Adriana Albini

Cardiotoxicity of Anticancer Drugs: The Need for Cardio-Oncology and Cardio-Oncological Prevention

Due to the aging of the populations of developed countries and a common occurrence of risk factors, it is increasingly probable that a patient may have both cancer and cardiovascular disease. In addition, cytotoxic agents and targeted therapies used to treat cancer, including classic chemotherapeutic agents, monoclonal antibodies that target tyrosine kinase receptors, small molecule tyrosine kinase inhibitors, and even antiangiogenic drugs and chemoprevention agents such as cyclooxygenase-2 inhibitors, all affect the cardiovascular system. One of the reasons is that many agents reach targets in the microenvironment and do not affect only the tumor. Combination therapy often amplifies cardiotoxicity, and radiotherapy can also cause heart problems, particularly when combined with chemotherapy. In the past, cardiotoxic risk was less evident, but it is increasingly an issue, particularly with combination therapy and adjuvant therapy. Todays oncologists must be fully aware of cardiovascular risks to avoid or prevent adverse cardiovascular effects, and cardiologists must now be ready to assist oncologists by performing evaluations relevant to the choice of therapy. There is a need for cooperation between these two areas and for the development of a novel discipline, which could be termed cardio-oncology or onco-cardiology. Here, we summarize the potential cardiovascular toxicities for a range of cancer chemotherapeutic and chemopreventive agents and emphasize the importance of evaluating cardiovascular risk when patients enter into trials and the need to develop guidelines that include collateral effects on the cardiovascular system. We also discuss mechanistic pathways and describe several potential protective agents that could be administered to patients with occult or overt risk for cardiovascular complications.

‘Angioprevention’: angiogenesis is a common and key target for cancer chemopreventive agents

The potential to block tumor growth by inhibition of the neoangiogenic process represents an intriguing approach to the treatment of solid tumors. The high proliferation rate in the tumor deprived of proper vascularization would be balanced by cell death due to lack of diffusion of nutrients and oxygen. Matrix metalloproteinases (MMPs), angiogenic growth factors, and their receptors are the main targets of an increasing number of clinical trials approved to test the tolerance and therapeutic efficacy of antiangiogenic agents. We observed that a series of substances proposed as possible cancer chemopreventive agents show antiangiogenic properties when tested in in vitro and in vivo angiogenesis models. We demonstrated that N‐acetyl‐L‐cysteine is able to reduce the invasive and metastatic potential of melanoma cells, and to inhibit endothelial cell invasion by direct inhibition of MMP activity. We also showed that epigallocatechin gallate (EGCG), a flavonoid from green tea that possesses chemopreventive activity in experimental and epidemi‐ological studies, is a potent inhibitor of MMP‐2 and MMP‐9. Angiogenesis has also been demonstrated to be a target for nonsteroidal anti‐inflammatory drug che‐mopreventive activity. Based on these data, we hypothesize that other chemopreventive agents, including natural or synthetic retinoids, steroid hormone antagonists, peroxisome proliferator‐activated receptor γ li‐gands, vitamin D, and protease inhibitors, might have antiangiogenesis as an important mechanism of action, a novel concept we will term ‘angioprevention’. We analyze the mechanisms on how and why chemopreven‐tive agents could exert antiangiogenic effects aimed at controlling tumor growth, and their potential use in the clinic.—Tosetti, F., Ferrari, N., De Flora, S., Albini, A. ‘Angioprevention’: angiogenesis is a common and key target for cancer chemopreventive agents. FASEB J. 16, 2–14 (2002)

TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis

The matrix metalloproteinase (MMP) inhibitor TIMP‐2 has a high specificity for gelatinase A/MMP‐2. An imbalance between gelatinase A and TIMP‐2 in favor of enzymatic activity is linked to the degradation of the extracellular matrix (ECM) associated with several physiologic and pathologic events, including angiogenesis, invasion and metastasis. Since TIMPs are secreted molecules, they have the potential to be used for gene therapy of certain tumors. We transfected B16F10 murine melanoma cells, a highly invasive and metastatic cell line, with an expression vector harboring a cDNA encoding for human TIMP‐2. The clones obtained were isolated and examined for TIMP‐2 over‐expression and changes in tumor cell phenotype. The amount of recombinant TIMP‐2 produced correlated with a reduction in invasion. In an in vivo angiogenesis assay, TIMP‐2‐transfected clones showed reduced levels of blood vessel formation, and in vitro conditioned media from TIMP‐2 transfectants showed diminished induction of endothelial cell migration and invasion. TIMP‐2 over‐expression limited tumor growth in vivo and neoangiogenesis when cells were injected subcutaneously in mice in the presence of Matrigel. However, TIMP‐2 over‐expressing clones were found to be more resistant to apoptosis than parental and control melanoma cells, while necrosis was increased. Our data confirm the role of TIMP‐2 in the down‐regulation of metastasis and angiogenesis but indicate a possible involvement in tumor cell survival. Int. J. Cancer 75:246–253, 1998.

Neutrophil restraint by green tea: Inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (−)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.

Tumors and inflammatory infiltrates: Friends or foes?

The recognition of a role for inflammation in the natural history of a tumor has a long record, stretching from the mid-19th century. From the times of Virkow, who postulated that cancer originates from inflamed tissues, to Metchnikoff and many others, this field has continued to excite (and divide) the scientific community. The question as to whether the inflammatory infiltrate helps or hinders tumors is still open. In a sense, modern molecular biology has, if anything, worsened this dualism, and the literature on this issue shows a plethora of conflicting reports. We would like to provide another contribution to this topic, which was the subject of a recent brilliant review (Balkwill F and Mantovani A. Lancet 2001; 357: 539–45 [1]), by focussing more specifically to the relation between inflammation and tumor invasion and how this could drive rational therapeutic approaches.

Inflammation, inflammatory cells and angiogenesis: decisions and indecisions

Endothelial-immune cell cross-talk goes well beyond leukocyte and lymphocyte trafficking, since immune cells are able to intimately regulate vessel formation and function. Here we review the evidence that most immune cells are capable of polarization towards a dichotomous activity either inducing or inhibiting angiogenesis. In addition to the well-known roles of tumor associated macrophages, we find that neutrophils, myeloid-derived suppressor and dendritic cells clearly have the potential for influencing tumor angiogenesis. Further, the physiological functions of NK cells suggest that these cells may also show a potentially important role in pro- or anti-angiogenesis regulation within the tumor microenvironment. At the same time many angiogenic factors influence the activity and function of immune cells that generally favor tumor survival and tolerance. Thus the immune system itself represents a major pharmaceutical target and links angiogenesis inhibition to immunotherapy.

The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A

The angiogenic activity of CXC-ELR+ chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-α in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck−/−fgr−/−), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck−/−fgr−/− mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck−/−fgr−/− mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck−/−fgr−/− mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

Interaction of HIV-1 Tat Protein with Heparin ROLE OF THE BACKBONE STRUCTURE, SULFATION, AND SIZE

Human immunodeficiency virus type 1 (HIV-1) Tat protein is released from infected cells. Extracellular Tat enters the cell where it stimulates the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Benelli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialunga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) and transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10, 1733-1739) activity of extracellular Tat. Here we demonstrate that heparin binds specifically to recombinant HIV-1 Tat produced as glutathione S-transferase (GST) fusion protein and immobilized on glutathione-agarose beads. Heparin and heparan sulfate (HS), but not dermatan sulfate, chondroitin sulfates A and C, hyaluronic acid, and K5 polysaccharide, competed with 3H-labeled heparin for binding to immobilized GST-Tat and inhibited HIV-LTR transactivation induced by extracellular GST-Tat. Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was ≥1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity.

Tumor invasion: molecular shears blunted by green tea.

To the editor–The recent press, both popular and scientific, has given wide coverage of the beneficial properties of green tea, most commonly used in Asian countries. Consumption has been associated with prevention of cancer development and metastasis. The main flavonol of green tea, epigallocatechin-3-gallate (EGCG), inhibits urokinase, one of the hydrolases implicated in tumor invasion. Moreover, green tea consumption by mice significantly limits angiogenesis, crucial for the growth of all solid tumors.