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Beni-Suef University Journal of Basic and Applied Sciences
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Severe carbohydrate restriction augments the antiproliferative effect of hormonal therapy in a murine model of Ehrlich breast adenocarcinoma: histological and immunohistochemical investigations

Beni-Suef University Journal of Basic and Applied Sciences volume 13, Article number: 94 (2024) Cite this article

Abstract

Background

Malignant tumors of the breast are the most diagnosed cancers in females globally. Recent evidence suggests that carbohydrate restriction (CR), especially ketogenic diets, has become a potential treatment approach for many malignancies, including breast cancer. Tamoxifen (TAX) is a selective estrogen receptor modulator (ERM) that can reduce the risk of cancer recurrence. The current work was designed to assess the impact of CR on the proliferation of breast adenocarcinoma cells and to compare this impact with that of TAX. Study groups included: group 1: vehicle-treated mice; group 2: the Ehrlich group: injected Ehrlich ascites carcinoma (EAC) cells (2.5 × 106) in 0.25 ml isotonic saline; group 3: CR group: mice were supplied with a diet regimen of severe CR throughout the study and injected EAC at week 7; group 4: hormonal therapy (HT) group: mice in this group injected with EAC at week 7 and then received TAX at a dose of 20 mg/kg 3 times/week orally for 3 weeks; and lastly group 5: the group of combined intervention. The mice in the CR, HT, and the combined groups received Ehrlich cancer cells at the same dose and route as the Ehrlich group.

Results

CR and HT groups demonstrated a significant decrease in levels of insulin-like growth factor (IGF-1), carbohydrate antigen (CA 15–3), hexokinase 2 (HK2), hypoxia-inducible factor-1 (HIF-1) α, and malondialdehyde (MDA) compared to the Ehrlich group. Additionally, the mean area % of caspase-3 was significantly increased, and the mean area % of Ki67 and estrogen receptor (ER)α was significantly decreased.

Conclusions

The combined treatment demonstrated the most advantageous outcome, as evidenced by reduced CA 15–3 levels, tumor size, and the mean area % of Ki67. This suggests that the addition of severe CR to the conventional therapy of breast cancer has a beneficial effect.

1 Background

The most prevalent malignant tumor in women worldwide is breast cancer that has been associated with persistent hyperinsulinemia and insulin resistance. Chronic hyperinsulinemia is thought to elevate the risk of developing breast cancer [1]. Consumption of carbohydrates (CHO) has been hypothesized to be a potential risk for cancer progression. It promotes cancer development [2] due to its ability to stimulate insulin production and glycolysis, leading to increased glucose release, the primary energy source for cancer cells [3].

TAX has been widely recognized as a revolutionary medication in medical oncology over the last forty years [4]. It is an essential part of saving many lives and is considered a vital component of cancer treatment [5]. Till now, TAX is the most effective medication for breast cancer therapy. It acts as a selective ERM that can be utilized for the treatment of estrogen-dependent breast cancer in addition to minimizing the risk of its recurrence in both pre- and postmenopausal females [6].

In vivo experimental laboratory animal models include the Ehrlich solid tumor and the adenocarcinoma of the mouse breast. These models have demonstrated the ability to develop breast adenocarcinoma in both ascitic and solid forms [7]. Ehrlich adenocarcinoma is characterized as an undifferentiated malignant tumor with a short life span, high proliferative capacity, rapid growth, and no regression, and it lacks a tumor-specific transplantation antigen [8]. Consequently, both the solid and ascitic types of EAC are frequently utilized to investigate tumor pathogenesis and evaluate the anticancer effects of various interventions [8].

In vivo, solid tumors exhibit three major phases: the avascular, vascular, and metastatic stages [9]. Due to deficient nutrition, progressive tumor growth leads to the death of its central cells, resulting in a necrotic core. However, after some time, the tumor can undergo an angiogenesis process to provide its vasculature secondary to the release of hypoxia-inducible factor (HIF). In the metastasis stage, some cells travel from the primary cancer, invade the blood vessels, and consequently form colonies in other sites [10].

Therefore, this investigation sought to assess the impact of severe carbohydrate restriction (CR) on the proliferation and progression of breast adenocarcinoma and compare its effect with TAX. Additionally, molecular pathways, biomarkers, and potential roles of insulin-like growth factor-1 (IGF-1) and hypoxia-inducible factor-1 (HIF-1) α in tumor growth and angiogenesis were investigated.

2 Methods

2.1 Induction of Ehrlich breast adenocarcinoma

Breast adenocarcinogenesis was induced in mice by administering a single intra-thigh injection of EAC cells derived from the ascitic fluid of BALB/c mice-bearing ascitic tumors that were 8–10 days old at a dose of 2.5 × 106 cells [11].

2.2 A regimen of diet restriction

Severe CR was implemented for 6 weeks before the induction of Ehrlich breast adenocarcinoma and was continued for 3 weeks after induction until the day of scarification at the end of week 9. Mice in the CR and combined groups were fed a diet with a composition of carbohydrate (C):protein (P):fat (F) = 12:43:45, whereas mice in the control, Ehrlich, and HT groups received a normal diet with a composition of C:P:F = 65:20:15 [12]. Additionally, the vitamin–mineral premix provided per kilogram was per Tuzcu et al. [13].

