Background:Terpinen-4-ol (T4O), a key constituent of tea tree essential oil and various aromatic plants, has shown promising antiproliferative and pro-apoptotic effects in melanoma and other cancer types. However, its efficacy against cutaneous squamous cell carcinoma (cSCC) remains unclear. Thus, in this study, we investigated the in vivo and in vitro effects of T4O on cSCC cell lines and preliminarily explored its impacting pathways. Methods:Using CCK8 and assay colony formation, we assessed the viability of cSCC A431, SCL-1, and COLO-16 cells treated with T40 at varying concentrations (0, 1, 2, and 4 μM). Flow cytometry was employed to evaluate T4O's effect on cSCC cell's cycle progression and apoptosis induction. Additionally, western blotting was utilized to examine the expression intensities of N-cadherin and E-cadherin, two indicative markers of the epithelial-mesenchymal transition (EMT) pathway. T4O's in vivo effect on inhibiting tumor progression was evaluated on an established xenograft tumor model. Then, the molecular mechanisms of T4O's antitumor effect were explored by an integrated genome-wide transcriptomics and proteomics study on cSCC A431c cells. Finally, calpain-2's potential mediator role in T4O's anti-tumor mechanism was investigated in calpain-2 knockdown cell lines prepared via siRNA transfection. Result:It's demonstrated that T4O treatment inhibited cSCC proliferation, clonogenicity, migration, and invasion while inducing apoptosis and suppressing the EMT pathway. T4O administration also inhibited cSCC tumorigenesis in the xenograft tumor model. RNA-sequencing and iTRAQ analysis detected significant upregulation of calpain-2 expression in T4O-treated cSCC cells. Western blotting confirmed that T4O significantly increased calpain-2 expression and promoted proteolytic cleavage of β-catenin and caspase-12, two calpain-2 target proteins. Importantly, siRNA-mediated calpain-2 knockdown relieved T4O's suppressive effect on cSCC cell proliferation and motility. Mechanistically, T4O upregulates calpain-2 expression and promotes the cleavage of β-catenin and caspase-12, with siRNA-mediated calpain-2 knockdown mitigating T4O's suppressive effects. Conclusion:These findings suggest that T4O's antitumor activity in cSCC is mediated through the upregulation of calpain-2 expression and subsequent modulation of β-catenin and caspase-12.

BackgroundSkin Cutaneous Melanoma (SKCM) incidence is continually increasing, with chemotherapy and immunotherapy being among the most common cancer treatment modalities. This study aims to identify novel biomarkers for chemotherapy and immunotherapy response in SKCM and explore their association with oxidative stress.MethodsUtilizing TCGA-SKCM RNA-seq data, we employed Weighted Gene Co-expression Network Analysis (WGCNA) and Protein-Protein Interaction (PPI) networks to identify six core genes. Gene co-expression analysis and immune-related analysis were conducted, and specific markers associated with oxidative stress were identified using Gene Set Variation Analysis (GSVA). Single-cell analysis revealed the expression patterns of Oxidative Stress-Associated Genes (OSAG) in the tumor microenvironment. TIDE analysis was employed to explore the association between immune therapy response and OSAG, while CIBERSORT was used to analyze the tumor immune microenvironment. The BEST database demonstrated the impact of the Oxidative Stress signaling pathway on chemotherapy drug resistance. Immunohistochemical staining and ROC curve evaluation were performed to assess the protein expression levels of core genes in SKCM and normal samples, with survival analysis utilized to determine their diagnostic value.ResultsWe identified six central genes associated with SKCM metastasis, among which the expression of DSC2 and DSC3 involved in the oxidative stress pathway was closely related to immune cell infiltration. DSC2 influenced drug resistance in SKMC patients. Furthermore, downregulation of DSC2 and DSC3 expression enhanced the response of SKCM patients to immunotherapy.ConclusionThis study identified two Oxidative Stress-Associated genes as novel biomarkers for SKCM. Additionally, targeting the oxidative stress pathway may serve as a new strategy in clinical practice to enhance SKCM chemotherapy and sensitivity.

