Oleandrin Promotes Apoptosis in an Autophagy-Dependent Manner in Gastric Cancer

Abstract

Background:

The medicinal phytochemical oleandrin (Ole) is obtained from the Nerium oleander plant. The exact relationship between Ole-induced apoptosis and autophagy in gastric cancer (GC) is unclear despite the fact that it has outstanding anti-tumor capabilities. This research aimed to demonstrate how autophagy and Ole-induced apoptosis interact in GC.

Methods:

The Cell Counting Kit (CCK)-8 assay and colony formation assays were employed to evaluate cell proliferation. Cellular apoptosis was evaluated with Calcein/Propidium Iodide (PI) assays and flow cytometry. Confocal and electron microscopes were employed to examine the morphology of autophagy. Protein concentrations were assessed by western blotting. Luciferase-positive HGC-27 cells were administered subcutaneously to Balb/c nude mice to evaluate Ole’s anti-tumor activity. Immunohistochemistry assessed Ki67 expression and H&E staining in tumor tissue.

Results:

Ole causes GC cells to undergo intracellular apoptosis and autophagy at low nanomolar doses, halting the cell cycle at the G0/G1 phase. Whereas 3-methyladenine (3-MA), the inhibitor of autophagy, counteracts the apoptosis generated by Ole in vitro and in vivo.

Conclusions:

Ole may trigger apoptosis through the activation of autophagy in GC. It offers a secure and efficacious candidate drug for the treatment of tumors in the digestive system.

Graphical abstract

Keywords

oleandrin / gastric cancer / apoptosis / autophagy / G0/G1 phase

1. Introduction

Globally, gastric cancer (GC) ranks as the fourth main cause of cancer-related deaths and the fifth most common cancer type [1]. It is one of the most common malignancies of the digestive system. China accounts for nearly half of all gastric cancer cases (43.8%) and nearly half of all gastric cancer deaths (47.5%) [2]. Surgery and chemotherapy are currently the gold standards for treating gastric cancer. Surgery causes a lot of damage, and chemotherapy has certain unavoidable side effects that reduce its effectiveness [3]. As a potential new approach to treating gastric cancer, natural substances show a wide variety of anticancer properties with little toxicity [4].

A wide range of natural product anti-cancer effects were investigated, including immunosurveillance, DNA damage linked to carcinogenesis, cell cycle regulation, and inhibition of cell proliferation [5, 6, 7]. For instance, Costunolide, a compound derived from the root of Auckiandialappa Decne, has the ability to trigger autophagy and apoptosis in GC cells [8]. It has been found that Honokiol, a compound derived from the bark of the Magnolia officinalis tree, reduces angiogenesis and peritoneal metastasis in GC [9]. Consequently, plant-derived natural compounds are regarded as significant for medication discovery in cancer therapy.

Oleandrin (Ole) is a foremost compound of Nerium oleander plant extracted from the Nerium oleander leaves [10]. Numerous researchers have documented that Ole exhibits anti-inflammatory and anti-cancer properties against colon, lung, pancreatic, and breast cancers [11, 12, 13, 14]. Previous study indicated that oleandrin prompted apoptosis [15]. Additionally, the report indicated that oleandrin may trigger death-promoting autophagy in breast cancer [16]. Nonetheless, the role and molecular mechanism of Ole in GC remains ambiguous. Our study sought to reveal the role and molecular mechanism.

2. Materials and Methods

2.1 Cell Culture

Human gastric cancer (GC) cell lines HGC-27 (Cat NO., CL-0107), SNU-1 (Cat NO., CL-0474), and human gastric mucosa epithelial cells GES-1 (CL-0563) were supplied by Procell Life Science & Technology (Wuhan, China). Mycoplasma testing was negative for all cell lines that underwent validation by Short Tandem Repeats (STR) profiling. At 37 °C in a 5% CO₂ atmosphere, cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), penicillin (100 U/mL).

