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. 2021 Oct;22(4):726.
doi: 10.3892/ol.2021.12987. Epub 2021 Aug 10.

Osthole induces apoptosis of the HT-29 cells via endoplasmic reticulum stress and autophagy

Xing-Hua Zhou  1 , Jian Kang  1 , Zhen-Dong Zhong  2 , Yue Cheng  1
Affiliations

Osthole induces apoptosis of the HT-29 cells via endoplasmic reticulum stress and autophagy

Xing-Hua Zhou et al. Oncol Lett. 2021 Oct.

Abstract

Endoplasmic reticulum stress (ERS) and autophagy are important pathways, which induce apoptosis of tumor cells. Osthole has been demonstrated to exert anticancer effects via the induction of apoptosis in several human colon cancer lines, but the mechanism underlying its involvement in the induction of ERS and autophagy in the human HT-29 colorectal cancer cell line remains unknown. The present study aimed to identify the possible signaling pathways involved in osthole-induced apoptosis of HT29 cells. Methodologically, colony formation and Cell Counting Kit-8 assays were used to assess cell proliferation and viability, respectively, while flow cytometry was performed to investigate apoptosis. Signaling pathways, including apoptosis, autophagy and ERS, were also investigated in the HT-29 cell line using western blot analysis. The results demonstrated that osthole inhibited cellular proliferation and viability in a dose-dependent manner. In addition, osthole induced the expression level of proteins associated with mitochondria-mediated cell apoptosis, autophagy and ERS. The association between autophagy and ERS in osthole-induced apoptosis in the HT-29 cell line was further clarified. Inhibiting cell autophagy with the inhibitor, 3-methyladenine, suppressed osthole-induced cell apoptosis and enhanced osthole-induced ERS. By contrast, alleviating ERS with the inhibitor, 4-phenylbutyric acid attenuated osthole-induced cell apoptosis and autophagy. In conclusion, osthole could significantly suppress the proliferation and viability of the HT-29 colorectal cancer cell line and induce cell apoptosis via autophagy and ERS. Furthermore, ERS may play a more important role in osthole-induced cell apoptosis.

Keywords: apoptosis; autophagy; colon cancer; endoplasmic reticulum stress; osthole.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Osthole inhibits proliferation and viability, and induces apoptosis of the HT-29 cell line. The proliferation and viability of the HT-29 cells treated with osthole (0, 100, 50 and 25 µM) for 24 h was detected using (A and B) colony formation and (C) Cell Counting Kit-8 assays, respectively. (D and E) HT-29 cell apoptosis was detected using flow cytometry. (F) The expression levels of the apoptosis-associated proteins Bax, Bcl-2 and cleaved caspase-3 were determined using western blot analysis, with β-actin as a loading control. *P<0.05, **P<0.01, ***P<0.001 vs. control group (0 µM).
Figure 2.
Figure 2.
Effects of osthole on the protein expression levels of autophagy- and endoplasmic reticulum stress-related markers in the HT-29 cell line. The protein expression levels of (A) p62 and LC3-II/LC3-I, (B) GRP78, p-PERK/PERK, p-elF2α/elF2α and CHOP were determined using western blot analysis. β-actin was used as a loading control. *P<0.05, **P<0.01, ***P<0.001 vs. control group (0 µM). LC3, microtubule-associated protein light chain 3; GRP78, 78 kDa glucose-regulated protein; p-, phosphorylated; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; elF2α, eukaryotic initiation factor 2α.
Figure 3.
Figure 3.
Inhibition of autophagy by 3MA modulates osthole-induced apoptosis and endoplasmic reticulum stress in the HT-29 cell line. (A and B) Cell proliferation was measured using a colony formation assay. (C) Cell viability was measured using a Cell Counting Kit-8 assay. (D and E) Apoptosis analysis was performed using flow cytometry. (F) The protein expression levels of GRP78, p-PERK/PERK, p-elF2α/elF2α and CHOP were determined using western blot analysis, and β-actin was used as a loading control. *P<0.05, **P<0.01, ***P<0.001 vs. control group. #P<0.05, ###P<0.001 vs. 3MA group. &&P<0.01, &&&P<0.001 vs. osthole group. GRP78, 78 kDa glucose-regulated protein; p-, phosphorylated; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; elF2α, eukaryotic initiation factor 2α; 3MA, 3-methyladenine.
Figure 4.
Figure 4.
Suppression of endoplasmic reticulum stress with 4-PBA attenuates osthole-induced apoptosis and autophagy in the HT-29 cell line. (A and B) Cell proliferation was measured using a colony formation assay. (C) Cell viability was measured using a Cell Counting Kit-8 assay. (D and E) Apoptosis analysis was performed using flow cytometry. (F) The protein expression levels of p62 and LC3 were determined using western blot analysis, and β-actin was used as a loading control. **P<0.01, ***P<0.001 vs. control group. #P<0.05, ##P<0.01, ###P<0.001 vs. 4-PBA group. &&P<0.01, &&&P<0.001 vs. osthole group. LC3, microtubule-associated protein light chain 3; 4-PBA, 4-phenylbutyric acid.
Figure 5.
Figure 5.
Combined effects of osthole, 3MA and 4-PBA on apoptosis in the HT-29 cell line. ***P<0.001 vs. control group. &&P<0.01, &&&P<0.001 vs. 3MA + osthole group. #P<0.05, ##P<0.01, ###P<0.001 vs. 4-PBA + osthole group. 3MA, 3-methyladenine; 4-PBA, 4-phenylbutyric acid.
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Grants and funding

The present study was supported by the National Natural Science Foundation of China (grant no. 81873073) and Science and Technology Development Fund of Hospital of Chengdu University of Traditional Chinese Medicine (grant no. 18MZ27).