目的 探讨松油烯-4-醇对宫颈癌 Siha 细胞凋亡的影响以及其可能的机制.方法 采用含 10%胎牛血清的DMEM培养基培养Siha细胞,并将其分为对照组、顺铂组(10 μmol/L)和 5、7.5、10 μmol/L松油烯-4-醇组.MTT法检测不同浓度松油烯-4-醇对Siha细胞增殖的影响,TUNEL荧光染色法和Annexin V-FITC/PI法检测Siha细胞凋亡情况,JC-1染色检测线粒体膜电位变化,RT-qPCR法检测Siha细胞Bax、cytochrome c、caspase9、HPV E6/E7 mRNA表达,Western blot法检测Siha细胞Bax、Bcl-2、cytochrome c、cleaved-caspase9 蛋白表达.结果 松油烯-4-醇(5、7.5、10 μmol/L)处理Siha细胞 24h后,细胞增殖能力减弱(P<0.05),细胞凋亡率升高(P<0.05),线粒体膜电位降低(P<0.05),细胞Bax、cytochrome c、cleaved-caspase9 蛋白表达升高(P<0.05),Bcl-2 蛋白表达降低(P<0.05),细胞Bax、cytochrome c、caspase9 mRNA表达升高(P<0.05),HPV E6/E7 mRNA表达降低(P<0.05),并呈剂量依赖性.结论 松油烯-4-醇能抑制Siha细胞体外增殖,诱导细胞凋亡,其作用机制可能与线粒体途径调控细胞Bax、Bcl-2、cytochrome c、cleaved-caspase9 的表达,并降低HPV E6/E7 mRNA表达有关.

例1男,68岁,右侧肩颈、上肢皮疹伴疼痛、麻木无力4 d.皮肤科情况:右侧肩颈部、右上肢可见暗红斑片上密集分布米粒至绿豆大水疱,疱壁紧张,疱液清,呈带状分布,未见破溃及渗出.电生理检查提示:右上肢臂丛(上干)神经损害.诊断:带状疱疹伴右上肢运动性麻痹.予抗病毒、营养神经、止痛等治疗,11个月后肌力基本恢复正常.例2男,52岁,左上肢疼痛半个月,加重伴皮疹10 d.皮肤科情况:左手、左前臂肿胀,左手可见深在绿豆大小水疱,左上肢、肩胛部可见暗红斑片上密集分布粟粒至黄豆大水疱,疱壁紧张,疱液浑浊,呈带状分布.肌电图神经传导速度(NVC)检查提示:左尺神经运动传导速度减慢.诊断:带状疱疹伴左上肢运动性麻痹.予以抗病毒、抗炎、康复训练、针灸等治疗,4个月后左上肢近端肌力基本恢复正常.

目的:研究芥子碱硫氰酸盐(ST)对人皮肤鳞状细胞癌SCL-1细胞增殖、上皮间质转化(EMT)和转移的影响,并考察其可能的作用机制.方法:将人皮肤鳞状细胞癌SCL-1细胞分为空白对照组(0.1％二甲基亚砜)和ST不同浓度组(5、10、20μmol/L),分别通过CCK-8实验、5-乙炔基-2'脱氧尿嘧啶核苷染色实验、细胞划痕实验和Transwell小室侵袭实验测定各组细胞的增殖、迁移和侵袭能力,通过Western blot实验和免疫荧光实验分别测定各组细胞中EMT相关指标N-钙黏着蛋白(N-cadherin)、E-钙黏着蛋白(E-cadherin)的表达水平.另将SCL-1细胞分为空白对照组(0.1％二甲基亚砜)、ST单用组(20μmol/L)、ST+NSC228155组[20μmol/L ST+100μmol/L NSC228155(EGFR激动剂)]和ST+SC79组[20μmol/L ST+20μmol/L SC79(PI3K/Akt激动剂)],分别通过CCK-8实验、细胞划痕实验和Transwell小室侵袭实验测定各组细胞的增殖、迁移和侵袭能力,并通过Western blot实验测定空白对照组(0.1％二甲基亚砜)和ST不同浓度组(5、10、20μmol/L)组细胞中表皮生长因子受体(EGFR)、磷脂酰肌醇3激酶(PI3K)、磷酸化磷脂酰肌醇3激酶(p-PI3K)、蛋白激酶B(Akt)和磷酸化Akt(p-Akt)蛋白的表达水平,以验证ST的作用与EGFR/PI3K/Akt信号通路的关系.另将SCL-1细胞和人正常皮肤成纤维细胞WS1分别分为空白对照组(0.1％二甲基亚砜)、ST组(20μmol/L)、ZD1839组(阳性对照,20μmol/L,EGFR抑制剂)和LY294002组(阳性对照,20μmol/L,PI3K/Akt抑制剂),采用CCK-8实验测定各组细胞的增殖能力,以评价ST的细胞毒性.结果:与空白对照组比较,5、10、20μmol/L ST组SCL-1细胞的增殖、迁移和侵袭能力均显著减弱(P<0.05);Western blot和免疫荧光实验结果显示,5、10、20μmol/L ST组SCL-1细胞中N-cadherin蛋白的表达均显著下调(P<0.05),E-cadherin蛋白的表达均显著上调(P<0.05),并且细胞中EGFR、p-PI3K、p-Akt蛋白的表达水平均显著降低(P<0.05).与ST单用组比较,ST+NSC228155组和ST+SC79组SCL-1细胞的增殖、迁移和侵袭能力均显著增强(P<0.05).与空白对照组比较,ST组WS1细胞的增殖能力差异无统计学意义(P>0.05),SCL-1细胞增殖能力显著减弱(P<0.05),ZD1839组和LY294002组两种细胞的增殖能力均显著减弱(P<0.05);与ST组比较,ZD1839组和LY294002组WS1细胞的增殖能力均显著减弱(P<0.05),但SCL-1细胞的增殖能力差异无统计学意义(P>0.05).结论:ST可能通过抑制EGFR/PI3K/Akt信号通路的活化而抑制人皮肤鳞癌SCL-1细胞的增殖、EMT和转移,且其毒副作用较小.

