背景:宫颈癌,主要由HPV感染引起,仍然是全球健康问题。当前的治疗面临包括耐药性和毒性的挑战。这项研究调查了E5-siRNA与化疗药物的结合,奥沙利铂和异环磷酰胺,增强HPV-16阳性宫颈癌细胞的治疗效果,靶向E5癌蛋白以克服现有疗法的局限性。
方法:用E5-siRNA转染CaSki宫颈癌细胞系,随后用奥沙利铂/异环磷酰胺治疗。定量实时PCR用于评估相关基因的表达,包括p53,MMP2,Nanog,和Caspases.细胞凋亡,细胞周期进程,使用膜联蛋白V/PI染色评估细胞活力,DAPI染色,和MTT测试,分别。此外,干性能力是通过集落形成试验确定的,通过伤口愈合试验评估细胞运动性。
结果:与对照组相比,E5-siRNA转染显着降低了CaSki细胞中E5mRNA的表达。MTT分析显示,用E5-siRNA单药治疗,奥沙利铂,或异环磷酰胺对细胞活力有中等影响。然而,联合治疗显示出协同作用,奥沙利铂的IC50从11.42×10-8M(45.36μg/ml)降至6.71×10-8M(26.66μg/ml),异环磷酰胺从12.52×10-5M(32.7μg/ml)降至8.206×10-5M(21.43μg/ml)。流式细胞术分析表明,联合治疗的细胞凋亡显着增加,细胞凋亡率从11.02%(单独的奥沙利铂)和16.98%(单独的异环磷酰胺)上升到24.8%(奥沙利铂+E5-siRNA)和34.9%(异环磷酰胺+E5-siRNA)。亚G1细胞数量从15.7%(单独的奥沙利铂)和18%(单独的异环磷酰胺)增加到21.9%(奥沙利铂E5-siRNA)和27.1%(异环磷酰胺E5-siRNA),表明细胞周期停滞。集落形成测定显示,在组合处理后,集落数量显著减少。qRT-PCR分析显示干性相关基因CD44和Nanog的表达降低,联合组中的迁移相关基因MMP2和CXCL8。凋亡相关基因Casp-3、Casp-9和pP53在联合治疗后表达增加,而BAX表达相对于对照增加而BCL2表达降低。
结论:该研究表明,E5-siRNA与奥沙利铂或异环磷酰胺联合使用可增强化疗对HPV-16阳性宫颈癌细胞的疗效。这种协同方法有效地针对癌细胞行为的多个方面,包括扩散,凋亡,迁移,和干劲。研究结果表明,这种组合策略可能允许较低的化疗剂量,从而在保持治疗功效的同时降低毒性。这项研究为靶向HPVE5提供了有价值的见解,作为针对E6和E7癌蛋白的现有疗法的补充方法,为宫颈癌综合治疗开辟新的途径。
BACKGROUND: Cervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This
study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies.
METHODS: The CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay.
RESULTS: E5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10-8 M (45.36 μg/ml) to 6.71 × 10-8 M (26.66 μg/ml) and Ifosfamide from 12.52 × 10-5 M (32.7 μg/ml) to 8.206 × 10-5 M (21.43 μg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control.
CONCLUSIONS: The
study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.