Abstract
Background/Aim: Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Standard treatment options include surgery, radiation therapy, chemotherapy, and targeted therapies. Despite advancements over the past 25 years, the prognosis of patients with lung cancer remains poor. This study evaluated the synergistic anticancer effects of fenbendazole (FZ) and diisopropylamine dichloroacetate (DADA) on A549 lung cancer cells. Materials and Methods: Fenbendazole (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl) carbamate) is a broad-spectrum benzimidazole anthelmintic commonly used in veterinary medicine. Diisopropylamine Dichloroacetate (DADA), an over-the-counter treatment for chronic liver disease, has demonstrated anti-tumor properties as an inhibitor of pyruvate dehydrogenase kinase. Results: The combination of FZ and DADA exhibited a synergistic effect on inhibiting the proliferation of A549 lung cancer cells. After 48 h of treatment, the FZ-DADA combination produced reactive oxygen species (ROS) and promoted apoptosis by down-regulating Bcl2 and up-regulating BAX protein expression. The combination activated caspase-3, caspase-7, and PARP, further driving apoptosis in A549 cells. The FZ-DADA treatment also induced cell cycle arrest, as evidenced by the inhibition of Cyclin A and Cyclin E proteins. Conclusion: The synergistic anticancer effects of the FZ-DADA combination were confirmed at both cellular and protein levels in A549 lung cancer cells. The combination modulates key apoptotic proteins, induces cell cycle arrest, and increases mitochondrial ROS production, suggesting a promising approach for lung cancer treatment that warrants further investigation and development.
Lung cancer is the leading cause of cancer-associated mortality worldwide, accounting for an estimated 2 million diagnoses and 1.8 million deaths (1). Lung cancer is divided into two broad histologic classes, which grow and spread differently: small-cell lung carcinomas (SCLC) and non-small-cell lung carcinomas (NSCLC). Treatment options for lung cancer include surgery, radiation therapy, chemotherapy, immunotherapy, and targeted therapy. Despite the improvements in diagnosis and therapy made during the past 25 years, the prognosis for patients with lung cancer is still unsatisfactory (2). This research aimed to investigate the anti-cancer effect of Diisopropylamine Dichloroacetate and Fenbendazole in lung cancer models.
Fenbendazole [methyl N-(6-phenylsulfanyl-1H-benzimi-dazol-2-yl) carbamate] is a broad-spectrum benzimidazole anthelmintic approved for use in numerous animal species. Recent studies have shown that fenbendazole (FZ) exhibits cytotoxicity to human cancer cells at micromolar concentrations by inducing mitochondrial translocation of p53, inhibiting glucose uptake, and disrupting other cellular pathways (3). While most reported cases of FZ self-administration have noted reductions in tumor size (4, 5), in some cases, FZ has been found to cause liver injury (6, 7), in which patients promptly recovered after discontinuing FZ. Combining FZ with another drug could mitigate this side effect and support the use of FZ in cancer treatment, enabling pilot clinical trials to be conducted. Therefore, identifying a safe, non-toxic drug synergistic with FZ, preferably a glycolysis inhibitor, is crucial for developing an effective anticancer combination therapy.
Diisopropylamine dichloroacetate (DADA), an over-the-counter drug for chronic liver disease, has demonstrated anti-tumor effects by inhibiting pyruvate dehydrogenase kinase (8). Additionally, DADA is a hepatoprotective pharmaceutical that would support the use of FZ in patients with liver cancer, bile duct cancer, or compromised liver function. FZ-DADA therapy could potentially reduce hepatotoxicity, enhance therapeutic efficacy, and improve tolerability within the framework of comprehensive metabolic therapy for proliferative disorders. This research investigated the effects of the FZ-DADA combination in A549 lung cancer cell line.
Materials and Methods
Cell culture. A549 cells were cultured in an 18 cm2 petri dish with Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) and incubated in a 5% CO2 incubator at 37°C. The cells were frequently maintained, subcultured, and checked for contamination.
