Opposed arsenite-induced signaling pathways promote cell proliferation or apoptosis in cultured lung cells

Arsenic is a well-known carcinogen that possibly promotes tumors and the development of various types of cancer in individuals chronically exposed to arsenic in their work or living environment. Many studies have demonstrated the activation of mitogen-activated protein kinase (MAPK) in several cell types by using lethal concentrations of arsenic in the range of 50-500 micro M. Since the exposure of humans to arsenic is normally at a much lower level in the workplace or in daily life, it is more relevant to study the effects of arsenic at this lower exposure level. In the present study we aimed at redefining the role of signal transduction pathways in arsenic-induced malignant transformation as well as apoptosis using our established in vitro rat lung epithelial cell model system. Our results indicate a molecular mechanism by which MAPK pathways might differentially contribute to cell growth regulation and cell death in response to different dosages of arsenite. A low level (2 micro M) of arsenite stimulated extracellular signal-regulated kinase (ERK) signaling pathway and enhanced cell proliferation, and this arsenite-induced ERK activity was blocked by MEK inhibitor, PD98059. In contrast, a high level (40 micro M) of arsenite stimulated the c-Jun N-terminal kinase (JNK) signaling pathway and induced cell apoptosis, and this arsenite-induced JNK activity was blocked by JNK inhibitor II, SP600125. The implications of these findings are that a high concentration of arsenic exposure causes apoptosis, whereas a low concentration of arsenic exposure is carcinogenic and may result in aberrant cell accumulation.     

Arsenic induces apoptosis through a c-Jun NH2-terminal kinase-dependent, p53-independent pathway.

Arsenic has been used as an effective chemotherapy agent for some human cancers, such as acute promyelocytic leukemia. In this study, we found that arsenic induces activation of c-Jun NH2-terminal kinases (JNKs) at a similar dose range for induction of apoptosis in JB6 cells. In addition, we found that arsenic did not induce p53-dependent transactivation. Similarly, there was no difference in apoptosis induction between cells with p53 +/+ or p53 -/-. In contrast, arsenic-induced apoptosis was almost totally blocked by expression of a dominant-negative mutant of JNK1. These results suggest that the activation of JNKs is involved in arsenic-induced apoptosis of JB6 cells. Taken together with previous findings that p53 mutations are involved in approximately 50% of all human cancers and nearly all chemotherapeutic agents kill cancer cells mainly by apoptotic induction, we suggest that arsenic may be a useful agent for the treatment of cancers with p53 mutation.     

Activation of the AKT and STAT3 pathways and prolonged survival by a mutant EGFR in human lung cancer cells

To clarify the pathogenic and biological significance of EGFR mutations in lung cancer, we compared the status of ERBB family receptors, their downstream signal transductions and biological phenotypes between lung cancer cell lines with mutant and wild type EGFR. We initially analyzed expression and phosphorylation of ERBB family receptors and their major downstream proteins, AKT, p44/42 MAPK and STAT3, in a series of lung cancer cell lines with or without EGFR mutation. The expression levels of EGFR as well as of ERBB2 and ERBB3 proteins in cells with EGFR mutation tended to be higher than those in cells with wild type EGFR. There was no difference in stability between mutant and wild type EGFR proteins. EGF induced phosphorylation of EGFR, AKT, p44/42 MAPK and STAT3 to various extents, but the level of induction was not associated with the existence of EGFR mutation. These results implied that the activation of AKT, p44/42 MAPK and STAT3 signaling transmitted by EGFR would be critical for the growth and survival of lung cancer cells, but specific features of mutant EGFR in lung cancer cells was not discriminated by these approaches. We therefore performed transfection studies using PC-13 cells with no detectable endogenous EGFR expression. Exogenous expression of wild type and mutant EGFR (delE746-A750) in the cells revealed that only in the mutant EGFR transfected cells, EGFR itself as well as AKT and STAT3 were highly phosphorylated after 24h of serum deprivation. The survival time of mutant EGFR transfected cells was prolonged under serum-free culture conditions, but not under standard culture conditions with 10% serum. These results suggest that cells with a mutant EGFR survive through the activation of the AKT and/or STAT3 pathways, even in low EGF microenvironments. This specific property due to EGFR mutation could be an important step of multistage lung cancer progression.     

