Mitochondrial alteration in malignantly transformed human small airway epithelial cells induced by α-particles.

Human small airway epithelial cells (SAECs) immortalized with human telomerase reverse transcriptase were exposed to either a single or multiple doses of α-particles. Irradiated cells showed a dose-dependent cytotoxicity and progressive neoplastic transformation phenotype. These included an increase in saturation density of growth, a greater resistance to N-phosphonoacetyl-L-aspartate, faster anchorage-independent growth, reinforced cell invasion and c-Myc expression. In addition, the transformed cells formed progressively growing tumors upon inoculation into athymic nude mice. Specifically, α-irradiation induced damage to both mitochondrial DNA (mtDNA) and mitochondrial functions in transformed cells as evidenced by increased mtDNA copy number and common deletion, decreased oxidative phosphorylation activity as measured by cytochrome C oxidase (COX) activity and oxygen consumption. There was a linear correlation between mtDNA copy number, common deletion, COX activity and cellular transformation represented by soft agar colony formation and c-Myc expression. These results suggest that mitochondria are associated with neoplastic transformation of SAEC cells induced by α-particles, and that the oncogenesis process may depend not only on the genomes inside the nucleus, but also on the mitochondrial DNA outside the nucleus.

Sodium arsenite exposure inhibits AKT and Stat3 activation, suppresses self-renewal and induces apoptotic death of embryonic stem cells.

Sodium arsenite exposure at concentration >5 µM may induce embryotoxic and teratogenic effects in animal models. Long-term health effects of sodium arsenite from contaminated drinking water may result in different forms of cancer and neurological abnormalities. As cancer development processes seem to be originated in stem cells, we have chosen to examine the effects of sodium arsenite on signaling pathways and the corresponding transcription factors that regulate cell viability and self-renewal in mouse embryonic stem cells (ESC) and mouse neural stem/precursor cells. We demonstrated that the crucial signaling pathway, which was substantially suppressed by sodium arsenite exposure (4 µM) in ESC, was the PI3K-AKT pathway linked with numerous downstream targets that control cell survival and apoptosis. Furthermore, the whole core transcription factor circuitry that control self-renewal of mouse ESC (Stat3-P-Tyr705, Oct4, Sox2 and Nanog) was strongly down-regulated by sodium arsenite (4 µM) exposure. This was followed by G2/M arrest and induction of the mitochondrial apoptotic pathway that might be suppressed by caspase-9 and caspase-3 inhibitors. In contrast to mouse ESC with very low endogenous IL6, mouse neural stem/precursor cells (C17.2 clone immortalized by v-myc) with high endogenous production of IL6 exhibited a strong resistance to cytotoxic effects of sodium arsenite that could be decreased by inhibitory anti-IL6 antibody or Stat3 inhibition. In summary, our data demonstrated suppression of self-renewal and induction of apoptosis in mouse ESC by sodium arsenite exposure, which was further accelerated due to simultaneous inhibition of the protective PI3K-AKT and Stat3-dependent pathways.

The role of TGFBI in mesothelioma and breast cancer: association with tumor suppression.

BACKGROUND: Transforming growth factor ß induced (TGFBI) product, an extracellular matrix (ECM) protein, has been implicated as a putative tumor suppressor in recent studies. Our previous findings revealed that expression of TGFBI gene is down-regulated in a variety of cancer cell lines and clinical tissue samples. In this study, ectopic expression of TGFBI was used to ascertain its role as a tumor suppressor and to determine the underlying mechanism of mesothelioma and breast cancer. METHODS: Cells were stably transfected with pRc/CMV2-TGFBI and pRc/CMV2-empty vector with Lipofectamine Plus. Ectopic expression of TGFBI was quantified by using quantitative PCR and Western-blotting. Characterization of cell viability was assessed using growth curve, clonogenic survival and soft agar growth. The potential of tumor formation was evaluated by an in vivo mouse model. Cell cycle was analyzed via flow cytometry. Expressions of p21, p53, p16 and p14 were examined using Western-blotting. Senescent cells were sorted by using a Senescence ß-Galactosidase Staining Kit. Telomerase activity was measured using quantitative telomerase detection kit. RESULTS: In this study, an ectopic expression of TGFBI in two types of cancer cell lines, a mesothelioma cell line NCI-H28 and a breast cancer cell line MDA-MB-231 was found to have reduced the cellular growth, plating efficiency, and anchorage-independent growth. The tumorigenicity of these cancer cell lines as determined by subcutaneous inoculation in nude mice was similarly suppressed by TGFBI expression. Likewise, TGFBI expression reduced the proportion of S-phase while increased the proportion of G1 phase in these cells. The redistribution of cell cycle phase after re-expression of TGFBI was correspondent with transiently elevated expression of p21 and p53. The activities of senescence-associated ß-galactosidase and telomerase were enhanced in TGFBI-transfected cells. CONCLUSION: Collectively, these results imply that TGFBI plays a suppressive role in the development of mesothelioma and breast cancer cells, possibly through inhibitions of cell proliferation, delaying of G1-S phase transition, and induction of senescence.

