Study: Diclofenac and metformin synergistic dose dependent inhibition of hamster fibrosarcoma, rescued with mebendazole. Biomed Pharmacother. 2023 Nov;167:115528. doi: 10.1016/j.biopha.2023.115528. Epub 2023 Sep 20. PMID: 37738800.
Background and Rationale
Both diclofenac and metformin have been shown to possess anticancer properties. Diclofenac is recognized for its inhibition of cyclooxygenase-2 (COX-2) and suppression of the nuclear factor-kappa B (NF-κB) signaling pathway, which plays a crucial role in the development and progression of cancer. Metformin has been widely studied for its ability to regulate glucose metabolism, inhibit NF-κB, and impact various oncogenic pathways. Given their individual effects, the combination of these drugs could act synergistically to inhibit cancer cell proliferation more effectively than either drug alone.
Methods
The study employed an established hamster fibrosarcoma model using BHK-21/C13 cells inoculated into Syrian golden hamsters. The experimental design included several treatment groups: control (saline), diclofenac alone, metformin alone, a combination of diclofenac and metformin, and a triple combination of diclofenac, metformin, and mebendazole (an antiparasitic drug used as a rescue experiment to stimulate NF-κB).
The study evaluated the following aspects of tumor growth:
Tumor volume
Tumor weight
Tumor surface area
Tumor burden (relative to body weight)
The tumors' histological and immunohistochemical characteristics included markers of proliferation (Ki-67, PCNA), glucose metabolism (GLUT-1), nitric oxide production (iNOS), angiogenesis (CD34, CD31), and apoptosis (COX4, cytochrome C).
The primary objective was to assess the efficacy of the diclofenac-metformin combination compared to the control and single-agent treatments.
Results
Tumor Reduction with Diclofenac and Metformin
The study's most significant finding was the marked tumor reduction in the combination treatment group. Hamsters treated with the combination of diclofenac and metformin showed the following improvements over single-agent treatments and the control group:
Tumor Volume: The combination therapy significantly decreased tumor volume, with mean tumor sizes smaller than those observed in the diclofenac or metformin monotherapy groups. For example, tumors in the combination group exhibited approximately a 90% reduction in volume compared to the control.
Tumor Weight and Surface Area: Similarly, tumors' weight and surface area were reduced by more than 80% in the combination group. In contrast, single-agent treatments showed moderate reductions but were not statistically significant compared to the control.
Tumor Burden: The combination therapy also reduced tumor burden (tumor weight as a percentage of total body weight), demonstrating that the diclofenac-metformin regimen effectively inhibited tumor growth.
Single-Agent Treatments: Diclofenac and Metformin
While diclofenac and metformin showed some potential to reduce tumor growth, their effects were modest compared to the combination. Neither diclofenac nor metformin as monotherapies significantly reduced tumor volume, weight, or burden. This highlights the importance of combining these two agents to achieve a potent anticancer effect.
Diclofenac Alone: In the diclofenac treatment group, tumor volume reductions were observed, but they were less pronounced and did not reach statistical significance. The drug's anti-inflammatory properties may have contributed to a slight inhibition of tumor progression, but the drug alone was insufficient to halt tumor growth.
Metformin Alone: Metformin alone had minimal effects on tumor size and volume. While metformin is known to modulate glucose metabolism and inhibit NF-κB, its influence on tumor growth in this model was limited without the addition of diclofenac.
Histological and Immunohistochemical Findings
The combination of diclofenac and metformin not only reduced tumor size but also exerted significant effects on several tumor growth processes:
Proliferation Markers (Ki-67, PCNA): Tumors treated with the combination therapy had significantly lower levels of Ki-67 and PCNA, indicating reduced cell proliferation. These markers are commonly used to assess tumor growth rate, and their reduction suggests that the combination therapy effectively slowed down the replication of cancer cells.
Glucose Metabolism (GLUT-1): GLUT-1 is a marker of glucose uptake and metabolism in cancer cells. The reduced expression of GLUT-1 in the combination therapy group implies that the tumor's metabolic activity was impaired, potentially due to metformin’s effect on glucose regulation.
Nitric Oxide Production (iNOS): The combination therapy also resulted in a significant decrease in iNOS expression, suggesting that the tumor's inflammatory environment was suppressed. This could be attributed to diclofenac’s anti-inflammatory properties.
Angiogenesis (CD34, CD31): the formation of new blood vessels to supply the tumor with nutrients was significantly reduced in the combination therapy group. This is a critical finding, as the inhibition of angiogenesis can starve the tumor of the necessary resources to grow and spread.
Apoptosis (COX4, Cytochrome C): Markers of apoptosis, including COX4 and cytochrome C, were significantly increased in the combination therapy group. This suggests that diclofenac and metformin together induced programmed cell death, a desirable outcome in cancer treatment.
The Role of Mebendazole
Interestingly, the triple therapy that included mebendazole did not provide additional benefits. In fact, adding mebendazole to the diclofenac-metformin combination "rescued" tumor growth; mebendazole blocked the anticancer activity of the diclofenac and metformin combination.
Mebendazole is known to stimulate NF-κB, a pathway inhibited by diclofenac and metformin, which may explain why its addition negated the anticancer effects. This finding underscores the importance of careful consideration when combining multiple drugs in cancer therapy, as certain combinations may lead to antagonistic effects.
This study provides compelling evidence that combining diclofenac and metformin can significantly reduce tumor growth in a hamster fibrosarcoma model. The synergistic effects of the two drugs were evident across multiple parameters, including tumor volume, weight, and key markers of proliferation, metabolism, and apoptosis. Importantly, this combination was well-tolerated and did not induce significant toxicity in the treated animals, making it a promising candidate for further investigation in cancer therapy.
Single-agent treatments with diclofenac or metformin were relatively ineffective, highlighting the power of combination therapy to target multiple pathways simultaneously. The role of NF-κB inhibition appears to be crucial, as demonstrated by the failure of the triple therapy with mebendazole, which activated NF-κB and reversed the anticancer effects of diclofenac and metformin.
In sum, the combination of diclofenac and metformin represents a promising approach to cancer treatment. By repurposing these affordable, well-established drugs, this strategy offers a potential low-toxicity, cost-effective solution to improving cancer patients' outcomes.
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