通过慢病毒样颗粒载体递送九香虫来源细胞色素c基因诱导线粒体凋亡抑制HER2阳性乳腺癌OA

Objective Human epidermal growth factor receptor 2(HER2)-positive breast cancer accounts for approximately 20%of all breast cancer cases.Persistent activation of the HER2/PI3K/AKT signaling pathway enables tumor cells to evade mitochondrial apoptosis through upregulation of Bcl-2/Bcl-xL and suppression of endogenous cytochrome c(Cyt c)release.Cyt c derived from Aspongopus chinensis(A.chinensis)Dallas exhibits potential pro-apoptotic activity targeting mitochondria.This study aims to construct lentivirus-like particles(VLPs)delivering the A.chinensis-derived Cyt c and systematically evaluate their anti-tumor effects and underlying mechanisms in HER2-positive breast cancer.Methods The full-length coding sequence of Cyt c was amplified from A.chinensis tissues and cloned into the lentiviral vector pWSLV containing mCherry reporter.Recombinant plasmids were co-transfected with packaging plasmids into HEK293T cells to construct mCherry-Cyt c-VLPs(titer≥1×108 TU/mL).P24 protein was detected with Western blotting and sequencing to confirm successful packaging.Primary cells isolated from HER2-positive breast cancer tissues using a modified tissue block culture method were phenotypically characterized by immunofluorescence staining for MUC1,Ki67,HER2,estrogen receptor(ER),and progesterone receptor(PR).In vitro experiments included a PBS group,an mCherry-VLP group,and an mCherry-Cyt c-VLP group(MOI=100).① RT-qPCR and Western blotting were used to evaluate Cyt c delivery efficiency;② cell viability and migration were assessed by MTT assay(24 h and 48 h)and Transwell assay(24 h),respectively;③ cell cycle distribution was analyzed by flow cytometry,and CDK2 and Cyclin A expression levels were detected by Western blotting;④ apoptosis was detected using Annexin V-FITC/PI double staining,and the expression levels of Bcl-2,Bcl-xL,Bax,cytosolic Cyt c,cleaved Caspase-9,and cleaved Caspase-3 were determined by Western blotting.A HER2-positive patient-derived xenograft(PDX)model was further established(n=3 per group).The mice received intratumoral injections(50 µg/kg,20 µL per injection,once every 2 days for 20 d).Tumor volume and body weight were recorded,and HE staining,Western blotting,blood routine test,and liver and kidney function tests were applied to evaluate in vivo antitumor efficacy and preliminary safety.Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test,with P<0.05 considered statistically significant.Results mCherry-Cyt c-VLPs were successfully constructed,and positive P24 protein expression confirmed intact VLP viral particle packaging.The isolated primary breast cancer cells exhibited typical epithelial cobblestone morphology and positive expression of MUC1,Ki67,HER2,ER,and PR,confirming them as HER2-positive luminal B primary breast cancer cells.In vitro results:① Gene delivery:compared with the PBS and mCherry-VLP groups,mCherry-Cyt c-VLP treatment significantly increased Cyt c at both mRNA(P<0.0001)and protein levels(P<0.000 1),confirming successful delivery.②Anti-proliferation and anti-migration:mCherry-Cyt c-VLP treatment significantly reduced cell viability(P<0.05)and the number of migrated cells(P<0.000 1),whereas the empty-particle group showed no significant effect(P>0.05).③ Cell cycle arrest:flow cytometry revealed a significantly increased proportion of cells in G1 phase and a decreased proportion in S phase in the mCherry-Cyt c-VLP group(P<0.05),and Western blotting showed significantly downregulated CDK2 and Cyclin A protein levels(P<0.000 1).④Apoptosis induction:the mCherry-Cyt c-VLP group exhibited approximately 20%apoptosis rate(P<0.000 1),with significant downregulation of Bcl-2 and Bcl-xL(P<0.000 1)and significant upregulation of Bax,cytosolic Cyt c,cleaved Caspase-9 and cleaved Caspase-3(P<0.000 1),indicating that exogenous Cyt c expression activated the cytosolic Caspase cascade and thereby the mitochondrial apoptotic pathway.In vivo PDX experiments:the tumor volume and weight in the mCherry-Cyt c-VLP group were significantly lower than those in the control groups(P<0.01).HE staining of tumor tissues showed decreased cell density and disordered architecture,with mitochondrial apoptosis-related protein changes in tumor tissue consistent with in vitro results.No significant differences were found across groups in mouse body weight or in hematological,liver and kidney function indicators(P>0.05).Conclusion The A.chinensis-derived Cyt c delivered by mCherry-Cyt c-VLPs is sustainably expressed in HER2-positive breast cancer cells and directly activates the Caspase-9/3 cascade,leading to suppressed proliferation and apoptosis of tumor cells.No obvious systemic toxicity is observed in either in vitro or in vivo experiments.