Effect of Agkistrodon acutus venom (AAVC-I) on apoptosis through modulation of the Keap1/Nrf2 pathway in HSC-3 oral squamous cell carcinoma cells

Introduction

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial regions, accounting for more than 300,000 new cases worldwide each year. The prognosis of the disease is poor, with a 5-year survival rate of only 50–60%, despite significant efforts to improve clinical outcomes for patients with OSCCs (1-3). Patients with early-stage oral cancer are treated with a comprehensive series of surgeries. Those with advanced localized cancer require preoperative induction chemotherapy, whereas patients who cannot tolerate surgery are treated with chemoradiotherapies. However, the use of chemoradiotherapies can cause a number of adverse effects, including liver and kidney damage, bone marrow suppression, and so on, thus deteriorating the quality of life of the patients (4). Therefore, there is an urgent need to identify effective treatment options for patients with OSCCs.

Snake venoms are complex mixtures of toxins, containing a variety of active ingredients such as proteins, peptides, enzymes, and other bioactive substances with diverse biological activities (5,6). For example, Agkistrodon acutus venom component I (AAVC-I), an anti-tumor compound, is purified from the crude venom of Agkistrodon acutus. AAVC-I can inhibit proliferation by inducing apoptosis in various tumor cells, including lung and laryngeal cancer (7-9). However, its mechanisms of action have yet to be identified.

Oxidative stress is the imbalance between the production and consumption of reactive oxygen species (ROS), which plays crucial roles in the pathogenesis and progression of various cancers (10). Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) signaling is the main regulatory pathway for cancer cells to resist ROS generation and protection against its harmful effects. Sustained activation of Nrf2 followed by Keap1 inactivation leads cancer cells to growth, proliferation, angiogenesis, and resistance to chemo-radiotherapies (11-14).

Here, we first investigated the effects of AAVC-I on OSCC cells using the HSC-3 cell line as a model, and then examined the effects of AAVC-I on the Keap1/Nrf2 signaling pathway. Then, this study explored the mechanism of AAVC-I -induced apoptosis in cells derived from OSCCs, which could be helpful in developing new chemotherapeutics for treating patients with OSCCs. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-182/rc).

Methods

Isolation and purification of AAVC-I

The venom of Agkistrodon acutus was procured from the Qimen Snakebite Research Institute (Huangshan, China). The lyophilized venom from Agkistrodon acutus was dissolved in a 5 mM sodium phosphate buffer (pH 8.0) and then centrifuged at 10,000 g for 15 minutes at 4 ℃. The supernatant was loaded into a DE-52 ion exchange chromatography column and equilibrated with the same 5 mM sodium phosphate buffer solution (pH 8.0). Gradient elution was performed using the same sodium phosphate buffer solution and 1 mol/L NaCl, with a flow rate set at 1.5 mL/min. Following desalting and dehydration, the fraction (peak I) was collected (Figure 1).

Figure 1 Results of using DEAE-cellulose column chromatography to separate the components of Agkistrodon acutus venom. Conc means the concentration of buffer solution; it will rise over time and eventually reach 1 mol/L. The red numbers on the horizontal axis represent the fraction numbers. UV, ultraviolet; mAu, milli-absorbance unit.

Cell culture and treatment

The Tongji Medical College of Huazhong University of Science and Technology provided the HSC-3 cells for the study. The cells were stored in the Central Laboratory of the First Affiliated Hospital of Wannan Medical College until use. They were grown on the 90% minimum essential medium (MEM) plus 10% fetal bovine serum (FBS) and 1% double antibiotic (penicillin/streptomycin), and were cultured under 5% CO₂, 37 ℃ constant temperature, and humidity conditions. When the cell growth reached the logarithmic phase, they were sub-cultured at a ratio of 1:4 and were used for subsequent experiments.

First, treatment of AAVC-I at different concentrations (0, 0.25, 0.5, 1, 2, 4, 8, 12, 16 µg/mL) were set up for cytotoxicity experiments. Based on the results of the cytotoxicity experiments, the concentrations for subsequent experiments were selected.

