melphalan and Necrosis

Cancer cells generally exhibit high levels of reactive oxygen species (ROS) that stimulate cell proliferation and promote genetic instability. Since this biochemical difference between normal and cancer cells represents a specific vulnerability that can be selectively targeted for cancer therapy, various ROS-generating agents are currently in clinical trials, either as single agents or in combination with standard therapy. However, little is known about the potential consequences of an increased oxidative stress for the efficacy of standard chemotherapeutic agents. In this context, we have assessed the influence of an oxidative stress generated by the combination of ascorbate and the redox-active quinone menadione on the capacity of melphalan, a common alkylating agent, to induce apoptosis in a chronic myelogenous leukemia cell line. Our data show that oxidative stress did not inhibit but rather promoted cancer cell killing by melphalan. Interestingly, we observed that, in the presence of oxidative stress, the type of cell death shifted from a caspase-3 dependent apoptosis to necrosis because of an ATP depletion which prevented caspase activation. Taken together, these data suggest that ROS-generating agents could be useful in combination with standard chemotherapy, even if all the molecular consequences of such an addition remain to be determined.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Apoptosis; Ascorbic Acid; Caspase 3; Drug Interactions; Humans; K562 Cells; Melphalan; Necrosis; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Vitamin K 3

We have recently demonstrated that cytolysis of human melanoma cells by immune effectors (both NK and T cells) is associated with release of the nuclear chromatin protein, high mobility group box I (HMGB1). Extracellular HMGB1 mediates a number of important functions including endothelial cell activation, stromagenesis, recruitment and activation of innate immune cells, and also dendritic cell maturation that, in the setting of cancer, lead to a chronic inflammatory response. This reparative inflammatory response promotes tumor cell survival, expansion, and metastases. Release of HMGB1 after chemotherapy-induced cytotoxicity has not been well characterized. We measured the release of HMGB1 after chemotherapy or immune cytolysis and demonstrated that this did not correlate with conventional markers of apoptosis and necrosis in several human colorectal, pancreatic, and melanoma tumor cell lines. Rather, we observed that tumor cells incubated with the platinating agent oxaliplatin, retained HMGB1 within the nucleus for significantly longer periods than other agents used at comparable cytotoxic concentrations or even with potent cytolytic cells. Thus, release of HMGB1 from dying tumor cells treated with chemotherapy or cells with lymphokine activated killer cell activity is not dependent solely on the mode of cell death. Sequestration of the damage associated molecular pattern molecule, HMGB1, may play a role in the clinical efficacy of platinating agents and suggests this as a superior agent for coupling with immunotherapeutic strategies, possibly enhancing their effectiveness.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; Combined Modality Therapy; HMGB1 Protein; Humans; Immunotherapy; Killer Cells, Lymphokine-Activated; Melanoma; Melphalan; Microscopy, Confocal; Necrosis; Neoplasms; Organoplatinum Compounds; Oxaliplatin; Paclitaxel; Pancreatic Neoplasms

The treatment of disseminated aspergillus infections in neutropenic patients remains a major challenge in spite of several new antifungal drugs. We report the case of a patient with multiple myeloma in prolonged neutropenia after primary failure of an autologous stem cell graft who developed invasive aspergillosis despite voriconazole monotherapy. He responded to a combination of voriconazole and caspofungin, supported by granulocyte transfusions and surgery. A subsequent allogeneic peripheral blood stem cell transplantation did not lead to recurring aspergillus infection. The patient is well and free of clinical disease with respect to the fungal infection and myeloma more than 18 months after the allogeneic transplantation.

Topics: Antifungal Agents; Aspergillosis; Caspofungin; Drug Therapy, Combination; Echinocandins; Granulocytes; Humans; Leukocyte Transfusion; Lipopeptides; Magnetic Resonance Imaging; Male; Melphalan; Middle Aged; Multiple Myeloma; Necrosis; Peptides, Cyclic; Pyrimidines; Radiography; Stem Cell Transplantation; Transplantation, Autologous; Transplantation, Homologous; Triazoles; Voriconazole

