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. 2020 Nov 17;11(11):988.
doi: 10.1038/s41419-020-03194-2.

Discovery of a novel ferroptosis inducer-talaroconvolutin A-killing colorectal cancer cells in vitro and in vivo

Yong Xia  1   2 , Shuzhi Liu  3 , Changlin Li  4 , Zhiying Ai  4 , Wenzhi Shen  4 , Wenqi Ren  4 , Xiaolong Yang  5
Affiliations

Discovery of a novel ferroptosis inducer-talaroconvolutin A-killing colorectal cancer cells in vitro and in vivo

VSports - Yong Xia et al. Cell Death Dis. .

Abstract

Ferropotsis is among the most important mechanisms of cancer suppression, which could be harnessed for cancer therapy. However, no natural small-molecule compounds with cancer inhibitory activity have been identified to date VSports手机版. In the present study, we reported the discovery of a novel ferroptosis inducer, talaroconvolutin A (TalaA), and the underlying molecular mechanism. We discovered that TalaA killed colorectal cancer cells in dose-dependent and time-dependent manners. Interestingly, TalaA did not induce apoptosis, but strongly triggered ferroptosis. Notably, TalaA was significantly more effective than erastin (a well-known ferroptosis inducer) in suppressing colorectal cancer cells via ferroptosis. We revealed a dual mechanism of TalaA' action against cancer. On the one hand, TalaA considerably increased reactive oxygen species levels to a certain threshold, the exceeding of which induced ferroptosis. On the other hand, this compound downregulated the expression of the channel protein solute carrier family 7 member 11 (SLC7A11) but upregulated arachidonate lipoxygenase 3 (ALOXE3), promoting ferroptosis. Furthermore, in vivo experiments in mice evidenced that TalaA effectively suppressed the growth of xenografted colorectal cancer cells without obvious liver and kidney toxicities. The findings of this study indicated that TalaA could be a new potential powerful drug candidate for colorectal cancer therapy due to its outstanding ability to kill colorectal cancer cells via ferroptosis induction. .