Cell cytotoxicity assay. A549 cells were cultured in 96-well plates (2.5×104 cells/well) and incubated overnight in a 5% CO2 incubator at 37°C. Cells were treated with various concentrations of FZ alone, DADA alone and FZ-DADA combination for 24, 48, and 72 h. The cell viability of A549 cells was measured using the MTT method. Violet crystals were dissolved in isopropanol, and the absorbance was measured with a spectrophotometer at 570nm using a SpectraMax ID5 microplate reader. The CI value was calculated using CompuSyn software.
ROS assay. A549 cells were seeded in 96-well plates (2.5×104 cells/well) at 37°C, 5% CO2 for 24 hours. Cells were then treated with various concentrations of FZ alone, DADA alone, and FZ-DADA combination for 48 h. ROS levels were measured with a Reactive Oxygen Species (ROS) Detection Assay Kit (ab287839) (Abcam, Cambridge, UK) at Ex/Em=495/529 nm in endpoint mode using SpectraMax ID5 microplate reader.
Hoechst staining. A549 cells were cultured in 6 cm plates (5×105 cells/well) at 37°C, 5% CO2 for 24 h. Cells were then treated with Paclitaxel 1 μM, FZ 1 μM, DADA 5 mM and FZ-DADA combination for 48 h at 37°C, 5% CO2. After treatment, the supernatant was removed. Cells were washed 3 times with cold PBS.
Cells were incubated with the staining solution containing Hoechst 33342 (Thermo Fisher) for 15 min while protected from light. After 15 min, the staining solution was removed. Cells were washed three times with cold PBS before imaging with a fluorescent microscope.
Cell cycle arrest. A549 cells were seeded in 6-well plates for 24 h before being treated with or without FZ, DADA, or an FZ-DADA combination for 48 h. Cells adhering to the plate were collected and washed in PBS twice. After fixing with ice-cold 70% ethanol at −20°C for 2 h, cells were washed with PBS and treated in PBS for 30 min with 1 mg/ml propidium iodine (PI) (Thermo Fisher) and 20 μg/ml RNase (Thermo Fisher). Cell cycle analysis was performed using a Novocyte 2000 flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA) and NovoExpress software (ACEA Biosciences Inc.).
Apoptosis assay. A549 cells were seeded in 6-well plates for 24 h before being treated with FZ, DADA, or an FZ-DADA combination for 48 h in experiments. In time-dependent experiments, cells were treated with or without the FZ-DADA composition for 24, 48, and 72 h. The supernatant, cell death washes with PBS, and adhering cells were collected and washed twice with PBS. Apoptotic cells were stained with annexin-V (1 mg/ml) and PI (1 mg/ml) using an annexin-V/PI staining kit. Apoptosis analysis was performed using a Novocyte 2000 flow cytometer (ACEA Biosciences Inc.) and NovoExpress software (ACEA Biosciences Inc.).
Western blot. A549 cells were lysed using RIPA buffer containing protease inhibitor and then sonicated for complete cell disruption. The Bradford assay (SERVA Electrophoresis GmbH, Heidelberg, Baden-Württemberg, Germany) determined the total protein lysis concentration. SDS-PAGE was used to separate proteins, which were transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA, USA) and blocked in TBS-T (50 mmol/l Tris-HCL pH 7.6), 150 mmol/l NaCl containing 0.1% Tween-20 (containing 5% BSA) for 1 h. Membranes were incubated with different antibodies (cell signaling) overnight at 4°C. Membranes were washed in TBST, labeled with an HRP-conjugated secondary antibody for 15 min (Thermo Fisher), washed thrice, and measured using an ECL luminescence enhancer (GE Healthcare, Chalfont St Giles, UK). Tubulin (Invitrogen, Waltham, MA, USA) was used as a loading control to ensure even protein distribution among the samples. Images of protein expression were captured by ImageQuant LAS 500.
