Qilian Formula Inhibits Tumor Cell Growth in a Bone Metastasis Model of Lung Cancer

Abstract

Background:

Bone metastasis is frequently common in advanced lung cancer with the major issue of a pathological fracture. Previous studies suggested that Astragalus membranaceus (Qi) and Ampelopsis japonica (Lian), which are used as folk medicine in China, have potential effects on inhibiting tumor growth and protecting bones, respectively. In this study, an experiment on the inhibitory effect of the Qilian formula (AAF) in vivo was designed to examine tumor growth in bone and osteoclast formation.

Materials and Methods:

The bone metastasis xenograft models were established by implanting NCI-H460-luc2 lung cancer cells into the right tibiae bones of mice. After confirming the model’s viability through optical imaging 7 days post-implantation, 2 groups, namely the AAF group and the control group, were administered 0.3 mL of AAF extract (9 g/kg/day) or normal saline via intragastric delivery for a duration of 4 weeks. Throughout the study, we longitudinally assessed tumor burden, bone destruction, and weight-bearing capacity in vivo using reporter gene bioluminescence imaging (BLI), micro-CT, and dynamic weight-bearing (DWB) tests. Mechanistic insights were gained through Hematoxylin-eosin (H&E) staining, immunohistochemical (IHC) analysis, western blotting, and flow cytometry.

Results:

Qilian formula produced significant inhibition to the progress of bone destruction and tumor burden in the right tibiae bone in the treatment group. It was further evidenced by molecular imaging in vivo via small animal micro-CT and BLI with parametric quantification, characterizing significantly lower uptake of BLI signal in the treated tumor lesions and improving the pathological changes in the microstructure of bone. Furthermore, DWB tests revealed that Qilian formula treatment significantly maintained the weight-bearing capacity. According to immunohistochemical analysis, the effect of the Qilian formula appeared to involve the suppression of osteoclast formation by lower expression of the tartrate-resistant acid phosphatase. Cell apoptosis and death induction were evidenced by a higher percentage of Bal2、BAX and caspase 3 expressions of Qilian formula-treated tumor tissues.

Conclusions:

Our study demonstrated a significant inhibitory effect of the Qilian formula on the progression of osteolytic invasion in vivo by suppressing osteoclastogenesis and promoting apoptotic cell death.

Background

Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality, and it poses the greatest threat to health and life of the population. The 5-year relative survival rate for lung and bronchus cancer with distant stage was 6% at diagnosis during 2012 through 2018. ¹ NSCLC metastasis is frequently common in bone, brain, lung, and liver with bone accounting for nearly 39% of total incidence. ² Skeletal metastases can cause bones to become weakened, which can result in the development of skeletal-related events (SREs) . These events may include fractures, compression of the spinal cord, bone pain, and disability. ³ They can significantly contribute to the morbidity and mortality of patients with advanced cancer. ⁴ Therefore, for bone metastasis, in addition to conventional anti-tumor therapy, bone conservation therapy must also be initiated in a timely manner.

Bone-targeted agents such as bisphosphonates and denosumab have been shown to be very effective in preventing and reducing SREs and are now the standard of care for the treatment of patients with malignant bone disease. ⁵ The mechanism of action of the 2 drugs is different. Bisphosphonate can impair the function of osteoclasts by acting on them, causing osteoclast apoptosis, while denosumab prevents osteoclast formation/proliferation/survival through high affinity/specific binding of RANKL. In clinical studies, the administration of the RANKL inhibitor denosumab to patients with early-stage breast cancer or non-metastatic castration-resistant prostate cancer (CRPC) did not demonstrate any impact on disease recurrence in women with breast cancer. ⁶ Additionally, the use of denosumab only resulted in a modest improvement in bone metastasis-free survival for CRPC patients, ⁷ implying that the RANK/RANKL pathway does not play a significant role in tumor cell colonization in bone. ⁸

