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

alpha-Phellandrene (alpha-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 alpha-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 alpha-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 mu M alpha-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, Delta psi m) were lower in the 100 or 250 mu M alpha-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 mu M alpha-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 mu M alpha-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 mu M alpha-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 alpha-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.