Glucose uptake assay. Glucose uptake was conducted using 2-deoxyglucose (2-DG), which is structurally similar to glucose (glucose uptake kit, AB136955) (Abcam, Cambridge, UK). 2-DG is taken by glucose transporters and converted to 2-DG-6-phosphate (2-DG6P), which cannot be further metabolized and consequently accumulates inside cells. The accumulation of 2-DG6P is proportional to cells’ absorption of 2-DG (or glucose). In this assay, 2-DG6P is oxidized to produce NADPH, the concentration of which is measured using an enzymatic recycling amplification reaction.
A549 cells were seeded in a 96-well plate (5×104 cells/well) for 24 h. Cells were then starved overnight in high glucose DMEM without FBS. Next, cells were treated for 24 h with various FZ, DADA, and FZ-DADA combination concentrations. Cells were incubated with Krebs-Ringer Phosphate-Hepes (KRPH) buffer for 40 min and insulin 1 μM for 20 min. Then, 10 mM 2-DG was added and incubated for 20 min. Cells were lysed with an extraction buffer, frozen at −80°C for 10 min, and heated at 85°C for 40 min. The lysates were neutralized by adding a neutralization buffer and centrifuged to collect the NADPH supernatant samples. The products were amplified following the glucose uptake kit protocol. The absorbance was measured at 412 nm using the SpectraMax ID5 microplate reader.
Lactate assay. Lactate Colorimetric Assay kits (AB65331) (Abcam) were used to measure lactate in the medium and cell lysates according to the manufacturer’s instructions. A549 cells were seeded in 96-well plates for 24 h, then treated with various concentrations of FZ, DADA and FZ-DADA combination. At 48 h, the lactate concentration in the culture medium or cell lysates was measured at 450 nm using the SpectraMax ID5 microplate reader based on a standard curve generated with known concentrations of lactate solution.
Statistical analysis. Statistical analysis was performed using GraphPad Prism (GraphPad, Boston, MA, USA) and SPSS 22.0 (IBM, Chicago, IL, USA). Means±SD or % were calculated as appropriate. Experiments were performed at least in triplicate, and the average was calculated. The statistical significance of experimental observations was determined using ANOVA with a significance level of p<0.05.
Results
Cytotoxic effects of FZ-DADA against A549 lung cancer cell lines. To evaluate the impact of FZ and DADA on cell viability, A549 lung cancer cells were treated with various concentrations of FZ and DADA for 48 h. IC50 values of FZ and DADA were determined to be 1 μM and 5 mM, respectively. The A549 cell death rate was determined to be dose-dependent, with approximately 30% of cells dying after treatment with the FZ, DADA, and FZ-DADA combination (Figure 1A). The results of combination doses were compared with single drug doses to determine the appropriate, effective combination ratio of FZ and DADA. Our results showed that the FZ-DADA significantly reduced cell cytotoxicity compared to FZ and DADA alone. Therefore, this combination ratio (1 μM FZ and 5 mM DADA) was chosen for further cytotoxicity experiments.
(A) The combination of fenbendazole and diisopropylamine dichloroacetate (FZ-DADA) inhibited the proliferation of A549 cells. A549 cells were treated with different concentrations of fenbendazole (FZ), diisopropylamine dichloroacetate (DADA), and the combination of FZ-DADA for 48 h in a 96-well plate. The MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay was used to measure the cell viability of A549 cells. The color absorbance was measured using a microplate reader at 570 nm. (B) Effect of FZ and DADA combination on the survival of A549 cells. The FZ-DADA composition showed a synergetic effect on A549 cell lines. (A) FZ -DADA composition was applied for 48 h, after which cell viability was determined using the MTT assay. The combination index (CI) and fraction affected (Fa) were calculated using CompuSyn software. The CI <1 indicates antagonism, and the CI >1 indicates synergism. The color absorbance was measured using a microplate reader at 570 nm. Data are mean±SD. *p<0.05, **p<0.01, ***p<0.001. All treatments were performed in triplicate.
