Targeting the AMP-Activated Protein Kinase for Cancer Prevention and Therapy

Despite the advances in biomedical research and clinical applications, cancer remains a leading cause of death worldwide. Given the limitations of conventional chemotherapeutics, including serious toxicities and reduced quality of life for patients, the development of safe and efficacious alternatives with known mechanism of action is much needed. Prevention of cancer through dietary intervention may hold promise and has been investigated extensively in the recent years. AMP-activated protein kinase (AMPK) is an energy sensor that plays a key role in the regulation of protein and lipid metabolism in response to changes in fuel availability. When activated, AMPK promotes energy-producing catabolic pathways while inhibiting anabolic pathways, such as cell growth and proliferation – thereby antagonizing carcinogenesis. Other anti-cancer effects of AMPK may include promoting autophagy and DNA repair upon UVB damage. In the last decade, interest in AMPK has grown extensively as it emerged as an attractive target molecule for cancer prevention and treatment. Among the latest developments is the activation of AMPK by naturally occurring dietary constituents and plant products – termed phytochemicals. Owing to their efficacy and safety, phytochemicals are considered as an alternative to the conventional harmful chemotherapy. The rising popularity of using phytochemicals for cancer prevention and therapy is supported by a substantial progress in identifying the molecular pathways involved, including AMPK. In this article, we review the recent progress in this budding field that suggests AMPK as a new molecular target in the prevention and treatment of cancer by phytochemicals.

AMP-activated protein kinase is a well-conserved energy sensor that plays a key role in the regulation of protein and lipid metabolism in response to changes in fuel availability ( 9 – 11 ). AMPK exists as heterotrimeric complexes comprising a catalytic α-subunit and regulatory β- and γ-subunits. In mammals, each subunit occurs as multiple isoforms encoded by multiple genes that can be assembled to form up to 12 heterotrimeric combinations ( 12 ). Although AMPK is traditionally thought to play a major role in the regulation of cellular metabolism, it is now widely recognized to have antineoplastic efficacy and as a target for chemotherapy. A key characteristic of tumor cells is their ability to rapidly grow and divide, thus requiring a tremendous demand for energy. An extensive body of evidence has demonstrated that AMPK inhibits essentially all anabolic pathways that promote cell growth, such as synthesis of fatty acid, phospholipid, protein, and ribosomal RNA synthesis ( 12 , 13 ). Thus, it is not surprising that AMPK antagonizes cancer cell growth. The first hint that AMPK may be linked to cancer was provided by the finding that liver kinase B1 (LKB1), a known tumor suppressor, acted as an upstream kinase of AMPK ( 14 , 15 ). LKB1 is mutated in the inherited cancer disorder Peutz–Jeghers syndrome and in many lung and cervical cancers, suggesting that AMPK could play a role in tumor suppression ( 16 , 17 ). Activators of AMPK AMP-activated protein kinase can be activated by various types of metabolic stress that lead to ATP depletion, such as conditions of low nutrient supply or prolonged exercise, or via an increase in intracellular Ca 2+ concentration ( 9 ). The upstream kinases, LKB1 and calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) activate AMPK by phosphorylating Thr172 in the activation loop of the catalytic α-subunit ( 18 – 20 ). The finding that CaMKKβ can also activate AMPK, independently of LKB1, broadened the potential for AMPK to be used for therapy in cancers that have mutant LKB1 and thus low AMPK activation. Loss of function of