Introduction
Mitochondria are cellular organelles that are present in the majority of eukaryotic cells. They play an important role in energy production, metabolism, and cell signaling. Eukaryotic mitochondria have their own DNA, which is referred to as mitochondrial DNA (mtDNA), and it encodes various critical proteins involved in oxidative phosphorylation and the synthesis of adenosine triphosphate (ATP).1 mtDNA is a tiny, circular, double-stranded genome, measuring about 16.6 kbp in humans, and is maternally inherited,2 passed from mother to child. This is due to the fact that the egg cell supplies the growing embryo with the vast majority of its cytoplasm, including its mitochondrion.
The mtDNA is partitioned into two distinct strands, namely the heavy (H) strand and the light (L) strand, which may be differentiated based on their nucleotide content. The H-strand has higher guanine content, while the L-strand shows a higher cytosine content. mtDNA is structured into three regions: the coding region, the D-loop region, and the non-coding region. The coding region contains 37 genes responsible for encoding proteins, transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs), crucial for mitochondrial protein translation. These proteins derived from mtDNA are essential for the electron transport chain (ETC), where they regulate the oxidative respiratory chain and preserve its functional integrity. The D-loop region serves as the regulatory area of mtDNA, hosting replication origins and transcription promoters. The non-coding region, between the coding and D-loop regions, contains regulatory elements important for the replication and transcription of mtDNA.3 mtDNA is a kind of DNA that exists in multiple copies within cells. Their number of copies may range from 100 to 10,000, and this variation is influenced by cellular energy needs governed by mitochondrial biogenesis.4 The process encompasses mitochondria proliferation which includes the development of inner and outer mitochondrial membranes, the initiation and movement of proteins encoded by nuclear genes, and the replication of mtDNA. The whole process is governed by several components, particularly peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factors (NRFs), and mitochondrial transcription factor A signaling pathways.5 Mitochondrial transcription factor A, a member of the high mobility group box subfamily, is the principal nuclear protein that interacts with the promoter region of mitochondria and regulates the replication of mtDNA.6 PGC-1α, a member of the transcriptional cofactor family, operates as a facilitator of mitochondrial biogenesis, exerting a positive regulatory effect. NRF-1/2 continues to function as a transcription factor downstream of PGC-1α, regulating mitochondrial proteins encoded by mtDNA. Several crucial upstream regulators, such as AMPK (AMP-activated protein kinase), and downstream effectors including sirtuins (SIRT), NRF-1/2, mTOR, and HIF-1α, have been shown to activate PGC-1α, promoting mitochondrial biogenesis.5,7,8 AMPK is responsible for keeping a check on mitochondrial homeostasis by direct phosphorylation of the mitochondrial fission factor, controlling mitochondrial fission through dynamin-like protein-1.9 AMPK is considered a tumor suppressor by negatively regulating aerobic glycolysis, known as the Warburg effect, in cancer cells, ultimately suppressing tumor growth.10 Given the pivotal role of mitochondrial damage in the initiation and progression of many diseases, comprehensive knowledge of the underlying processes and regulatory signals involved in biogenesis is essential. Therefore, this review will concentrate on the significance of mtDNA in the context of liver cancer. It will also summarize how mtDNA genes, proteins, and microRNA (miRNA) are implicated in mtDNA dysfunction. Additionally, the study will discuss existing research on phytochemicals and chemicals targeting mitochondrial biogenesis and mtDNA disruption in liver cancer.
