D-mannose downregulates IDH2 protein level

It was reported that D-mannose treatment can increase reactive oxygen species (ROS) production [12], while IDH2 is important for the generation of NADPH, the crucial reducing factor. These findings prompted us to determine whether D-mannose can regulate IDH2. To test this hypothesis, we treated MDA-MB-231 cells with D-mannose and examined the expression level of IDH2. Interestingly, we found that D-mannose treatment significantly decreased the expression level of IDH2 in human breast cancer cell line MDA-MB-231 and MCF-7 (Fig. 1A). To confirm this result, MDA-MB-231 cells were treated with D-mannose for different time and dose. In line with previous results, D-mannose can reduce IDH2 in both time- and dose- dependent manners (Fig. 1, B and C). Notably, the mRNA level of IDH2 showed no response to D-mannose treatment in both cell lines (Fig. 1D). What’s more, block of D-mannose uptake into cells by GLUT transporter inhibitor WZB117 rescued the IDH2 degradation by D-mannose, which further validate our finding (Fig. 1E). In conclusion, D-mannose can downregulate IDH2 protein levels.

Fig. 1

D-mannose decreased the protein level of IDH2. (A) Western blot analysis of IDH2 level in MDA-MB-231 cells and MCF-7 cells treated with or without D-mannose. (B) Western blot analysis of IDH2 level in MDA-MB-231 cells treated with 100 mM D-mannose for different time as indicated. (C) Western blot analysis of IDH2 level in MDA-MB-231 cells treated with different concentrations of D-mannose as indicated for 48 h. (D) qPCR analysis of IDH2 mRNA level in MDA-MB-231 cells and MCF-7 cells treated with or without D-mannose. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ns, not significant. (E) Western blot analysis of IDH2 level in MDA-MB-231 cells and MCF-7 cells treated with or without D-mannose and WZB117.

D-mannose promotes the ubiquitination and degradation of IDH2

Next, we determined whether D-mannose impairs the protein stability of IDH2. Cycloheximide (CHX) was used to switch off protein synthesis in MDA-MB-231 cells, followed by the detection of IDH2 protein levels in the subsequent 0, 2, 4, and 6 h. Results showed that D-mannose accelerated the degradation of IDH2 protein, and the half-life of IDH2 protein was much shorter under D-mannose treatment (Fig. 2A). To explore the pathway through which IDH2 protein level was decreased by D-mannose treatment, the D-mannose treated cells were subjected to proteasome inhibitor MG132 and lysosome inhibitor NH4Cl treatment, respectively, and the protein level of IDH2 was examined. As a result, we found that MG132 treatment blocked the D-mannose mediated IDH2 degradation (Fig. 2B). Furthermore, we evaluated the ubiquitination level of over-expressed Flag-IDH2 protein in 293T cells upon D-mannose treatment and found that D-mannose significantly increased the ubiquitination level of Flag-IDH2, especially under MG132 treatment (Fig. 2C). What’s more, to further illustrate our findings, ubiquitination level of endogenous IDH2 was detected, results showed that IDH2 ubiquitination level was upregulated under D-mannose treatment in MDA-MB-231 cells (Fig. 2D). Together, these results suggest that D-mannose mediates the degradation of IDH2 protein through the ubiquitin-proteasome pathway.

Fig. 2

D-mannose promotes IDH2 ubiquitination and proteasomal degradation. (A) The half-life of IDH2 under D-mannose treatment was determined by CHX-chase assay in MDA-MB-231 cells. (B) Western blot analysis of IDH2 level in MDA-MB-231 cells treated with D-mannose in the absence or presence of proteasome inhibitor MG132, or lysosome inhibitor NH4Cl. (C) Western blot analysis of IDH2 ubiquitination level under D-mannose treatment in 293T cells with overexpressed Flag-IDH2. (D) Western blot analysis of IDH2 ubiquitination level under D-mannose treatment in MDA-MB-231 cells

D-mannose decreases the NADPH level and inhibits mammary cancer cell proliferation

