Research Article |
Corresponding author: Wamidh H. Talib ( altaei_wamidh@yahoo.com ) Academic editor: Danka Obreshkova
© 2023 Ayah Kamel Saeed Kamal, Wamidh H. Talib.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kamal AKS, Talib WH (2023) Combination of ketogenic diet and probiotics inhibits breast cancer in mice by immune system modulation and reduction of Insulin growth factor-1. Pharmacia 70(4): 1411-1422. https://doi.org/10.3897/pharmacia.70.e111822
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Breast cancer (BC) is the most prevalent malignancy in women and the first tumor type in the world. Ketogenic diets (KD), which are high in fat, low in carbohydrates, and sufficient in protein, can be used alone or as adjuvants with cancer drug medication as cancer therapy or prevention methods. Probiotics are nonpathogenic microorganisms or groups of bacteria that live in the gut and nourish the host body. In this work, we tested a new KD-probiotic combination against breast cancer implanted in mice. Several combination of probiotics (1×109 CFU/0.5ml) and KD (14.1 kcal/2g) reduced tumor size and enhanced cure rate. KD and combination therapy groups increased beta-hydroxybutyrate (β-OHB) while decrease blood glucose, and IGF-1.IFN-γ, IL-2, IL-4, and IL-10 serum levels were measured to assess immune reaction to various therapies. Combination and probiotics therapy raised IFN-γ, and IL-4 levels, however IL-10 level did decrease in all treated group with highest decrease in combination group. In the safety profile, probiotics, ketogenic diet, and their combination were safe. Overall, the combination of a ketogenic diet and probiotics has the potential to be utilized in the future development of anti-cancer nutrition to augment conventional therapies.
Graphical abstract. Summary of the effect of Ketogenic diet, Probiotics and its combination on EMP-6 breast cancer cell and in vivo:
antitumor, combination therapy, breast cancer, high fat diet
Breast cancer (BC) is the most prevalent malignancy in women and the first tumor type in the world (organization). Breast cancer is metastatic cancer that may often spread to distant organs such as the bone, liver, lung, and brain (
Nutrition is a salient component of breast cancer treatment, recovery, and quality of life. As a result of cancer and its treatment side effects, such as nausea and vomiting, many cancer patients are at risk for poor nutritional status. Nutrition education is critical due to increasingly diverse populations of the United States and many other Western countries, such as Canada, Australia, and European countries, as well as the rising costs of cancer treatment (
Several lines of the study suggest the assumption of low-toxicity therapeutic techniques based on specialized diets like ketogenic diets (KD), which comprise high fat, moderate to low protein, and extremely low carbohydrate, forcing the body to use fat instead of glucose to produce adenosine triphosphate (ATP) (
Probiotics are nonpathogenic microorganisms or groups of bacteria that live in the gut and nourish the host body (
A combination of ketogenic diet with probiotics has not been evaluated in the literature to treat breast cancer. Accordingly, this study was designed to test the therapeutic potential of a new combination consisting of ketogenic diet and probiotics against breast cancer implanted in mice.
One mouse mammary cell line was used in this study, the parent (EMT6/P) cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, United Kingdom). The cell line was cultured using a minimum essential medium supplemented with 10% fetal calf serum, 1% L-glutamine, 0.1% gentamycin, 1% penicillin-streptomycin solution, and 0.5 mL of 0.1% Non-Essential Amino acids 100×. Perfect cell culture conditions were provided for cell growth using complete tissue culture media (MEM).Cell was incubated at 37 °C, 5% carbon dioxide, and 95% humidity.
Commercially available probiotics (Jamieson) used in this study were suspended in a phosphate buffer solution (PBS) to obtain the desired bacterial cell concentration of 1×109 CFU/0.5 ml, and supplied by oral gavage route (
Commercially available ketogenic diet “Ketocal” used in this study. Each mouse was delivered daily 14.1 kcal/2g (
This study was conducted according to standard ethical guidelines. The Research and Ethical Committee approved all the experimental protocols at the Faculty of Pharmacy, Applied Science Private University. This study was conducted using 24 healthy female Balb/C mice, ranging between 21–25 grams of weight and 6–8 weeks of age. Mice were supplied by the animal house in the Applied Science Private University, Amman, Jordan. All protocols of animal experiments were validated by the Research and Ethical Committee of Applied Science University with Standard ethical guidelines. Mice were accommodated in well ventilated rooms, at room temperature 25 °C and 50–60% humidity, as well as alternating cycles of dark and light every 12 hours, in order to accomplish all required environmental conditions. They were raised in cages equipped with wooden shavings for bedding, a special water bottle, and food.
