Targeting Warburg effect: involvement of lactate transporter MCT1 and its chaperone in cancer cell killing by 18β-glycyrrhetinic acid.

Studies of lactate metabolism in vivo in humans and rodents have shown that lactate is not only an end product of glycolysis, but is an important fuel for active muscles and other tissues. The operation of lactate shuttles within and among cells, tissues and organs such as retina, brain, testis, liver, and cardiac and skeletal muscle under fully aerobic conditions is well established. Lactate is produced in significant amounts in cancer cells under fully aerobic conditions due to enhanced glycolysis, described as a "Warburg Effect," but the role of lactate and its transporters in cancer growth is poorly understood. PURPOSE: In this study, we sought to identify differences in the expression of monocarboxylate transporters (MCT) and lactate dehydrogenase isoforms (LDH) in two breast cancer cell lines (MCF-7 and MDA-MB-231) and a normal control untransformed primary breast cell line (184), to understand the role of lactate transporters in cancer growth. METHODS: Immunoblotting (IB) and Confocal laser scanning microscopy (CLSM) were used to examine the expression and the localization of LDH and MCT proteins in normal and cancer cell lines. Oxygen consumption and lactate production per hour were measured using a Clark-type oxygen electrode and spectrophotometry, respectively. RESULT: Our data show that MCT (1, 2, and 4), and LDH isoforms (A and B) are expressed in both normal and cancerous breast cells, except that MDA-MB-231 did not express MCT1. MCT1 was highly expressed in control 184l cells when compared to cancer cells. MCT4 was highly expressed in MDA-MB-231, and MCT2 was highly expressed in MCF-7. LDH was highly expressed in both cancerous cell lines compared to the normal cell line, and MCF-7 expressed mainly LDHB, while MDA-MB-231 and 184 expressed mainly LDHA. MCT2, MCT4, and LDH were localized in mitochondria in addition to the plasma membrane and cytosol, respectively, whereas MCT1 was localized in plasma membrane. This localization was the same in cancerous and normal cell lines. CONCLUSION: We report changes in the expression of MCT and LDH in breast cancer cells with no change in their localization. These changes corresponded to the breast cancer cells' oxidative capacity. Our data support the existence of the previously reported lactate shuttle in tumors. Lessons from exercise physiology may prove useful in terms of targeting and killing cancer cells. Conversely, metabolic adaptations to support the hypermetabolism of cancer cells may provide models for understanding metabolic adaptations to exercise training. Supported by NIH grant AR050459 and a gift from CytoSport, Inc.

Cancer Cell Metabolism: Warburg and Beyond

Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS) resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA), which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.

Hodgkin Lymphoma: Metabolic Symbiosis Between Malignant Cells and Cancer-Associated Stromal Tissue

Molecular-targeted therapy has been developed for cancer chemoprevention and treatment. Cancer cells have different metabolic properties from normal cells. Normal cells mostly rely upon the process of mitochondrial oxidative phosphorylation to produce energy whereas cancer cells have developed an altered metabolism that allows them to sustain higher proliferation rates. Cancer cells could predominantly produce energy by glycolysis even in the presence of oxygen. This alternative metabolic characteristic is known as the “Warburg Effect.” Although the exact mechanisms underlying the Warburg effect are unclear, recent progress indicates that glycolytic pathway of cancer cells could be a critical target for drug discovery. With a long history in cancer treatment, traditional Chinese medicine (TCM) is recognized as a valuable source for seeking bioactive anticancer compounds. A great progress has been made to identify active compounds from herbal medicine targeting on glycolysis for cancer treatment. Herein, we provide an overall picture of the current understanding of the molecular targets in the cancer glycolytic pathway and reviewed active compounds from Chinese herbal medicine with the potentials to inhibit the metabolic targets for cancer treatment. Combination of TCM with conventional therapies will provide an attractive strategy for improving clinical outcome in cancer treatment.

