Effects of Xylitol on Tumor Progression in Syngeneic Mice Cancer Models

1. Introduction

Xylitol is a well-known, preventative product used in dentistry for decades [1]. Erythritol and xylitol are polyols extensively researched and demonstrated to have notable anti-cariogenic and anti-periodontal disease properties with appropriate use [2,3,4]. Polyols have been used traditionally (for about 80 years) to replace sugar in sweet foods to block tooth enamel demineralization and to reduce postprandial blood glucose surges. However, the benefits of added dietary polyols go beyond removing sugar. Emerging evidence shows that xylitol and erythritol can play several functional roles in actively supporting oral and systemic health maintenance with anti-biofilm, antioxidant, and anti-diabetic effects. Xylitol and erythritol disrupt keystone oral disease initiators such as S. mutans (caries) and P. gingivalis (periodontal disease) while acting as prebiotics and helping to balance and maintain a healthy microbiome, beginning with the oral gateway microbiome, which supports innate immunity and disease resistance [5,6,7,8,9,10,11,12,13,14].

The role of the microbiome in cancer development and treatment is now well-recognized 15. Indeed, the interactions between microbiome and cancer have generated research into the complex microbial communities and the possible mechanisms through which the microbiota influence cancer prevention, carcinogenesis, and anti-cancer therapy. In addition, developing next-generation prebiotics and probiotics designed to target specific diseases is considered extremely urgent [16]. Ideally, healthcare providers will one day optimally implant effective prebiotics, probiotics, and the derived postbiotics to ameliorate disease [17]. Immune elimination and immune escape are reported as hallmarks of cancer, and both can be partly bacteria-dependent, with the shaping of immunity by mediating host immunomodulation. In addition, host immunity regulates the microbiome by altering bacteria-associated signaling to influence tumor surveillance. Cancer immunotherapy, including immune checkpoint blockade (ICB), appears to have heterogeneous therapeutic effects in different individuals, partially attributed to the microbiota [18]. In the era of personalized medicine, the microbiota and its interactions with cancer must be better understood, and manipulating the gut microbiota to improve cancer therapeutic responses could be necessary for future cancer treatment [19].

Research demonstrates the inhibitory property of xylitol with many cancer cell lines when administered both dietary and systemically [20,21,22]. Because xylitol exhibits almost no side effects and is safely utilized by healthy human cells, it could be a beneficent natural supplement for potentially inhibiting cancer cell proliferation [23,24]. Additionally, xylitol inhibits angiogenesis, thereby decreasing the vascularization of the tumor [25]. Increased vascularization would support tumor growth and possibly cancer metastasis. Interestingly, the typical human is reported to endogenously produce approximately 15 gm per day in the liver, making xylitol endogenous [26]. Indeed, xylitol is utilized by the mitochondria of cells as the precursor to the first step in the tricarboxylic acid cycle (also known as the citric acid cycle or the Krebs cycle), with xylitol dehydrogenase on the cristae converting xylitol to d-xylulose which then converts NADP to NADPH [27]. In addition to the liver production of xylitol, many plants, such as blueberries, strawberries, plums, cauliflower, and even oats, have substantial amounts of xylitol naturally present [28]. There is considerable overlap when comparing the list of xylitol-containing foods to the American Heart Association's “heart-friendly” foods list. Xylitol has been utilized even in diabetes prevention and as an anti-inflammatory [29,30].

Previous research with xylitol supplementation in animal models has had positive results in inhibiting cancer cell lines and xenografts [31,32]. Combination treatments with xylitol have also been reported as successful [33]. Animal models are considered the first phase of cancer research, looking for potential therapeutic agents [34]. This research study intends to use two mouse models to evaluate the efficacy of different concentrations and the modality of delivery of xylitol in cancer.

Metabolomics can be used to identify cancer biomarkers and the drivers of tumorigenesis [35]. Metabolism is dysregulated in cancer cells to support uncontrolled cell proliferation [36]. This dysregulation of cellular metabolism leads to specific metabolic phenotypes. These metabolic phenotypes can be used for earlier cancer diagnosis, clinical trials, patient selection, and/or as biomarkers of treatment response. Drugs that selectively target metabolic enzymes and, with precision medicine and nutrition, can affect cancer-related unique metabolic dependencies [37]. Cancer outcomes and the patient's quality of life may be affected by cancer and cancer treatments in individual and complex manners due to the whole-body interaction and the influences of diet and exercise [38].

