Abstract
Background/Aim: Melanoma is a prevalent and severe disease, making the development of new treatments essential. Nowata110 has demonstrated significant therapeutic efficacy in phase II clinical study against plantar warts. It has shown promising anti-cancer effects in both in vitro and in vivo studies using HPV-16 and HPV-18-induced cervical cancer models. This study evaluated the effectiveness of Nowarta110 in murine melanoma-implanted animals. Materials and Methods: Nowata110 is a novel compound composed of colloidal silver and fig extract. Male and female immunodeficient C(Cg)-Cd 79 atm 1 (cre) and immunocompetent BALB/c mice were used. Murine melanoma cancer cells (B16-F10) were implanted subcutaneously in experimental animals. Once the top cross-sectional area of tumors reached a minimum of 5 mm2 and approximately 4 mm depth, mice were anesthetized and treated with Nowarta110 intravenously or intratumorally. Control mice received no treatment. Results: In non-treated immunodeficient mice, tumor growth progressed over five weeks, whereas Nowarta110-treated mice drastically reduced tumor volume until five weeks after treatment was initiated. Similarly, in untreated BALB/c mice, tumor growth persisted over five weeks. However, Nowarta110-treated mice exhibited a notable reduction in tumor size. Although intravenous administration of Nowarta110 did not result in complete tumor regression in immunodeficient mice, it did lead to reduced tumor growth. In BALB/c immunocompetent mice, intravenous treatment generally stalled tumor progression. Conclusion: Intratumoral and intravenous administration of Nowarta110 effectively treated murine melanoma in immunodeficient and immunocompetent mice. The Nowarta110 antitumoral efficacy was more pronounced in immunocompetent mice treated intravenously than immunodeficient mice. Clinical studies to examine the efficacy of Nowarta110 in human melanoma and skin cancers are warranted.
The increasing number of worldwide cases of skin cancer pose a significant healthcare challenge. Malignant melanoma, an incredibly aggressive type of cancer, is becoming more prevalent and is resistant to many standard treatments (1, 2). Therefore, understanding the role of natural compounds in preventing and treating malignant melanoma is extremely important (3). The current treatment methods for melanoma include cryotherapy, surgery, radiation therapy, chemotherapy, photodynamic therapy, biological therapy, and targeted drug therapy (4). Unfortunately, these treatments are often inadequately effective, expensive, and have many side effects. The development of multidrug resistance, multiple side effects, and the high costs of conventional therapies for malignant melanoma are prompting the discovery of innovative, safe, inexpensive, and more effective therapeutics (5).
Plants are an abundant source of novel chemical pharmaceutical compounds (6). More than 75% of the current antineoplastic drugs are derived from natural sources, including marine, microbial, and botanical sources (7). Throughout history, natural products have played a significant role in treating both animal and human illnesses. These products have contributed to the development of many current pharmaceutical agents, particularly for cancer therapy (8).
The fig (Ficus carica) is commonly used in food and medicine in the Middle East. It has been historically used to treat warts and skin tumors (9, 10). Fig latex has shown effectiveness against specific human cancer cells, including hepatocellular carcinoma and glioblastoma cell lines (11). Fig leaves have high peroxidation inhibitory activity, likely due to a higher concentration of flavonoids, furanocoumarins, and linolenic acid methyl ester. This could be valuable in developing therapies for skin cancer (12). In the form of colloidal silver or silver nanoparticles, silver has demonstrated antitumor and antiviral activity in various in vitro and in vivo studies, including those on melanomas (13-15).
Nowarta110 is a promising product with potential antiviral and anticancer properties. It contains fig extract and colloidal silver and shows promise as an effective treatment for skin cancer. Our previous phase II clinical study on human plantar warts demonstrated its excellent efficacy and safety profile, which gives hope for its future application in skin cancer treatment (16).
In vitro and in vivo studies have demonstrated that Nowarta110 effectively triggers cell death in human cancer cells transformed by HPV-16 and HPV-18. It has also been shown to induce tumor regression in mouse models with cancer cells implanted intravaginally or subcutaneously. These findings suggest that Nowarta110 could be further studied and utilized for the treatment of cervical dysplasia and cervical cancer (17).
This study aimed to provide valuable insights into Nowarta110’s potential as an effective treatment for murine malignant melanoma. If successful, this research could significantly contribute to developing innovative and safe therapeutics for melanoma and possibly other types of skin cancer.
