Selective apoptosis induction by the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide is achieved by modulating mitochondrial bioenergetics in premalignant and malignant human prostate epithelial cells

Prostate tumorigenesis is coupled with an early metabolic switch in transformed prostate epithelial cells that effectively increases their mitochondrial bioenergetic capacity. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) inhibits prostate cancer development in vivo, and triggers reactive oxygen species (ROS)-dependent prostate cancer cell apoptosis in vitro. The possibility that 4HPR-induced ROS production is associated with mitochondrial bioenergetics and required for apoptosis induction in transformed prostate epithelial cells in vitro would advocate a prospective mechanistic basis for 4HPR-mediated prostate cancer chemoprevention in vivo. We investigated this tenet by comparing and contrasting 4HPR’s effects on premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. 4HPR promoted a dose- and/or time-dependent apoptosis induction in PWR-1E and DU-145 cells, which was preceded by and dependent on an increase in mitochondrial ROS production. In this regard, the PWR-1E cells were more sensitive than the DU-145 cells, and they consumed roughly twice as much oxygen as the DU-145 cells suggesting oxidative phosphorylation was higher in the premalignant cells. Interestingly, increasing the [Ca²⁺] in the culture medium of the PWR-1E cells attenuated their proliferation as well as their mitochondrial bioenergetic capacity and 4HPR’s cytotoxic effects. Correspondingly, the respiration-deficient derivatives (i.e., ρ⁰ cells lacking mitochondrial DNA) of DU-145 cells were markedly resistant to 4HPR-induced ROS production and apoptosis. Together, these observations implied that the reduction of mitochondrial bioenergetics protected PWR-1E and DU-145 cells against the cytotoxic effects of 4HPR, and support the concept that oxidative phosphorylation is an essential determinant in 4HPR’s apoptogenic signaling in transformed human prostate epithelial cells.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.     

Involvement of Akt2/protein kinase B β (PKBβ) in the 8‐Cl‐cAMP‐induced cancer cell growth inhibition

8‐chloro‐cyclic AMP (8‐Cl‐cAMP), which induces differentiation, growth inhibition, and apoptosis in various cancer cells, has been investigated as a putative anti‐cancer drug. However, the exact mechanism of 8‐Cl‐cAMP functioning in cancer cells is not fully understood. Akt/protein kinase B (PKB) genes (Akt1, Akt2, and Akt3) encode enzymes belonging to the serine/threonine‐specific protein kinase family. It has been suggested that Akt/PKB enhances cell survival by inhibiting apoptosis. Recently, we showed that 8‐Cl‐cAMP and 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) inhibited cancer cell growth through the activation of AMPK and p38 MAPK. Therefore, we anticipated that the phosphorylation of Akt/PKB would be decreased upon treatment with 8‐Cl‐cAMP. However, treatment with 8‐Cl‐cAMP and AICAR induced the phosphorylation of Akt/PKB, which was inhibited by ABT702 (an adenosine kinase inhibitor) and NBTI (an adenosine transporter inhibitor). Furthermore, whereas Compound C (an AMPK inhibitor), AMPK‐DN (AMPK‐dominant negative) mutant, and SB203580 (a p38 MAPK inhibitor) did not block the 8‐Cl‐cAMP‐induced phosphorylation of Akt/PKB, TCN (an Akt1/2/3 specific inhibitor) and an Akt2/PKBβ‐targeted siRNA inhibited the 8‐Cl‐cAMP‐ and AICAR‐mediated phosphorylation of AMPK and p38 MAPK. TCN also reversed the growth inhibition mediated by 8‐Cl‐cAMP and AICAR. Moreover, an Akt1/PKBα‐targeted siRNA did not reduce the phosphorylation of AMPK and p38 MAPK after treatment with 8‐Cl‐cAMP. These results suggest that Akt2/PKBβ activation promotes the phosphorylation of AMPK and p38 MAPK during the 8‐Cl‐cAMP‐ and AICAR‐induced growth inhibition. J. Cell. Physiol. 228: 890–902, 2013. © 2012 Wiley Periodicals, Inc.     