Reprogramming of energy metabolism is a key hallmark of cancer. Most cancer cells display a glycolytic phenotype, with increased glucose consumption and glycolysis rates, and production of lactateas the end product, independently of oxygen concentrations. This phenomenon, known as "Warburg Effect", provides several survival advantages to cancer cells and modulates the metabolism and function of neighbour cells in the tumour microenvironment. However, due to the presence of metabolic heterogeneity within a tumour, cancer cells can also display an oxidative phenotype, and corruptible cells from the microenvironment become glycolytic, cooperating with oxidative cancer cells to boost tumour growth. This phenomenon is known as "Reverse Warburg Effect". In either way, lactate is a key mediator in the metabolic crosstalk between cancer cells and the microenvironment, and lactate transporters are expressed differentially by existing cell populations, to support this crosstalk.In this review, we will focus on lactate and on lactate transporters in distinct cells of the tumour microenvironment, aiming at a better understanding of their role in the acquisition and maintenance of the direct/reverse "Warburg effect" phenotype, which modulate cancer progression.

Many cancer cells exhibit an altered metabolism characterized by increased aerobic glycolysis known as the Warburg effect. To elucidate transcriptional events necessary for the Warburg effect in cancer, we compared gene expression profiles from glycolytic versus less glycolytic breast tumors and cancer cell lines. Amongst the mRNAs that significantly correlated with glycolytic phenotype was one encoding monocarboxylate transporter 1 (MCT1). MCT1 facilitates proton‐linked transport of lactate, pyruvate, and other monocarboxylates across the plasma membrane. We found that MCT1 levels are elevated in malignant breast lesions, and high MCT1 expression predicts poor prognosis in breast cancer patients. MCT1 LOF results in decreased glucose consumption rates in multiple breast cancer cell lines; however, MCT1 LOF does not consistently result in altered lactate production rates. Surprisingly, our data suggests that MCT1 primarily functions to export pyruvate, and that breast cancer cell lines export significant amounts of pyruvate. MCT1 LOF dramatically reduces breast cancer cell proliferation by causing a G1 cell cycle arrest, and reduces mammary fat pad tumor xenograft formation. Together, these data show MCT1 to be critical for breast cancer cell metabolism and proliferation, and suggest that MCT1 inhibitors may be effective anti‐cancer agents.

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.

Introduction: Despite the improvement of survival in localized osteosarcoma patients, patients with metastasis still carry a poor prognosis. Thus, identification of factors contributing to metastasis is needed. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell-surface glycoprotein which plays multiple roles in physiologic and pathologic conditions. EMMPRIN is highly expressed in several cancers and stimulates adjacent stromal cells or tumor cells to produce matrix metalloproteinase (MMP). EMMPRIN also stimulates expression of vascular endothelial growth factor (VEGF) which leads to angiogenesis. These findings have made EMMPRIN an attractive therapeutic target in many cancer types. The goals of this study were; to investigate the expression of EMMPRIN in osteosarcoma; to examine if EMMPRIN, via tumor-stroma interaction, is associated with metastatic potential in osteosarcoma. Methods: Level of EMMPRIN mRNA expression was evaluated by RT-PCR in 6 tumor-derived osteosarcoma cell lines and by immunohistochemistry prechemotherapy biopsies from 52 patients. Clinical data of these patients were reviewed to examine the association of EMMPRIN expression and clinical outcome. Transfection of EMMPRIN-targeting siRNA in SaOS-2 was performed to test the role of EMMPRIN in osteosarcoma progression. To study the role of EMMPRIN in tumor-stromal interaction, co-culture of SaOS-2 with osteoblast (hFOB) was experimented. Functional in vitro assays that reflect metastatic potential of osteosarcoma cells were performed. MMP production, VEGF production, cell invasion and proliferation were studied by gelatin zymography, VEGF ELISA, Matrigel invasion assay and WST-1 assay, respectively. Results: EMMPRIN was expressed in 90% by immunohistochemistry with strong accentuation along the cell membrane. Level of EMMRIN mRNA expression was significantly higher in 5 of 6 tumor-derived cell lines compared to MG63. Patients with high EMMPRIN expression had significantly worse metastasis-free survival. EMMPRIN mRNA levels was significantly down-regulated by siRNA transfection compared to control-siRNA transfected cell. Co-culture of osteoblast and SaOS-2 enhanced stimulation of pro-MMP2. This stimulation was reversed by transfection of SaOS-2 with EMMPRIN-targeting siRNA. Number of cells crossing the chamber was statistically lower in EMMPRIN-siRNA transfected cells. When osteoblast and SaOS-2 were co-cultured, VEGF expression was increased compared to SaOS-2 only culture. EMMPRIN-targeting siRNA transfection resulted in decrease of VEGF expression. SaOS-2 cells transfected with EMMPRIN-siRNA showed decreased proliferation potential compared to control-siRNA transfected cells. Conclusion: Our data suggest that highly expressed EMMPRIN plays an important role in metastatic potential of osteosarcoma. EMMPRIN could serve as a potential therapeutic target in osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1434.

