cells) were treated with 25 µg/mL T sinensis or 40 µg/mL A camphorata or combination of both for 24 hours and then the cell viability was measured. Values were expressed as mean ± standard deviation (n = 3). Statistical significance was assigned as ***P < .001 compared with untreated control cells and ^###P < .001 compared with T sinensis or A camphorata alone treated cells.

Figure 2.

Effect of Toona sinensis and/or Antrodia camphorata treatment on sub-G₁ cell cycle of HL-60 cells. (A) HL-60 cells were treated with 25 µg/mL T sinensis or 40 µg/mL A camphorata or in combination for 24 hours and then analyzed by flow cytometry. (B) Percentage of sub-G₁ cell distribution after T sinensis, A camphorata, treatments were presented. All values are expressed as mean ± standard deviation (n = 3). Statistical significance was assigned as ***P < .001 compared with untreated control cells and ^###P < .001 compared with T sinensis or A camphorata alone treated cells.

Figure 3.

Toona sinensis and/or Antrodia camphorata treatment induced the release of cytochrome c in HL-60 cells. HL-60 cells were treated with 25 µg/mL T sinensis or 40 µg/mL A camphorata or in combination for 24 hours. The expression of cytosolic cytochrome c, caspase-3, and PARP proteins were measured by Western blot method using β-actin as an internal control. Densitometric analysis was performed using the AlphaEase (Genetic Technology Inc) with the value of control assigned as 1.

Figure 4.

Effect of Toona sinensis and/or Antrodia camphorata treatment on Bax/Bcl-2 ratio in HL-60 cells. HL-60 cells were treated with 25 µg/mL T sinensis or 40 µg/mL A camphorata or in combination for 24 hours. The expression of Bax, Bcl-2 proteins were measured by Western blot using β-actin as an internal control. Densitometric analysis was performed using the AlphaEase (Genetic Technology Inc) with the value of control assigned to be 1.

Figure 5.

Effect of Toona sinensis and/or Antrodia camphorata treatment on HL-60 cell mitochondria membrane potential. (A, B) HL-60 cells were treated with 25 µg/mL T sinensis and/or 40 µg/mL A camphorata for 24 hours, followed by measurement of mitochondrial membrane potential by flow cytometry. The percentage of mitochondrial membrane potential was indicated by DiOC₆ fluorescence. All the values are expressed in mean ± standard deviation (n = 3). Statistical significance was assigned as ***P < .001 compared with untreated control cells and ^###P < .001 compared with T sinensis or A camphorata alone treated cells.

Figure 6.

Effects of Toona sinensis and/or Antrodia camphorata treatment on the expression of various tumorigenic proteins in HL-60 cells. HL-60 cells were treated with 25 µg/mL T sinensis or 40 µg/mL A camphorata or in combination for 24 hours. Western blot method was used to measure (A) Fas, FasL, and Bid proteins; and (B) MMP-2, MMP-9, and uPAR proteins. All values were expressed as mean ± standard deviation (n = 3). β-actin was used as an internal control. Densitometric analysis was performed using the AlphaEase (Genetic Technology Inc) with the value of control assigned to be 1.

Figure 7.

Toona sinensis and Antrodia camphorata co-treatment has downregulated the expression of COX-2 and PGE₂ production in HL-60 cells. HL-60 cells were treated with T sinensis (25 µg/mL), A camphorata (40 µg/mL), and T sinensis + A camphorata for 24 hours. (A) Western blot method was used to measure the expression of COX-2 protein. AlphaEase (Genetic Technology Inc) was used for the densitometric analysis. Control value was assigned as one. (B) Using the ELISA kit method, PGE₂ concentration in the culture media was also determined. All values were expressed as mean ± standard deviation (n = 3). Statistical significance was assigned as ***P < .001 compared with untreated control cells and ^##P < .05; ^###P < .001 compared with T sinensis or A camphorata alone treated cells.