Plasma Levels of Inflammatory Biomarkers in Peripheral Arterial Disease: Results of a Cohort Study.

Previous research analyzed the level of plasma inflammatory markers in patients with coronary disease, but very few studies have evaluated these markers in patients with peripheral arterial disease (PAD). The objective of this study was to investigate the plasma levels of inflammatory markers in patients with PAD and in healthy controls. The following plasma levels of biomarkers were measured in 80 patients with PAD (mean age 68 ± 5 years) and in 72 healthy participants (mean age 67 ± 6 years): interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), L-selectin (LS), neopterin (N), P-selectin (PS), E-selectin (ES), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and matrix metalloproteinase 2 (MMP-2), and 9 (MMP-9). Significantly higher levels of IL-6 (P < .001), TNF-α (P < .0001), ES (P < .0001), LS (P < .0001), PS (P < .0001), ICAM-1 (P < .001), VCAM-1 (P < .001), N (P < .001), MMP-2 (P < .001), and MMP-9 (P < .005) were found in the patients with PAD. Patients with PAD show a inflammation marker profile different from that of control participants. Reducing the high plasma levels of inflammatory markers could be a new therapeutic approach both for the prevention and the treatment of PAD.

Combining chemo-, hormonal and targeted therapies to treat breast cancer (Review).

Breast cancer ranks as the second most common cause of cancer death among women in the United States. Anticancer agents are an important component of breast cancer therapy. Drugs frequently used to treat breast cancer include methotrexate, 5-fluorouracil (5-FU), cyclophosphamide, anthracyclines, taxanes, trastuzumab, tamoxifen and aromatase inhibitors. These agents inhibit breast cancer progression by a variety of different mechanisms. Mutations may occur in cancer cells, which result in the elevated expression or constitutive activation of various growth factor receptors. The Raf/MEK/ERK and PI3K/Akt pathways are often activated by mutations in these growth factor receptors. These pathways are regulated by upstream Ras, which is mutated in 20-30% of human cancers. Downstream B-Raf and PI3K are also activated by mutation. Many of the events elicited by the Raf/MEK/ERK and PI3K/Akt pathways have direct effects on survival and the proliferative pathways. Aberrant regulation of the Raf/MEK/ERK and PI3K/Akt pathways can contribute to uncontrolled cell growth and lead to malignant transformation. Effective targeting of these pathways may result in the suppression of cell growth and the death of malignant cells. This review focuses on the targeting of the Raf/MEK/ERK and PI3K/Akt pathways with small molecule inhibitors, as well as on the effects of conventional chemo- and hormonal therapies in the treatment of breast cancer.

Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance.

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis. Components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt). Also, mutations occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. These pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of elevated activated Akt levels to phosphorylate and inactivate Raf-1. We have investigated the genetic structures and functional roles of these two signaling pathways in the malignant transformation and drug resistance of hematopoietic, breast and prostate cancer cells. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell-lineage-specific effects. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines (FDC-P1 and TF-1), a trait associated with tumorigenesis. In contrast, expression of activated PI3K or Akt does not abrogate the cytokine dependence of these hematopoietic cell lines, but does have positive effects on cell survival. However, activated PI3K and Akt can synergize with activated Raf to abrogate the cytokine dependence of another hematopoietic cell line (FL5.12) which is not transformed by activated Raf expression by itself. Activated Raf and Akt also confer a drug-resistant phenotype to these cells. Raf is more associated with proliferation and the prevention of apoptosis while Akt is more associated with the long-term clonogenicity. In breast cancer cells, activated Raf conferred resistance to the chemotherapeutic drugs doxorubicin and paclitaxel. Raf induced the expression of the drug pump Mdr-1 (a.k.a., Pgp) and the Bcl-2 anti-apoptotic protein. Raf did not appear to induce drug resistance by altering p53/p21Cip-1 expression, whose expression is often linked to regulation of cell cycle progression and drug resistance. Deregulation of the PI3K/PTEN/Akt pathway was associated with resistance to doxorubicin and 4-hydroxyl tamoxifen, a chemotherapeutic drug and estrogen receptor antagonist used in breast cancer therapy. In contrast to the drug-resistant breast cancer cells obtained after overexpression of activated Raf, cells expressing activated Akt displayed altered (decreased) levels of p53/p21Cip-1. Deregulated expression of the central phosphatase in the PI3K/PTEN/Akt pathway led to breast cancer drug resistance. Introduction of mutated forms of PTEN, which lacked lipid phosphatase activity, increased the resistance of the MCF-7 cells to doxorubicin, suggesting that these lipid phosphatase deficient PTEN mutants acted as dominant negative mutants to suppress wild-type PTEN activity. Finally, the PI3K/PTEN/Akt pathway appears to be more prominently involved in prostate cancer drug resistance than the Raf/MEK/ERK pathway. Some advanced prostate cancer cells express elevated levels of activated Akt which may suppress Raf activation. Introduction of activated forms of Akt increased the drug resistance of advanced prostate cancer cells. In contrast, introduction of activated forms of Raf did not increase the drug resistance of the prostate cancer cells. In contrast to the results observed in hematopoietic cells, Raf may normally promote differentiation in prostate cells which is suppressed in advanced prostate cancer due to increased expression of activated Akt arising from PTEN mutation. Thus in advanced prostate cancer it may be advantageous to induce Raf expression to promote differentiation, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK-induced proliferation. These signaling and anti-apoptotic pathways can have different effects on growth, prevention of apoptosis and induction of drug resistance in cells of various lineages which may be due to the expression of lineage-specific factors.

