Correction: Crosstalk from Non-Cancerous Mitochondria Can Inhibit Tumor Properties of Metastatic Cells by Suppressing Oncogenic Pathways.

[This corrects the article DOI: 10.1371/journal.pone.0061747.].

Crosstalk from non-cancerous mitochondria can inhibit tumor properties of metastatic cells by suppressing oncogenic pathways.

Mitochondrial-nucleus cross talks and mitochondrial retrograde regulation can play a significant role in cellular properties. Transmitochondrial cybrid systems (cybrids) are an excellent tool to study specific effects of altered mitochondria under a defined nuclear background. The majority of the studies using the cybrid model focused on the significance of specific mitochondrial DNA variations in mitochondrial function or tumor properties. However, most of these variants are benign polymorphisms without known functional significance. From an objective of rectifying mitochondrial defects in cancer cells and to establish mitochondria as a potential anticancer drug target, understanding the role of functional mitochondria in reversing oncogenic properties under a cancer nuclear background is very important. Here we analyzed the potential reversal of oncogenic properties of a highly metastatic cell line with the introduction of non-cancerous mitochondria. Cybrids were established by fusing the mitochondria DNA depleted 143B TK- ρ0 cells from an aggressive osteosarcoma cell line with mitochondria from benign breast epithelial cell line MCF10A, moderately metastatic breast cancer cell line MDA-MB-468 and 143B cells. In spite of the uniform cancerous nuclear background, as observed with the mitochondria donor cells, cybrids with benign mitochondria showed high mitochondrial functional properties including increased ATP synthesis, oxygen consumption and respiratory chain activities compared to cybrids with cancerous mitochondria. Interestingly, benign mitochondria could reverse different oncogenic characteristics of 143B TK(-) cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, and colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis suggested that several oncogenic pathways observed in cybrids with cancer mitochondria are inhibited in cybrids with non-cancerous mitochondria. These results suggest the critical oncogenic regulation by mitochondrial-nuclear cross talk and highlights rectifying mitochondrial functional properties as a promising target in cancer therapy.

Transmitochondrial cybrids: tools for functional studies of mutant mitochondria.

Mitochondrial functions are controlled by both mitochondrial DNA (mtDNA) and nuclear DNA. Hence, it is difficult to identify whether mitochondrial or nuclear genome is responsible for a particular mitochondrial defect. Cybrid is a useful tool to overcome this difficulty, where we can compare mitochondria from different sources in a defined nuclear background. Cybrids are constructed by fusing enucleated cells harboring wild type or altered mtDNA of interest with ρ(0) cells (cells lacking mtDNA) in which the endogenous mtDNA has been depleted. Therefore, cybrids are very useful in studying consequences of mtDNA alterations or other mitochondrial defects at the cellular level by excluding the influence of nuclear DNA mutations.

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties.

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.

The mechanical stress-activated serum-, glucocorticoid-regulated kinase 1 contributes to neointima formation in vein grafts.

RATIONALE: Mechanical stress plays an important role in proliferation of venous smooth muscle cells (SMCs) in neointima, a process of formation that contributes to failure of vein grafts. However, it is unknown what intracellular growth signal leads to proliferation of venous SMCs. OBJECTIVE: The objective of this study is to identify mechanisms of mechanical stretch on neointima formation. METHODS AND RESULTS: By a microarray analysis, we found that mechanical cyclic stretch (15% elongation) stimulated the transcription of SGK-1 (serum-, glucocorticoid-regulated kinase-1). Mechanical stretch-induced SGK-1 mRNA expression was blocked by actinomycin D. The mechanism for the SGK-1 expression involved MEK1 but not p38 or JNK signaling pathway. SGK-1 activation in response to stretch is blocked by insulin-like growth factor (IGF)-1 receptor inhibitor and mammalian target of rapamycin complex (mTORC)2 inhibitor (Ku-0063794) but not mTORC1 inhibitor (rapamycin). Mechanical stretch-induced bromodeoxyuridine incorporation was reduced by 83.5% in venous SMCs isolated from SGK-1 knockout mice. In contrast, inhibition of Akt, another downstream signal of PI3K resulted in only partial inhibition of mechanical stretch-induced proliferation of venous SMCs. Mechanical stretch also induced phosphorylation and nuclear exportation of p27(kip1), whereas knockout of SGK-1 attenuated this effect of mechanical stretch on p27(kip1). In vivo, we found that placement of a vein graft into artery increased SGK-1 expression. Knockout of SGK-1 effectively prevented neointima formation in vein graft. There is significant lower level of p27(kip1) located in the nucleus of neointima cells in SGK-1 knockout mice compared with that of wild-type vein graft. In addition, we also found that wire injury of artery or growth factors in vitro increased expression of SGK-1. CONCLUSIONS: These results suggest that SGK-1 is an injury-responsive kinase that could mediate mechanical stretch-induced proliferation of vascular cells in vein graft, leading to neointima formation.

Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids.

Reprogramming of energy metabolism is one of the hallmarks of cancer. In normal conditions, cells rely on mitochondrial oxidative phosphorylation to provide energy for cellular activities. Cancer cells are characterized by increased glycolysis and reduced mitochondrial respiratory function. In the past decade, somatic mitochondrial DNA alterations are found to be common in all types of cancers. However, the functional significance of the altered cancer mitochondria is largely unknown. This is because the bulk of cancer properties are regulated by nuclear encoded genes. To overcome this problem, the transmitochondrial cybrid system, which allows the study of the effect of cancer mitochondria in a common nuclear background, has been used. Here we review the accumulating evidence that altered cancer mitochondria affect the respiratory chain function and oncogenic properties in vitro and in vivo using cybrid technologies.

Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis.

The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P < 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

Mitochondrial dysfunction in human breast cancer cells and their transmitochondrial cybrids.

Somatic mitochondrial DNA alterations have been found in all types of cancer. To better understand the role of mitochondria and their involvement in the pathogenic mechanisms of cancer development, the effects of cancer mitochondria were investigated in a defined nuclear background using a transmitochondrial cybrid system. Our results demonstrated that cancer mitochondria confer a significant reduction in cell growth when cells are metabolically stressed in a galactose medium. Activities of the respiratory chain complexes, cellular oxygen consumption, and ATP synthesis rates were found to be much lower in breast cancer cells, than those in normal breast epithelial cells of MCF-10A (10A). These results suggest that there is reduced mitochondrial function in the studied breast cancer cell lines. Similarly reduced mitochondrial function was observed in cybrids containing cancer mitochondria. Novel tRNA mutations were also identified in two breast cancer cell lines, possibly responsible for the observed mitochondrial dysfunction. We conclude that altered mitochondria in cancer cells may play a crucial role in tumor development.

Angiotensin II stimulates transcription of insulin-like growth factor I receptor in vascular smooth muscle cells: role of nuclear factor-kappaB.

Increased expression of the IGF-I receptor (IGF-IR) is associated with proliferation and survival of vascular smooth muscle cells (VSMCs). In cultured VSMCs, we reported that angiotensin II (Ang II) increases transcription and expression of IGF-IR. Now, we show that mesenteric arteries of rats infused with Ang II develop thickening and increased IGF-IR expression. To determine how Ang II transcriptionally regulates IGF-IR expression in VSMCs, we generated 5'-end deletions of the IGF-IR promoter and measured Ang II-induced promoter-luciferase activity in VSMCs. Activities from these promoter sequences suggested that the Ang II-responsive region is located between -270 and -135 of the IGF-IR promoter. Using a DNase I foot printing analysis, we identified two putative nuclear factor-kappaB (NF-kappaB)-like sequences located in the same region of the IGF-IR promoter. When we mutated either of these NF-kappaB-like sites, Ang II-induced IGF-IR promoter activity decreased sharply. Electrophoretic mobility gel shift, anti-p50 of NF-kappaB supershift and chromatin immunoprecipitation assays demonstrated that both the p65 and p50 subunits of NF-kappaB will bind to this Ang II response element in the IGF-IR promoter. When we blocked the Ras/MAPK kinase 1 pathway or the inhibitory-kappaB kinase pathway, both Ang II-induced IGF-IR promoter activity and expression of IGF-IR protein significantly declined. Our results indicate that the mechanism by which Ang II stimulates IGF-IR expression in VSMCs involves NF-kappaB binding to NF-kappaB sites in the IGF-IR promoter, leading to expression of IGF-IR through both Ras/MAPK kinase 1-and inhibitory-kappaB kinase-dependent pathways. Because IGF-IR is a major factor associated with thickening of mesenteric vessels, our results provide potential therapeutic targets.

Dual pathways for nuclear factor kappaB activation by angiotensin II in vascular smooth muscle: phosphorylation of p65 by IkappaB kinase and ribosomal kinase.

