Anti-Tumor Effect and Mechanism Study of Caloric Restriction,Achieved by Time-Restricted Feeding,in Mice

Abstract

Objective

To evaluate the therapeutic effects and explore the mechanisms behind caloric restriction achieved through time-restricted feeding (CR) in inhibiting mouse tumors, providing a theoretical basis and data support for future CR diet-assisted anticancer treatment protocols.

Methods

C57BL/6 and BALB/c mice were divided into four cell line groups. Each group was further split into normal diet (ND) and a CR diet groups. The ND groups had free access to water and a normal diet, while the CR diet groups had access to water but were only fed from 9 a.m. to 11 a.m., fasting for the remaining 22 h. Food intake was recorded daily starting on day 1 of the experiment. Tumor models were established and assessed every 2 days. Blood biochemical indicators, serum pyruvic acid levels, and cytokine expression were measured.

Results

The CR diet inhibited tumor growth in mice. Colorimetric assays and ELISAs showed a reduction in pyruvic acid levels and in key upstream and downstream rate-limiting enzymes in the sera of CR mice. Routine blood and blood biochemistry tests suggested minor effects of the CR diet on these parameters. Western blotting revealed that the CR diet suppressed mTOR and AKT protein expression in tumor tissues. ELISA showed that various mTOR-related signaling pathways were downregulated. Immunohistochemistry staining indicated reduced expression of P53, P-AKT, EGFR, and IGF-1 in tumor tissues. TUNEL staining confirmed that the CR diet promoted tumor apoptosis.

Conclusion

The CR diet inhibited tumor growth by suppressing mTOR and its related upstream and downstream gene signaling pathways, reducing tumor glycolysis, and accelerating tumor cell apoptosis.

Keywords

caloric-restricted tumor inhibition Mammalian target of rapamycin phosphorylated protein kinase B tumor glycolysis apoptosis

Introduction

Cancer is a major public health challenge threatening human health. According to the World Health Organization (WHO), global cancer cases are expected to exceed 20 million by 2023, with 4,824,700 new cases in China, marking a 10% increase from the previous year.^1-5 Dietary restriction (DR), specifically Caloric restriction, has emerged as an effective complementary approach in the treatment of malignant tumors. A Caloric-restriction diet, involves a moderate reduction in fat and carbohydrate intake based on the satisfaction of five major nutrients, namely protein, vitamins, minerals, dietary fiber, and water, by eliminating 25% to 50% of normal energy intake.^6-9 Research shows that a Caloric-restriction diet can lower blood lipid levels, delay aging, and reduce damage in patients with acute craniocerebral injury.¹⁰ Additionally, it has been found to inhibit breast and cervical cancer progression in subcutaneous tumor-bearing model, by decreasing plasma lipid levels and reducing stearoyl coenzyme A desaturase (SCD) activity. This results the cancer cells’ ability to access dietary and intracellular lipids, thereby restricting tumor growth,^6,11,12 suggesting Caloric-restriction could be a promising strategy for oncology treatment.^13-16 One study demonstrated that a short-term, strictly Caloric-restriction diet was safe for patients with cancer receiving standard treatment. When combined with standard antitumor therapy to reduce blood glucose and growth factor concentrations, decrease peripheral blood immunosuppressive cells, and enhance T cell infiltration in tumors, thus slowing tumor progression.¹⁷ Caloric-restriction diets have also been effective in treating type 2 diabetes, with more than one-third of participants experiencing diabetic remission, improved beta cell function, and restored insulin secretory capacity for up to eight years, as shown in a study by Sattar et al¹⁸

Caloric-restriction has emerged as a promising strategy for the prevention and treatment of cancers, particularly age-related cancers. Its anti-cancer effects involve various mechanisms, such as inhibition of growth signaling, modification of cellular metabolism, induction of autophagy and apoptosis, and activation of stress resistance pathways. Although direct human trials are limited, preclinical studies and observations in human populations indicate that caloric restriction and its mimetics have a significant potential for enhancing cancer outcomes. Future research should aim to clarify the exact molecular mechanisms underlying these effects and refine CR-based interventions for clinical use.^19-22 Large amounts of experimental animal data are required to validate this finding in the early stages. Currently, there is limited discussion on the broad-spectrum therapeutic effects of a Caloric-restriction diet on various types of cancers. In this study, we investigated the inhibitory effects of a Caloric-restriction diet on colon, kidney, lung, and breast cancer in tumor-bearing mouse models. We also elucidated the mechanism by which the Caloric-restriction diet inhibited the mammalian target of rapamycin (mTOR) signaling pathway to provide a theoretical basis and data support for the implementation of Caloric-restriction diet-assisted clinical anticancer treatments in the future.

