Novel role of NADPH oxidase in ischemic myocardium: a study with Nox2 knockout mice.

Several potential sources of reactive oxygen species (ROS) in cells exist. One source is NADPH oxidase, which is especially important for superoxide radical production. Nox2 is a primary regulatory subunit of NADPH oxidase. In the present study, we examined the role of ROS and NADPH oxidase in ischemic preconditioning (IP)-mediated cardioprotection by using Nox2(-/-) mice. Both wild-type (WT) and Nox2(-/-) mice were subjected to either 30 min of ischemia followed by 2 h of reperfusion (IR) or IP prior to 30 min ischemia and 2 h of reperfusion. Reduction in left ventricular developed pressure (60.1 versus 63 mmHg), dp/dt (max) (893 versus 1,027 mmHg/s), and aortic flow (0.9 versus 1.8 ml/min) was observed in Nox2(-/-)IPIR compared to WTIPIR along with increased infarct size (33% versus 22%) and apoptosis after 120 min of reperfusion. Differentially regulated genes were demonstrated by comparing gene expression in WTIPIR versus Nox2(-/-) IPIR hearts. Selected differentially regulated genes such as ß-catenin, SRPK3, ERDR1, ACIN1, Syntaxin-8, and STC1 were validated by real-time PCR. Taken together, this is the first report identifying important, differentially expressed genes during ischemic preconditioning in Nox2(-/-) mice by using microarray analysis.

HIF-prolyl hydroxylases and cardiovascular diseases.

Prolyl hydroxylases belong to the family of iron- and 2-oxoglutamate-dependent dioxygenase enzyme. Several distinct prolyl hydroxylases have been identified. The hypoxia-inducible factor (HIF) prolyl hydroxylase termed prolyl hydroxylase domain (PHD) enzymes play an important role in oxygen regulation in the physiological network. There are three isoforms that have been identified: PHD1, PHD2 and PHD3. Deletion of PHD enzymes result in stabilization of HIFs and offers potential treatment options for many ischemic disorders such as peripheral arterial occlusive disease, myocardial infarction, and stroke. All three isoforms are oxygen sensors that regulate the stability of HIFs. The degradation of HIF-1α is regulated by hydroxylation of the 402/504 proline residue by PHDs. Under hypoxic conditions, lack of oxygen causes hydroxylation to cease HIF-1α stabilization and subsequent translocation to the nucleus where it heterodimerizes with the constitutively expressed ß subunit. Binding of the HIF-heterodimer to specific DNA sequences, named hypoxia-responsive elements, triggers the transactivation of target genes. PHD regulation of HIF-1α-mediated cardioprotection has resulted in considerable interest in these molecules as potential therapeutic targets in cardiovascular and ischemic diseases. In recent years, attention has been directed towards identifying small molecule inhibitors of PHD. It is postulated that such inhibition might lead to a clinically useful strategy for protecting the myocardium against ischemia and reperfusion injury. Recently, it has been reported that the orally absorbed PHD inhibitor GSK360A can modulate HIF-1α signaling and protect the failing heart following myocardial infarction. Furthermore, PHD1 deletion has been found to have beneficial effects through an increase in tolerance to hypoxia of skeletal muscle by reprogramming basal metabolism. In the mouse liver, such deletion has resulted in protection against ischemia and reperfusion. As a result of these preliminary findings, PHDs is attracting increasing interest as potential therapeutic targets in a wide range of diseases.

Diabetes, oxidative stress, molecular mechanism, and cardiovascular disease--an overview.

In recent years, diabetes and its associated complications have come to represent a major public health concern. It is a complex disease characterized by multiple metabolic derangements and is known to impair cardiac function by disrupting the balance between pro-oxidants and antioxidants at the cellular level. The subsequent generation of reactive oxygen species (ROS) and accompanying oxidative stress are hallmarks of the molecular mechanisms responsible for cardiovascular disease. Among several oxidative stress-mediated mechanisms that have been proposed, ROS-mediated oxidative stress has received the most attention. ROS have been shown to interact with proteins, lipids, and DNA, causing damage to the cellular macromolecules and subsequently, deterioration of cellular function. Induction of thioredoxin-1 (Trx1) gene expression has been demonstrated to protect the diabetic myocardium from dysfunction by reducing oxidative stress and enhancing the expression of heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF). The failure of antioxidants to consistently demonstrate clinical benefit necessitates further investigation of the role of oxidative stress in diabetes-mediated cardiovascular disease.