2.3 Inhibition of estrogen by using TAX

TAM was given orally at a dosage of 20 mg/kg three times per week for three weeks, specifically from week 7 to week 9 of the study [14].

2.4 Animal grouping and study design

Forty female Swiss mice, approximately 10–12 weeks old and weighing 25 to 30 g, were enrolled in this study. Following a 7-day acclimatization period in the Animal House to adjust to environmental conditions such as humidity, temperature, and light/dark cycles, they were placed in wire mesh cages with ad libitum access to food and water. The mice were then randomly assigned to five groups. The study lasted a total of 9 weeks, with the induction of solid Ehrlich tumors in the left thigh occurring at the beginning of week 7. The mice were euthanized via cervical dislocation at the end of week 9.

Mice were randomly divided into five groups according to the study design and experimental procedures outlined in Fig. 1:

Fig. 1
figure 1

Timeline represents the experimental groups and the study design. CR refers to CHO restriction, HT refers to hormonal therapy, and W refers to week

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2.5 Group 1 (control group, n = 8)

Normal mice received a single 0.25 ml dose of isotonic saline injected into the left thigh as a vehicle.

2.6 Group 2 (Ehrlich group, n = 8)

Mice in this group were injected with EAC cells at a dose of 2.5 × 106 suspended in 0.25 ml of isotonic saline (11‏), administered once into the left thigh.

2.7 Group 3 (CR group, n = 8)

Mice in this group were supplied with a severe CHO restriction throughout the study (12‏). On the first day of the 7th week, they were injected with EAC cells at the same dose and route as Group 2, while continuing with CHO restriction for 3 weeks.

2.8 Group 4 (HT group, n = 8)

Mice in this group received a normal diet and were injected with EAC cells at the same dose and route as Group 2 on the first day of the 7th week. They were then treated with hormonal therapy (TAX) orally at a dose of 20 mg/kg three times per week for 3 weeks (14‏).

2.9 Group 5 (combined group, n = 8)

Mice in this group underwent severe CHO restriction as in Group 3, followed by injection of EAC cells at the same dose and route as Group 2, and hormonal therapy as in Group 4.

Regular body weight measurement was taken, and data were recorded at baseline, after 3 weeks, after 6 weeks, and at the end of the study.

2.10 Sample collection and scarification

On the final day of the experiment, after the 9 weeks, blood samples were collected just before scarification, using a capillary tube from the retrobulbar plexus of the mice. These blood samples were then transferred to 5-ml Eppendorf tubes to assess cancer antigen (CA) 15–3 levels and IGF-1 levels.

At the planned time, the mice were killed by cervical dislocation [15]. After scarification, the animals' left thighs were dissected and divided into two samples. One sample was used for biochemical analysis, including hexokinase 2 (HK2), hypoxia-inducible factor-1 (HIF-1) α, and matrix metalloproteinases (MMP-9) using quantitative real-time PCR, along with assessing reduced glutathione (rGSH) and malondialdehyde (MDA) levels using calorimetry. Each mouse's other sample of thigh tissue was reserved for histological examination using hematoxylin and eosin (H&E) stain and immunostaining of caspase-3, ER, and Ki67 (a marker for cell proliferation).

2.11 Determination of serum carbohydrate antigen (CA 15–3) and IGF-1

Serum CA 15–3 and IGF-1 levels were determined using specific assays. CA 15–3 levels were measured using a mice CA 15–3 (Catalog #: MBS023512, MyBioSource, MyBioSource, Inc. San Diego, CA 92195–3308) for serum analysis. On the other hand, IGF-1 levels were quantified using Mouse IGF-1 ELISA Kit (Catalog #: MBS175810, MyBioSource, MyBioSource, Inc. San Diego, CA 92195–3308).

2.12 Determination of HK2, HIF-1 α, MMP-9 using real-time polymerase chain reaction (real-time PCR)

Using the SV Total RNA Isolation System from Promega, Madison, WI, USA, RNA was extracted from thigh tissues in order to determine HK2, HIF-1 α, and MMP-9 using real-time PCR., following our previously published data [16]. The primers used for real-time PCR quantification hermo-cycler (T100, Bio-Rad, Hercules, CA, USA) were as follows:

HK2:

Forward primer: 5′-ATTGTCCAGTGCATCGCGGA-3′

Reverse primer: 5′-AGGTCAAACTCCTCTCGCCG-3′

HIF-1 α:

Forward primer: 5′-TGCTTGGTGCTGATTTGTGA-3′

Reverse primer: 5′-GGTCAGATGATCAGAGTCCA-3′

MMP-9:

Forward primer: 5′-GGGCAACTCGGCAGGAGAGC-3′

Reverse primer: 5′-CCAGGTGACGGGCTGCTGT-3′

The following primers were used to normalize these primers to the housekeeping gene beta-actin:

Forward primer: 5′-TCTGGCACCACACCTTCTACAATG-3′

Reverse primer: 5′-AGCACAGCCTGGATAGCAACG-3′

2.13 Estimating of MDA and GSH levels in the solid tumor of the thigh by colorimetry

After tissue homogenization, samples were prepared, and the supernatant was collected to assay MDA levels (Catalog #: MBS2540407, MyBioSource, MyBioSource, Inc. San Diego, CA 92195–3308). The method used for estimating GSH relied on reducing 5, 5′-dithiobis [2-nitrobenzoic acid] (DTNB) by GSH (Catalog #: MBS2540433, MyBioSource, MyBioSource, Inc. San Diego, CA 92195–3308).