mtDNA in disease pathology
mtDNA is a significant part of cellular bioenergy synthesis and metabolic reactions, and thus alterations in mtDNA have been associated with various diseases, particularly mitochondrial diseases. Mitochondrial diseases include a collection of disorders resulting from genetic abnormalities in either mtDNA or nuclear DNA, which subsequently impair mitochondrial functionality. These disorders are characterized by persistent depletion of cellular energy, leading to noticeable disease traits due to the inability to fulfill cellular energy requirements. The clinical range of mitochondrial disease is rather wide, often involving tissues with higher metabolic requirements, such as the central nervous system and cardiac tissue.11 Moreover, empirical evidence has shown that alterations in mtDNA have a significant role in the pathogenesis of several diseases, including neurological disorders, diabetes, and the aging process.12,13 mtDNA alterations have been extensively involved in the etiology of Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.14 These mutations can affect mitochondrial function, leading to increased oxidative stress, which contributes to the development of these disorders. Besides, these alterations can significantly impact cellular functioning and downstream events too. The comprehension of the involvement of mtDNA in the pathophysiology of diseases has significant importance in the advancement of focused therapeutic approaches for various ailments. Further, it has been shown that modifications in mtDNA have a significant impact on the proliferation, advancement, and metastasis of cancer cells, as well as their interactions with the immune system and the tumor microenvironment (TME).15 Mutations in mtDNA have been observed in various cancer types, including breast, ovarian, colon, and rectum cancer, affecting the functionality of the ETC and leading to oxidative phosphorylation dysfunction and increased reactive oxygen species (ROS) production, promoting cancer development. Moreover, it has been observed that mutations in mtDNA contribute to the development of several types of cancer in different organs, including the lungs, stomach, and liver. In addition to mutations, changes in mtDNA copies are linked to the cancer occurrence and unfavorable prognosis across diverse cancer types, such as lung, ovarian, and breast cancer. Additionally, mtDNA damage and apoptosis are interconnected processes (Fig. 1). Apoptotic factor APAF-1 can inhibit anti-apoptotic proteins, allowing pro-apoptotic factors to cause membrane permeabilization in the outer mitochondrial membrane, leading to cytosolic release of oxidized mtDNA. This exudence of distorted mtDNA can trigger various receptors, including Toll-like receptors and inflammasomes.15 mtDNA defects can also result in reduced levels of respiratory complexes along with ATP content, which leads to mitigation of cell growth and induction of apoptotic activities.16 Hence mtDNA defects contribute to mitochondrial deregulation, leading to increased inflammation, apoptosis, and ultimately, cell death.17
mtDNA alteration in liver cancer
Hepatic cell carcinoma (HCC), also known as liver cancer, ranks as the fifth most prevalent form of cancer on a global scale.18 Furthermore, it stands as the fourth leading cause of cancer-related mortality.19 The etiology and pathogenesis of liver cancer are complex, involving multiple genetic and environmental factors. The prognosis for liver cancer is characterized by a lower survival rate in cases of advanced-stage illness, owing to the poor prediction of liver cancer propagation.20 Hence, it is essential to prioritize the development of novel and efficacious therapeutic strategies for the treatment of liver cancer. HCC, the predominant type of principal liver cancer, often exhibits abnormalities in mtDNA. The two most prevalent forms of mtDNA abnormalities seen in HCC are somatic point mutations and depletions.21 Previous studies have shown a notable association between decreased mtDNA quantity in HCC and adverse clinical factors, including increased tumor dimensions, liver cirrhosis, and reduced 5-year survival rate.22 Furthermore, numerous experimental and clinical investigations have shown a significant correlation between mtDNA and HCC. The precise mechanism behind the contribution of mtDNA alterations and mitochondrial deregulation to the advancement of HCC remains incompletely elucidated. Hypermethylated mtDNA is reported to deregulate mitochondrial genes and metabolism in liver cancer cell lines.23 Frequent occurrences of mutations in the D-loop regulatory region of mtDNA have been observed in HCC patients.24 Furthermore, the presence of circulating mtDNA in the bloodstream is related to the disease.25 These alterations may impact the expression and functionality of mitochondrial proteins that are implicated in oxidative phosphorylation and ATP production.22,26 Consequently, this can result in metabolic reprogramming and changes in energy metabolism within cancerous cells.