A spindle cell variant of diffuse large B-cell lymphoma possesses genotypic and phenotypic markers characteristic of a germinal center B-cell origin.

Lymphoma with prominent spindle cell features, the so-called spindle cell lymphoma, is an unusual morphological variant of diffuse large B-cell lymphoma. Five new cases of spindle cell lymphoma have been analyzed by a multiparameter approach in order to clarify its clinical and biological features. All patients presented advanced stage disease with extranodal involvement. Vagina was the most common extranodal site. All patients received chemotherapy and are alive in complete remission. Morphologically, all five cases exhibited proliferation of spindle cells with a vaguely storiform pattern highly suggestive of spindle cell neoplasms of nonlymphoid origin. In contrast, the results of immunohistochemical analysis indicated that all five cases were hematolymphoid neoplasms of the B-cell lineage. These lymphomas consisted of a B-cell clonal population which exhibited somatic immunoglobulin and BCL-6 mutations as well as BCL-6 protein expression. The neoplastic spindle cells therefore closely resemble B cells residing in the germinal center. The absence of MUM1 expression in neoplastic spindle cells suggested that neoplastic spindle cells may be related to the early phases of intragerminal center maturation of B cells. The germinal center phenotype, with restricted expression of BCL-6, was associated with the presence of a primary extranodal origin, normal lactate dehydrogenase levels, and good response to treatment.

Pharmacological breast cancer therapy (review).

Breast cancer ranks as the second most common cause of cancer death among women in the United States. Anticancer agents are an important component of breast cancer therapy. Drugs frequently used to treat breast cancer include methotrexate, 5-fluorouracil (5-FU), cyclophosphamide, anthracyclines, taxanes, trastuzumab, tamoxifen, and aromatase inhibitors. These agents inhibit breast cancer progression by a variety of different mechanisms. This review describes each of these drugs and the varying effects each of them have upon breast cancer cells.

Analysis of BRAF mutation in primary and metastatic melanoma.

Mutation of BRAF has been proposed to contribute to melanoma development. However, it remains unclear whether or not BRAF mutation is associated with any particular stage of melanoma progression. Tumor biopsy specimens from patients with melanoma were analyzed to determine whether the frequency of BRAF mutation in metastatic melanoma differed from primary melanoma. BRAF mutation was present in 15 of 23 (61%) patients with primary melanoma and in 7 of 12 (58%) patients with metastatic melanoma. These results suggest that BRAF mutation in melanoma is most likely to occur prior to the development of metastatic disease.

Plasma levels and zymographic activities of matrix metalloproteinases 2 and 9 in type II diabetics with peripheral arterial disease.

Deregulation of matrix metalloproteinases (MMPs) is an important factor contributing to the development of vascular lesions. Plasma levels and zymographic activities of MMP-2 and MMP-9 were investigated in type II diabetics with (n = 51) or without (n = 42) peripheral artery disease (PAD) and in normal volunteers (n = 23). Plasma MMP-2 levels were higher in type II diabetics with (p < 0.01) or without (p > 0.05) PAD in comparison with normal volunteers. Similarly, type II diabetics with (p < 0.0001) or without (p > 0.05) PAD had higher plasma MMP-9 levels than normal volunteers. Plasma zymographic activities of both MMP-2 and MMP-9 were positively correlated with their plasma levels. Plasma MMP-2 zymographic activity was higher in type II diabetics with PAD than type II diabetics without PAD (p > 0.05). Plasma MMP-9 zymographic activity was higher in type II diabetics with (p < 0.0001) or without (p < 0.0001) PAD in comparision with normal volunteers. Together, these results indicate that increased plasma levels and zymographic activities of MMP-2 and MMP-9 may contribute to PAD in type II diabetics. In particular, plasma MMP-9 may be a useful marker for the development of vascular disease in type II diabetics.