2.14 Histopathological examination

Histopathological examination involved the preparation of thin slices, typically 5–7 μm thick, which were then subjected to the following procedures:

(A) Staining with hematoxylin and eosin (H&E).

(B) Immunohistochemical staining using the avidin–biotin–peroxidase complex method [17] for the following antibodies: anticaspase-3 rabbit polyclonal antibody and (RB-1197-R7) from NEO markers (Thermo Scientific) Laboratories (USA); anti-ER α a rabbit monoclonal antibody (ab271827) from Abcam (USA); and anti-Ki67 rabbit monoclonal antibody (ab16667) from Abcam (USA).

Sections were deparaffinized with xylene and then rehydrated with progressively lower alcohol grades. H2O2 was applied to the sections for a duration of 15 minutes in order to inhibit endogenous peroxidase activity. The "Ultra V Block" approach blocked the non-specific background for five minutes. The primary antibody was then applied to the sections and incubated for 60 min. The next step was applying a goat antipolyvalent secondary antibody that had been biotinylated for ten minutes, followed by a ten-minute application of streptavidin–peroxidase. HRP polymer, DAB Plus Chromogen, and the Ultravision One detection system were used to visualize the reaction. The counterstain employed was Mayer's hematoxylin. After that, the slides were mounted, cleaned with xylene, and dehydrated using progressively higher alcohol concentrations.

For caspase-3, human tonsil sections were used as the positive (+ve) control, showing a cytoplasmic response. The positive (+ve) control for Ki67, demonstrating a nuclear reaction, was human colon carcinoma. Lastly, human endometrial carcinoma was the positive control for ER α, exhibiting both cytoplasmic and nuclear reactions.

2.15 Statistical analysis

The Statistical Package for the Social Sciences (SPSS) program, especially version 28, from IBM Corp., Armonk, NY, USA, was used to code and enter the data. To characterize the data, summary statistics like mean and standard deviation were employed. Analysis of variance (ANOVA) with multiple comparisons post hoc test was used to compare groups [18]. To evaluate correlations between quantitative variables, the Pearson correlation coefficient was utilized [19]. A significance level of p < 0.05 was deemed statistically meaningful.

3 Results

3.1 Changes in mice body weight throughout the study duration

Table 1 displays the mean body weight values for the Ehrlich, CR, HT, control, and combination groups at baseline with no substantial differences being observed. The CR group showed a considerably lower body weight after three weeks of the trial compared to the Ehrlich and control groups. In contrast, the HT group's body weight increased significantly more than that of the CR group. When compared to the control, Ehrlich, and HT groups, the combined group showed a statistically significant drop in body weight.

Table 1 Comparison of body weight of mice between all studied groups
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The CR group showed a considerably lower body weight after 6 weeks of the trial compared to the Ehrlich and control groups. Conversely, the HT group exhibited a noticeably higher body weight than the CR group. When compared to the control, Ehrlich, and HT groups, the combined group showed a statistically significant drop in body weight.

As indicated in Table 1, at the end of the study, the Ehrlich group exhibited a notable decrease in body weight compared to the control group. Furthermore, the body weights of animals in the CR, HT, and combined groups showed a significant decrease compared to the control group.

3.2 Biochemical results

3.2.1 Levels of CA 15–3 and IGF-1 in all groups at the end of the study

Comparing the Ehrlich group to the control group, Table 2 shows a significant increase in CA 15–3 and IGF-1 levels. By comparison, the CR group's levels of CA 15–3 and IGF-1 were considerably higher than those of the control group, but they were significantly lower than those of the Ehrlich group. Furthermore, as compared to the control and CR groups, the HT group's levels of CA 15–3 and IGF-1 significantly increased, whereas they significantly decreased when compared to the Ehrlich group. CA 15–3 and IGF-1 levels were significantly lower in the combination group as compared to the Ehrlich, CR, and HT groups.

Table 2 Serum levels of CA 15–3 and IGF-1 in the studied groups at the end of the study
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3.2.2 Tissue levels of HK2, HIF-1 α, and MMP-9 in all studied groups at the end of the study

Comparing the Ehrlich group to the control group, Table 3 shows a significant increase in the levels of HK2, HIF-1 α, and MMP-9. Comparing the CR and HT groups to the Ehrlich group, however, the levels of HK2, HIF-1 α, and MMP-9 were significantly lower. In addition, the levels of these variables were much lower in the combination group than in the Ehrlich, CR, and HT groups.