Activation of nuclear factor (NF)-kappaB by angiotensin II (Ang II) plays an essential role in stimulating expression of vascular adhesion molecules, which are essential for vascular inflammation. We report that Ang II activates NF-kappaB by phosphorylating its p65 subunit via a pathway mediated partially by ribosomal S6 kinase (RSK). In investigating other pathway(s) that may be involved, we found that the ability of Ang II to activate NF-kappaB in mouse embryonic fibroblast is suppressed (approximately 70%) either by deletion of IkappaB Kinase (IKK) or by inhibiting or knocking down IKK in vascular smooth muscle cells using a dominant-negative IKK adenovirus or small interference RNA to IKKbeta. Thus, Ang II also stimulates NF-kappaB via IKK. In vitro, we found that Ang II stimulates IKK to phosphorylate myelin basic protein and the p65 subunit of NF-kappaB. The mechanism by which Ang II activates IKK is to increase phosphorylation of IKKbeta in its activation loop (Ser181) rather than IkappaB phosphorylation. Inhibiting both the RSK and IKK pathways completely blocks the Ang II-induced p65 phosphorylation and NF-kappaB activation. These 2 pathways are independent: inhibiting IKK does not block Ang II-induced phosphorylation of RSK, whereas inhibiting mitogen-activated protein kinase 1 does not affect phosphorylation of IKK. Finally, we found that Ang II can induce expression of vascular adhesion molecules by 2 pathways; both IKK and RSK lead to phosphorylation of the p65 subunit of NF-kappaB to increase vascular cell adhesion molecule-1 transcription. The 2 pathways are functionally important because inhibiting IKK and RSK in vascular smooth muscle cells blocks Ang II-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to limit vascular inflammation.

A new cellular signaling mechanism for angiotensin II activation of NF-kappaB: An IkappaB-independent, RSK-mediated phosphorylation of p65.

OBJECTIVE: Angiotensin II (Ang II) promotes vascular inflammation and remodeling via activation of nuclear factor kappaB (NF-kappaB)-mediated transcription of proinflammatory genes such as interleukin-6 (IL-6). We examined the signaling mechanism whereby Ang II activates NF-kappaB in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Ang II treatment did not increase phosphorylation of inhibitor of kappaBalpha (IkappaBalpha) or IkappaBbeta or decrease their levels. In contrast, mitogen-activated protein kinase kinase-1 (MEK1) inhibition (dominant-negative MEK1 adenovirus or inhibitor U0126) suppressed Ang II-induced NF-kappaB promoter activity, NF-kappaB DNA-binding activity, p65 phosphorylation, and led to 70% reduction in IL-6 transcription/production. The mechanism involved Ang II activation of Ras and MEK1. Signaling distal to MEK1 involved extracellular signal-regulated kinase (ERK) because inhibition of MEK1 suppressed the Ang II-induced activation of ribosomal S6 kinase (RSK), a substrate of ERK. Downregulation of RSK by small interfering RNA (SiRNA) in VSMCs was found to suppress Ang II-induced activation of NF-kappaB and p65 phosphorylation. Immunopurified RSK from Ang II-treated VSMCs phosphorylated recombinant glutathione S-transferase-p65 in vitro. CONCLUSIONS: We uncovered a nonclassical signaling pathway (Ras/MEK1/ERK/RSK) from Ang II to activation of NF-kappaB, a mechanism by which Ang II stimulates RSK-mediated phosphorylation of p65 to participate in vascular inflammation.

[Isolation of E1A-related drug-sensitive new genes by suppression subtractive hybridization].

BACKGROUND & OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug-sensitivity of tumor cells to several antitumor agents, and also increase the radio-sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer-related genes,which were differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH). METHODS: To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed-gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over-expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over-expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi-quantitative RT-PCR analysis, and the difference of mRNA expression was approximately 3-8 times. CONCLUSION: Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full- length genes and studying their functions.

[Selective myelo-protection by MDR1 and MnSOD genes regulated by a specific promoter].

