Black ginseng extract suppresses lung cancer growth in a xenograft mouse model

Introduction

Lung cancer is the most commonly diagnosed cancer worldwide and accounts for 18% of all cancer-related deaths, with over 2.2 million new cases reported annually (1). It is broadly categorized into two main types: small cell lung cancer (SCLC), which accounts for 15%, and non-small cell lung cancer (NSCLC), which comprises 85% (2). In its early stages, lung cancer can be treated effectively with surgery to remove non-metastatic lesions completely, thereby improving patient survival (3). However, most lung cancer cases are diagnosed at advanced stages, requiring chemotherapy, radiation therapy, or targeted therapy (4). These non-surgical treatments often result in significant adverse effects, including chemotherapy-induced toxicity and drug resistance (5), both of which impair patient quality of life and threaten survival. Therefore, the development of drugs with high selectivity and efficiency—targeting cancer cells while sparing normal tissue—is essential. Such agents must also demonstrate low toxicity and reduced potential for drug resistance to ensure improved patient outcomes.

Plant-derived compounds constitute an important source of novel pharmaceuticals, with several plants exhibiting inherent anticancer properties (6). The identification and investigation of these compounds represent a key strategy for discovering new anticancer agents that are both highly effective and minimally toxic (7). In lung cancer, numerous natural products have been reported to exert inhibitory effects through multiple mechanisms, including suppression of tumor cell proliferation, induction of apoptosis, inhibition of metastasis, modulation of angiogenesis, and regulation of the tumor immune microenvironment (8). Representative phytochemicals include polyphenols such as curcumin (9), resveratrol (10), and epigallocatechin-3-gallate (EGCG) (11), saponins like ginsenoside Rg3 (12), and alkaloids such as berberine (13), all of which have been shown in preclinical studies to affect key signaling pathways involved in cell survival, migration, and immune modulation. Collectively, these findings highlight the promise of naturally derived compounds as potential therapeutic agents for lung cancer and provide a rationale for further investigation.

Ginseng, a traditional herb widely used in East Asian countries, exerts diverse pharmacological effects primarily through ginsenosides (14). These compounds have been reported to exhibit a broad spectrum of bioactivities, including anticancer (15), anti-inflammatory (16), antioxidant (17), antidiabetic (18), cardioprotective (19), neuroprotective (20), and immunomodulatory effects (21). Recent studies have shown that ginsenoside Rg3 inhibits lung cancer cell proliferation and induces apoptosis through activation of the mitochondrial-mediated and caspase-dependent pathways (22,23). In addition, ginsenoside Rg3 suppresses tumor formation, cell survival, epithelial-to-mesenchymal transition, and invasion in lung cancer cells, and enhances radiosensitivity (24-26). Furthermore, ginsenoside Rg5 has been identified as another ginsenoside that promotes apoptosis in lung cancer cells (23).

Ginseng is classified into white, red, and black types based on processing methods (27). White ginseng is produced by air-drying fresh ginseng, whereas red ginseng is prepared by steaming and then drying it once (28). Black ginseng undergoes nine repeated cycles of steaming and drying, resulting in more extensive chemical modifications (29). These repeated processes induce the Maillard reaction, altering the molecular structure of ginsenosides and increasing the levels of low-molecular-weight ginsenosides such as Rg3, Rg5, Rk1, and Rh1 (30,31). Since ginsenosides are saponins composed of a triterpene backbone with attached sugar moieties, those with fewer sugar units (i.e., lower molecular weight) tend to exhibit stronger anticancer activity (32). Compared with white and red ginseng, black ginseng contains higher concentrations of these pharmacologically active compounds and exhibits distinct anticancer properties (33).

To date, the pharmacological evaluation of ginsenoside complexes in black ginseng against lung cancer has been explored primarily through in vitro studies, with limited findings and no in vivo evidence. Therefore, this study aimed to assess the potential of black ginseng extract (BGE) in lung cancer prevention and treatment by examining its anti-proliferative effects on human gastric cancer cells in vitro and its effects on the growth of solid human lung tumors in vivo. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2901/rc).

Methods

Animals

Five-week-old male Athymic NCr-nu/nu mice were purchased from Koatech (Pyeongtaek, Korea). All animal experiments were performed under a project license (No. KW-240116-1) granted by Ethics Committee of the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University, in compliance with national or institutional guidelines for the care and use of animals.