The purpose was to evaluate the effects, including cell line inhibition of xylitol on tumor progression, in two syngeneic mouse cancer models. Specifically, this pilot study measured tumor inhibition from two cancer cell line implantations in two syngeneic mouse cancer models, 4T1 mammary carcinoma and B16F10 melanoma, with 20% xylitol (prebiotic) solution intratumorally administered daily. Institutional (IACUC) approval was obtained for this study.

2. Materials and Methods

This study included two strains of immunocompetent female mice: 20 C57BL/6 and 20 BALB/c mice for a total of 40 mice. The BALB/c group was injected with 4T1 mammary carcinoma cells (1 x 10^6 cells into the 4th mammary gland), and the C57BL/6 group was injected with B16F10 melanoma cells (1 x 10^6 cells into the flank). When tumor sizes reached 50 to 100 mm3, both treatment groups (10 each) were injected daily with 20% xylitol solution intratumorally and subcutaneously. Control mice received sterile saline (10 each) with the same daily treatment frequency (Monday – Sunday) as the xylitol group. The treatment route was subcutaneous (SQ) - ~75% of volume Intra-tumoral (IT)- with the remaining 25% peri-tumorally. Tumor tissue and terminal blood were collected for pharmacokinetic, metabolomic, and histopathologic analyses upon mouse euthanasia.

Euthanization protocol:

All mice were euthanized when control tumor volumes were equal to or larger than 2000 mm3 or if mice lost more than 20% of their original body weight. In addition, euthanization was performed if there are other severe clinical health issues (e.g., paralysis) that would cause undue discomfort. Xylitol was given daily until significant changes in the tumor sizes were observed (the study terminated if no changes were seen). Euthanasia was performed according to IACUC protocol. Animals were not combined from different cages, and when euthanizing some of the mice from a cage, the rest of the animals remained in their original cage. The maximum number of mice per cage was five, and the CO2 flow rate per mouse cage was 3 L/min until one minute after breathing stopped. Euthanasia was confirmed by cervical dislocation.

Data:

All mice's body weight and Tumor volume were measured every other day. After the study terminated, tumor tissue and blood were collected for the following analyses: pharmacokinetic, metabolomic (tissue flash frozen in liquid nitrogen), and histopathology—tissue placed in formalin (for H&E staining for general morphology, IHC staining for microvascular density).

3. Results

After five days of 20% xylitol injections, tumor volumes were reduced by 40% in the C57BL/6 group injected with B16F10 melanoma cells (see Figure 1), but with the BALB/c + 4T1 cancer line (adaptive immunity), the tumor reduction was insignificant. With repeated intra-tumoral injections, the tumor stroma deteriorated in the B16F10 tumors, resulting in substantial xylitol leaking onto the skin surface. Afterward, experimental and control tumor volumes would become clinically comparable by study termination at day 14 (see Table 1). Interstitial tumor pressure increased, preventing sufficient injection of the solution after five days. Metabolomic analysis revealed apparent differences between experimental and control tumor cellular metabolism. Lymph node histological analysis demonstrated metastasis in both groups by the time of euthanasia. The metabolomic analysis demonstrates that intratumoral xylitol reduces tumor cell production of histamine, NADP+, ATP, and glutathione, thereby affecting the availability of reactive oxidative species and the host immune response (see Figure 2). The xylitol group showed significantly decreased phosphocreatine, citrate, and pyruvic acid levels, signifying metabolic stress likely due to inhibiting mitochondrial metabolism within the tumor cells. Notably, xylitol levels were increased in tumors, demonstrating that xylitol accumulates in cancer cells and suggests that the effects of xylitol are likely due to cancer cell-intrinsic effect on metabolism (see Table 2). In addition, a decrease in tumor glutathione may affect the innate immune response, enhancing the effect of reactive oxidative species.

4. Discussion

The data denotes that xylitol inhibits both cancer cell lines, although only significantly in the melanoma group (syngeneic model C57L/C, B16F10- innate immunity). Cancer cells cannot utilize xylitol as can normal human cells (as do rodents such as rats and mice) in their mitochondria for energy [24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39]. Most cancer cells utilize glycolysis and mitochondrial metabolism to sustain growth in vivo [40]. Certain animal species cannot correctly utilize xylitol, especially carnivores that have not been evolutionarily exposed to plants as foods 41. Tubers were once a significant source of food for homo sapiens and still a survivor food for hunter-gatherers, such as the Hadza tribe [42,43]. The microbiome of hunter-gatherers and traditional tribal people maintains specific bacterial taxa that can utilize complex carbohydrates and polyols [44,45]. One can theorize that homo sapiens cell mitochondria may have rapidly evolved to metabolize xylitol due to its presence in several survival foods, especially during periods of scarcity [46]. Cockroaches, mice, rats, swine, and humans can metabolize xylitol [41,42 and 46].