OBJECTIVE: To study the specific protection of myeloid cells from chemotherapeutic agents and radiation. METHODS: The recombinant retroviral vectors containing MDR1 gene and MnSOD gene regulated by APN myeloid promoter were constructed and introduced into myeloblastic cell line KG1a and hepatoma cell line BEL7402. The resistance of the cells to antitumor drugs and radiation were analyzed by cell survival assay. In vivo, the murine bone marrow cells were isolated and infected by the retroviral particles, which were transplanted into recipient mouse treated with paclitaxel or X-ray. The murine white blood cell (WBC) was counted in order to assay the effects of MDR1 or MnSOD gene on hematopoiesis in the course of chemotherapy and radiotherapy. RESULTS: The resistance to chemotherapeutic agents such as cochicine, Vp-16, vincristine, doxorubcin and paclitaxel were elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in KG1a cell line transduced with MDR1 gene. The resistance to radiation increased 3.7 folds at the dose of 10 Gy compared with parental cells in KGla cell line transduced with MnSOD gene derived by APN promoter. In contrast, the chemosensitivity and the radiosensitivity showed no significant change in BEL 7402 cell line transduced with MDR1 gene and MnSOD gene. In vivo, the WBC counts in the mouse introduced with MDR1 gene or MnSOD gene were higher than those in the control mouse (P < 0.01). CONCLUSION: The expression of MDR1 gene and MnSOD gene regulated by APN myeloid promoter is effective on myelo-specific protection without enhancing the resistance of tumor cells in vitro. The hematopoiesis can be reconstituted in vivo during anticancer drug or radiation treatment. This study may provide experimental evidence and new clues for myeloprotection of cancer patients being treated with chemotherapy and/or radiotherapy.

Expression of adenovirus type 5 E1A in the methylotrophic yeast Pachia pastoris and the inhibitory effect on S-180 tumor growth.

The human adenovirus type 5 (Ad5) early-region 1A (E1A) proteins have been shown to have strong tumor-suppressive activities in human tumor cells and to enhance the sensitivity of a variety of malignant tumors to apoptosis induced by ionizing radiation and chemotherapeutic agents. However, the inherent limitations of E1A gene therapy prevent its application, such as the efficiency of expression, precision of targeting, and toxicity of vector. This prompted us to construct an E1A expression vector (pPIC9/E1A) and express the E1A protein in the methylotrophic yeast Pichia pastoris. The E1A protein was purified using two steps of ion-exchange column chromatography on HiTrap Q and HiTrap SP. The analysis indicated that the E1A protein/liposome inhibited S-180 tumor growth and also rendered the S-180 tumor strongly susceptible to the anticancer drug bleomycin in vivo. Furthermore, tunnel assay clearly revealed that the mechanism was induction of cellular apoptosis. Importantly, the E1A protein overcame the limitations of gene therapy. Thus the E1A protein may be a useful therapeutic agent for some malignant tumors.

[MnSOD gene regulated by aminopeptidase N promoter specifically protects bone marrow from radiation].

BACKGROUND & OBJECTIVE: It is an effective way to induce radio-tolerant gene into hematopoietic cells in bone marrow for overcoming the suppression of radiotherapy on hematopoietic system. However, this also increases the radiation tolerance of tumor cells. This study was designed to investigate a method to specifically protect bone marrow cell from being damaged by radiation, along without increasing resistance of tumor cell to radiation. METHODS: The retrovirus vector of manganese superoxide dismutase (MnSOD) gene regulated by aminopeptidase N (APN) bone marrow-specific gene promoter was constructed and induced into myeloblastic KG1a and cancer cell BEL7402. MnSOD mRNA level was analyzed by PT-PCR; MnSOD activity in the cells was determined; the sensitivity of bone marrow cell and hepatic carcinoma cell to x-ray was detected by cell survival test; the cell apoptosis was analyzed with flow cytometry and fractural DNA electrophoresis. RESULTS: The MnSOD mRNA level and enzyme activity in KG1a cells transferred with the gene was obviously increased. Expression of MnSOD mRNA drove by APN myelo-specific promoter effectively inhibited apoptosis of KG1a cells induced by radiation and endowed KG1a cell line with the enhancement of tolerance to radiation, which increased by 3.7 folds compared to parental cells at the dose of 10 Gy. In contrast, the level of MnSOD mRNA, the enyme activity of MnSOD and the radiosensitivity had no significant change in BEL 7402 cells transduced with MnSOD gene. CONCLUSION: APN bone marrow-specific promoter could control MnSOD gene expression highly in myeloid cell and lower in cancer cell. In the process of killing of cancer cell by x-ray, MnSOD gene regulated by APN bone marrow-specific promoter could specifically protect myeloid cell. This study provides a new clue to solve the bone marrow suppression in high dose radiotherapy.