To investigate the synergistic effects of FZ-DADA, the combination index (CI) value was calculated (Figure 1B and C, and Table I). The results revealed that FZ-DADA had synergistic effects after 48 h, which improved when the combination dosage was increased. This was proved by the CI value of all test combination dosages being less than one, with the maximum number affected index (Fa) recorded being approximately 0.8. A previous study on combining FZ with the DADA-similar compound dichloroacetate (DCA) also showed synergistic effects consistent with this result (3).
The combination of fenbendazole and diisopropylamine dichloroacetate (FZ-DADA) showed a synthetic effect in A549 cell lines.
FZ-DADA induced ROS in A549 cells. Reactive oxygen species (ROS) are chemically reactive chemicals containing oxygen (9). ROS are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis (10). ROS and mitochondria play pivotal roles in the induction of apoptosis under physiological and pathological conditions. A high level of ROS may lead to increased cell damage through the oxidative process of DNA, carbohydrates, lipids, and proteins (11). To evaluate the effects of FZ and DADA on ROS productions, A549 cells were treated with various ratios of their combination. The level of ROS in the supernatant was then measured after 48 h. The results indicated that FZ-DADA increased ROS levels in A549 cells compared to the single treatment with FZ or DADA (Figure 2).
The combination of fenbendazole and diisopropylamine dichloroacetate (FZ-DADA) induced the production of reactive oxygen species (ROS) in A549 cells. A549 cells were treated with different concentrations of FZ, DADA, and the combination of FZ-DADA for 48 h in a 96-well plate. ROS levels were evaluated using the ROS Detection assay kit. Data was measured by mean±SD (****p<0.0001 compared with vehicle-treated control, n=3).
FZ-DADA combination induced apoptosis in A549 cells. Fluorescence-activated cell sorting (FACS) analysis employing Annexin-V-FITC and propidium iodide staining was used to investigate the effect of FZ, DADA, and their combination on apoptosis in A549 cells (Figure 3A and B). The flow cytometry results after 48 h treatments revealed that FZ and DADA alone substantially triggered apoptosis at their IC50, with apoptosis occurring in 38.83% (14.62% early apoptosis and 24.01% late apoptosis) and 38.48% (16% early apoptosis and 22.48% late apoptosis), respectively. At the dose 1 μM and 5 mM, FZ-DADA combination showed a dramatic percentage of 71.54% (13.1% early apoptosis and 58.44% late apoptosis) of apoptotic cells. This combination significantly increased the proportion of cells in the late apoptosis phase compared with the single-dose treatment with FZ or DADA.
(A, B) The combination of fenbendazole and diisopropylamine dichloroacetate (FZ-DADA) induced apoptosis in A549 cells. Cells were treated with fenbendazole (FZ), diisopropylamine dichloroacetate (DADA), and their combinations for 48 h, then incubated with Annexin V and propidium iodine to detect the apoptotic stage. (C) Cells were treated with DADA alone, FZ alone, as well as the combination of DADA and FZ composition for 48 h. After removing the supernatant, cells were stained with a Hoescht staining solution for 15 min and observed under a fluorescent microscope (20×). (D) A549 cells were seeded in 6-cm plates for 24 h. Cells were treated with DADA alone, FZ alone, or FZ-DADA for 48 h. Cells were then harvested to extract proteins for western blot with the indicated antibodies. The expression of PARP, BCl2 and BAX, caspase-3, and caspase-7 was normalized to the housekeeping a-tubulin; data are mean±SD (n=3), *p<0.05, **p<0.01 *** p<0.001, ****p<0.0001 compared with the negative control.
