Anticancer Effects of Key Constituents of Pileostegia tomentella via ROS-Mediated Classical Apoptosis in Non-Small Cell Lung Cancer Cells

Summary

This protocol presents a method to evaluate reactive oxygen species (ROS)-mediated apoptosis induced by natural compounds in non-small cell lung cancer (NSCLC) cells.

Abstract

This study explores the anticancer potential of Pileostegia tomentella, with a particular focus on its ability to induce classical apoptosis in H1299 non-small cell lung cancer (NSCLC) cells through reactive oxygen species (ROS)-mediated pathways. Using a bioactivity-guided isolation approach, two active compounds, umbelliferone (UMB) and epi-Vogeloside, were identified as the major cytotoxic constituents. Both compounds exhibited significant inhibitory effects on cell viability and effectively triggered apoptotic cell death in H1299 cells. Mechanistic investigations demonstrated that treatment with UMB and epi-Vogeloside led to a marked increase in intracellular ROS levels, implicating oxidative stress as a key mediator. Western blot analysis revealed increased levels of cleaved caspase-3 and decreased expression of the anti-apoptotic protein Bcl-2, indicating activation of the intrinsic mitochondrial apoptotic pathway. Additionally, transcriptomic profiling showed widespread alterations in gene expression, further supporting disruption of cellular homeostasis. These results suggest that Pileostegia tomentella and its active constituents hold promise as potential therapeutic agents for lung cancer.

Introduction

Cancer presents a formidable global public health challenge, characterized by a substantial annual mortality rate. Non-small cell lung cancer (NSCLC) is the principal global driver of cancer-related fatalities, constituting approximately 85% of lung cancer incidences¹. Carcinogenesis, an inherently complex interplay of molecular and cellular processes, is critically influenced by disruptions in reactive oxygen species (ROS) homeostasis. ROS, which include unstable oxidants such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, are byproducts of normal cellular metabolism and execute essential functions in signaling, proliferation,....

Protocol

The reagents and equipment used in this study are listed in the Table of Materials.

1. Chemical composition of Pileostegia tomentella and NSCLC target prediction

The chemical constituents of Pileostegia tomentella were analyzed using high-performance liquid chromatography combined with linear ion trap-Orbitrap mass spectrometry (HPLC-LTQ/MS). Extracts were dissolved in appropriate solvents and filtered before injection. Chromatographic separation was achieved using a reversed-phase column under a gradient elution program tailored for the compound classes. Mass spe....

Results

Screening of key components of Pileostegia tomentella against lung cancer
An overview of the experimental design is presented in Figure 1. HPLC-LTQ/Orbitrap MS was employed to characterize seven principal bioactive constituents present in Pileostegia tomentella (Table 3). Subsequently, an in silico target prediction was conducted on these compounds using the SwissTargetPrediction database

Discussion

Non-small cell lung cancer (NSCLC) remains a pressing global health concern, characterized by its aggressive biology, rapid clinical progression, and high metastatic potential, frequently resulting in poor patient outcomes¹². Although standard interventions, such as surgical resection, radiotherapy, and chemotherapy, offer therapeutic benefit in early-stage disease, their effectiveness diminishes in advanced stages due to the emergence of drug resistance and the burden of systemic toxicity. The un.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V–PE / 7-AAD Apoptosis Detection Kit	BD Biosciences	556547	Flow cytometry-based apoptosis detection
Antibody: BAX	Abcam	ab32503	Western blot for pro-apoptotic protein
Antibody: Bcl-2	Abcam	ab182858	Western blot/immunofluorescence
Antibody: Cleaved Caspase-3	Abcam	ab32042	Western blot / IF
Antibody: GAPDH	Abcam	ab8245	Internal control in Western blot
Antibody: PARP1	Abcam	ab32064	Western blot for apoptosis marker
Antibody: Tubulin	Abcam	ab78078	Internal control in Western blot
ChemDraw	PerkinElmer Informatics	CCG821391	Structure drawing of small molecules
Chemiluminescence Imaging System	Tanon Science & Technology	Tanon 5200	For visualizing protein bands
Cytoscape	Cytoscape.org	https://cytoscape.org	Network construction and analysis
DAPI Nuclear Stain	Sigma	D9542	Blue nuclear counterstain for IF
DAVID	NIH	https://david.ncifcrf.gov	Functional annotation and KEGG pathway enrichment
DMSO	Sigma	MFCD00002089	Used to dissolve formazan crystals
Ethanol (for extraction)	Sigma	MFCD00003568	Ultrasonic-assisted extraction solvent
Ethyl Acetate	Sigma	MFCD00009171	Used for bioactive fractionation
FITC-conjugated Secondary Antibody	Abcam	ab6785	Used in immunofluorescence
Flow Cytometer	Thermo Scientific	A29001	Used for apoptosis and ROS detection
Fluorescence Microscope	Olympus Corporation	BX53F	For imaging immunofluorescence slides
Freeze Dryer	Labconco Corporation	7670520	For sample lyophilization and preservation
GeneCards	Weizmann Institute	https://www.genecards.org	Gene database for disease relevance
HPLC-LTQ-Orbitrap Mass Spectrometry	Thermo Scientific	IQLAAEGAAVFACZMAIK	Compound identification by MS
Illumina NovaSeq Sequencing Platform	Illumina Inc.	20012850	Used for RNA-seq
LDH Cytotoxicity Assay Kit	Beyotime	C0016	For measuring LDH release as cytotoxicity indicator
Microplate Reader	Thermo Scientific	VL0000D0	For absorbance measurements in MTT and LDH assays
MTT Cell Viability Assay Kit	Beyotime	C0009S	Used for MTT assay to assess cell viability
qPCR Machine	Thermo Scientific	4485701	For gene expression analysis
R software	R Foundation	https://www.r-project.org	Statistical computing and pathway plotting
Reverse Transcription Kit (RT Kit)	Vazyme	MR201	For cDNA synthesis
RNA-seq Library Prep Kit	Illumina Inc.	20020594	Library preparation for sequencing
SwissTargetPrediction	Swiss Institute	http://www.swisstargetprediction.ch	Compound-target prediction tool
SYBR Green qPCR Mix	Vazyme	Q311-02	For quantitative PCR
TRIzol Reagent (RNA extraction)	Sigma	T3934	For total RNA extraction
Ultrasonic Cleaner	Branson	M1800-CPXH	For ultrasonic-assisted extraction
Western Blot Apparatus	Thermo Scientific	A56727	Gel electrophoresis and protein transfer