α‑Phellandrene enhances the apoptosis of HT‑29 cells induced by 5‑fluorouracil by modulating the mitochondria‑dependent pathway

Abstract

α‑Phellandrene (α‑PA), a natural constituent of herbs, inhibits cancer cell viability and proliferation. 5‑Fluorouracil (5‑FU) is a frequently utilized chemotherapeutic medicine for the treatment of colon cancer, which works by triggering cancer cell apoptosis. The present study examined how the combination of α‑PA and 5‑FU affects the suppression of human colon cancer cells by promoting apoptosis. The impact of this treatment on cell viability, apoptosis, and the expression levels of Bcl‑2 family members, caspase family members and mitochondria‑related molecules in HT‑29 cells was assessed by the MTT assay, immunocytochemistry, western blotting and quantitative PCR. The combination of 5‑FU and α‑PA had a synergistic inhibitory effect on cell viability, as determined by assessing the combination index value. Bax protein expression levels were higher in the 50, 100 or 250 µM α‑PA combined with 5‑FU groups compared with those in the 5‑FU alone group (P<0.05). By contrast, Bcl‑2 protein expression levels and mitochondrial membrane potential (MMP, ΔΨm) were lower in the 100 or 250 µM α‑PA combined with 5‑FU groups than those in the 5‑FU alone group (P<0.05). In addition, hexokinase‑2 (HK‑2) protein expression levels were lower in the 50, 100 or 250 µM α‑PA combined with 5‑FU groups than those in the 5‑FU alone group (P<0.05). Compared with 5‑FU alone, after HT‑29 cells were treated with 50, 100 or 250 µM α‑PA combined with 5‑FU, the mRNA expression levels of extrinsic‑induced apoptotic molecules, including caspase‑8 and Bid, were higher (P<0.05). Treatment with 50, 100 or 250 µM α‑PA combined with 5‑FU also increased the mRNA expression levels of cytochrome c, caspase‑9 and caspase‑3, regulating intrinsic apoptosis (P<0.05). These results showed that α‑PA and 5‑FU had a synergistic effect on reducing the viability of human colon cancer HT‑29 cells by inducing extrinsic and intrinsic apoptosis pathways. The mechanism by which apoptosis is induced may involve the intrinsic apoptosis pathway that activates the mitochondria‑dependent pathway, including regulating the expression levels of Bcl‑2 family members, including Bax, Bcl‑2 and Bid, regulating MMP and HK‑2 expression levels, and increasing the expression of caspase cascade molecules, including caspase‑9 and caspase‑3. In addition, it may involve the extrinsic apoptosis pathway that activates caspase‑8 and caspase‑3 leading to apoptosis.

Keywords: 5‑fluorouracil; Bcl‑2 family; HT‑29 cells; apoptosis; caspase family; mitochondria; α‑phellandrene.

Figure 1.

Effect of α-PA combined with 5-FU treatment on HT-29 cell viability and apoptosis. HT-29 cells (1.0×10⁵ cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Viability of HT-29 cells. (B) Morphological examination of HT-29 cells (×400 magnification). (C) Immunocytochemical staining with Annexin V (green) and PI (red) (×400 magnification). Quantitative analysis of the relative intensities of (D) Annexin V and (E) PI staining after treatment. Untreated cells and cells treated with 5 µM 5-FU or 250 mM α-PA for 72 h served as control groups. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; ^#P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

Figure 2.

Effect of α-PA combined with 5-FU treatment on p53, Bax and Bcl-2 protein expression, and MMP levels in HT-29 cells. HT-29 cells (1.0×10⁵ cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Western blotting was performed to determine the expression levels of (A) p53, (B) Bax and (C) Bcl-2 in HT-29 cells after treatment. (D) Quantitative analysis of (E) immunocytochemical staining with DiOC6 (green) and Hoechst 33342 (blue) to analyze the relative MMP levels (×400 magnification). Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; ^#P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; MMP, mitochondrial membrane potential.

Figure 3.

Effect of α-PA combined with 5-FU treatment on VDAC-1 and HK-2 protein expression in HT-29 cells. HT-29 cells (1.0×10⁵ cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. (A) Immunocytochemical staining was performed to determine the expression levels of HK-2 (green) and VDAC-1 (red) (Hoechst 33342 (blue) (×400 magnification). Quantitative analysis of the relative expression levels of (B) HK-2 and (C) VDAC-1 after treatment. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; ^#P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene; HK-2, hexokinase-2; VDAC, voltage-dependent anion channel.

Figure 4.

Effect of α-PA combined with 5-FU treatment on cytochrome c and caspase mRNA expression in HT-29 cells. HT-29 cells (1.0×10⁵ cells/30-mm plate) were treated with 50, 100 or 250 µg/mM α-PA combined with 5-FU for 72 h. Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA expression levels of (A) caspase-8, (B) Bid, (C) cytochrome c, (D) caspase-9 and (E) caspase-3. T. Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. Control; ^#P<0.05 vs. 5-FU. 5-FU, 5-fluorouracil; α-PA, α-phellandrene. 5-FU, 5-fluorouracil; α-PA, α-phellandrene.

Figure 5.

Possible mechanisms by which α-phellandrene combined with 5-FU treatment induces apoptosis in human colon cancer HT-29 cells by regulating the mitochondria-dependent pathway. 5-FU, 5-fluorouracil; HK-2, hexokinase-2; MMP, mitochondrial membrane potential; VDAC, voltage-dependent anion channel.