The results of Hoescht staining confirmed the effects of FZ and DADA on A549 cells (Figure 3C). At the protein level, FZ-DADA increased approximately 17 times the expression of BAX and reduced 4 times the expression of Bcl2. The Bcl2 family proteins are key regulators of apoptotic cell death. High expression of Bcl2 in various human cancers mediates the resistance of cancers to a wide range of chemotherapeutic drugs and γ-irradiation, which act by inducing apoptosis in tumor cells. Therefore, the blocking of Bcl2 can restore the apoptotic process in tumor cells (12). In contrast, the expression of BAX in cancer cells activated cell death (13).
The morphological changes in apoptosis are primarily due to caspases, a family of cysteine proteases that act as effectors in the cell death pathway (14). As the most downstream enzyme in the apoptosis-inducing protease pathway, caspase 3 plays a pivotal role in cell death by cleaving key proteins in the cell repair process. Caspase 3 cleaves at an aspartate residue, producing p12 and p17 subunits, forming the active cleaved caspase 3. This enzyme is crucial for the morphological and biochemical changes characteristic of apoptosis (15-18). At the protein level, the combination of 1μM FZ and 5 mM DADA exhibited synergistic effects on cleaved caspase-3 protein, resulting in approximately 3.3 and 4 times increase in their levels compared with the treatment with 1 μM FZ or 5 mM DADA alone, respectively. Similar to caspase 3, caspase-7 is universally activated during apoptosis. Interestingly, the combination of FZ-DADA with the dose 1 μM and 5 mM increased the expression of cleaved caspase-7 by 12-fold, which is 3 times and 12 times higher than the levels induced by FZ and DADA alone, respectively. The caspase family, especially caspase-3 and caspase-7, cleave the 116 kDa form of PARP into 85 kDa and 24 kDa fragments (19, 20). PARP has been suggested to contribute to cell death by depleting the cells of NAD and ATP (21). PARP-1 cleavage is a switch point that directs death receptor signaling toward either apoptosis or necrosis (20). Our results indicated that the combination significantly increased about eight times the levels of cleaved PARP. Our results (Figure 3) provide valuable evidence demonstrating the activity of FZ and DADA and their synergistic effects in the apoptosis of A549 lung cancer cells.
FZ-DADA composition arrested the cell cycle at G2/M in A549 cells by down-regulating Cyclin A and E. We also examined the effect of FZ and DADA in the cell cycle regulation of A549 cells. Treatment with FZ and DADA alone at IC50 for 48 h substantially enhanced cell number at G2/M and sub G1 compared to control (Figure 4A and B). Following treatment with the FZ-DADA combination 1 μM and 5 mM G2/M and sub-G1 levels increased while G1 levels decreased, which was statistically significant. When paired with earlier apoptosis findings, these results suggested that the FZ-DADA combination synergistically triggered apoptosis via the G2/M cell cycle arrest block (3). The expression of several cell cycle checkpoints under the influence of FZ and DADA was investigated to assess cell cycle regulation. At the protein level, the FZ-DADA combination decreased the expression of Cyclin A and Cyclin E about 3.5 times and 6.6 times compared to treatments with FZ and DADA alone, respectively (Figure 4E).
The combination of fenbendazole and diisopropylamine dichloroacetate (FZ-DADA) induced G2/M cell cycle arrest in the A549 cell line when compared to the treatment with each component of the combination alone. Cells were treated with FZ, DADA, or FZ-DADA for 48 h, then incubated with RNAse and propidium iodine to detect the apoptotic stage. (A) measurement of various cell cycle stages in untreated and treated A549 cells. (B) The bar graph represents the cell cycle results at the combination of FZ-DADA compared with each agent alone. The result was analyzed using ANOVA. *p<0.05, ***p<0.001. Error bars represent the standard deviation of three experiments. (C) A549 cells were treated with a single agent or the combination of DADA and FZ for 48 h. Total cells were harvested for the western blot experiment with indicating antibodies. The expression of cyclin A (D) and cyclin E (E) was normalized to the housekeeping a-tubulin; data are mean±SD (n=3), *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001 compared with negative control.
