Matrine Reverses Sorafenib Resistance By Inhibiting The Notch1 Signaling Pathway In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common pathological type of primary liver cancer and the third leading cause of cancer-related death.Sorafenib is the first tyrosine kinase inhibitor approved for the first-line treatment of advanced HCC.This drug can prolong median survival in HCC patients but only by 3-5 months because of drug resistance.Although the combined application of sorafenib with immunotherapy has been reported to reduce sorafenib resistance to a certain extent,because of the superposition of adverse rections,some patients have developed intolerance.Therefore,exploring the predictive markers of sorafenib resistance and survival prognosis,screening patients who can benefit from sorafenib treatment,exploring the potential mechanism underlying sorafenib resistance,and developing novel combination therapy strategies for improving the outcomes of advanced HCC patients are essential.The inflammatory state of the body is closely associated with the response to tumor treatment.The systemic inflammation response index(SIRI)is an effective indicator of inflammation and is increasingly crucial for predicting drug resistance and survival prognosis.However,the prognostic value of SIRI in sorafenib-treated HCC patients remains unclear.Abnormal activation of the Notch1 signaling pathway is tightly associated with tumor initiation,development,invasion,and metastasis.Notch1 overexpression induces epithelial–mesenchymal transition(EMT)in ovarian and breast cancer,thereby leading to cytotoxic drug resistance.However,whether the Notch1 signaling pathway mediates sorafenib resistance in HCC remains unclear.Matrine is an effective alkaloid extracted from Sophora alopecuroides.It could inhibit the proliferation,metastasis,and invasion of tumors and reverse multidrug resistance.In our previous study,matrine was found to prevent HCC and alleviate liver function injury by inhibiting the Notch1 signaling pathway.Whether matrine combined with sorafenib can enhance efficacy and reduce toxicity and its underlying mechanism need to be elucidated further.We here first evaluated the ability of SIRI to predict sorafenib resistance and survival prognosis to explore new predictive markers for sorafenib resistance and survival prognosis.Second,the activation of the Notch1 signaling pathway in sorafenib-resistant HCC cells and parental HCC cells was compared.Then,the Notch1 signaling pathway was blocked to observe whether this pathway was involved in sorafenib resistance development.Third,we evaluated whether matrine could reverse sorafenib resistance and explored the underlying mechanisms.Finally,a xenograft HCC model in nude mice was established for in vivo verification.Part One Systemic inflammatory response index predicts sorafenib resistance and survival prognosis in patients with HCCObjective: To explore the predictive value of SIRI in sorafenib resistance and survival prognosis of sorafenib-treated HCC patients and screen effective biomarkers for predicting sorafenib resistance and survival prognosis.Methods: In this retrospective study conducted at the Fourth Hospital of Hebei Medical University,352 HCC patients were treated with sorafenib alone and/or immune checkpoint inhibitors(ICIs).The receiver operating characteristics curve was used to determine the best cutoff value.Independent risk factors were analyzed using the Cox proportional risk model.Results:1.The OS,PFS,ORR and DCR in the high baseline SIRI group were significantly lower than those in the low baseline SIRI group.After treatment,the OS and PFS of patients with increased SIRI were significantly lower than those with decreased SIRI.2.SIRI was an independent predictor of OS and PFS.3.High SIRI was associated with high tumor load,poor liver reserve capacity,poor general physical status and late disease stage.4.SIRI was significantly and negatively correlated with CD3+,CD4+and CD8+ T-cell counts.Summary: SIRI can serve as a potential biomarker for predicting sorafenib resistance and survival prognosis.Patients with high baseline SIRI and increased SIRI after treatment were more likely to develop sorafenib resistance and have a