Table 3 Tissue levels of HK2, HIF-1 α, MMP-9, GSH, and MDA in all groups at the end of the study
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3.2.3 Tissue levels of GSH and MDA in the studied groups at the end of the study

In comparison with the control mice, the Ehrlich group showed a large increase in MDA levels and a significant drop in GSH levels, as given in Table 3. As opposed to the Ehrlich group, the CR and HT groups displayed a considerable rise in GSH levels and a significant drop in MDA levels. Furthermore, in comparison with the Ehrlich, CR, and HT group, the combined group showed a large drop in MDA levels and a considerable increase in GSH levels.

3.3 Correlation between variables

As shown in Fig. 2, IGF-1 levels and the levels of CA 15–3 (r = 0.973, p < 0.001), HK2 (r = 0.970, p < 0.001), HIF-1 α (r = 0.953, p < 0.001), and MDA (r = 0.920, p < 0.001) showed a substantial positive correlation. Moreover, a significant inverse correlation was discovered between GSH and IGF-1 levels (r = − 0.955, p < 0.001).

Fig. 2
figure 2

A, B, C, and D A positive correlation between IGF-1 from one side and the levels of Ca15-3 (r = 0.973 and p < 0.001), HK2 (r = 0.970 and p < 0.001), HIF-1 α (r = 0.953 and p < 0.001), and MDA (r = 0.920 and p < 0.001) from the other side. E Represents a negative correlation between IGF-1 and the levels of GSH (r = − 0.955 and p < 0.001)

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As illustrated in Fig. 3, levels of CA 15–3 were found to strongly positively correlate with HK2 (r = 0.969, p < 0.001), HIF-1 α (r = 0.972, p < 0.001), and MDA (r = 0.960, p < 0.001). Furthermore, there was a negative correlation (r = − 0.941, p < 0.001) between the levels of GSH and CA 15–3.

Fig. 3
figure 3

A, B, and C A positive correlation between Ca15-3 from one side and the levels of HK2 (r = 0.969 and p < 0.001), HIF-1 α (r = 0.972 and p < 0.001), and MDA (r = 0.960 and p < 0.001) from the other side. (D): represents a negative correlation between CA15-3 and the levels of GSH (r − 0.941 and p < 0.001)

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3.4 Histological results

3.4.1 Gross picture (Fig. 4)

Fig. 4
figure 4

Gross picture presenting the tumor mass in all studied groups. A Control group revealing normal skeletal muscle. B Ehrlich group showing an enlarged mass in the thigh. C CR group demonstrating a smaller mass. D HT group exhibiting a moderate-sized mass. E Combined group showing a small mass. F Mean surface area of thigh muscle in the studied groups at the end of the study. Values are presented as mean ± SD, *: statistically significant compared to the control group, #: statistically significant compared to Ehrlich group, $: statistically significant compared to CR group, @: statistically significant compared to HT group, the p value is < 0.05

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The control group showed normal thigh skeletal muscles. In contrast, the Ehrlich group revealed a large mass in the thigh. On the other hand, the CR and HT groups demonstrated smaller mass in comparison with Ehrlich group. The combined group showed a marked reduction in tumor size compared to both CR and HT groups.

3.4.2 Hematoxylin and eosin-stained sections (Fig. 5, Supplementary Figs. 1 and 2)

Fig. 5
figure 5

A Photomicrograph of H&E-stained sections in A control group revealing longitudinal (LS) and transverse (circle) sections of muscle fibers separated by connective tissue perimysium (triangle). Within the muscle bundles, the muscle fibers are separated by thin connective tissue endomysium (bifid arrows). Longitudinal muscle fibers show multiple peripheral pale nuclei (arrows) and transverse striations of the cytoplasm (wavy arrows). Transverse sections reveal heterogeneous cytoplasm with pale peripheral nuclei (arrows). B Ehrlich group demonstrating areas of tumor cells in glandular pattern (asterisk). C CR group revealing longitudinal section of skeletal muscles (LS) in between different types of tumor cells; D HT group showing sections of skeletal muscles (R) in between tumor cells. E Combined group exhibiting transverse (TS) and longitudinal (LS) sections of skeletal muscles having multiple pale nuclei (arrows) [mononuclear lymphocytes; bifid arrows, tumor cells with notched nuclei containing clumps of chromatin material; curved arrows, ghost cells with homogenous cytoplasm; g, and multinucleated giant cells; arrowheads, cells have nuclear ghost (ng) with inconspicuous nucleoli] (magnification: ×400)

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Sections from the control group showed longitudinal and transverse sections of muscle fibers separated by perimysium connective tissue. Within the muscle bundles, endomysium connective tissue separates the muscle fibers. Longitudinal muscle fibers exhibited multiple peripheral pale nuclei and transverse striations in the cytoplasm. Transverse sections displayed heterogeneous cytoplasm with pale peripheral nuclei.

In the Ehrlich group, proliferative adenocarcinoma areas exhibited tumor cells arranged in glandular patterns and areas of mononuclear infiltration. Tumor cells had vacuolated cytoplasm, with some displaying notched nuclei containing clumps of chromatin material, others having enlarged nuclei with folds and invaginations, and some showing nuclear ghosts. Additionally, multinucleated giant cells and ghost cells with homogeneous cytoplasm were observed.

