Role of phloretin as a sensitizer to TRAIL-induced apoptosis in colon cancer

Abstract

Phloretin is one of the apple polyphenols with anticancer activities. Since tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves important roles in inducing apoptosis, the present study examined the effect of phloretin on TRAIL-induced apoptosis in colon cancer cells. Treatment with both phloretin and TRAIL markedly suppressed the survival of cancer cells from several colon cancer cell lines compared with that of cells treated with either TRAIL or phloretin. Additionally, decreased numbers of colonies were observed following addition of phloretin and TRAIL. Furthermore, TRAIL- and phloretin-treated HT-29-Luc cells exhibited decreased luciferase activity. Increased apoptosis was observed in phloretin- and TRAIL-treated HT-29-Luc colon cancer cells, accompanying elevated levels of cleaved poly(ADP-ribose) polymerase, and caspase-3, -8 and -9. The expression levels of MCL1 apoptosis regulator BCL2 family member (Mcl-1) were decreased following addition of phloretin in colon cancer cells. In addition, overexpression of Mcl-1 in phloretin- and TRAIL-treated HT-29-Luc cells resulted in increased cell survival. Treatment of HT-29-Luc cells with a combination of cycloheximide (CHX) and phloretin led to a more prominent decrease in Mcl-1 expression compared with that in cells treated with CHX alone, while Mcl-1 expression was recovered by treatment with MG132. Binding of ubiquitin with Mcl-1 was verified using immunoprecipitation. Intraperitoneal injection of both TRAIL and phloretin into tumor xenografts was associated with a decreased tumor volume compared with that following injection with either TRAIL or phloretin. Overall, the present results suggest a synergistic effect of phloretin on TRAIL-induced apoptosis in colon cancer cells.

Keywords: BCL2 family member; MCL1 apoptosis regulator; apoptosis; colon cancer; phloretin; tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1.

Antitumor effects of phloretin, TRAIL, and a combination of phloretin and TRAIL on colon cancer cells. (A) Structure of phloretin. (B) Effects of phloretin on the survival of colon cancer cells (DLD-1, HT-29-Luc, HCT116 and SNU283) and normal colon cells (CCD18-Co). (C) TRAIL inhibited the survival of colon cancer cells. (D) Survival of normal colon cells (CCD18-Co) was not affected by phloretin or TRAIL. (E) Viability was decreased in colon cancer cells treated with a combination of phloretin and TRAIL compared with that in cells treated with either phloretin or TRAIL alone. Data are presented as the mean ± SD (n=3). *P<0.05; **P<0.01; ***P<0.0001. n.s., non-significant; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 2.

(A) Morphology of colonies, (B) colony formation assay and (C) bioluminescent assay of HT-29-Luc cells treated with phloretin, TRAIL, or a combination of phloretin and TRAIL. (A) Colony formation was most prominently inhibited in HT-29-Luc cells treated with both TRAIL and phloretin. (B) The fewest colonies were formed by HT-29-Luc cells treated with both phloretin and TRAIL (***P<0.0001). (C) Luciferase activity was decreased in HT-29-Luc cells treated with a combination of phloretin and TRAIL compared with either treatment alone (***P<0.0001). Data are presented as the mean ± SD (n=3). Scale bar, 100 µm. Con, control; n.s., non-significant; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 3.

Mechanism for the synergistic effect of phloretin on TRAIL-induced apoptosis. (A) Annexin V assay showing the induction of apoptosis was greater in phloretin- and TRAIL-treated HT-29-Luc cells compared with either treatment alone (***P<0.0001). (B) Increased levels of c-PARP, and c-caspase-3, −8 and −9 in HT-29-Luc cells treated with a combination of phloretin and TRAIL. (C) Decreased expression levels of Mcl-1 in phloretin-treated HT-29-Luc cells. (D) Decreased expression levels of Mcl-1 under treatment with phloretin in DLD-1 and SNU283 colon cancer cells. (E) Time-dependent decreased expression levels of Mcl-1 in HT-29-Luc cells after treatment with phloretin. (F) Immunofluorescence staining showing decreased expression levels of Mcl-1 in phloretin-treated HT-29-Luc cells. (G) Overexpression of Mcl-1 reversed decreased survival in phloretin- and TRAIL-treated HT-29-Luc cells (***P<0.0001). (H) Increased c-PARP expression after treatment with both phloretin and TRAIL was reversed by overexpression of Mcl-1 in HT-29-Luc cells. Data are presented as the mean ± SD (n=3). Bid, BH3 interacting domain death agonist; Bim, Bcl-2-like protein 11; c-, cleaved; Con, control; DR, death receptor; Mcl-1, MCL1 apoptosis regulator BCL2 family member; n.s., non-significant; PARP, poly (ADP-ribose) polymerase; Puma, p53-upregulated modulator of apoptosis; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; XIAP, X-linked inhibitor of apoptosis protein.

Figure 4.

Mechanism by which phloretin suppresses Mcl-1 expression in colon cancer cells. (A) Mcl-1 mRNA expression levels did not change in phloretin-treated HT-29-Luc cells compared with those in control cells. (B) Combined treatment with CHX and phloretin suppressed Mcl-1 expression more effectively than treatment with CHX alone in HT-29-Luc cells (*P<0.05). (C) Suppressed Mcl-1 expression in phloretin-treated HT-29-Luc cells was reversed by MG132. (D) Ubiquitin-mediated degradation of Mcl-1 in phloretin-treated HT-29-Luc cells was demonstrated by immunoprecipitation. Data are presented as the mean ± SD (n=3). CHX, cycloheximide; IP, immunoprecipitation; Mcl-1, MCL1 apoptosis regulator BCL2 family member; Ub, ubiquitin; WB, western blot.

Figure 5.

Synergistic effect of phloretin on TRAIL-induced apoptosis in in vivo models. (A) Suppression of tumor growth was the most prominent in xenografts treated with a combination of phloretin and TRAIL. Gains in (B) tumor volume and (C) weight were most markedly inhibited in xenografts treated with a combination of phloretin and TRAIL. (D) Apoptosis was the most prominent in tumors from mice treated with both phloretin and TRAIL. ***P<0.0001. Scale bar, 20 mm. Data are presented as the mean ± SD (n=5 in each treatment group). Con, control; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; n.s., non-significant.

Figure 6.

Synergistic effect of phloretin on TRAIL-mediated apoptosis signaling in colon cancer cells. Combined treatment with phloretin and TRAIL acts on colon cancer cells by activating both the DR pathway and the stress pathway. The stress pathway is modulated by phloretin through suppression of Mcl-1 expression via proteasomal degradation, leading to increased expression of cleaved caspase-3. Cyt c, cytochrome c; DR, death receptor; Mcl-1, MCL1 apoptosis regulator BCL2 family member; MMP, mitochondrial membrane potential; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Ub, ubiquitin.