Synergistic anti-hepatoma effect of triptolide and quercetin co-inhibition and interaction with Janus kinase and mammalian target of rapamycin signal pathway.

[BACKGROUND] Liver carcinoma, as a major global health concern due to its high incidence and mortality rates. Despite advancements in diagnostic and treatment methodologies, outcomes for hepatocellular carcinoma remain unsatisfactory. In response to these limitations, patients increasingly turn to alternative therapies such as traditional Chinese medicine, which has demonstrated potential in enhancing quality of life and prolonging survival in combination with conventional treatments. Triptolide (TP) and quercetin, as two broad-spectrum antitumor activities, act through multiple mechanisms, and whether the combination of them can provide a synergistic effect to improve the treatment effect. To optimize the dosage combination of TP and quercetin to maximize their therapeutic benefits in treating liver cancer, potentially advancing the field of drug combination therapy. This approach seeks to explore new treatment strategies and elucidate the underlying mechanisms that could lead to improved outcomes for hepatocellular carcinoma patients facing limited effective treatment options.

[AIM] To investigate the synergistic anti-hepatoma effect of TP and quercetin and elucidate the underlying molecular mechanism involving the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and mammalian target of rapamycin (mTOR) signaling pathways.

[METHODS] The study utilized 5-week-old female BALB/c-nu mice for establishing a liver cancer subcutaneous transplant tumor model. TP and quercetin were administered intraperitoneally over 21 days to evaluate the effectiveness of the combination, with monitoring of tumor growth. IncuCyte Zoom and CompuSyn software were employed to analyze drug effects for different dose combination on cell proliferation and synergy. Various assays such as CCK-8 cell proliferation analysis, plate cell clone formation, cell scratch experiments, Transwell migration and invasion assays, Annexin V-FITC flow cytometry, and western blotting using specific antibodies were employed to assess cell apoptosis, migration, invasion. Then transcriptome analysis was used RNA sequencing to find the potential synergistic mechanisms and proved by western blotting.

[RESULTS] , the combination therapy significantly slowed down tumor growth compared to the control group, quercetin alone group, and TP alone group. The tumor inhibition rates were 28.91% (quercetin), 28.8% (TP), and 59.3% (combination therapy), respectively. The determination of IncuCyte Zoom and CCK-8 confirmed that there is a concentration gradient and time gradient effect on tumor inhibition, with the synergistic effect of 25 nmol/L TP and 100 μmol/L quercetin being the best. Platelet cell clone formation and cell wound scratch assay showed that the combination group had better inhibitory effects. Transwell analysis showed a decrease in migration and invasion in the combination therapy group. Flow cytometry showed that over time, cell apoptosis increased after combination therapy. Transcriptome analysis emphasizes unique pathways influenced by the combination (JAK-STAT and mTOR signaling pathways) and has been validated at the protein level.

[CONCLUSION] Compared with a single drug, the specific metering combination of TP and quercetin has enhanced anti-tumor effects, mediated by inhibition of cell proliferation, inducing cell apoptosis and inhibiting migration/invasion. This synergistic effect is closely related to the simultaneous inhibition of signaling pathways JAK-STAT and mTOR concurrently.