Furthermore, mitochondrial dysfunction resulting from mtDNA mutations may contribute to DNA damage, genomic instability, and altered signaling pathways that promote cancer cell survival and proliferation. Moreover, mitochondrial dysfunction may also affect the immune response and contribute to tumor immune evasion.27 Nevertheless, a growing body of evidence suggests that the unintended release of mtDNA into the cytoplasm serves as a damage-associated molecular pattern, activating the innate immune response and eliciting an inflammatory reaction.28 The innate immune system functions as the first barrier against infections, effectively identifying and removing them, playing a crucial role in preserving internal equilibrium and being strongly associated with the initiation of inflammation. The potential activation of self-mediated innate immune responses cannot be disregarded when considering the release of mtDNA by liver cells, which have the largest mitochondrial content in the human body, under pathological situations.28
Additionally, AMPK is reported to promote innate immunity and antiviral defense by modulating the signaling pathway of Stimulator of Interferon Genes (STING).29 Emerging research has shown that the use of STING pathway agonists for regulatory purposes might potentially counteract the progression of HCC and serve as a viable therapeutic approach.30 Undoubtedly, the cGAS-STING pathway plays a crucial role in the evolution of HCC. A publication highlights an association between diminished levels of STING in tumor tissues and worse prognosis among patients with HCC.31 Moreover, the re-establishment of IFI16 expression in neoplastic cells significantly facilitates tumor regression, although this process may be somewhat hindered by the suppression of p53 signaling activation or the induction of the inflammasome.32 Along with this, the Toll-like receptor 9 has received significant attention in the field of liver diseases, with several studies dedicated to its investigation. Hypoxia-induced translocation of mtDNA and high mobility group box 1 into the cytoplasm of cancer cells activates Toll-like receptor 9, promoting tumor cell proliferation.33 AMPK upregulation was seen as involved in increasing the drug sensitization in HCC.34 Its activation was observed as a result of the knockdown of a critical enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, in HCC.35 In general, the involvement of mtDNA modifications in HCC is intricate and diverse, necessitating further investigation to comprehensively comprehend the fundamental processes and establish efficacious treatment approaches focusing on liver cancer’s mitochondrial function.
mtDNA genes and their implications in liver cancer
Mitochondria are considered the powerhouse of cells and are involved in various metabolic and cell survival activities, as they possess their own genome. This genome contains 13 polypeptides associated with the ETC, along with 22 tRNA genes and 2 rRNA genes, facilitating protein synthesis inside the mitochondria. The remaining protein subunits participate in the ETC complexes. A study was conducted on a significant liver cancer C57BL/6J mice strain model to access the whole genomic sequence. The overall length of the mitogenome was determined to be 16,308 bp, bearing protein-coding genes (13), rRNA genes (2), tRNA genes (22), and the D-loop region (1). Additionally, evidence of mutations was documented.36 Similar sequencing details were also reported by Zhang et al.37
The ATP synthase enzyme complex plays a pivotal role in the synthesis of ATP, which serves as the principal form of cellular energy. Situated inside the internal mitochondrial membrane, the ATP synthase enzyme plays a crucial role in the ultimate stage of oxidative phosphorylation, converting glucose and other nutrients into ATP, the primary energy currency of the cell.38 Recent scientific research has revealed that ATP synthase has a significant role in cancer development and progression. Overexpression of ATP synthase has been associated with increased cancer cell growth, propagation, metastasis, and resistance to chemotherapy. Furthermore, ATP synthase is involved in modulating the TME. The involvement of ATP synthase in the control of the pH of the TME has been determined, which potentially influences the growth and viability of cancerous cells. Additionally, ATP synthase has been implicated in modulating immune response inside the TME has also been shown. Cytochrome c oxidase (COX) serves as a crucial component of the mitochondrial ETC, playing a pivotal role in ATP synthesis and the formation of ROS.39 During the process of apoptosis, it oxidizes cytochrome c for its cytosolic release, and thus its dysfunction may impact the apoptotic process. NADH dehydrogenase, referred to as complex I, is an essential component of the mitochondrial ETC. The process under consideration is accountable for transferring electrons from NADH to ubiquinone. This electron transport results in the creation of a proton gradient along the inner mitochondrial membrane. This proton gradient is responsible for facilitating ATP synthesis. Additionally, this process has been linked to the formation of hepatic malignancies.40 The mitochondrial ribosome consists of two distinct subunits, the large subunit (mtLSU) and the small subunit (mtSSU), composed of rRNA and protein components. Dysregulation of mitochondrial ribosome biogenesis and function has been observed in the pathogenesis and advancement of liver cancer.41 Mitochondrial transfer RNA (mt-tRNA) is responsible for delivering amino acids to the ribosome during protein synthesis,42 and mutations in mt-tRNA are linked to diverse mitochondrial diseases, including certain forms of liver disease.