Aggressive forms of non-Hodgkin's lymphoma in two patients bearing coinfection of Epstein-Barr and hepatitis C viruses.

Although epidemiologic and experimental data suggest an etiopathogenetic role for both hepatitis C virus (HCV) and Epstein-Barr virus (EBV) infection in development of B-cell non-Hodgkin's lymphoma (NHL), potential interactions between EBV and HCV during progression of B-cell NHL have not yet been fully investigated. In the present study, tumor biopsy specimens from patients with both B-cell NHL and chronic HCV infection (HCV(+)) were analyzed for the presence of EBV-encoded RNA (EBER) by in situ hybridization (ISH). VH and VL gene segments were amplified from tumor biopsy specimen DNA by PCR. EBV infection (EBV(+)) was detected in tumors from 2 of 31 (6%) HCV(+) B-cell NHL patients. Clinical histories of these two EBV(+)/HCV(+) B-cell NHL patients indicated a particularly aggressive course of disease. Chemotherapy failed to induce long lasting remission for either of these EBV(+)/HCV(+) B-cell NHL patients. Amplification of CDR3 of the Ig heavy chain gene from DNA isolated from each EBV(+)/HCV(+) B-cell NHL indicated the presence of monoclonal B-cell expansion. Rearrangement of Ig genes in neoplastic B-cell clones from both EBV(+)/HCV(+) patients was similar to that previously reported for EBV(-)/HCV(+) B-cell NHL patients. Additionally, neoplastic B-cell clones from these two EBV(+)/HCV(+) B-cell NHL patients did not exhibit intraclonal variation. Previous studies have demonstrated that intraclonal variation is common among neoplastic B-cell clones from EBV(-)/HCV(+) patients. EBV infection may have prevented evolution of variant neoplastic B-cell clones by suppressing antibody affinity maturation. Together, these data suggest that EBV infection may cooperate with HCV infection during progression of B-cell NHL in immunocompetent individuals. Such an interaction may accelerate the course of disease in B-cell NHL patients.

Detection of bcl-2 rearrangement in mucosa-associated lymphoid tissue lymphomas from patients with hepatitis C virus infection.

It has been shown that t(14;18)(q32;q21) involving fusion of IGH with MALT1 occurs frequently in mucosa-associated lymphoid tissue (MALT) lymphomas. Results of the present study indicate that the classical form of t(14;18)(q32;q21) involving fusion of IGH with bcl-2 can be detectable in a subset of MALT lymphomas in patients with hepatitis C virus (HCV) infection.

Thymidylate synthetase mRNA levels are increased in liver metastases of colorectal cancer patients resistant to fluoropyrimidine-based chemotherapy.

BACKGROUND: Fluoropyrimidines such as 5-fluorouracil (5-FU) and 5-fluoro-2'deoxyuridine (FUDR) are among the most effective chemotherapeutic agents for treatment of metastatic colorectal cancer (CRC). Increased expression of thymidylate synthetase (TS) in CRC metastases has been proposed to be an important mechanism of resistance to fluoropyrimidine-based chemotherapy. METHODS: The present study investigated whether TS mRNA levels in liver metastases of 20 CRC patients before treatment with FUDR by hepatic arterial infusion (HAI) correlated with frequency of clinical response or survival duration. RESULTS: Median survival duration of patients with TS mRNA levels above and below the median was 15 and 18 months, respectively (p > 0.05). Clinical response was achieved in 40% of patients with low TS mRNA levels, but in only 20% of patients with high TS mRNA levels (p = 0.01). TS mRNA levels were also measured for liver metastases of 7 of the patients that did not achieve a clinical response. A statistically significant increase in expression of TS mRNA was observed for liver metastases resistant to chemotherapy (21 +/- 14) in comparison to liver metastases of the same patients before chemotherapy (8 +/- 4) (p = 0.03). CONCLUSION: This is the first report to demonstrate increased TS expression in liver metastases from CRC patients resistant to fluoropyrimidine based chemotherapy. These findings are consistent with previous studies indicating that increased TS expression is associated with resistance to fluoropyrimidine-based chemotherapy.

Fibroblastic, hematopoietic, and hormone responsive epithelial cell lines and culture conditions for elucidation of signal transduction and drug resistance pathways by gene transfer.