the tumor suppressor LKB1 occurs in 30–50% of lung adenocarcinomas. Memmott et al. ( 21 ) found that lipid-based AKT inhibitors, phosphatidylinositol ether lipid analogs (PIA), activate AMPK independently of LKB1 in LKB1-mutant non-small cell lung cancer (NSCLC) cell lines. The more well-known activators of AMPK include several pharmacological agents that stimulate the LKB1 pathway. Metformin, the most widely prescribed Type-2 diabetes drug for more than 30 years, has been shown to activate AMPK in an LKB1-dependent manner ( 22 , 23 ). Metformin mimics an energetic stress because it inhibits the mitochondrial complex I in hepatocytes and cancer cells which leads to the decrease in intracellular ATP and an increase in glycolysis and lactate production ( 24 , 25 ). Consistent with this, diabetic patients treated with metformin had a lower incidence of cancer compared to those on other medications ( 26 ). In light of this, other retrospective studies have been performed, one of which showed that breast cancer patients on metformin for diabetes responded significantly better to chemotherapy than other diabetic patients not on metformin and non-diabetic patients ( 27 ). Since then, several studies have shown that metformin exerts antineoplastic effects in other cancer cells and animal models. Phenformin, a biguanide more potent than metformin, and A-769662, a direct AMPK activator developed by Abbott also delayed tumorigenesis in a mouse cancer model ( 28 ). 5-Amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide, or AICAR, is an analog of AMP and widely used to activate AMPK in experiments. Interestingly, AMPK is also activated by ionizing radiation (IR) in lung, prostate, and breast cancer cells, independent of LKB1. This suggests that AMPK may play a role as a target for radiosensitization of human cancer cells ( 29 ). Since the discovery of antineoplastic effects of AMPK, the number of patents describing potential AMPK activators has grown rapidly ( 30 , 31 ). Among the most recent developments is the activation of AMPK by naturally occurring dietary constituents and plant products, to be reviewed in the latter part of this article.

Since the AMPK cascade has emerged as an important pathway implicated in cancer control, many discoveries have been made in the past decade revealing robust anti-cancer effects of AMPK in vitro and in vivo , including animal cancer models of breast, lung, colorectum, skin, and hematological malignancies. We review the role of AMPK in major cancers, as outlined below. Breast cancer Observations that diabetics treated with biguanide drugs have a reduced risk of developing cancer have prompted an enthusiasm for these agents as anti-cancer therapies. Metformin, which activates AMPK, was shown to inhibit cell proliferation and induce apoptosis in the triple-negative breast cancer cell line, as well as the estrogen receptor (ER) α-positive and the human epidermal receptor (HER) 2-positive cell lines. Moreover, synergistic inhibition of the G1 phase of the cell cycle was demonstrated by the combination treatment of metformin and chemotherapeutic agents carboplatin, paclitaxel, and doxorubicin ( 44 ). Moreover, a novel small molecule AMPK activator, OSU-53 derived from inactive peroxisome proliferator-activated receptor gamma (PPARγ), was reported to inhibit the proliferation of the triple-negative breast cancer, a disease for which there are limited therapeutic options ( 45 ). Phenformin, a stronger biguanide than metformin and also a direct activator of AMPK, was demonstrated to be effective in the prevention and treatment of ER-positive and receptor triple-negative xenografts in immunocompromised mice ( 46 ). In another study, AICAR and phenformin elicited clear anti-proliferative effects in ER-positive, ER-negative, and triple-negative breast cancer cell lines ( 47 ).