Materials and Methods
Mice. Male and female immunodeficient C(Cg)-Cd 79 atm 1 (cre) and immunocompetent BALB/c mice were housed in individually ventilated cages under pathogen-free conditions. Mice were allowed ad libitum access to sterilized water and standard chow. Once mice reached 5-8 weeks of age and a weight of 15-25 g, they were used for subsequent tumor implantation experiments.
Tissue culture. Murine melanoma cancer cells (B16-F10) were obtained from ATCC (Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with 1% sodium pyruvate (Fisher Scientific, Hampton, NH, USA), 10% fetal bovine serum, and 1% penicillin/streptomycin cocktail. Cells were maintained in continuous culture for no longer than three months.
Murine melanoma cancer cell implantation into mice. At approximately 80% confluence, cultured murine melanoma cancer cells were washed with phosphate-buffered saline (PBS) and detached from a culture flask with 0.25% trypsin-EDTA (Fisher Scientific). Cells were resuspended in a 50/50 mixture of PBS and Matrigel High Concentration (BD Biosciences, Franklin Lakes, NJ, USA) at 1×108 cells/ml concentration. Mice were anesthetized via 3% isoflurane (Abbott Laboratories, Alpharetta, GA, USA) inhalation during induction and 2% isoflurane during maintenance. Before cell inoculation (at the flank), fur was shaved (BALB/c mice), and skin was sterilized with isopropyl alcohol (BALB/c and immunodeficient mice). Mice were then subcutaneously inoculated with a 100 μl bolus of cells.
Tumor treatment with Nowarta110. Tumor growth was measured using calipers. Once the top cross-sectional area of tumors reached a minimum of 5 mm2 and approximately 4 mm depth, mice were anesthetized as described earlier and treated with Nowarta110 intravenously or intratumorally. For intravenous treatment, 200 μl Nowarta110 was administered twice weekly, whereas for intratumoral treatment, 100 μl Nowarta110 was administered twice weekly. Control mice received no treatment.
Tumor monitoring. Upon initiation of Nowarta110 treatments, tumor sizes were recorded twice weekly. Tumor volumes were calculated using the ellipsoid equation: V=4/3 •π•a•b•c, wherein a is the long dimension of the tumor ellipsoid, b is the short dimension perpendicular to the axis along a, and c is the height of the ellipsoid.
Results
Intratumoral Nowarta110 treatment dramatically reduces tumor size. To determine the direct effects of Nowarta110 on melanoma cell-derived tumors, we first injected the drug intratumorally in immunodeficient mice or BALB/c mice implanted with murine melanoma cancer cells. The treatments were initiated once tumors reached a minimum of 5 mm2 in the top cross-sectional area and 4 mm depth. In non-treated immunodeficient mice, tumor growth continuously progressed over 5 weeks, whereas a drastic reduction in tumor volume until five weeks after treatment initiation was observed in Nowarta110-treated mice (Figure 1A and B). At this point, tumors were barely detectable. However, some degree of skin necrosis was observed at the tumor site. Similarly, in non-treated BALB/c mice, tumors continuously grew over five weeks. However, in Nowarta110-treated mice a dramatic reduction in tumor sizes was observed over the same timespan (Figure 1C and D). Skin necrosis was also present at the tumor site, but to a greater degree. This may indicate that inflammation may contribute to the increased collateral tissue damage. Nonetheless, mortality was not observed in any of the animals throughout the duration of treatment. These results demonstrate that Nowarta110 can potently attenuate tumor burden when delivered directly to the tumor.
Intratumoral Nowarta110 treatment dramatically reduces tumor size. (A) Average tumor volumes in immunodeficient mice subcutaneously implanted with murine melanoma cancer cells. Mice received either intratumoral Nowarta110 treatment or no treatment. Dimensions were measured weekly and are presented as percent relative to the Week 1 no-treatment group. (B) Representative images of immunodeficient mice at Week 5 after the start of Nowarta110 treatment. White arrows indicate the site of the tumor. (C) Average tumor volumes in BALB/c mice subcutaneously implanted with murine melanoma cancer cells. Mice received either intratumoral Nowarta110 treatment or no treatment. Dimensions were measured weekly and are presented as percent relative to the Week 1 no-treatment group. (D) Representative images of BALB/c mice at Week 5 after the start of Nowarta110 treatment. White arrows indicate the site of the tumor.