Tumor Microenvironment and Metabolic Synergy in Breast Cancers: Critical Importance of Mitochondrial Fuels and Function

See related article, pages 1973–1979. The lack of effective and widely applicable pharmacological treatments for ischemic stroke patients may explain a growing interest in traditional medicines, for which extensive observational and anecdotal experience has accumulated over the past thousand years. The World Health Organization (WHO) defines traditional medicine as “health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being”.1 Unlike Western medicine, which focuses on disease, traditional medicine takes the approach that the body provides external clues to an internal imbalance that can be addressed by interventions such as herbs and acupuncture (holistic treatment approach).2 According to a 2003 WHO report,1 traditional medicine is very popular in all developing countries, and its use is rapidly increasing in industrialized countries. For example, traditional herbal preparations account for 30% to 50% of the total medicinal consumption in China. In Europe, North America and other industrialized regions, over 50% of the population have used traditional medicine at least once. The global market for herbal medicines currently stands at over US $60 billion annually and is growing steadily.1 In recent years, several reviews have been published on the effect and potential benefits of traditional Eastern medicine in stroke.3–7 It has been suggested that some herbal medicines, or their products, may improve microcirculation in the brain,4,8 protect against ischemic reperfusion injury,8,9 possess neuroprotective properties3,4 and inhibit apoptosis,10 thus justifying their use in ischemic stroke patients. However, unlike industrially manufactured pharmacological drugs used in Western medicine, the active (potent) components of herbal medicines often have not been specified and measured precisely, although there have been recent attempts to regulate dosages and use of …

The preferential use of aerobic glycolysis for energy production by cancer cells, a phenomenon known as the 'Warburg effect', is well recognized and is being considered for therapeutic applications. However, whether inhibition of glycolysis will be effective in all types of cancer is unclear. The current study shows that a glycolytic inhibitor, 2-deoxy-D-glucose (2DG), exhibits the cytotoxic effect on non-small cell lung cancer in a p53-dependent manner. 2DG significantly inhibits ATP production in p53-deficient lung cancer cells (H358) but not in p53-wt cells (A549). In contrast to p53-wt cells, p53-defective cells are unable to compensate for their need of energy via oxidative phosphorylation (OXPHOS) when glycolysis is inhibited. In the presence of p53, increased ROS from OXPHOS increases the expression of p53 target genes known to modulate metabolism, including synthesis of cytochrome c oxidase 2 (SCO2) and TP53-induced glycolysis and apoptosis regulator (TIGAR). Importantly, 2DG selectively induces the expression of the antioxidant enzymes manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx1) in a p53-dependent manner. The results demonstrate that the killing of cancer cells by the inhibitor of glycolysis is more efficient in cancer cells without functional p53 and that p53 protects against metabolic stress by up-regulation of oxidative phosphorylation and modulation of antioxidants.