While participating in the TCA cycle for energy production, IDH2 converts isocitrate to α-KG by reducing NADP + to NADPH. Therefore, we measured the levels of NADPH in MDA-MB-231 cells and found that D-mannose treatment can significantly decrease the cellular levels of NADPH (Fig. 3A). The colony formation and proliferation of cells were also tested, the results showed that D-mannose treatment significantly inhibited the colony formation of MDA-MB-231 cells (Fig. 3B). Consistently, the proliferation of both MDA-MB-231 cells and MCF-7 cells was also inhibited by D-mannose treatment (Fig. 3C). NADPH can serve as the donor of reductive power to neutralize the ROS accumulated in the rapid growth process in tumor cells. We thus evaluated the combination effect of a pro-oxidant, buthionine sulfoximine (BSO) and D-mannose, and found that, while both single agents decreased the tumor cell survival, the combination treatment showed the best effect of inhibition in both MDA-MB-231 cells and MCF-7 cells (Fig. 3D). To validate the role of ROS in D-mannose mediated suppression on cell proliferation, the anti-oxidant N-acetyl cysteine was supplemented into the D-mannose treated breast cancer cells and results showed that D-mannose mediated cell proliferation suppression was rescued by N-acetylcysteine treatment in both MDA-MB-231 and MCF-7 cells (Fig. 3E). Collectively, D-mannose can synergize with BSO to inhibit breast cancer cell proliferation through ROS accumulation.

Fig. 3

D-mannose synergizes with BSO to inhibit tumor cell proliferation. (A) Relative levels of NADPH in MDA-MB-231 cells treated with or without D-mannose. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (B) Colony formation assay of the control and D-mannose treated MDA-MB-231 cells. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (C) Cell proliferation assay of the control and D-mannose treated MDA-MB-231 cells and MCF-7 cells. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (D) Relative survival of MDA-MB-231 cells and MCF-7 cells under the indicated treatments for 48 h. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. * p < 0.05, *** p < 0.001, ****p < 0.0001. (E) Relative survival of MDA-MB-231 cells and MCF-7 cells under the indicated treatments for 48 h. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. *** p < 0.001, ****p < 0.0001

D-mannose promotes the expression of RNF185

The E3 ubiquitin ligase plays an essential role in the protein ubiquitination and proteasomal degradation process by attaching ubiquitin to the lysine sites of targeted proteins [17]. Therefore, we tried to identify the E3 ubiquitin ligase involved in the D-mannose mediated IDH2 ubiquitination and degradation. The human mammary cancer cell line MDA-MB-231 was subjected to D-mannose treatment followed by RNA-seq analysis. The top 10 upregulated and top 10 downregulated genes by D-mannose treatment were identified and shown (Fig. 4A). Cross analysis of the RNA-seq identified D-mannose regulated genes and IDH2 interactome data from the BioGRID database highlighted the genes RNF185 and ENAH (Fig. 4B). Given that Ring Finger Protein 185 (RNF185) is an E3 ubiquitin ligase, which plays essential roles in the ubiquitination and proteasomal degradation of proteins, we focused on RNF185, which was sharply elevated after D-mannose treatment in MDA-MB-231 cells according to the RNA-seq data. (Fig. 4A). What’s more, the result of RNA-seq was validated in MDA-MB-231 cells and MCF-7 cells through qPCR, the mRNA level of RNF185 increased about 3 folds after D-mannose treatment (Fig. 4C). Consistently, the protein level of RNF185 also showed a significant increase (Fig. 4D). Then we tried to find the pathways involved in D-mannose mediated RNF185 upregulation, RNA-seq showed the upregulated pathways in D-mannose treatment cells (Fig. 4E). Among the upregulated pathways, AMPK signaling pathway caught our attention because it has been reported that D-mannose can activate AMPK in cancer cells [13, 16]. Therefore, we tested the effect of AMPK inhibitor on D-mannose mediated RNF185 upregulation and found that AMPK inhibitor compound C can inhibit the upregulation of RNF185 by D-mannose (Fig. 4F), suggesting an important role of AMPK signaling in this process. Taken together, D-mannose increases the expression of RNF185 in breast cancer cells through AMPK acitivation.