Cell culture

Immortalized human Calu-3 and NCI-1975 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Welgene) and 1% (v/v) penicillin-streptomycin solution (Welgene). The cells were maintained in a humidified incubator at 37 °C with 5% (v/v) CO₂. The culture medium was replaced every 2 days, and when the cells reached approximately 80% confluence, they were detached using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Welgene). The dissociated cells were then reseeded onto 100 mm culture dishes (SPL, Pocheon, Korea) and maintained under the same conditions. Subculturing was performed at 5-day intervals.

Experimental designs

The stock solution of BGE, obtained from Truth & Ginseng Biotechnology Research Cooperation (Jinan, Korea), was prepared by dissolving the extract in warm distilled water (DW) to a concentration of 100 mg/mL. In the first experiment (Experiment 1), the cytotoxicity of BGE in Calu-3 and NCI-H1975 cells was assessed by measuring cell viability after treatment. Cells were incubated for 48 hours in high-glucose DMEM supplemented with different concentrations of BGE (0, 0.5, 1, 2, 5, or 10 mg/mL). Cell viability was evaluated using the colorimetric Alamar blue assay. Subsequently, cells were treated with concentration of BGE that significantly reduced cell viability and cultured for 24 hours, after which apoptosis was assessed by flow cytometry using Annexin V and propidium iodide (PI) double staining. In the second experiment (Experiment 2), the antitumor effects of BGE were examined in a lung tumor-bearing mouse model established by inoculating Calu-3 cells into nude mice. The mice received oral doses of either BGE-free DW (control) or DW containing 50 mg/kg BGE at one-day intervals for 21 days. Tumor volume and body weight were recorded every seven days during the treatment period, and tumor weight was measured on day 21. To assess the non-toxicity of BGE in the organs of the treated mice (Experiment 3), the absolute weights and histopathological characteristics of the liver and spleen were evaluated using tissues collected from Calu-3 cell xenograft nude mice administered either with or without BGE.

Colorimetric Alamar blue assay

Calu-3 and NCI-H1975 cells were seeded in 24-well culture plates (SPL) at a density of 5×10⁴ cells and 1×10⁴ cells per well, respectively, and incubated for 48 hours at 37 °C in high glucose DMEM according to the experimental design. The cells were then washed once with Dulbecco’s Phosphate-Buffered Saline (DPBS; Welgene) and treated with 10% (v/v) alamarBlue^® (Thermo Fisher Scientific, Waltham, MA, USA) for 4 hours at 37 °C. Absorbance was measured at 570 and 600 nm using a microplate reader (Epoch Microplate Spectrophotometer; BioTek Instruments Inc., Winooski, VT, USA).

Apoptosis analysis

Calu-3 or NCI-H1975 cells were plated in 60-mm dishes (SPL) at a density of 1×10⁵ cells per dish and incubated for 24 hours at 37 °C in culture medium supplemented with BGE (10 mg/mL for Calu-3 cells and 0.5 mg/mL for NCI-H1975 cells). Cells were then harvested by scraping, collected, and washed twice with DPBS containing Ca²⁺ and Mg²⁺ (herein referred to as Ca²⁺/Mg²⁺-DPBS). Subsequently, cells were stained for 30 minutes at room temperature with fluorescein isothiocyanate (FITC)-conjugated Annexin V (Cat. No. A13199; Thermo Fisher Scientific) diluted 1:50 in Ca²⁺/Mg²⁺-DPBS. Afterward, cells were incubated for an additional 15 minutes at room temperature in Ca²⁺/Mg²⁺-DPBS containing 20 µg/mL PI (Thermo Fisher Scientific). Double-stained cells were analyzed using a FACSymphony A3 flow cytometer (BD Biosciences, San Jose, CA, USA), and data were processed using BD CellQuest Pro software (BD Biosciences).

Lung cancer xenograft mouse model

Fifty microliters of high glucose medium containing 2×10⁶ Calu-3 cells were mixed with 50 µL of Matrigel (Corning, NY, USA). The resulting mixture was subcutaneously injected into the right and left dorsal flanks of anesthetized mice, with 100 µL administered at each site. Once the tumors reached an average volume of 100 mm³, a total of 8 mice were randomly assigned to two experimental groups, with 4 mice in each group: a control group and a BGE-treated group.

