Mitochondrial bioenergetic background confers a survival advantage to HepG2 cells in response to chemotherapy

Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (ΔΨm). It thus may be predicted that ΔΨm regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O₂ consumption/ATP production coupling, ΔΨm maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered ΔΨm, leading to an increased energy request to regenerate ΔΨm in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization. © 2009 Wiley‐Liss, Inc.     

Hodgkin and Reed–Sternberg cells of classical Hodgkin lymphoma are highly dependent on oxidative phosphorylation

The metabolic properties of lymphomas derived from germinal center (GC) B cells have important implications for therapeutic strategies. In this study, we have compared metabolic features of Hodgkin–Reed–Sternberg (HRS) cells, the tumor cells of classical Hodgkin's lymphoma (cHL), one of the most frequent (post‐)GC‐derived B‐cell lymphomas, with their normal GC B cell counterparts. We found that the ratio of oxidative to nonoxidative energy conversion was clearly shifted toward oxidative phosphorylation (OXPHOS)‐linked ATP synthesis in HRS cells as compared to GC B cells. Mitochondrial mass, the expression of numerous key proteins of oxidative metabolism and markers of mitochondrial biogenesis were markedly upregulated in cHL cell lines and in primary cHL cases. NFkappaB promoted this shift to OXPHOS. Functional analysis indicated that both cell growth and viability of HRS cells depended on OXPHOS. The high rates of OXPHOS correlated with an almost complete lack of lactate production in HRS cells not observed in other GC B‐cell lymphoma cell lines. Overall, we conclude that OXPHOS dominates energy conversion in HRS cells, while nonoxidative ATP production plays a subordinate role. Our results suggest that OXPHOS could be a new therapeutic target and may provide an avenue toward new treatment strategies in cHL.     

Mitochondrial DNA mutations in ageing and cancer

Advancing age is a major risk factor for malignant transformation and the development of cancer. As such, over 50% of neoplasms occur in individuals over the age of 70. The pathologies of both ageing and cancer have been characterized by respective groups of molecular hallmarks, and while some features are divergent between the two pathologies, several are shared. Perturbed mitochondrial function is one such common hallmark, and this observation therefore suggests that mitochondrial alterations may be of significance in age‐related cancer development. There is now considerable evidence documenting the accumulation of somatic mitochondrial DNA (mtDNA) mutations in ageing human postmitotic and replicative tissues. Similarly, mutations of the mitochondrial genome have been reported in human cancers for decades. The plethora of functions in which mitochondria partake, such as oxidative phosphorylation, redox balance, apoptosis and numerous biosynthetic pathways, manifests a variety of ways in which alterations in mtDNA may contribute to tumour growth. However, the specific mechanisms by which mtDNA mutations contribute to tumour progression remain elusive and often contradictory. This review aims to consolidate current knowledge and describe future direction within the field.     

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the V600EBRAF oncogene

Oncogene addiction describes how cancer cells exhibit dependence on single oncogenes to escape apoptosis and senescence. While oncogene addiction constitutes the basis for new cancer treatment strategies targeting individual kinases and pathways activated by oncogenic mutations, the biochemical basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to ^V600EBRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that ^V600EBRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to ^V600EBRAF. Finally, the senescence response associated with inhibition of ^V600EBRAF is rescued by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing direct evidence that oncogene addiction rests on a metabolic foundation.     

BCAT1 knockdown-mediated suppression of melanoma cell proliferation and migration is associated with reduced oxidative phosphorylation