We started to prepare the inoculation of mice with EMT6/P (parent breast cancer cell line). Briefly, EMT-6/P in MEM medium was prepared for the whole 24 mice. Tumor inoculation was achieved by injecting a tumor induction dose of 1000000 cells (in 0.1 mL) subcutaneously in the abdominal area of each female Balb/C mouse. Injected cancer cells were left to grow and form new tumors.
Twenty-four mice were used in this treatment, mice were divided into four groups (n = 6/group). Control group, Ketogenic diet group, Probiotics group, and combination of Probiotics and Ketogenic diet group Table
Where: V, L, and W are the volume, length, and width of the tumor, respectively.
F, I represent the final and initial tumor volumes, respectively.
Finally, after 25 days from the beginning of the experiment, mice were sacrificed by cervical dislocation, and dissected tumors were weighed and stored in 10% formalin to preserve their morphology.
No. of groups | Group 1 | Group 2 | Group 3 | Group 4 |
---|---|---|---|---|
No. of mice | 6 mice | 6 mice | 6 mice | 6 mice |
Treatment | Unrestricted diet (SD) | Ketogenic diet (KD) | Probiotics | Combination of Probiotics and Ketogenic diet |
Daily dose | 12 kcal/5g | 14.1 kcal/2g | 1×109 CFU/0.5 ml | (1×109 CFU/0.5ml) of Probiotics and (14.1 kcal/2g) of Ketogenic diet |
Blood levels of glucose and β-HB were assessed on days: 10, 13, 17, 20, and 23, and compared with normal-untreated mice bearing no tumor. Blood glucose levels were measured using the Accu-Chek blood glucose monitoring system (Roche, Basel, Switzerland). β-HB Assay Kit (Sigma, United States) was used to measure the levels of β-HB in the serum (
Mouse IGF-1 ELISA kits (Invitrogen, United States) had been used to assess IGF-1 levels. IGF-1 ELISA kits were investigated to negative control (untreated mice with tumor), and different treatment groups.
The effect of each treatment on the immune response of tumor-bearing mice was determined using Mouse Th1/Th2 ELISA kit (Thermo Fisher Scientific, Toronto, Canada). Blood samples were collected from mice subjected to different treatments and serum samples were prepared. Interferon (IFN)-γ, interleukin (IL)-2, IL-4 and IL-10 were detected using kit instructions (
Serum lipid profile had been evaluated by measuring total cholesterol, TG, LDL, and HDL using Beckman Coulter AU480 (Beckman Coulter, Inc, USA) for negative control (untreated mice with tumor), positive control (normal mice without tumor), and different treatment groups.
Serum levels of alanine Transaminase (ALT), aspartate Transaminase (AST), and creatinine were measured in negative control (untreated mice with tumor), positive control (normal mice without tumor), and different treatment groups using Beckman Coulter AU480 (Beckman Coulter, Inc, USA) to assess any relative nephrotoxicity and liver toxicity.
Data were presented using the mean ± SEM (Standard Error of Mean). The statistical significance among the groups were determined using SPSS (Statistical Package for the Social Science, Chicago, Illinois, version 22) by one-way analysis of variance (ANOVA; post hoc test: Tukey), and Kruskal-Wallis test. Differences between groups were considered significant when the p-value was less than 0.05 (p < 0.05).
Generally, treatment of tumor bearing mice with a combination of probiotics (1×109 CFU/0.5 ml) and ketogenic diet (14.1 kcal/2g) caused significant (p < 0.05) decrease in tumor size with a percentage change in tumor size of (-55.68%) compared with an increase of (106.82%) in the negative control group. A slightly lower percentage change (-38.53%) was observed in the group treatment with Ketogenic diet group (14.1 kcal/2g). Interestingly, probiotic group (1×109 CFU/0.5 ml) resulted in the highest reduction in tumor size (-58.89%).
Table
Effect of different treatments on tumor size, cure percentage, tumor and body weight. Where (mm³) is a cubic millimeter, (g) is gram, (av.) is an average, and (n) is a number.