Increased susceptibility of human small airway epithelial cells to apoptosis after long term arsenate treatment.

Inorganic arsenic (arsenate and arsenite) are well known human carcinogens. Apoptosis is a normal biological process that is involved in regulating cell development and differentiation, and is an important protective response to cell injury. The aim of this study was to determine the long term arsenic effect on human small airway epithelial cells (SAEC) by analyzing two distinct apoptosis-inducing agents, Fas ligand (Fas L), which evokes death receptor-mediated apoptosis, and hydrogen peroxide H2O2, which induces apoptosis mediated by reactive oxygen species (ROS). The SAEC were continuously exposed to 0.5 microg/mL arsenic for 28 weeks, and apoptosis was examined after 24 h treatment with either Fas L or H2O2. SAEC displayed decreased cell viability and increased apoptosis after treatment with Fas L and H2O2, compared to non-arsenic treated control cells. Furthermore, treatment of these arsenic-exposed SAEC with Fas L or H2O2 induced cleavage of the DNA damage recognition protein, poly (ADP-ribose) polymerase (PARP), and the 'effector' caspase, Caspase-3, both canonical indicators of apoptosis. We observed increased phosphorylation of p38, a member of the MAP kinase family, following treatment with Fas L or H2O2. To confirm the involvement of p38 in the regulation of apoptosis we pretreated cells with the p38 kinase inhibitor, SB 203580 and observed a significant decrease in apoptosis.

Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2.

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.

Effect of curcumin on irradiated and estrogen-transformed human breast cell lines.

Curcumin (diferuloyl methane) is a well known antioxidant that exerts antiproliferative and apoptotic effects. Curcumin effect was evaluated in a breast cancer model that was developed using the immortalized breast epithelial cell line MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150 keV/µm) of radiation, and subsequently cultured in the presence of 17ß-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) MCF-10F; ii) Estrogen cell line; iii) a malignant Alpha3 cell line; iv) a malignant and tumorigenic, Alpha5 cell line; and v) a cell line derived from Alpha5 injected into the nude mice that gave rise to Tumor2 cell line. Curcumin decreased anchorage-independent growh in transformed breast cancer cell lines in comparison to their counterparts and increased the percentage of cells from G0/G1 with a concomitant increase in G2/M phases, as well as a decrease in PCNA and Rho-A protein expression. Among the oncogenes, c-Ha-Ras and Ras homologous A (Rho-A) are important cell signaling factors for malignant transformation and to reach their active GTP bound state, Ras proteins must first release bound GDP mediated by a guanine nucleotide releasing factor (GRF). Then curcumin decrease RasGRF1 protein expression in malignant cell lines. Further, differential expression levels of cleaved (ADP) ribose polymerase 1 (PARP-1) and phosphorylated histone H2AX (γ-H2AX) were observed after curcumin treatment. It seems that PARP-1 similar to H2AX, confers cellular protection against radiation and estrogen-induced DNA damage mediated by curcumin. Therefore, targeting either PARP-1 or H2AX may provide an effective way of maximizing the therapeutic value of antioxidants for cancer prevention.

Protective role of curcumin in oxidative stress of breast cells.

Curcumin (diferuloylmethane) is a well known antioxidant that exerts anti-proliferative and apoptotic effects. The effects of curcumin were evaluated in a breast cancer model that was developed with the immortalized breast epithelial cell line, MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150 keV/µm) of radiation, and subsequently cultured in the presence of 17ß-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) a control cell line, MCF-10F, ii) an estrogen-treated cell line, named Estrogen, iii) a malignant cell line, named Alpha3 and iv) a malignant and tumorigenic, cell line named Alpha5. Curcumin decreased the formation of hydrogen peroxide in the control MCF-10F, Estrogen and Alpha5 cell lines in comparison to their counterparts. Curcumin had little effect on NFκB (50 kDa) but decreased the protein expression in the Estrogen cell line in comparison to their counterparts. Curcumin enhanced manganese superoxide dismutase (MnSOD) protein expression in the MCF-10F and Alpha3 cell lines. Results indicated that catalase protein expression increased in curcumin treated-Alpha3 and Alpha5 cell lines. Curcumin slightly decreased lipid peroxidation in the MCF-10F cell lines, but significantly (P<0.05) decreased it in the Alpha5 cell line treated with curcumin in comparison to their counterparts as demonstrated by the 8-iso-prostaglandin F2α (8-iso-PGF2α) levels. It can be concluded that curcumin acted upon oxidative stress in human breast epithelial cells transformed by the effect of radiation in the presence of estrogen.

Transforming growth factor-ß-induced protein (TGFBI) suppresses mesothelioma progression through the Akt/mTOR pathway.