AMP-activated protein kinase has been shown to inhibit cancer cell growth in various hematological cancers. In acute lymphoblastic leukemia (ALL) cell lines, AICAR induced dose- and time-dependent cell growth inhibition ( 53 ), leading to increased AKT phosphorylation and decreased mTOR phosphorylation ( 54 ). When used in combination with methotrexate or pemetrexed, AMPK showed a synergistic cytotoxic effect and cell growth inhibition ( 53 ). Sengupta et al. ( 55 ) reported that the apoptotic effect of AMPK was mediated by activation of the p38 MAPK pathway, increased expression of cell-cycle inhibitory proteins p27 and p53, and the downstream effects of the mTOR pathway. In B-cell chronic lymphocytic leukemia (B-CLL) cells, AMPK-induced apoptosis in a p53-independent manner ( 56 ). In mantle cell lymphoma (MCL), a clinically aggressive B-cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) and overexpression of cyclin D1, stimulation of the AMPK kinase activity using AICAR inhibited phosphorylation of critical downstream effectors of mTOR signaling, such as 4E-BP1 and ribosomal protein s6 (rps6) ( 57 ). In BCR-ABL-expressing chronic myeloid leukemia (CML) precursors and ALL cells that are positive for the Philadelphia chromosome (Ph+), metformin, and AICAR suppressed the mTOR pathway and cell growth ( 58 ). Lastly, induction of the LKB1/AMPK tumor suppressor pathway demonstrates a strong potential for the treatment of acute myeloid leukemia (AML). Green et al. showed that the LKB1/AMPK/TSC tumor suppressor axis could lead to a specific inhibition of the mammalian target of rapamycin (mTOR) catalytic activity, inducing 4E-BP1 dephosphorylation, which inhibits the initiation step of mRNA translation. Metformin consequently reduced the recruitment of mRNA molecules encoding oncogenic proteins to the polysomes, resulting in a strong anti-leukemic activity against primary AML cells while sparing normal hematopoiesis ex vivo and significantly reducing the growth of AML cells in nude mice ( 59 ).

It was not until about 5 years ago that AMPK began to be recognized as a target for various phytochemicals. In this section, we review the studies that have demonstrated the role of AMPK in the chemopreventive effects of phytochemicals (Table 1 ), the majority of which have been reported in the last few years. Table 1 Chemopreventive/chemotherapeutic phytochemicals that activate AMPK . Phytochemical Effect Reference CURCUMIN Curcumin (diferuloylmethane), from turmeric ( Curcuma longa L.) Activates AMPK to induce cell death in CaOV3 ovarian cancer cells in a p38 MAPK-dependent manner ( 67 ) Stimulates AMPK, resulting in downregulation of PPARγ in 3T3-L1 adipocytes and in COX-2 in MCF-7 breast cancer cells, inhibiting differentiation and growth ( 65 ) Inhibits mTOR, independent of AMPK ( 64 ) Downregulates COX-2 and pAKT in an AMPK-dependent manner, leading to apoptosis of H29 colon cancer cells ( 66 ) GRAPE POLYPHENOLS Resveratrol Induces apoptosis in chemoresistant HT-29 colon cancer cells via modulation of AMPK signaling pathway ( 74 ) Activates AMPK and suppresses LPS-induced NF-κB-dependent COX-2 activation in RAW 264.7 macrophage cells ( 75 ) Promotes autophagy-mediated cell death in chronic myelogenous leukemia cells in an AMPK-dependent manner ( 76 ) 3,4-DMS, a methylated resveratrol derivative, induced autophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway ( 80 ) Activates AMPK via SIRT1 in both ER-positive and ER-negative breast cancer cells, leading to inhibition of 4E-BP1 signaling and mRNA translation via mTOR ( 105 ) Enhances prostate cancer cell response to ionizing radiation by modulation of AMPK ( 78 ) Inhibits AKT/mTOR signaling via AMPK and potentiates the effects of gefitinib in breast cancer ( 77 ) Enhances anti-tumor effects of temozolomide in glioblastoma via ROS-dependent AMPK-TSC-mTOR signaling pathway ( 79 ) FLAVONOIDS Apigenin Induces AMPK