Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate

It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient ρ⁰ cells that highly express HKII. Interestingly, the dissociation of HKII itself did not directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death.     

Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells

Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC) transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2), KG-1 (ABCB1) and HepG2 cells (ABCB1 and ABCG2). Interestingly, although side population (SP) cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.     

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.     

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.     

Inhibition of TACE activity enhances the susceptibility of myeloma cells to TRAIL

Background

TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. However, the susceptibility of MM cells to TRAIL is largely low in most of MM cells by yet largely unknown mechanisms. Because TNF-α converting enzyme (TACE) can cleave some TNF receptor family members, in the present study we explored the roles of proteolytic modulation by TACE in TRAIL receptor expression and TRAIL-mediated cytotoxicity in MM cells.

Methodology/Principal Findings

MM cells preferentially expressed death receptor 4 (DR4) but not DR5 on their surface along with TACE. Conditioned media from RPMI8226 and U266 cells contained a soluble form of DR4. The DR4 levels in these conditioned media were reduced by TACE inhibition by the TACE inhibitor TAPI-0 as well as TACE siRNA. Conversely, the TACE inhibition restored surface levels of DR4 but not DR5 in these cells without affecting DR4 mRNA levels. The TACE inhibition was able to restore cell surface DR4 expression in MM cells even in the presence of bone marrow stromal cells or osteoclasts, and enhanced the cytotoxic effects of recombinant TRAIL and an agonistic antibody against DR4 on MM cells.

Conclusions/Significance

These results demonstrate that MM cells post-translationally down-modulate the cell surface expression of DR4 through ectodomain shedding by endogenous TACE, and that TACE inhibition is able to restore cell surface DR4 levels and the susceptibility of MM cells to TRAIL or an agonistic antibody against DR4. Thus, TACE may protect MM cells from TRAIL-mediated death through down-modulation of cell-surface DR4. It can be envisaged that TACE inhibition augments clinical efficacy of TRAIL-based immunotherapy against MM, which eventually becomes resistant to the present therapeutic modalities.     

Compromising the unfolded protein response induces autophagy-mediated cell death in multiple myeloma cells

Objective

To determine whether the Unfolded Protein Response (UPR) sensors (PERK, ATF6 and IRE-1) can be targeted to promote death of Multiple Myeloma (MM) cells.

Methods

We have knocked-down separately each UPR stress sensor in human MM cell lines using RNA interference and followed MM cell death by monitoring the membrane, mitochondrial and nuclear alterations. Involvement of caspases in MM cell death consecutive to UPR sensor knock-down was analyzed by western blotting, measurement of their enzymatic activity using fluorigenic substrates and susceptibility to a pan-caspase inhibitor. Activation of the autophagic process was measured directly by detection of autophagosomes (electronic microscopy), monodansylcadaverine staining, production of the cleaved form of the microtubule-associated protein 1A/1B light chain 3 (LC3) and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3MA and bafilomycin A1.

Results

We show that extinction of a single UPR stress sensor (PERK) induces a non-apoptotic form of cell death in MM cells that requires autophagy for its execution. We also show that this cytotoxic autophagic process represses the apoptosis program by reducing the cytosolic release of the apoptogenic factors Smac/DIABLO and cytochrome c.

Interpretation

Altogether our findings suggest that autophagy can contribute to execution of death in mammalian cells that are exposed to mild ER stress. They also suggest that the autophagic process can regulate the intrinsic apoptotic pathway by inhibiting production of death effectors by the mitochondria, thus preventing formation of a functional apoptosome. Altogether these findings give credit to the idea that UPR sensors can be envisaged as therapeutic targets for the treatment of MM.     

NF-kappa B as a therapeutic target in multiple myeloma

We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As(2)O(3) act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As(2)O(3) inhibit nuclear factor (NF)-kappaB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-kappaB blockade in mediating anti-MM activity. To characterize the effect of specific NF-kappaB blockade on MM cell growth and survival in vitro, we used an IkappaB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFalpha-induced NF-kappaB activation in a dose- and time-dependent fashion in MM cells through inhibition of IkappaBalpha phosphorylation and degradation of IkappaBalpha, respectively. Dexamethasone (Dex), which up-regulates IkappaBalpha protein, enhances blockade of NF-kappaB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNFalpha-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (20-50%) inhibits MM cell proliferation, suggesting that NF-kappaB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFalpha induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-kappaB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-kappaB.     