Malignant melanoma has a high mutational rate. As a result, resistance to current therapies is common. Consequently, there is an unmet medical need to develop novel therapies. Recent data suggest that branched-chain amino acid transaminase 1 (BCAT1) is overexpressed in multiple cancers, and such overexpressed BCAT1 is necessary for individual cancer progression. Therefore, BCAT1 appears to be a good target in cancer treatment. Additionally, because its expression in healthy tissues is highly restricted in adults and is limited to the brain, ovary, and placenta, BCAT1 is especially an ideal target in cancer therapies. Currently, the function of BCAT1 in malignant melanoma has not been demonstrated. Therefore, we investigated the role of BCAT1 in the proliferation and migration of malignant melanomas using human samples and mouse malignant B16 melanoma cell line. Our data showed that BCAT1 was overexpressed in malignant melanoma tissues both in humans and mice. Besides, BCAT1 knockdown suppressed melanoma cell proliferation and migration, which was associated with reduced oxidative phosphorylation. Collectively, our data indicate that BCAT1 is a promising therapeutic target for the treatment of malignant melanomas.     

氧化应激对蛋白酶体抑制剂诱导甲状腺癌细胞凋亡影响的研究

目的:探讨氧化应激在启动蛋白酶体抑制剂诱导甲状腺癌细胞凋亡中的作用.方法:选取4种人甲状腺未分化癌细胞系ARO、FRO、KTC2和8305C,分别设空白对照组、万珂处理组和万珂+钛剂联合组;流式细胞仪(FCM)检测细胞凋亡情况;Caspase-3活性测试法检测各组细胞Caspase-3活性;用荧光分光光度计法检测各组细胞蛋白酶体活性和细胞内ROS水平.结果:与空白对照组相比,蛋白酶体抑制剂万珂对人甲状腺癌细胞系FRO和KTC2具有很强的细胞凋亡诱导作用;使FRO和KTC2细胞中Caspase-3的活性显著增高,P＜0.01;可持续快速抑制蛋白酶体活性,但在4组细胞中差异无统计学意义,P＞0.05;万珂可显著提高活性氧簇在FRO和KTC2甲状腺癌细胞中的表达.万珂组与万珂+钛剂组活性氧簇水平在4种甲状腺癌细胞中差异均有统计学意义,P＜0.05;在FRO和KTC2甲状腺癌细胞中,万珂组与万珂+钛剂组中的细胞凋亡率差异有统计学意义,P＜0.01.结论:蛋白酶体抑制剂可以提高活性氧簇在甲状腺未分化癌细胞中的表达,促进肿瘤细胞的凋亡,并且这种效应可以被抗氧化剂所抑制.     

Susceptibility of human melanoma cells to oxidative stress including UVA radiation

Ultraviolet radiation, and in particular UVA (320–400 nm), induces significant oxidative stress to human skin. Ferritin and glutathione have been shown to be among the more important molecules within human skin cells providing protection against this damage, the presence of lower levels of these anti-oxidants giving rise to increased cellular sensitivity to stress. We compared endogenous levels of ferritin and glutathione in human melanoma cells with normal human skin fibroblasts and keratinocytes, also the response of melanoma cells to oxidative stress with fibroblasts and keratinocytes. Ferritin levels were heterogenous in the untreated melanoma cell lines tested and remained the same following oxidative stress (UVA radiation) or hemin treatment. Epidermal keratinocytes were unaffected, as were the melanoma cell lines, but skin fibroblasts showed dose-dependent ferritin depletion. Similar results were seen for glutathione alterations resulting from UVA radiation: melanoma cell lines and epidermal skin keratinocytes remained unchanged following UVA radiation, while skin fibroblasts showed dose-dependent depletion. Our results show that human melanoma cells have low ferritin and glutathione levels, yet are resistant to oxidative stress. © 1996 Wiley-Liss, Inc.     

Activating oxidative phosphorylation by a pyruvate dehydrogenase kinase inhibitor overcomes sorafenib resistance of hepatocellular carcinoma

Background:

Sorafenib is the only drug approved for the treatment of hepatocellular carcinoma (HCC). The bioenergetic propensity of cancer cells has been correlated to anticancer drug resistance, but such correlation is unclear in sorafenib resistance of HCC.