Xylitol enhances the innate immune system response to cancer cells. Cancer cell lines are effectively more sensitive to ROS the killer T-cells produce [47]. Results from metabolomics support the theory. The metabolomic values were different between the vehicle and xylitol groups. Specifically, tumor cell production of glutathione and histamine was reduced by xylitol. Glutathione is an antioxidant that reduces the reactive oxygen species (ROS) effect on tumor cells [48,49]. Xylitol reduction of histamine may hypothetically reduce metastasis by reducing vascularization and proliferation [50,51]. In addition, mitochondrial metabolism is necessary to determine stem cell fate. Mitochondrial metabolism is responsible for the production of ATP and maintains the tricarboxylic acid (TCA) cycle. The metabolites of the TCA cycle support stem cell survival and growth. Recent evidence shows that mitochondria control mammalian stem cell’s fate and function through reactive oxygen species (ROS) generation, TCA cycle metabolite production, NAD+/NADH ratio regulation, pyruvate metabolism, and mitochondrial dynamics [52]. Xylitol affects the mitochondria and the TCA cycle, apparently with anti-oncogenic properties.

Our first protocol utilized intra-tumor injections, which proved less effective due to loss of stroma. Lack of structural integrity, interstitial fluid pressure, and possibly tumor trauma resulted in xylitol solution leaking from the tumor, allowing for tumor progression [53]. This exact mechanism was reported with previous intra-tumoral injections using chemotherapeutics, such as 5 fluorouracil, until the development of smart hydrogels [54]. Studies in progress at the Developmental Therapeutics Core utilize Alzet mini osmotic pumps that deliver a consistent concentration of xylitol to the subject animals [55,56]. Our research team is now cautiously optimistic that this approach will advance techniques used in previously published studies with IV xylitol, which could be more challenging to implement in an animal group [21].

The human microbiota and microbiome have many biological functions, including food digestion, synthesis of various vitamins, protection from pathogen colonization, and resistance against systemic infections [57]. The gut microbiota also plays a crucial role in immune system development, and its alteration can cause immune dysregulation, which can lead to autoimmune diseases [58]. The role of microbiome in cancer development/progression via several mechanisms, including immune response modulation and the promotion of a pro-inflammatory tumor environment, has been very well studied and established [59].

It has also been noted that xylitol significantly affects the composition of the gut microbiota, stimulates the propagation of beneficial bacteria, and produces short-chain fatty acids (SCFAs) [60,61]. This altered metabolism of xenobiotic xylitol can potentially influence cancer growth and progression [62].

5. Conclusions

Our study offers novel insights into the potential therapeutic role of xylitol in cancer treatment, specifically in melanoma. Treatment with a 20% xylitol solution demonstrated a significant reduction in the initial growth of B16F10 melanoma tumors in syngeneic mouse models, highlighting xylitol's potential as an adjunctive treatment in oncology. Notably, this effect was predominantly observed in the melanoma model, indicating a potential cancer-type specificity or differential mechanism of action. The efficacy of the xylitol injections decreased after five days due to melanoma tumor stroma degradation, and the tumors re-commenced growth.

The findings underscore the intricate relationship between metabolic interventions and tumor progression. Xylitol's impact on tumor metabolism, specifically its influence on reducing tumor cell production of crucial metabolites like histamine, NADP+, ATP, and glutathione, paves the way for further exploration into its role as a metabolic modulator in cancer therapy. This is particularly relevant given the growing interest in metabolic pathways as targets for cancer treatment.

Moreover, the differential responses observed between the 4T1 mammary carcinoma and B16F10 melanoma models illuminate the complex nature of cancer biology and the necessity for targeted therapeutic strategies. Our study also brings to light the challenges associated with intratumoral drug delivery, as evidenced by the complications in maintaining the structural integrity of the tumor stroma during xylitol administration.

Future research should focus on optimizing the delivery method of xylitol to enhance its therapeutic efficacy. Extending these findings to other cancer models and eventually to clinical trials will be crucial to fully understanding xylitol's potential in cancer treatment. Our research lays the groundwork for such future investigations, with the hope of contributing to more effective and targeted cancer therapies.