In bone metastasis, research reports focused on 2 different aspects of colonization: homing mechanisms and interaction of carcinoma cells with the bone/bone marrow stroma. ⁹ Integrin antagonists in the treatment of tumor bone metastasis can directly target tumor cells while inhibiting osteoclast activity, exerting a dual inhibitory effect. ¹⁰ In one respect, cancer cells that are already within the bone microenvironment play an impactful role on the further development of these metastatic lesions.^11,12 There is systemic crosstalk between tumors and bones. Carcinoma cells can remotely activate osteoblasts in bones even in the absence of local metastasis. Osteoblasts could supply tumors with neutrophils, which foster cancer progression. ⁹ Therefore, the treatment of bone metastasis cannot only focus on bone protection.

In another respect, dual-target drugs are increasingly being used in clinic. A dual pan class I PI3K and mTOR inhibitor used for treating osteolytic bone disease has been shown to decrease MM plasma cell proliferation, osteoclast formation and function and promote osteoblast formation and function. ¹³

The aim of the current study was to investigate the anti-osteoclast and the apoptosis-promoting effects of the mixture of Astragalus membranaceus and Ampelopsis japonica derived from traditional Chinese medicines that have been reported to have anti-cancer effects.

Astragalus membranaceus (AM) is a traditional herbal medicine that has been used for cancer treatment in herbal decoctions. AM has drawn the attention of researchers for its ability to restore the function of immune cells such as T-cells that are impaired by tumor-mediated mechanisms in cancer patients. ¹⁴ In animal models, AM has shown anti-tumor effects by enhancing immune responses, reducing tumor size or growth, and restoring mitogenic activity. ¹⁵ In osteoporosis related studies, AM has been shown to preserve trabecular bone microarchitecture by down-regulating the expression of TRAF6 and NFATc1 in RANKL-RANK pathway. ¹⁶

Ampelopsis japonica (AJ) is another oriental herb that has been used for fever, pain, and wound healing. AJ possesses anti-1,1-diphenyl-2-picryl-hydrazyl (DPPH) and anti-reactive oxygen species (ROS) properties. ¹⁷ Moreover, AJ has demonstrated anti-metastatic and anti-invasive effects on MDA-MB-231 breast cancer cells by downregulating metalloproteinase (MMP)-2 and MMP-9 expression. ¹⁸ AJ also inhibits HCT-116/SW480 colorectal cancer cells by targeting STAT3 and beta-catenin signaling pathways. ¹⁹

In the current study, we attempted to investigate the potential of Qilian formula to inhibit growth of the highly metastatic NCI-H460-luc2 lung cancer cells implanted into right tibiae bone of mice.

Materials and Methods

Human Lung Cancer Cell Line: NCI-H460-luc2

NCI-H460-luc2 (Bioware^® Ultra-Light Producing Cell Line), a highly metastatic lung cancer cell line stably transfected with the firefly luciferase gene (luc2) was established by transducing lentivirus containing luciferase 2 gene under the control of human ubiquitin C promoter.

Animals and Model Establishment

A total of 20 athymic female nude mice (BALB/c nu/nu; 6-week-old; Shanghai Laboratory Animal Center, CAS (SLACCAS) Service Shanghai) were divided into 2 groups: a normal saline (NS) control group (n = 10) and a Qilian formula treatment group (n=10). Animals were acclimated to the animal facility for 1 week before surgery on a 12 h light–dark cycle with access to food and water ad libitum in specific pathogen free (SPF) barrier. For each mouse, 1.0 × 10⁸/mL NCI-H460 cells were inoculated into right tibiae using our optimized established procedure. ²⁰ Briefly, after ensuring adequate anesthesia, the mice were immobilized with their right tibia fully exposed and bent at a 90° angle with the femur. A BD insulin syringe (U40;1 mL; 29 g × 12.7 mm; diameter, 0.30 mm; BD Biosciences. Franklin Lakes, NJ, USA) without any contents was used to inject the needle through the lateral edge of the tibia into the bone marrow from the middle of the tibial plateau. The needle was carefully withdrawn to relieve the pressure of the marrow cavity. Then, a gaseous micro syringe (25 μL diameter; 0.50 mm) was employed to inject a mixture of NCI-H460 cell suspension (10 μL; 1 × 10⁸ cells/mL), air (1 μL) and gel foam (2 μL) along the same injection site. The injection site was pressed for a few seconds to stop bleeding. The use of animals and the experimental protocol were approved by the Institutional Animal Care and Use Committee of Longhua Hospital, Shanghai University of Traditional Chinese Medicine.