Intravenous Nowarta110 delivery limits tumor growth. Although intratumoral injection of Nowarta110 led to a profound regression in tumor size, it remained unclear whether direct treatment was required for this, or if systemic infusion of the drug could also be effective. To examine this, we administered Nowarta110 intravenously and assessed its effects on tumor growth in immunodeficient and BALB/c mice. Unlike intratumoral injection, tumor growth was reduced in immunodeficient mice (Figure 2A and B), whereas it was mostly stalled in BALB/c mice (Figure 2C and D). These results show that Nowarta110 can limit the growth of melanoma cell-derived tumors when delivered into circulation, but antitumoral effects are not as potent as direct intratumoral injection.
Intravenous Nowarta110 delivery limits tumor growth. (A) Average tumor volumes in immunodeficient mice subcutaneously implanted with murine melanoma cancer cells. Mice received either intravenous Nowarta110 treatment or no treatment. Dimensions were measured weekly and are presented as percent relative to Week 1 no treatment group. (B) Representative images of immunodeficient mice at Week 5 after the start of Nowarta110 treatment. White arrows indicate site of tumor. (C) Average tumor volumes in BALB/c mice subcutaneously implanted with murine melanoma cancer cells. Mice received either intravenous Nowarta110 treatment or no treatment. Dimensions were measured weekly and are presented as percent relative to Week 1 no treatment group. (D) Representative images of BALB/c mice at Week 5 after the start of Nowarta110 treatment. White arrows indicate the site of the tumor.
Discussion
Melanoma, a prevalent and life-threatening form of skin cancer, remains a significant challenge for both prevention and treatment. While notable progress has been made in comprehending its origins and treatment, the quest for effective prevention techniques and therapies is still ongoing (18). Melanoma arises from the malignant transformation of pigment-producing cells called melanocytes. It can develop on the skin (cutaneous melanoma) or in other areas, such as the eyes, ears, gastrointestinal tract, genitalia, urinary system, and meninges, leading to mucosal or other melanomas (19). The main challenge in treating melanoma is the low response rate to current treatments, as melanoma cells often develop resistance and show incomplete responses (20).
This study investigated the potential for an improved anti-tumor response in immunocompetent mice following treatment with Nowarta110 compared to immunodeficient mice. We found that subcutaneous tumors in immunocompetent mice treated with Nowarta110 showed a significant reduction compared to treated immunodeficient mice. We also observed substantial differences between the intravenous and intratumoral administration treatment in immunodeficient mice, leading to smaller tumors than in the respective immunodeficient mice. There were some similarities in the intratumoral administration between the two strains, likely due to the direct injection of Nowarta110 into the tumor, which resulted in necrosis or apoptosis. In the experiments with intravenously administered Nowrata110, the antitumor response in the immunocompetent mice was significantly more robust than in the immunodeficient mice. This suggests that Nowarta110 may potentially augment the immune response against implanted tumors.
The results of our current study and the data from our prior human and animal investigations indicated that Nowarta110 could be explored in a control clinical trial as a topical and systemic potential treatment for skin cancer, including melanomas. Most melanomas (about 90%) are diagnosed as primary tumors without metastasis, which makes Nowarta110 a more practical solution for local or transdermal therapy (21-23).
Melanoma is a severe disease, and it is crucial to develop new ways to prevent and treat this cancer. Natural remedies are promising because they are affordable and may have fewer side effects. Further clinical studies of Nowarta110 are needed to examine its effectiveness in treating skin cancers, including melanomas, in humans and animals.
Conclusion
Intratumoral and intravenous administration of Nowarta110 effectively treated murine melanoma implanted in immunodeficient and immunocompetent mice. The Nowarta110 antitumoral efficacy was more remarkable in immunocompetent mice treated intravenously compared to immunodeficient mice. Clinical studies are warranted to support the potential application of Nowarta110 in treating human melanoma and other skin cancers.
Acknowledgements
The Authors thank Mr. Vu Duy Van and Mrs. Nguyen Thu Ha of Fobic Pharma of Hanoi, Vietnam, for their invaluable support in completing this manuscript writing and publication.
Footnotes
Authors’ Contributions
JS: Participated in research design, data analysis, and manuscript writing. MK: Participated in research design, performed animal study, data analysis, and manuscript writing. NK: Participated in research design, performed animal study, and data analysis. KM: Conceptualization, data analysis, and manuscript writing. FA: Conceptualization and editing. HANT: Review and editing. BH: conceptualization, writing, and editing. BXH: Conceptualization, study design, data analysis, and manuscript writing.
Conflicts of Interest
All Authors declare no conflicts of interest related to the writing and publishing the manuscript.
Funding
Nowarta Biopharma Inc. funded the study.
- Received October 31, 2024.
- Revision received November 4, 2024.
- Accepted November 5, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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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