Thioredoxin 1 enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction: a study using thioredoxin 1 transgenic mice.

Oxidative stress plays a crucial role in disruption of neovascularization by alterations in thioredoxin 1 (Trx1) expression and its interaction with other proteins after myocardial infarction (MI). We previously showed that Trx1 has angiogenic properties, but the possible therapeutic significance of overexpressing Trx1 in chronic MI has not been elucidated. Therefore, we explored the angiogenic and cardioprotective potential of Trx1 in an in vivo MI model using transgenic mice overexpressing Trx1. Wild-type (W) and Trx1 transgenic (Trx1(Tg/+)) mice were randomized into W sham (WS), Trx1(Tg/+) sham (TS), WMI, and TMI. MI was induced by permanent occlusion of LAD coronary artery. Hearts from mice overexpressing Trx1 exhibited reduced fibrosis and oxidative stress and attenuated cardiomyocyte apoptosis along with increased vessel formation compared to WMI. We found significant inhibition of Trx1 regulating proteins, TXNIP and AKAP 12, and increased p-Akt, p-eNOS, p-GSK-3ß, HIF-1α, ß-catenin, VEGF, Bcl-2, and survivin expression in TMI compared to WMI. Echocardiography performed 30days after MI revealed significant improvement in myocardial functions in TMI compared to WMI. Our study identifies a potential role for Trx1 overexpression and its association with its regulatory proteins TXNIP, AKAP12, and subsequent activation of Akt/GSK-3ß/ß-catenin/HIF-1α-mediated VEGF and eNOS expression in inducing angiogenesis and reduced ventricular remodeling. Hence, Trx1 and other proteins identified in our study may prove to be potential therapeutic targets in the treatment of ischemic heart disease.

Thioredoxin-1 gene therapy enhances angiogenic signaling and reduces ventricular remodeling in infarcted myocardium of diabetic rats.

BACKGROUND: The present study evaluated the reversal of diabetes-mediated impairment of angiogenesis in a myocardial infarction model of type 1 diabetic rats by intramyocardial administration of an adenoviral vector encoding thioredoxin-1 (Ad.Trx1). Various studies have linked diabetes-mediated impairment of angiogenesis to dysfunctional antioxidant systems in which thioredoxin-1 plays a central role. METHODS AND RESULTS: Ad.Trx1 was administered intramyocardially in nondiabetic and diabetic rats immediately after myocardial infarction. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. Myocardial function was measured by echocardiography 30 days after the intervention. The Ad.Trx1-administered group exhibited reduced fibrosis, oxidative stress, and cardiomyocyte and endothelial cell apoptosis compared with the diabetic myocardial infarction group, along with increased capillary and arteriolar density. Western blot and immunohistochemical analysis demonstrated myocardial overexpression of thioredoxin-1, heme oxygenase-1, vascular endothelial growth factor, and p38 mitogen-activated protein kinase-beta, as well as decreased phosphorylated JNK and p38 mitogen-activated protein kinase-alpha, in the Ad.Trx1-treated diabetic group. Conversely, we observed a significant reduction in the expression of vascular endothelial growth factor in nondiabetic and diabetic animals treated with tin protoporphyrin (SnPP, a heme oxygenase-1 enzyme inhibitor), even after Ad.Trx1 therapy. Echocardiographic analysis after 4 weeks of myocardial infarction revealed significant improvement in myocardial functional parameters such as ejection fraction, fractional shortening, and E/A ratio in the Ad.Trx1-administered group compared with the diabetic myocardial infarction group. CONCLUSIONS: This study demonstrates for the first time that impairment of angiogenesis and myocardial dysfunction can be regulated by Ad.Trx1 gene therapy in streptozotocin-induced diabetic rats subjected to infarction.

Transcriptome sequencing of malignant pleural mesothelioma tumors.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

Niacin bound chromium treatment induces myocardial Glut-4 translocation and caveolar interaction via Akt, AMPK and eNOS phosphorylation in streptozotocin induced diabetic rats after ischemia-reperfusion injury.

Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr(3+)) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia+NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia+NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia+NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia+NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.

Comparative effects of a novel plant-based calcium supplement with two common calcium salts on proliferation and mineralization in human osteoblast cells.