FZ-DADA combination inhibited glucose uptake and lactate production. In studies where A549 cells were treated with FZ alone, the anticancer effect of FZ was linked to the inhibition of glucose uptake, resulting in changes in glucose metabolism and cell death (3). DADA was identified as a PDK-4 inhibitor, which reduces lactate generation (8, 23). As a result, the synergistic effects of the FZ-DADA combination, being a glucose uptake inhibitor and PDK-4 inhibitor, were chosen for the glucose metabolism study. Similar to earlier findings, FZ and high-dosage DADA alone dramatically inhibited glucose uptake in A549 cells after 24 h of treatment (Figure 5A). Compared to treatment with either agent alone, the combination treatment resulted in significantly less 2-DG absorption. As expected, FZ and DADA alone reduced lactate generation after 48 h, and the effect was further enhanced when these drugs were combined (Figure 5B). Thus, the FZ-DADA combination demonstrated a synergistic effect in inhibiting glucose uptake and lactate production.
The combination of fenbendazole and diisopropylamine dichloroacetate (FZ-DADA) reduced glucose uptake and lactate production in A549 cells. (A) A549 cells were treated with FZ, DADA, or FZ-DADA combination for 24 h. 2-deoxyglucose (2-DG) uptake was examined using the Glucose Uptake Assay Kit (Colorimetric) (Abcam, Cambridge, UK). (B) A549 cells were left untreated or treated with FZ, DADA, and their combination for 48 h. The lactate levels in the culture medium were collected for detection using the L-Lactate Assay Kit (Colorimetric) (Abcam). The L-lactate inhibition ratio was calculated by comparing it with the untreated group. (C, D, E, F) A549 cells were treated with a FZ-DADA or 1, 3, 5, 7 h. Cells were harvested, and protein was extracted for western blot using antibodies against AKT, pAKT, PI3K, and p-PI3K. The expression of these proteins was normalized to the housekeeping protein b-tubulin. The results were analyzed using ANOVA. *p<0.05, ***p<0.001. All treatments were performed in triplicate.
Glucose uptake is a critical physiological process regulated by several mechanisms, with insulin playing the most prominent role. This powerful anabolic hormone facilitates glucose transport into cells, primarily in metabolically active tissues, such as skeletal muscles, adipose tissue, and the liver, via a specialized transporter known as GLUT4. The process involves a complex sequence of events, primarily mediated by the PI3K/AKT signaling pathway (24). In time-dependent experiments, the FZ-DADA combination at a ratio of 1 μM and 5 mM reduced the levels of both pAKT and pPI3K (Figure 5C-F). The most significant inhibition of AKT and PI3K phosphorylation occurred after 5 and 7 h of treatment with the FZ-DADA combination. For the concentration-dependent experiment, A549 cells were treated with FZ, DADA, or their combination for 7 h. The results showed inhibition of AKT and PI3K phosphorylation by FZ, DADA, and their combination (Figure 5G-K). These findings suggest that FZ and DADA inhibit glucose uptake and lactate production through the PI3K/AKT pathway.
Discussion
In the current study, the FZ and DADA combination exhibits a robust synergistic effect against A549 lung cancer cells. This combination enhances cytotoxicity, induces apoptosis, and disrupts cellular metabolism and cell cycle progression. Our study highlights the potential of the FZ-DADA combination as a therapeutic strategy for lung cancer treatment. These findings provide a solid foundation for further investigation in preclinical and clinical settings to optimize the usage of FZ and DADA in cancer therapy and determine the underlying molecular therapeutic mechanisms of the composition.