Cytotoxicity test

HSC-3 cells were collected and seeded at a concentration of 5,000 cells per well in 96-well plates (n=5). After 12 hours of initial seeding, the cells were treated with AAVC-I at different doses and incubated for an additional 24 and 48 hours. After that, 10 µL of Cell Counting Kit-8 (CCK-8) solution (Biosharp, Hefei, China) was added to the each well and incubated for 2 hours. Then, the 96-well plate was placed on a microplate reader (BioTek, Winooski, VT, USA) and measured the absorbance at 450 nm. Finally, the cell survival rates were calculated as follows: average optical density (OD) value of drug treatment group/average OD value of control group × 100%.

Detection of apoptosis and mitochondrial membrane potential

HSC-3 cells were collected and seeded at a density of 2.0×10⁵ cells per well in a 6-well plate. The cells were treated with different concentrations of AAVC-1 (0, 2, 4, and 8 µg/mL) after 12 hours of initial seeding. Apoptosis of cells following the treatment was analyzed using Annexin V-FITC kit (Servicebio, Wuhan, China) by a flow cytometry (Beckman Coulter, Brea, CA, USA). The mitochondrial membrane potential of treated and untreated cells was examined with a flow cytometry and a fluorescent microplate reader followed by staining the cells with mitochondrial membrane potential detection kit (JC-1) (Beyotime, Haimen, China) according to the manufacturers’ guidelines.

Western blot

After treatment of HSC-3 cells at various doses of AAVC-I for 12 hours, cells were collected and washed with phosphate-buffered saline (PBS) 3 times. Then, the cytosolic and nuclear proteins were collected according to the instructions of nuclear and cytoplasmic proteins extraction kits (Beyotime). The total proteins were collected using Western and IP Cell Lysis Buffer (Beyotime) containing 1 mM protease inhibitor. A bicinchoninic acid (BCA) kit (Beyotime) was used to determine the concentration of protein. After that, 30 µg of protein was loaded into each well of 6%, 10%, and 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with Bax (Beyotime), Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-3 (Wanleibio, Shenyang, China), cleaved caspase-3 (Wanleibio), caspase-9 (Wanleibio), cleaved caspase-9 (Wanleibio), Cyt-c (Servicebio), Keap1 (Servicebio), Nrf2 (Servicebio), HO-1 (Servicebio), NQO1 (Servicebio), β-actin (Servicebio), and Lamin-b (Wanleibio) antibodies overnight at 4 ℃. Afterwards, they were washed with PBS with Tween 20 (PBST) for 30 minutes and then incubated with horseradish peroxidase (HRP) coupled secondary antibody for 1 hour at room temperature. Finally, the signals of the target proteins were detected using an enhanced chemiluminescence (ECL) detection system, and the grayscale values of the target proteins bands were analyzed using Image J software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA). β-actin was used as the total proteins, and Lamin-b was used as the nuclear proteins loading controls.

Detection of oxidative stress

HSC-3 cells were seeded at a density of 2.0×10⁵ cells per well in 6-well plates. After 12 hours of seeding, cells were treated with different concentrations (0, 2, 4, 8 µg/mL) of AAVC-I for an additional 12 hours. For the estimation of ROS, DCFH-DA (Jiancheng Bioengineering Institute, Nanjing, China) and Bodipy-C11 (Maokang Biotechnology, Shanghai, China) fluorescent probes were used. In short, the cells were incubated with the probes for 30 minutes followed by removal of treatment and the ROS levels were measured according to the instructions using flow cytometry and fluorescence microscope.

Measurement of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) was performed according to the reagent kits (Wanleibio). For this experiment, samples were collected from HSC-3 cells and the protein concentration of the samples were determined using the BCA assay kit and the activity of SOD, GSH, and MDA were measured according to the manufacturer’s guidelines.