ATP synthase
The administration of Etomoxir resulted in an elevated oxidative state and impaired mitochondrial metabolic system, as evidenced by a notable reduction in reduced glutathione, the reduced/oxidized glutathione ratio, mitochondrial membrane potential, and ATP levels. Additionally, there was a concurrent increase in oxidized glutathione and superoxide generation.43 The role of ATP synthase in cancer development was also substantiated by preparing an antisense of hAS-e and exploring its potential to inhibit cell proliferation via the MAPK pathway.44 Chrysophanol-induced necrotic cellular death was associated with depletion in ATP levels, resulting in a reduction of energy production.45 The synthetic metal complexes (1 and 2) derived from α-N-heterocyclic thiosemicarbazones were shown to cause mitochondrial damage, ROS generation, a decrease in ATP content, and mitochondrial membrane depolarization.46
COX
The role of COX has been explored from a very early time. Long back in 1990, thyroid hormone and/or dexamethasone were given for the treatment of hepatoma. This experimentation led to an estimated 4-fold upregulation of various RNAs ciphered in the mitochondrial genomic sequence, which includes subunit II of COX. The study inferred the potent role of modification in transcription and RNA stability in elevated mitochondrial genomic expression.47 Then, in 1998, hepatitis B virus (HBV) infection and its role in altering cellular metabolic energy, resulting in mitochondrial dysfunction were associated with the ATP synthase 6 and COX III genes.48 A study conducted on HL-7702 cells displayed that co-localization of HBx and COX III resulted in an increase in mitochondrial function as well as ROS production, which was further linked to hepatic cell carcinogenesis associated with HBV.49 Single nucleotide polymorphisms (SNPs) aggregated mtDNA is disposed to cancer development. Wang et al.50 have studied SNPs in the COX genes of the mtDNA coding region in hepatic cancer patients and healthy controls. The 9545G allele was observed to be a risk for malignant conditions. Recently, Zhao and his co-workers have investigated the amplified expression of several genes, including from the COX family (mt-CO1) in HCC. A positive correlation between elevated gene expression and poor survival rates was observed.51 A 9-base pair deletion polymorphism in mitochondrial CoII/tRNA was studied to have pathogenic activities in hepatic carcinoma.52
mt-rRNA
Polymorphism in 12S rRNA (mt-RNR1) G709A has been considered to have an impact on metastasis-free survival and general survival. A study was conducted on a patient suffering from cirrhosis in HBV-related hepatic carcinoma, where a direct association between hexokinase 2 expression and poor diagnosis in liver cancer patients was observed.53
mt-tRNA
Nucleotide sequencing of mt-tRNAAsp in Morris hepatoma-5123D was explored in 1981, revealing many abnormal characteristics accompanying the normal features when compared to healthy controls. Lacking GC loop adjoining to loop-IV was observed. Further, several loci such as 4, 16, 23, and 24 were totally different in the hepatoma model.54 Several insertions (AH-130 tRNACys) and deletions (YS tRNATyr, AH-7974-tRNATrp) in tRNA mutations have already been documented in the past when compared to tumor and healthy rat models (Yoshida Sarcoma’s, Ascites hepatoma; AH-7974 & AH-130).55 Alterations in mt-tRNA-modifying enzymes have been reported to be connected with certain disorders such as MTU1 in cases of acute infantile liver failure.56 Very recently, Zhao and his co-workers in 2023 have conducted a study to unravel the role of tRNA methyltransferase 5 in liver cancer progression. It was observed that mitigating tRNA methyltransferase 5 has a suppressive effect on the hypoxia-inducible factor-1 (HIF-1) signaling pathway by amplifying the oxygen quantity of cells, as well as decreasing doxorubicin resistance in liver cells.57
Role of miRNA in the functional activity of mitochondrial proteins
miRNAs are small non-coding RNAs with a length of about 19–25 nucleotides and are involved in many physiological and pathological cellular processes. They can act as both oncogenic and tumor suppressor miRNAs, controlling the proliferation, dissemination, and propagation of cancer cells (Table 1).58–66