紫丁香苷调控PI3K/Akt/mTOR信号通路诱导乳腺癌细胞凋亡的研究

目的 研究紫丁香苷的抗乳腺癌作用及分子机制，为紫丁香苷的临床应用提供理论依据。方法 MTT检测紫丁香苷对乳腺癌细胞增殖的抑制作用；台盼蓝、TUNEL和Annexin V-FITC/PI染色检测细胞的凋亡状况，Western bolt检测Caspase-3的活化情况，判断细胞凋亡是否发生；检测凋亡相关蛋白B淋巴细胞瘤2（Bcl-2）的表达，结合JC-1染色探讨紫丁香苷对线粒体凋亡途径的影响；运用PI3K激动剂Recilisib做对比，qRT-PCR和Western bolt检测紫丁香苷调控PI3K/Akt/mTOR通路诱导癌细胞凋亡的作用。结果 紫丁香苷对乳腺癌细胞的增殖具有时间和剂量依赖的抑制作用，能诱导癌细胞发生凋亡。进一步研究发现，紫丁香苷处理后，细胞内Caspase-3被激活，Bcl-2表达下降，线粒体膜电位明显丧失，PI3K、Akt和mTOR的mRNA与蛋白质水平表达无明显变化，但蛋白质磷酸化水平明显下降；Recilisib处理后部分抵消了紫丁香苷对乳腺癌细胞凋亡的作用。结论 紫丁香苷对乳腺癌细胞MDA-MB-231和MCF-7具有良好的抑制作用，其通过抑制PI3K/Akt/mTOR信号通路的活化来抑制细胞增殖并诱导细胞发生线粒体途径的凋亡。紫丁香苷是具有开发潜力的抗乳腺癌药物。     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin,doxorubicin and taxol

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.     

Inhibitory effects of Bcl-2 on mitochondrial respiration

In contrast to the well-established anti-apoptotic effect of Bcl-2 protein, we have recently demonstrated that Bcl-2 overexpression by vaccinia virus causes apoptosis in BSC-40 cells, while it prevents apoptosis in HeLa G cells. Given the key role of mitochondria in the process of apoptosis, we focused on effects of Bcl-2 expression on mitochondrial energetics of these two cell lines. In this study we present data indicating that BSC-40 cells derive their ATP mainly from oxidative phosphorylation whereas HeLa G cells from glycolysis. More importantly, we show that in both cell lines, Bcl-2 inhibits mitochondrial respiration and causes a decrease of the ATP/ADP ratio. However, it appears that BSC-40 cells cannot sustain this decrease and die, while HeLa G cells survive, being adapted to the low ratio of ATP/ADP maintained by glycolysis. Based on this observation, we propose that the outcome of Bcl-2 expression is determined by the type of cellular ATP synthesis, namely that Bcl-2 causes apoptosis in cells relying on oxidative phosphorylation.     

Galphaq/11 signaling induces apoptosis through two pathways involving reduction of Akt phosphorylation and activation of RhoA in HeLa cells

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.     

NLRX1 regulates TNF-α-induced mitochondria-lysosomal crosstalk to maintain the invasive and metastatic potential of breast cancer cells

An increased level of proinflammatory cytokines, including TNF-α in tumor microenvironment regulates the bioenergetic capacity, immune evasion and survival of cancer cells. Emerging evidences suggest that mitochondrial immune signaling proteins modulates mitochondrial bioenergetic capacity, in addition to the regulation of innate immune response. The optimal oxidative phosphorylation (OxPhos) capacity is required for the maintenance of functional lysosomes and autophagy flux. NLRX1, a mitochondrial NOD family receptor protein, regulates mitochondrial function during apoptosis and tissue injury. However, its role in regulation of mitochondrial and lysosomal function to modulate autophagy flux during inflammatory conditions is not understood. In the current study, we investigated the role of NLRX1 in modulating TNF-α induced autophagy flux and mitochondrial turnover and its implication in regulating the invasive and metastatic capability of breast cancer cells. Expression analyses of clinical breast cancer samples and meta-analysis of multiple public databases revealed that NLRX1 expression is significantly increased in basal-like and metastatic breast carcinoma as compared to non-basal-like and primary breast cancer. Depletion of NLRX1 expression in triple-negative breast cancer cells, altered the organization and activity of OxPhos complexes in presence of TNF-α. NLRX1 depletion further impaired lysosomal function and hence the turnover of damaged mitochondria through mitophagy in presence of TNF-α. Importantly, loss of NLRX1 decreased OxPhos-dependent cell proliferation and migration ability of triple-negative breast cancer cells in presence of TNF-α. These evidences suggest an essential role of NLRX1 in maintaining the crosstalk of mitochondrial metabolism and lysosomal function to regulate invasion and metastasis capability of breast cancer cells.     

Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells

We explored the role of low mitochondrial membrane potential (DeltaPsim) and the lack of oxidative phosphorylation in apoptosis by assessing the susceptibility of osteosarcoma cell lines with and without mitochondrial DNA to staurosporine-induced death. Our cells without mitochondrial DNA had low DeltaPsim and no functional oxidative phosphorylation. Contrary to our expectation, these cells were more resistant to staurosporine-induced death than were the parental cells. This reduced susceptibility was associated with decreased activation of caspase 3 but not with the mitochondrial permeability transition pore or cytochrome c release from the mitochondria. Apoptosis in both cell lines was associated with an increase in DeltaPsim. Bcl-x(L) could protect both cell types against caspase 3 activation and apoptosis by a mechanism that does not appear to be mediated by mitochondrial function or modulation of DeltaPsim. Nevertheless, we found that Bcl-x(L) expression can stimulate cell respiration in cells with mitochondrial DNA. Our results showed that the lack of functional oxidative phosphorylation and/or low mitochondrial membrane potential are associated with an antiapoptotic effect, possibly contributing to the development of some types of cancer. It also reinforces a model in which Bcl-x(L) can exert an antiapoptotic effect by stimulating oxidative phosphorylation and/or inhibiting caspase activation.     

In vitro identification of mitochondrial oxidative stress production by time-resolved fluorescence imaging of glioma cells

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker? Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.     

Essential roles of mitochondrial and heme function in lung cancer bioenergetics and tumorigenesis

Contrary to Warburg’s hypothesis, mitochondrial oxidative phosphorylation (OXPHOS) contributes significantly to fueling cancer cells. Several recent studies have demonstrated that radiotherapy-resistant and chemotherapy-resistant cancer cells depend on OXPHOS for survival and progression. Several cancers exhibit an increased risk in association with heme intake. Mitochondria are widely known to carry out oxidative phosphorylation. In addition, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in OXPHOS and oxygen consumption. Further, lung cancer cells exhibit heme accumulation and require heme for proliferation and invasion in vitro. Abnormalities in mitochondrial biogenesis and mutations are implicated in cancer. This review delves into mitochondrial OXPHOS and lesser explored area of heme metabolism in lung cancer.     

Metformin rescues cell surface major histocompatibility complex class I (MHC-I) deficiency caused by oncogenic transformation

Active avoidance by tumor cells from attack and elimination by immune cells is an emerging cancer hallmark that is achieved primarily through decreasing the levels of major histocompatibility complex class I (MHC-I) at the cancer cells’ surface. Deficiencies in MHC-I antigen-restricted immunosurveillance may be intertwined with an altered, Warburg-like cancer cell-intrinsic metabolism, another emerging hallmark of cancer that involves a switch from mitochondrial respiration to glycolysis to efficiently support large-scale biosynthetic programs that are required for active cell proliferation. We recently envisioned that intervention strategies aimed at reversing the bioenergetic signature of cancer cells (e.g., the antidiabetic biguanide metformin) should correct oncogene (e.g., HER2)-driven MHC-I defects, thus preventing immune escape of oncogene transformants. First, we explored how metformin treatment impacted mitochondrial biogenesis in cultured breast cancer cells overexpressing the membrane tyrosine kinase receptor HER2, the best-characterized downregulator of MHC-I. Metformin exposure was found to dose-dependently increase the expression levels of cytochrome c oxidase I and mitochondrial succinate dehydrogenase, which are encoded by mitochondrial and nuclear DNA, respectively. Second, we explored whether metformin-enhanced mitochondrial biogenesis might significantly alter the MHC-I status in breast carcinoma cells. MHC-I expression, as assessed by flow cytometry using an anti-HLA-ABC monoclonal antibody, was fully restored (up to ~25-fold upregulation) in MHC-I-negative HER2 gene-amplified carcinoma cells. These findings may help delineate a previously unrecognized mechanism through which metformin (and metformin-like drugs) may enable a cancer patient’s own immune system to mount an efficient anti-metastasis response that can prevent or delay disease recurrence. Restored antigenicity and immunogenicity of tumor cells may represent a previously unrecognized primary mode of action underlying the cancer-preventive effects of metformin.