Examination of sections from the CR group revealed longitudinal sections of skeletal muscles interspersed with mononuclear inflammatory cellular infiltration and small areas displaying a glandular pattern of adenocarcinoma. Various cell types were identified, including mononuclear lymphocytes, multinucleated giant cells, and macrophages with kidney-shaped nuclei. Tumor cells exhibited notched nuclei containing clumps of chromatin material, enlarged nuclei with folds and invaginations, or ghost cells with homogeneous cytoplasm.

In the HT group, longitudinal sections of skeletal muscles were observed surrounded by mononuclear inflammatory cellular infiltration. Higher magnification revealed multinucleated giant cells and mononuclear lymphocytes. Tumor cells with vacuolated cytoplasm were present, with some cells displaying notched nuclei containing clumps of chromatin material, others having enlarged nuclei with folds and invaginations, and others showing nuclei with inconspicuous nucleoli.

In the combined group, both transverse and longitudinal sections of skeletal muscles were observed within mononuclear inflammatory cellular infiltration. A small glandular pattern of adenocarcinoma was observed in a small area. Sections of skeletal muscles exhibited multiple pale vesicular nuclei, distinguishable from the dark nuclei of satellite cells.

3.4.3 Ki67 immunostained sections (Fig. 6)

Fig. 6
figure 6

A photomicrograph of Ki67 immunostained sections. A Negative control. B Control group revealing negative caspase-3 immunostaining of transverse and longitudinal sections of skeletal muscles. C, D Ehrlich group showing many positive nuclear Ki67 immunostaining. E, F CR and HT groups revealed few Ki67 immunostained nuclei. G Combined group exhibiting scanty Ki67 immunostained nuclei. H Mean area% of Ki67 immunostaining in the studied groups at the end of the study. Values are presented as mean ± SD, *: statistically significant compared to the control group, #: statistically significant compared to Ehrlich group, $: statistically significant compared to CR group, @: statistically significant compared to HT group, the p value is < 0.05 (magnification: ×400)

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Immunohistochemical staining for Ki67 in the control group showed negative immunostaining of muscle fibers. However, in the Ehrlich group, thyroid sections displayed numerous Ki67 immunopositive nuclei. A significant reduction was detected in the CR and HT groups compared to the Ehrlich group. Furthermore, the combined group exhibited few Ki67 immunopositive nuclei.

3.4.4 Caspase-3 immunostained section (Fig. 7)

Fig. 7
figure 7

A photomicrograph of caspase-3 immunostained sections. A Control group revealing negative caspase-3 immunostaining of transverse and longitudinal sections of skeletal muscles. B Ehrlich group showing large areas of negative cytoplasmic caspase-3 immunostaining (asterisk) with small areas of positive immunostaining (arrows). C, D CR and HT groups exhibiting large areas of cells with positive cytoplasmic caspase-3 immunostaining (arrows) among weak caspase-3 positive immunostained small skeletal muscles (bifid arrows). Note the presence of small areas of negative caspase-3 immunostaining (asterisk). E Combined group demonstrating marked widespread cytoplasmic caspase-3 immunostaining (arrows) among skeletal muscle fibers revealing weak cytoplasmic immunostaining (bifid arrows). F Mean area% of caspase-3 immunostaining in the studied groups at the end of the study. Values are presented as mean ± SD, *: statistically significant compared to the control group, #: statistically significant compared to Ehrlich group, $: statistically significant compared to CR group, @: statistically significant compared to HT group, the p value is < 0.05 (magnification: A, D; ×200, B, C, E; ×100, Inset; ×400)

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Examination of control animals revealed negative caspase-3 immunostaining. In contrast, the Ehrlich group exhibited large areas of negative cytoplasmic caspase-3 immunostaining, with small areas showing positive immunostaining. However, there were noticeable regions of cells in both the CR and HT groups with cytoplasmic caspase-3 immunostaining, along with weak caspase-3 positive immunostained small skeletal muscles. The combined group demonstrated widespread cytoplasmic caspase-3 immunostaining.

3.4.5 Estrogen receptor α (Er α) immunostained sections (Fig. 8)

Fig. 8
figure 8

A photomicrograph of estrogen receptors-α (ER α) immunostained sections. A Control group revealing negative ER α immunostaining of longitudinal sections of skeletal muscles. B Ehrlich group exhibiting widespread positive cytoplasmic and nuclear ER α immunostaining. C CR showing moderate positive cytoplasmic ER α immunostaining. D HT groups demonstrating minimal positive cytoplasmic ER α immunostaining. E The combined group revealed negative ER α immunostaining. Mean area % of ER α immunostaining in the studied groups at the end of the study Values are presented as mean ± SD, *: statistically significant compared to the control group, #: statistically significant compared to Ehrlich group, $: statistically significant compared to CR group, @: statistically significant compared to HT group, the p value is < 0.05 (magnification: ×400)

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Examination of the control group revealed limited ER α immunopositivity. In contrast, the Ehrlich group showed widespread cytoplasmic and nuclear ER α immunopositivity. However, the CR and HT groups displayed a significant decline in the mean area percentage of ER α immunopositivity, accompanied by weak ER α immunopositivity in small skeletal muscles. The combined group demonstrated minimal ER α immunopositivity.