Our findings determined the potent synergy between FZ and DADA in inducing apoptosis in A549 cells. The significant modulation of key apoptotic markers, BAX and BCL2, underscores the mechanism through which this combination operates (12, 14, 26). The pro-apoptotic protein BAX promotes mitochondrial outer membrane permeabilization, facilitating the release of cytochrome c and the subsequent activation of the caspase cascade (12, 13). In contrast, Bcl2, an anti-apoptotic protein, prevents this permeabilization, thereby inhibiting apoptosis. The observed down-regulation of Bcl2 and up-regulation of BAX suggest a shift in balance towards apoptosis (12, 14, 27). Furthermore, our study showed up-regulation of the executioner caspases caspase-3 and caspase-7. Once activated, these caspases cleave various substrates, leading to the dismantling of the cell (14-18). This up-regulation indicates that the FZ-DADA combination effectively triggers the apoptotic machinery, reinforcing the mechanism of cell death.
Additionally, the combination treatment effectively arrested the cell cycle at the G2/M phase. Cyclins and cyclin-dependent kinases (CDKs) are pivotal in cell cycle regulation. Cyclin A and Cyclin E are crucial for the G1 to S phase transition and the G2/M transition, respectively. The marked reduction in Cyclin A and Cyclin E levels upon treatment with the FZ-DADA combination aligns with the observed cell cycle arrest, reinforcing that disrupting these transitions is critical for inducing cell death (14, 22).
Another important aspect of this study is the role of mitochondrial ROS production in mediating apoptosis. Reactive oxygen species (ROS) are involved in various cellular processes, including apoptosis (9, 11). The mitochondrial pathway of apoptosis is particularly sensitive to changes in ROS levels. The increase in mitochondrial ROS production observed with a combination of 1 μM and 5 mM FZ-DADA combination likely contributes to the disruption of mitochondrial function, further promoting apoptosis.
In the metabolic system, FZ and DADA demonstrated their capacity to inhibit glucose uptake and lactate synthesis by inhibiting the PI3K/AKT pathway. The FZ-DADA combination effectively prevented glucose uptake and lactate synthesis in A549 cells. Metabolic reprogramming is a hallmark of cancer, with glucose uptake and lactate production being key components of the altered metabolic pathways that support cancer cell proliferation and survival (28-31). The ability of FZ and DADA to inhibit glucose uptake and lactate synthesis highlights their potential to disrupt the metabolic flexibility of cancer cells, thereby impairing their growth and survival.
FZ-DADA’s inhibition of the PI3K/AKT pathway is particularly noteworthy. The PI3K/AKT pathway is a critical regulator of cell metabolism, promoting glucose uptake and glycolysis while inhibiting apoptosis (32-35). By targeting this pathway, FZ-DADA effectively reduces glucose availability for energy production and biosynthesis, leading to decreased lactate production, a byproduct of aerobic glycolysis commonly known as the Warburg effect in cancer cells. This disruption of metabolic processes could sensitize cancer cells to apoptosis and reduce their proliferative capacity.
These findings provide valuable insights into the potential therapeutic applications of FZ and DADA. The ability to synergistically induce apoptosis through multiple mechanisms, including modulation of apoptotic proteins, cell cycle arrest, and ROS production, makes this combination a promising candidate for further investigation in lung cancer therapy. This combination warrants future studies exploring the detailed molecular pathways involved and assessing their efficacy in in vivo models.
Conclusion
The FZ-DADA combination therapy demonstrates a robust synergistic effect in inducing apoptosis in A549 cells, notably at the 1 μM and 5 mM ratios. By modulating key apoptotic proteins, arresting the cell cycle, and increasing mitochondrial ROS production, this combination offers a promising approach to lung cancer treatment, warranting further exploration and development.
Footnotes
Authors’ Contributions
TQN: Participated in research design, research, data analysis, and manuscript writing. DHN: Participated in research, data analysis, and manuscript writing. UTTP, PTTT, HTL, SHL: Participated in research design and execution. JN: conceptualization, writing, and editing. BH: conceptualization, writing, and editing. BXH: conceptualized and participated in research design, data analysis, and manuscript writing.
Conflicts of Interest
All Authors declare no conflicts of interest in writing and publishing the manuscript.
Funding
No external funding was received for this work.
- Received August 18, 2024.
- Revision received September 10, 2024.
- Accepted September 16, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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