α-hederin induces autophagic cell death in colorectal cancer cells through reactive oxygen species dependent AMPK/mTOR signaling pathway activation

Abstract

α-hederin, a monodesmosidic triterpenoid saponin, had previously demonstrated strong anticancer effects. In the current study, the pharmacological mechanism of autophagic cell death induced by α-hederin was investigated in human colorectal cancer cells. First, through cell counting kit-8 and colony formation assays, it was demonstrated that α-hederin could inhibit the proliferation of HCT116 and HCT8 cell. Results of flow cytometry using fluorescein isothiocyanate Annexin V/propidium iodide and Hoechst 33258 staining indicated that α-hederin could induce apoptosis. Western blotting demonstrated that α-hederin could activate mitochondrial apoptosis signal pathway. Then, using light chain 3 lentiviral and electron microscope assay, it was demonstrated that α-hederin could induce autophagy in colorectal cancer cells. In addition, immunohistochemistry results from in vivo experiments also demonstrated that α-hederin could induce autophagy. AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling was demonstrated to be activated by α-hederin, which could be blocked by reactive oxygen species (ROS) inhibitor NAC. Furthermore, NAC could inhibit apoptosis and autophagy induced by α-hederin. Finally, 3-MA (autophagy inhibitor) reduced the inhibition of α-hederin on cell activity, but it had no significant effect on apoptosis. In conclusion, α-hederin triggered apoptosis through ROS-activated mitochondrial signaling pathway and autophagic cell death through ROS dependent AMPK/mTOR signaling pathway activation in colorectal cancer cells.

Total flavones of Rhododendron simsii Planch flower protect rat hippocampal neuron from hypoxia-reoxygenation injury via activation of BKCa channel

“…After H/R experiment, the neurons in each well were incubated with a final concentration of 0.5 mg/ml MTT solution for 4 h. 100 μl of DMSO was then added into each well to dissolve the generated formazan. MTT assay was performed using a microplate reader to measure optical density (OD) of each well at 490 nm; LDH activity and MDA content in cultured supernatant of the neurons were, respectively, detected at 532 and 450 nm by a spectrophotometry according to the procedures provided by the assay kits; NSE activity in the supernatants was measured at 450 nm by ELISA method; The neurons were stained with 15 μg/ml Hoechst 33258 at 37°C in dark for 30 min, and H/R‐induced apoptosis was examined under a fluorescence microscope …”

“…MTT assay was performed using a microplate reader to measure optical density (OD) of each well at 490 nm; LDH activity and MDA content in cultured supernatant of the neurons were, respectively, detected at 532 and 450 nm by a spectrophotometry according to the procedures provided by the assay kits; NSE activity in the supernatants was measured at 450 nm by ELISA method; The neurons were stained with 15 lg/ml Hoechst 33258 at 37°C in dark for 30 min, and H/R-induced apoptosis was examined under a fluorescence microscope. [20] Western blotting analysis Western blotting analysis was performed as previously described. [21] Briefly, the cultured rat hippocampal neurons were washed twice with PBS, lyzed with cell lysate containing protease inhibitor and centrifuged at 3000 g. Protein content in the supernatant was measured by using the BCA protein assay.…”

α‐Hederin induces the apoptosis of gastric cancer cells accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway

“…Meanwhile, the above changes were amplified when pretreated with BSO, while attenuated pretreatment with NAC than α-hederin alone. In addition, researches in colorectal cancer cells indicated that α-hederin could trigger apoptosis through ROS-activated mitochondrial signaling pathway and induce autophagic cell death through ROS dependent adenosine monophosphate-activated protein kinase/mammalian target of rapamycin signaling pathway activation (Sun and Feng et al, 2019).…”

“…Moreover, a growing body of researches showed that α‐hederin exhibited promising antitumor potential, in that it revealed significant cytotoxicity by inhibiting growth and promoting apoptosis against a variety of human cancer cell lines, for instance, liver cancer, breast cancer, lung cancer, and pancreas cancer in vitro and xenografted tumor in vivo (Cheng et al, ; Rooney & Ryan, ; Zhu et al, ). Further researches indicated that α‐hederin could induce autophagic cell death through activation of reactive oxygen species (ROS) dependent adenosine monophosphate‐activated protein kinase/mammalian target of rapamycin signaling pathway and inhibit interleukin 6‐induced epithelial‐to‐mesenchymal transition through disruption of JAK2/STAT3 signaling pathway (Sun et al, ; Sun et al, ). Additionally, α‐hederin has been shown ability to potentiate the efficacy of widely prescribed 5‐fluorouracil in a human colon cancer cell line HT‐29, particularly for the membrane alterations as a biological membrane‐perforating saponin (Bun et al, ).…”

<p>AMPK/mTOR/ULK1 Axis-Mediated Pathway Participates in Apoptosis and Autophagy Induction by Oridonin in Colon Cancer DLD-1 Cells</p>

“…23 Furthermore, accumulating evidence has indicated that AMP-activated protein kinase (AMPK) and mTOR play a decisive function in autophagy and apoptosis. [24][25][26][27] More interestingly, it has been reported that oridonin augmented cisplatin sensitivity through its pro-apoptotic activity, which was regulated by AMPK/ Akt/mTOR-dependent autophagosome stimulation in A549 cells. 28 However, the detailed mechanisms by which oridonin acts against CRC cells need further research, specifically regarding the targeting mTORrelated modulators via the AMPK signaling pathway.…”