Fig 1: The table of content (TOC figure).In normal healthy cells the ROS is low, and REDOX reaches intracellular homeostasis; but in cancer cells, due to vigorous cell metabolism and proliferation, the ROS level is much higher. However, a set of antioxidant system against ROS is derived by tumor cells, so that tumor cells cannot be harmed by ROS, but utilize ROS as a positive regulatory signal for advanced survival and proliferation. When ROS level continues to rise beyond the tolerance threshold of tumor cells, a programmed death (such as ferroptosis) will be triggered. TalaA was able to strongly induce ferroptpsis at least via the following mechanism: (1) TalaA elevates the ROS level in colorectal cells; (2) TalaA down regulates the SLC7A11 and GSS expressions, which suppresses the synthesis of important antioxidant molecule—GSH, and in turn enhance ferroptosis; (3) oxidation of arachidonic acid is an important cause of iron death, and TalaA increases the arachidonic acid oxidase—ALOXE3, which accelerates ferroptosis. (4) TalaA causes upregulation of HMOX1 which lead to the degradation of heme and the release of free iron, accumulating in mitochondria and giving rise to lipid peroxidation.
Fig 2: TalaA enhanced ferroptosis in CRC cells by up-regulation of ALOXE3.A ALOXE3 mRNA was increased by TalaA dose-dependently; *p < 0.05, **p < 0.01, N = 3 independent repeats. B The protein level of ALOXE3 was elevated by TalaA in a dose-dependent manner. C The mRNA level was decreased via lenti-shALOXE3 infection. **p < 0.01 versus ShCon, N = 3 independent repeats. D The ALOXE3 protein level was reduced by lenti-shALOXE3. E Although 10 µM TalaA violently caused cell membrane destroy in wild type SW480 cells, same concentration TalaA only led to mild membrane destroy in ALOXE3 knocked down SW480 cells. The yellow arrows indicated broken cells. F The lipid peroxidation was detected by cell-based lipid peroxidation assay kit. The stained cells were recorded with a fluorescence microscope. When the lipids were peroxidized, the fluorescence shifted from red to green. G The cell activity curve right shifted as ALOXE3 was knocked down. The black points represented wild type SW480, and purple triangles represented ALOXE3 knocked down SW480 cells. For each concentration point, three repeats were performed.
Fig 3: TalaA treatment-induced transcriptome alteration.The SW480 cells were treated with two concentrations of TalaA for 12 h. A Volcano plot showing the differences in RNA expression after 5.0 µM TalaA treatment. VPL means low concentration (5.0 µM) TalaA treatment; VPC means no chemical treatment (DMSO Control). B The volcano plot showing the differences in RNA expression after 10.0 µM TalaA treatment. VPH means high concentration (10.0 µM) TalaA treatment; VPC means no chemical treatment (DMSO control). C Through KEGG-enrichment analysis, it was found that the ferroptosis pathway molecules were up-regulated by 5.0 µM TalaA. D KEGG-enrichment analysis showed that the ferroptosis pathway molecules were up-regulated by 10.0 µM TalaA. E The heatmap data showed 5.0 µM TalaA resulted in gene expression alteration of ferroptosis-correlated molecules including FTL, SAT2, ALOXE3, GSS, ALOX12, HMOX1, ACSL5, and so on. F The heatmap data showed 10.0 µM TalaA resulted in gene expression alteration of ferroptosis-correlated molecules including FTL, SLC7A11, HMOX1, ALOXE3, SAT1, SAT2, GSS, ACSL5, ALXO12, PCBP1, MAP1LC3P, and so on.
Fig 4: The correlation of YAP with ALOXE3 in HCC. (A) The correlation between expression levels of YAP and ALOXE3 in HCC tissues were analyzed using the TIMER database. (B) Representative images of immunohistochemical staining of YAP and ALOXE3 in HCC tissues and normal liver tissue on tissue microarray sections are shown. Scale bars, 200 μm. (C) Expression scores of ALOXE3 in HCC tissues (n = 174) and normal liver tissue (n = 16) assessed by immunohistochemistry. (D,E) ALOXE3 expression in tumor tissues of HCC patients with different tumor grades or with different TNM classification. I, n = 3 patients. II, n = 61 patients. III, n = 110 patients. G1, n = 9 patients. G2, n = 140 patients. G3, n = 20 patients. (F) Positive correlation between the nuclear YAP score and the ALOXE3 score in human HCC tissues (n = 174). (G) Percentage of tumors with positive or negative staining of nuclear YAP or ALOXE3.
Fig 5: The cancer therapeutic potential of ferroptosis induction with sorafenib in HCC with enhanced activation of YAP. Parental or YAPS127A-overexpressing LM3 cells (2 × 106) were injected subcutaneously into nude mice. The average volume of tumors was allowed to reach ~100 mm3, mice bearing tumors were orally administered with 50 mg/kg sorafenib daily for 10 days. (A) Growth curves of tumors of each group. n = 8, on the linear scale for actual tumor size. (B) Images of resected tumors from mice xenografted with parental or YAPS127A-overexpressing LM3 cells. (C) Representative H&E and immunostaining images of YAP, ALOXE3, 4HNE, PTGS2 and Ki67. Scale bars for 4HNE, 100 µm. Scale bars for others, 200 µm. (D) Comparison of the survival curves of YAP with high and low expression in HCC with or without sorafenib treatment in the GEPIA database. (E) Comparison of the survival curves of GPX4 with high and low expression in HCC with or without sorafenib treatment in the GEPIA database.
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