Materials and methods

Animals

Female specific pathogen-free (SPF) C57BL/6 and BALB/c mice (6-8 weeks old) weighing 20-22 g were purchased from Shanghai Bikai Keyi Biotechnology Co, Ltd [SCXK (Shanghai) 2023-0009]. The mice were housed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition, in an SPF facility maintained at 20°C-26°C and 40-70% relative humidity.²³ The number of animals used was minimized and measures were taken to reduce their suffering. Ethics approval for the study was obtained under number 202301102, and reporting adhered to the ARRIVE 2.0 guidelines.²⁴ At the end of the experiment, the mice were first induced into a coma with 40% carbon dioxide, followed by euthanasia at an increased concentration of 60% carbon dioxide. After confirming death, blood samples were collected via cardiac puncture and the tumor tissues were removed and weighed.

Cell Lines

The following mouse-derived colon cancer cell line were used: colon cancer cell line MC38(OBIO No.HYC3401), renal cancer cell line RENCA(ATCC No. CRL-2947™), lung cancer cell line LL/2(ATCC No.CRL-1642™) and breast cancer cell line 4T1(National Collection of Authenticated Cell Cultures No.SCSP-5056) were purchased from the National Collection of Authenticated Cell Cultures. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS at 37°C with 5% CO₂. Cells in the logarithmic growth phase were collected, counted after digestion with 0.25% EDTA-containing trypsin, centrifuged, and cell density was adjusted to 1 × 10⁷/mL.

Experimental Reagents

Reagents included anti-rabbit mTOR antibody (dilution:1/10000; ab134903), AKT antibody (dilution:1/10000; ab179463), and protein assay kit (ab287853) were purchased from Abcam. Anti-mouse antibodies for EGFR (JM-11695M1), P53 (JM-02303M1), IGF-1 (JM-12005M1), AMPK (JM-02303M1), IGF-1 (JM-12005M1), AMPK (JM-03142M1), P-AKT(JM-12301M1), and TNF-α (JM-02415M1) were purchased from JINGMEI Biotechnology; LDH-A(CB12799-M), PKM2(CB11550-Mu), and HK(CB10393-Mu) were purchased from COIBO Biotechnology. Anti-mouse antibody for β-actin (dilution:1/200;sc-47778) was purchased from Santa Cruz Biotechnology. Anti-mouse (dilution:1/10000;926-32210) and anti-rabbit (dilution:1/10000;926-32211) secondary antibodies were purchased from LiCOR Biosciences. RIPA lysis and extraction buffer (89900) and the HiMark™ Prestained Protein Standard (LC5699) were purchased from Thermo Fisher Scientific. The In Situ Cell Death Detection Kit (11684795910) was purchased from Shanghai Roche Pharmaceutical Co, Ltd A Pyruvic Acid (PA) Content Detection Kit (D799449) was purchased from Sangon Biotech (Shanghai), and Irradiated Laboratory Mouse Growth and Reproduction Feed (1010038) was purchased from Jiangsu Xietong Pharmaceutical Bioengineering Co, Ltd.

Model Preparation and Tumor Measurements

After 1 week of acclimatization, to a normal diet and water, 20 C57BL/6 and BALB/c mice with similar body weights were inoculated with 1 × 10⁶ cells/100 μl in the groin to establish tumor models. MC38 and RENCA cell lines were inoculated into 10 C57BL/6 mice, while the LL/2 and 4T1 cell lines were inoculated into 10 BALB/c mice respectively. The mice were randomly divided into four normal diet (ND) groups and four Caloric-restriction diet (CR) groups with five mice in each group. The day of cell inoculation was considered day 0 of the experiment. The long (L) and short (S) diameters of subcutaneous tumors were measured every 2 days. For irregularly shaped tumors, L was considered the longest diameter and S was considered the median of the short diameters perpendicular to the longest diameter. The tumor volume (V, mm³) was calculated according to the following formula: V = 0.5×L × S × S. To avoid malnutrition in mice with tumors that were too large and were subjected to the CR diet for too long in violation of animal welfare, the endpoint of the experiment was considered when the tumor size of any mouse in any group was greater than 1000 mm³ to avoid the effects of different CR times on physiological indices.

