Department of Biotechnology, Alagappa University, Karaikudi, 630 003, Tamil Nadu, India.
Anticancer Agents Med Chem. 2024;24(10):773-788. doi: 10.2174/0118715206289038240214102951.
Non-Small Cell Lung Cancer (NSCLC) is a malignancy with a significant prevalence and aggressive nature, posing a considerable challenge in terms of therapeutic interventions. Autophagy and apoptosis, two intricate cellular processes, are integral to NSCLC pathophysiology, each affecting the other through shared signaling pathways. Phytol (Phy) and α-bisabolol (Bis) have shown promise as potential anticancer agents individually, but their combined effects in NSCLC have not been extensively investigated.
The present study was to examine the synergistic impact of Phy and Bis on NSCLC cells, particularly in the context of autophagy modulation, and to elucidate the resulting differential protein expression using LCMS/ MS analysis.
The A549 cell lines were subjected to the patented effective concentration of Phy and Bis, and subsequently, the viability of the cells was evaluated utilizing the MTT assay. The present study utilized real-time PCR analysis to assess the expression levels of crucial apoptotic genes, specifically Bcl-2, Bax, and Caspase-9, as well as autophagy-related genes, including Beclin-1, SQSTM1, Ulk1, and LC3B. The confirmation of autophagy marker expression (Beclin-1, LC3B) and the autophagy-regulating protein SQSTM1 was achieved through the utilization of Western blot analysis. Differentially expressed proteins were found using LC-MS/MS analysis.
The combination of Phy and Bis demonstrated significant inhibition of NSCLC cell growth, indicating their synergistic effect. Real-time PCR analysis revealed a shift towards apoptosis, with downregulation of Bcl-2 and upregulation of Bax and Caspase-9, suggesting a shift towards apoptosis. Genes associated with autophagy regulation, including Beclin-1, SQSTM1 (p62), Ulk1, and LC3B, showed significant upregulation, indicating potential induction of autophagy. Western blot analysis confirmed increased expression of autophagy markers, such as Beclin-1 and LC3B, while the autophagy-regulating protein SQSTM1 exhibited a significant decrease. LC-MS/MS analysis revealed differential expression of 861 proteins, reflecting the modulation of cellular processes. Protein-protein interaction network analysis highlighted key proteins involved in apoptotic and autophagic pathways, including STOML2, YWHAB, POX2, B2M, CDA, CAPN2, TXN, ECHS1, PEBP1, PFN1, CDC42, TUBB1, HSPB1, PXN, FGF2, and BAG3, emphasizing their crucial roles. Additionally, PANTHER pathway analysis uncovered enriched pathways associated with the differentially expressed proteins, revealing their involvement in a diverse range of biological processes, encompassing cell signaling, metabolism, and cellular stress responses.
The combined treatment of Phy and Bis exerts a synergistic inhibitory effect on NSCLC cell growth, mediated through the interplay of apoptosis and autophagy. The differential protein expression observed, along with the identified proteins and enriched pathways, provides valuable insights into the underlying molecular mechanisms. These findings offer a foundation for further exploration of the therapeutic potential of Phy and Bis in the management of NSCLC.
非小细胞肺癌（NSCLC）是一种发病率高且侵袭性强的恶性肿瘤，在治疗干预方面构成了相当大的挑战。自噬和细胞凋亡是两种复杂的细胞过程，是 NSCLC 病理生理学的组成部分，它们通过共享的信号通路相互影响。植物醇（Phy）和α- 姜黄烯（Bis）已被证明是有前途的潜在抗癌药物，但它们在 NSCLC 中的联合作用尚未得到广泛研究。
本研究旨在探讨 Phy 和 Bis 对 NSCLC 细胞的协同影响，特别是在自噬调节方面，并通过 LCMS/MS 分析阐明由此产生的差异蛋白表达。
将 A549 细胞系暴露于专利有效浓度的 Phy 和 Bis 下，然后使用 MTT 测定法评估细胞活力。本研究利用实时 PCR 分析评估关键凋亡基因的表达水平，特别是 Bcl-2、Bax 和 Caspase-9，以及自噬相关基因，包括 Beclin-1、SQSTM1、Ulk1 和 LC3B。通过 Western blot 分析证实自噬标志物表达（Beclin-1、LC3B）和自噬调节蛋白 SQSTM1 的表达。使用 LC-MS/MS 分析发现差异表达的蛋白质。
Phy 和 Bis 的联合应用显著抑制 NSCLC 细胞生长，表明其具有协同作用。实时 PCR 分析显示向细胞凋亡转变，Bcl-2 下调，Bax 和 Caspase-9 上调，表明向细胞凋亡转变。与自噬调节相关的基因，包括 Beclin-1、SQSTM1（p62）、Ulk1 和 LC3B，表达显著上调，表明可能诱导自噬。Western blot 分析证实自噬标志物如 Beclin-1 和 LC3B 的表达增加，而自噬调节蛋白 SQSTM1 的表达显著减少。LC-MS/MS 分析显示 861 种蛋白质的差异表达，反映了细胞过程的调节。蛋白质-蛋白质相互作用网络分析突出了涉及细胞凋亡和自噬途径的关键蛋白质，包括 STOML2、YWHAB、POX2、B2M、CDA、CAPN2、TXN、ECHS1、PEBP1、PFN1、CDC42、TUBB1、HSPB1、PXN、FGF2 和 BAG3，强调了它们的关键作用。此外，PANTHER 途径分析揭示了与差异表达蛋白相关的丰富途径，表明它们参与了广泛的生物学过程，包括细胞信号转导、代谢和细胞应激反应。
Phy 和 Bis 的联合治疗对 NSCLC 细胞生长具有协同抑制作用，通过细胞凋亡和自噬的相互作用介导。观察到的差异蛋白表达以及鉴定的蛋白质和丰富的途径提供了对潜在分子机制的宝贵见解。这些发现为进一步探索 Phy 和 Bis 在 NSCLC 管理中的治疗潜力奠定了基础。