Methods:

Six sorafenib-naive HCC cell lines and one sorafenib-resistant HCC cell line (Huh-7R; derived from sorafenib-sensitive Huh-7) were used. The bioenergetic propensity was calculated by measurement of lactate in the presence or absence of oligomycin. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, and siRNA of hexokinase 2 (HK2) were used to target relevant pathways of cancer metabolism. Cell viability, mitochondrial membrane potential, and sub-G1 fraction were measured for in vitro efficacy. Reactive oxygen species (ROS), adenosine triphosphate (ATP) and glucose uptake were also measured. A subcutaneous xenograft mouse model was used for in vivo efficacy.

Results:

The bioenergetic propensity for using glycolysis correlated with decreased sorafenib sensitivity (R²=0.9067, among sorafenib-naive cell lines; P=0.003, compared between Huh-7 and Huh-7 R). DCA reduced lactate production and increased ROS and ATP, indicating activation of oxidative phosphorylation (OXPHOS). DCA markedly sensitised sorafenib-resistant HCC cells to sorafenib-induced apoptosis (sub-G1 (combination vs sorafenib): Hep3B, 65.4±8.4% vs 13±2.9% Huh-7 R, 25.3± 5.7% vs 4.3±1.5% each P<0.0001), whereas siRNA of HK2 did not. Sorafenib (10 mg kg⁻¹ per day) plus DCA (100 mg kg⁻¹ per day) also resulted in superior tumour regression than sorafenib alone in mice (tumour size: −87% vs −36%, P<0.001).

Conclusion:

The bioenergetic propensity is a potentially useful predictive biomarker of sorafenib sensitivity, and activation of OXPHOS by PDK inhibitors may overcome sorafenib resistance of HCC.     

REST‐dependent expression of TRF2 renders non‐neuronal cancer cells resistant to DNA damage during oxidative stress

REST is a neuronal gene silencing factor ubiquitously expressed in non‐neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non‐neuronal cancers. Conversely, recent findings on REST‐dependent tumorigenesis in non‐neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non‐neuronal cancers. We observed abundant expression of REST in various types of non‐neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non‐neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere‐binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down‐regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST‐dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress‐resistant REST mutants found in specific human cancers, may account for REST‐dependent tumorigenesis.     

Cellular response to oxidative stress and ascorbic acid in melanoma cells overexpressing gamma-glutamyltransferase

The extracellular gamma-glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. This study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H(2)O(2). Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents.     

DHCR24 gene expression is upregulated in melanoma metastases and associated to resistance to oxidative stress-induced apoptosis

The DHCR24 gene encoding for the 3beta-hydroxysterol delta24-reductase, an oxidoreductase involved in cholesterol biosynthesis, was isolated by subtractive hybridization as highly expressed in a short-term melanoma cell line derived from a cutaneous metastases (S/M2) compared to that obtained from the autologous primary tumor (S/P). DHCR24 (alias seladin-1, diminuto/dwarf1 homolog) has been reported to act as an antiapoptotic factor in neurons. Gene expression analysis by Northern blot confirmed that DHCR24 was 5-fold upregulated in S/M2 compared to S/P cells. High levels of DHCR24 gene expression were detected in 13/25 melanoma metastases and in 1/7 primary melanomas by real-time PCR, indicating that upregulation of this gene may occur in melanoma progression. In S/M2 cells, high DHCR24 gene expression associated with resistance to apoptosis triggered by oxidative stress induced by exposure to hydrogen peroxide. DHCR24 gene transfer was shown to protect melanoma cells from H2O2-induced cytotoxicity. Although higher cholesterol levels were shown in S/M2 cells compared to S/P cells, DHCR24 gene transfer did not increase cholesterol content. To evaluate whether DHCR24 acts as an antiapoptotic factor in melanoma metastases, the cytotoxic effect of chemotherapeutic agents was tested in DHCR24 transfectants and in the presence of a DHCR24 inhibitor, U18666A. High DHCR24 gene expression in transfectants did not result in a higher resistance to cytotoxic agents; treatment with U18666A was cytotoxic in S/P cells with a lower DHCR24 content and showed additive cytotoxic effect only when associated with H2O2 and not with cysplatin or etoposide, indicating that the DHCR24 protective effect is exerted through an oxidative stress-specific mechanism.     

