Synergistic anti-tumor effect of fenbendazole and diisopropylamine dichloroacetate in immunodeficient BALB/c nude mice transplanted with A549 lung cancer cells

Introduction

According to GLOBOCAN 2020, lung cancer ranks among the highest cancer incidence and mortality rates worldwide. In 2020, 2.2 million new lung cancer cases and 1.8 million lung cancer-related deaths approximately represented approximately represented approximately 11.4% and 18.0% of the total cancer cases and total cancer deaths, respectively (1). Lung cancer is broadly categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for 85% of all cases (2,3). In recent years, many treatments for NSCLC patients have seen advancement, including surgical resection, chemotherapy, radiation therapy, targeted therapy, and immunotherapy (4-6). With the development of personalized, targeted therapies, NSCLC patients now benefit from individualized treatment options. However, despite progress in genetic understanding, diagnostics, and therapeutic strategies, the prognosis for lung cancer remains poor, with only about 20% of patients achieving an overall survival longer than 5 years post-diagnosis, and the figure has shown minimal improvement (7). This underscores the urgent need for new, effective therapeutic strategies for NSCLC patients.

Fenbendazole (FZ) or [5-(phenylthio)-1H-benzimidazol-2-yl] carbamic acid methyl ester is a benzimidazole commonly used as an antiparasitic agent in veterinary medicine. Like several anticancer drugs, FZ inhibits the microtubules’ formation and function by binding with tubulin (8,9). Recent studies have explored the anticancer activity of FZ. Han et al. improved the anti-cancer effect of FZ by inhibiting the reactive oxygen species (ROS) in HL-60 cells (10). In addition, previous investigations indicated that FZ can arrest the G2/M phase in the cell cycle of H4IIE hepatocellular cells (11) and SNU-C5 colorectal cancer cells (12). Peng et al. research demonstrated that FZ interferes with HeLa cervical cancer cell proliferation and glucose metabolism (13). Although most reported cases of FZ self-administration by oral treatment have shown their anti-cancer effects, FZ has been associated with drug-induced liver injury in some cases. However, patients generally recover after discontinuation of FZ (14,15). Furthermore, due to absorption limitation in the intestine, the systemic level of FZ and its metabolites in blood and tissue is lower than the administrated dose (16,17). To optimize FZ’s anticancer potential while minimizing this adverse effect, combining it with a synergistic, safe, and non-toxic agent may facilitate clinical translation. Specifically, identifying a partner compound that can act as a glycolysis inhibitor could be a key to developing an effective anticancer combination therapy.

By inhibiting pyruvate dehydrogenase kinase, diisopropylamine dichloroacetate (DADA), a therapeutic agent for chronic liver disease, has shown anti-tumor properties (18). DADA also acts as a hepatoprotective agent (19), which could support the use of FZ in patients with liver cancer, bile duct cancer, or compromised liver function. Combining FZ and DADA could potentially reduce liver toxicity, increase the effectiveness of the therapy, and improve patient tolerance within a comprehensive metabolic therapy framework for treating proliferative disorders. Thus, this study aimed to investigate the effects of the FZ-DADA combination in a mouse model of lung cancer. We present this article in accordance with the ARRIVE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1272/rc).

Methods

Cells culture

A549 cells were purchased from ATCC (ATCC-CCL-185) and cultured in an 18 cm² culture dish with Roswell Park Memorial Institute (RPMI) medium (Sigma-Aldrich Solution, Merck, Germany) containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen, USA), 1% penicillin/streptomycin solution (penicillin 10,000 U/mL and streptomycin 10,000 µg/mL) (Gibco, Invitrogen, USA) and incubated at 37 ℃ with 5% CO₂. The cells were routinely maintained, subcultured, and checked for contamination. For animal injections, cells were washed twice with 1× phosphate buffer saline (PBS) solution and trypsinization with 1× Trypsin-EDTA solution. The numbers were evaluated using an Invitrogen Countess^® II FL cell counter and analyzer. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Animal models

Immunodeficient BALB/c nude mice (Foxn1nu) were utilized for the in vivo assessment of anticancer activity. A total of 72 mice were imported from BioLASCO (Taiwan) for the study. The mice were housed in a sterile environment following the standard operating procedures for nude mouse care provided by the Center for Experimental Animal Research, Military Medical Academy. All animals were acclimated to the facility’s conditions for one week before the initiation of the experiment, with ad libitum access to food and water. Room temperature and humidity were carefully controlled, and a 12-hour light/dark cycle was maintained. Human lung cancer cells (A549) were collected and diluted in culture media to 2×10⁷/mL, and each immunodeficient mouse received 2×10⁶ cells/0.1 mL into the right flank’s subcutaneous site. Tumor sizes were monitored. When the tumor reached 50 mm³, the mice were randomized into eight groups, as shown in Table 1.