Tumor volume, tumor size, and organ weight

The long and short axes of the tumors were measured using a digital caliper (Traceable Carbon Fiber Calipers; Control Company, Webster, TX, USA), and tumor volume was calculated using the formula: volume (mm³) = (long axis × short axis²)/2. In addition, the body weight of the mice and the weights of excised tumors or organs were measured using a scale (Mettler Toledo, Columbus, OH, USA).

Histological analysis

The liver and spleen were fixed in 4% (v/v) formaldehyde (Sigma-Aldrich, Waltham, MA, USA) diluted in phosphate-buffered saline (PBS; Thermo Fisher Scientific) at 4 °C for 48 hours. After washing with PBS, the fixed organs were embedded in paraffin (Leica, Wetzlar, Germany). The paraffin-embedded tissues were sectioned into 4 µm slices using a microtome (Leica) and mounted onto microscope slides (Matsunami, Kishiwada, Japan). Hematoxylin and eosin (H&E) staining of the sections was performed automatically using a TP1020 (Leica) according to the manufacturer’s instructions. Finally, the stained tissues were covered with a coverslip using Permount (Thermo Fisher Scientific) and examined under an optical microscope (Axio Imager A2; Carl Zeiss, Oberkochen, Germany).

Statistical analysis

All statistical analyses for the experimental groups were performed using the Statistical Analysis System (version 9.4; SAS Institute, Cary, NC, USA). Comparisons between treatment groups were conducted using the least-squares or DUNCAN method, and the significance of main effects was evaluated by analysis of variance within the statistical analysis system (SAS) software. A P value of less than 0.05 was considered statistically significant.

Results

Experiment 1: in vitro antitumor effects of BGE on human lung cancer cells

To evaluate the potential antitumor effects of BGE on human lung cancer cells, its cytotoxic and apoptotic activities were assessed in Calu-3 cells or NCI-H1975 cells, representative human lung cancer models. In Calu-3 cells, a significant reduction in cell viability (91.35%±3.00%, P=0.002) was observed at 10 mg/mL BGE (Figure 1A). Moreover, no Annexin V/PI double-positive cells (0%) were detected in the untreated (0 mg/mL) control group (Figure 1B), whereas treatment with 10 mg/mL BGE resulted in 26.97%±3.89% double-positive cells (Figure 1C). Similarly, NCI-H1975 cells showed a significant reduction in cell viability beginning at 0.5 mg/mL BGE (91.35%±3.00%, P<0.001), with cell viability declining significantly in dose-dependent manner thereafter (Figure S1A). Moreover, no Annexin V/PI double-positive cells (0%) were observed in the untreated (0 mg/mL) control groups (Figure S1B), whereas treatment with 0.5 mg/mL BGE resulted in 17.69%±2.62% double-positive cells (Figure S1C). These results demonstrate the apoptosis-associated cytotoxic effects of BGE on lung cancer cells and highlight its potential as an antitumor agent against human lung cancer at the cellular level.

Figure 1 BGE-induced cytotoxicity and apoptosis in Calu-3 human lung cancer cells. Calu-3 cells were cultured for 48 hours in culture medium containing various concentrations of BGE (0, 0.5, 1, 2, 5, or 10 mg/mL). Cell viability was subsequently assessed using the Alamar blue assay. For apoptosis analysis, Calu-3 cells were incubated for 24 hours in either BGE-free medium or medium supplemented with 10 mg/mL BGE, followed by Annexin V/PI double staining and flow cytometric analysis. Treatment with 10 mg/mL BGE significantly reduced cell viability (A), and 26.97%±3.89% of the cells were double-positive for Annexin V/PI (C). In contrast, no double-positive population was detected in untreated control cells (B). Data are presented as the mean ± SD of three independent experiments. *, P=0.002. BGE, black ginseng extract; PI, propidium iodide; SD, standard deviation.

Experiment 2: in vivo antitumor effects of BGE on human lung cancer cells

To evaluate the in vivo antitumor effects of BGE, nude mice bearing Calu-3 cell-derived lung tumors were orally administered BGE. Tumor volume, tumor weight, and body weight were measured and compared between the BGE-treated group and control mice that did not receive BGE. From day 7 onward, tumor volume increased in the Calu-3 xenograft mice; however, tumor growth was suppressed in BGE-treated mice, and from day 14 onward, tumor volume was significantly smaller than in untreated controls (Figure 2A). No significant differences in body weight (P=0.37) were observed between the BGE-treated (25.22±2.59 g) and control groups (26.92±2.29 g) throughout the experiment (Figure 2B). On day 21, evaluation of tumor weight (BGE-treated: 0.15±0.06 vs. control: 0.30±0.10 g, P=0.042) and size (BGE-treated: 436.22±118.33 vs. control: 1281.16±418.10 mm³, P=0.008) revealed a significant reduction in tumor weight (Figure 2C) and visibly smaller tumors (Figure 2D) in the BGE-treated mice. These results provide strong evidence that oral administration of BGE substantially inhibits lung tumor growth in vivo, supporting its potential as an effective antitumor agent against human lung tumors.