Herb

Qilian formula comprises 2 herbs: Astragalus membranaceus (Batch number: 140714, Gansu province) and Ampelopsis japonica (Batch number: 2009112513, Anhui province). The raw herbs used to prepare Qilian formula were purchased from Longhua Hospital, China (Shanghai Huapu Chinese Herbal Medicine Co., LTD., and Shanghai Kangqiao Chinese Medicine Tablet Co., LTD, Shanghai, China) and mixed at a ratio of 1:1; the weight of each herb (gram, dry weight) is 30 g, respectively.

Preparation of Drugs

The Qilian Formula is composed of 30 g of Huangqi (Astragalus membranaceus) and 30 g of Bailian (Ampelopsis japonica), and is decocted at the Tumor Research Institute of Longhua Hospital. The concentration of the decoction is 0.6 g/mL. According to the conversion method of traditional Chinese medicine standards, the dosage is 9 g/kg/day.

Quality Control

Quality Control of the active components of Astragalus membranaceus and Ampelopsis japonica was determined by HPLC. The Astragalus, Ampelopsis, and compound (Astragalus: Ampelopsis, 1:1) were added to 40, 40, and 60 mL of methanol/ethanol and refluxed for 1 h. Samples were filtered and concentrated to 4, 4, and 6 mL and analyzed. Compound purity analysis was performed in on a Waters H-CLASS UPLC system. Standards (0.98-1.92 mg) were analyzed with 1 mL methanol, followed by elution. Acetonitrile, 0.01% trifluoroacetic acid solution was used as mobile phase A and 0.01% trifluoroacetic acid solution as mobile phase B. The detection wavelength was 330 nm. The sample, control and negative control were measured as 10 μL each for the test. The solution was further analyzed by HPLC. The extracted bioactive components of AAF included gallic acid, oleanolic acid, emodin and total flavonoids of Astragalus, as shown in Figure 1C. The active ingredient of Astragalus membranaceus methanol extract is total flavonoids of Astragalus membranaceus (see (Figure 1A). The active components of the methanol extract of Ampelopsis japonica are gallic acid, oleanolic acid, and resveratrol (see Figure 1B).

Figure 1.

The methanol extracts of Astragalus membranaceus, Ampelopsis japonica, and AAF were analyzed by high performance liquid chromatography (HPLC) in 203 nm. (A) Methanol extract of Astragalus membranaceus. (B) Methanol extract of Ampelopsis japonica. (C) Methanol extract of AAF.

Treatment Protocol

Following 7 days after the tumor cell inoculation in mice, confirmation of tumor growth in bone was performed by optical imaging. Seven days after confirmation of the bone metastasis model, the random number method was used based on weight to randomly divide the subjects into the AAF group and the control group. Mice in in the 2 groups were intragastrically injected with 0.3 mL AAF extract or normal saline. Each group of mice received at the time points of 0, 1, and 3 weeks over a 4-week period the following gavage: treatment with AAF or saline for 5 days on, 2-days-off cycle with body weight and tumor growth in right tibia monitored per week. At the endpoint, the right tibia was removed. Mice were euthanized by cervical dislocation following overdose of isoflurane anesthesia. All animal procedures were performed in compliance with Shanghai University of Traditional Chinese medicine Institutional Animal Care and Use Committee (IACUC) guidelines.