poor prognosis.SIRI was closely related to lymphocyte subsets and can be used to noninvasively assess immunoinflammatory responses and predicte the efficacy of sorafenib.For patients with increased SIRI and susceptibility to sorafenib resistance,other interventions need to be taken earlier to improve their prognosis.Part Two Abnormal activation of the Notch1 signaling pathway inducesEMT and leads to sorafenib resistance in HCC cellsObjective: To investigate the role of the Notch1 signaling pathway and its internal relationship with EMT in sorafenib resistance development in HCC patients.Methods: The parental Hep G2/Huh7 cells,sorafenib-resistant Hep G2-SR/Huh7-SR cells,and the Hep G2-SR/Huh7-SR cells with an inhibited Notch1 signaling pathway were treated with sorafenib.The colony formation,Transwell,and cell scratch assays were used to detect cell invasion,metastasis,and proliferation.Flow cytometry was used to detect the cell cycle and apoptosis.The expression levels of Notch1,Jagged1,Hes1,and EMTrelated markers(E-cadherin,N-cadherin,and vimentin)were detected through RT-PCR,western blotting,and immunofluorescence.Results:1.Compared with the Hep G2/Huh7 cells,sorafenib-mediated inhibition of the proliferation,invasion,and metastasis of the Hep G2-SR/Huh7-SR cells was significantly weakened.2.N-cadherin and vimentin levels were significantly higher in the Hep G2-SR/Huh7-SR cells than in the Hep G2/Huh7 cells,whereas the E-cadherin levels were significantly lower than those in the Hep G2/Huh7 cells.The sorafenib-resistant HCC cells exhibited an enhanced EMT phenotype.3.Compared with the Hep G2/Huh7 cells,Notch1,Jagged1,and Hes1 expression levels were significantly increased in the Hep G2-SR/Huh7-SR cells.The Notch1 signaling pathway was activated in the sorafenib-resistant HCC cells.4.When the Notch1 signaling pathway was blocked using the Notch1 signaling pathway inhibitor,the inhibitory actions of sorafenib on the proliferation,invasion,and metastasis of Hep G2-SR/Huh7-SR cells were enhanced.Blocking the Notch1 signaling pathway successfully reversed EMT,increased E-cadherin levels,and decreased N-cadherin and vimentin levels in the Hep G2-SR/Huh7-SR cells.Summary: Abnormal activation of the Notch1 signaling pathway induced EMT,a key factor involved in sorafenib resistance development in HCC.Inhibition of the Notch1 signaling pathway could reverse EMT and enhance the therapeutic efficacy of sorafenib in sorafenib-resistant HCC cells.The mechanism underlying the Notch1 signaling pathway-mediated sorafenib resistance may offer a new target for HCC treatment.Part Three Matrine overcomes sorafenib resistance by inhibiting the Notch1 signaling pathway and reversing EMT in HCC cellsObjective: To investigate whether matrine could increase the therapeutic effect of sorafenib on sorafenib-resistant HCC cells and the underlying mechanism.Methods: Hep G2-SR/Huh7-SR cells were treated with sorafenib/matrine monotherapy,matrine combined with sorafenib,and matrine combined with sorafenib and the Notch1 signaling pathway activator.Cell proliferation,invasion,and metastasis were detected through the clonal formation,cell scratch,and Transwell assays in different treatment groups.The cell cycle and apoptosis in different treatment groups were detected through flow cytometry.The expression levels of Notch1 signaling pathway-related indices(Notch1,Jagged1,and Hes1)and EMT-related indices(E-cadherin,N-cadherin,and vimentin)were detected through western blotting,immunofluorescence,and RT-PCR.Results:1.Compared with the sorafenib treatment group,the combination treatment group exhibited stronger inhibitory effects on the proliferation,invasion,and metastasis of the Hep G2-SR/Huh7-SR cells,as well as a stronger effect on apoptosis promotion.Matrine and sorafenib exhibited a synergistic antitumor effect.2.Compared with the sorafenib treatment group,N-cadherin and vimentin levels in the Hep G2-SR/Huh7-SR cells were significantly decreased and E-cadherin level was significantly increased in the combination treatment group.Matrine combined with sorafenib reversed the EMT phenotype of the sorafenib-resistant HCC cells.3.Compared with the sorafenib treatment group,Notch1,Jagged1,and Hes1 expression