The Pro-Apoptotic Effect of Glucose Restriction in NSCLC via AMPK-Regulated Circadian Clock Gene Bmal1

Abstract

The circadian clock is a crucial regulator of mammalian physiology, controlling daily oscillations in key biological processes, such as cell proliferation, apoptosis, and DNA damage repair. Disruption of circadian rhythms has been identified as a significant risk factor for cancer development and progression, yet the specific molecular mechanisms linking circadian dysfunction to cancer remain poorly understood. Recent studies have increasingly focused on the role of diet in modulating circadian rhythms, highlighting the potential for dietary interventions in cancer management. However, how dietary factors like glucose restriction interact with circadian rhythms to influence cancer cell behavior remains an open question. Here, we investigate the mechanisms underlying glucose restriction-induced apoptosis in non-small cell lung cancer (NSCLC) cells, with a focus on the role of circadian clock genes. Analysis of the GEPIA database revealed that the circadian gene Bmal1 is highly expressed in normal tissues and associated with better prognosis in lung adenocarcinoma patients. In NSCLC cells, Bmal1 expression correlated with proapoptotic gene activity. In a tumor xenograft model using severe combined immunodeficiency (SCID) mice, a glucose-restricted (ketogenic) diet significantly delayed tumor growth and increased the expression of Bmal1 and proapoptotic genes. These findings suggest that glucose restriction promotes apoptosis in NSCLC cells through a Bmal1-mediated pathway, providing novel insights into the intersection between circadian regulation and cancer biology. Targeting core circadian clock genes like Bmal1 may represent a promising therapeutic strategy for managing lung cancer, broadening our understanding of how circadian rhythms can be harnessed for cancer prevention and treatment.

Keywords: AMPK; Bmal1; NSCLC; apoptosis; glucose restriction.

FIGURE 1

Glucose restriction promotes apoptosis in tumor cells. (A, B) The mRNA levels of Bax, Bcl‐2, Caspase‐3 and Caspase‐8 in cells were detected by qRT‐PCR after treated with different concentrations of glucose (11, 5.5, 2.5 mM) for 12 h. (C, D) Western Blot analysis of Bax, Bcl‐2, Cleaved Caspase‐3, Caspase‐8 in cells cultured with different concentrations of glucose for 24 h.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

Glucose restriction promotes apoptosis in NSCLC cells through AMPK involvement. (A) mRNA expression of AMPK after transfected with AMPKα1 overexpression plasmid. (B) mRNA expression of AMPK after AMPK inhibitor compound C (40 μM) used. (C) mRNA expression of Bax, Bcl‐2, Caspase‐3 and Caspase‐8 after AMPK overexpression. (D) mRNA expression of Bax, Bcl‐2, Caspase‐3 and Caspase‐8 after AMPK inhibition. Protein expression of Bax, Bcl‐2, Cleaved Caspase‐3 and Caspase‐8 after AMPK overexpression (E) and AMPK inhibition (F).

FIGURE 3

Expression levels of circadian clock genes in lung adenocarcinoma and normal tissue. (A) Analysis of circadian gene expression in lung adenocarcinoma tumor samples and adjacent noncancerous tissues from the GEPIA database. (B) Analysis of circadian gene expression in tumor tissue samples patients from the GEPIA database compared to normal lung tissue samples from the GTEx database. (C) Surviavl analysis of differential expression levels of clock genes. *p < 0.05.

FIGURE 4

Regulation of apoptosis gene expression by Bmal1 in NSCNC cells. (A, D) mRNA expression of Bmal1, Bax, Bcl‐2, Caspase‐3 and Caspase‐8 after transfected with Bmal1 overexpression plasmid. (B) Protein expression of Bmal1, Bax, Bcl‐2, Cleaved Caspase‐3 and Caspase‐8 after transfected with Bmal1overexpression plasmid. (C) The impact of AMPK overexpression or inhibition on Bmal1 expression. (E) Flow cytometry measured after Bmal1 overexpression. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Glucose restriction increased the expression of apoptosis‐related genes in vivo. (A) Grouping of xenograft tumor mice (by Figdraw) and the proportions of the three macronutrients in a normal diet and a ketogenic diet. (B) mRNA expression of Bax, Bcl‐2, Caspase‐3, Caspase‐8 on normal diet and ketogenic diet groups. (C) The rhythmic expression levels of Bmal1 on blank control, normal diet and ketogenic diet groups. (D) Protein expression of Bax, Bcl‐2, Caspase‐8 on normal diet and ketogenic diet groups. *p < 0.05, ***p < 0.001.