Cooperative and redundant effects of STAT5 and Ras signaling in BCR/ABL transformed hematopoietic cells

The Akt, Ras and STAT5 signaling pathways have each been linked to transformation of hematopoietic cells by BCR/ABL. However the relative contributions of these signaling pathways to BCR/ABL mediated cytokine-independent survival, proliferation and resistance to DNA damage-induced apoptosis have not been systematically defined. Here we report that activation of either Akt, Ras or STAT5 confers cytokine-independent survival to IL-3 dependent BaF3 cells. Ras or STAT5, but not Akt, also drives cytokine-independent proliferation and imparts sustained resistance to DNA damage-induced apoptosis. We also show that dominant negative (DN) inhibition of STAT5, but not Ras or Akt, significantly reduces resistance to DNA damage-induced apoptosis in BCR/ABL transformed BaF3 cells. Whereas inhibition of STAT5 or Ras alone does not compromise cytokine-independent proliferation of BaF3-BCR/ABL cells, simultaneous blockade of both STAT5 and Ras reduces proliferation and maximally sensitizes BaF3-BCR/ABL cells to DNA damage induced by gamma-irradiation, suggesting a cooperative role for these two signaling pathways in BCR/ABL transformation. The anti-apoptotic properties of BCR/ABL can be partly explained by an increase in the expression of Pim-1 and Bcl-XL, as ectopic expression of these STAT5 target genes imparts both cytokine-independent survival and partial gamma-radiation resistance. These data illustrate both cooperative and redundant effects of STAT5 and Ras signaling in BCR/ABL transformed cells, with STAT5 playing a dominant role in resistance to DNA damage-induced apoptosis.     

Interleukin-7 in T-cell acute lymphoblastic leukemia: an extrinsic factor supporting leukemogenesis?

The malignant transformation and expansion of tumor cells involve both cell-autonomous mechanisms and microenvironment signals that regulate viability, nutrient utilization, metabolic activity and cell growth. In T-cell acute lymphoblastic leukemia (T-ALL), the co-culture of leukemic cells with stroma or the addition of particular cytokines prevents ex vivo spontaneous apoptosis. Interleukin-7 (IL-7), a cytokine produced by thymic and bone marrow stroma, increases the viability and proliferation of T-ALL cells. IL-7 induces the activation of Jak/STAT, MEK/Erk and PI3K/Akt signaling pathways in T-ALL cells. PI3K/Akt is the dominant pathway that mediates the effects of IL-7 on T-ALL. PI3K signaling is required for the induction of Bcl-2, the down-regulation of p27(kip1) and cell cycle progression. PI3K signaling is also required for the expression of the glucose transporter Glut1, uptake of glucose, activation of the metabolic machinery, increase in cell size, and maintenance of mitochondrial integrity. These observations suggest that substrates of molecular pathways activated by microenvironmental factors represent attractive molecular targets for the regulation of the viability and proliferation of T-ALL cells and provide the means for the development of novel treatment strategies.     

The nitric oxide prodrug, V-PYRRO/NO, mitigates arsenic-induced liver cell toxicity and apoptosis

Arsenite is an important cancer chemotherapeutic. The liver is a major target tissue of arsenic toxicity and hepatotoxicity may limit its chemotherapeutic efficacy. O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO)-producing prodrug metabolized by hepatic P450 enzymes to release NO locally. V-PYRRO/NO protects against various organic or inorganic hepatotoxicants but any role in arsenic hepatotoxicity is undefined. Thus, we studied the effects of V-PYRRO/NO (0-1000muM) pretreatment on inorganic arsenic-induced toxicity in cultured rat liver (TRL 1215) cells. These cells metabolized the prodrug to release NO, producing extracellular nitrite levels to 41.7-fold above control levels (7.50+/-0.38 microM) after 24h V-PYRRO/NO (1000 microM) exposure. The effect of pretreatment with V-PYRRO/NO (24h) on the cytolethality of arsenic (as NaAsO(2)) exposure (24h) was assessed. Arsenic was markedly less toxic in V-PYRRO/NO pretreated cells (LC(50)=30.3 microM) compared to control (LC(50)=20.1 microM) and the increases in LC(50) showed a direct relationship to the level of NO produced (measured as nitrite). Consistent with the cytolethality data, V-PYRRO/NO pretreatment markedly reduced arsenic-induced apoptosis as assessed by DNA fragmentation. Activation of the c-Jun N-terminal kinase (JNK) pathway can be critical to apoptosis and pretreatment with V-PYRRO/NO suppressed arsenic-induced JNK activation. V-PYRRO/NO pretreatment modestly increased metallothionein (MT), a metal-binding protein, but greatly enhanced arsenic induction of MT. Thus, V-PYRRO/NO pretreatment directly mitigates arsenic toxicity in cultured liver cells, reducing cytolethality, apoptosis and related JNK pathway activation, apparently through generation of NO. The role of NO in reducing the hepatotoxicity of arsenical chemotherapeutics in vivo deserves additional study.     