Table 1Effect of microRNAs upon various genes/proteins in hepatic cell carcinoma
| MicroRNA | Targeted sites | Effect | Model used for the study | Reference |
|---|
| MiR-34a, miR-26a & miR-145 upregulation | Sirtuins | p53 – mediated suppression of SIRT1, 2, 6, & 7 after transcription; PGC-1α/β mediated regulation of SIRT3, 4 & 5 while transcription | Livers of telomerase knockout male G4 mice (mice between the age of 8 and 16 weeks | 58 |
| MiR-181a-5p | Mitochondrial DNA genes of the COX family (mt-CYB & mt-CO2) | MMP decreased, increased expressions of HK-2 & GLUT-1 enhanced release of glucose and lactic acid, Lactate dehydrogenase upregulated | SMMC-7721, HepG2 cells, 4-week-old nude mice | 59 |
| MiR-199a-5p downregulation | ANRIL (A long non-coding RNA) served as a competitive endogenous RNA that stimulated the production of ARL2 through miR-199a-5p/ARL2 axis. | Increased mtDNA copy numbers, as well as ATP synthesis | Huh7, SMMC7721, HepG2, Hep3B cells; LO2 (Normal human hepatic cells) | 60 |
| MiR-329-3p upregulation | Lysine (K)-specific demethylase 1A | Demethylation of myocyte-specific enhancer factor-2, activation of Programmed Death Ligand-1 expression | HepG2, SMMC7721; H22 (murine cancer); LO2 (Normal Human Hepatic cells) | 61 |
| MiR-342-3p upregulation | MCT-1 | Decrease in cell proliferation, migration, colony formation | LT2/MYC animal tumor model | 62 |
| MiR-214 upregulation | Wnt2a | Decrease in cell proliferation | SMMC-7721, HepG2, Hep3B; HL-7702 (Normal Human Hepatic cells) | 63 |
| MiR-552-5p downregulation | MiR-552-5p interaction to 3′ UTR of ACSL4 mRNA | Glutathione quantity was reduced, intracellular Fe2+ amount was enhanced, increased ACSL4 expression, overexpression of ZNF8, and increased ferroptosis | Huh-7, Hep3B cells | 64 |
| Mir-122 upregulation | Decrease in PKM2 expression | Glycolytic activity suppression, reduction in lactate generation, and elevated oxygen consumption | HepG2, Hep3B, Huh-7, MHCC97L, MHCC97H, Hepatic cancer cells from primary tumor (H2P) and its matched metastasis (H2M) | 65 |
| Mir-520 downregulation | PFKP regulation | TAT-activated regulatory DNA-binding protein repression impaired glycolysis | Female athymic nude mice | 66 |
Natural products targeting mitochondrial proteins in liver cancer
The promising prospective of plant-based natural products to be explored as potent novel anticancer agents has always been an area of interest for researchers.67,68 Phytochemicals specifically targeting mitochondrial proteins included in mitochondrial biogenesis and mutation have been summarized below. A schematic illustration of targeted proteins has been depicted in Figure 2.
Curcumin, a natural polyphenol from Curcuma longa,69 exerted mitochondrial as well as nuclear DNA damage in human liver carcinoma (HepG2) cells in a dose-dependent mode.70 Curcumin also demonstrated an inhibitory effect on hepatic cancer cells through the mitigation of ATP synthase activity.71 Resveratrol, a well-known and extensively studied polyphenol, demonstrated the induction of apoptosis in HepG2 cells via upregulating phospho-AMPK expressions while simultaneously downregulating surviving signaling.72 Resveratrol also demonstrated tumor suppressor liver kinase B1 mediated SIRT1 phosphorylation and activation.73 A combinatorial exposure of resveratrol and sorafenib exerted a significant antiproliferative effect in mice-bearing HepG2 xenograft. The pathway involved was PKA/AMPK/eEF2K in HepG2 and Huh7 cells.74 Likewise, Xu et al.75 have studied recently the synergistic efficacy of resveratrol along with fibroblast growth factor 1 in hepatic cancer cells. An AMPK/NRF2-mediated noticeable repression of oxidative stress and liver dysfunction was observed in both in vitro and in vivo models.75 PLGA nanoparticles of resveratrol were also studied and observed to have an enhanced activity profile compared to their native form. They were found to alleviate lipid formation and reduce cancer cells more efficiently.76
