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. 2020 Dec 8;11(49):4593-4604.
doi: 10.18632/oncotarget.27835.

The inhibitory effect of TU-100 on hepatic stellate cell activation in the tumor microenvironment

Yuma Wada  1   2 , Kazunori Tokuda  1   2 , Yuji Morine  1 , Shohei Okikawa  1 , Shoko Yamashita  1 , Tetsuya Ikemoto  1 , Satoru Imura  1 , Yu Saito  1 , Shinichiro Yamada  1 , Mitsuo Shimada  1
Affiliations

The inhibitory effect of TU-100 on hepatic stellate cell activation in the tumor microenvironment

Yuma Wada et al. Oncotarget. .

Abstract

Introduction: The tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation.

Materials and methods: The HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 μg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC's culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression.

Results: Colon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death.

Conclusions: TU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.

Keywords: CRC; CRLM; HSC; IL-6; TU-100.

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Conflict of interest statement

CONFLICTS OF INTEREST We state any potential conflicts of interest regarding our study as follows; Mitsuo Shimada received grant support from Tsumura & Co. The other authors declare no conflicts of interest in association with the present study.

Figures

Figure 1
Figure 1. TU-100 inhibited HSC activation and cytokine’s secretions from HSCs.
(A) HSC was stimulated with cancer-conditioned medium (cancer-CM) derived from the 24 hours cancer cells (HT29 and HCT116) culture or cultured with normal medium (control; Ctrl). α-SMA and IL-6 mRNA expressions of activated HSCs in cancer-CM were analyzed by PCR analysis. (B) HSCs were cultured in cancer-CM, with simultaneous TU-100 treatment: (90: 90 μg/mL; 270: 270 μg/mL; and 900: 900 μg/mL), as described in Supplementary Figure 1. α-SMA and IL-6 mRNA expressions were analyzed by PCR analysis. (C) Cytokine arrays of HSC’s culture supernatants (aHSC-CM) with or without TU-100 (900) treatment. (D) Pixel density of dot plots was calculated by using ImageJ software. (E) IL-6 and VEGF secretions from HSCs with or without TU-100 treatment were analyzed by ELISA. (*significantly different from Ctrl, P < .05, n = 4. Mann-Whitney U test).
Figure 2
Figure 2. TU-100 modified HSC-CM reduced cancer cell migration and proliferation.
Cancer cells (HT29 and HCT116) were cultured for 24 hours with aHSC-CM or modified HSC-CM as described in Supplementary Figure 2. (A) Proliferation assay and (B and C) migration assay of cancer cells were monitored for 3 days and 24 hours, respectively. (*significantly different from aHSC-CM group without TU-100 treatment, P < .05, n = 4, The one-way ANOVA with Turkey-Kramer’s test (proliferation assay), Mann-Whitney U test (migration assay).
Figure 3
Figure 3. HSC derived from IL-6 promoted cancer cell proliferation and migration.
(A) Proliferation assay and (B and C) migration assay of cancer cells in aHSC-CM with or without an IL-6 antibody were monitored for 3 days and 24 hours, respectively. (*significantly different from aHSC-CM group without IL-6 neutralization, P < .05, n = 4, The one-way ANOVA with Turkey-Kramer’s test (proliferation assay), Mann-Whitney U test (migration assay).
Figure 4
Figure 4. TU-100 directly reduced cancer malignant potential in activated HSC conditioned.
Colon cancer cells (HT29 and HCT116) were cultured for 24 hours in aHSC-CM with or without simultaneous TU-100 (90, 270, 900) treatment as described in Supplementary Figure 3. (A) Proliferation assay and (B, C) migration assay of cancer cells cultured in aHSC-CM with or without TU-100 treatment were monitored for 3 days and 24 hours, respectively. (*significantly different from aHSC-CM group without TU-100 treatment, P < .05, n = 4, The one-way ANOVA with Turkey-Kramer’s test (proliferation assay), Mann-Whitney U test (migration assay).
Figure 5
Figure 5. TU-100 induced the autophagic cell death.
(A) To assess autophagosomes, colon cancer cells (HT29 and HCT116) in aHSC-CM with or without TU-100 (900) treatment were stained with blue dots in autophagic vacuoles (blue) and DAPI (blue). Arrowhead; bright blue dots indicate autophagy. Scale bar, 10 μm. (B) Western blotting of LC3-I and LC3-II in HT29 cultured HSC’s supernatants (aHSC-CM) with or without TU-100 (900) treatment. (C) Beclin-1 mRNA expressions of cancer cells were detected by PCR analysis. (*significantly different from aHSC-CM group without TU-100 treatment, P < .05, n = 4, Mann-Whitney U test).
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