Repurposing Metformin for Lung Cancer Management

2.1.1. Metformin corrects hyperglycemia

Metformin can accumulate within the matrix of mitochondria, and it could exert the inhibition of the complex I of the mitochondrial electron transport chain. Further, reduction of nicotinamide adenine dinucleotide hydride (NADH) oxidation can also cause the reduction of synthesis of adenosine triphosphate (ATP). After the activation of AMP-activated protein kinase (AMPK), it enhances catabolic activity instead of the anabolic process. It initiates transcriptional signal transduction, inhibition of gluconeogenesis, and induction of glucose uptake into muscle cells via glucose transporters (GLUT2) [12]. Moreover, metformin can indirectly cause the induction of insulin receptor expression to facilitate insulin sensitivity and reduce insulin resistance, which is associated with tumor growth [13].

2.1.2. Metformin upregulates AMPK

Metformin can activate AMPK to initiate the downstream signal transduction to affect the transcription of tumor suppressor liver kinase B1 (LKB1) [14]. Once AMPK is activated, it negatively regulates the mTOR pathway by phosphorylation and activation of tuberous sclerosis complex 2 (TSC2) and inhibition of downstream small GTPase (RHEB). The mTOR pathway is crucial for tumor cell survival because mTOR plays a vital role in cell growth, proliferation and protein synthesis. Moreover, the mTOR pathway could be activated via mitogenic responsive phosphoinositide 3-kinase/protein kinase B/AKT (PI3K/PKB/AKT) pathway. When metformin-related AMPK dependent pathway is affected, the inhibition of mTOR signal transduction and reduction of cancer cell proliferation are achieved [15].

2.1.3. AMPK and p53 pathways

It is known that p53 can activate numerous genes to negatively regulate the AKT and mTORC1 pathways, resulting in cancer cell quiescence, senescence and further apoptosis. Thus, once AMPK phosphorylates p53, it could lead to p53-mediated cell cycle arrest in p53-expressing cells and cell apoptosis for cells with mutated p53 [11].

2.1.4. Inflammatory pathway and metformin

For tumorigenesis, chronic inflammation is attributed to tumor growth and development. Once the inflammatory process is stimulated, it causes DNA adduct formation and increases the amount of inflammation biomarkers (such as cytokine/chemokines, immune-related effectors, acute phase proteins, reactive oxygen and nitrogen species, prostaglandins, cyclooxygenase-related factors and transcription factors and growth factors) [16]. Like tumor necrosis factor-alpha (TNF-α), nuclear factor-kappa B (NF-kB) and signal transducer and activator of transcription 3 (STAT3) are important components of the inflammation reaction. Arai et al. elucidated that metformin reduces the process and production of TNF-α in human monocytes [17].

Reactive oxygen species (ROS) play a vital role in the formation of advanced glycation end products to enrich oxidative stress. Metformin can reduce the production of endogenous reactive oxygen species via inhibition of mitochondrial complex I [18].

2.1.5. Cell cycle pathway and metformin

Sahra et al. found that metformin has an antiproliferation effect, which is mediated by G1 cell cycle arrest. In a study with prostate cancer cells, metformin-induced cell cycle arrests by inhibiting the expression of cyclin D1 and retinoblastoma-protein (pRb) [19].

2.1.6. Angiogenesis and metformin

For tumor cells growth, vast amounts of nutrition and oxygen are needed. As the tumor enlarges and begins to invade and cause distant metastasis, angiogenesis is the cornerstone. Tumor cells easily develop pro-angiogenic agents once exposed to a hypoxic environment [8]. The vascular endothelial growth factor (VEGF) formation is the key step. The VEGF group is consisted of four members (VEGF-A, B, C and D), and VEGF-A is the most potent. VEGF-A has four isoforms: VEGF-A₁₂₁, VEGF-A₁₆₅, VEGF-A₁₈₉ and VEGF-A₂₀₆. VEGF-A₁₆₅ functions in both the angiogenic process and cell growth. Moreover, the angiogenesis process needs a cofactor (neuropilin; NRP-1) to facilitate the VEGF ligand interaction with the VEGF receptor (VEGF-R2) [16,20]. In our previous lung cancer cell line (A549) experiments, the expression of NRP-1 decreased after the addition of metformin.