Berberine, isolated from the rhizome of Rhizoma coptidis, is known to induce up-regulation of p38 MAPK signaling in both hepatic cancer cells (HepG2, MHCC97-L) via stimulation of the AKT/Beclin-1/mTOR-signaling transduction pathway.77 Further, berberine and rotenone were explored for their effect on mitochondrial pathways in C57BL/6J male rats as well as primary hepatocytes isolated from rats. It was observed that berberine has a repressive effect on mitochondrial swelling and mitochondrial complex I. Additionally, it was found to mitigate ATP and citrate synthesis. Furthermore, mtDNA copy numbers were also found to be reduced in hepatic cancer cells.78 Higher expressions of AMPK by downregulating mTOR 4E-binding protein-1 along with repressing mRNA translation in p53-negative Hep 3B cells were observed by exposing the cells to Epigallocatechin gallate, a major catechin in green tea.79 Epigallocatechin gallate treatment given to both male and female BALB/c nude mice injected with doxorubicin-resistant BEL-7404 cells also resulted in a subsequent decrease in cellular growth and proliferation with a simultaneous decrease in drug efflux and an increase in doxorubicin sensitivity in cells.80 AMPK activation is reported to repress mTOR by suppressing HIF-1α expressions, which is one of the key regulators of glycolysis in the cells.81 Quercetin (a promising flavonoid) and gemcitabine/doxorubicin combination, in doxorubicin-resistant BEL-7404 cells, demonstrated an increase in sensitization of cells to the drug. There was downregulation of drug-resistant proteins and HIF-1α, with an increase in apoptotic activities.82 Restoration of SIRT6 followed by repression of Frizzled-4 and H3K9AC proteins were observed in hepatic cancer cell lines (HepG2, HuH-6, and HepT1) by treating them with quercetin.83 Kaempferol, a known flavonol, was studied in SK-HEP-1 (human liver carcinoma cells) and found to cause G2/M repression and a decrease in expressions of CDK1/cyclin B via the AKT/AMPK signaling pathway.84,85 The antioxidant potential of kaempferol was also explored in HepG2 cells to find out that it could amplify the NRF2 expression and subsequently stimulate its target genes (SOD1 and GPX3).86 Apigenin, when given together with the known anticancer drug paclitaxel to HepG2 cells, exerted enhanced anticancer profiling of paclitaxel via reducing HIF-1α and phosphorylated as well as whole AKT.87 AMPK activation and repression of downstream mTOR/NF-κBp65 interaction were also observed with Naringenin treatment of fatty acids-induced damages in HepG2 cells.88 A distinctive anthocyanin pigment, Cyanidin-3-O-β-glucoside, was examined and observed to exert its significant effects on AMPK activation and fatty acid oxidation in HepG2 cells. Further, an increase in CPT-1 expression was also observed.89 Anthracenone T-514, abundantly available in Karwinski aparvifolia, demonstrated mitochondrial alterations prior to apoptosis. This was substantiated by increased expressions of cytochrome oxidase, p53, and decreased PCNA levels. Simultaneously, proapoptotic biomarkers were upregulated with the downregulation of anti-apoptotic Bcl-2 proteins.90 Cryptotanshinone, a diterpene extracted from Salvia miltiorrhiza Bunge, demonstrated an activated pAMPK/SIRT1/NRF2 pathway in HepG2 and AML-12 cells.91 AMPK activation and inhibition of HCC in vitro and in C57BL/6 mice were observed with dietary intake of Genistein.92 Carnosol, a phenolic diterpene, inhibited the proliferation, propagation, and metastasis of HepG2 and Huh7 cell lines by activating the AMPK-p53 pathway.93
Miscellaneous
A PLGA nanoformulation named PPCu, comprising Cu12Sb4S13 in hyperthermic conditions, was observed to induce apoptosis by downregulating mt-CO1 through the RAS/MAPK/MT-CO1 signaling pathway, eventually resulting in mitochondrial deregulation and consequent apoptosis.94 Metformin, working as an adjuvant to sorafenib, helped repress sorafenib resistance in Huh7 and Hep3B hepatic cancer cell lines via AMPK/CEBPD-mediated autophagy.95 Very recently, metformin was investigated to affect deregulated glycolysis, ROS generation, and apoptotic activities via AMPK/p38MAPK upregulation.96 Diallyl trisulfide, a powerful antioxidant, displayed activation of the AMPK/SIRT1 signaling pathway in the HepG2 cell line.97 A combination of Empagliflozin with metformin was investigated in male albino mice, and an AMPK-dependent inhibition of NFкβ, p38, and ERK1/2 proteins was observed.98 An antiplatelet drug cilostazol inhibited hepatic cancer cell proliferation via AMPK and AKT/ERK signaling.99 Propofol inhibited HepG2 proliferation by activating the AMPK signaling.100