Animals in treatment groups (groups 4, 5, 6, 7, 8) received a daily treatment administered orally, while mice in the positive control group received 5 mg/kg of cisplatin daily by injection directly around the tumor.

Evaluate the toxicity of FZ and DADA treatment

After 60 days of treatment, mice were anesthetized with isoflurane and euthanized by cervical dislocation. Serum, lung, and tumor samples were collected for assays. Post-tumor inoculation, the following parameters were monitored to assess overall health and treatment effects, including body weight, appearance, tumor growth, and survival time. Mice’s health was evaluated based on the following criteria:

• Weight: recorded twice weekly using an electronic scale TE3102S Sartorius to track mice’s weight changes before and after treatment;
• Movement: observations were made on movement patterns (normal, hyperactive, hypoactive, or immobile);
• Response to stimuli: response to tactile and environmental stimuli was categorized as normal, heightened, reduced, or absent;
• Skin color: evaluated for changes, including normal color, purple, and bleeding;
• Mice feces: monitored for consistency and appearance (normal, loose, bloody stool).

The safety assessment of FZ and DADA and their combination

The safety profile of FZ and DADA, individually and in combination, was evaluated through the following metrics:

• Mice’s general condition and body weight: determine hematopoietic function by counting red blood cells, hemoglobin, and white blood cells;
• Determine the amount of liver cell damage by measuring enzyme activity in the blood [alanine aminotransferase (ALT), aspartate transaminase (AST), and histological pictures];
• Evaluate renal function using serum urea and creatinine levels and histopathological images.

Histopathological imaging

Immunodeficient BALB/c nude mice (Foxn1nu) were housed in a sterile environment following the standard operating procedures for nude mouse care. A549 were grown on cell culture flasks in RPMI 1640 culture media supplemented with 10% FBS, 1% penicillin and streptomycin at 37 ℃ and CO₂. After 1 week of housing, A549 cells were injected into mice with 2×10⁶ cells/0.1 mL/mice into the right flank’s subcutaneous site. The manipulation is done in a sterile environment. When the tumor reached 50 mm³, the mice were randomized into eight groups: healthy control, tumor control, cisplatin, FZ 40 mg/kg, DADA 20 mg/kg, DADA 100 mg/kg, FZ 40 mg/kg + DADA 20 mg/kg, FZ 40 mg/kg + DADA 100 mg/kg. Animals were observed after 60 days. Hematoxylin and eosin staining were applied to liver and kidney tissues after harvesting.

Antitumor effect

The tumor volume was calculated using the following formula:

V=(D×R2)×0.5[1]

While: V: tumor volume (mm³); D: tumor length as measured (mm); R: tumor width as measured (mm).

The survival time of each mouse, the average survival time of the groups of mice, the cumulative survival of the treatment and control groups of mice, and the correlation comparison were all monitored until the end of the experiment.

Statistical analysis

Data were analyzed using SPSS version 22.0 (IBM Corp.) and GraphPad Prism 10 (GraphPad Software, USA). The Chi-square test (χ² test) was used to compare two observed proportions. For normally distributed data, a t-test is used to compare the means between two independent groups. In comparison, a one-way analysis of variance (ANOVA) is used to compare the means among three or more groups, with a Dunnett test used to determine which differences are significant. A two-way ANOVA compares the means among three or more groups at different time points. Comparison of medians between two independent groups was performed using the Mann-Whitney U test, with the significance determined based on the P value (P>0.05: no statistically significant difference and P<0.05: statistically significant difference). The log-rank test was applied to compare the cumulative survival time between different treatment methods.

Ethical considerations

All experimental procedures involving animals followed institutional ethical guidelines for the laboratory animal use and care. The protocol was approved by the Dinh Tien Hoang Institute of Medicine’s review board (operating code IRB-VN02010, approval No. IRB-A-2200) before the study. The protocol was prepared before the study without registration.

