Butyrate up-regulates the expression of CDKN1A to block the cell cycle and inhibits the PI3K-AKT signaling pathway to promote apoptosis to play an anti-Acute Myeloid Leukemia role

Abstract

Abstract BACKGROUND Our previous research indicated a significant reduction in the beneficial metabolite butyrate acid, derived from gut microbiota, in the intestines and blood of patients with primary Acute Myelocytic Leukemia (AML). To investigate the key molecules through which butyrate acid influences AML progression, this study aimed to elucidate the anti-tumor mechanisms by which butyrate inhibits AML cell viability, promotes apoptosis, and blocks the cell cycle in vitro, while also assessing its efficacy in delaying AML progression in vivo.METHODS The effect of butyrate acid on the proliferation activity of AML cells was measured using the Cell Counting Kit-8 assay. Flow cytometry was used to detect the effect of butyrate acid on apoptosis and cell cycle of AML cells. Differentially expressed genes (DEGs) were screened by cellular transcriptomics and analyzed for functional enrichment. The mRNA of DEGs was verified by qRT-PCR, and the protein levels were detected by Western blot (WB). Patient-derived xenograft (PDX) AML models were constructed by luciferase labeling AML cells. The changes of gut microbiota were detected by 16S rRNA in mouse feces, and the SCFAs content were detected by targeted GC-MS in mouse feces and serum. Blood routine tests, peripheral blood and bone marrow blast cell tests, and in vivo fluorescence tests in mice were conducted to verify the tumor cell burden. HE was used to observe the pathological changes of organs, and immunohistochemistry (IHC) was used to detect the expression levels of key molecules.RESULTS Butyrate acid inhibited AML cell proliferative viability, promoted apoptosis, and blocked the G1 phase of the AML cell cycle. Butyrate acid significantly up-regulated the mRNA and protein expression of cyclin-dependent kinase inhibitor 1A (CDKN1A), and inhibited the phosphorylation levels of PI3K and AKT, but did not alter the expression of apoptosis-related proteins BAX and BCL-2. 16S-rRNA sequencing and SCFAs measurements showed that butyrate acid restored the abundance of some beneficial intestinal microbiota and directly replenished butyrate concentration in mice. The butyrate acid intervention prolonged the survival time of the mice, slowed down the spleen enlargement and colon shortening of AML mice, and reduced the AML cell load of AML mice; the Ki67 positivity rate of AML mice was significantly higher than that of the butyrate-intervened AML group. IHC and WB confirmed that butyric acid significantly up-regulated the expression of CDKN1A in the spleen.CONCLUSION Dysbiosis of gut microbiota accelerates AML disease progression, and AML disease alters the structure of intestinal flora. Butyrate plays an anti-AML tumor role as a key intermediate metabolite regulating CDKN1A expression, inhibiting the PI3K-AKT pathway while blocking the cell cycle and promoting apoptosis. Exogenous butyrate is a feasible new therapeutic strategy for AML and has good clinical prospects.