4 Discussion

In this study, we successfully induced solid breast adenocarcinoma in the left thigh of female mice. For the induction of the solid tumor, we utilized a tumor cell line that had been maintained in our laboratory where Swiss female mice were given multiple intraperitoneal passages every 7–10 days.

We ensured aseptic conditions throughout all procedures, following protocols established in previous studies by Mahmoud et al. [20] and Debnath et al. [21]. We adhered the protocol outlined by Abdel-Maksoud [11] to obtain the suspension of EAC cells. According to Radulski et al. [22], we obtained the tumor cells from Swiss mice (mice-bearing tumors) and it was transplanted to the same type of mice.

In order to consistently produce a concentration of 2.5 million EAC cells in 0.1 mL of solution, all processes were carried out under sterile conditions. Tumorigenesis was initiated by injecting these tumor cells into the thigh rather than the breast tissues of the animals. It is worth noting that the mammary glands of female mice (Mus musculus) share similarities in mammae distribution and anatomical location with rats, except for the total number of mammary glands [23].

During the study period, we monitored changes in body weight. Our observations indicated a continuous increase in body weight among the control group. However, the rats in the Ehrlich group revealed a steady and consistent increase in body weight until week 6 of the study. Subsequently, their body weight declined following tumor induction, attributed to cancer development in this group [24]. Notably, the loss of body weight, a key prognostic marker for breast cancer, has been included as a criterion in the latest guidelines for breast cancer surveillance by the American Society of Clinical Oncology [25].

Based on the higher levels of the tumor marker CA 15–3 in the Ehrlich tumor group than in the control group, the Ehrlich tumor model was confirmed. Since it is noninvasive and reasonably priced, serum-detectable tumor marker CA 15–3 is frequently used in clinical practice for early identification, monitoring, and prognosis of breast cancer at different stages [26].

The current results reported a significant rise in the CA 15–3 level in the Ehrlich group. This is following a previous report that revealed a marked elevation in serum CA 15–3 in Ehrlich-induced breast cancer in mice [27].

A significant increase in serum IGF-1 levels was detected in the Ehrlich breast adenocarcinoma group in comparison with control animals suggesting the involvement of IGF-1 in the pathogenesis of mammary carcinoma. IGF-1 is a growth factor that is important for both normal and cancerous cells. It is involved in antiapoptotic and mitogenic pathways in different types of cells. It acts as a "cell cycle progression factor," facilitating cell cycle evolution from the G1 to the S phase [28]. Over the past two decades, extensive research has demonstrated the involvement of IGF-1 in various pathophysiological pathways, including the development of solid tumors such as breast cancer [29].

Elevated IGF-1 expression has been linked to a higher risk of breast cancer [30], as it can stimulate cell proliferation and migration [31]. Consequently, IGF-1 has become a potential target for the management of breast cancer [32]. Additionally, IGF-1 may act as a pro-angiogenic factor in the breast cancer microenvironment by inducing the production of vascular endothelial growth factor (VEGF) or nitric oxide (NO) [33].

In our study, rats in the CR group revealed a relevant decline in body weight in comparison with control animals over time. Recent evidence supports the positive impact of CR diets, including low-, moderate-, and very low-carbohydrate regimens, on various metabolic processes [34]. Severe CR diets can lead to decreased insulin levels in circulation and elevated glucagon levels, resulting in regression of lipogenesis and fat accumulation [35]. This promotes the mobilization of fatty acids from adipose tissue, resulting in the ketone bodies being produced. Ketone bodies function as a substitute energy source, particularly for extrahepatic tissues such as the brain and muscles, in place of glucose [36]. Furthermore, ketone bodies preserve lean body mass, explaining why lean tissue is maintained during extremely low-carbohydrate diets [37].

The ketogenic diet (KD) is becoming more and more popular as a potential additional or alternative treatment strategy for a number of cancers, including breast cancer [38]. Malignant cells exhibit inefficiency in synthesizing ATP through oxidative phosphorylation using ketone bodies or fatty acids, primarily due to defects in their mitochondria's structure, function, and number [32]. Ketone bodies disrupt glycolysis, a critical energy-producing process for malignant cells [39]. A low-carbohydrate diet, such as the KD, can downregulate the insulin-like growth factor-1 (IGF-1)/insulin–PI3K–Akt–mTOR signaling pathways associated with significant tumor growth [40]. Additionally, the KD reduces inflammation and edema around tumors, inhibiting malignant cell growth and metastasis [41]. Our study observed the effects of CR on cancer progression. In TAX-resistant breast cancer cells (MCF-7), upregulation of HK2 and mTOR was reported, indicating enhanced glycolysis. Blocking HK2 could potentially help overcome TAX resistance [42]. In our current work, IGF-1 and HK2 levels significantly decreased in response to CR compared to the Ehrlich tumor group, suggesting a potential therapeutic effect of CR in breast cancer management.

