Ethanolic extract of Thevetia peruviana flowers enhances TNF-α and TRAIL-induced apoptosis of human cervical cancer cells via intrinsic and extrinsic pathways

Abstract

Tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) are promising candidates for cancer treatment due to their ability to induce apoptosis through death receptor stimulation. However, their usage may be limited due to the resistance of cancer cells to TNF-α- and TRAIL-induced apoptosis. Currently, there is interest in screening for natural products that can sensitize cancer cells to TNF-α- and TRAIL-induced apoptosis for their use in combination with TNF-α or TRAIL. It was previously reported that the bark extract of Thevetia peruviana showed a reversal effect on TRAIL-resistance in human gastric adenocarcinoma cell lines. In the present study, the effects of the ethanolic extract of T. peruviana flowers on TNF-α- and TRAIL-induced apoptosis of human cervical cancer HeLa cells were investigated in vitro by determining cell viability and apoptosis using a WST-1 cell proliferation assay and immunoblot analysis, respectively. The ethanolic extract of T. peruviana flowers promoted TNF-α and TRAIL-mediated cell death through the activation of the caspase cascade, poly(ADP-ribose) polymerase and BH3-interacting domain death agonist cleavage. Combined treatment using the extract plus TNF-α resulted in downregulation of anti-apoptotic protein, including myeloid cell leukemia sequence-1, B-cell lymphoma-extra large (Bcl-XL), X-linked inhibitor of apoptosis protein and survivin, while the combined treatment with TRAIL downregulated Bcl-XL. Thus, the ethanolic extract of T. peruviana flowers has potential in sensitizing the TNF-α- and TRAIL-induced apoptosis of HeLa cells via the intrinsic and extrinsic pathways.

Keywords: B-cell lymphoma-extra large; HeLa cells; anti-apoptotic protein; cardiac glycosides; cervical cancer; death receptor; plant extract.

Figure 1.

Effect of the ethanolic extract of Thevetia peruviana flowers on TNF-α-induced cell death. HeLa cells were pretreated with the extract for 30 min, and further incubated with and without TNF-α (20 ng/ml) for 24 h, after which cell viability was determined using the WST-1 Cell Counting kit. Data are expressed as the mean ± standard deviation from three independent experiments. *P<0.01. TNF-α, tumor-necrosis factor-α.

Figure 2.

Enhancement of TNF-α-induced apoptosis by the ethanolic extract of Thevetia peruviana flowers. HeLa cells were pretreated with the extract (100 µg/ml) for 30 min, and further incubated in the presence or absence of TNF-α (20 ng/ml) for 12 h. (A) Images were captured under a microscope at an original magnification of ×40. (B) Whole cell lysates were analyzed by immunoblot for apoptotic proteins, caspase-8 and −3, and PARP, using actin as the loading control. TNF-α, tumor-necrosis factor-α; PARP, poly(ADP-ribose) polymerase.

Figure 3.

Inhibitory effect of the ethanolic extract of Thevetia peruviana flowers on apoptotic and anti-apoptotic proteins. HeLa cells were pretreated with the extract (100 µg/ml) for 30 min, and further incubated in the presence or absence of TNF-α (20 ng/ml) for 12 h. (A) Whole cell lysates were analyzed by immunoblot for apoptotic proteins, Bid, caspase-9 and-3, and PARP. (B) Whole cell lysates were analyzed by immunoblotting for anti-apoptotic proteins Mcl-1, Bcl-XL, XIAP and survivin using actin as a loading control. TNF-α, tumor-necrosis factor-α; PARP, poly(ADP-ribose) polymerase; Mcl-1, myeloid cell leukemia sequence-1; Bcl-XL, B-cell lymphoma-extra large; XIAP, X-linked inhibitor of apoptosis protein.

Figure 4.

Time-dependent decrease in survivin level by the treatment of the ethanolic extract of Thevetia peruviana flowers. HeLa cells were pretreated with the extract (100 µg/ml) for 30 min, and further incubated with and without TNF-α (20 ng/ml) for 3,6 and 12 h. Whole cell lysates were analyzed by immunoblotting for the anti-apoptotic protein survivin using actin as a loading control. TNF-α, tumor-necrosis factor-α; PARP, poly(ADP-ribose) polymerase.

Figure 5.

Effect of the ethanolic extract of Thevetia peruviana flowers on TRAIL-induced cell death in HeLa cells. Cells were pretreated with the extract for 30 min, and further incubated with and without TRAIL (200 ng/ml) for 24 h, after which cell viability was determined using the WST-1 Cell Counting kit. Data are expressed as the mean ± standard deviation from three independent experiments. *P<0.01. TNF-α, tumor-necrosis factor-α; TRAIL, TNF-related apoptosis-inducing ligand.

Figure 6.

Enhancement of TRAIL-induced apoptosis by the ethanolic extract of Thevetia peruviana flowers. (A) HeLa cells were pretreated with the extract (10 µg/ml) for 30 min, and further incubated with and without TRAIL (200 ng/ml) for 3,6 and 12 h. Images were captured at an original magnification of ×20. (B) Whole cell lysates at 12 h were analyzed by immunoblotting for the apoptotic proteins Bid, caspase-3, and PARP, using actin as a loading control. TNF-α, tumor-necrosis factor-α; PARP, poly(ADP-ribose) polymerase; TRAIL, TNF-related apoptosis-inducing ligand.

Figure 7.

Correlation of the dose-dependent decrease in Bcl-XL level by the ethanolic extract of Thevetia peruviana flowers. HeLa cells were pretreated with the extract for 30 min, and further incubated with and without TRAIL (200 ng/ml) for 3 h. Whole cell lysates were analyzed by immunoblotting for the anti-apoptotic proteins Bcl-XL and XIAP, using actin as a loading control. PARP, poly(ADP-ribose) polymerase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Bcl-XL, B-cell lymphoma-extra large; XIAP, X-linked inhibitor of apoptosis protein