Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.     

Effects of hyperthermia and tumour necrosis factor on inflammatory cytokine secretion and procoagulant activity in endothelial cells

The application of hyperthermia (HT) and tumour necrosis factor alpha (TNF) in isolation perfusion of the limb or liver results in regression of advanced cancers confined to these regions of the body in most patients and are thought to exert anti-tumour effects primarily on tumour neovasculature. However, the individual contribution of either treatment factor on endothelial cells (EC) are not known. In this study, we investigated the in vitro effects of moderate and severe HT on human umbilical vein EC (HUVEC) with and without TNF in clinically relevant doses. HUVEC were exposed to normothermia (37 degrees C) or moderate (39 degrees C) and severe (41 degrees C) HT for 90 or 180 min with or without TNF (1 microg/ml). Cell viability, cytokine secretion (IL-6, IL-8, VEGF, ICAM-1, VCAM-1, RANTES, E-selectin, P-selectin, L-selectin, and PECAM-1), and induction of procoagulant activity as reflected in tissue factor (TF) production were assessed at the end of the treatment period and at several time points thereafter. Neither HT nor TNF exerted significant cytotoxic effects on EC at the doses and temperatures used. HT resulted in increased production of PECAM-1 with little or no additional effect when combined with TNF. TNF caused increased secretion of IL-6, IL-8, ICAM-1, and VCAM-1 with little or no additional effect from HT. Increased E-selectin and RANTES levels were observed with TNF and HT only at 24 h after treatment. HT and TNF had mainly antagonistic effects on VEGF secretion with HT causing primarily decreased production and TNF causing increased VEGF secretion under all temperatures. Most notably, there was a rapid, prolonged and synergistic peak increase in procoagulant activity when TNF and HT were used in combination compared to TNF or HT treatment alone. These results indicate that TNF and HT exert primarily independent effects on inflammatory cytokine production in EC but synergistically increase procoagulant activity as reflected in TF production. These data provide a possible mechanism for the thrombotic effects in tumour neovasculature seen following isolation perfusion with these agents and provide a rationale for their combined use in this treatment setting.     

Immunotherapy: a way to improve the therapeutic outcome of photodynamic therapy?

Photodynamic therapy (PDT) is a treatment for cancer and non-cancerous lesions involving light and a sensitizing drug, a so-called photosensitizer. Photosensitizers for PDT usually accumulate in tumour tissues with some selectivity. Thus, malignant and abnormal cells can be destroyed by PDT which acts by producing singlet oxygen and possible other reactive oxygen species. However, the efficiency of PDT is often limited by shallow light penetration into tissue. In some cases one treatment modality cannot cure a patient because of treatment limitations and/or side effects. In recent years, many preclinical studies have indicated that the therapeutic outcome of PDT can be improved, doses and side effects lowered by combination with immunotherapy. Most experiments have been done with animals and cell lines. This review summarizes the current knowledge about different immunotherapeutic approaches which can be used to improve effectiveness and extend the applications of PDT in clinics.     

Use of the radiation-inducible WAF1 promoter to drive iNOS gene therapy as a novel anti-cancer treatment

BACKGROUND: Inducible nitric oxide synthase (iNOS) gene therapy has been identified as a potential anti-tumour strategy. A major problem common to most gene therapy strategies is targeting of treatment to the tumour volume. In this study we report on the use of the X-ray-inducible WAF1 promoter to achieve targeting of iNOS expression to the tumour volume. METHODS: A WAF1/iNOS/liposome complex was injected directly into RIF-1 and HT29 tumours in mice. A 4 Gy dose of X-rays was applied to induce the WAF1 promoter followed, 8 h later, by treatment doses of 10 or 20 Gy. Tumour volume was measured, and growth curves plotted. RESULTS: Intra-tumoural injection of WAF1/iNOS combined with a priming dose of X-rays to induce the WAF1 promoter, followed by a treatment dose, resulted in sensitiser enhancement ratios of 2.0 and 1.3 in RIF-1 and HT29 tumours, respectively, compared with radiation treatment alone. PCR analysis of organ tissue after intra-tumoural injection of WAF1/iNOS showed that vector sequences were detected in all tissue tested; however, Western blot analysis revealed that iNOS protein levels were significantly increased only in tumour and the surrounding dermal tissue that had been exposed to the 4 Gy inducing dose. CONCLUSIONS: iNOS gene therapy in combination with an inducible promoter results in significant tumour cell radiosensitisation. The WAF1 promoter may be a good candidate for a gene therapy as it is silent in normal tissue yet can be induced by the tumour environment.     