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles’ heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.     

Increased expression of transglutaminase 2 drives glycolytic metabolism in renal carcinoma cells

Transglutaminase 2 (TGase 2) expression and glycolysis are increased in most renal cell carcinoma (RCC) cell lines compared to the HEK293 kidney cell line. Although increased glycolysis and altered tricarboxylic acid cycle are common in RCC, the detailed mechanism by which this phenomenon occurs remains to be elucidated. In the present study, TGase 2 siRNA treatment lowered glucose consumption and lactate levels by about 20–30 % in RCC cells; conversely, high expression of TGase 2 increased glucose consumption and lactate production together with decreased mitochondrial aconitase (Aco 2) levels. In addition, TGase 2 siRNA increased mitochondrial membrane potential and ATP levels by about 20–30 % and restored Aco 2 levels in RCC cells. Similarly, Aco 2 levels and ATP production decreased significantly upon TGase 2 overexpression in HEK293 cells. Therefore, TGase 2 leads to depletion of Aco 2, which promotes glycolytic metabolism in RCC cells.     

Blockade of SDF-1/CXCR4 reduces adhesion-mediated chemoresistance of multiple myeloma cells via interacting with interleukin-6

Resistance to chemotherapy represents a major cause for treatment failure in multiple myeloma (MM). Herein, this study was conducted to explore the effect of SDF-1/CXCR4 and interleukin-6 (IL-6) in MM cell adhesion-mediated chemoresistance. Enzyme-linked immunosorbent assay was applied to detect expressions of SDF-1α and IL-6 in MM patients and healthy controls. RPMI-8226 cells and isolated bone marrow stromal cells (BMSCs) were stimulated using recombinant SDF-1α and IL-6. Effect of cocultured BMSCs and RPMI-8226 cells on chemosensitivity and apoptosis of RPMI-8226 cells was analyzed. Effect of doxorubicin on the adhesion rate of RPMl-8226 cells to BMSCs was analyzed by calcitonin test. Effect of SDF-1α-induced upregulation of IL-6 on chemotherapeutic resistance and apoptosis of RPMI-8226 cells in adhesion state was analyzed. Cell adhesion model was treated with recombinant protein SDF-1α and phosphoinositide 3-kinase (P13K) inhibitor Wortmarmin. The levels of P13K and protein kinase B (AKT) and its phosphorylation as well as the expression of IL-6 were analyzed. SDF-1α was positively correlated with IL-6. Recombinant human SDF-1α increased IL-6 expression and induced IL-6 secretion in a time- and dose-dependent manner in BMSCs, which was inhibited by IL-6 and SDF-1α neutralizing antibodies. Coculture of MM cells with BMSCs increased the drug resistance and inhibited the apoptosis on MM cells. SDF-1α-induced IL-6 upregulation mediates chemoresistance and apoptosis of RPMI-8226 cells in adhesion state. SDF-1α may up-regulate the expression of IL-6 by activating the P13K/AKT signaling pathway. SDF-1/CXCR4 may up-regulate the expression of IL-6 through the activation of the P13K/AKT signaling pathway, thereby affecting the chemoresistance mediated by adhesion in MM cells.     

The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression

Cancer cells heavily rely on the glycolytic pathway regardless of oxygen tension. Hexokinase II (HKII) catalyses the first irreversible step of glycolysis and is often overexpressed in cancer cells. 3-Bromopyruvate (3BP) has been shown to primarily target HKII, and is a promising anti-cancer compound capable of altering critical metabolic pathways in cancer cells. Abnormal vasculature within tumours leads to heterogeneous microenvironments, including glucose availability, which may affect drug sensitivity. The aim of the present study was to elucidate the mechanisms by which 3BP acts on colorectal cancer (CRC) cells with focus on the HKII/Akt signalling axis. High HKII-expressing cell lines were more sensitive to 3BP than low HKII-expressing cells. 3BP-induced rapid Akt phosphorylation at site Thr-308 and cell death via both apoptotic and necrotic mechanisms. Cells grown under lower glucose concentrations showed greater resistance towards 3BP. Cells with HKII knockdown showed no changes in 3BP sensitivity, suggesting the effects of 3BP are independent of HKII expression. These results emphasize the importance of the tumour microenvironment and glucose availability when considering therapeutic approaches involving metabolic modulation.     

Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.     

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.