目的 探讨芥子碱硫氰酸盐(sinapine thiocyanate,ST)对皮肤鳞癌A431和Colo-16细胞恶性生物学行为的影响及机制.方法 以20μmol·L-1的ST与紫杉醇处理皮肤成纤维细胞,CCK-8比较两者细胞毒性;以0、5、10和20μmol·L-1的ST处理A431和Colo-16细胞,CCK-8、克隆平板、划痕和Transwell实验检测各组细胞增殖、克隆形成、迁移和侵袭;应用分子对接技术明确ST与AKT蛋白的结合;采用Western blot检测各组细胞AKT、p-AKT(S473)、β-catenin、E-cadherin、N-cadherin、Vimentin和PCNA的表达;以DMSO、ST以及ST联合AKT激动剂SC79处理A431和Colo-16细胞,采用CCK-8、克隆平板、划痕和Transwell实验检测各组细胞增殖、克隆形成、迁移和侵袭.结果 同浓度的ST毒性比紫杉醇小;ST抑制A431和Colo-16细胞增殖、克隆形成、迁移和侵袭;ST可与AKT稳定结合;ST减少A431和Colo-16细胞中p-AKT(S473)、β-catenin、N-cadherin、Vimentin和PC-NA的表达以及增加E-cadherin的表达;SC79可减轻ST的抑制作用.结论 ST通过AKT/β-catenin通路抑制皮肤鳞癌A431和Colo-16细胞的恶性生物学行为.

The prognosis of patients with human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is poorer than those with HPV-positive HNSCC. The present study aimed to identify novel and specific biomarkers of HPV-negative HNSCC using bioinformatics analysis and associated experiments. The gene expression profiles of HPV-negative HNSCC tissues and corresponding clinical data were downloaded from The Cancer Genome Atlas database and used in a weighted gene co-expression network analysis. Genes in clinically significant co-expression modules were used to construct a protein-protein interaction (PPI) network. The genes demonstrating a high degree score in the PPI network and a high correlation with tumor grade were considered hub genes. The diagnostic value of the hub genes associated with HPV-negative and HPV-positive HNSCC was analyzed using differential expression gene (DEG) analysis, immunohistochemical (IHC) staining and a receiver operating characteristic (ROC) curve analysis. Seven genes [Serrate RNA effector molecule (SRRT), checkpoint kinase 2 (CHEK2), small nuclear ribonucleoprotein polypeptide E (SNRPE), proteasome 26S subunit ATPase 2 (PSMC2), origin recognition complex subunit 5 (ORC5), S100 calcium binding protein A7 and keratinocyte differentiation associated protein (KRTDAP)] were demonstrated to be hub genes in clinically significant co-expression modules. DEG, IHC and ROC curve analyses revealed that SRRT, CHEK2 and SNRPE were significantly upregulated in HPV-negative and HPV-positive HNSCC tissues compared with in adjacent tissues, and these genes demonstrated a high diagnostic value for distinguishing HNSCC tissues. However, PSMC2, ORC5 and KRTDAP were the only differentially expressed genes identified in HPV-negative HNSCC tissues, and these genes demonstrated a high diagnostic value for HPV-negative HNSCC. PSMC2, ORC5 and KRTDAP may therefore serve as novel and specific biomarkers for HPV-negative HNSCC, potentially improving the diagnosis and treatment of patients with HPV-negative HNSCC.