Calcium is an essential mineral to support bone health and serves as a major therapeutic intervention to prevent and delay the incidence of osteoporosis. Many individuals do not obtain the optimum amount of calcium from diets and depend on bioavailable calcium supplements. The present study was conducted to examine the effect of a novel plant-based calcium supplement, derived from marine algae, and contains high levels of calcium, magnesium, and other bone supporting minerals [commercially known as AlgaeCal (AC)], on proliferation, mineralization, and oxidative stress in cultured human osteoblast cells, and compared with inorganic calcium carbonate and calcium citrate salts. Cultured human fetal osteoblast cells (hFOB 1.19) were treated with AC (0.5 mg/ml, fixed by MTT assay), calcium carbonate, or calcium citrate. These cells were harvested after 4 days of treatment for ALP activity, PCNA expression, and DNA synthesis, and 2 days for Ca(2+) deposition in the presence and absence of vitamin D3 (5 nM). The ability of AC to reduce H(2)O(2) (0.3 mM)-induced oxidative stress was assessed after 24 h of treatment. ALP activity was significantly increased with AC treatment when compared to control, calcium carbonate, or calcium citrate (4.0-, 2.0-, and 2.5-fold, respectively). PCNA expression (immunocytochemical analysis), DNA synthesis (4.0-, 3.0-, and 4.0-fold, respectively), and Ca(2+) deposition (2.0-, 1.0-, and 4.0-fold, respectively) were significantly increased in AC-treated cells when compared with control, calcium carbonate, or calcium citrate treatment. These markers were further enhanced following additional supplementation of vitamin D3 in the AC-treated group cells. AC treatment significantly reduced the H(2)O(2)-induced oxidative stress when compared to calcium carbonate or calcium citrate (1.5- and 1.4-fold, respectively). These findings suggest that AC may serve as a superior calcium supplement as compared to other calcium salts tested in the present study. Hence, AC may be developed as a novel anti-osteoporotic supplement in the near future.

Strategic targets to induce neovascularization by resveratrol in hypercholesterolemic rat myocardium: role of caveolin-1, endothelial nitric oxide synthase, hemeoxygenase-1, and vascular endothelial growth factor.

Endothelial dysfunction and impaired angiogenesis constitute a hallmark of hypercholesterolemia. This study was designed to examine the effects of resveratrol, an antioxidant with lipid-lowering properties similar to those of statins, on neovascularization along with caveolar interaction with proangiogenic molecules in hypercholesterolemic rats. Animals were divided into: rats maintained on a normal diet (control group); rats maintained on a 5% high-cholesterol diet for 8 weeks (HC group); and rats maintained on a 5% high-cholesterol diet for 8 weeks and administered resveratrol (20 mg/kg) orally for 2 weeks (HCR group). Myocardial infarction was induced by ligating the left anterior descending artery. Herein we examined a novel method for stimulating myocardial angiogenesis by pharmacological preconditioning with resveratrol at both the capillary and arteriolar levels and the potential role of hemeoxygenase-1, endothelial nitric oxide synthase and caveolin-1 in mediating such a response. We also investigated the functional relevance of such treatment by assessing whether the induced neovascularization can help preserve left ventricle-contractile functional reserve in the setting of a chronic hypercholesterolemic condition. Four weeks after sham surgery and left anterior descending artery occlusion, rats underwent echocardiographic evaluation, which revealed improvement in ejection fraction and fractional shortening in the HCR group compared with the HC group. Left ventricular tissue sections displayed increased capillary and arteriolar density in the HCR group compared with the HC group. Western blot analysis revealed downregulation of vascular endothelial growth factor and hemeoxygenase-1 and increased association of caveolin-1 eNOS in the HC group, decreasing the availability of eNOS to the system; which was reversed with resveratrol treatment in the HCR group. This study was further validated in cardiac-specific hemeoxygenase-1-overexpressed mice assuming molecular cross-talk between the targets. Hence, our data identified potential regulators that primarily attenuate endothelial dysfunction by resveratrol therapy in hypercholesterolemic myocardium.

