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, differentiation, and apoptosis². An imbalance in ROS concentrations can precipitate oxidative stress, potentially activating programmed cell death pathways, notably autophagy and apoptosis, thereby impeding cancer cell proliferation and survival³. Consequently, ROS exhibit a dual role in oncology, acting as both promoters and suppressors of tumor progression. In the last decade, therapeutic strategies for NSCLC have advanced significantly, with the development of novel anticancer agents designed to exploit this dichotomous nature of ROS representing a particularly vibrant area of investigation⁴.

Numerous chemically synthesized anticancer agents, such as adriamycin (doxorubicin), cisplatin, and cyclophosphamide, are known to induce apoptosis in cancer cells by promoting ROS production⁵. Despite advancements in targeted therapies and immunotherapeutic antibodies, platinum-based chemotherapy continues to be the cornerstone of treatment for NSCLC patients. Nevertheless, the emergence of resistance and adverse side effects from chemotherapy necessitates the development of novel anti-NSCLC drugs. Consequently, exploring natural sources for anticancer compounds is increasingly recognized for its importance and potential. Recent research has highlighted that certain plant extracts and their active components exhibit potent anticancer properties, characterized by low toxicity, high specificity, and the ability to target multiple pathways⁶. Notably, some of these natural compounds can modulate ROS levels and signaling⁷, thereby inducing autophagy or apoptosis in cancer cells and exerting therapeutic effects^8,9.

Pileostegia tomentella Hand. Zucc, a perennial herb from the Clematis genus within the Buttercupaceae family, is predominantly found in regions like Japan, North Korea, and China. The plant's various components, including roots, stems, leaves, and flowers, are known for their medicinal properties, offering benefits such as heat clearance, detoxification, swelling reduction, pain relief, and antibacterial and anti-inflammatory effects. Recent research has shed light on the anticancer activities of Hoshi Hairy Vine extracts or active ingredients, demonstrating tumor-suppressive effects on a range of cancers, including glioma, breast, colon, and cervical cancer¹⁰. However, the specific anticancer mechanisms of Pileostegia tomentella, particularly its potential role in modulating ROS to induce autophagy or apoptosis in cancer cells, remain largely unexplored.

Despite emerging interest in natural compounds for cancer therapy, the pro-apoptotic mechanisms of key constituents from Pileostegia tomentella in lung cancer remain unexplored. This study pioneers the identification and mechanistic dissection of umbelliferone and epi-Vogeloside as selective ROS-dependent apoptosis inducers in NSCLC.

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 spectrometry data were collected in positive electrospray ionization mode across a mass range of 100-1500 m/z. Molecular structures of identified compounds were drawn using generic chemical drawing software and saved in SDF format. These structural files were submitted to an online target prediction tool to identify potential biological targets, with the top 20 targets selected for further validation. NSCLC-associated targets were retrieved from a publicly accessible gene database. The overlapping targets from both datasets were identified as potential therapeutic targets of Pileostegia tomentella.

2. Component-target network construction

Protein-protein interaction data for predicted targets were obtained from an open-access protein interaction database. Interaction data were imported into general network visualization software to construct the compound-target interaction network. Network topological properties, including degree centrality, betweenness, and closeness, were analyzed using built-in analytical tools to identify key regulatory nodes.

3. KEGG pathway enrichment analysis

Functional enrichment of predicted target proteins was performed using KEGG pathway analysis via publicly available enrichment tools and statistical software. Pathways with adjusted p-values less than 0.05 were considered significant. Significant pathways were integrated with compound-target data to build a "compound-target-pathway" interaction network using generic network visualization software. Network topology analysis highlighted key biological regulatory hubs, illustrating the multi-component and multi-target mechanisms of Pileostegia tomentella.

4. Extraction of Pileostegia tomentella constituents

Dried vine stems of Corydalis spinosa were grounded into a fine powder. The powdered material was extracted with 70% ethanol under ultrasonic treatment at room temperature for a defined period. The crude extract was filtered and concentrated under reduced pressure. Liquid-liquid partitioning was performed using solvents of increasing polarity, including petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate fraction, which showed the highest biological activity in preliminary assays, was collected, concentrated, freeze-dried, and stored at -20 °C until further use.

5. Cell line and treatment conditions

The human lung cancer cell line H1299 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were seeded at an appropriate density and allowed to adhere overnight before treatment. The cells were exposed to varying concentrations (500 µg/mL, 750 µg/mL, and 1,000 µg/mL) of two test compounds, UMB and epi-Vogeloside, as listed in Table 1. After 24 h of incubation under standard culture conditions, the cells were harvested for subsequent analyses to assess the biological effects of the compounds.