Statistical analysis

All experiments were carried out independently at least 3 times. Statistical analysis was performed using SPSS 18.0 statistical software (IBM Corp., Armonk, NY, USA). Measurement data were expressed as x¯±s, and t-tests were used for comparison between two groups. Analysis of variance (ANOVA) was used for multiple group comparisons. A P value <0.05 was considered a significant difference.

Results

Cytotoxicity and apoptosis

The effects of AAVC-I on the survival rates of HSC-3 cells are presented in Figure 2A. AAVC-I induced HSC-3 cell death in a concentration-dependent manner with a 50% inhibiting concentration (IC₅₀) of 8.86 µg/mL after 24 hours of treatment. It was noted that at 2 µg/mL of AAVC-I, the survival rates of HSC-3 cells started to decrease notably, and at a concentration of 4 µg/mL, it reached a statistically significant difference. Therefore, in subsequent experiments, the treatment groups were used as follows: 2, 4, and 8 µg/mL as low, medium, and high concentration groups, respectively (Table 1).

Figure 2 Effect of AAVC-I on the survival rate and apoptosis of HSC-3 cells. (A) Changes in survival rate of HSC-3 cells treated with different concentrations of AAVC-I. **, compared with the control group, P<0.01. (B) Apoptosis of HSC-3 cells treated with AAVC-I. AAVC-I, Agkistrodon acutus venom component I; Q1, the selected cell range; UL, upper left quadrant; UR, upper right quadrant; LL, lower left quadrant; LR, lower right quadrant; PI, propidium iodide; FITC, fluorescein isothiocyanate.

The treatment of AAVC-I induced apoptosis of HSC-3 cells (Figure 2B) and the proportion of apoptotic and necrotic cells were increased with the increment of AAVC-I concentration.

AAVC-I induced reduction of mitochondrial membrane potential

Reduction of mitochondrial membrane potential is a hallmark of apoptosis. Thus, we used a JC-1 kit to measure the mitochondrial membrane potential of HSC-3 cells followed by AAVC-I treatment. It was noted that AAVC-I caused a significant reduction of mitochondrial membrane potential of HSC-3 cells (Figure 3). Interestingly, with the increasing of AAVC-I concentration, the intensity of red fluorescence decreased, whereas the intensity of green fluorescence increased. These results indicated the higher loss of mitochondrial membrane potential with higher doses of AAVC-I treatment (Figure 3).

Figure 3 Mitochondrial membrane potential of HSC-3 cells treated with AAVC-I. (A) Fluorescent microplate reader; (B) flow cytometry. *, compared with the control group, P<0.05; **, compared with the control group, P<0.01. AAVC-I, Agkistrodon acutus venom component I; Q1 represents the selected cell range. UL, upper left quadrants; UR, upper right quadrants; LL, lower left quadrants.

Regulation of apoptosis-related proteins by AAVC-I treatment

The upregulation or downregulation of apoptosis-related proteins in HSC-3 cells followed by AAVC-I were analyzed using western blots. The expressions of Bax, Bcl2, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and Cyt-c were examined. It was found that with the increasing of AAVC-I concentrations, the expression levels of Bax, cleaved caspase-3, cleaved caspase-9, and Cyt-c increased whereas the expression level of Bcl2 decreased significantly (P<0.05). The expression of caspase-3 at various doses remained the same whereas caspase-9 expression increased significantly at 4 µg/mL or higher doses of AAVC-I treatment (Figure 4).

Figure 4 Effect of AAVC-I on the expression of apoptosis related proteins in HSC-3 cells. *, compared with the control group, P<0.05; **, compared with the control group, P<0.01; ns, compared with the control group, there was no significant difference. AAVC-I, Agkistrodon acutus venom component I.