Low‐dose angiostatic tyrosine kinase inhibitors improve photodynamic therapy for cancer: lack of vascular normalization

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro‐angiogenic side effects, which may counteract its angio‐occlusive mechanism. The combination of PDT with anti‐angiogenic drugs offers a possibility for improved anti‐tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti‐VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low‐dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR‐2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib‐treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti‐angiogenic agents to further improve the efficacy of photodynamic anti‐cancer therapy.     

Synergistic effect of angiopoietin-1 and vascular endothelial growth factor on neoangiogenesis in hypercholesterolemic rabbit model with acute hindlimb ischemia

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are essential for vascular integrity and development. The purpose of the study was to test the hypothesis that Ang1 will promote angiogenic response to VEGF in the spontaneous Watanabe heritable hypercholesterolemic (WHHL) rabbit model of acute hindlimb ischemia. Immediately after the ligation of the external iliac artery and the excision of the common and superficial femoral artery in one female WHHL rabbit, 250 microg of phVEGF(165) (n = 8), 500 microg of pAng1* (n = 8), or 250 microg of phVEGF(165) plus 500 microg of pAng1* (n = 8) was injected intramuscularly into the ischemic hindlimb muscles. Gross appearance of ischemic limb, collateral vessel formation and limb perfusion were assessed 30 days after treatment. The incidence of ischemic limb necrosis was higher in the animals treated by phVEGF(165) or by pAng1* than in those treated by phVEGF(165) plus pAng1* (100%, 75% and 14.3%, respectively; P = 0.002). Animals in the combination therapy group had a significantly higher calf blood pressure ratio at day 30 (VEGF plus Ang1* = 0.84 +/- 0.06; VEGF = 0.54 +/- 0.01; Ang1* = 0.59 +/- 0.05; P < 0.01). A combination therapy of VEGF plus Ang*1 had a significantly higher (P < 0.01) angiographic score than either therapy alone. Capillary density (P < 0.05) and capillary/muscle fiber ratio (P < 0.01) of the combination therapy group were also significantly higher than that of either therapy alone. In conclusion, Ang1 can potentiate the angiogenic response to VEGF in the hyperlipidemic rabbit model of acute hindlimb ischemia. Intramuscular administration of cytokines on revascularization of the ischemic hindlimb model of hyperlipidemic rabbit is feasible.     

Hypericin-mediated photodynamic therapy in combination with Avastin (bevacizumab) improves tumor response by downregulating angiogenic proteins.

Photodynamic therapy (PDT) is a therapeutic modality in which a photosensitizer is locally or systemically administered followed by light irradiation of suitable wavelength to achieve selective tissue damage. In addition, PDT is an oxygen-consuming reaction, that causes hypoxia mediated destruction of tumor vasculature that results in effective treatment. However, the hypoxic condition within tumors can cause stress-related release of angiogenic growth factors and cytokines and this inflammatory response could possibly diminish the efficacy of PDT by promoting tumor regrowth. In such circumstances, PDT effectiveness can be enhanced by combining angiogenesis inhibitors into the treatment regimen. Avastin (bevacizumab), a vascular endothelial growth factor (VEGF) specific monoclonal antibody in combination with chemotherapy is offering hope to patients with metastatic colorectal cancer. In this study we evaluated the combination of hypericin-mediated PDT and Avastin on VEGF levels as well as its effect on overall tumor response. Experiments were conducted on bladder carcinoma xenografts established subcutaneously in Balb/c nude mice. Antibody array, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) were performed to assess VEGF concentrations in the various treatment groups. Our results demonstrated that the targeted therapy by Avastin along with PDT can improve tumor responsiveness in bladder tumor xenografts. Immunostaining showed minimal expression of VEGF in tumors treated with combination therapy of PDT and Avastin. Angiogenic proteins e.g., angiogenin, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and interleukins (IL-6 and IL-8) were also found to be downregulated in groups treated with combination therapy.     

Treatment of human lung carcinoma xenografts with a combination of 131I-labelled monoclonal antibody Po66 and doxorubicin