Preclinical studies of the proteasome inhibitor bortezomib in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm that is resistant to chemotherapy. Bortezomib is an FDA-approved proteasome inhibitor that is currently under clinical investigation in multiple neoplasms but has not been studied extensively in MPM. In this report, we determine the biological and molecular response of cultured MPM cells to bortezomib alone and in combination with cisplatin or pemetrexed. We used four MPM cell lines (MS589, H28, H2052, JMN), a normal mesothelial cell line (HM3), and a lung cancer cell line (H23) in survival studies utilizing bortezomib, cisplatin, and pemetrexed alone and in combination by administering concurrently or by varying the order of administration. We determined the effect of bortezomib on the cell cycle, apoptosis, and on the expression of cell cycle proteins p21/WAF1 and p27/KIP1 and on apoptosis-related proteins IAP-1, IAP-2, survivin, and XIAP. Bortezomib was highly cytotoxic to MPM cells and induced both G(2)/M and G(1)/S cell cycle arrest. Apoptosis increased in a concentration- and time-dependent manner in 3 of 4 MPM cell lines. Bortezomib stabilized or increased protein levels of p21/WAF1 and IAP-1 and to a lesser degree p27/KIP1, IAP-2, XIAP, and survivin. In combination studies with cisplatin, bortezomib was generally synergistic at high concentrations and antagonistic at low concentrations. Bortezomib increased the cytotoxicity of cisplatin and pemetrexed in a concentration-dependent manner when administered prior to either. Bortezomib may improve outcome in MPM patients alone or in combination with standard chemotherapy but the order of administration is likely to be important. This study justifies further evaluation of bortezomib in MPM.

Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition.

Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.

c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions.

Small cell lung cancer (SCLC) is an aggressive cancer, and most patients present with cancer already spread beyond the lung. The receptor tyrosine kinase (RTK) c-MET has been implicated in various solid tumors, including SCLC, and is involved in mediating tumorigenesis, cell motility, scattering, invasion and metastasis. Mutations of c-Met have been described in renal papillary carcinoma and gastrointestinal cancers including hepatocellular carcinoma. The sequence of c-MET was examined for possible mutations in the 10 SCLC cell lines and 32 paired-SCLC/normal tissues. Novel c-MET alterations were identified among 3 of 10 separate SCLC cell lines and in 4 of 32 SCLC tumor tissue samples. These include two different c-MET missense mutations in the juxtamembrane (JM) domain (R988C found in NCI-H69 and H249 cell lines; and T1010I in SCLC tumor sample T31). Also, there are one Sema domain missense mutation (E168D in SCLC tumor sample T5), two-base-pair insertional mutations (IVS13- (52-53)insCT in both SCLC tumor samples T26 and T27) within the pre-JM intron 13, as well as an alternative transcript involving exon 10 (H128 cell line). c-MET receptors are expressed at various levels among the 10 SCLC cell lines studied (high expression: H69, H345, H510, and H526; medium-expression: H128 and H146; and low/no-expression: H82, H209, H249, and H446). The level of c-MET expression does not have any apparent correlation with presence or absence of mutations of c-MET in the cell lines. We show that the two identified JM mutations (R988C and T1010I), when introduced into the interleukin-3 (IL-3)-dependent BaF3 cell line, regulated cell proliferation resulting in a small but significant growth factor independence. When introduced into a SCLC cell line (H446, with minimal endogenous wild-type c-MET expression), the JM mutations also regulated cell morphology and adhesion, as well as causing enhanced tumorigenicity by both increases in focus-formation and soft-agar colony-formation assays. Both of the JM mutations also increased cell motility and migration evident in wound healing assay and time-lapse video-microscopy speed analysis. The JM mutations also altered the c-MET RTK signaling, resulting in preferentially increased constitutive tyrosine phosphorylation of various cellular proteins, including the key focal adhesion protein paxillin on tyrosine residue Y31 (first CRKL-binding site), correlating with increased motility. These results suggest a novel and unique role of the JM domain in c-MET signaling in SCLC with significant implications in cytoskeletal functions and metastatic potential. The novel JM gain-of-function somatic mutations described are the first to be reported in SCLC, and may be associated with a more aggressive phenotype. It would now be useful to study the inhibition of c-MET as a therapeutic target against SCLC.

Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cancer cells.

