Breast cancer (BC) is the most common cancer among women [1]. The more aggressive triple-negative breast cancer (TNBC), a subtype lacking the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2, has a 5-year survival rate of only 12% [2]. TNBC is a prevalent form of BC in women. Chemotherapy is one of the most widely used treatment modalities in oncology clinics [3]. Despite advances in surgery, chemotherapy, targeted therapy, and immunotherapy, the prognosis of many BC patients remains poor [4], primarily due to the significant side effects and poor tolerance of current therapeutic regimens. Researchers at home and abroad are exploring postoperative treatment strategies for BC to improve the postoperative quality of life of patients.

Epidemiological evidence indicates that diet not only influences cancer risk but also exerts a significant impact on treatment outcomes [5]. Most notably, Warburg first described the association between carbohydrates and cancer in 1,923, when he reported a marked increase in glucose consumption by tumor cells [6,7]. This phenomenon is referred to as the Warburg effect. In recent years, a low-glucose ketogenic diet (KD) has also been shown to reverse redox signaling pathways that promote tumor malignancy [8,9]; when used as an adjuvant therapy for gliomas, it can alter transgene expression patterns and reduce reactive oxygen species (ROS) levels [10]. Hopkins et al. [11] also demonstrated that combined KD administration improves survival in mice bearing various PI3K-driven tumors. In most preclinical tumor models, KD has been proven to inhibit glycolysis and cancer cell proliferation [12]. Given that cancer cells exhibit dysregulated glucose metabolism, resulting in higher glucose uptake in tumors compared to adjacent normal tissues [13], KD may serve as a diet-based postoperative adjuvant strategy for cancer treatment.

Glucose starvation is one of the major forms of metabolic stress in tumor cells [14]. Glucose deprivation impairs glycolysis and the pentose phosphate pathway [14], and increases ROS production, thereby inducing oxidative stress and subsequent cell death. ROS are a group of diverse molecular oxygen derivatives generated during normal aerobic metabolism [15]. Under glucose-limiting conditions, ROS are regarded as positive regulators of AMPK [15]. A reduction in cellular energy status, along with elevated ADP/ATP and AMP/ATP ratios, activates AMP-activated protein kinase (AMPK) [16]—a key regulator of cellular metabolic homeostasis [17,18]. AMPK is also capable of inhibiting the mTOR pathway [19,20], particularly under energy-deficient conditions. Although previous studies have demonstrated that KD or glucose restriction can modulate ROS levels and activate AMPK in various cancer types, including TNBC [21], the precise molecular crosstalk between KD-induced ROS generation and the AMPK–mTOR signaling pathway in 4T1 breast tumors has not been directly elucidated.

The potential of KD as a complementary strategy for cancer therapy has been highlighted by several preclinical and clinical studies [22]. By inducing a metabolic state characterized by ketosis, this dietary regimen creates an unfavorable metabolic microenvironment for cancer cells, which may impede their growth and proliferation; however, its underlying mechanism of action in 4T1 BC remains unclear. In the present study, we investigated whether the KD exerts tumor-suppressive effects on 4T1 tumor-bearing mice in vivo via the ROS-AMPK-mTOR pathway and whether glucose restriction regulates mTOR expression in 4T1 cells in vitro through the ROS-AMPK pathway. Notably, our study suggests potential links between KD-induced ROS accumulation, AMPK activation, and mTOR inhibition, complementing previous studies focused solely on metabolic stress or AMPK activation in tumor suppression.