 Po66, a mouse monoclonal antibody, is directed against an intracytoplasmic antigen present in human lung squamous cell carcinoma cells. In previous work it was found that the co-administration of ¹²⁵I-radiolabelled Po66 and doxorubicin strongly enhanced the uptake of radioactivity by the tumour. The present-work was designed to evaluate, in a tumour-bearing mouse model of lung carcinoma, the ability of ¹³¹I-labelled Po66 to retard tumour growth when injected alone, or in combination with doxorubicin (8 mg kg ^– 1 at 1-week intervals). A single dose of 550 μCi ¹³¹I-Po66 alone had no effect on tumour growth, whereas three fractionated doses of 250 μCi ¹³¹I-Po66 decreased it over two doubling times from 14.5±1.5 days for untreated control mice to 24.8±2.7 days. Mice treated with doxorubicin alone had a double tumour doubling time of 22.6±4.9 days, compared to 35.2±2.9 days (1.55-fold increase) in mice treated with doxorubicin and a single dose of 550 μ Ci ¹³¹I-Po66. Doxorubicin combined with three fractionated doses of 250 μCi ¹³¹I-Po66 provoked a twofold decrease in tumour growth compared to mice treated with doxorubicin alone. The administration of fractionated doses of ¹³¹I-Po66 simultaneously with doxorubicin resulted in a highly delayed mortality, which was not observed when ¹³¹I-Po66 was administered after doxorubicin. Thus, in a non-small-cell lung tumour model, a ¹³¹I-radiolabelled monoclonal antibody, directed against an intracellular antigen, significantly potentiated the effect of chemotherapy. Such a therapeutic approach could be used as an adjuvant therapy and improve the effect of chemotherapy on distant small metastases. Received: 20 June 1996 / Accepted: 3 October 1996     

Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.     

Photoinduced antitumour effect of hypericin can be enhanced by fractionated dosing

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80mm3 the mice were intraperitoneally injected with hypericin, either in a single dose (5mg/kg; 1 or 6h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70mW/cm2, 168 J/cm2). All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05). Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.     

Delayed tumor growth caused by a combined treatment with BCG and Pseudomonas aeruginosa

Summary Sera from mice treated i.v. with 1 mg BCG, followed 14 days later by 0.1 ml (10⁸ killed organisms) of Pseudomonas aeruginosa have shown the capacity to induce tumor necrosis when injected into mice bearing subcutaneous transplants either of a methyl-cholanthrene-induced sarcoma or of the P815 mastocytoma. Furthermore, immunotherapeutic trials were performed in mice bearing a subcutaneous transplanted sarcoma by combining BCG and low doses (0.01 to 0.05 ml) of Pseudomonas. Tumor necrosis was detectable 24 hours later only in the group treated by both BCG and Pseudomonas. In this group, we have also observed a significant decrease of tumor size in comparison with the groups of mice receiving BCG or Pseudomonas alone or no treatment.     

Cell proliferation and cell cycle alterations in oesophageal p53‐mutated cancer cells treated with cisplatin in combination with photodynamic therapy

Objectives: The major goal of anti‐cancer therapies is selective destruction of tumour cells with minimum side effects on normal cells. Towards this aim, combination of different therapeutic modalities has been evaluated for improving control of neoplastic diseases and quality of life for the patient. Photodynamic therapy (PDT) is a procedure for treatment of various types of cancer, but its combination with other established treatments has not been evaluated in detail. We have used KYSE‐510 cells from a human oesophageal carcinoma as an in vitro model to investigate whether cisplatin (CDDP) could be combined with PDT to increase cell death with respect to single treatments. Materials and methods: p53‐mutated KYSE‐510 cells were treated with CDDP alone or in combination with PDT. Analyses of cell viability, cell cycle progression and apoptosis induction were carried out at specific times after treatments. Results: Decrease in cell viability, cell cycle arrest at the G₂/M‐ and S‐phases boundary, and apoptosis induction were observed after single and combined treatments. Conclusions: Our results show that low CDDP doses (0.25–1 μm ) induce cell mortality and cell cycle perturbation, which were more evident when given in combination with PDT, but in contrast to work of other authors no synergistic activity was found. Apoptosis occurred via intrinsic pathways in treated cells, although it did not represent the predominant mode of cell death.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Synergistic anti-tumor effects of combination of photodynamic therapy and arsenic compound in cervical cancer cells: in vivo and in vitro studies

The effects of As(4)O(6) as adjuvant on photodynamic therapy (PDT) were studied. As(4)O(6) is considered to have anticancer activity via several biological actions, such as free radical production and inhibition of VEGF expression. PDT or As(4)O(6) significantly inhibited TC-1 cell proliferation in a dose-dependent manner (P<0.05) by MTT assay. The anti-proliferative effect of the combination treatment was significantly higher than in TC-1 cells treated with either photodynamic therapy or As(4)O(6) alone (62.4 and 52.5% decrease compared to vehicle-only treated TC-1 cells, respectively, P<0.05). In addition, cell proliferation in combination of photodynamic therapy and As(4)O(6) treatment significantly decreased by 77.4% (P<0.05). Cell survival pathway (Naip1, Tert and Aip1) and p53-dependent pathway (Bax, p21(Cip1), Fas, Gadd45, IGFBP-3 and Mdm-2) were markedly increased by combination treatment of photodynamic therapy and As(4)O(6). In addition, the immune response in the NEAT pathway (Ly-12, CD178 and IL-2) was also modulated after combination treatment, suggesting improved antitumor effects by controlling unwanted growth-stimulatory pathways. The combination effect apparently reflected concordance with in vitro data, in restricting tumor growth in vivo and in relation to some common signaling pathways to those observed in vitro. These findings suggest the benefit of combinatory treatment with photodynamic therapy and As(4)O(6) for inhibition of cervical cancer cell growth.     