Results

Toxicity of FZ and DADA in BALB/c nude mice

After A549 cell transplantation, mice maintained normal behaviors, including consistent eating, weight gain, active movement, and responses to stimuli. There were no signs of loose stools, and the anal area remained dry. The mice’s skin at the injection location was normal, with no bleeding or infection. The average weight of all 7 groups of mice was similar before treatment, and the difference was not statistically significant (P>0.05) (Table S1). After treatment, the body weights of the seven groups were measured twice a week. The body weight tended to increase compared to the baseline, except for group DADA, which was 20 mg/kg (Figure 1).

Figure 1 Mice’s body weight after 60 days. Immunodeficient BALB/c nude mice (Foxn1nu) were housed in a sterile environment following the standard operating procedures for nude mouse care. A549 were grown on cell culture flasks in RPMI 1640 culture media supplemented with 10% FBS, 1% penicillin, and streptomycin at 37 ℃ and CO₂. After 1 week of housing, A549 cells were injected into mice with 2×10⁶ cells/0.1 mL/mice into the right flank’s subcutaneous site. The manipulation is done in a sterile environment. When the tumor reached 50 mm³, the mice were randomized into eight groups: healthy control, tumor control, cisplatin, FZ 40 mg/kg—9 mice, DADA 20 mg/kg, DADA 100 mg/kg, FZ 40 mg/kg + DADA 20 mg/kg, FZ 40 mg/kg + DADA 100 mg/kg. The body weight of seven groups of mice was recorded twice a week by an electronic scale TE3102S Sartorius. Data are presented as mean ± SD. One-way ANOVA for the comparison of means among untreated and the other groups, **, P<0.01. ANOVA, analysis of variance; DADA, diisopropylamine dichloroacetate; FBS, fetal bovine serum; FZ, fenbendazole; SD, standard deviation; RPMI, Roswell Park Memorial Institute.

Hematological and kidney function analysis

To further evaluate the side effects of our treatment in mice, we continued to examine the hematological indices of the mice, which play a vital role in assessing the impact of FZ and DADA on BALB/c mice. Table 2 indicates that after 60 days of treatment, there was no statistically significant difference in hematopoietic function (red blood cell count, hemoglobin content, white blood cell count) among eight groups of mice (P>0.05). On the other hand, urea and creatinine levels in mice’s blood were measured to confirm the effect of FZ and DADA on kidney function. After 60 days of therapy, kidney function tests in all 6 treatment groups (5 treatment groups, 1 positive control group) showed no statistically significant difference from the tumor non-treatment control group (P>0.05) (Table 3). Histological analysis of the kidney tissues again confirmed that FZ and DADA treatment did not adversely affect kidney function (Figure 2).

Figure 2 Histology sections of kidney in nude mice. Hematoxylin and eosin staining were applied to liver and kidney tissues after harvesting (×40). (A) Healthy control; (B) tumor control; (C) cisplatin 5 mg/kg; (D) FZ 40 mg/kg; (E) DADA 20 mg/kg; (F) DADA 100 mg/kg; (G) FZ 40 mg/kg + DADA 20 mg/kg; (H) FZ 40 mg/kg + DADA 100 mg/kg. The arrows represent the glomerulus. DADA, diisopropylamine dichloroacetate; FZ, fenbendazole.

Liver function and metabolic assessment

ALT and AST levels were measured to elucidate the impact of FZ and DADA in the liver, and liver tissue was analyzed histologically after 60 days of treatment (Figure 3). These evaluations showed no significant hepatocellular damage due to FZ and DADA treatment. Since cancer treatment can influence metabolic processes, glucose, and lactate were also measured in the blood samples, and our results showed nonsignificant differences in glucose and lactate levels in mice’s blood among eight groups (P>0.05) (Figure 4).

Figure 3 Effect of drug formulation on liver function. (A,B) The AST and ALT index in eight groups was measured at day 60 after the treatment. Data present mean ± SD. One-way ANOVA for the comparison of means among untreated and the other groups. (C-J) Hematoxylin and eosin staining were applied to liver tissues after harvesting (×40): (C) healthy control; (D) tumor control; (E) cisplatin 5 mg/kg; (F) FZ 40 mg/kg; (G) DADA 20 mg/kg; (H) DADA 100 mg/kg; (I) FZ 40 mg/kg + DADA 20 mg/kg; (J) FZ 40 mg/kg + DADA 100 mg/kg. The arrows represent for the central lobule vein. ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate transaminase; DADA, diisopropylamine dichloroacetate; ns, not significant; FZ, fenbendazole; SD, standard deviation.