Diet Therapy

The regular chow (irradiated mouse growth and reproduction) used in this study consisted of 24% protein, 15% fat, and 61% carbohydrates with added vitamins, minerals, and amino acids. In this study design, “time-restricted feeding” refers to a protocol in which the animals are allowed access to food only during a specific time window. Specifically, the normal diet (ND) group had ad libitum access to food and water throughout the day. In contrast, the caloric restriction through time-restricted feeding (CR) groups were allowed regular chow only from 9:00 a.m. to 11:00 a.m. each day and fasted for the remaining 22 hours, starting on the first day of the study. It is important to note that this temporal restriction is not exactly equivalent to traditional caloric restriction, as the animal receives enough food during the feeding window to maintain its nutritional requirements. Food and drink consumption was recorded for all groups. Other feeding conditions were the same as those used in the ND group.

Weight and Diet Measurements

Chow for the ND group was recorded at 11:00 am every day. The chow in the CR group was removed simultaneously. The weights of all the mice were recorded before the start of the CR group feeding at 9:00 a.m. the following day. Subsequently, 150 g regular chow was added to each cage in the CR group. The amount of ND groups’ 24-hour feed and CR groups’ 2-hour feed were recorded 2 h later at 11:00 a.m. Subsequently, all the chow in the CR group was removed. This procedure was repeated until the end of the experiment to calculate the dietary decline rate by comparing the reduction in feed intake between the CR and ND groups.

Routine Blood Tests and Blood Biochemistry

Whole blood was collected from the mice at the end of the experiment. A portion of whole blood was placed in heparin anticoagulation tubes for routine blood tests. The remaining whole blood was centrifuged and serum was collected for blood biochemistry tests.

Colorimetric Determination of Serum Pyruvic Acid (PA) Levels

Ice-bath homogenization was performed using a serum/pyruvic acid extract volume ratio of 1:10. The supernatants were collected and centrifuged at room temperature. The colorimetric reagent I was added to each well and mixed thoroughly. After incubation at room temperature for 2 min, colorimetric reagent II was proportionally added to each well. The optical density (OD) was measured at 520 nm to detect the concentration of pyruvic acid (PA) in the serum, with a blank well used to determine the zero value.

Enzyme-Linked Immunosorbent Assay (ELISA) for Serum Multifactor Levels

Serum samples and standards were diluted 5-fold with PBS diluent. An aliquot of the diluted samples and standards was then combined with the enzyme reagent in the designated wells of the microplate. The microplate was incubated for 60 minutes at 37°C to enable the enzyme-linked antigen or antibody to bind to the immobilized antigen or antibody on the plate. After incubation, the microplate was washed five times with washing solution to remove any unbound reagents. Each wash was followed by gentle patting to ensure the complete removal of the washing solution. After the final wash and drying, a chromogen substrate was added to each well to initiate a color development reaction. The plate was then incubated for a specified period to allow color development, which indicated the presence and quantity of the target antigen or antibody in the serum samples.

The optical density (OD) of each well was measured at 450 nm to detect the levels of AMPK, P53, P-AKT, EGFR, IGF-1, TNF-α, HK, LDHA and PKM2 in the serum, with a blank well used to determine the zero value.

Protein Immunoblotting (Western Blot) Determination of mTOR and AKT Expression Levels in Tumor Tissues

Tumor tissues were lysed using RIPA buffer containing 1% PMSF and centrifuged at 4°C. Total protein concentration was determined using a Pierce BCA protein assay kit. Proteins were separated by 10% SDS‒PAGE, electrotransferred to a PVDF membrane, and incubated with mTOR and AKT antibodies. The membrane was scanned using an imaging system and the results were analyzed.

Immunohistochemistry (IHC) Staining of P53, P-AKT, EGFR, and IGF-1 in Tumor Tissues

Tumor tissue were paraffin-embedded, sectioned, and dewaxed. P53, P-AKT, EGFR, and IGF-1 antibodies were diluted proportionally and incubated with the sections. The sections were then incubated with a secondary antibody at room temperature and washed. After hematoxylin staining, the sections were photographed and analyzed. For each section, ten images were obtained at a resolution of 20 × . The immunostained areas were statistically analyzed using the ImageJ 1.54d software.

TdT-Mediated dUTP Nick-End Labelling (TUNEL)

Some paraffin sections of the tumor tissue were washed with ethanol after dewaxing, treated with proteinase K, and incubated with the TUNEL reaction mixture. After washing with PBS, the sections were stained with an anti-quenching mounting medium containing DAPI and observed under a fluorescence microscope. For each section, ten images were obtained at a resolution of 20×. The average fluorescence intensity of the TUNEL-stained areas was analyzed using Image-Pro Plus.

Statistical Analysis

Data was analyzed using GraphPad 8.0 and Image-Pro Plus software, and are presented as the mean ± standard deviation (±s). A t test was used for statistical comparison, with P values <0.05 indicating statistical significance. All study data are available.