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. TalaA killed colorectal cancer cells.
A The structure of TalaA. B CRC cells were incubated with TalaA in DMEM media containing 10% FBS for 24 h, then the CCK8 kit was employed to examine the cells activities. From left to right, the cells were HCT116, SW480, and SW620, respectively. For each concentration point, three repeats were performed. C CRC cells were incubated with TalaA in DMEM media containing 1% FBS for 24 h, then the CCK8 kit was employed to examine the cells activities. From left to right, the cells were HCT116, SW480, and SW620, respectively. For each concentration point, three repeats were performed. D After HCT116 cells were incubated with TalaA in 10% FBS contained media for 48 h, Edu solution was added and cells were stained according to manufacturer’s instruction. Red spots meant Edu-positive cells, and blue spots meant Hoechst33342-positive cells. E After SW480 cells were incubated with TalaA in 10% FBS contained media for 48 h, Edu solution was added and cells were stained according to manufacturer’s instruction. Red spots meant Edu-positive cells, and blue spots meant Hoechst33342-positive cells. F The crystal violet staining results for the clonogenicity of SW480 cells. The SW480 cells were cultured with 0–10 μM TalaA for 12 days.
Fig. 2
Fig. 2. TalaA elevated ROS in CRC cells.
A The SW480 cells were co-incubated with or without TalaA for 24 h, and the cells were stained by PI/RNase. And the stained cells were detected by flow cytometry to examine the cell cycle. The two red peaks represent G1 and G2 stage, and the cross-court area represents S stage. The blue arrow means the dead cell debris. B The SW480 cells were co-incubated with or without TalaA for 24 h, and the cells were stained by PI and Annexin V-FITC. The stained cells were detected by flow cytometry to examine the apoptosis. C The SW480 cells were treated by 0–10 μM TalaA for 24 h, and the cellular morphology was recorded by microscope. Note: cells with membranes perforated were marked with yellow arrows; and the dead cells were marked with blue arrows. D The SW480 cells were treated by 8.0 μM for 24 h, and the cell membrane and nucleus were stained by DiO (green) and Hoechst 33342 (blue). Note: the yellow arrow indicated damaged membrane and the blue indicated membrane fragment without nucleus. E The SW480 cells were treated by 8.0 μM for 4 h, and the ROS was detected by H2DCFDA. The yellow arrow indicated ROS increased cells. F The SW480 cells treated with TalaA or H2O2 were incubated with 5 µM H2DCFDA in PBS in the dark for 30 min at 37 °C. After being digested, the H2DCFDA-stained cells were detected by flow cytometer.
Fig. 3
Fig. 3. TalaA-induced ferroptosis in CRC cells.
A Transmission electron microscopy is used to observe the microscopic substructure of cells: The SW480 cells were treated by 5.0 μM TalaA for 24 h and fixed by 2.5% glutaraldehyde. The fixed cells were taken photos using transmission electron microscope (Hitachi HT7800, Japan) with different magnifications (the magnification was shown in picture). The green arrow indicates mitochondria and red arrow indicates membrane. After being treated by TalaA, the mitochondria are wrinkled, with the internal crest disappearing; and the cell membrane broken. B The SW480 cells pretreated by 0.1 μM ferrostatin-1 were co-incubated with 10.0 μM TalaA for 12 h. Then the cellular morphology was recorded by microscope. (The cells that look like membranes perforated were marked with yellow arrows.) C The SW480 cells pretreated by 0–0.5 μM ferrostatin-1 were co-incubated with 10 μM TalaA for 24 h, and the cells were tested by CCK8 kit. Error bars means SD, N = 3 independent repeats. p values were calculated using two-tailed unpaired Student’s t-test, * means p < 0.05; *** means p < 0.001 versus TalaA treatment. D The lipid peroxidation was detected by cell-based lipid peroxidation assay kit. The lipid peroxidation sensor changes its fluorescence from red to green upon peroxidation by ROS in cells. The stained cells were taken photos in a fluorescence microscope. E The ratio of green fluorescence to red fluorescence was calculated with Image J software to show the degree of lipid peroxidation. F Comparison of the anti-cancer effect between erastin and TalaA on colon cancer cells. Colorectal cancer SW480 cells were treated by different concentrations of TalaA and erastin respectively, for 24 h, and the relative cell activity was detected with CCK8 kit. Red points represent TalaA treatment group, and blue triangles represent erastin treatment group. For each concentration point, three repeats were performed. G SW480 cells were co-incubated with 0, 7.5, 15 μM TalaA and erastin, respectively. After 48 h, the cultured cells were taken pictures with phase contrast microscope. To compare the morphological alteration, the photos of 15 μM TalaA and erastin-treated SW480 cells were amplified. The red arrows indicated dead cells with obvious morphological alteration.
Fig. 4
Fig. 4. 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. 5
Fig. 5. TalaA accelerated ferroptosis in CRC cells by down-regulation of SLC7A11.
A SLC7A11 mRNA was decreased by TalaA dose-dependently; *p < 0.05, **p < 0.01, N = 3 independent repeats. B SLC7A11 protein level was decreased by TalaA in a dose-dependent manner. C The SLC7A11 protein level was increased by SLC7A11 overexpression plasmid (SLC7A11 OVX) transfection. D The relative cell activities of SLC7A11-overexpressed cells and control cells after treated by 5.0 μM TalaA. **p < 0.01, N = 3 independent repeats. E The mRNA expression was suppressed by SLC7A11-specific lenti-shRNA; **p < 0.01 versus shCon, N = 3 independent repeats. F The SLC7A11 protein level was decreased by lenti-shSLC7A11. G Total glutathione and reduced glutathione was decreased as SLC7A11 being knocked down; **p < 0.01, N = 3 independent repeats. H 5.0 μM TalaA induced slight cell membrane to get destroyed in wild type SW480 cells. However same concentration of TalaA induced strong membrane to get destroyed in SLC7A11 knocked down SW480 cells. The yellow arrows indicate membrane-damaged cells. I 5.0 μM TalaA-treated SLC7A11 knocked-down SW480 cells had lower cell activity than wild type SW480 with same concentration TalaA treatment; **p < 0.01, N = 3 independent repeats. J The scatter plot of TalaA inhibited cell growth. The blue points represented wild type SW480, and red squares represented SLC7A11 knocked down SW480 cells. For each concentration point, three repeats were performed. K The SLC7A11 knockdown SW480 and wild type SW480 were treated by 5.0 μM TalaA with or without Ferrostatin-1. The cell activity of SW480 cells was detected with CCK8 kit; **p < 0.01, N = 3 independent repeats.
Fig. 6
Fig. 6. 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. 7
Fig. 7. TalaA inhibited xenografted tumor growth in vivo.
A Tumor column was recorded. The black points represented blank control group (corn oil), and the red squares TalaA treatment group (six mice for each group). B The final tumor weight was compared between the two groups: ***p < 0.001 indicated the significant difference. C Mice body weight was recorded. The black points represented blank control group and red squares TalaA treatment group. D The final body weight was compared between the two groups: no significant difference between the two groups; “ns” represent no significant difference. E Pathological staining for xenografted tumors of the above two groups: H&E staining photos and IHC staining for Ki67, SLC7A11, and HMOX1 photos for both control group and TalaA treatment group. F The mice liver and kidney were fixed in the formalin and stained with H&E dye for both control group and TalaA treatment group.
Fig. 8
Fig. 8. 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.
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