Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, China; Guangzhou University of Chinese Medicine, Guangzhou, China.
Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, China; The First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, China; Guangzhou University of Chinese Medicine, Guangzhou, China.
Phytomedicine. 2023 Jan;108:154491. doi: 10.1016/j.phymed.2022.154491. Epub 2022 Oct 17.
Non-small cell lung cancer (NSCLC) accounts for almost 85% of lung cancer-related deaths worldwide. Xihuang Pill (XHP) is a representative anticancer Chinese patented medicine used to treat NSCLC in China. However, to date, a systematic analysis of XHP's antitumour effects and its impact on the immune microenvironment has not been performed.
Based on the systems biology strategy and experimental validation, the present study aimed to investigate the pharmacological mechanisms involved in treating NSCLC with XHP.
A subcutaneous tumour model was established to evaluate XHP's tumour-inhibitory effect in BALB/c nude mice. RNA sequencing (RNA-seq) and bioinformatics analysis were conducted to identify differentially expressed genes (DEGs) and signalling pathways related to XHP treatment. Network analysis based on network pharmacology and protein-to-protein networks was applied to identify the compounds and genes targeted by XHP. External data from the TCGA-NSCLC cohort were used to verify the clinical significance of XHP-targeted genes in NSCLC. The expression of survival-related candidate genes after XHP treatment was verified via qPCR. The protein expression of calcium voltage-gated channel subunit alpha 1C (CACNA1C) in different NSCLC cell lines was analysed in the Human Protein Atlas database (HPA) and DepMap Portal. Using the Estimation of STromal and Immune cells in MAlignant Tumour tissues using Expression data (ESTIMATE) algorithm and the single-sample gene set enrichment analysis (ssGSEA) algorithm uncovered the role of CACNA1C in the NSCLC tumour microenvironment (TME).
XHP (2 g/kg/d) significantly inhibited the growth of transplanted A549 tumours. RNA-seq identified a total of 529 DEGs (189 upregulated and 340 downregulated). In addition, 542 GO terms, 41 significant KEGG pathways, 9 upregulated hallmarks pathways, and 18 downregulated hallmark pathways were enriched. These GO terms and signalling pathways were closely related to cell proliferation, immunity, energy metabolism, and the inflammatory response of NSCLC. In addition, XHP's network pharmacology analysis identified 301 compounds and 1,432 target genes. A comprehensive strategic analysis identified CACNA1C as a promising gene by which XHP targets and regulates the TME of NSCLC, benefiting patient survival. CACNA1C expression was positively correlated with both the immune score and stromal score but negatively correlated with the tumour purity score. Additionally, CACNA1C expression was significantly correlated with the infiltration levels of 15 types of immune cells and the expression levels of 6 well-known checkpoint genes.
Our results show that by regulating the pathways associated with cell proliferation and immunity, XHP can suppress cancer cell growth in NSCLC. Additionally, XHP may increase the expression of CACNA1C to suppress immune cell infiltration and regulate the expression of checkpoint-related genes, thereby improving the overall survival of NSCLC patients.
非小细胞肺癌（NSCLC）占全球与肺癌相关死亡人数的近 85%。西黄丸（XHP）是一种治疗 NSCLC 的代表性抗癌中药，在中国被广泛应用。然而，迄今为止，尚未对 XHP 的抗肿瘤作用及其对免疫微环境的影响进行系统分析。
基于系统生物学策略和实验验证，本研究旨在探讨 XHP 治疗 NSCLC 的药理学机制。
建立 BALB/c 裸鼠皮下肿瘤模型，评估 XHP 的肿瘤抑制作用。通过 RNA 测序（RNA-seq）和生物信息学分析，鉴定与 XHP 治疗相关的差异表达基因（DEGs）和信号通路。基于网络药理学和蛋白质-蛋白质网络的网络分析，鉴定 XHP 靶向的化合物和基因。利用 TCGA-NSCLC 队列的外部数据，验证 XHP 靶向基因在 NSCLC 中的临床意义。通过 qPCR 验证 XHP 处理后与生存相关的候选基因的表达。在人类蛋白质图谱数据库（HPA）和 DepMap 门户中分析不同 NSCLC 细胞系中钙电压门控通道亚基 alpha 1C（CACNA1C）的蛋白表达。使用 ESTIMATE 算法和 ssGSEA 算法估计肿瘤组织中基质和免疫细胞，揭示 CACNA1C 在 NSCLC 肿瘤微环境（TME）中的作用。
XHP（2 g/kg/d）显著抑制了移植的 A549 肿瘤的生长。RNA-seq 共鉴定出 529 个 DEGs（189 个上调，340 个下调）。此外，还富集了 542 个 GO 术语、41 个显著的 KEGG 通路、9 个上调的标志性通路和 18 个下调的标志性通路。这些 GO 术语和信号通路与 NSCLC 的细胞增殖、免疫、能量代谢和炎症反应密切相关。此外，XHP 的网络药理学分析鉴定出 301 种化合物和 1432 个靶基因。综合策略分析鉴定出 CACNA1C 是一个有前途的基因，XHP 通过该基因靶向并调节 NSCLC 的 TME，有利于患者的生存。CACNA1C 的表达与免疫评分和基质评分呈正相关，与肿瘤纯度评分呈负相关。此外，CACNA1C 的表达与 15 种免疫细胞的浸润水平以及 6 个已知的检查点基因的表达水平显著相关。
本研究结果表明，XHP 通过调节与细胞增殖和免疫相关的途径，抑制 NSCLC 中的癌细胞生长。此外，XHP 可能通过增加 CACNA1C 的表达来抑制免疫细胞浸润，并调节检查点相关基因的表达，从而提高 NSCLC 患者的总生存率。