Elucidation of signal transduction pathways involved in proliferation, cell cycle progression and the regulation of apoptosis has shown great promise in the treatment of various diseases including neoplastic, inflammatory, autoimmune, immunodeficiency, arthritic and neurodegenerative disorders. By understanding how these signal transduction pathways function, chemotherapeutic targets may be identified which will suppress or eliminate the disease. This information may eventually be translated into therapy, which would either eliminate or safely contain the patient's disease. This chapter will focus on basic tissue culture techniques which are used to elucidate signal transduction pathways. Furthermore, this chapter will provide a general background for understanding how gene transfer techniques can be used to elucidate signal transduction pathways as well as various pitfalls commonly encountered with their usage.

Elucidation of signal transduction pathways by transfection of cells with modified oncogenes.

This chapter will focus on introduction of various wild type (WT) and mutant genes into cells by DNA transfection. Techniques for analysis of the inheritance, expression, and biological effects of the introduced genes will be described. Various strong and weak points about three different techniques of stable gene transfer, including calcium-phosphate DNA precipitation, transfection via liposomes, and transfection via electroporation, will be discussed.

Elucidation of signal transduction pathways by retroviral infection of cells with modified oncogenes.

This chapter will focus on understanding how various wild type (WT), dominant negative (DN), constitutively active (CA), and conditionally active (COND) oncogenes, as well as antisense (AS) genes contained in retroviral vectors may be used to elucidate signal transduction pathways. We will describe methods to introduce these genes into cells and subsequent analysis of inheritance, expression, and biological effects of the genes introduced. Furthermore, we will discuss various strong points about each of these different types of constructs, how they can be used to elucidate signal transduction, apoptotic, and drug resistance pathways as well as various pitfalls commonly encountered with their usage.

Raf-1 and Bcl-2 induce distinct and common pathways that contribute to breast cancer drug resistance.

Overexpression of Bcl-2 plays a role in the development of drug resistance in leukemia and other apoptosis-prone tumors. Raf isoforms areserine/threonine kinases that act as signal transducers in cascades initiated by many growth factors and mitogens. Raf isoform activation has been linked to drug resistance in leukemia. In this study we investigated effects of Bcl-2 and Raf-1 on doxorubicin-induced growth inhibition of MCF-7 breast cancer cells. In the absence of doxorubicin, overexpression of Bcl-2 or a constitutively active form of Raf-1 in MCF-7 cells did not affect proliferation rate. Overexpression of Bcl-2 increased resistance of MCF-7 cells to doxorubicin in 2-day, 5-day, and 8-week assays. Analysis of doxorubicin sensitivity of individual MCF/Bcl-2 clones showed that doxorubicin resistance was positively correlated with level of Bcl-2 overexpression. Overexpression of constitutively active Raf-1 also increased resistance to doxorubicin. Induction of Raf-1 activity in MCF-7 cells overexpressing Bcl-2 resulted in greater doxorubicin resistance than induction of Raf-1 activity in MCF-7 cells lacking Bcl-2 overexpression. Furthermore, levels of P-glycoprotein mRNA were increased in MCF-7 cells overexpressing a constitutively active Raf-1. MCF-7 cells overexpressing constitutively active Raf-1 were also more resistant to paclitaxel, which, like doxorubicin, is a substrate of P-glycoprotein. These observations suggest both independent and overlapping roles for Raf-1 and Bcl-2 oncogenes in the resistance to growth inhibition by doxorubicin.

Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review).

The Ras/Raf/MEK/ERK signal transduction pathway regulates cell cycle progression and apoptosis in diverse types of cells. Mutations in this pathway are often observed in transformed cell lines and frequently linked with human cancers. The Ras/Raf/MEK/ERK pathway can induce events both associated with cell proliferation and cell cycle arrest. The particular course chosen may depend on the strength and the particular Raf gene activated by Ras. This pathway also is involved in maintaining cell survival by modulating the activity of apoptotic molecules including Bad and Bcl-2. This review will discuss the regulation of the Ras/Raf/MEK/ERK pathway and how it modulates cell cycle progression and cell survival.

EGFR family signaling and its association with breast cancer development and resistance to chemotherapy (Review).

Epidermal growth factor (EGF) receptors (EGFRs) and signaling pathways activated by these receptors have been associated with development of breast cancer as well as its resistance to treatment with cytotoxic drugs. This review describes the current understanding of EGFRs and their downstream signaling pathways. Emphasis is placed upon Raf/MEK/ERK and PI3K/PDK1/Akt signaling pathways and their relationship to regulation of apoptosis and cell cycle progression. Also discussed is the relationship between these signaling pathways and response of breast cancer to chemotherapeutic treatment. An appreciation of how these signaling pathways relate to development of breast cancer and its response to chemotherapy may lead to improved prevention, diagnosis, and treatment of this disease.