Inhibitor for protein disulfide‑isomerase family A member 3 enhances the antiproliferative effect of inhibitor for mechanistic target of rapamycin in liver cancer: An in vitro study on combination treatment with everolimus and 16F16

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Abstract
MTOR is involved in the proliferation of liver cancer. However, the clinical benefit of treatment with mTOR inhibitors for liver cancer is controversial. Protein disulfide isomerase A member 3 (PDIA3) is a chaperone protein, and it supports the assembly of mTOR complex 1 (mTORC1) and stabilizes signaling. Inhibition of PDIA3 function by a small molecule known as 16F16 may destabilize mTORC1 and enhance the effect of the mTOR inhibitor everolimus (Ev). The aim of the present study was to elucidate the usefulness of combination treatment with Ev and 16F16 in liver cancer using cultured Li‑7 and HuH‑6 cells. The proliferation of cultured cells was examined following treatment with 0.01 µM Ev, 2 µM 16F16 or both. The expression levels and phosphorylation of S6 kinase (S6K) and 4E‑binding protein 1 (4E‑BP1) were examined by western blotting. Li‑7 was susceptible to Ev, and proliferation was reduced to 69.5±7.2% by Ev compared with that of untreated cells. Proliferation was reduced to 90.2±10.8% by 16F16 but to 62.3±12.2% by combination treatment with Ev and 16F16. HuH‑6 cells were resistant to Ev, and proliferation was reduced to 86.7±6.1% by Ev and 86.6±4.8% by 16F16. However, combination treatment suppressed proliferation to 57.7±4.0%. Phosphorylation of S6K was reduced by Ev in both Li‑7 and HuH‑6 cells. Phosphorylation of 4E‑BP1 was reduced by combination treatment in both Li‑7 and HuH‑6 cells. Immunoprecipitation assays demonstrated that PDIA3 formed a complex with 4E‑BP1 but not with S6K. The small molecule 16F16 increased susceptibility to Ev in cultured liver cancer cells, which are resistant to Ev. The inhibition was associated with reduction of 4E‑BP1 phosphorylation, which formed a complex with PDIA3. Combination treatment with Ev and 16F16 could be a novel therapeutic strategy for liver cancer.

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