Results

Reduction in Body Weight and Diet in Mice

Mouse experiments were conducted for 19 consecutive days. The experimental procedure is illustrated in Figure 1. The daily dietary reduction rate of mice in each CR group remained stable between 25% and 50% of the ND (Figure 1(A)–(D)). The dietary regimen adhered to animal welfare and CR dietary requirements.²⁵ Compared to the corresponding ND control groups, the body weights of mice in the 4T1-CR, LL/2-CR, MC38-CR, and RENCA-CR groups decreased rapidly in the first week of the experiment. Using the body weights of the mice in the ND groups as a reference, the body weights in the 4T1-CR, MC38-CR, LL/2-CR, and RENCA-CR groups dropped by 11.29 ± 1.01%, 9.95 ± 1.39%, 9.46 ± 0.88%, and 8.08 ± 1.24%, respectively, at the lowest point. one week after the start of the experiment, the body weights began to stabilize, and the average body weight reduction was consistently between 2.31% and 11.29% (Figure 1(E)–(H)).

Figure 1.

Comparison of feed consumption in each group of mice (A-D n = 5), Body weight changes in each group of mice (E-H n = 5).

CR Diet Inhibits Tumor Volume Growth in Mice

The day of cell inoculation was designated as Day 0. Tumor sizes were measured every two days until the end of the experiment, and five mice reached the humane endpoint within 19 days. The tumor growth curves revealed that the rate of tumor growth was significantly lower in the 4T1-CR group (431.78 ± 51.48 mm³), LL/2-CR group (314.22 ± 10.27 mm³), MC38-CR group (248.42 ± 29.29 mm³) and RENCA-CR group (342.37 ± 33.91 mm³) compared to the corresponding ND control groups (4T1-ND group (950.52 ± 52.15 mm³), LL/2-ND group (645.79 ± 120.46 mm³), MC38-ND group (702.42 ± 76.79 mm³) and RENCA-ND group (866.87 ± 155.83 mm³)) (Figure 2(A), (D), (G), (J)).

Figure 2.

CR diet inhibits tumor volume growth in mice. Tumor growth curves, tumor weights and tumor tissues images from the ND and CR groups are shown from left to right for 4T1 (A-C), LL/2 (D-F), MC38 (G-I), and RENCA (J-L) tumors. n = 5, *P < 0.05, **P < 0.01.

At the end of the experiment tumor tissues were removed and weighed. A significant reduction in tumor weight in the 4T1-CR group (0.616 ± 0.047 g), LL/2-CR group (0.284 ± 0.028 g), MC38-CR group (0.207 ± 0.030 g) and RENCA-CR group (0.318 ± 0.025 g) was observed compared to their corresponding ND control groups (4T1-ND group (0.972 ± 0.079 g), LL/2-ND group (0.747 ± 0.105 g), MC38-ND group (0.601 ± 0.094 g) and RENCA-ND group (0.837 ± 0.167 g)) (Figure 2(B), (C), (E), (F), (H), (I), (K), (L). These findings are consistent with the tumor growth curves, indicating that the CR diet effectively inhibited tumor growth in mice.

CR Diet Affects the Levels of PA and Its Upstream and Downstream Key Rate-Limiting Enzymes in Mouse Serum

Mouse serum was analyzed for PA levels using a colorimetric assay and for HK, LDHA, and PKM2 levels using enzyme immunoassay. Compared to the corresponding ND control groups, the serum PA (***P < 0.001), LDHA (*P < 0.05) and PKM2 (*P < 0.05) levels were significantly lower in the 4T1-CR group, the serum PA (*P < 0.05), HK (**P < 0.01) and LDHA (**P < 0.01) levels were significantly lower in the LL/2-CR group, the serum PA (**P < 0.01), HK (**P < 0.001) and PKM2 (*P < 0.05) levels were significantly lower, in the MC38-CR group. While, serum HK level (**P < 0.01) was significantly lower in the RENCA-CR group (Figure 3(A)–(D)). The results suggest that the CR diet significantly reduced serum PA levels in mice and influenced the levels of key rate-limiting enzymes upstream and downstream of PA.^26,27

Figure 3.

Serum expression of PA (A), HK (B) LDHA (C) and PKM2 (D) in each group. n = 5, *P < 0.05, **P < 0.01, ***P < 0.001.