and autophagy, inhibiting mTOR, and further inducing autophagy in both HaCaT cell line and primary normal human epidermal keratinocytes. This effect was independent of AKT and LKB1 but dependent on CaMMKβ ( 90 ) Anthocyanin Activates AMPK, leading to a reduction in mTOR phosphorylation and inhibition of HT-29 colon cancer cell growth ( 106 ) Fisetin Activates AMPK to induce apoptosis in multiple myeloma cells ( 85 ) Inhibits PI3K/Akt and mTOR and activates AMPK in non-small cell lung cancer ( 86 ) Induces autophagy-mediated cell death by suppressing mTOR in prostate cancer cells ( 87 ) Quercetin Induces apoptosis via AMPK activation and p53 in HT-29 colon cancer cells ( 88 ) Suppresses cell viability via AMPK-induced Hsp70 and EGFR downregulation ( 89 ) Baicalein Induces apoptosis and AMPK in human tumor cells ( 107 ) Luteolin Induces cell death in HepG2 cells and reduces tumor volume in a tumor xenograft model ( 91 ) Hispidulin Activates AMPK and inhibits downstream mTOR, which induces apoptosis in glioblastoma multiforme cells by p53 and p21 induction ( 108 ) Genistein Decreases reactive oxygen species levels and induces antioxidant enzymes manganese superoxide dismutase and catalase in a AMPK and PTEN-dependent manner in prostate cancer cells ( 82 ) Potentiates arsenic trioxide-induced apoptosis in human leukemia cells by activation of AMPK ( 83 ) Deguelin Activates AMPK and inhibits UVB-induced tumorigenesis in the SKh-1 hairless mouse model ( 92 ) Tephrosin (plant rotenoid) Enhances cytotoxicity of anti-cancer agent via ATP depletion and reducing autophagy by activation of AMPK and inactivation of mTOR expression ( 109 ) Chrysin Leads to cell growth inhibition and apoptosis in lung cancer cells via activation of AMPK and inhibition of AKT/mTOR ( 94 ) Celastrol Suppresses breast cancer MCF-7 cell viability via the AMP-activated protein kinase (AMPK)-induced p53-polo like kinase 2 (PLK-2) pathway ( 93 ) GREEN TEA POLYPHENOLS Epigallocatechin gallate (EGCG) EGCG analogs activate AMPK, leading in inhibition of cell proliferation, up-regulation of the cyclin-dependent kinase inhibitor p21, downregulation of the mTOR pathway, and suppression of stem cell population in human breast cancer cells ( 96 ) Enhances 5-fluorouracil-induced cell growth inhibition of hepatocellular carcinoma cells, associated with AMPK hyperactivation and COX-2 inhibition ( 98 ) Activates AMPK in the liver and prevents diethylnitrosamine-induced liver tumorigenesis in obese and diabetic mice ( 97 ) Induces apoptosis in HT-29 colon cancer cells via the AMPK/COX-2 pathway ( 95 ) Catechin Induces apoptosis in colon cancer cells by attenuation of H 2 O 2 -stimulated COX-2 expression via AMPK ( 110 ) OTHER CATEGORY p -HPEA-EDA, phenolic compound of virgin olive oil Activates AMPK to suppress COX-2 and inhibit cell survival in HT-29 colon cancer cells ( 102 ) 24-Hydroxyursolic acid from persimmon Activates AMPK and induces apoptosis in HT-29 colon cancer cells; Also block EGF-induced ERKs phosphorylation and inhibits AP-1

Flavonoids can be found in many different sources, including soy, berries, tea, wine, beer, chocolate, many vegetables, and most fruits. While there are several 1000 types, they can be categorized as flavones (quercetin, fisetin, luteolin), isoflavonoids (genistein, deguelin), and neoflavonoids ( 81 ). Epidemiological evidence reveals a lower incidence of prostate cancer in Asian countries, where soy products are more frequently consumed than in Western countries. The chemopreventive effects of a soy isoflavonoid genistein can be attributed to its ability to activate AMPK (Figure 4 ) and PTEN, leading to the induction of the antioxidant enzymes manganese superoxide dismutase and catalase ( 82 ). In another study, genistein was demonstrated to potentiate arsenic trioxide-induced apoptosis in human leukemia cells by activation of AMPK ( 83 ). Moreover, anthocyanin – belonging to