Metformin Injection

Metformin competitively binds to the ubiquinone binding site of mitochondrial complex I, blocking the initial step of the electron transfer chain (ETC). This inhibition leads to the collapse of the proton gradient (Δ PSI m), a decrease in ATP synthase activity, and a sharp drop in the intracellular ATP/AMP ratio. A study conducted by Northwestern University in 2023 showed that metformin treatment can reduce ATP levels in tumor cells by 60% -70%, triggering rapid activation of AMPK (adenosine monophosphate activated protein kinase).

AMPK, as a cellular energy sensor, triggers multiple metabolic reprogramming upon activation:
Enhanced fatty acid oxidation: phosphorylates ACC (acetyl CoA carboxylase), inhibits fatty acid synthesis, and promotes β - oxidation.
Upregulation of glucose uptake: Increased glucose uptake by muscle and adipose tissue through GLUT4 translocation.
Protein synthesis inhibition: Phosphorylation of mTORC1 (mammalian rapamycin target protein complex 1), blocking downstream S6K1 and 4EBP1 signaling, and inhibiting tumor cell proliferation.

Tumor cells rely on aerobic glycolysis (Warburg effect) to produce ATP, but metformin induced metabolic switching significantly weakens their energy supply:
Glycolysis inhibition: AMPK activation reduces the production of fructose-2,6-diphosphate (F2,6BP) and lowers the rate of glycolysis by inhibiting PFKFB3 (6-phosphofructose-2-kinase/fructose-2,6-diphosphatase 3).
Oxidative phosphorylation blockade: Complex I inhibition directly disrupts the mitochondrial respiratory chain, forcing cells to switch to an inefficient glycolytic pathway, but ATP production is less than 10% of the original level.

Reduced lactate accumulation: Glycolysis inhibition reduces lactate production, improves tumor microenvironment acidification, and enhances the damage effect of radiation on DNA.
Clinical evidence: In SW480 colorectal cancer cells, the combination of metformin and radiotherapy resulted in a 40% decrease in the expression of the key glycolytic enzyme HK2 (hexokinase 2), a 55% decrease in intracellular ATP levels, and a 70% decrease in colony forming ability.

Ionizing radiation generates free radicals through direct ionization and indirect hydrolysis, leading to DNA double strand breaks (DSBs), single strand breaks (SSBs), and base damage. Tumor cells rely on two major repair systems to respond to damage:
Homologous recombination repair (HR): It uses BRCA1/2 as the core and sisters chromatids as the template to precisely repair DSB.
Non homologous terminal junction (NHEJ): Quickly connecting broken ends through proteins such as Ku70/Ku80 and DNA PKcs can easily introduce errors.

Directly inhibiting the expression of HR repair proteins is achieved by metformin downregulating the expression of key HR proteins through the AMPK/mTOR pathway
Rad51 inhibition: In pancreatic cancer cells, metformin reduces the Rad51 protein level by 60%, resulting in a 50% reduction in HR repair efficiency.
BRCA1/2 ubiquitination degradation: Metformin activates E3 ligase MDM2, promoting proteasomal degradation of BRCA1/2 and weakening HR repair ability.

Metformin injection interferes with NHEJ repair through oxidative stress:
Phosphorylation inhibition of DNA PKcs: ROS (reactive oxygen species) accumulation leads to dephosphorylation of serine 2609 in DNA PKcs (DNA dependent protein kinase catalytic subunit), making it unable to recruit repair factors.
Ku70/Ku80 dissociation: High concentrations of ROS oxidize Cys58 and Cys155 of Ku70, disrupting their binding ability to DNA ends.
Animal experiments: In a nude mouse transplant tumor model, the combination of metformin and radiotherapy prolonged the duration of γ - H2AX (DSB marker) foci by three times and reduced the expression of repair signaling pathway proteins (p-ATM, p-ATR) by 70%.

Metformin injection increases ROS levels through a dual pathway of mitochondrial complex I inhibition and metabolic reprogramming:
Inhibition of complex I: Blocking electron transfer leads to electron leakage, which reacts with O ₂ to generate superoxide anions (O ₂⁻⁻), which are then converted to H ₂ O ₂ by SOD (superoxide dismutase).
Enhanced fatty acid oxidation: Acetyl CoA produced by β - oxidation enters the TCA cycle, promoting NADH production and further exacerbating complex I inhibition.
Glutathione (GSH) depletion: Metformin inhibits GCL (glutamate cysteine ligase) and GS (glutathione synthase), causing a 50% decrease in GSH levels and weakening cellular antioxidant capacity.

Direct DNA damage is caused by the Fenton reaction of H ₂ O ₂ to generate hydroxyl radicals (· OH), which directly attack the DNA sugar phosphate backbone and bases, leading to SSB and base oxidation damage. Each Gy of radiation can generate approximately 10 ⁵ · OH, and metformin pretreatment increases the amount of · OH generated by 2 times.

ROS regulates radiosensitivity through redox sensitive signaling molecules:
ASK1/JNK/c-Jun pathway activation: ROS oxidizes Cys250 of ASK1 (apoptotic signal regulated kinase 1), releasing its self inhibition, activating JNK (c-Jun N-terminal kinase) and c-Jun, promoting cell apoptosis.

NF - κ B inhibition: ROS oxidizes Cys179 of I κ B alpha (nuclear factor kappa B inhibitory protein alpha), promoting its degradation and releasing NF - κ B into the nucleus. However, metformin inhibits IKK (I κ B kinase) activity through AMPK, blocks NF - κ B nuclear translocation, and reduces the expression of anti apoptotic proteins such as Bcl-2 and XIAP.
Animal experiment: In the osteosarcoma U2OS cell transplantation tumor model, metformin combined with radiotherapy increased ROS levels in the tumor by three times and increased cell apoptosis rate by 50%.