Low Level Of 3,3′-diindolylmethane Promotes Gastric Cancer Progression Through Autocrine Wnt4 Signaling

Objective: To explore the effects of low level of DIM on the gastric cancer cells and the underlying mechanism. This study will provide new evidence for the roles of low level of DIM in gastric cancer progression and new target for gastric cancer therapy.Methods: Colony formation assay was used to analyze the growth ability of gastric cancer cells treated with low level of DIM. Western blot and immunofluorescence were applied to determine the proliferation, migration, and stemness associated proteins in gastric cancer cells. The migrating ability of low level of DIM treated gastric cancer cells was evaluated by wound healing and transwell migration assay. The effect of low level of DIM on the differentiation potential of gastric cancer cells was examined by adipogenic differentiation assay. The β-catenin signaling inhibitor ICG001 was used to identify the β-catenin activation in low level DIM-treated gastric cancer cells. TOP/FOP Flash luciferase reporter assay was used to assess the activation of β-catenin signaling pathway. Western blot and immunofluorescence were conducted to detect the expression of Wnt4 and β-catenin in gastric cancer cells treated with low level of DIM. The expression of PORCN or WNT4 in HGC-27 gastric cancer cells was inhibited by using lentivirus containing PORCN or WNT4 shRNA, and the efficiency of PORCN or Wnt4 knockdown in HGC-27 cells were verified by qRT-PCR and Western blot. Colony formation assay, Western blot, immunofluorescence and adipogenic differentiation assay were applied to evaluate growth, migration, stemness and differentiation potential of PORCN or WNT4 knockdown HGC-27 cells treated with low level of DIM. The effects of low level of DIM on gastric cancer growth in vivo was investigated by subcutaneous tumor xenograft model in nude mice. The expression of metastasis-associated genes Snail and N-cadherin in the tumor tissues was detected by q RT-PCR. Immunohistochemistry and Immunofluorescence were used to analyze the expression of Wnt4, β-catenin and CD44 in tumor tissues.Results: Low level of DIM could obviously promote gastric cancer cell proliferation, enhance the migrating ability and induce differentiation potential of gastric cancer cells. Low level of DIM promoted the expressions of proliferation, migration, and stemness associated proteins in gastric cancer cells. The effects of low level of DIM on gastric cancer cells could be reversed by ICG001, indicating that low level of DIM enhanced the proliferation, migration and stemness of gastric cancer cell through activation of β-catenin signaling. Knockdown of Wnt4 inhibited proliferation, migration, stemness and differentiation potential of HGC-27 cells. In addition, low level of DIM also promoted subcutaneous xenograft tumor growth in vivo. The expression of Wnt4, β-catenin, and CD44 was obviously increased in low level of DIM-treated mouse tumor tissues.Conclusions: Our findings indicated that low level of DIM promoted gastric cancer cells proliferation, migration, stemness and pluripotency through inducing the secretion of Wnt4 and the activation of β-catenin signaling, which provides new evidence for understanding the role of low level of DIM in gastric cancer.