2.2 Cell Counting Kit-8 (CCK-8) Assay

We used a Multiskan™ FC microplate reader (Thermo Fisher, Carlsbad, CA, USA) to measure the optical density at 450 nm after exposing GC cells to Ole at various doses (0, 2.5, 5, 10, 20, 40, 80, and 160 nM) [17] for 24 hours after overnight culture in 96-well plates. Then, we tested the cells using a CCK-8 assay.

2.3 Colony Formation Assay

Following the injection of 500 GC cells into plates for a duration of 24 hours, the cells underwent a ten-day treatment with Ole at several doses (0, 10, 20, 40 nM). Before fixation with 4% paraformaldehyde, the cells were washed twice with PBS (0.2 M, pH 7.8). Subsequently, they were subjected to staining with 1% crystal violet for 15 minutes at ambient temperature. Colonies comprising over fifty cells were enumerated and analyzed. The colony formation rate can be determined by dividing the number of treatments by the number of controls and subsequently multiplying the result by 100%.

2.4 Calcein/PI Assays

After seeding GC cells onto 96-well plates, they were treated with Ole at multiple concentrations (0, 10, 20, 40 nM) for a duration of 24 hours. In order to stain the cells, calcein AM and PI were diluted a thousand times with the working solution. Olympus Corporation’s BX41 fluorescence microscope (Tokyo, Japan) was used to examine and capture images of the fluorescence.

2.5 Flow Cytometry

GC cells were cultured for 24 hours with gradient concentration of Ole (0, 10, 20, 40 nM) or 3-methyladenine (3-MA) (30 µM). The cells were incubated with 75% ethanol at 4 °C overnight, and the following day, they were stained with PI for cell cycle analysis. Apoptosis assessment was conducted on cells treated with Ole and stained with Annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI).

2.6 Western Blotting

Ole (0, 10, 20, 40 nM) or 3-MA (30 µM) were applied to GC cells during 24 hours. Various proteins involved in cell cycle regulation, intrinsic and extrinsic apoptosis, and autophagy were examined using western blotting. To summarize, the proteins from these cells were isolated by lysing them on ice with Radio Immuno Precipitation Assay (RIPA) lysis buffer. Beyotime Biotech’s Bicinchoninic Acid (BCA) Protein Assay Kit (P0011, Shanghai, China) was used to quantify the supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to extract proteins of the same grade, which were then transferred to nitrocellulose membranes. Skim milk in PBS (0.2 M, pH 7.8) was used to block the membranes for 2 hours. The next step was to incubate the primary antibodies (rabbit anti-human cell division cyclin 25 homolog C (Cdc25c) (1:1000, cat. No. 4866S), rabbit anti-human Cdk1(1:1000, cat. No.77055S), rabbit anti-human Cyclin B1 (1:1000, cat. No. 12231S), rabbit anti-human Caspase 3 (1:1000, cat. No. 9662S), rabbit anti-human Bak (1:1000, cat. No. 12105S), rabbit anti-human Bax (1:1000, cat. No. 2774S), rabbit anti-human Bcl-2(1:1000, cat. No. 4223S), rabbit anti-human PARP (1:1000, cat. No. 9532S), rabbit anti-human Caspase 8 (1:1000, cat. No. 4790S), rabbit anti-human Fas (1:1000, cat. No. 4233S), rabbit anti-human Fas ligand (FasL) (1:1000, cat. No. 68405S), rabbit anti-human microtubule-associated protein1 light chain3B (LC3B) (1:1000, cat. No. 3868S), rabbit anti-human Beclin (1:1000, cat. No. 34) at 4 °C throughout the night. Membranes were incubated at room temperature for 1 hour with HRP-conjugated secondary antibodies (Proteintech, SA00001-2, Chicago, IL, USA) not long ago. Alpha’s FluorChem FC2 (92-13779-00, Center Valley, CA, USA) was used to detect the blots. Using GAPDH, the data was standardized.