Immunohistochemical Staining and Histologic Procedures

All animal specimen collection and CT examination procedures in this experiment were conducted after euthanizing the animals. Mice were deeply anesthetized with 5% isoflurane. After the last imaging session, bone specimens were removed and placed in a histologic cassette and postfixed overnight at 4℃ in neutral formalin buffer (10%). Along with hematoxylin and eosin staining (Servicebio Co., Ltd, No. G1005, Wuhan, China), TRAP immunolabeling (Sinopharm Chemical Reagent Co., Ltd, No.10017818, Shanghai, China) was examined on 3 -µm bone sections. All regions shown on the histologic slices in Figure 3A are located in the metaphysis, anterior to the growth plate in bone regions where resorption was important.

Western Blotting

Protein samples were extracted from xenografts generated from the upper tibial epiphysis that crossed the bone cortex and extended into the soft tissue. Total protein was extracted by radioimmunoprecipitation assay lysis buffer (cat. no. G2002; Wuhan Servicebio Co., Ltd.); Protein concentration was determined by bicinchoninic acid protein assay using a quantification kit (cat. no. G2026; Wuhan Servicebio Co., Ltd). Proteins (40 µg) were separated by 10% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA).

After blocking with 5% skim milk for 2 hours at room temperature, membranes were assayed with the following primary antibodies (1:1000 dilution): caspase 3 (cat. no. BS1518; Bioworld Technology, Inc., St. Louis Park, MN, USA), b-cell lymphoma 2 (Bcl-2; cat. no. 2870; Cell Signaling Technology, Inc., Danvers, MA, USA) and bcl-2-associated X protein (Bax; cat. no. GB11690; Wuhan Ursa Bio Co., Ltd.) at 4˚C overnight. Subsequently, membranes were incubated with enzyme-labeled immunoglobulin G secondary antibody (1:3 000 dilution, cat. No. GB23303; semi-quantitative of band intensity using an enhanced chemiluminescence kit (cat. No. G2019; using Alpha Ease FC (ProteinSimple, San Jose, CA, USA). β-actin (cat. no. GB11001, Wuhan Seri Biological Co., Ltd.) was used as a loading control.

Apoptosis Assay

Apoptosis was measured by flow cytometry method using Annexin V FITC Apoptosis Detection Kit I (Cat. No. 556547, BD Pharmingen™). Briefly, each excised xenograft was cut into multiple 1 × 1 × 1 mm ³ pieces within 30 minutes of surgical removal at 4°C, then washed with aseptic salt water and filtered by 200-micron mesh sieve to prepare single cell suspension. Cells were washed twice with cold PBS and then resuspended in 1 × binding buffer at a concentration of 1 × 10⁶ cells/mL. 100 µL of the solution (1 × 10⁵) was transferred to a 5 mL culture tube, and 5 µL of FITC Annexin V and 5 uL PI were added. Cells were gently vortexed and incubated for 15 minutes at RT (25℃) in the dark.400 µL of 1X Binding Buffer was added to each tube, and samples were analyzed immediately by flow cytometry (BD FACSuite™) within 1 hour.

Bioluminescence Imaging

Bioluminescence images were acquired using IVIS Lumina II spectra (Caliper Corp., Alameda, CA, USA). In preparation for in vivo imaging, mice were anesthetized in 2% to 3% isoflurane in oxygenation-induced chambers. Once anesthetized, mice were transferred to an isolation room, then placed in the imaging chamber and connected to the room anesthesia delivery system and maintained at 1% to 2% isoflurane. The bioluminescence signal is shown in a pseudo color scale in the image, from red (the strongest) to violet (the weakest) for the signal intensity. Scaling was manually adjusted to the same values for each comparable image (in vivo and in vitro) to standardize the bioluminescence intensity across time points.