Figure 2 Antitumor effect of BGE in a Calu-3 cell xenograft nude mouse model. Calu-3 cells (2×10⁶) were inoculated into nude mice. Once tumor volume reached 100 mm³, oral administration of DW (without BGE, control) or DW containing BGE (with BGE) was initiated (day 0) and continued daily for 21 days. Tumor volume (A) and body weight (B) were measured at 7-day intervals. On day 21, tumors were harvested, weighed (C), and photographed (D). Tumor volume increased from day 7 onward in control mice; conversely, mice receiving BGE exhibited significantly smaller tumor volumes (A). No significant differences in body weight were observed between groups throughout the experiment (B). Tumor weight (C) and size (D) at day 21 showed that tumors from BGE-treated mice were significantly smaller than those from controls. (D) Representative images of harvested tumors, with BGE-treated tumors displayed below and control tumors above. Data in (A,B) represent means ± SD of four independent experiments. Data in (C) represent the mean (solid line) from four independent experiments. *, P<0.05. (A) P=0.008, (B) P=0.37, (C) P=0.042. BGE, black ginseng extract; DW, distilled water; Rep., replication; SD, standard deviation.

Experiment 3: evaluation of BGE toxicity in non-cancerous mouse organs

To evaluate the potential toxicity of BGE on non-cancerous tissues, we examined the effects of oral BGE administration on the liver and spleen of lung tumor-bearing mice over a 21-day period (34). Absolute organ weights and histopathological features of the liver and spleen were compared between mice treated with BGE and those that did not receive BGE. No significant differences were observed in the weights of the liver (BGE-treated: 1.53±0.33 vs. control: 1.58±0.25 g, P=0.67) and spleen (BGE-treated: 0.11±0.03 vs. control: 0.10±0.01 g, P=0.41) between the BGE-treated and control groups (Figure 3A). Moreover, histopathological analysis revealed no notable abnormalities in the liver and spleen tissues of BGE-treated mice compared to those in untreated mice (Figure 3B). These results indicate that BGE does not induce detectable toxicity in non-cancerous tissues.

Figure 3 Absolute weight and histopathological analysis of liver and spleen from Calu-3 cell xenograft nude mice orally administered with BGE. Calu-3 cells (2×10⁶) were inoculated into nude mice. Once tumor volume reached 100 mm³, oral administration of DW (without BGE, control) or DW containing BGE (with BGE) was initiated and continued daily for 21 days. During necropsy, the liver and spleen were excised and weighed. Organs were then sectioned, fixed, processed, paraffin-embedded, and stained with H&E. No significant differences in liver (P=0.67) or spleen (P=0.41) weights were observed between BGE-treated and control mice (A). Representative micrographs show no BGE-related histopathological abnormalities in the liver or spleen compared to controls (B). Data in Figure (A) represent means ± SD of three independent experiments. Scale bar =50 µm. BGE, black ginseng extract; DW, distilled water; H&E, hematoxylin and eosin; SD, standard deviation.

Discussion

The development of safer and more effective cancer therapies is essential to address the limitations of conventional chemotherapy, including severe side effects and drug resistance. Natural compounds, many with long-standing use in traditional medicine, represent a valuable source for anticancer drug discovery. In this study, we show that BGE exhibits significant antitumor activity against lung cancer. In vitro, BGE demonstrated pronounced cytotoxicity toward Calu-3 human lung cancer cells. In vivo, oral administration of BGE markedly suppressed tumor growth in a Calu-3 xenograft mouse model without causing observable toxicity in major organs, even with repeated dosing. These findings highlight BGE as a safe and promising candidate for lung cancer therapy and support its further development as a potential therapeutic agent.