The histological evaluation of the CR group showed small areas of glandular adenocarcinoma along with mononuclear inflammatory cellular infiltration. Both food restriction and ketogenic diets have been reported to significantly reduce tumor growth by inducing apoptosis and decreasing microvascular density.

In contrast, the HT group exhibited a persistent and notable elevation in body weight compared to the control mice. Recent studies indicated no direct association between TAX use and weight gain [43], suggesting that TAX does not influence changes in body weight.

The development and metastasis of breast cancer are significantly influenced by the existence of hypoxia. Hypoxia triggers an increase in glycolysis, leading to acidification within the tumor microenvironment. The protein HIF-1 α is closely coupled with accelerated tumor growth, metastasis, and reduced treatment feedback [44]. Notably, the mean expression of HIF-1 α rises significantly from normal breast tissue to ductal carcinoma in situ and invasive breast cancer [40]. Additionally, MMP-9, a matrix metalloproteinase, is involved in the breakdown of extracellular matrix [13], facilitating tumor invasion. Our findings revealed a significant elevation in HIF-1 α and MMP-9 levels in the Ehrlich group in comparison with the control mice, highlighting the impact of hypoxia on breast cancer progression.

For the treatment of estrogen-positive breast cancer, TAX has been utilized extensively. Our study found that TAX regulates HIF-1 α levels. Specifically, our results showed that TAX reduced the level of HIF-1 α, which is consistent with the findings reported by Cortes in 2019 [45]. This suggests that HT could serve as a therapeutic strategy by promoting the production of small molecules that inhibit the formation of the active HIF-1 α complex, potentially contributing to managing breast cancer [46].

HK2 plays a crucial role in humans as a glycolysis regulator, linking metabolism and proliferation in cancerous cells. The interaction between HK2 and the outer mitochondrial membrane is essential for its oncogenic activity [47, 48]. The majority of cancerous cells predominantly depend on aerobic glycolysis instead of oxidative phosphorylation, a process that is frequently known as the "Warburg effect." This metabolic strategy allows cancer cells to utilize glucose efficiently to meet their energy demands for rapid growth and proliferation [49].

There are five well-studied isoforms of HK in mammalian cells [50]. HK2 is typically found in embryonic cells and aggressive malignancies, including cancers affecting the lung, liver, breast, and prostate. Its expression is specific to cancers, where it has an impact in promoting cellular proliferation and inhibiting cell death [51]. Therefore, our study reported a relevant elevation in tissue expression levels of HK2 in the Ehrlich group in comparison with the control group.

The ideal molecular marker for assessing tumor cell proliferative activity is Ki67. Ki67 is a nuclear antigen connected to the growth of cells and is expressed in various tumor cells, including those found in breast, ovarian, esophageal, and lung cancers. The degree of tumor malignancy is directly associated with the expression level of Ki67 [52]. In our study, we observed a relevant increase in the mean area percentage of Ki67 in the Ehrlich group in comparison with the control group. Conversely, in the CR group, the mean area percentage of Ki67 was significantly decreased compared to the Ehrlich group.

Transcription factors (TFs) such as ER α and estrogen receptor β (ER β) are essential for controlling a number of physiological functions in humans. Abnormalities in ER signaling can lead to the development of several diseases, including metabolic disorders, cancer, and inflammation. Estradiol activates the ligand-dependent transcription factor ER α, which is present in approximately 80% of breast tumors [53, 54]. Our study demonstrated a noticeable increase in the mean area percentage of ER α immunoreactivity in the Ehrlich group in comparison with the control group.

The mean area percentage of ER α immunoreactivity in the CR group was significantly lower than in the Ehrlich group, according to the current study. Another study reported that a 30% decrease in caloric intake led to the suppression of mammary epithelial cell density, the "proliferative index," and ER and ErbB2 communication [55]. The HT group exhibited a significant reduction in ER α immunostaining in comparison with the Ehrlich group. TAX acts as an estrogen antagonist in breast tissue and is classified as a selective ERM. It inhibits the transcription of estrogen-responsive genes, thus controlling estrogenic effects by competitively binding to ER in tumors and target tissues [56].

Furthermore, a significant reduction in GSH was noted in all mice of the Ehrlich-induced breast adenocarcinoma group in comparison with the control animals in this study. The disturbance of GSH homeostasis substantially impacts cellular physiology and has been implicated in various pathologies, including diabetes, neurodegenerative diseases, and malignant tumors [57]. In fact, oxidative damage to DNA can occur during the creation of the tumor phenotype due to elevated amounts of reactive oxygen species (ROS) that are not adequately countered by antioxidant defenses. Chromosome rearrangements, base damage, and single- and double-strand breaks are possible examples of this damage. The formation and spread of malignant tumors may be aided by these DNA changes, which may stimulate or inhibit tumor suppressor genes in an oncogenic manner [58, 59]. The significant elevation in tissue MDA levels observed in all mice belonging to the Ehrlich-induced breast adenocarcinoma group compared to the control group suggests increased lipid peroxidation (LPO). MDA is considered a marker of LPO and is known to act as a tumor enhancer and co-carcinogenic agent. This is because MDA is highly cytotoxic and has the potential to inhibit antioxidant enzymes, leading to cellular damage and promoting the development and progression of cancer [60, 61]. Several studies revealed that breast cancer patients had greater MDA levels than healthy controls [62]; they linked this to excessive ROS production and lack of antioxidant defenses.