Mutations of multiple tumor suppressor genes, such as PTEN and p53, have been proposed to play important roles in the development of prostate cancer. Loss of one allele of PTEN occurs in 70-80% of human primary prostate tumors and homozygous inactivation of PTEN is associated with advanced disease. Similarly, p53 is found completely lost or mutated almost exclusively in advanced human prostate cancer. Thus, selective killing of prostate cancer cells harboring mutations of PTEN and p53 may prove to be a promising strategy for the treatment of advanced prostate cancer. The Warburg effect of aerobic glycolysis has now been generally accepted as a key metabolic hallmark of cancer. In this study, we investigated the molecular target leading to the Warburg effect in the growth and aggressiveness of prostate cancer cells harboring inactivation of PTEN and p53 and delineated the underlying mechanism. We identified that expression of hexokinase II (HK2), an enzyme involved in the first step of glycolysis, is preferentially elevated in human prostate cancer cells bearing mutations of both PTEN and p53 (PC3). Functional studies demonstrated that HK2 expression is crucial for the Warburg effect in PC3 cells and knockdown of HK2 inhibits tumor growth in PC3 xenograft mouse model. These novel findings prompted us to test whether 2-de-oxyglucose (2-DG), an inhibitor of HK2, could potentially suppress prostate cancer growth by targeting the Warburg effect. We found that an induction of AMPK-dependent autophagy prevents cancer cells from apoptosis upon 2-DG treatment, thereby limiting therapeutic efficacy on prostate cancer in vivo. Consistent with cell survival function of autophagy, its inhibition by chloroquine (a small molecule inhibitor of autophagy) or individual knockdown of the essential genes involved in autophagy (Atg5, Atg7, Beclin1, and ULK1) induced massive cell death when combined with 2-DG. This cell death can be rescued by overexpression of anti-apoptotic protein Bcl-2 or downregulation of pro-apoptotic protein Bax in PC3. More importantly, we demonstrated that combination of chloroquine and 2-DG could specifically kill prostate cancer cells, leaving normal prostate epithelial cells untouched. This specificity is due to the preferential induction of HK2 through the activated Rictor-AKT-mTOR pathway in cancer cells. Finally, combination of chloroquine and 2-DG caused synthetic lethality in prostate cancer cells and effectively suppressed tumor growth in PC3 xenograft mouse model. Towards therapeutic translation, we have observed that expression of HK2 by staining primary human prostate tumor samples with HK2 antibodies correlated with the stages of prostate cancer. Given our findings, we therefore propose that targeting the Warburg effect and autophagy pathways may serve as an effective and selective treatment for patients with advanced prostate cancer, in particular those with PTEN and p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3213. doi:1538-7445.AM2012-3213

BackgroundTwenty percent of patients with classical Hodgkin Lymphoma (cHL) have aggressive disease defined as relapsed or refractory disease to initial therapy. At present we cannot identify these patients pre-treatment. The microenvironment is very important in cHL because non-cancer cells constitute the majority of the cells in these tumors. Non-cancer intra-tumoral cells, such as tumor-associated macrophages (TAMs) have been shown to promote tumor growth in cHL via crosstalk with the cancer cells. Metabolic heterogeneity is defined as high mitochondrial metabolism in some tumor cells and glycolysis in others. We hypothesized that there are metabolic differences between cancer cells and non-cancer tumor cells, such as TAMs and tumor-infiltrating lymphocytes in cHL and that greater metabolic differences between cancer cells and TAMs are associated with poor outcomes. MethodsA case-control study was conducted with 22 tissue samples of cHL at diagnosis from a single institution. The case samples were from 11 patients with aggressive cHL who had relapsed after standard treatment with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or were refractory to this treatment. The control samples were from 11 patients with cHL who achieved a remission and never relapsed after ABVD. Reactive non-cancerous lymph nodes from four subjects served as additional controls. Samples were stained by immunohistochemistry for three metabolic markers: translocase of the outer mitochondrial membrane 20 (TOMM20), monocarboxylate transporter 1 (MCT1), and monocarboxylate transporter 4 (MCT4). TOMM20 is a marker of mitochondrial oxidative phosphorylation (OXPHOS) metabolism. Monocarboxylate transporter 1 (MCT1) is the main importer of lactate into cells and is a marker of OXPHOS. Monocarboxylate transporter 4 (MCT4) is the main lactate exporter out of cells and is a marker of glycolysis. The immunoreactivity for TOMM20, MCT1, and MCT4 was scored based on staining intensity and percentage of positive cells, as follows: 0 for no detectable staining in > 50% of cells; 1+ for faint to moderate staining in > 50% of cells, and 2+ for high or strong staining in > 50% of cells. ResultsTOMM20, MCT1, and MCT4 expression was significantly different in Hodgkin and Reed Sternberg (HRS) cells, which are the cancerous cells in cHL compared with TAMs and tumor-associated lymphocytes. HRS have high expression of TOMM20 and MCT1, while TAMs have absent expression of TOMM20 and MCT1 in all but two cases. Tumor-infiltrating lymphocytes have low TOMM20 expression and absent MCT1 expression. Conversely, high MCT4 expression was found in TAMs, but absent in HRS cells in all but one case. Tumor-infiltrating lymphocytes had absent MCT4 expression. Reactive lymph nodes in contrast to cHL tumors had low TOMM20, MCT1, and MCT4 expression in lymphocytes and macrophages. High TOMM20 and MCT1 expression in cancer cells with high MCT4 expression in TAMs is a signature of high metabolic heterogeneity between cancer cells and the tumor microenvironment. A high metabolic heterogeneity signature was associated with relapsed or refractory cHL with a hazard ratio of 5.87 (1.16–29.71; two-sided P < .05) compared with the low metabolic heterogeneity signature. ConclusionAggressive cHL exhibits features of metabolic heterogeneity with high mitochondrial metabolism in cancer cells and high glycolysis in TAMs, which is not seen in reactive lymph nodes. Future studies will need to confirm the value of these markers as prognostic and predictive biomarkers in clinical practice. Treatment intensity may be tailored in the future to the metabolic profile of the tumor microenvironment and drugs that target metabolic heterogeneity may be valuable in this disease.