Previous studies have demonstrated that ginsenosides Rg3 and Rg5 induce apoptosis and autophagy in lung cancer cells by activating caspase-9 and caspase-3 and promoting poly(ADP-ribose) polymerase (PARP) cleavage (23). They also suppress the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and epidermal growth factor receptor (EGFR)/vascular endothelial growth factor (VEGF) signaling pathways, thereby inhibiting metastasis and reducing lung cancer cell proliferation (23). In particular, ginsenoside Rg3 has been shown to modulate the Notch/hairy and enhancer of split-1 (Hes1) signaling pathway, resulting in increased apoptosis, G2/M cell cycle arrest, and inhibition of tumor spheroid formation (35). Additionally, Rg3 effectively suppresses the migration and invasion of lung cancer cells, ultimately inhibiting epithelial-mesenchymal transition (EMT) (24). In the present study, BGE enriched with ginsenosides Rg3 and Rg5 exhibited significant antitumor effects at both the cellular and histological levels. These effects are likely attributed to the synergistic interactions of the ginsenoside complex within BGE, which effectively inhibits cellular proliferation while simultaneously promoting apoptosis and autophagy.

It is well established that smoking accounts for 80–90% of lung cancer cases (36). Carcinogenic compounds in tobacco not only increase cancer risk through direct exposure but also contribute to the development of lung inflammation (37). Inhalation of tobacco smoke triggers an inflammatory response, leading to the generation of reactive oxygen species (ROS) (38). These ROS induce oxidative stress, which exacerbates chronic inflammation (39). Persistent inflammation causes cellular damage and the accumulation of genetic mutations, promoting the transformation of normal cells into cancerous ones (40). This ongoing inflammatory response not only facilitates the initiation of lung cancer but also drives the progression and aggressiveness of established tumors (41). In summary, lung inflammation plays a central role in both the initiation and progression of lung cancer, and the continuous cellular damage associated with chronic inflammation underscores the importance of strategies aimed at managing and preventing inflammation to reduce lung cancer risk.

Previous studies on major inflammatory airway diseases, including asthma and chronic obstructive pulmonary disease (COPD), have shown that treatment with ginsenoside Rg3 significantly reduces eosinophil infiltration, oxidative responses, airway inflammation, and airway hyperresponsiveness in asthma mouse models (42). Furthermore, Rg3 decreases the expression of inflammatory cytokines and reduces ROS production. In a mouse model of acute exacerbation of COPD exposed to cigarette smoke for 14 weeks, Rg3 improved lung function and morphology and attenuated neutrophil migration and inflammation by inhibiting PI3K activation (43). Additionally, both ginsenosides Rg3 (44) and Rg5 (45) suppress the expression of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, as well as inflammatory enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the lungs of lipopolysaccharide (LPS)-induced models. These ginsenosides also inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and the nuclear translocation of p65 (44). Notably, ginsenoside Rg5 alleviates pulmonary inflammation by blocking the binding of LPS to toll-like receptor 4 (TLR-4) on macrophages (45). These findings indicate that BGE exerts anti-inflammatory effects by mitigating inflammatory responses. Consequently, the observed anticancer effects against lung cancer in this study may, in part, result from the combined anti-inflammatory actions of BGE, suggesting that regular consumption could potentially contribute to the prevention of de novo lung cancer development.

Conclusions

In conclusion, BGE exhibited anticancer activity against lung tumors at both the cellular and histopathological levels, without inducing toxicity in major organs. These findings underscore BGE’s potential as an effective therapeutic agent for lung cancer and highlight its promise as both an adjunctive treatment and a preventive strategy for lung cancer development. However, this study has limitations, including a small sample size, the inherent differences between xenograft mouse models and human lung tumors, and toxicity assessments restricted to H&E staining and organ weights, without evaluation of serum biochemical markers or comprehensive organ pathology. Future studies should address these limitations by conducting long-term safety evaluations, including liver and kidney function tests and more extensive organ pathology. In addition, mechanistic studies using clinically relevant models are required to further validate the therapeutic potential of BGE.

Acknowledgments

We acknowledge that the study was conducted collaboratively between the company and the university. English language editing was performed by Textcheck. A certificate is available at: http://www.textcheck.com/certificate/r02FBT.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2901/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2901/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2901/prf

Funding: This research was supported by grants from Truth & Ginseng Biotechnology Research Co., Ltd.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2901/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. All animal experiments were performed under a project license (No. KW-240116-1) granted by Ethics Committee of the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University, in compliance with national or institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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