Enhancement of the photodynamic antitumor effect by streptococcal preparation OK-432 in the mouse carcinoma

 Biological response modifier antitumor effects are enhanced by the activation of the host defense mechanisms. We have investigated the antitumor effect of photodynamic therapy (PDT) and/or local administration of a biological response modifier, the streptococcal preparation OK-432, on transplanted NR-S1 mouse squamous cell carcinoma. Hematoporphyrin oligomers (20 mg/kg body weight) were used to photosensitize PDT. A pulsed Nd:YAG dye laser, tuned at 630 nm, was used as the light source. The laser power was 15 mJ cm⁻² pulse⁻¹, and the irradiation time was 40 min. The photosensitizer was injected intraperitoneally 48 h before laser irradiation. Where used, OK-432 was injected into the tumor either 3 h prior to PDT or immediately afterwards. The antitumor effects were evaluated 48 h after each protocol by (a) estimating the area of tumor necrosis (%) in hematoxylin/eosin-stained specimens, and (b) bromodeoxyuridine immunohistochemistry. Furthermore, the tumor sizes were evaluated 3, 7 and 10 days after each protocol, and the survival time after each protocol was evaluated as well. The anti-tumor effect of PDT was enhanced by administration of OK-432 3 h before PDT, whereas the administration of OK-432 immediately after PDT did not potentiate a PDT antitumor effect. Treatment with OK-432 alone had little effect on tumors. Photodynamic therapy in combination with local administration of OK-432 3 h before PDT is considered to be a useful treatment modality. Received: 23 July 1999 / Accepted: 31 May 2000     

AdVEGF165 gene transfer increases survival in overdimensioned skin flaps

BACKGROUND: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. VEGF A also plays an important role in wound healing of the skin by promoting angiogenesis and by stimulating blood vessel growth. Therefore we tested the hypothesis that flap survival could be increased by the preoperative injection of AdVEGF(165). METHODS: We studied the effect of AdVEGF(165) in an overdimensioned ischemic random-pattern-flap model in the rat (n = 50) with a length-to-width ratio of 4 : 1. VEGF cDNA was administered in two concentrations of 5 x 10(8) plaque-forming units (pfU) and 1 x 10(9) pfU using a recombinant adenoviral vector. Recombinant virus was injected subdermally 7, 3 or 0 days prior to flap harvest for the lower concentration and 7 days prior for the higher concentration. Flap survival and necrosis were observed at day 7, the day the animals were sacrificed. RESULTS: Adenoviral gene transfer with VEGF(165) 3 and 7 days before flap harvest showed a significantly increased flap survival of 50% together with a significantly reduced necrosis (p < 0.01). Injection using a titer of 1 x 10(9) pfU 7 days prior to surgery increased flap survival even more, though failing to reach statistical significance compared to the lower concentration. VEGF protein concentration in the injected skin was significantly higher than in controls (p < 0.01). Flap perfusion was increased as well, demonstrated by indocyanine green (ICG) fluoroscopy (p < 0.001). CONCLUSIONS: Our results confirm the important role of VEGF(165) on angiogenesis in ischemic flaps. Indeed by injecting VEGF(165) at 3 to 7 days preoperatively in a concentration of 1 x 10(9) pfU our data show that length-to-width ratio for random-pattern-flaps could be increased from 2 : 1 to 3 : 1 and therefore may allow a wider range of applications of this simple flap technique.     

Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion

Targeted angiostatic therapy receives major attention for the treatment of cancer and exudative age‐related macular degeneration (AMD). Photodynamic therapy (PDT) has been used as an effective clinical approach for these diseases. As PDT can cause an angiogenic response in the treated tissue, combination of PDT with anti‐angiogenic compounds should lead to improved therapy. This study was undertaken to test the clinically used small molecule kinase inhibitors Nexavar® (sorafenib), Tarceva® (erlotinib) and Sutent® (sunitinib) for this purpose, and to compare the results to the combination of Visudyne®‐PDT with Avastin® (bevacizumab) treatment. When topically applied to the chicken chorioallantoic membrane at embryo development day (EDD) 7, a clear inhibition of blood vessel development was observed, with sorafenib being most efficient. To investigate the combination with phototherapy, Visudyne®‐PDT was first applied on EDD11 to close all <100 μm vessels. Application of angiostatics after PDT resulted in a significant decrease in vessel regrowth in terms of reduced vessel density and number of branching points/mm². As the 50% effective dose (ED50) for all compounds was approximately 10‐fold lower, Sorafenib outperformed the other compounds. In vitro, all kinase inhibitors decreased the viability of human umbilical vein endothelial cells. Sunitinib convincingly inhibited the in vitro migration of endothelial cells. These results suggest the therapeutic potential of these compounds for application in combination with PDT in anti‐cancer approaches, and possibly also in the treatment of other diseases where angiogenesis plays an important role.     

Potential impact of silymarin in combination with chlorogenic acid and/or melatonin in combating cardiomyopathy induced by carbon tetrachloride

The aim of this study was to investigate the effective role of silymarin either alone or in combination with chlorogenic acid and/or melatonin against the toxic impact of carbon tetrachloride (CCl4) induced cardiac infarction. CCl4 (l.2 ml/kg body weight) was administered as a single dose intraperitoneally. The results revealed that the administration of silymarin alone or in combination with chlorogenic acid (CGA) and/or melatonin for 21 consecutive days, 24 h after CCl4 injection to rats, markedly ameliorated the increases in serum markers of cardiac infarction, including troponin T and creatine kinase-MB (CK-MB), as well as increases in the pro-inflammatory biomarkers, including interleukin-6 (IL-6), interferon-γ (IFN-γ) in serum and tumor necrosis factor-α (TNF-α) and C-reactive protein in cardiac tissue compared to CCl4 intoxicated rats. The used agents also successfully modulated the alteration in vascular endothelial growth factor (VEGF) in serum and the oxidative DNA damage and the increase in the apoptosis marker caspase 3 in cardiac tissue in response to CCl4 toxicity. The present biochemical results are supported by histo-pathological examination. The current results proved that treatment with silymarin in combination with CGA and melatonin was the most effective one in ameliorating the toxicity of CCl4 induced cardiac damage and this may support the use of this combination as an effective drug to treat cardiac damage induced by toxic agents.     

Expression of high molecular weight cysteine proteinase inhibitor in ovarian cancer tissues: regulation of cathepsin B expression by placental CPI

Mouse polyclonal antibodies against placental cysteine proteinase inhibitor (CPI) react with the placental 67 kDa CPI on Western blots, and CPI present in ovarian cancer homogenate and serum was shown by double immunodiffusion to react with the same antiserum. By immunohistochemical staining, positive expression of high molecular weight CPI was observed on the tumour cell surface in serous and endometrioid ovarian carcinomas with metastasis. Normal endometrioid tissue was not stained with anti-placental CPI antibodies. Cathepsin B and pro-cathepsin B median levels in ovarian cancer tissue homogenates increased progressively with FIGO stage of the disease. The enzyme level decreased 22-fold after treatment of tissue homogenates with 5 nM purified CPI. These results provide evidence that addition of CPI reduces the levels of cysteine-type cathepsins to those of normal non-cancerous values.     

Two combined photosensitizers: a goal for more effective photodynamic therapy of cancer

Photodynamic therapy (PDT) is a clinically approved therapeutic modality for the treatment of diseases characterized by uncontrolled cell proliferation, mainly cancer. It involves the selective uptake of a photosensitizer (PS) by neoplastic tissue, which is able to produce reactive oxygen species upon irradiation with light, leading to tumor regression. Here a synergistic cell photoinactivation is reported based on the simultaneous administration of two PSs, zinc(II)-phthalocyanine (ZnPc) and the cationic porphyrin meso-tetrakis(4-N-methylpyridyl)porphine (TMPyP) in three cell lines (HeLa, HaCaT and MCF-7), using very low doses of PDT. We detected changes from predominant apoptosis (without cell detachment) to predominant necrosis, depending on the light dose used (2.4 and 3.6 J/cm², respectively). Analysis of changes in cytoskeleton components (microtubules and F-actin), FAK protein, as well as time-lapse video microscopy evidenced that HeLa cells were induced to undergo apoptosis, without losing adhesion to the substrate. Moreover, 24 h after intravenous injection into tumor-bearing mice, ZnPc and TMPyP were preferentially accumulated in the tumor area. PDT with combined treatment produced significant retardation of tumor growth. We believe that this combined and highly efficient strategy (two PSs) may provide synergistic curative rates regarding conventional photodynamic treatments (with one PS alone).