levels in the Hep G2-SR/Huh7-SR cells were significantly reduced in the combination treatment group.Matrine inhibited the Notch1 signaling pathway in the sorafenib-resistant HCC cells.4.The Notch1 signaling pathway was activated by the Notch1 signaling pathway activator,thereby weakening the inhibitory effects of combination treatment on the proliferation,invasion,and metastasis of the Hep G2-SR/Huh7-SR cells.Meanwhile,the ability of the combination treatment to reverse the EMT phenotype of the sorafenib-resistant HCC cells was weakened.Summary: Matrine combined with sorafenib could effectively enhance the therapeutic effect of sorafenib on the sorafenib-resistant HCC cells,and its underlying mechanism was the inhibition of the Notch1 signaling pathway and EMT reversal.Part Four Matrine can overcome sorafenib resistance by inhibiting the Notch1 signaling pathway and reversing EMT in an animal model of HCCObjective: To investigate the effect of the Notch1 signaling pathway on EMT and sorafenib resistance,the effect of matrine on this pathway,and whether the combination of matrine and sorafenib has the same antitumor effect in vivo as at the cellular level.Methods: Hep G2 cell and Hep G2-SR cell xenograft tumor models were established in nude mice.Tumor volume and tumor weight of the two cell groups(Hep G2 cell group and Hep G2-SR cell group)and two treatment groups(sorafenib treatment group and sorafenib combined with matrine group)were monitored to evaluate tumor proliferation and anti-HCC efficacy in vivo.Changes in the body weight and liver function of the tumor-bearing mice in different treatment groups were dynamically monitored to evaluate drug tolerance.Histopathological differences were analyzed through HE staining.E-cadherin,N-cadherin,and vimentin expressions in tumor tissues were detected through immunohistochemistry.The Notch1,Jagged1,and Hes1 expression levels were detected through RT-PCR and western blotting.Results:1.At the same sorafenib concentration,tumor volume and tumor weight were significantly higher in the Hep G2-SR cell group than in the Hep G2 cell group.HE staining revealed that the necrotic area of the tumor tissue was significantly smaller in the Hep G2-SR cell group than in the Hep G2 cell group.The expression levels of mesenchymal markers(N-cadherin and vimentin)were significantly increased in the Hep G2-SR cell group,whereas that of the epithelial marker(E-cadherin)was significantly decreased.Expression levels of the Notch1 signaling pathway markers(Notch1,Jagged1,and Hes1)were also significantly higher in the Hep G2-SR cell group than in the Hep G2 cell group.2.In the Hep G2-SR tumor-bearing mice,the tumor volume and tumor weight of the sorafenib treatment group were significantly higher than those of the combination treatment group.HE staining revealed that the combination treatment group had a larger necrotic area of the tumor tissue.Compared with the combination treatment group,N-cadherin and vimentin expression levels were increased,whereas the E-cadherin expression level was significantly decreased in the sorafenib treatment group.Moreover,Notch-1,Jagged-1,and Hes-1 expression levels were significantly higher in the sorafenib treatment group than in the combination treatment group.3.No significant loss of weight was observed in the mice during treatment.The combination treatment could alleviate sorafenib-induced liver injury.The tumor-bearing mice tolerated the combination treatment well.Summary: This part of the study confirmed that abnormally activated Notch1 signaling pathway-induced EMT was the key factor leading to sorafenib resistance,and matrine could reverse EMT by inhibiting the Notch1 signaling pathway to overcome sorafenib resistance with good safety in vivo.Conclusions: SIRI is an effective biomarker for predicting sorafenib resistance and survival prognosis in HCC patients,providing a satisfactory risk assessment in HCC patients receiving sorafenib-based therapy.Abnormal activation of the Notch1 signaling pathway induces EMT,a key factor leading to sorafenib resistance.Matrine reverses EMT by inhibiting the Notch1 signaling pathway to overcome sorafenib resistance and alleviate sorafenib-related liver injury with good safety.