Micro CT Imaging

Micro CT scans were performed using Inveon Micro CT (Siemens AG, Munich, Germany) on weeks 0, 1, and 3 after therapy initiation (Figure 1A). After euthanizing the mice, their tibiae were extracted. The Inveon analysis workstation (V2: Siemens AG) was used to perform ex vivo Micro-CT analysis from the tibial plateau to the inner condyle of the tibia and to reconstruct 3D structures according to the according to the same protocol version of 1.5.0.28. The region of interest for quantitative measurements was the cancellous bone area of the femoral head. The Inveon analysis workstation was also used to calculate bone volume/total volume, trabecular thickness. trabecular number and trabecular separation.

Weight Bearing Test

The dynamic weight-bearing (DWB) device consists of a small Plexiglas chamber (11 × 11 cm) with a floor sensor containing 1936 pressure sensors. A video camera was aligned to the side of the fence to assist in the data analysis. To test for load-bearing capacity, mice were placed in a chamber and allowed to move freely within the instrument for a 2-minute test period. The weight passing through each paw is automatically assessed by the pressure sensor. The operator manually verified that the footprints used as data during each test period correspond to the claws in the synchronized video as a reference.

Statistical Analysis

SPSS statistical software (PASW^® Statistics18, Chicago) was used for data management and analysis. Data are presented as mean ± SD or otherwise specified. In vitro experiments were performed in triplicate. Differences in in vitro experiments were determined by Student’s t test and Wilcoxon rank sum test. All of the statistical tests are 2-sided. P < .05 was considered to indicate a statistically significant difference.

Result

Tumor Growth In Vivo Was Inhibited Significantly by AAF

To evaluate the inhibitory effect of AAF in a mouse model of lung cancer bone metastasis, we measured the tibial tumor volume and the tumor bioluminescence intensity by using an in vivo imaging system. After 1 week of drug administration, the tibial tumor volume in the control group nude mice was 848.38 ± 192.48 mm ³ , while the volume in the AAF group was 279.36 ± 84.89 mm ³ . The AAF group had a significantly smaller tumor volume compared to the control group (P = .003). By the 3rd week, the tumor volumes for the control and AAF groups were 1562.57 ± 445.49 mm ³ and 462.52 ± 123.59 mm ³ , respectively. In this model, AAF administration resulted in a significant slowing of tumor growth (P = .001). Tumor activity was assessed using a small animal live imaging system at the start of the intervention, 1 week and 3 weeks after drug administration. Quantitative assessment was based on the fluorescence intensity values at the tumor site. The results showed that the fluorescence intensity values of both groups gradually increased over time, with a more pronounced increase in the control group. Three weeks after drug administration, the fluorescence intensity values of the AAF group were significantly lower than those of the control group. The specific drug administration process and its effect on tumors are shown in Figure 2. These findings suggest that AAF has anti-tumor effects on the proliferation of NCI-H460 cells in mouse bone metastasis.

Figure 2.

AAF treatment reduces lung cancer growth. NCI-H460 Luc2 cells at a concentration of 1.0 × 10⁸/mL were surgically implanted into the right tibiae of mice, and after 7 days, the tumor was exposed to AAF treatment. (A) Schematic representation of the AAF therapeutic and scan strategy of the animal model. (B-1) Distribution of tumor cells after injection was visualized by bioluminescence imaging 10 minutes after injection. Subsequent imaging experiments revealed the presence of metastasis in the right tibiae at the designated time points. The bioluminescence signal strength was represented using pseudo-colors, with red indicating a heavier tumor burden and blue indicating a lighter tumor burden. Quantitative analysis was performed on the bioluminescence signal strength. (B-2) AAF treatment at the indicated time points resulted in significant tumor growth reduction of NSCLC implanted xenografts particularly 3 weeks after treatment (volume of bone metastasis tumors = 462.5 vs 1562.6 mm ³ , P = .001). (B-3) The strength of the bioluminescence signal was subject to quantitative comparison between AAF treated and control group. P value: AAF group versus Blank group. Number of samples = 3, Data: Mean ± SD, Statistical Analysis: T test.

