Exploring the Molecular Therapeutic Mechanisms of Gemcitabine through Quantitative Proteomics.

Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.

miR-125b-5p-MAPK1-C/EBPα feedback loop regulates all-trans retinoic acid resistance in acute promyelocytic leukemia.

BACKGROUND: Various studies have highlighted the significance of miR-125b-5p in tumour chemotherapy resistance; However, whether miR-125b-5p is associated with all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukemia (APL) has not been reported. METHODS: Drug-resistance-related factors in APL were predicted using the DRESIS database. The expression levels of miR-125b-5p in ATRA-sensitive and ATRA-resistant APL cells were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A nitrotetrazolium blue (NBT) reduction assay and flow cytometry (FCM) were used to detect the effect of miR-125b-5p on ATRA resistance in APL cells. An APL xenograft tumour mouse model was established to determine the effect of miR-125b-5p on ATRA resistance. A dual-luciferase gene reporter assay, qRT-PCR, and western blotting verified the regulation by miR-125b-5p of its target gene, MAPK1, and the MAPK1 downstream factor, C/EBPα. An NBT reduction assay and FCM were used to detect the effect of C/EBPα on ATRA resistance in APL cells. Western blotting and qRT-PCR were used to assess the regulation of miR-125b-5p and MAPK1 by C/EBPα. RESULTS: miR-125b-5p expression levels were dramatically increased in ATRA-resistant APL cells. Both in vitro and in vivo experiments revealed that miR-125b-5p overexpression enhanced ATRA resistance in APL. miR-125b-5p promoted ATRA resistance by sponging MAPK1. C/EBPα was negatively regulated by miR-125b-5p, which in addition, regulated ATRA resistance in APL cells. C/EBPα also regulated the miR-125b-5p-MAPK1 axis. CONCLUSION: The findings of this study indicate that the miR-125b-5p-MAPK1-C/EBPα feedback loop regulated ATRA resistance in APL. Thus, miR-125b-5p may be a promising target for treating ATRA resistance in APL.

Aristolocholic acid I promotes renal tubular epithelial fibrosis by upregulating matrix metalloproteinase-9 expression via activating the C3a/C3aR axis of macrophages.

Aristolochic acid I (AAI) can cause nephrotoxicity and is characterized by interstitial fibrosis. The C3a/C3aR axis of macrophages and matrix metalloproteinase-9 (MMP-9) play important roles in fibrosis, but whether they are involved in AAI-induced renal interstitial fibrosis and are related remains to be elucidated. In this study, we investigated whether C3a/C3aR axis of macrophages promotes renal interstitial fibrosis by regulating MMP-9 in aristolochic acid nephropathy (AAN). Intraperitoneal injection of AAI for 28 days successfully induced AAN in C57bl/6 mice. The content of C3a in the kidney of AAN mice was increased, and there was a significant distribution of macrophages in the renal tubules. The same results were observed in the in vitro experiment. We also explored the role and mechanism of macrophages after AAI administration in the epithelial-mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) and found that AAI could activate the C3a/C3aR axis of macrophages to upregulate p65 expression in macrophages. p65 upregulated MMP-9 expression in macrophages not only directly but also by promoting the secretion if interleukin-6 by macrophages and then activating STAT3 in RTECs. The upregulation of MMP-9 expression could promote the EMT of RTECs. Taken together, our study demonstrated that the AAI-activated the C3a/C3aR axis of macrophages, which induced MMP-9 production, was one of the causes of renal interstitial fibrosis. Therefore, targeting the C3a/C3aR axis of macrophages is an effective therapeutic strategy for the prevention and treatment of renal interstitial fibrosis in AAN.

Cinobufagin induces acute promyelocytic leukaemia cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway.

CONTEXT: Acute promyelocytic leukaemia (APL) is a malignant hematological tumour characterized by the presence of promyelocytic leukaemia-retinoic acid receptor A (PML-RARA) fusion protein. Cinobufagin (CBG) is one of the main effective components of toad venom with antitumor properties. However, only a few reports regarding the CBG treatment of APL are available. OBJECTIVE: We explored the effect and mechanism of action of CBG on NB4 and NB4-R1 cells. MATERIALS AND METHODS: We evaluated the viability of NB4 and NB4-R1 cells treated with 0, 20, 40, and 60 nM CBG for 12, 24, and 48 h. After treatment with CBG for 24 h, Bcl-2 associated X (Bax), B-cell lymphoma 2 (Bcl-2), ß-catenin, cyclin D1, and c-myc expression was detected using western blotting and real-time polymerase chain reaction. Caspase-3 and PML-RARA expression levels were detected using western blotting. RESULTS: CBG inhibited the viability of NB4 and NB4-R1 cells. The IC50 values of NB4 and NB4-R1 cells treated with CBG for 24 h were 45.2 nM and 37.9 nM, respectively. CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner and inhibited the ß-catenin signalling pathway. DISCUSSION AND CONCLUSION: CBG induced NB4 and NB4-R1 cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the ß-catenin signalling pathway. This study proposes a novel treatment strategy for patients with APL, particularly those with ATRA-resistant APL.