CR Diet has a Minor Effect on Routine Blood and Blood Biochemistry Parameters in Mice

The results from routine blood and blood biochemistry tests revealed that, compared with the corresponding ND groups, the MCHC levels in the 4T1-CR group increased significantly (*P < 0.05), and both the decrease in PLC and the increase in the LYMPH percentage were significant in the LL/2-CR group (*P < 0.05). The remaining routine blood tests (Table 1) and blood biochemistry indices (Figure 4) showed no significant changes. These results suggest that the CR diet had a minimal effect on blood indices and liver and kidney functions in mice.

Table 1.

Changes in Routine Blood Indicators in the Four Groups of Mice (n = 5).

Group	4T1 ND			4T1 CR			LL/2 ND			LL/2 CR
WBC (10^9/L)	869.4	±	62.7	834.0	±	28.2	999.2	±	200.7	964.0	±	56.9
RBC (10^12/L)	1081.6	±	14.7	1081.8	±	19.5	1060.8	±	51.4	1085.6	±	10.3
HGB (g/L)	164.6	±	1.9	166.2	±	3.1	162.2	±	8.6	164.8	±	2.2
HCT (%)	475.2	±	5.5	472.2	±	8.0	474.2	±	35.4	495.2	±	4.3
MCV (fL)	439.4	±	1.2	436.6	±	2.7	446.6	±	17.6	456.2	±	6.0
MCH (pg)	152.0	±	0.6	153.4	±	0.4	152.8	±	5.5	152.0	±	0.6
MCHC (g/L)	346.4	±	1.4	351.8	±	1.7	342.4	±	9.3	333.0	±	5.1
PLT (10^9/L)	1332.4	±	60.9	1251.0	±	82.6	1430.4	±	288.3	1301.2	±	95.0*
RDW-SD (fL)	291.8	±	2.7	291.8	±	2.6	273.6	±	39.8	288.8	±	2.1
RDW-CV (%)	224.8	±	0.9	225.0	±	1.3	202.0	±	43.4	214.6	±	3.5
PDW (fL)	98.4	±	4.9	90.4	±	4.9	84.6	±	12.8	76.0	±	1.7
MPV (fL)	80.4	±	1.9	77.0	±	1.3	75.6	±	3.4	69.0	±	1.3
P-LCR (%)	107.2	±	4.6	96.2	±	5.6	136.8	±	85.8	76.2	±	14.5
LYMPH# (10^9/L)	720.4	±	51.0	694.0	±	23.7	791.2	±	116.6	854.2	±	57.0
EO# (10^9/L)	10.6	±	1.1	14.2	±	2.2	15.8	±	12.3	13.6	±	3.6
BASO# (10^9/L)	0.6	±	0.2	0.6	±	0.2	1.8	±	2.9	0.8	±	0.2
NEUT% (%)	123.8	±	7.7	113.2	±	12.0	133.4	±	52.7	74.8	±	13.2
LYMPH% (%)	829.0	±	9.5	832.6	±	15.1	798.8	±	60.9	885.0	±	18.1*
EO% (%)	12.2	±	1.0	17.0	±	2.5	16.0	±	11.4	14.0	±	3.4
BASO% (%)	0.6	±	0.2	0.6	±	0.2	0.4	±	0.5	0.8	±	0.2

Group	MC38 ND			MC38 CR			RENCA ND			RENCA CR
WBC (10^9/L)	787.0	±	169.6	814.8	±	140.1	863.8	±	215.0	924.0	±	143.8
RBC (10^12/L)	1075.2	±	51.3	1055.8	±	38.8	1121.8	±	47.2	1117.4	±	18.3
HGB (g/L)	162.8	±	4.7	161.6	±	2.5	168.4	±	7.5	168.4	±	2.6
HCT (%)	459.0	±	18.0	460.4	±	7.8	469.4	±	23.2	468.4	±	8.6
MCV (fL)	427.2	±	4.8	436.2	±	11.4	418.2	±	4.9	419.0	±	1.9
MCH (pg)	151.6	±	4.0	153.4	±	5.0	150.2	±	0.8	150.8	±	2.4
MCHC (g/L)	354.6	±	7.4	350.8	±	4.5	358.8	±	3.4	359.8	±	6.9
PLT (10^9/L)	1110.4	±	414.0	910.2	±	460.2	1337.6	±	134.4	1236.0	±	282.7
RDW-SD (fL)	287.8	±	4.6	268.6	±	31.7	279.2	±	4.0	277.4	±	6.8
RDW-CV (%)	225.2	±	5.6	204.6	±	37.3	228.4	±	2.9	225.0	±	4.7
PDW (fL)	89.4	±	3.7	96.8	±	18.6	99.8	±	5.7	90.6	±	7.6
MPV (fL)	75.8	±	1.6	78.4	±	5.6	78.4	±	0.9	76.2	±	3.0
P-LCR (%)	84.6	±	32.4	72.6	±	38.7	105.0	±	9.9	95.0	±	22.5
LYMPH# (10^9/L)	630.6	±	153.3	641.4	±	174.0	634.6	±	108.3	677.2	±	108.1
EO# (10^9/L)	24.0	±	15.4	23.4	±	15.7	21.2	±	7.1	19.6	±	7.6
BASO# (10^9/L)	0.6	±	0.6	0.6	±	0.6	1.0	±	0.7	1.2	±	0.5
NEUT% (%)	129.6	±	67.2	123.0	±	29.2	201.0	±	41.4	203.0	±	47.8
LYMPH% (%)	800.8	±	80.8	774.2	±	104.3	744.8	±	53.3	735.6	±	74.8
EO% (%)	32.0	±	22.9	32.4	±	28.8	24.4	±	4.5	20.8	±	6.1
BASO% (%)	0.6	±	0.6	0.6	±	0.6	1.0	±	0.7	1.2	±	0.5

