Takahashi Shuhei, Yoshida Koki, Paudel Durga, Morikawa Tetsuro, Uehara Osamu, Harada Fumiya, Ariwansa Dedy, Giri Sarita, Sato Jun, Nagayasu Hiroki, Abiko Yoshihiro
Division of Oral Medicine and Pathology, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan.
Advanced Research Promotion Center, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan.
Discov Oncol. 2025 Jul 9;16(1):1293. doi: 10.1007/s12672-025-02928-y.
This study explored the potential of Zebularine (Zeb), a DNA methyltransferase inhibitor (DNMTi), and Valproic acid (Vpa), a histone deacetylase inhibitor (HDACi), as a combined treatment strategy for OSCC.
OSCC cell lines, HSC4 (well-differentiated type) and SAS (poorly differentiated type), were cultured and treated with Zeb, Vpa, and their combinations. Cell viability, mRNA expression of P16, P21, NPY, and RASSF1 using quantitative reverse transcription polymerase chain reaction (qRT-PCR), DNA methylation using methylation-specific PCR (qMSP), and in situ HDAC activity were analyzed in vitro. In vivo, a xenograft tumor formation assay was conducted using male BALB/Slc-nu nude mice, in accordance with the Basel Declaration and The ARRIVE guidelines 2.0. Tumor samples were analyzed by qRT-PCR, and qMSP.
In vitro experiments using the HSC4 and SAS cell lines revealed significant cytotoxic effects and upregulation of tumor suppressor genes (P16, P21, NPY, and RASSF1) after treatment with Zeb + Vpa. In vivo xenograft assay in nude mice treated with Zeb + Vpa demonstrated reduced tumor volume in HSC4 cell-transplanted tumors without significant adverse effects on the body weights of the mice, whereas no significant reduction in tumor size was observed in SAS cell-transplanted tumors compared with controls. Molecular analysis confirmed elevated gene expression levels and reduced DNA methylation percentages in the treated tumors, with a more pronounced effect in HSC4 compared to SAS.
These findings suggest that the combination of Zeb and Vpa holds promise as an effective and low-toxicity therapeutic strategy for well-differentiated type of OSCC.
本研究探讨了DNA甲基转移酶抑制剂（DNMTi）泽布替尼（Zebularine，Zeb）和组蛋白去乙酰化酶抑制剂（HDACi）丙戊酸（Valproic acid，Vpa）联合治疗口腔鳞状细胞癌（OSCC）的潜力。
培养口腔鳞状细胞癌细胞系HSC4（高分化型）和SAS（低分化型），并用Zeb、Vpa及其组合进行处理。体外分析细胞活力、使用定量逆转录聚合酶链反应（qRT-PCR）检测P16、P21、NPY和RASSF1的mRNA表达、使用甲基化特异性PCR（qMSP）检测DNA甲基化以及原位HDAC活性。在体内，根据《巴塞尔宣言》和《ARRIVE指南2.0》，使用雄性BALB/Slc-nu裸鼠进行异种移植瘤形成试验。通过qRT-PCR和qMSP分析肿瘤样本。
使用HSC4和SAS细胞系进行的体外实验显示，用Zeb + Vpa处理后具有显著的细胞毒性作用，并上调了肿瘤抑制基因（P16、P21、NPY和RASSF1）。在接受Zeb + Vpa处理的裸鼠体内异种移植试验中，HSC4细胞移植瘤的肿瘤体积减小，且对小鼠体重无明显不良影响，而与对照组相比，SAS细胞移植瘤的肿瘤大小未观察到显著减小。分子分析证实，处理后的肿瘤中基因表达水平升高，DNA甲基化百分比降低，与SAS相比，HSC4中的效果更明显。
这些发现表明，Zeb和Vpa的组合有望成为高分化型口腔鳞状细胞癌的一种有效且低毒的治疗策略。