Stromal fibroblasts synergize with hypoxic oxidative stress to enhance melanoma aggressiveness

On the basis of recent advances indicating a key role of microenvironment for tumor progression, we investigated the role of fibroblasts, macrophages and hypoxia, for primary melanoma aggressiveness. Our data indicate a key role of hypoxia in stromal reactivity, acting on both myofibroblasts and machrophages differentiation. Hypoxic myofibroblasts are more active than macrophages in inducing melanoma invasiveness and exploit their oxidative stress due to hypoxia to secrete soluble factors favouring melanoma invasion and chemotaxis. We underscore the key role of microenviroment on melanoma malignancy, highlighting reactive fibroblasts, intratumoral hypoxia and oxidative stress as promising targets for melanoma antimetastatic strategies.     

Colocalization of MnSOD expression in response to oxidative stress

The loss of manganese superoxide dismutase function has been associated with increased incidence of Barrett's esophagus and esophageal adenocarcinoma. In previous studies, we have demonstrated that loss of MnSOD resulted in severe esophageal damage by both endogenous and exogenous bile. However, the alterative manner of MnSOD in esophageal epithelium is largely unknown. In this study, we investigated the expression and localization of MnSOD in response to the exposure to bile salts in an esophageal epithelial cell line. Het‐1A cells were seeded at 5 × 10⁵ and 10⁷ and incubated with taurocholate, cholate, glycochlate, deoxycholate, and the mixture of these bile salts. Mitochondria and cytoplasma were separated, and the expression and localization of MnSOD was determined by Western blot and immunocytochemical assay. Proliferation rates were strongly inhibited in the groups with taurocholate and bile salts mixture at 4 h, with 0.367 ± 0.042 and 0.396 ± 0.046, respectively, compared to 0.684 ± 0.054 in untreated groups (P < 0.05). An increased apoptotic rate compared to untreated group (3.65 ± 0.59) were significantly increased in taurocholate group and in bile salts mixture group were 33.62 ± 10.25 and 31.52 ± 8.97 at 4 h, respectively (P < 0.05). The protein level of MnSOD in mitochodria was increased at 4 h, but with a decreased enzymatic activity after bile salts treatment. Cytoplasmic MnSOD was detected in the cells with bile salts treatment. Immunocytochemical staining demonstrated that esophageal epithelial cell underwent morphological alteration and MnSOD relocalization after bile salts treatment. This is the first study to demonstrate cellular cytosolic MnSOD expression and that this relocalization to the cytosol is a cause for decreased MnSOD enzymatic activity. This suggests that bile salts may contribute to the dysfunction of mitochondria, by enzymatically inhibiting of MnSOD localization and thus activation in the mitochondria. © 2009 Wiley‐Liss, Inc.     

Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib‐resistant BRAFV600E mutation bearing metastatic melanoma cells

Treatment with vemurafenib, a potent and selective inhibitor of mitogen‐activated protein kinase signaling downstream of the BRAF^V600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib‐resistant metastatic melanomas. We have conducted high‐throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib‐resistant melanoma cell lines, A2058R and A375R (containing the BRAF^V600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E‐BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib‐resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug‐resistant melanomas harboring BRAF ^V600E mutations.     

Genetic determinants of oxidative stress-mediated sensitization of drug-resistant cancer cells

Drug resistance in cancer is an overwhelming problem, because drug-resistant cancer cells are harder to kill with the same drug. The mechanism of drug resistance differs for various cancers based on the type of drug being used for its treatment. Most current drugs are shown to increase reactive oxygen species (ROS) in respective cancer cells that induces apoptosis, but continuous treatment with the same drug may reduce cellular ROS levels and may convert drug sensitive cancer cells into drug resistant cells. In addition, exogenous elevation of ROS in conjunction with drug resensitizes drug-resistant cancer cells. Thus, constant maintenance of higher ROS level in cancer cells may be a prerequisite for drug efficacy in certain type of cancer cells. Thus, modulation of ROS-mediated genetic pathway genes could be an efficient alternative to maintain higher ROS level in cancer cells for "combinational chemotherapy" with the drug. In this review, I discuss whether ROS reduction in drug-resistant cancer cells could be a general mechanism of drug resistance for most cancers with its specific drug, and whether elevation of ROS levels with the drug could be a valuable strategy for increasing drug efficacy in most cancers.     