Metabolic adaptation to CR can occur by decreasing growth factors [63], enhancement of antioxidant systems, resulting in reduced free radical-induced DNA disruption [64], reduction in the inflammation [65], and delaying aging-associated regression of host immunosurveillance [63].

In our study, we observed that substantial MDA levels were lower and GSH levels were noticeably higher in the CR and HT groups compared to the Ehrlich group. The KD is recognized for elevating mitochondrial GSH and antioxidative lipoic acid levels, thereby protecting against excessive DNA methylation and consequent DNA damage [66].

As per the Warburg effect, cancerous cells can alter their metabolism to prioritize glucose as their primary energy source, facilitating their growth, development, and survival [67]. These consequences can be countered by CR and fasting, with CR enhancing antiproliferative effects, reducing DNA synthesis, and promoting apoptosis. In our study, there was a notable rise in caspase-3 immunostaining observed in the CR group in comparison with the Ehrlich group, highlighting the potential impact of CR on apoptosis in cancer cells.

TAX causes mitochondrial lipid peroxidation by inhibiting mitochondrial respiration and lowering cytochrome c levels, ultimately stimulating tumor apoptosis [68]. Our study revealed a significant reduction in Ki67 immunostaining in both the CR and HT groups compared to the Ehrlich group. This observation aligns with Gabrielson's findings, indicating that TAX, administered at low or high doses following a brief presurgical course, diminishes the expression of Ki67 or proliferation markers in tumors and adjacent tissues [69].

5 Conclusion

Collectively, CR and HT effectively regulated tumor micromovement, leading to the suppression of cancer growth by reducing levels of HIF-1 α, HK2, and MDA. The combined treatment exhibited the most beneficial effect, notably decreasing serum levels of IGF-1, CA 15–3, tissue HK2, HIF-1 α, MMP-9, and MDA compared to CR or HT alone.

Lower serum levels of IGF-1 and CA 15–3 signify a favorable prognosis and therapeutic response, whereas elevated levels of IGF-1, CA 15–3, HIF-1 α, and HK2 indicate proliferation, metastasis, and poor prognosis. Furthermore, the combined treatment demonstrated a significant elevation in serum GSH levels compared to individual treatments, underscoring its efficacy.

Additionally, the combined treatment resulted in minimal tumor cell presence between normal muscle fibers, contrasting with the CR and HT groups, which exhibited numerous tumor cells. This observation indicates a favorable prognosis and therapeutic outcome.

The strong positive correlation observed between IGF-1 and CA 15–3 levels on the one side and the levels of HK2, HIF-1 α, and MDA on the other side emphasizes the significance of these factors in cancer proliferation.

Availability of data and materials

The data supporting this study's findings are available upon request.

Abbreviations

Ca15-3:

Carbohydrate antigen

CR:

Carbohydrate restriction

DTNB:

5, 5′-dithiobis [2-nitrobenzoic acid]

EAC:

Ehrlich adenocarcinoma

ER:

Estrogen receptor

HK2:

Hexokinase 2

HIF-1 α:

Hypoxia-inducible factor-1 α

HT:

Hormonal therapy

IGF-1:

Insulin-like growth factor

MDA:

Malondialdehyde

MMP-9:

Matrix metalloproteinases

NO:

Nitric oxide

rGSH:

Reduced glutathione

ERM:

Estrogen receptor modulator

Tamoxifen:

TAX

TFs:

Transcription factors

VEGF:

Vascular endothelial growth factor

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Authors and Affiliations

  1. Department of Physiology, Faculty of Medicine, October 6 University, Cairo, Egypt

    Ashraf Kotb, Reham Abdelnaby, Ahmed Desoky & Asmaa Mohammed ShamsEldeen

  2. Department of Physiology, Faculty of Medicine, Cairo University, Cairo, Egypt

    Asmaa Mohammed ShamsEldeen

  3. Histology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

    Sara Adel Hosny

  4. Department of Biochemistry, Faculty of Medicine, October 6 University, Cairo, Egypt

    Ahmed Bahgat Eldemery

  5. Department of Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt

    Laila Ahmed Rashed

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AK, RA, and AS were involved in conceptualization. RA, AD, AE, LR, SH, and AS were responsible for methodology and validation. RA, SH, and AS took part in writing—original draft and figure preparation. AK, RA, AD, AE, SH, and AS participated in writing—review and editing. AK and AS contributed to supervision. All authors contributed to the article and approved the submitted version.

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Kotb, A., Abdelnaby, R., Hosny, S.A. et al. Severe carbohydrate restriction augments the antiproliferative effect of hormonal therapy in a murine model of Ehrlich breast adenocarcinoma: histological and immunohistochemical investigations. Beni-Suef Univ J Basic Appl Sci 13, 94 (2024). https://doi.org/10.1186/s43088-024-00560-3

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