The regulation of biological functions including cell growth, viability, migration, and adhesion of small cell lung cancer (SCLC) cells depends largely on the autocrine or paracrine stimulation of growth factor receptors and chemokine receptors. Stem cell factor (SCF) and its receptor c-Kit have been identified as important regulators of SCLC viability and are coexpressed in approximately 40-70% of SCLC specimens. In vitro, the inhibition of c-Kit tyrosine kinase activity by the small molecule tyrosine kinase inhibitor STI571 (Gleevec) abrogates cell growth. We have investigated the role of c-Kit and chemokine receptors in the regulation of cell migration and adhesion of SCLC cells. CXCR4, the chemokine receptor for stromal cell-derived factor-1alpha (SDF-1alpha), was found to be the major chemokine receptor commonly expressed in all of the 10 SCLC cell lines tested. SCF and SDF-1alpha increased cellular proliferation over a course of 72 h in both the c-Kit- and the CXCR4-positive NCI-H69 SCLC cell line. Recently, SDF-1alpha and CXCR4 have been shown to be important regulators of migration and metastasis in breast and ovarian cancer. We found that SDF-1alpha dramatically increased cell motility and adhesion in CXCR4-expressing NCI-H446 SCLC cells. In addition, SDF-1alpha altered cell morphology with increased formation of filopodia and neurite-like projections. In NCI-H69 SCLC cells, SCF and SDF-1alpha cooperatively induced morphological changes and activated downstream signaling pathways. Treatment of NCI-H69 cells with STI571 specifically inhibited the c-Kit signaling events of Akt and p70 S6 kinase, whereas SDF-1alpha-mediated activation of Akt or p70 S6 kinase was normal. In contrast, the phosphatidylinositol 3-kinase inhibitor, LY294002, prevented these cells from adhering and completely blocked SCF- and/or SDF-1alpha-induced Akt or p70 S6 kinase phosphorylation. These results demonstrate that the CXCR4 receptor is functionally expressed in SCLC cells and may, therefore, be involved in the pathogenesis of SCLC in vivo. Inhibition of both the CXCR4 and the c-Kit downstream events could be a promising therapeutic approach in SCLC.

Modulation of the c-Met/hepatocyte growth factor pathway in small cell lung cancer.

The c-Met receptor tyrosine kinase and its ligand HGF (hepatocyte growth factor) have been shown to be involved in angiogenesis, cellular motility, growth, invasion, and differentiation. The role of c-Met/HGF axis in small cell lung cancer (SCLC) has not been reported previously. We have determined the expression of p170(c-Met) precursor and p140(c-Met) beta-chain in seven SCLC cell lines by immunoblotting. We used the SCLC cell line H69, which expressed an abundant amount of c-Met to study the function and downstream effects of c-Met activation. Stimulation of H69 cells with HGF (40 ng/ml, 6-h stimulation) significantly altered cell motility of the SCLC cells with increased formation of filopodia and membrane ruffling, characterized as membrane blebbing, as well as increased migration of the cellular clusters were seen. We have further studied the signal transduction pathways of HGF/c-Met in the H69 cell line. The stimulation of H69 with HGF (40 ng/ml, >24 h, maximal at 1 h) increased the amount of reactive oxygen species formed by 34%. HGF stimulation (40 ng/ml, 7.5-min stimulation) of H69 cells showed increased tyrosine phosphorylated bands identified at M(r) 68,000, 120,000-140,000, and 200,000. Some of these tyrosine-phosphorylated bands were identified as the focal adhesion proteins paxillin, FAK, PYK2, and the c-Met receptor itself. Phospho-specific antibodies show that tyrosines at amino acid (a.a.) 31 of paxillin, and autophosphorylation sites at a.a. 397 of p125FAK, and a.a. 402 of PYK2 are phosphorylated in response to HGF/c-Met signaling. We also demonstrate that the Hsp90 inhibitor geldanamycin, which also affects c-Met, reduced the growth and viability of four of four SCLC cell lines by 25% to 85%, over a 72-h time period. Geldanamycin caused apoptosis of SCLC cells, as well as led to increased levels of Hsp70 but not Hsp90. These results demonstrate that c-Met/HGF pathway is functional in SCLC, and it would be useful to target this pathway toward novel therapy.

Flow cytometric determination of lipid peroxidation using fluoresceinated phosphoethanolamine.

In conclusion, we describe the use of fluor-DHPE as a flow cytometric probe to assess lipid peroxidation in erythrocytes. The stability and nonexchangeability of this probe make it suitable for monitoring lipid peroxidation in a particular cell type via flow cytometry, which can be done in the presence of other cells. Application of the probe in erythrocytes from rats fed a vitamin E-deficient diet demonstrated a higher susceptibility to lipid peroxidation among these cells than among erythrocytes from rats fed a normal diet. The current flow cytometric lipid peroxidation detection method can be interfaced directly with several standard techniques that are available to measure specific blood cell populations via flow cytometry.