AAF Promotes Early Apoptosis of Non-small Cell Lung Cancer Cells

We used flow cytometry to assess the apoptotic rate of the tibial tumor cells and to elucidate the anti-tumor mechanisms of AAF. After 3 weeks of administration, the 2 groups of tumors were homogenized and analyzed by flow cytometry. As shown in Figure 3, the lower right quadrant represents early apoptosis. The results showed that the proportion of early apoptosis in the AAF group was 41.7%, while that in the control group was 23.7%. Statistical analysis showed a significant increase in the proportion of early apoptotic cells in the AAF group compared to the control group(P = .008).

Figure 3.

AAF induces apoptosis in metastatic NCI-H460 cells in vivo. (A) Tumor cells from obtained randomly from either AAF-treated or untreated mice groups 3 weeks post-treatment, were subjected to annexin V and PI staining, followed by analysis using the fluorescence-activated cell sorting (FACS) technique. The percentage of annexin V +/ PI- cells were analyzed where early apoptotic cells were higher in Astragalus & Ampelopsis radix treated cells compared to untreated control (P = .008). Number of samples = 5. (B) Whole protein cell lysates were also prepared randomly from 3 tumors in each group for Western blotting. At week 3, the expression of Caspase 3 was increased(P = .008). β-actin was used as a loading control to ensure equal loading between samples. Number of samples = 3. (C). Tibia sections of intraosseous NCI-H460 tumors obtained from AAF and control group mice at 3 weeks were stained with TUNEL and DAPI. Blue fluorescence indicated nuclei stained with DAPI. Number of samples = 3. Green fluorescence indicated TUNEL positive NCI H460 cells in the bone marrow cavity. The magnification was 400 times. Quantification of TUNEL positive NCI H460 cells from both groups of bone tumor bearing mice at the end of study. HE: Hematoxylin-eosin staining, Tunnel: TdT-mediated dUTP Nick-End Labeling). P value: AAF group versus Blank group. Data: Mean ± SD, Statistical Analysis: T test.

The Effects of AAF on Apoptosis-Related Proteins in Non-small Cell Lung Cancer Bone Metastasis Tumor Cells

To explore the apoptosis-related pathways, we analyzed the apoptosis-related response proteins. Three weeks after drug administration, the tumor was tested for apoptotic pathway proteins using western blot analysis. The results showed that the expression levels of Bax, Bcl-2, and Caspase3 proteins were increased after treatment with the AAF. There was no significant statistical difference in the Bax/Bcl-2 protein ratio between the 2 groups in this study, while the Caspase3 expression in the Qilian formula group was significantly higher than that in the control group (P = .008). See Figure 3.

The Protective Effect of AAF on the Tibial Structure and Its Inhibitory Effect on Osteoclasts

To investigate the effects of AAF on bone and osteoclasts, we used micro-CT to examine the bone structure and measured the osteoclast ratio in the model limbs. By using micro-CT analysis of relevant bone parameters, it was found that the number of bone trabeculae in the control group was 2.48 ± 0.70, and in the AAF group was 4.39 ± 0.48. The cortical thickness of the 2 groups of bones was 0.19 ± 0.01 mm and 0.34 ± 0.02 mm, respectively. The bone volume was 0.09 ± 0.04 mm and 0.25 ± 0.05 mm, respectively. Compared with the control group, the AAF group had a higher number of residual bone trabeculae (P = .034), thicker cortical bone (P = .001), and larger bone volume (P = .003).

Observation of HE-stained sections of the model tibia embedded in paraffin showed that the bone trabecular structure in the AAF group was better preserved and the cortical bone was thicker compared to the control group. TRAP staining revealed that the control group had more osteoclasts attached to the surface of bone trabeculae compared to the AAF group, suggesting that AAF may suppress bone resorption by inhibiting the formation of osteoclasts. See Figure 4.

Figure 4.

