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Effects of 17-allylamino-17-demethoxygeldanamycin on the induction of apoptosis and cell cycle arrest in HCT-116 cells.

ABSTRACT: The present study investigated the effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on apoptosis and the cell cycle of the HCT-116 human colon carcinoma cell line, with the aim of elucidating their underlying mechanisms. MTT was used to examine the inhibitory effects of 17-AAG on the proliferation of HCT-116 cells at various time points and doses. The cells were stained with Annexin V-fluorescein isothiocyanate/propidium iodide and evaluated by flow cytometry. The expression of signal transducer and activator of transcription (STAT)3, cyclin D1, cytochrome c (cyt-c), caspase 9 and caspase 3 at the mRNA and protein level was determined using reverse transcription-polymerase chain reaction and western blotting. Treatment with 17-AAG at a concentration of 1.25-20 mg/l for 24 and 48 h significantly inhibited the proliferation of HCT-116 cells in a time-dependent and concentration-dependent manner. Treatment with 17-AAG at concentrations of 1.25, 2.5 and 5 mg/l for 48 h significantly induced apoptosis and cell cycle arrest in HCT-116 cells. Exposure to 17-AAG at concentrations of 1.25, 2.5 and 5 mg/l for 48 h significantly downregulated the mRNA and protein expression of STAT3 and cyclin D1, but upregulated cyt-c, caspase 9 and caspase 3 in a concentration-dependent manner in HCT-116 cells. Therefore 17-AAG is able to inhibit cell proliferation, inducing apoptosis and G1 stage cell cycle arrest by downregulating the expression of cyclin D1, and promoting the mitochondria apoptosis by downregulating STAT3 in HCT-116 cells.

SUBMITTER: Zhao X

PROVIDER: S-EPMC5530076 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:Colorectal cancer (CRC) ranks as the second leading cause of cancer‑related death worldwide due to its aggressive nature. After surgical resection, >50% of patients with CRC require adjuvant therapy. As a result, eradicating cancer cells with medications is a promising method to treat patients with CRC. In the present study, a novel compound was synthesized, which was termed compound 225#. The inhibitory activity of compound 225# against CRC was determined by MTT assay, EdU fluorescence labeling and colony formation assay; the effects of compound 225# on the cell cycle progression and apoptosis of CRC cells were detected by flow cytometry and western blotting; and the changes in autophagic flux after the administration of compound 225# were detected using the double fluorescence fusion protein mCherry‑GFP‑LC3B and western blotting. The results demonstrated that compound 225# exhibited antiproliferative properties, inhibiting the proliferation and expansion of CRC cell lines in a time‑ and dose‑dependent manner. Furthermore, compound 225# triggered G2/M cell cycle arrest by influencing the expression of cell cycle regulators, such as CDK1, cyclin A1 and cyclin B1, which is also closely related to the activation of DNA damage pathways. The cleavage of PARP and increased protein expression levels of PUMA suggested that apoptosis was triggered after treatment with compound 225#. Moreover, the increase in LC3‑II expression and stimulation of autophagic flux indicated the activation of an autophagy pathway. Notably, compound 225# induced autophagy, which was associated with endoplasmic reticulum (ER) stress. In accordance with the in vitro findings, the in vivo results demonstrated that compound 225# effectively inhibited the growth of HCT116 tumors in mice without causing any changes in their body weight. Collectively, the present results demonstrated that compound 225# not only inhibited proliferation and promoted G2/M‑phase cell cycle arrest and apoptosis, but also initiated cytoprotective autophagy in CRC cells by activating ER stress pathways. Taken together, these findings provide an experimental basis for the evaluation of compound 225# as a novel potential medication for CRC treatment.

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Effects of 17-allylamino-17-demethoxygeldanamycin on the induction of apoptosis and cell cycle arrest in HCT-116 cells.

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