Heterogeneous Responses of Gastric Cancer Cell Lines to Tenovin-6 and Synergistic Effect with Chloroquine

Abstract

Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer death. Approximately 15% of GC is associated with Epstein-Barr virus (EBV). GC is largely incurable with a dismal five-year survival rate. There is an urgent need to identify new therapeutic agents for the treatment of GC. Tenovin-6 was initially identified as a p53 activator, but it was later found to inhibit autophagy flux, and the protein deacetylase activity of sirtuins. Tenovin-6 shows promising therapeutic effect in various malignancies. However, it remains unknown whether Tenovin-6 is effective for GC. In this study, we found that EBV-positive and -negative GC cell lines were sensitive to Tenovin-6 but with different response times and doses. Tenovin-6 suppressed anchorage-independent growth of GC cells. Tenovin-6 induced different levels of apoptosis and phases of cell-cycle arrest depending on the cell lines with some manifesting gap 1 (G1) and others showing synthesis (S) phase cell-cycle arrest. Mechanistically, Tenovin-6 induced autophagy or p53 activation in GC cells depending on the status of TP53 gene. However, initiation of autophagy following treatment with Tenovin-6 conferred some protective effect on numerous cells. Combined treatment with Tenovin-6 and autophagy inhibitor chloroquine increased the cytotoxic effect by inducing microtubule-associated protein 1 light chain 3B (LC3B)-II accumulation, and by enhancing apoptosis and cell-cycle arrest. These results indicated that Tenovin-6 can be used as a potential therapeutic agent for GC, but the genetic background of the cancer cells might determine the response and mechanism of action. Treatment with Tenovin-6 alone or in combination with chloroquine could be a promising therapeutic approach for GC.

Keywords: Epstein–Barr virus (EBV); Tenovin-6; autophagy; chloroquine; gastric cancer; p53 activation.

Figure 1

Tenovin-6 inhibits cell proliferation and anchorage-independent growth of gastric cancer (GC) cells. (A) Examination of cell proliferation following treatment with Tenovin-6. Cells seeded at 2.5 × 10⁴ or 5 × 10⁴ cells/well were treated with the indicated concentrations of Tenovin-6 and counted at 24, 48, and 72 h post treatment. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) The half maximal inhibitory concentration (IC₅₀) values were calculated using SPSS software based on the relative cell numbers at 72 h post treatment in all GC cell lines. (C) Suppression of anchorage-independent growth of AGS, AGS-EBV, and HGC-27 cells by Tenovin-6. Representative pictures captured at 5× magnification are presented in the left panel. Colonies with diameter >50 μm were counted, and colony numbers in each field are presented in the right panel. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar: 500 μm.

Figure 2

Tenovin-6 induces apoptosis and cell-cycle arrest of GC cells. (A) Percentages of apoptotic cells in the indicated cell lines after Tenovin-6 treatment based on annexin V and propidium iodide (PI) staining. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Percentages of cells at gap 1 (G1), synthesis (S), and G2/mitotic (M) phases of the indicated cell lines after Tenovin-6 treatment based on PI staining. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Tenovin-6 induces p53 activation or initiated but failed to induce full program of autophagy in GC cells. The levels of microtubule-associated protein 1 light chain 3B (LC3B), sequestosome-1 (SQSTM1)/p62 (p62), acetylated-p53 (Ac-p53 (K382)), phospho-p53 (Ser15) (P-p53 (S15)), p53, and p21 were examined in GC cell lines following treatment with Tenovin-6 for 24 and 48 h. Tenovin-6 was used at 0.5 μM for AGS, AGS-EBV, and SNU-1 cells, 6 μM for SNU-719 cells, 1 μM for HGC-27 and KATO-III cells, and 2 μM for N87 cells.

Figure 4

Chloroquine (CQ) enhances the inhibitory effect of Tenovin-6 (T6) on cell proliferation in GC cells. (A–D) Examination of cell proliferation in AGS (A), AGS-EBV (B), SNU-719 (C), and SNU-1 (D) cells following treatment with different concentrations of T6 or CQ alone, or in combination. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Tenovin-6 (T6) and chloroquine (CQ) synergistically induce apoptosis and cell-cycle arrest in GC cells. (A) Examination of apoptotic cells by annexin V and propidium iodide (PI) staining in AGS, AGS-EBV, SNU-719, and SNU-1 cells following treatment with different concentrations of T6 or CQ alone or in combination. * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Examination of cell-cycle phases in AGS, AGS-EBV, SNU-719, and SNU-1 cells following treatment with different concentrations of T6 or CQ alone or in combination. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Chloroquine induces microtubule-associated protein 1 light chain 3B (LC3B)-II but blocks autophagy flux to enhance cytotoxicity of Tenovin-6. (A–D) Examination of LC3B and sequestosome-1 (SQSTM1)/p62 (p62) protein levels following treatment with Tenovin-6 or chloroquine alone, or in combination for 48 h in AGS (A), AGS-EBV (B), SNU-719 (C), and SNU-1 (D) cells.