A dominant role for the c-Jun NH2-terminal kinase in oncogenic ras-induced morphologic transformation of human lung carcinoma cells

Oncogenic (activated) Ras is a signal transducer that activates multiple effector-mediated signaling pathways leading to altered cell morphology, growth and differentiation, and neoplastic transformation. Activating mutations of Ras family genes have been detected in many types of human cancers, including lung cancer. However, the signaling mechanisms by which oncogenic Ras controls cancer cell growth is poorly characterized. This study evaluates the role of two specific signaling pathways, the c-Jun NH2-terminal kinase (JNK) pathway, and the extracellular signal-regulated kinase (ERK) pathway, in oncogenic Ras-induced morphological transformation of NCI-H82 human small cell lung cancer cells. In the NCI-H82 cell line, oncogenic Ras causes a marked and sustained activation of JNK but only has a modest effect on activation of the ERK pathway. The persistent JNK activation is associated with Ras-induced changes in cell morphology and enhanced transforming activity. Furthermore, JNK activation correlates with the induction of c-Jun expression, c-Jun phosphorylation on serines 63 and 73, and increased AP-1 activity. Deregulation of the JNK pathway using a dominant-negative mutant of JNK1, JNK1(APF), completely reverses the oncogenic Ras-induced transformed phenotype, including morphological reversion and inhibition of anchorage-independent growth and low-serum growth. Moreover, expression of JNK1(APF) leads to a decrease in c-Jun/AP-1 activity. In contrast, inhibition of ERK activation via a pharmacological approach using a mitogen-activated protein kinase/ERK kinase-specific inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one is unable to reverse the Ras-induced transformed morphology and c-Jun/AP-1 induction. These results demonstrate that the JNK/c-Jun/AP-1 pathway plays an essential role in mediating oncogenic Ras function in lung carcinoma cells.     

Mitochondrial damage mediates genotoxicity of arsenic in mammalian cells

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. For decades, arsenic was considered a nongenotoxic carcinogen. Using the highly sensitive A(L) mutation assay, we previously showed that arsenic is, indeed, a potent gene and chromosomal mutagen and that its effects are mediated through the induction of reactive oxygen species. However, the origin of these radicals and the pathways involved are not known. Here we show that mitochondrial damage plays a crucial role in arsenic mutagenicity. Treatment of enucleated cells with arsenic followed by rescue fusion with karyoplasts from controls resulted in significant mutant induction. In contrast, treatment of mitochondrial DNA-depleted (rho(0)) cells produced few or no mutations. Mitochondrial damage can lead to the release of superoxide anions, which then react with nitric oxide to produce the highly reactive peroxynitrites. The mutagenic damage was dampened by the nitric oxide synthase inhibitor, N(G)-methyl-L-arginine. These data illustrate that mitochondria are a primary target in arsenic-induced genotoxic response and that a better understanding of the mutagenic/carcinogenic mechanism of arsenic should provide a basis for better interventional approach in both treatment and prevention of arsenic-induced cancer.     

An inhibitory function for JNK in the regulation of IGF-I signaling in breast cancer

Insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed in a variety of cancer types. Since many breast tumors and cancer cell lines overexpress IGF-IR, we tested IGF-I effects on chemotherapy-treated breast cancer cells. IGF-I protects from chemotherapy-induced apoptosis, suggesting that overlapping signaling pathways modulate IGF-I and chemotherapy treatment outcomes. Taxol and other chemotherapy drugs induce c-Jun N-terminal kinase (JNK), a kinase that conveys cellular stress and death signals. Notably, in this paper we show that IGF-I alone induces a potent JNK response and this activity is reversed by inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with LY294002 in MCF-7 but not T47D cells. Cotreatment of cells with chemotherapy and IGF-I leads to additive JNK responses. Using cells overexpressing Akt, we confirm that IGF-I-mediated survival is Akt dependent. In contrast, overexpression of JNK significantly enhances Taxol-induced apoptosis and inhibits IGF-I survival effects. Further, JNK attenuates anchorage-independent growth of MCF-7 cells. The inhibitory effect of JNK appears to be mediated by serine phosphorylation of IRS-1 (insulin receptor substrate) since both Taxol and IGF-I treatment enhanced Ser(312) IRS-1 phosphorylation, while LY294002 blocked IGF-I-mediated phosphorylation. Taken together, these data provide a mechanism whereby stress or growth factors activate JNK to reduce proliferation and/or survival in breast cancer cells.     

6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells

STAT3 is persistently activated and contributes to malignant progression in various cancers. Janus activated kinases (JAK) phosphorylate STAT3 in response to stimulation by cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anticancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO)-mediated growth inhibition of human melanoma cells and assessed 6BIO as a potential anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induces apoptosis of human melanoma cells. 6BIO directly inhibited JAK-family kinase activity, both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, expression of the antiapoptotic protein Mcl-1 was downregulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the Akt and mitogen-activated protein kinase (MAPK) signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results show that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induces tumor cell apoptosis. Our findings support further development of 6BIO as a potential anticancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.