As treasures of the traditional Chinese medicine (TCM), acupuncture and Chinese herbal medicine have a history of more than 2,500 years and have achieved sound effects in the clinical practice [1]. The effects of acupuncture and Chinese herbs have usually been demonstrated by biological regulations of physiological and pathological processes [2, 3], which are inherent responses of human beings and of great importance in the life science research. In recent years, the researches of acupuncture and Chinese herbal medicine have improved significantly due to the prosperity and development of modern life science technology [4–7]. At the same time, the studies of acupuncture and Chinese herbal medicine have in turn enhanced the development of biomedical science as well as the understanding of life [8–10] and offered new perspectives that can benefit modern medicine [11, 12]. However, the major challenge of the integration of TCM with modern medicine is the inadequate understanding of the biological foundation of TCM experience and concepts. In order to deepen our knowledge of TCM, it is important to clarify the biological value and mechanistic action of acupuncture and Chinese herbal medicine, which may turn hypothesis to solid data-based research and creative discovery of life science. We are excited to present our readers with this special issue that summarizes recent discoveries and knowledge of acupuncture and Chinese herbal medicine. This specialized issue collects 24 original research articles and 4 reviews that provide the clinical or animal-based evidence elucidating the impacts of acupuncture and Chinese herbal medicine on the life science. Among these original research articles, the biological functions and potential mechanisms of acupuncture and Chinese herbal drugs in disease treatment were demonstrated in animal models of diseases such as focal cerebral ischemia, pain, atopic dermatitis, insomnia, fatty liver, and Crohn's disease. Two papers are on cancer cells: Han-Peng Kuo et al. reported that Ganoderma tsugae extract inhibited the growth of HER2-overexpressing cancer cells and enhanced the growth inhibitory effect of antitumor drugs via the modulation of HER2/PI3K/Akt signaling pathway; Xiu-Feng Wang et al. showed that PC-SPESII herbal extract could impair human breast cancer metastasis by regulating proteolytic enzymes and matrix dynamics through the p38MAPK and SAPK/JNK pathway. Additionally, You Ning et al. reported the molecular mechanisms underlying the antiproliferative and prodifferentiative effects of psoralen on adult neural stem cells using DNA microarray. Interestingly, two research articles (Lei-Miao Yin et al. and Xiang-Gen Zhong et al.) simultaneously focused on the same issue, specific link between the lung and the large intestine, which provide new lines of evidence for the modern biological theory of “exterior-interior correlation between Zang and Fu organs.” The review by Yu Wang et al. presented a comprehensive overview of the researches on acupuncture effective biomolecules, covered diverse carriers such as cerebrospinal fluid, serum, organs, and tissues, and discussed how to promote the development of biological medicine based on the discovery of acupuncture effective biomolecules. The other 3 review articles also provided us with current understanding of biological values of acupuncture and Chinese herbal medicine on inflammatory bowel diseases, amblyopia, and glucose and lipids metabolism, respectively. In summary, this special issue provides up-to-date and valuable information about the role of acupuncture and Chinese herbs in the life science. Accordingly, future researches with modern technologies would further advance our understanding of the biological effects and cellular/molecular bases for acupuncture and Chinese herbal medicine in diverse diseases.