Compared with the ND group, *P < 0.05.

WBC: White blood cell count. RBC: Red blood cell count. HGB: Hemoglobin. HCT: Hematocrit. MCV: Mean corpuscular volume. MCH: Mean corpuscular hemoglobin. MCHC: Mean corpuscular hemoglobin concentration. PLT: Platelet count. PDW: Platelet distribution width. RDW: Red blood cell distribution width. MPV: Mean platelet volume. P-LCR: platelet-larger cell ratio. LYMPH: Lymphocyte. EO: Eosinophil. BASO: Basophil. NEUT: Neutrophilic granulocyte.

Figure 4.

Blood biochemical tests. Alanine aminotransferase (A), albumin (B), aspartate aminotransferase (C), creatinine (D), and urea (E) in the serum for liver and kidney functions in the ND and CR groups. n = 5.

CR Diet Inhibits mTOR and AKT Protein Expression in Tumors

The protein levels of mTOR and AKT in the ND and CR groups for each tumor model were analyzed by western blotting. Figure 5 shows that the protein expression of mTOR and AKT was significantly lower than that in the corresponding ND groups (*P < 0.05). These results were statistically normalized using the internal control reference β-actin. This indicates that the CR diet can inhibit mTOR and AKT protein expression in tumors.

Figure 5.

Western blot detection of mTOR (A) and AKT (B) protein expression in mouse tumors across each group. Histograms illustrating the differences in the levels of the mTOR (C) and AKT (D) proteins between the ND and CR groups. n = 4, *P < 0.05.

CR Diet Reduces the Serum Levels of AMPK, P53, P-AKT, EGFR, IGF-1, and TNF-α

Mouse serum was analyzed using ELISA. Compared with the corresponding ND control groups, the serum levels of AMPK (**P < 0.01), P-AKT (*P < 0.05), EGFR (*P < 0.05), IGF-1 (*P < 0.05), and TNF-α (**P < 0.01) were significantly lower in the 4T1-CR group, the serum levels of AMPK (***P < 0.001), P53 (**P < 0.01), P-AKT (*P < 0.05), EGFR (**P < 0.01), IGF-1 (*P < 0.05), and TNF-α (**P < 0.01) were significantly lower in the LL/2-CR group, and the serum levels of AMPK (**P < 0.01), P53 (**P < 0.01), P-AKT (***P < 0.001), EGFR (*P < 0.05), and TNF-α (***P < 0.001) showed a significant decrease in the RENCA-CR group at the endpoint of the experiment (Figure 6). However, the MC38-CR group showed no significant differences in the serum levels of AMPK, P53, P-AKT, EGFR, or IGF-1 compared to the MC38-ND group (Figure 6). These findings revealed that the CR diet could downregulate a variety of mTOR-related signaling pathways and that this effect varies among different tumors.

Figure 6.

Protein expression levels detection. AMPK (A), P53 (B), P-AKT (C), EGFR (D), IGF-1 (E), and TNF-α (F) in the serum of each group were displayed in the figure. n = 5, *P < 0.05. **P < 0.01, ***P < 0.001.