2.7 Immunofluorescence Confocal Microscopy

After a 24-hour Ole treatment at 20 nM, GC cells were collected and fixed with paraformaldehyde. Cell blocking and permeabilization were done with 5% BSA and Triton X-100 (Beyotime Biotech, Shanghai, China). Overnight, the cells were exposed to anti-microtuble-associated protein light chain 3 (LC3) antibody from Cell Signaling Technology (A32731, A11072, Cambridge, MA, USA) at 4 °C. Nuclear DNA was counterstained with DAPI for 15 minutes in the dark after cells were incubated with Alexa-Fluor 488/594-conjugated secondary antibodies (Thermo Fisher, Carlsbad, CA, USA) in 1% bovine serum for 1 hour at 37 °C. A confocal laser scanning microscope (Olympus Corporation, Center Valley, CA, USA) was used to take the pictures.

2.8 Transmission Electron Microscopy

1 \times 10⁶ GC cells were treated with 20 nM Ole for 24 hours and then rinsed twice with pre-cooled PBS (0.2 M, pH 7.8). After removing the cells and fixing them with 2.5% glutaraldehyde at 4 °C for the night, electron microscopy slices were prepared. Using an ultramicrotome (50–60 nm), the fixed cells were embedded, sectioned, and then dried. The slices were then double-stained with 3% uranyl acetate and lead citrate. Ultimately, cellular ultrastructures were examined and captured using HITACHI transmission electron microscopy (HT7700-SS, Tokyo, Japan).

2.9 Tumour Model and Therapeutic Treatment In Vivo

All animal experiments adhered to the National Institutes of Health (Bethesda, MD, USA) guidelines for laboratory animal care and utilization. Weighing 90–120 grams, 10 female Balb/c nude mice were supplied by the Laboratory Animal Center of Xi’an Jiaotong University. The mice were aged 4–6 weeks. All mice were fed normal diet during the test period. A subcutaneous injection was made into the right flank of nude Balb/c mice containing luciferase-positive HGC-27 cell suspensions (5 \times 10⁶ cells per mouse). Two groups of mice were randomly assigned after the tumor volume reached 50 mm³. Mice in the Ole group got 50 µg/kg Ole intraperitoneally every three days, while the control group received 1% Dimethyl Sulfoxide (DMSO) in PBS (pH 0.2 M, pH 7.8). Mouse weight, breadth (W) and tumor length (L) were measured every three days. On days 6 and 18, we used Perkin Elmer’s IVIS imaging equipment (Perkin Elmer, Waltham, MA, USA) for bioluminescence imaging. Mice were intraperitoneally given 100 µL of PBS (0.2 M, pH 7.8) with 25 mM D-luciferin (Caliper Life Sciences, Hopkinton, MA, USA) ten minutes before luciferase detection. The formula for tumor volume is L \times (W)²/2. On the 21st day after Ole injection, all mice were euthanized by inhaling 5% isoflurane for 5 minutes, and their tumors, hearts, spleens, livers, kidneys, and lungs were removed for immunohistochemistry and H&E staining.

2.10 Immunohistochemistry

After being fixed in 4% formaldehyde, the tumor tissues were subjected to alcohol gradient drying, embedded in paraffin at 60 °C for 3 hours, sawn into pieces, and then baked. At ambient temperature, the antigen was repaired for 15 minutes in a citrate buffer (0.01 M, pH 6.0), and then, to inhibit endogenous peroxidase, it was incubated for another 15 minutes in 3% hydrogen peroxide. For 30 minutes, we let the serum block at room temperature. After being treated overnight with anti-Ki67 antibodies (1:500, Proteintech, 98224-1-RR, Wuhan, China), the slices were incubated with HRP-conjugated goat anti-rabbity IgG (1:2000, Sigma-Aldrich, AP-307P, Darmstadt, Germany) for 2 hours at room temperature. Using 3, 3-diaminobenzidine (DAB), the chromogen may be observed under a microscope. A phase contrast microscope (CKX53, Olympus, Tokyo, Japan) was used to examine the slices after dehydrating them and staining them with hematoxylin for three minutes.