Haptoglobin alpha-subunit and hepatocyte growth factor can potentially serve as serum tumor biomarkers in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) frequently presents as metastatic disease. It would be useful to detect serum tumor biomarkers at an earlier stage in order to improve the overall survival. MATERIALS AND METHODS: Serum samples from SCLC patients (6 limited disease, 7 extensive disease, 4 relapsed disease, 4 no evidence of disease post-treatment) and 5 normal controls were used to identify tumor biomarkers utilizing proteomics. Serum hepatocyte growth factor was also studied using standard ELISA. RESULTS: Utilizing MALDI-TOF-Mass Spectrometry (MS) based protein identification techniques, a SCLC specific overexpressed protein was identified to be haptoglobin alpha-subunit, with its serum level correlating with the disease stage. The mean level of alpha-haptoglobin was increased in SCLC serum as compared to the normal controls. Serum HGF was also studied as potential tumor biomarker and was found to correlate with the disease status. Either serum alpha-haptoglobin relative level (above 1.9 U), or HGF level (above 500 pg/ml) was associated with a trend towards worse survival. CONCLUSION: Our current findings suggest that serum levels of alpha-haptoglobin and HGF may serve as useful serum tumor biomarkers in SCLC. It would now also be useful to determine if these serum biomarkers are altered in response to therapy for this disease.

Role of receptor tyrosine kinases in lung cancer.

Receptor tyrosine kinases are important in normal cellular physiology as well in the pathogenesis of a variety of tumors, including lung cancer. RTKs are a target for novel therapies currently being investigated. In the clinics, EGFR inhibitors and c-Kit inhibitors are already being utilized, and c-Met inhibitors are in development. Even though the RTK inhibitors provide a novel mechanism, it is important to realize that lung cancer etiology is a complex process, and eventually standard chemotherapy may need to be used in conjunction with these novel therapies to make an important difference in response rates.

Molecular alterations in lung cancer. Impact on prognosis.

There are multiple molecular abnormalities that can occur in lung cancer. Based on the aberrancies described previously, many investigators and drug companies are designing novel therapies. The molecular markers can also be used as prognostic variables for future clinical trials and therapeutic interventions. It will not be an easy task to make an impact on lung cancer using these methods, since multiple pathways are abnormal in its pathogenesis. It is hoped that with the advent of novel and directed therapeutics, we may soon show some impact on the survival of this devastating disease.

Mechanisms of metastasis as related to receptor tyrosine kinases in small-cell lung cancer.

Small-cell lung cancer (SCLC) is an aggressive, malignant neoplasm with a 5-year survival of less than 10%. This poor survival rate is related to a high propensity for recurrence and a high rate of metastases. Metastases initially occur in the lymph nodes and thereafter in other organs such as the lung itself, liver, adrenal glands, brain, bone, and bone marrow. The mechanisms of metastases have been better understood recently and are described in this review. Receptor tyrosine kinases (RTKs) have been identified as important therapeutic targets in non-small-cell lung cancer (such as the EGF-receptor). We have begun to identify RTKs in SCLC and have shown that c-Kit and c-Met are expressed and functional in SCLC. RTKs have also been shown to be important in the metastasis of cancer cells. The roles of RTKs in the mechanism of metastasis are detailed in this review, with special emphasis on downstream signal transduction from RTK signaling.

Modulation of c-Kit/SCF pathway leads to alterations in topoisomerase-I activity in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, for which cytotoxic chemotherapy appears to have reached its maximal efficacy. This neoplasm is characterized by the overexpression of several receptor tyrosine kinases (RTKs), especially c-Kit. The ligand for c-Kit is stem cell factor (SCF). In SCLC, SCF can influence c-Kit activation by autocrine or paracrine mechanisms. We have recently shown that the c-Kit/SCF pathway is operational in SCLC and can be inhibited by Glivec (STI571). Because the inhibition of topoisomerase-I (topo-I) is one approach used to treat SCLC, we determined the effects of c-Kit/SCF signaling on topo-I activity. A unique phosphorylation of c-Kit on amino acid 823 and amino acid 703 was identified with the SCF stimulation of H526 cells. We demonstrate that with SCF stimulation over 16 hours (dose response 0-100 ng/mL) in H526 SCLC cells (c-Kit positive, SCF responsive), a decrease in topo-I activity was observed, whereas in H82 SCLC cells (c-Kit negative, SCF unresponsive) there was no modulation of topo-I activity by SCF. Using STI571 (5 microM, 16 hours) to inhibit the c-Kit pathway following stimulation with SCF (100 ng/mL), an upregulation of topo-I activity was observed in H526 cells but not in H82 cells. Performing viability assays, we show that STI571 in combination with topo-I inhibition by camptothecin or SN38, the active metabolite of irinotecan, can cooperatively inhibit H526 cell viability (but not H82 cell viability) for 72 hours. We also show that STI571 does not directly inhibit topo-I activity in SCLC. The combination of STI571 with topo-I inhibition could provide a useful combination in the treatment of SCLC.