As an uncommon cancer, mesothelioma is very hard to treat with a low average survival rate owing to its usual late detection and being highly invasive. The link between asbestos exposure and the development of mesothelioma in humans is unequivocal. TGFBI, a secreted protein that is induced by transforming growth factor-ß in various human cell types, has been shown to be associated with tumorigenesis in various types of tumors. It has been demonstrated that TGFBI expression is markedly suppressed in asbestos-induced tumorigenic cells, while an ectopic expression of TGFBI significantly suppresses tumorigenicity and progression in human bronchial epithelial cells. In order to delineate a potential role of TGFBI in mediating the molecular events that occur in mesothelioma tumorigenesis, we generated stable TGFBI knockdown mutants from the mesothelium cell line Met-5A by using an shRNA approach, and secondly created ectopic TGFBI overexpression mutants from the mesothelioma cell line H28 in which TGFBI is absent. We observed that in the absence of TGFBI, the knockdown mesothelial and mesothelioma cell lines exhibited an elevated proliferation rate, enhanced plating efficiency, increased anchorage-independent growth, as well as an increased cellular protein synthesis rate as compared with their respective controls. Furthermore, cell cycle regulatory proteins c-myc/cyclin D1/phosphor-Rb were upregulated; a more active PI3K/Akt/mTOR signaling pathway was also detected in TGFBI-depleted cell lines. These findings suggest that TGFBI may repress mesothelioma tumorigenesis and progression via the PI3K/Akt signaling pathway.

TGFBI expression reduces in vitro and in vivo metastatic potential of lung and breast tumor cells.

Controversy has arisen as to the role of transforming growth factor-ß-induced protein (TGFBI) in the regulation of tumor metastasis. Using lung and breast cancer cell lines (H522 and MCF-7, respectively), we established that TGFBI induced cell adhesion to extracellular matrix proteins by activating adhesion-associated signaling and subsequent structure reformation, ultimately leading to cells less motile; whereas TGFBI reduced abilities of colony formation in soft agar, penetration through matrix gel, and activation of matrix metalloproteinases 2 and 9. Furthermore, injection of TGFBI-expressing cells into immuno-deficient mice resulted in a significant reduction in tumor metastasis in vivo. Taken together, these data suggest that TGFBI moderates the metastatic potential of cancer cells.

TGFBI deficiency predisposes mice to spontaneous tumor development.

Loss of TGFBI, a secreted protein induced by transforming growth factor-beta, has been implicated in cell proliferation, tumor progression, and angiogenesis by in vitro studies. However, in vivo antitumor functions of TGFBI as well as the underlying molecular mechanism are not well understood. To these aims, we have generated a mouse model with disruption of TGFBI genomic locus. Mice lacking TGFBI show a retarded growth and are prone to spontaneous tumors and 7,12-dimethylbenz(a)anthracene-induced skin tumors. In relation to wild-type (WT) mouse embryonic fibroblasts (MEF), TGFBI(-/-) MEFs display increased frequencies of chromosomal aberration and micronuclei formation and exhibit an enhanced proliferation and early S-phase entry. Cyclin D1 is up-regulated in TGFBI(-/-) MEFs, which correlates with aberrant activation of transcription factor cyclic AMP-responsive element binding protein (CREB) identified by chromatin immunoprecipitation and luciferase reporter assays. TGFBI reconstitution in TGFBI(-/-) cells by either retroviral infection with WT TGFBI gene or supplement with recombinant mouse TGFBI protein in the culture medium leads to the suppression of CREB activation and cyclin D1 expression, and further inhibition of cell proliferation. Cyclin D1 up-regulation was also identified in most of the tumors arising from TGFBI(-/-) mice. Our studies provide the first evidence that TGFBI functions as a tumor suppressor in vivo.

Neoplastic transformation of human small airway epithelial cells induced by arsenic.

Human small airway epithelial cells (SAECs) previously immortalized with human telomerase reverse transcriptase (h-TERT) were continuously treated with sodium arsenite at a dose of 0.5 microg/mL in culture for up to 6 months. Arsenic-treated cells progressively displayed an increase in transformed phenotype including enhanced growth saturation density, plating efficiency, and anchorage-independent growth and invasion capability compared with their nontreated control cells. To determine whether arsenic-induced cell transformation was associated with genomic instability, treated and control cells were also analyzed for micronuclei formation. A 4.8-fold increase in micronuclei incidence in arsenic-treated cells was detected in conjunction with increased N-phosphonacetyl-l-aspartate (PALA)-resistant characteristics. In addition, arsenic-treated cells showed an increase in c-H-ras, c-myc, and c-fos protein expression relative to controls. The change in oncoprotein expression correlated with a decrease in wild-type p53 expression and hyperphosphorylated retinoblastoma. Taken together, these results strongly suggest that h-TERT immortalized human small airway epithelial cells underwent step-wise transformation after inorganic arsenic treatment.

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment.

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.