2.11 H&E and TUNEL Staining

The tumors, hearts, livers, spleens, lungs, kidneys, and other tissues were preserved by fixing, drying, embedding in paraffin, sectioning, and baking. Xylene was employed for dewaxing and hydration. The slices were subsequently stained with hematoxylin and differentiated using 0.1% hydrochloric acid ethanol. Subsequently, the sequential processes of eosin staining, alcohol dehydration, xylene transparency, and resin sealing were executed. The slices were finally examined and photographed using a phase contrast microscope. For morphological evaluation, slices were stained with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL).

2.12 Statistical Analysis

Data is presented as mean \pm standard error of the mean (SEM). A parametric or nonparametric Student’s t-test was used to determine the statistical significance of two groups, whereas for multiple pairwise comparisons involving more than two groups, one-way ANOVA with Tukey’s post-hoc testing was utilized. In every instance, * p 0.05, ** p 0.01, *** p 0.001. Utilizing GraphPad Prism 9 software (Dotmatics, Boston, MA, USA), statistical analysis was carried out.

3. Results

3.1 Ole Inhibited the Proliferation of Gastric Cancer Cells Arresting at G0/G1 Phase

The inhibition effect of Ole (chemical structure featured in Fig. 1A) on proliferation of HGC-27 and SNU-1 cells was tested by CCK-8. The results showed that the proliferation of HGC-27 and SNU-1 cells were significantly inhibited by Ole in a dose-dependent manner compared to GES-1 cells (p 0.05, Fig. 1B). The half-maximal inhibitory concentration (IC50) for HGC-27 cell was 6.36 n M at 24 h and 4.13 n M at 48 h, IC50 for AGS cell was 5.94 n M at 24 h and 3.36 n M at 48 h, and IC50 for GES-1 was more than 10 uM (Fig. 1C) both at 24 h and 48 h. According to these results, we choose 0, 10, 20, 40 n M for the following assays. The results of colony formation assay showed that Ole could evidently inhibit the proliferation of HGC-27 and SNU-1 cells even at 10 n M compared with GES-1 cells, and there were remarkable inhibitions at 20 n M and 40 n M (p 0.05, Fig. 1D). As a result, we confirmed that Ole possess an obvious inhibition on proliferation of HGC-27 and SNU-1 cells in a dose-dependent manner at low nanomolar concentrations. Then we tested the effect of Ole on cell cycles of HGC-27 and SNU-1 cells. The flow cytometry essay revealed that Ole could induce cell cycle arresting at G0/G1 phase of HGC-27 and SNU-1 cells in a dose-dependent manner ((p 0.05, Fig. 1E). And Western blot results also showed that cell cycle-related proteins such as cyclin B1, Cdc25c, Cdk1 in HGC-27 and SNU-1 cells treated with Ole were significantly down-regulated depending on Ole dosage (p 0.05, Fig. 1F). These results indicated that Ole could induce gastric cancer cells arresting at G0/G1 phase in a dose dependent manner.

3.2 Ole Induced Apoptosis of HGC-27 and SNU-1 Cells Through Endogenous Mechanism

Calcein/PI and flow cytometry was performed to verify the apoptosis effect of Ole on gastric cancer cells. The results shown that active cells stained by Calcein Acetoxymethyl Ester significantly decreased and apoptotic cells stained by Propidium Iodide increased depending on dosage in HGC-27 and SNU-1 cells (p 0.05, Fig. 2A). The flow cytometry also exhibited that apoptotic cells increased in a dose-dependently manner of HGC-27 and SNU-1 cells (p 0.05, Fig. 2B). These results suggested that Ole could induce apoptosis of gastric cancer cells depend on concentration. Further more, Western blot results revealed that cle-Caspase 3, Bax, Bak, cle-PARP were up-regulated, and Bcl-2 was down-regulated in a Ole dosage-dependent manner (p 0.05, Fig. 3A). However, no significant changes existed in cle-Caspase 8, Fas, FasL depending on Ole dosage (p > 0.05, Fig. 3B). Cle-Caspase 3, Bax, Bak, cle-PARP and Bcl-2 are endogenous apoptotic-related proteins, whereas cle-Caspase 8, Fas, FasL are extrinsic apoptosis-related proteins. These results indicated that Ole induces gastric cancer cells apoptosis with an intracellular mechanism.