CR Diet Reduces P53, P-AKT, EGFR, and IGF-1 Expression in Tumor Tissues

The expression of P53, P-AKT, EGFR and IGF-1 in the tumors was determined by immunohistochemical staining. Antigen-positive cells are indicated by red arrows (Figure 7(A)–(D)). Compared to those in the corresponding ND groups, P53 (**P < 0.01), P-AKT (**P < 0.01) and EGFR (**P < 0.01) were significantly lower in the 4T1-CR group, P53 (***P < 0.001), P-AKT (****P < 0.0001), and EGFR (**P < 0.01)) were significantly decreased in the LL/2-CR group. The MC38-CR group showed significant decrease in P53 (**P < 0.01), P-AKT (**P < 0.01), EGFR (*P < 0.05) and IGF-1 (**P < 0.01). In the RENCA-CR group, P53 (*P < 0.05), P-AKT (****P < 0.0001), EGFR (**P < 0.01) and IGF-1 (**P < 0.01) were also significantly (Figure 7(E–H)). These findings were consistent with the ELISA results for P53, P-AKT, EGFR, and IGF-1 protein expression levels in mouse serum.

Figure 7.

Immunohistochemistry staining. P53 (A), P-AKT (B), EGFR (C), and IGF-1 (D) in the tumors of the mice in each group. Images were organized by tumor types 4T1, LL/2, MC38, and RENCA from top to bottom, respectively, with a resolution of 20×. Histograms comparing the percentages of P53 (E), P-AKT (F), EGFR (G), and IGF-1 (H)-positive cells in each group of mice. n = 5, *P < 0.05. **P < 0.01, ***P < 0.001, ****P < 0.0001.

CR Diet Promotes Tumor Apoptosis

The TUNEL assay was performed to detect tumor apoptosis. Apoptotic cells are represented in green and nuclei are represented in blue (Figure 8(A)–(D)). The tumor apoptosis rate was significantly higher in the 4T1-CR group (***P < 0.001), LL/2-CR group (***P < 0.001), MC38-CR group (*P < 0.05), and RENCA-CR group (*P < 0.05) (Figure 8E). These results indicated that the CR diet promoted tumor cell apoptosis.

Figure 8.

TUNEL staining for 4T1 (A), LL/2 (B), MC38 (C), and RENCA (D) groups were displayed. The histogram compared the average fluorescence intensity of TUNEL signals in each group (E). n = 5, *P < 0.05, ***P < 0.001.

Discussion

In recent years, two major low-carbohydrate diets the CR and the ketogenic diets (KD), have been widely studied as promising dietary intervention therapies. Previous studies, a CR diet prolonged the lifespan of C57BL/6 mice and has been used as an adjunctive therapy in the clinic to reduce the incidence of diabetes, cancer and cardiovascular disease.^28-31 The relationship between dietary intervention and cancer pathogenesis is complex. While recent CR diet programs have provided insights into metabolic and immunomodulatory effects, the underlying therapeutic mechanisms remain largely unexplored.³² This lack of clarity often complicates the systematic application of these diets in treating these diseases.

In this study, we examined changes in mTOR and its upstream and downstream signaling pathways in various mouse tumor models following a CR diet. As a diet-sensitive protein kinase, mTOR regulates cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration to maintain cellular homeostasis.^33-35 Mutations and abnormal amplification of mTOR and its upstream and downstream genes lead to constitutive activation of the mTOR pathway, which causes aging, neurological disorders, and malignancies. The activation of mTOR is closely linked to tumor progression. Through ribosome profiling of the mTOR signaling pathway, Hsieh et al reported that mTOR is overactivated in prostate cancer, with mTOR inhibition suppressing prostate cancer invasion and metastasis. Fruman et al demonstrated that mTOR signaling is enhanced in all types of cancers, whereas solid tumor data suggested that mTOR signaling is dysregulated in nearly 30% of cancers.³⁶ Consequently, mTOR is recognized as a potent cancer biomarker.^37-42 Inhibition or blockade of mTOR and its associated signaling pathways is regarded as a major therapeutic strategy for cancer.^43,44

In this study, the effects of a CR diet were evaluated in a variety of established mouse subcutaneous tumor models. Based on the pre-experiments, the mice maintained a caloric intake of 50-75% of the normal diet without an abnormal physiological state when the daily CR diet was restriction to two hours per day. The tumor growth rates of mice in the CR group were significantly lower than those in the ND group. By analyzing the blood of the mice, we found that the levels of PA and its key rate-limiting enzymes, HK, LHDA, and PKM2, were significantly lower in the CR group than in the control group. Additionally, the expression of the mTOR-related signaling pathway components AMPK, P53, P-AKT, EGFR, and IGF-1 was lower in the CR group than in the ND group. These results indicated that the dietary regimen could reduce PA expression by inhibiting key rate-limiting enzymes and mTOR-related signaling pathways. Routine blood and blood biochemistry tests showed that the CR diet had a minor effect on blood indices and liver and kidney function in mice. Further analysis revealed that the protein expression levels of mTOR and AKT were significantly downregulated in each CR group of mouse tumor tissues. Moreover, immunohistochemical staining revealed that the expression of P53, P-AKT, EGFR, and IGF-1 in the tumor tissues of the CR group was significantly lower than that in the ND group, which was consistent with the ELISA results. TUNEL staining revealed that the percentage of apoptotic tumor cells in tumor tissues was significantly greater in the CR group than in the ND group, demonstrating that the CR diet promoted the apoptosis of tumor cells in mice.

