Novel strategies in HPV‑16‑related cervical cancer treatment: An in vitro study of combined siRNA-E5 with oxaliplatin and ifosfamide chemotherapy

• Published in: Gene 2025-01, Vol.932, p.148904, Article 148904
• Main Authors: Rasizadeh, Reyhaneh, Shiri Aghbash, Parisa, Mokhtarzadeh, Ahad, Poortahmasebi, Vahdat, Ahangar Oskouee, Mahin, Sadri Nahand, Javid, Amini, Mohammad, Zahra Bahojb Mahdavi, Seyedeh, Hossein Yari, Amir, Bannazadeh Baghi, Hossein
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Cell Line, Tumor; Cell Movement - drug effects; Cell Proliferation - drug effects; Cell Survival - drug effects; Cervical cancer; Combination therapy; E5 oncoprotein; Female; Gene Expression Regulation, Neoplastic - drug effects; Gene therapy; Human papillomavirus 16 - genetics; Humans; Ifosfamide; Ifosfamide - pharmacology; Oncogene Proteins, Viral - genetics; Oxaliplatin; Oxaliplatin - pharmacology; Papillomavirus Infections - drug therapy; Papillomavirus Infections - genetics; Papillomavirus Infections - virology; RNA, Small Interfering - genetics; siRNA; Uterine Cervical Neoplasms - drug therapy; Uterine Cervical Neoplasms - genetics; Uterine Cervical Neoplasms - virology
• ISSN: 0378-1119; 1879-0038; 1879-0038
• DOI: 10.1016/j.gene.2024.148904

[Display omitted] •E5-siRNA enhances sensitivity of cervical cancer cells to Oxaliplatin and Ifosfamide.•Combination therapy increases apoptosis and arrests cell cycle in sub-G1 phase.•E5-siRNA with chemotherapy reduces stemness features in CaSki cells.•Combined treatment significantly inhibits cell migration and metastasis-related genes.•Synergistic approach allows for lower chemotherapy doses, potentially reducing side effects.•Findings support combined gene silencing and chemotherapy as a promising cancer treatment. 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. 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. 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