Phytol and α-Bisabolol Synergy Induces Autophagy and Apoptosis in A549 Cells and
Additional Molecular Insights through Comprehensive Proteome Analysis via Nano
LC-MS/MS
Authors: Kiruthiga Chandramohan , Niharika Kambati , Devi Kasi Pandima 
Published: February 2024

Background:
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.

Objective:
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.

Methods:
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.

Results:
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.

Conclusion:
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.