6. MTT assay for cell viability

The cytotoxic effects of the compounds were evaluated using a colorimetric MTT assay. H1299 cells were seeded into 96-well plates at a defined density and allowed to adhere overnight. Cells were then treated with the test compounds for 72 h under standard culture conditions. Following treatment, a solution of MTT was added to each well and incubated to allow viable cells to convert MTT into insoluble purple formazan crystals. After incubation, the medium was removed, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm using a microplate reader. The absorbance values correspond to cell viability, providing a quantitative assessment of compound cytotoxicity.

7. Assessment of membrane integrity via LDH release

Cell membrane integrity was assessed by measuring lactate dehydrogenase (LDH) release as an indicator of cytotoxicity. H1299 cells were treated with the test compounds for 24 h, 48 h, or 72 h. At each time point, culture supernatants were collected and subjected to an LDH activity assay according to the manufacturer's protocol. Since LDH is released only from cells with damaged membranes, the absorbance values measured reflect the extent of membrane damage induced by the compounds over time.

8. Flow cytometric analysis of apoptosis

Apoptosis induction was assessed by flow cytometry using Annexin V and 7-Aminoactinomycin D (7-AAD) staining. Following treatment with the test compounds, H1299 cells were collected by centrifugation, washed with cold phosphate-buffered saline, and resuspended in binding buffer. Cells were then incubated with Annexin V conjugated to a fluorescent dye and 7-AAD according to the staining protocol. Samples were immediately analyzed by flow cytometry. This method allowed differentiation among viable cells (unstained), early apoptotic cells (Annexin V positive, 7-AAD negative), and late apoptotic or necrotic cells (positive for both stains).

9. Intracellular ROS quantification

Intracellular reactive oxygen species (ROS) levels were measured using a fluorescence-based assay. H1299 cells were seeded in 12-well plates at a density of 1 × 10⁵ cells per well and cultured until reaching approximately 80% confluence. Cells were then treated with the designated compounds for 72 h. Following treatment, cells were incubated with 1 µM of the ROS-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C for 30 min in the dark. After incubation, cells were harvested, and fluorescence intensity was measured at an emission wavelength of 525 nm using a fluorescence microplate reader. The fluorescence intensity corresponds to the intracellular ROS levels.

10. Real-time quantitative PCR (RT-qPCR)

H1299 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and cultured until reaching approximately 80% confluence. Cells were then treated with the designated compounds for 72 h. Total RNA was extracted using a widely used phenol-chloroform method. Complementary DNA (cDNA) synthesis was performed using a reverse transcription kit according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was conducted using a real-time PCR system with specific primers (listed in Table 2). Relative gene expression levels were calculated using the comparative Ct method, with housekeeping genes used as internal controls.

11. Immunoblotting for protein expression

Western blot analysis was conducted on H1299 cells cultured and treated. Total protein was extracted using an appropriate lysis buffer, and lysates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C. Protein concentrations were determined using a spectrophotometric assay. Equal amounts of protein from each sample were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% non-fat dry milk for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies specific for Bcl-2, caspase-3, PARP1, BAX, GAPDH, and Tubulin at optimized dilutions. After washing, membranes were incubated with appropriate secondary antibodies for 2 h at room temperature. Protein bands were visualized and imaged using a chemiluminescent substrate. Densitometric analysis was performed with image analysis software, and band intensities were normalized to GAPDH and Tubulin as loading controls.

12. Immunofluorescence microscopy of Caspase-3

The expression and subcellular localization of caspase-3, a key apoptotic protein, were evaluated by immunofluorescence staining. After treatment, H1299 cells were fixed with paraformaldehyde, permeabilized with a mild detergent solution, and incubated with a primary antibody against caspase-3. A fluorescently labeled secondary antibody was then applied to detect the primary antibody signal. Cell nuclei were counterstained using a DNA-binding fluorescent dye. The stained cells were visualized under a fluorescence microscope. The intensity and cellular distribution of the fluorescence signal provided qualitative and spatial information on caspase-3 activation, supporting its involvement in the compound-induced apoptotic response.

13. Transcriptomic sequencing

To profile the cellular transcriptional response to the test compounds, total RNA was extracted from treated H1299 cells and assessed for integrity using standard quality control methods. Sequencing libraries were prepared following established protocols and subjected to high-throughput sequencing using a next-generation sequencing platform. Raw sequencing data were processed through a conventional bioinformatics pipeline, which included quality control filtering, alignment to the reference genome, and quantification of transcript abundance. Differential gene expression analysis was conducted to identify genes with statistically significant changes in expression following treatment. Enrichment and pathway analyses of the differentially expressed genes were performed to elucidate the key biological processes affected by the compounds, thereby providing a comprehensive transcriptomic overview of their molecular effects.

14. Statistical analysis

All data are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical significance between groups was determined using standard methods, including Student's t-test or one-way ANOVA, where appropriate. Differences were considered statistically significant for P-values less than 0.05.

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