3.3 Ole Induced Endogenous Apoptosis by Promoting Autophagy in Gastric Cancer Cells

The variation of autophagy treated with Ole in HGC-27 and SNU-1 cells were investigated. Transmission electron microscopy revealed that the formation of autophagic vacuoles in HGC-27 and SNU-1 cells treated with Ole was obviously increased compared to control group (Fig. 4A). And the confocal microscopy also showed that Ole lead to aggregation of autophagosomes in HGC-27 and SNU-1 cells compared to control group (Fig. 4B). Western blot results also showed that autophagy related proteins such as LC3BⅡ/LC3BⅠ ratio and beclin-1 were increased and p62 was decreased (p 0.051, Fig. 4C). These results suggested that Ole could promote autophagy accumulation in gastric cancer cells. 3-MA, an autophagy inhibitor, was used to identify autophagy flux induced by Ole in gastric cancer cells. The flow cytometry revealed that the apoptosis of HGC-27 and SNU-1 cells in Ole-treated group was increased compared to Ole+3-MA treated group (p 0.05, Fig. 5A), and the autophagy-related proteins LC3BⅡ/LC3BⅠ ratio was up-regulated and p62 was down-regulated in Ole-treated group compared to Ole+3-MA treated group. Meanwhile, the endogenous apoptosis-related proteins cle-Caspase 3, Bax and cle-PARP were significantly up-regulated in Ole-treated group compared to Ole+3-MA treated group (p 0.05). Whereas extrinsic apoptotic associated proteins (Cle-Caspase 8, FasL) had no significant change in Ole-treated group compared to Ole-3-MA treated group (p > 0.05) (Fig. 5B). These results suggested that Ole induces gastric cancer cells apoptosis in an endogenous mechanism depending on activation of autophagy.

3.4 Ole Inhibited Tumour Growth and Induces Intracellar Apoptosis and Autophagy In Vivo

Subcutaneous xenograft model of gastric cancer cells were used to define effect of Ole on tumor growth. The tumour size and weight in Ole (50 μg/kg) group were significantly reduced compared to control group (p 0.05, Fig. 6A–C), and IVIS images exhibit the same changes in tumour size and weight (p 0.05, Fig. 6D). H&E staining shown tumor cells with irregular shape, uneven cell size, large nuclei, and deep cytoplasmic staining in Ole group compared to control group. And Tunel positive staining was increased in Ole group compared to control group (p 0.05, Fig. 6E). Protein expression level of tumor tissue revealed by western blot confirmed that endogenous apoptosis-related proteins (cleaved-Caspase 3, PARP, Bax) were up-regulated and autophagy-related proteins (LC3BⅡ/LC3BⅠ) were up-regulated and p62 were down-regulated in Ole (50 μg/kg) group compared to control group (p 0.05), whereas exogenous apoptosis-related proteins (Cle-Caspase 8, FasL) had no significant change in these two groups (p > 0.05, Fig. 6F). These in vivo results confirmed that Ole could inhibit tumor growth by inducing endogenous apoptosis and activating autophagy in gastric cancer.

3.5 The Toxicity of Ole on Mice

The toxicity of Ole on tumor bearing animals were evaluated. The body weight and liver weight has no significant changes in Ole group compared to control group (p > 0.05, Fig. 7A,B). And H&E staining of different organs uncovered that there was no evident damage on heart, liver, spleen, lung and kidney of mice (magnification: 200\times) in Ole group compared to control group (Fig. 7C). These results confirmed that the effective dosage of Ole on gastric tumor is safe for animals.

