Chandimali et al. (2018) investigated the effect a phenolic compound (hispidin) of Shanghuang mushroom (Phellinus linteus) on the proliferation, migration and invasion of pancreatic cancer (AsPC1 and BxPC-3) cells. The results shown that Hispidin inhibits the growth of pancreatic cancer cells, especially BxPC-3 cells, and is further studied for the mechanism involved.
Fig 1. Effect of hispidin on pancreatic cancer cell proliferation and stemness. A: Two-dimensional structure of hispidin. B: 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay revealed a dose-dependent reduction of viability of hispidin-treated AsPC-1 and BxPC-3 pancreatic cancer cells. C: Western blotting showed dose-dependent effects of hispidin on tumor-suppressor p53; apoptosis-related proteins B-cell lymphoma 2 (BCL2), cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP); cancer stem cell marker CD44; and stemness markers NANOG and Sex-determining region Y-box 2 (SOX2) in BxPC-3 cells. Data represent the mean±SEM (n=5 per group). Significantly different from untreated cells at *p<0.05, and **p<0.01. NFĸB: nuclear factor-kappa B; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
The results shown that Hispidin have antiproliferative effect of pancreatic cancer cells, especially BxPC-3 cells. BxPC-3 cells were selected for the mechanism involved. At 150 µL of hispidin inhibited the proliferation of pancreatic CSCs after treatment for 48 h. The mechanisms were to reduce the number of cells in the S phase and the G2 phase to increase the number of cell in G1 phase, inhibit NF-KB activation and antiproliferative of pancreatic cancer cells induce apoptosis.
Fig 2. Inhibition of pancreatic cancer stem-like properties by hispidin treatment. A: Colony-formation assay showing the effect of 150 μM hispidin on BxPC-3 cancer stem cell (CSC) proliferation. B: Effect of hispidin on cell mobility was assessed by transwell migration and invasion assays. C,D: Immunocytochemistry assay showed 150 μM hispidin suppressed the stemness of CD44+ BxPC-3 CSCs in a dose-dependent manner through reduction of expression of stemness markers. E: Western blotting confirming findings shown in C and D. F, G: Sphere-forming assay and sphere immunocytochemistry assay revealed that hispidin reduced the self-renewal ability of pancreatic CSCs. Data represent the mean±SEM (n=5 per group). SOX2: Sex-determining region Y-box 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; DAPI: 4’,6-diamidino-2-phenylindole staining.
It also significantly reduced the size, number and expression of CD44, NANOG and SOX2 , indicating the reduction of self-renewal ability. Subsequently, the efficaciency of pancreatic cancer drugs (Gemcitabine) was studied. The result shown both substances were inhibited the growth of pancreatic cancer cells better than using either. It was concluded that an extract of Sang hwang mushroom (Phellinus linteus) has the potential to enhance the effectiveness of gemcitabine in the treatment of pancreatic cancer.
Fig 3. Hispidin enhances the sensitivity of CD44+ BxPC-3 stem cells to gemcitabine by inhibiting stemness. A: Viability of cells treated with the combination of hispidin and gemcitabine was significantly reduced in a dose-dependent manner compared to cells subjected to either treatment alone. B: Cell viability of CD44+ BxPC-3 CSCs. The concentrations of hispidin and gemcitabine used were 150 μM and 1 μM, respectively; the combination therapy of hispidin and gemcitabine was performed for 48 h. C: Western blotting showed significantly reduced expression of stemness markers, the CD44 stem cell marker, and apoptosis-related proteins by synergistic treatment. Immunocytochemistry assays confirmed a greater reduction in D: CD44 and E: NANOG and Sex-determining region Y-box 2 (SOX2) expression after the combination treatment than that after either treatment alone. Data represent the mean±SEM (n=5 per group). Significantly different from untreated cells at *p<0.05, and **p<0.01. NFĸB: nuclear factor-kappa B; BCL2: B-cell lymphoma 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; DAPI: 4’,6-diamidino-2- phenylindole staining.
It was concluded that an extract of Sang hwang mushroom (Phellinus linteus) has the potential to enhance the effectiveness of gemcitabine in the treatment of pancreatic cancer.
Reference : Chandimali N, Huynh DL, Jin WY, Kwon T. Combination Effects of Hispidin and Gemcitabine via Inhibition of Stemness in Pancreatic Cancer Stem Cells. Anticancer Res. 2018 Jul;38(7):3967-3975. doi: 10.21873/anticanres.12683. PMID: 29970519.
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