Fig. 4

D-mannose elevates the expression of RNF185. (A) Heatmap showed the top 10 upregulated and top 10 downregulated genes by D-mannose treatment. (B) Venn diagram showed the cross analysis of RNA-seq data and BioGRID data. (C) qPCR analysis of RNF185 mRNA level in MDA-MB-231 cells and MCF-7 cells treated with or without D-mannose. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ** p < 0.01. (D) Western blot analysis of RNF185 protein level in MDA-MB-231 cells and MCF-7 cells treated with or without D-mannose. (E) KEGG enrichment analysis of upregulated pathways in D-mannose treatment cells. (F) qPCR analysis of RNF185 mRNA level in MDA-MB-231 cells and MCF-7 cells treated with or without D-mannose and Compound C. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ** p < 0.01

D-mannose promotes the degradation of IDH2 through RNF185

We then wondered whether the E3 ubiquitin ligase RNF185 plays a role in the D-mannose mediated IDH2 ubiquitination and degradation. The co-immunoprecipitation was conducted in 293T cells to test if there was any interaction between RNF185 and Flag-tagged IDH2. The result showed that RNF185 can bind to Flag-IDH2 (Fig. 5A). Furthermore, small interfering RNAs (siRNAs) were developed to disturb the expression of RNF185(Fig. 5B) and the effect of RNF185 knockdown on IDH2 protein level was examined. The result showed that knockdown of RNF185 led to a significant increase of IDH2 protein level (Fig. 5C). What’s more, knockdown of RNF185 effectively blocked D-mannose mediated IDH2 degradation (Fig. 5D). Taken together, these results suggest that RNF185 can directly bind to IDH2 and promote its degradation under D-mannose treatment.

Fig. 5

RNF185 binds with and degrades IDH2 under D-mannose treatment. (A) The interaction between Flag-IDH2 and RNF185 in 293T cells was determined by co-IP and western blot. (B) qPCR analysis of RNF185 mRNA level in MDA-MB-231 cells transfected with control or siRNF185. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (C)Western blot analysis of RNF185 and IDH2 protein levels in MDA-MB-231 cells transfected with control or siRNF185. (D)Western blot analysis of RNF185 and IDH2 protein levels in MDA-MB-231 cells transfected with control or siRNF185 and treated with or without D-mannose

RNF185 plays a key role in the D-mannose mediated inhibition of tumor cell proliferation through IDH2 degradation

To further validate the role of RNF185-IDH2 axis in D-mannose mediated inhibition of tumor cell proliferation, we knocked down RNF185 in MDA-MB-231 cells and examined its effects on NADPH production and cell proliferation. The results showed that D-mannose mediated decrease of cellular NADPH level can be recovered by knockdown of RNF185(Fig. 6A). Similarly, the inhibition of tumor cell proliferation by D-mannose was rescued under RNF185 knockdown (Fig. 6B). Furthermore, when RNF185 was knocked down, the inhibition effect of BSO and D-mannose combination on tumor cells was partially abrogated, and the relative survival of tumor cells showed significant improvement (Fig. 6C). Together, we concluded that D-mannose mediated RNF185 upregulation plays a key role in IDH2 degradation and tumor cell inhibition (Fig. 6D).

Fig. 6

Knockdown of RNF185 abrogates the effect of D-mannose on tumor cells. (A) Relative levels of NADPH in MDA-MB-231 cells with indicated treatment. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (B) Cell proliferation analysis of MDA-MB-231 cells with indicated treatment. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (C) Relative survival analysis of MDA-MB-231 cells under the indicated treatments for 48 h. Values are means ± SD from n = 3 independent experiments. Statistical differences were determined by t-test. ****p < 0.0001. (D) Schematic representation of the mechanism under D-mannose mediated IDH2 degradation