Groups (n = 6) | Av. initial tumor size (mm3) ± SEM | Av. final tumor size (mm3) ± SEM | % Change in tumor size | % cure | Av. tumor weight (g) | % Change in body weight |
---|---|---|---|---|---|---|
Control | 279.50 ±30.66 | 578.04 ± 118.76 | 106.82 | 0 | 0.445 | 2.543 |
Ketogenic diet | 292.87 ± 30.34 | 180.02 ± 63.50 | -38.53 | 33.33 | 0.257 | 0 |
Probiotics | 422.51 ± 56.79 | 173.69± 56.66 | -58.89 | 33.33 | 0.182 | -6.238 |
Combination | 401.13 ± 31.09 | 177.76 ± 40.50 | -55.68 | 16.67 | 0.069 | -1.275 |
Table
We used mice bearing EMT6/P tumor cells to examine the effect of each treatment in vivo. We should note that the beginning tumor sizes of each group were different from other. Variable tumor sizes within each group were observed at the end of the study in all groups as indicated by high standard deviation values. Such difference is mainly due to the difference in response toward each treatment. Although all mice were inoculated using the same concentration of EMT6/P cells. We found that ketogenic diet treatment showed significantly reduction in tumor size. Several studies showed that the effect of the ketogenic diet against implanted breast tumors can vary according to the duration of treatment or formula concentration of fat, carbohydrate, and protein. For example, in a previous study, KD (comprised of 6% calories from CHO, 19% from protein, and 55% from FAT) was provided to breast cancer patients for 90 consecutive days concurrent with the first 12 weeks of chemotherapy. KD led to a significant decrease in stage and tumor size compared to the control group. The tumor size in the KD group showed a significant reduction compared to the baseline; the reduction in tumor size was 27 mm in the intervention group compared to 6 mm in the control group (
In our study, we also examined the effect of probiotics on tumor growth in vivo, we found that probiotics treatment showed the highest reduction in tumor size. Our findings are in agreement with previous studies which reported the ability of probiotics to reduce tumor size in mice breast cancer cells (
In a different study, probiotics were taken for limited time for mice with cancer. Significant reductions in body weight were observed in treated group (
The subsequent analysis of glucose levels showed that treatments with combination therapy (ketogenic diet and probiotics) had the lowest level of glucose (p < 0.001). Also, ketogenic diet treated group had lower glucose levels than the control group, noteworthy the reduction was significant Fig.
Serum levels β-hydroxybutyrate for different treatments. Highest β- hydroxybutyrate levels was observed in group taking combination therapy. Results are expressed as means ± SEM (n = 3).*P < 0.05, *** P < 0.001. (Treatment groups compared with the control group). Test was performed in duplicate.
The use of ketogenic diet inhibits breast cancer in mice by reducing blood glucose. Also, the use of this diet resulted in an increase in blood levels of β-hydroxybutyrate which is known of its antitumor effects (
Remarkably, Treatment of Blab/C mice with ketogenic diet group (14.1 kcal/2g) caused decrease in the level of IGF-1 (14.815 ng/ml) compared with the negative control group (59.795 ng/ml). On the flip side, the levels of IGF-1 were (37.170 ng/ml) for probiotics group, and (59.795 ng/ml) for the negative control group. Regarding to the mice treated with combination (1×109 CFU/0.5 ml of probiotics, 14.1 kcal/2g of ketogenic diet), Reduction of IGF-1 levels to (16.069 ng/ml) was significant comparing with control group Fig.
The ability of a ketogenic diet to reduce serum IGF-1 levels could be a target mechanism for using it as a cancer adjuvant therapy. Increased IGF-1 levels are associated with an increased risk of several cancers such as breast, lung, colorectal, and prostate cancer (
To explore the effect of different treatments on the immune response in animals, the serum levels of IFN-γ, IL-2, IL-4 and IL-10 were detected Fig.
Effect of different treatments on serum levels of INF-γ, IL-2, IL-4, and IL-10. Concentration of serum cytokines (pg/ml) in different treated mice. The highest level of INF-γ, IL-4, IL-2, and lowest IL-10 were detected in probiotics group. The lowest level of IL-10 was detected in combination group. Results are expressed as means ± SEM (n = 3).*P < 0.05, *** P < 0.001. (Treatment groups compared with the control group). Test was performed in duplicate.
In cancer xenografts, the anti-proliferative action of IFN-γ, probably due to enhanced cell death by up-regulation of some caspases and anti-angiogenic activity, have been found (
IL-2 induces the proliferation of activated T cells and the differentiation of cytotoxic T lymphocytes (CTL); it also has effects on other immune cells including NK cells, B cells, monocyte/macrophages, and neutrophils (
Measured serum lipid distribution for different treatments using Beckman Coulter AU480. Serum lipid levels of the normal mice without any tumors were measured and used as a reference for normal levels. The changes in serum triglyceride levels were lower in all groups compared to healthy mice (125 mg/dl) Fig.