the family of flavones that occurs in all tissues of higher plants, including leaves, stems, roots, flowers, and fruits – is shown to be a powerful activator of AMPK. Lee and Park ( 84 ) demonstrated that anthocyanin activates AMPK, leading to a reduction in mTOR phosphorylation and ultimately inhibiting cancer cell growth. Fisetin, a flavonoid, activates AMPK to induce apoptosis in multiple myeloma cells ( 85 ), inhibits PI3K/AKT and mTOR and activates AMPK in non-small cell lung cancer ( 86 ), and induces autophagy-mediated cell death by activating AMPK and suppressing mTOR in prostate cancer cells ( 87 ). Quercetin induces apoptosis via AMPK activation and p53 in HT-29 colon cancer cells ( 88 ) and suppresses cell viability via AMPK-induced Hsp70 and EGFR downregulation ( 89 ). Apigenin induces AMPK, inhibiting mTOR and further inducing autophagy in both HaCaT cell line and primary normal human epidermal keratinocytes. This effect was independent of AKT and LKB1 but dependent on CaMMKβ ( 90 ). Luteolin showed its anti-tumor effects in an in vivo tumor model, in which its activation of AMPK reduced tumor volume in a tumor xenograft model ( 91 ). Deguelin, a plant-derived rotenoid with cancer chemopreventive activity, was shown to inhibit UVB-induced skin carcinogenesis with the SKh-1 hairless mouse model. Topically applied deguelin significantly inhibited the multiplicity of UVB-induced skin tumors by activating AMPK ( 92 ). Celastrol, another antioxidant flavonoid, suppressed the viability of breast cancer MCF-7 cells in an AMPK-dependent fashion. Celastrol also induced an increase in ROS levels, leading to AMPK phosphorylation and increased the pro-apoptotic p53 in an AMPK-dependent manner ( 93 ). Chrysin, a naturally occurring flavone chemically extracted from the passion flowers Passiflora caerulea and Passiflora incarnata , leads to growth inhibition and apoptosis of lung cancer cells via AMPK activation and inhibition of AKT/mTOR ( 94 ). Thus, flavonoids and related compounds act as direct activators of AMPK and demonstrate their promising potential to be used as chemotherapeutic agents. Figure 4 Schematic representation of AMPK-dependent anti-cancer effects of flavonoids and related compounds . This figure shows the major flavonoids and their AMPK-dependent effects on inhibition of cancer growth. Apigenin and anthocyanin activates AMPK, which inhibits mTOR signaling, leading to apoptosis of GBM, multiple myeloma (MM), and non-small cell lung cancer (NSCLC) cells. Activation of AMPK by fisetin and hispidulin also inhibits mTOR, resulting in autophagy-dependent cancer cell death and decreased growth of colon cancer cells, respectively. Quercetin inhibits survival of colon cancer cells by downregulation of epidermal growth factor receptor (EGFR) signaling and heat shock protein (hsp)70 expression. Activation of AMPK deguelin leads to UVB-induced tumorigenesis in a non-melanoma skin cancer mouse model. See text for effects of other flavonoids and related compounds.

Berberine, a traditional plant alkaloid used in Ayurvedic and Chinese medicine for its antimicrobial and antiprotozoal properties, strongly increased AMPK phosphorylation via ROS production, leading to inhibition of tumor cell adhesion, tumor invasion, and the expression of epithelial to mesenchymal transition (EMT)-related genes. Furthermore, berberine inhibited the metastatic potential of melanoma cells through a decrease in ERK activity and protein levels of cyclooxygenase-2 (COX-2) by a berberine-induced AMPK activation ( 99 ). In another study, berberine was shown to inhibit migration of colon cancer cells in an AMPK-dependent manner ( 100 ). Furthermore, 24-hydroxyursolic acid from persimmon, capsaicin, and p-HPEA-EDA, a phenolic compound of virgin olive oil, activate AMPK and inhibit cell survival in HT-29 colon cancer cells ( 100 – 103 ).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.