2.1.7. Models of metformin using different cell lines

In the beginning, anticancer studies were started from cell lines and animal models (commonly using a xenograft model). There are a vast number of antitumor studies with metformin on ovarian cancer, gastric cancer, pancreatic cancer and breast cancer. Some emphasize cell cycle-related proteins (CD1, CDK4 and CDK6) and some focus on microRNA (miR) regulation and signal transduction [21–24]. Moreover, cancer stem cells (CSC) were also found to be involved in the antitumor effect of metformin in both cell lines and xenograft models. The dosage and administration route of metformin were diverse. We provided a brief summary of previous findings of the antitumor effects of metformin on different cell lines in Table 1 [21, 22, 24, 25].

2.2.1. Preclinical studies

2.2.2. Observational studies

3.1.1. Background

When a tumor develops, it demands a vast amount of oxygen and nutrition for tumor growth. When a tumor is small in size, approximately 10⁵ to 10⁶ tumor cells, it depends on the diffusion effect for nutrition transport. As the tumor enlarges, it must overcome hypoxia and develop an angiogenic switch, including proangiogenic and angiogenic factors. Once vasculogenesis and angiogenesis are established, the tumor can invade extensively and cause distant metastasis [36]. Based on this theory, we demonstrate lung tumor formation via the angiogenesis model.

3.1.2. Materials and methods, see Ref. [37]

1. Create transgenic mice carrying the mccsp-Vegf-A₁₆₅-sv40 transgenic fusion gene (Figure 1A)

   • A 1975-bp mccsp-Vegf-A₁₆₅–sv40 transgene was directly microinjected into pronuclear stage FVB mouse embryos and then transferred into the fallopian tube of the recipient females mice.

   • Transgenic mice were mated with littermates or normal FVB mice to produce offspring.

   • The resulting 12-month transgenic offspring were the candidates for performing the lung cancer model.

2. Gross picture of homozygous transgenic mice compared with wild-type mice (Figure 1B)

   • Illustration of the whole picture of lung tissues from transgenic mice (homozygous) and wild type mice. We can observe the mass with a bulging appearance of lung tissue, with tumor formation in the transgenic mice (Figure 1B, upper panel).

3. Histopathologic analysis of the lung tissue in transgenic mice and wild-type mice (Figure 1B)

   • From the lower part of Figure 1B, we can easily find the lung tumor formation with bizarre cell shapes and increased nucleus/cytoplasm (N/C) ratios in the lung tissues of transgenic mice. The wild type mice showed no tumor-specific appearance.

4. Validation of hVEGF-A₁₆₅protein expression of transgenic mice was performed by western blot analysis

   • In the lung tissue of 12-month transgenic mice, the western blot data proved, there were more than 5-fold higher levels of VEGF expression compared with wild type mice (Figure 1C).

3.2.1. Background

In cancer research, it is important to perform in vivo animal experiments to further mimic the effects on human beings. During the study period, it is necessary to record images simultaneously. Real time imaging depends on the in vivo image system (IVIS). The creation of good IVIS images is necessary and demands comprehensive consideration. The property of good IVIS images requires an optimal imaging window. For the mammalian tissue study, obtaining deep optical images requires near-infrared (NIR) fluorescent probes. The NIR optical window is around from 650 to 900 nm. Under this optical window, mammalian tissue is considered more transparent to light due to the limited combined absorption of water, melanin and hemoglobin. Moreover, under this spectral region, it could eliminate autofluorescence and have low light scattering [38]. The common used near-infrared fluorescent proteins (iRFP) included iRFP670, iRFP682, iRFP702 and iRFP720 [39]. Based on this concept, we designed a near-infrared fluorescent mice tumor model to further evaluate the IVIS expression during lung tumor formation.

3.2.2. Material and methods, see Ref. [40]

3.2.2.1. Construction of dual fluorescence-expression vector

1. The construction map of the pCAG-iRFP-2A-venus transgene is shown (Figure 2A).

2. Transfection of lung adenocarcinoma cell line (A549) with pCAG-iRFP-2A-venus expression was performed. The iRFP can show red fluorescence. Venus can show green fluorescence. And DAPI (4’,6-diamidino-2-phenylindole) emits blue fluorescence in cell nuclei as a background control (Figure 2B).