Fluconazole toxicity is independent of oxidative stress and apoptotic effector mechanisms in Saccharomyces cerevisiae

Fluconazole toxicity was previously shown to require intact mitochondria in Saccharomyces cerevisiae, however, the mechanism of mitochondrial-dependent azole toxicity is unclear. Here we show that fluconazole toxicity is not attenuated by the overexpression of the mitochondrially-acting human antiapoptotic protein Bcl-2 nor is it increased by the presence of the oxidating agents in S. cerevisiae. Our data further support the notion that mitochondrial toxicity in the presence of azoles is modulated by the conversion of ergosterol precursors to toxic sterols.     

Polyethylene glycol derivative 9bw suppresses growth of neuroblastoma cells by inhibiting oxidative phosphorylation

Neuroblastoma (NB) is a childhood malignancy originating from the sympathetic nervous system, and accounts for approximately 15% of all pediatric cancer‐related deaths. As the 5‐y survival rate of patients with high‐risk NB is <50%, novel therapeutic strategies for NB patients are urgently required. Nonaethylene glycol mono(′4‐iodo‐4‐biphenyl)ester (9bw) is a polyethylene glycol derivative, synthesized by modifying a compound originally extracted from filamentous bacteria. Although 9bw shows remarkable inhibition of tumor cell growth, the underlying mechanisms remain unclear. Here, we examined the efficacy of 9bw on human NB‐derived cells, and investigated the molecular mechanisms underlying the cytotoxic effects of 9bw on these cells. Our results indicated that 9bw induced cell death in NB cells by decreasing the production of ATP. Metabolome analysis and measurement of oxygen consumption indicated that 9bw markedly suppressed oxidative phosphorylation (OXPHOS). Further analyses indicated that 9bw inhibited the activity of mitochondrial respiratory complex I. Moreover, we showed that 9bw inhibited growth of NB in vivo. Based on the results of the present study, 9bw is a good candidate as a novel agent for treatment of NB.     

姜黄素抑制乳腺癌MCF-7细胞增殖及其相关的氧化应激机制

目的：探讨姜黄素抑制乳腺癌MCF7细胞增殖及其相关的氧化应激机制。方法：用不同浓度(5～40 μmol/L) 的姜黄素作用于乳腺癌细胞株MCF7细胞,以MTT法检测在6、12、24、48 h的细胞增殖率,流式细胞术检测细胞凋亡率,DCFHDA染色流式检测细胞内活性氧(reactive oxygen species ROS)生成变化,黄嘌呤氧化酶法检测氧化物歧化酶（superoxide dismutase,SOD）,可见光分光光度计法检测过氧化氢酶（catalase, CAT）活性。结果：低浓度(5、10 μmol/L) 姜黄素作用后MCF7细胞增殖受抑制（P<0.01）,但无凋亡发生;伴随细胞内ROS短暂升高后随时间逐步下降,SOD、CAT先下降后逐步提高。高浓度（20、40 μmol/L）姜黄素作用后细胞增殖抑制率明显增加（P<0.01）,细胞出现大量凋亡;伴随细胞内ROS即刻明显升高,随时间延长无明显下降;SOD、CAT明显下降（P<0.01）。结论：姜黄素具有剂量依赖的抗氧化与促氧化双重作用,不同浓度的姜黄素通过对乳腺癌细胞内氧化还原状态的调节参与了其抗肿瘤细胞增殖作用的机制。