4. Discussion

Ole is among the most pharmacologically potent phytochemical constituents in Nerium oleander, exhibiting efficient and non-toxic anti-cancer properties. The anti-cancer efficacy of oleandrin on osteosarcoma [18], colon cancer [4, 11], and pancreatic cancer [19] has been documented by other researchers [10]. There has been little investigation into the role of Ole or its underlying mechanism in GC. This work initially demonstrated that Ole can reduce the growth of GC cells in a dose-dependent manner at low nanomolar doses, while showing no significant effect on GES-1. Cell cycle dysregulation is well recognized as a critical characteristic of malignancies. Prior investigations demonstrated that Ole had significant anti-cancer activity by disrupting the cell cycle [20, 21]. For example, Ole could suppress cells proliferation in colon cancer via blocking in the G2/M phase [22], and Ole also inhibit proliferation of pancreatic cancer via arresting cells cycle at G2/M phase [23]. Our finding suggested that Ole could block cell cycle at G0/G1 phase and thus suppressed the growth in GC cells.

Cancer cells have a remarkable ability to evade apoptosis. Ole could promote cell apoptosis in human melanoma cells by means of the toll-like receptor pathway and is associated with microRNAs [24]. Ole could trigger cell apoptosis in colorectal cancer patients by way of the mitochondrial pathway [22]. Our findings demonstrated that Ole substantially causes apoptosis in GC cells through initiating intrinsic apoptosis, with no noticeable modifications in extrinsic apoptosis.

An increase in acidic vesicle organelles during autophagy, a vital biological process requiring lysosomal breakdown within cells, is typically dysregulated in cancer cells, providing an alternative mode of programmed cell death [25, 26, 27]. Ole is claimed to trigger the death of the human pancreatic cancer cell line PANC-1 by activating autophagy and may also cause autophagic cell death in undifferentiated human colon cancer CaCO-2 cells [28]. Autophagy occurs during cell stress. To prevent apoptosis, the cell will autophagically respond to non-lethal stress [29, 30]. When cells exceed an intensity threshold, autophagy and related proteins may degrade biological components or activate apoptotic pathways, causing cell death [31, 32]. According to our findings, Ole may significantly increase gastric cancer cell autophagy. Moreover, we reasoned that Ole may induce endogenous apoptosis in gastric cancer cells by activating autophagy. According to the research, Bcl-2-interacting protein 1 (Beclin1) is involved in autophagy and apoptotic cell death pathway regulation [33]. We found that Beclin was significantly upregulated in Ole-induced autophagy in GC cells, while Bcl-2 was significantly downregulated in activated apoptosis, and 3-MA reversed the shift of apoptosis-related proteins. This suggests that Ole may induce apoptosis by stimulating autophagy through Beclin.

There are certain limitations on this research. First, the dual role of pharmacology and toxicology of Ole remains a major puzzle in phytomedicine. Therefore, the safe dose of Ole must be concerned in future studies. Second, we have not addressed Ole-induced autophagy-dependent intracellular apoptotic signaling. In next step, we will find the key proteins and related pathways by single-cell sequencing and proteome sequencing, and knock them down using siRNA or CRISPR to further clarify the relationship between the key proteins and signaling pathways with apoptosis and autophagy. Third, the effectiveness of Ole should be evaluated on patient-derived xenograft models to assess its potential in personalized medicine. We will construct the patient-derived organoid and organoid xenograft models. Last, we will analyze the impact of Ole on mitochondrial function and integrity, as mitochondria play a crucial role in both apoptosis and autophagy.

In summary, Ole can limit the proliferation of gastric cancer cells by stopping the cell cycle at the G0/G1 phase, and trigger apoptosis through the activation of autophagy in gastric cancer. It offers a secure and efficacious candidate drug for the treatment of tumors in the digestive system (Fig. 8).

5. Conclusion

This study revealed that Ole had strong pro-apoptotic capability on gastric carcinoma at low concentrations depending on activation of pre-death autophagy. It may offer a secure and efficacious candidate drug for the treatment of tumors in the digestive system.

Availability of Data and Materials

The analyzed data presented in the study are included in the article, which will be made available by the corresponding author.

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Funding

Shaanxi Province Innovation Capability Support Plan(2024RS-CXTD-84)

Shaanxi Province health gastrointestinal tumor organ precise diagnosis and treatment research innovation platform, the Science and Technology Program of Xi’an(23YXYJ0186)

Science and Technology Talent Support Program of Shaanxi Provincial People’s Hospital(2021LJ-02)