Inactivation of the Keap1/Nrf2 pathway by AAVC-I treatment

The expression levels of Keap1, Nrf2, HO-1, NQO1, and Nrf2 in the nuclear proteins were examined. The expression of Keap1 increased significantly with increasing concentrations of AAVC-I, whereas the expression of Nrf2 decreased in the nuclear proteins fraction (Figure 5). However, the expression of Nrf2 in total proteins did not change significantly. In addition, expression of heme oxygenase 1 (HO-1) slightly increased at low concentrations (2 µg/mL) yet decreased significantly at the concentrations of 4 µg/mL or higher doses. Furthermore, the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) significantly increased at low (2 µg/mL) and medium (4 µg/mL) concentrations, and on the contrary, it decreased at high concentrations (8 µg/mL). However, this reduction of NQO1 at higher doses of AAVC-I treatment did not reach the statistical significance level (Figure 5). These results implied that treatment of HSC-3 cells with AAVC-I induced inactivation of Keap1/Nrf2 signaling pathway.

Figure 5 Expression of oxidative stress pathway proteins in HSC-3 cells. *, compared with the control group, P<0.05; **, compared with the control group, P<0.01; ns, compared with the control group, there was no significant difference. AAVC-I, Agkistrodon acutus venom component I.

Induction of oxidative stress in HSC-3 cells by AAVC-I treatment

Production of ROS in HSC-3 cells after AAVC-I treatment at low, medium, and high concentrations is presented in Figure 6. The results showed that ROS level in HSC-3 cells increased significantly after AAVC-I treatment with a concentration-dependent manner (Figures 6,7). In addition, analysis of GSH and SOD showed that at lower dose (2 µg/mL) of AAVC-I, the level of GSH and SOD increased slightly, whereas it significantly decreased at high concentration of AAVC-I (8 µg/mL) treatment (Figure 8A,8B). Meanwhile, the level of MDA in HSC-3 cells increased with the increment of AAVC-I treatment, indicating a concentration-dependent pattern of elevation of MDA in HSC-3 cells receiving AAVC-I treatment (Figure 8C).

Figure 6 ROS levels in HSC-3 cells. After treatment of HSC-3 cells with different concentrations of AAVC-I, the production of ROS was detected by DCFH-DA probe. (A) Fluorescence staining; green: the production of ROS in HSC-3 cells; blue: nucleus. Magnification: 400×. (B) ROS levels in HSC-3 cells detected by flow cytometry. NC, control group; AAVC-I, Agkistrodon acutus venom component I; DAPI, 4',6-diamidino-2-phenylindole; ROS, reactive oxygen species.

Figure 7 Lipid ROS levels in HSC-3 cells. AAVC-I, Agkistrodon acutus venom component I; ROS, reactive oxygen species.

Figure 8 SOD, GSH, and MDA levels in HSC-3 cells. (A) SOD; (B) GSH; (C) MDA. *, compared with the control group, P<0.05; **, compared with the control group, P<0.01; ns, compared with the control group, there was no significant difference. SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde.

Discussion

Chemotherapy is an important option for the patients with OSCC in clinical settings. However, low response, adverse side effects, and resistance to the chemotherapies deteriorate the quality of life and limit their point-of-care application. Thus, it is an urgent need to develop effective drugs for the treatment of patients with OSCC. The present study revealed that AAVC-I inhibited the survival of OSCC (HSC-3) cells significantly through induction of the apoptosis of cancer cells. Also, it was noted that AAVC-I reduced the mitochondrial membrane potential and increased the ROS levels in HSC-3 cells. In addition, AAVC-I may regulate the Keap1/Nrf2 signaling pathway to increase oxidative stress levels in OSCC cells, resulting in effective elimination of cancer cells (Figure 9).

Figure 9 Possible mechanism of AAVC-I. AAVC-I induces apoptosis in HSC-3 cells through the classical mitochondrial pathway and enhances cellular oxidative stress levels by inhibiting the expression of Nrf2 in the nucleus. Green arrows represent a promoting effect on the next stage, red arrows represent the inhibitory effect, black arrows indicate an increase in expression level. Blue arrows mark the composition of the apoptotic complex (this figure was drawn by Adobe Illustrator). AAVC-I, Agkistrodon acutus venom component I; ROS, reactive oxygen species; MMP, mitochondrial membrane potential.