AAF inhibits osteoclast generation and bone destruction. (A) Tibiae sections of intraosseous NCI-H460 tumor obtained from AAF treated or untreated control mice were analyzed by H&E and Micro CT scan showing tumor and its adjacent bone occupying the bone marrow cavity with loss of trabecular (red arrows indicate tumor cells and the red dotted circle indicates the location of the marrow cavity). The expression levels of TRAP proteins were also examined by immunohistochemical (IHC) staining (red arrows point to positive staining). Activated osteoclast cells at the AAF treated tumor bone interface shown in TRAP (dark purple color) staining. (B) Parameter analysis of bone including trabecular number, cortical thickness and BV/TV (P < .05). (C) Serum biochemical analysis of alkaline phosphatase, serum calcium and serum phosphorus of the 2 groups. HE, hematoxylin-eosin staining; TRAP, Tartrate-resistant acid phosphatase; BV/TV, bone volume/total volume. P value: AAF group versus Blank group. Number of samples = 3, Data: Mean ± SD, Statistical Analysis: T test.

To support that AAF has a direct effect on osteoclast activity, the effect of AAF on osteoclast viability in vitro was assessed by Cell Counting Kit-8. The results showed that the osteoclast viability was significantly inhibited when the AAF concentration reached 200 ng/mL (P = .002). The osteoclast viability corresponding to the AAF concentrations of 200, 300, 400, 500, 600, 700, and 800 ng/mL were 94.08%, 92.24%, 89.7%, 85.64%, 79.02%, 74.32%, and 71.7%, respectively.

AAF Alleviated the Weight-Bearing Limitation of the Affected Limb in a Non-small Cell Lung Cancer Bone Metastasis Model

To observe the effect of AAF on weight-bearing of the affected limb in this model, we performed dynamic weight-bearing tests on both groups. Weight measurements were taken at the beginning of the intervention, 1 week after administration, and 3 weeks after administration for both groups of models. The results showed that there was no statistically significant difference in weight between the 2 groups based on the 3 weight measurements (P > .05). Using a small animal dynamic weight-bearing system, it was observed that as the tumor on the right tibia grew, the control group gradually exhibited differences in weight-bearing ratio between the healthy limb and affected limb, which were 0.91 ± 0.05, 1.66 ± 0.69, and 3.85 ± 0.85, respectively. However, there was no significant difference in weight bearing between the limbs of the AAF group, which were 1.00 ± 0.12, 1.08 ± 0.24, and 1.46 ± 0.67, respectively. See Figure 5.

Figure 5.

Body weight and the dynamic weight bearing (DWB). The dynamic weight bearing was also tested, the averaged values of the R/L raw pressure data were calculated and analyzed. P value: AAF group versus Blank group. Number of samples = 3, Data: Mean ± SD, Statistical Analysis: One Way ANOVA.

Discussion

In ancient Chinese literature from the Han dynasty, it is recorded that Astragalus membranaceus and Ampelopsis japonica were used together to treat “bone ulcers.” This combination has the effect of promoting tissue growth and healing and supplement deficiencies while eliminating pus. It reflects the characteristics of using the “supporting method” and “supplementing method” in the “3 internal treatment methods” of traditional Chinese surgery.

Modern pharmacology has shown that Astragalus membranaceus can effectively counteract the immunosuppressive effects of chemotherapy drugs and stimulate macrophages to produce IL-6 and tumor necrosis factor.^20,21 The major bioactive components in Astragalus membranaceus was proven to increase the number of lineage-CD11c+ dendritic cell (DCs) and enhance the immune checkpoint inhibitor anti-cancer effect. In clinical practice, we conducted a meta-analysis of chemotherapy with platinum-containing drugs combined with traditional Chinese medicine containing Astragalus membranaceus and compared it to chemotherapy alone. The results showed that the combined treatment significantly improved overall survival (HR = 0.64, 95% CI = 0.49-0.83, P = .001), 1-year survival rate (RR = 0.74, 95% CI = 0.67-0.81, P < .001), 2-year survival rate (RR = 0.44, 95% CI = 0.27-0.73, P = .001) and 3-year survival rate (RR = 0.38, 95% CI = 0.26-0.55, P < .001). ¹⁴