Figure 4 Effect of FZ and DADA on glucose metabolism. Glucose (A) and lactate levels (B) were determined in the blood of animals on day 60 of the experiment. Data presents mean ± SD. One-way ANOVA for the comparison of means among untreated and the other groups, Dunett’s test was run after one-way ANOVA to determine which different are significant. ANOVA, analysis of variance; DADA, diisopropylamine dichloroacetate; FZ, fenbendazole; ns, not significant; SD, standard deviation.

Anti-tumor effect of FZ and DADA

At the start of treatment, there was no significant difference in the tumor volume across the groups (P>0.05) (Table S2). Tumor growth in the combination treatment group was slower than tumors in the single-treatment groups, with group 7 showing a smaller mean tumor volume than groups 4 and 5. However, this difference was not statistically significant (P>0.05). Interestingly, the combination of FZ and DADA in a ratio of 40:100 resulted in a greater reduction in tumor volume than in single-dose groups across all time points. From day 14, group 8 significantly decreased tumor volume compared with the tumor control group, while the others did not show any statistical difference. At day 60 of the experiment, the combination treatment in group 8 performed a greater anti-tumor effect than their single-dose treatment (Figure 5A), indicating the synergistic anti-tumor effect of FZ and DADA at 40:100 mg/kg ratio.

Figure 5 Tumor volume results in groups of mice during treatment. (A) The tumor volume was calculated using the following formula: V = (D × R²) × 0.5 with V: tumor volume (mm³), D: tumor length as measured (mm), and R: tumor width as measured (mm). Data present mean ± SEM, two-way ANOVA for the comparison of means among untreated and the other groups, *, P<0.05; ***, P<0.001; ****, P<0.0001. (B,C) Tumor reduction rate in treated mice group. The number of tumor-loss mice in eight groups was recorded during 60 days. Chi-square test for comparison of two percentages. ANOVA, analysis of variance; DADA, diisopropylamine dichloroacetate; FZ, fenbendazole; SEM, standard error of the mean.

Tumor reduction rates of FZ and DADA in BALB/c nude mice

On day 7 following treatment, tumor reduction (tumor regression) began in the mice treated in groups 6, 7, and 8. The number of mice losing tumors in the therapy groups steadily rose. The tumor untreated group did not lose any tumor at any stage throughout the trial, whereas the number of dead mice progressively grew. Table 4 shows the number of mice that achieved tumor loss in treatment groups. From day 21 onward, the percentage of tumor-free mice in group 8 was significantly higher than that of the untreated group (P<0.0001). At the end of the experiment (day 60), the tumor reduction rate in the tumor control group, positive control, and group 6 was 0 (0%); groups 4, 5 was 1 (11.1%); group 7 was 2 (20%), group 8 was 5 (50%). The number of mice with tumor loss was 9, accounting for 15% of the total. Interestingly, from days 21 to 60, the proportion of tumor-losing mice in group 8 was significantly higher than that of groups 4 and 6 (Table 4, Figure 5B,5C). Our results indicated the synergic effect of FZ and DADA at a ratio of 40:100 mg/kg in promoting tumor regression in BALB/c nude mice.

Effect of FZ and DADA on the survival duration and death rate of BALB/c nude mice

Survival time was used as a criterion to assess the effectiveness of FZ and DADA treatment in A549 lung cancer-bearing nude mice. As shown in Figure 6A, the average survival time in all treatment groups was significantly higher than in the control group. Survival rates were compared across groups to validate treatment efficacy further. In the tumor control group, the first death occurred on day 21, with mortality increasing steadily throughout the time. The number and death rates of mice in the tumor control group were higher than in the other six treatment groups at all research time points. However, the difference was only statistically significant between the control and treatment groups from day 49 to the completion of the experiment (day 60) (Figure 6B and Table 5).