Snake venom is a complex mixture of proteins and peptides that exhibit biological activity during physiological processes. Therefore, further separation and detailed study of the components within this mixture is currently a research hotspot. Previous studies (15-17) have highlighted the potential applications of snake venom toxins in anti-tumor therapy. Ha et al. identified four proteins: PLA2, SVMP, CRiSP, and CTL/snalec in the venom of Agkistrodon acutus using methods such as mass spectrometry, electrophoresis, and N-terminal sequencing (16). Among these, PLA2 has been suggested to exhibit anti-tumor and angiogenic effects, although some reports (17,18) indicate that PLA2 lacks cytotoxicity. SVMP has been documented in multiple studies (15,16) as disrupting the extracellular matrix, altering the adhesion and migration of cancer cells, and has significant implications for the development of anticancer drugs. CRiSP has been recognized for biological functions such as antimicrobial activity and Ca²⁺ and K⁺ channel blockade, but its anti-tumor activity has yet to be reported (19). AAVC-I, an anti-tumor component extracted from the crude venom of Agkistrodon acutus, may enhance the functionality of each individual component in practical applications.

The balance between cell survival (proliferation) and death is crucial for maintaining normal growth and development. Once this balance is disrupted, it leads to the pathogenesis of cancers and other degenerative diseases. Apoptosis, a key programmed cell death phenomenon, is often considered an effective target for developing cancer therapeutics (20). In this study, considerable portion of cells treated with AAVC-I underwent apoptosis, thereby reducing the survival rate of HSC-3 cells in a dose-dependent manner. Similarly, AAVC-I causes reduction of growth and proliferation of cancer cells derived from laryngeal, lung, and other types of cancers (7,8). Additionally, at a higher dose (8 µg/mL), AAVC-I increased the proportion of necrotic cells along with an increase in the proportion of apoptotic cell populations.

Apoptosis is a type of programmed cell death mediated by various pathways, including exogenous (death receptors) and endogenous (mitochondria), ultimately leading to cell death. Related molecular events include changes in mitochondrial membrane potential, DNA breakage, and specific protease cascades. In this process, Bcl2, by binding to BH3, promotes the formation of pro-apoptotic effectors by interacting with Bax and Bak. This complex affects mitochondrial membrane permeability and releases Cyt-c into the cytoplasm. Subsequently, Cyt-c binds to Apaf-1, resulting in the formation of an apoptotic body, which triggers the caspase cascade reactions of apoptosis (21,22). Cyt-c is translocated to the cytoplasm, oxidative phosphorylation is decoupled, and higher levels of ROS such as H₂O₂, and OH− are generated (23). This excessive ROS generation and accumulation induce apoptosis of cells through the mitochondrial and endoplasmic reticulum pathways (24,25). AAVC-I treatment of HSC-3 cells caused a reduction in the anti-apoptotic protein Bcl2 and increased the expression of the pro-apoptotic protein Bax, which is consistent with a previous study on oropharyngeal cancer (7). Additionally, HSC-3 cells receiving AAVC-I showed a decrease in mitochondrial membrane potential and an increase in ROS levels. Next, we further examined the expression of cleaved caspase-9 and caspase-3, and noted that the expression of cleaved caspase-9 and cleaved caspase-3 significantly increased after AAVC-I treatment. Therefore, AAVC-I primarily induces cell apoptosis through endogenous pathways.