We found that there were no significant differences between the groups in total cholesterol, TG, HDL, and LDL. This study’s results showed that both combination, and ketogenic groups had in insignificant reduced serum LDL concentrations compared to the control group. Numerous clinical trials on cancer patients or obese patients reported that blood LDL or/and triglyceride concentration decreased after ketogenic diet intervention for a while (
The potential of developing toxicity associated with different treatments was investigated by measuring the serum levels of AST, and ALT liver enzymes, as well as creatinine. Serum levels were also measured for normal mice with no tumors (as a reference for normal liver and kidney function). The treated group exhibited insignificant differences in serum ALT (p > 0.997), and AST levels (p > 0.9909) compared to normal untreated mice. No significant change in the AST level was observed across untreated animals (229 U/l), healthy normal animals (242 U/l), and animals treated with a combination of ketogenic diet (14.1 kcal/2g), and probiotics (1×109 CFU/0.5ml) (171 U/l) Fig.
The effect of different treatments on serum levels of aspartate transaminase (AST), and alanine transaminase (ALT). The measured levels (U/l) of AST and ALT creatinine in animals with different treatment groups. Results are expressed as means ± SEM (n = 3). Test was performed in duplicate.
This study shows that the ketogenic diet and probiotics are important ways to treat breast cancer in animal model system. Gut microbiota and human health have already been linked in a clear way by the growing field of microbiome research. Microbial driven processes affect inflammation, promote metabolite exposures, impact gene expression through transcription, translation, epigenetic modifications, and modulate immunological signaling. The ketogenic diet is a common cancer therapy. In fact, the ketogenic diet has been tested in a variety of cancer animal models. The results of a ketogenic diet on its own vary depending on the kind of cancer, however the combination of a ketogenic diet with chemotherapy or radiation seems encouraging all pathways involved in the genesis, and pathophysiology of cancer. The evidence supporting the involvement of nutrition in cancer risk, development, and prognosis has a complex link. However, looking at the relationship between diet and cancer with the lens of microbes may give us new insights. This research found that combining a ketogenic diet with probiotics caused regression of breast cancer in mice. The combination of a ketogenic diet and probiotics has the potential to be utilized in the future development of anti-cancer nutrition. Understanding which microbial metabolites of dietary components provide the most anti-cancer benefits could aid in the development of new preventive or therapeutic strategies by optimizing exposure across individuals, either through microbiota manipulation or direct administration of bioactive microbiota-derived metabolites, also known as postbiotics. However, significant concerns about the relationships between food, microbiota, and cancer persist, posing obstacles to the development of preventive or therapeutic nutritional treatments for particular cancer types. At the most fundamental level, it is necessary to understand which components of microbial consortiums interact with certain dietary components to form compounds with anti-cancer properties, and then to demonstrate their frequency throughout diverse human populations and across the lifetime. Determining the biological mechanisms by which the ketogenic diet and microbial metabolites regulate cancer-related pathways is also necessary for establishing bioavailability, effective intake levels, and identifying possible interactions with chemotherapies treatment. Developing human studies that stratify test populations based on their microbiota composition and ability to produce targeted metabolites, while continuing to investigate cellular-level interactions, are the next steps required to define and refine the microbiota as a hub between nutrition and cancer.
Acronyms, abbreviations, symbols. Breast cancer (BC), Ketogenic diets (KD), Beta-hydroxybutyrate (β-OHB), Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), Breast cancer-associated gene 1 and 2 (BRCA1 and BRCA2), Human epidermal growth factor receptor 2 (HER2), Epidermal Growth Factor Receptor (EGFR), Cyclooxygenase-2 (COX-2), c-myelocytomatosis oncogene (c-Myc), Insulin-like growth factor (IGF-1), Adenosine triphosphate (ATP), Phosphate buffer solution (PBS), Unrestricted diet (SD), Interferon (IFN)-γ , Interleukin (IL), Alanine Transaminase (ALT), Aspartate Transaminase (AST), KetoCal (KC), High-density lipoprotein (HDL), low-density lipoprotein (LDL), Azoxymethane (AOM)
The authors are grateful to the Applied Science Private University, Amman, Jordan, for the full financial support granted to this research project.
This study was conducted according to standard ethical guidelines. The Research and Ethical Committee approved all the experimental protocols at the Faculty of Pharmacy, Applied Science Private University (approval number: 2022-PHA-38).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
This research project was fully financially supported by Applied Science Private University, Amman, Jordan.
AKSK and WHT both conceived and designed the study. AKSK carried out the study, interpreted the data, and drafted the manuscript. WHT supervised the study. Both authors read and approved the final manuscript to be published.