Figure 6 The survival duration and death rate death rate of nude mice. The number of survival mice in eight groups was recorded during 60 days of the experiment. (A) Mean of survival (day) data present mean ± SD; one-way ANOVA was used to compare the mean of treatment groups and untreated group, ****, P<0.0001. (B) Survival rate (%), the log-rank test was applied to compare the cumulative survival time between different treatment methods. ANOVA, analysis of variance; DADA, diisopropylamine dichloroacetate; FZ, fenbendazole; SD, standard deviation.

Discussion

This study confirmed the safety profile of FZ (40 mg/kg) and DADA (100 mg/kg) in BALB/c mice. The treatment did not alter body weight, blood sugar levels, or liver and kidney function. Our findings demonstrate that combining FZ 40 mg/kg and DADA 100 mg/kg effectively inhibits tumor growth in BALB/c nude mice transfected with A549 NSCLC cells. The combination treatment group showed significantly reduced tumor volume and higher tumor regression rates than the single-treatment groups. Additionally, combination therapy markedly extended survival time in treated mice compared to controls. Previous studies have highlighted the anti-cancer effects of FZ in many cancer types (8-13,16,17,20). Similarly, DADA has shown promise in enhancing efficacy in cancer treatment (21-24). Our findings suggested that the synergistic effects of oral treatment with FZ and DADA, a pyruvate dehydrogenase kinase inhibitor (18) and hepatoprotective compound (19,24,25), may represent a promising therapeutic strategy for NSCLC.

In our previous study, the combination of FZ and DADA showed a synergistic effect in inhibiting the proliferation of A549 lung cancer cells. The FZ-DADA combination induced ROS production and promoted apoptosis by downregulating B-cell lymphoma 2 (Bcl2) and upregulating BAX protein expression. The combination activated caspase-3, caspase-7, and poly (ADP-ribose) polymerase (PARP), further driving apoptosis in A549 cells. Additionally, FZ-DADA treatment also induced cell cycle arrest, as evidenced by the inhibition of Cyclin A and Cyclin E proteins. These findings provide valuable insights into the potential therapeutic applications of the FZ and DADA combination for lung cancer (25,26).

In the current study, we compared the therapeutic efficacy of FZ-DADA with that of peritumoral cisplatin injection to evaluate its potential as a treatment for NSCLC. It is important to note, however, that this comparison may not accurately reflect the true clinical advantage of FZ-DADA over cisplatin in human patients, as cisplatin is typically administered intravenously in clinical settings and is associated with severe adverse effects, including nephrotoxicity, bone marrow suppression, and increased susceptibility to infections—all of which can significantly impair quality of life and reduce overall survival. In contrast, FZ-DADA has demonstrated a favorable safety profile in both human preclinical and animal clinical studies.

Adding DADA to the potential therapeutic combination with FZ may thus serve a dual role in this combination therapy, enhancing FZ’s anticancer efficacy while offering hepatoprotective benefits. This synergistic effect is particularly advantageous for patients with compromised liver function or those requiring long-term cancer treatment, as DADA could mitigate the risk of liver injury and potentially enhance systemic tolerability. Our findings in the current study underscore the potential clinical utility of combining FZ and DADA, supporting its translational research and development as a safe and effective therapeutic option for lung cancer.

Conclusions

Combining 100 mg/kg DADA and 40 mg/kg FZ synergistically inhibited tumor growth in immunodeficient BALB/c nude mice transplanted with A549 lung cancer cells. A clinical study is warranted to prove the efficacy and safety of this well-characterized drug combination as a repurposing treatment for lung cancer.

Acknowledgments

The authors would like to thank Doctor Ngo Thi Thu Hang and the staff of the Center for Experimental Animal Research, Military Medical Academy, Hanoi, Vietnam, for all the assistance and technical help in animal experiments. We also express our gratitude to doctors Nguyen Khanh Hoa and Nguyen Thi Thanh Huong at the Dinh Tien Hoang Institute of Medicine for meaningful advice in experiment design.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1272/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1272/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1272/prf

Funding: The project was funded by Thai Minh Pharmaceuticals JSC, Hanoi, Vietnam.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1272/coif). All authors report that the project was funded by Thai Minh Pharmaceuticals JSC, Hanoi, Vietnam. The authors have no other 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 experimental procedures involving animals followed institutional ethical guidelines for the laboratory animal use and care. The protocol was approved by the Dinh Tien Hoang Institute of Medicine’s review board (operating code IRB-VN02010, approval No. IRB-A-2200) before the study. The protocol was prepared before the study without registration. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

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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