ROS accumulation in cancer cells can induce oxidative stress by modulating the activity of the Keap1/Nrf2 signaling pathway. Activation of Nrf2 promotes an antioxidant response and reduces intracellular ROS levels. Meanwhile, inactivation of Nrf2 is associated with reduced cell proliferation. Therefore, Nrf2 can promote oncogene-induced cancer growth and proliferation (26-28). Results from the present study suggest that AAVC-I appears to be a potential inhibitor of Nrf2, as the expression of Nrf2 in both total and nuclear proteins of HSC-3 cells decreased significantly after AAVC-I treatment. Most importantly, Nrf2 expression in the nucleus was inhibited even at low concentrations of AAVC-I (2 µg/mL). Under physiological conditions, Keap1 binds to Nrf2 and localizes it in the cytoplasm, resulting in its ubiquitination and degradation. However, under oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it interacts with the antioxidant response element (ARE), leading to the activation of transcription of a series of antioxidant response genes, thereby enhancing the cell’s antioxidant capacity (28). AAVC-I treatment induced excessive ROS generation and simultaneously reduced Nrf2 expression and upregulated Keap1 expression. Deng et al. recently reported that metformin can inhibit the Nrf2/HO-1 signaling pathway and increase oxidative stress levels in cancer cells. When oxidative stress levels in cells rise, MDA levels typically increase, whereas those of SOD and GSH decrease (29). This aligns with our research findings, which indicate that AAVC-I elevates oxidative stress levels in HSC-3 cells. In an earlier study, Park et al. found that viper venom can elevate oxidative stress levels in neuroblastoma cells; however, they only measured two indicators of oxidative stress, ROS and mitochondrial membrane potential, and their evaluation of the corresponding molecular events was incomplete (30). Thus, the Keap1/Nrf2 signaling pathway is the main antioxidant pathway in cells, and interference with this pathway by AAVC-I appears promising for developing better therapeutics. Interestingly, this finding contrasts with traditional chemotherapy drugs such as cisplatin, which increases the expression of Nrf2 in cancer cells, thereby leading to drug resistance (31,32). Furthermore, AAVC-I treatment induced the downregulation of HO-1 in HSC-3 cells, whereas NQO1 expression was mildly upregulated. It has been observed that overexpression of HO-1 and NQO1 is associated with drug resistance in cancer cells (31,32). However, overexpression of NQO1 alone does not affect the Keap1/Nrf2 pathway (33,34).

In this study, we primarily report that AAVC-I, a mixture isolated from Agkistrodon acutus venom, may regulate the oxidative stress pathway and induce tumor cell apoptosis. Considering the significant side effects of existing chemotherapy drugs, the extraction of anti-tumor agents from snake venom may offer new breakthroughs in future cancer treatment. However, our current research is limited to a single cell line in vitro and the lack of in vivo evidence; additionally, the biological safety of AAVC-I has not been evaluated. These aspects need to be addressed in subsequent studies.

Conclusions

In summary, this study demonstrates that AAVC-I inhibits the proliferation and survival of OSCC (HSC-3) cells by inducing the endogenous apoptosis pathway. Unlike traditional chemotherapy drugs, AAVC-I not only increases oxidative stress but also inhibits the Keap1/Nrf2 signaling pathway, potentially overcoming drug resistance caused by activation of this pathway. Under oxidative stress conditions, Nrf2 translocates to the nucleus and activates the transcription of a series of antioxidant genes, thereby mitigating the oxidative stress response. AAVC-I may inhibit this process by promoting ubiquitin-mediated degradation of Nrf2 in the cytoplasm. However, further studies involving animal models and the examination of AAVC-I’s biological safety in vivo and its host toxicity are required. Additionally, as only one cell line was used in this study, more research is necessary to support AAVC-I as a new therapeutic for treating oral cancer.

Acknowledgments

Funding: This study was supported by the Applied Basic Research Project of Wuhu Science and Technology Bureau (No. 2022jc68), Open Project of Anhui Provincial Key Laboratory for Active Biomacromolecules (No. LAB202206), Young and Middle-Aged Scientific Research Fund from Wannan Medical College (No. WK202202, No. WK2023ZQNZ41), and Major Scientific Research Project of Anhui Provincial Department of Education (No. KJ2021ZD0103).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-182/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-182/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-182/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-182/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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