Different mouse models exhibit unique metabolic and immune responses, which may influence the outcomes of CR diet interventions.⁴⁵ The observed differences in IGF-1 response to caloric restriction (CR) between C57BL/6 and BALB/c mouse strains suggest potential strain-specific effects. Specifically, C57BL/6 mice showed no IGF-1 response to CR, whereas BALB/c mice did exhibit such a response. This disparity suggests that genetic factors associated with the strain may influence the CR’s impact on IGF-1 regulation. Therefore, the possibility that strain-specific characteristics impact the response to CR, particularly regarding IGF-1, cannot be dismissed. Further research comparing the responses of multiple mouse strains to CR is needed to fully understand the extent of strain-specific effects on IGF-1 and other biomarkers associated with tumor growth and progression.

In future studies, we will conduct additional animal experiments to assess the effects of a CR diet on a broader range of tumors and explore the underlying mechanisms. Furthermore, while dietary changes usually produce fewer side effects than drug therapy³² and clinical trials have shown that a short-term CR diet for 2-3 weeks is sufficient to remodel metabolism and antitumor immunity in cancer patients,^17,46 it has been demonstrated that a CR diet in a mouse tumor model for more than 21 days can cause autophagic responses, leading to autophagic cell death and apoptosis in healthy cells due to nutrient deprivation.⁴⁷

Findings on tumor growth in response to caloric restriction (CR) in animal studies come with several important caveats. First, these studies were conducted exclusively using mouse models of preclinical cancer treatment. As seen in humans, cancer progression can vary significantly between species owing to differences in metabolism, physiology, and tumor biology. Therefore, the validation of these findings in human clinical studies is necessary. Second, the study period was relatively short (19 days). Although this duration was sufficient to observe that CR could slow tumor development, the long-term effects of CR on tumor growth and survival remain unclear. To gain a more comprehensive understanding, future research should include studies on chronic Caloric restriction and carefully assess the potential adverse effects. Additionally, the study focused on four types of cancer: lung, breast, renal, and colon. Although this study provides valuable insights into CR’s potential of CR as an anti-malignant agent across different systems, its effects on other cancer types have not been examined. Future studies should include a broader range of cancers and investigate the mechanisms underlying the effects of CR.

Therefore, the potential risks in tumor-bearing mice should not be overlooked. In subsequent experiments, we will extend the duration of the dietary intervention and systematically assess its safety, identifying any potential side effects and contraindications. This will help ensure the stability of the results, and the feasibility of translating these findings into adjuncts for human cancer therapy.

Currently, no universal treatment exists for all cancer types, regardless of the cancer type, region, or age. The CR diet has shown potential as a universal adjuvant treatment for tumors. Additionally, understanding the underlying mechanisms will offer guidance and ideas for nutrition in clinical oncology.

Conclusions

Collectively, our results suggest that a CR diet can slow the growth rate of a wide range of mouse tumors. This broad-spectrum therapeutic effect may result from the inhibition of mTOR and its upstream and downstream gene signaling pathways, which in turn slow tumor glycolysis and accelerate tumor cell apoptosis.

Footnotes

Author Contributions

LU Weisheng was involved in the experimental operation, data management, writing of the original draft and funding acquisition. WANG Jue contributed to cell line culture. WANG Chengji participated in the design of the study. WANG Haijie and GAO Wenhao contributed to the therapeutic analysis of serum samples. YE Shouchong helped to revise the article. SHEN Ruling was responsible for experimental design, experimental supervision, funding acquisition and manuscript revision. All authors have participated sufficiently in the work and have agreed to take responsibility for all aspects of the work. All authors have contributed to editorial changes in the manuscript and have read and approved the final manuscript.

Declaration of Conflicting Interests

The author(s) have declared that they have no potential conflicts of interest with regard to the research, writing and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the grants from the Science and Technology Commission of Shanghai Municipality (No. 22DZ2291200, No. 22140900102), and Shanghai Laboratory Animal Research Center 2023 “Science and Technology Innovation Plan” Nova project (No. 2023NS04).

Ethical Statement

ORCID iDs

Weisheng Lu

Ruling Shen

Appendix

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