Existing studies report on the related research of Ampelopsis japonica mainly focus on its anti-inflammatory and wound-healing properties as a topical medicine. Studies have concluded that Ampelopsis japonica has wound-healing properties through observation of its external use in treating burned skin and its symptoms. ¹⁷ This provides a clue for explaining the anti-tumor effect of Ampelopsis japonica from the perspective of inflammation and tumor correlation. There are also studies indicating that Ampelopsis japonica has a direct anti-tumor effect, and various extracts of Ampelopsis japonica have a certain anti-tumor effect.^18,22 However, there is no relevant research on the treatment of bone metastasis with Ampelopsis japonica.

This study found for the first time that the combination of Astragalus membranaceus and Ampelopsis japonica can slow down lung cancer bone metastasis by promoting the early apoptosis of tumor cells, reducing bone destruction and enhancing the weight bearing capacity of affected limbs.

In the present study, AAF markedly attenuated tumor volume and tumor fluorescence intensity values induced by NCI-H460-luc2, indicating an improvement in cancer-induced bone metastasis mice. Its protective effect on bone metastasis was further confirmed by histopathological examination. The histopathological result showed significant effects in inhibiting osteoclast growth and cell apoptosis, reducing the degree of bone erosion. Besides, 3D-micro-CT analysis showed protective effects of AAF on the structure and the density of tibial trabecular bone. Flow cytometry analysis showed that AAF could promote the early apoptosis of tumor cells. Caspase-3 is the most important terminal shear enzyme in the process of apoptosis. The ratio between the Bax/Bcl-2 proteins was a key factor for apoptosis inhibition. Western blot showed that AAF could promote an increase in the expression of caspase 3. The result of histopathological observation was consistent with the result of weight-bearing ratio, indicating AAF has a significant improvement against NCI-H460-luc2-induced bone metastasis mice.

This research article elucidated the role of AAF in promoting early apoptosis in a mouse model of bone metastasis of lung cancer, as well as its potential for bone protection. Additionally, TRAP staining revealed a lower number of osteoclasts in the treated group compared to the control group, and the CCK-8 detection demonstrated the inhibitory effect of AAF on osteoclast activity. These observations suggest a potential relationship between the bone-protective effect of AAF and its ability to promote apoptosis or inhibit osteoclasts. In the process of tumor bone metastasis, tumor cells first promote osteoclast maturation by secreting cytokines. Osteoclasts then release large amounts of tumor-promoting cytokines (CXCR-4/CXCL-12, CXCL-5, CXCL-10, CXCL-13, CX3CL-1, CCL-2) ⁸ from the bone matrix by destroying it. As an important “accomplice” of bone metastasis of tumor cells, osteoclasts form a vicious cycle with tumor cells, which plays a key role in bone metastasis of lung cancer.^23,24

Some traditional Chinese medicines, both classic and bone-specific, are widely used to enhance bone metabolism, stimulate osteoblast activity, and counteract the effects of osteoclasts.^25,26 In this study, we have identified a potential non-bone-specific drug that exhibits tumor inhibition properties and offers bone protection.

However, there are 2 limitations in our study. On the one hand, we did not utilize various gradient doses of AAF in vivo; therefore, the correlation between dosage and effectiveness remains unexplained. On the other hand, although this study demonstrated the impact of AAF in promoting tumor apoptosis and bone protection, it lacks research on the role of osteoclasts. Further experiments are necessary to address this aspect in the future.

Conclusions

Increasingly, research has centered on the interplay between osteoclasts and tumors. Targeting this interaction via AAF may represent a promising strategy for preventing or treating bone metastasis in lung cancer. While further investigations are required to validate these findings in human subjects and assess the potential adverse effects of such interventions, this research provides insight into a potential therapeutic target for this bone metastasis.