We have recently reported, in a murine tumor model, that apoptotic cells induced by different agents may vary in their ability to elicit host immunity. The basis for this observation is unclear but may involve varying efficiencies of cross-presentation and/or direct activation of immunity by different apoptotic preparations. As a first step in addressing this issue, we compared expression patterns of selected immune genes (MHC class I, class II, CD40, B7-1, B7-2) on viable and apoptotic populations induced by four different agents. The histone deacetylase inhibitor trichostatin A (TSA) induced MHC class II expression on viable and apoptotic cell populations, while LPAM, H2O2 and gamma-irradiation did not activate class II. Each agent employed elicited a different expression pattern of costimulatory molecules (CD40, B7-1, B7-2) on both apoptotic and 7-AAD+ 'necrotic' populations. In striking contrast to the TSA induction of MHC class II, class I cell surface protein was diminished on the apoptotic populations. These effects were not a result of changes in the cell cycle produced by the various treatments. The data demonstrate that distinctive gene expression patterns on viable and apoptotic cells are elicited by different apoptosis inducing agents. We discuss how expression patterns on dead or dying tumor cells could potentially affect the tumor's ability to elicit immunity.

Topics: Animals; Antigens, CD; Antineoplastic Agents, Alkylating; Apoptosis; B7-1 Antigen; B7-2 Antigen; CD40 Antigens; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, MHC Class I; Genes, MHC Class II; Histone Deacetylase Inhibitors; Hydroxamic Acids; Melphalan; Membrane Glycoproteins; Mice; Necrosis; Plasmacytoma

Topics: Antineoplastic Combined Chemotherapy Protocols; Biopsy; Chemotherapy, Cancer, Regional Perfusion; Combined Modality Therapy; Connective Tissue; Extremities; Heart-Lung Machine; Humans; Hyperthermia, Induced; Melphalan; Necrosis; Neoplasm Staging; Sarcoma; Soft Tissue Neoplasms; Tumor Necrosis Factor-alpha

We have previously shown how tumor response of isolated limb perfusion (ILP) with melphalan was improved when tumor necrosis factor alpha (TNF-alpha) was added. Taking into account that other vasoactive drugs could also improve tumor response to ILP, we evaluated histamine (Hi) as an alternative to TNF-alpha.. We used a rat ILP model to assess the combined effects of Hi and melphalan (n = 6) on tumor regression, melphalan uptake (n = 6), and tissue histology (n = 2) compared with Hi or melphalan alone. We also evaluated the growth of BN-175 tumor cells as well as apoptosis, necrosis, cell morphology, and paracellular permeability of human umbilical vein endothelial cells (HUVECs) after Hi treatment alone and in combination with melphalan.. The antitumor effect of the combination of Hi and melphalan in vivo was synergistic, and Hi-dependent reduction in tumor volume was blocked by H1 and H2 receptor inhibitors. Tumor regression was observed in 66% of the animals treated with Hi and melphalan, compared with 17% after treatment with Hi or melphalan alone. Tumor melphalan uptake increased and vascular integrity in the surrounding tissue was reduced after ILP treatment with Hi and melphalan compared with melphalan alone. In vitro results paralleled in vivo results. BN-175 tumor cells were more sensitive to the cytotoxicity of combined treatment than HUVECs, and Hi treatment increased the permeability of HUVECs.. Hi in combination with melphalan in ILP improved response to that of melphalan alone through direct and indirect mechanisms. These results warrant further evaluation in the clinical ILP setting and, importantly, in organ perfusion.

Topics: Animals; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Capillary Permeability; Chemotherapy, Cancer, Regional Perfusion; Drug Synergism; Endothelial Cells; Hindlimb; Histamine; Humans; Male; Melphalan; Necrosis; Rats; Rats, Inbred BN; Receptors, Histamine H1; Receptors, Histamine H2; Sarcoma, Experimental; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Umbilical Veins

Treatment with the DNA topoisomerase inhibitors etoposide, doxorubicin, and camptothecin, and with the alkylating agents cisplatin and melphalan, caused peroxide accumulation and apoptosis in U-937 human promonocytic cells. Preincubation with the reduced glutathione (GSH) synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO) always potentiated peroxide accumulation. However, although GSH depletion potentiated the toxicity of cisplatin and melphalan, occasionally switching the mode of death from apoptosis to necrosis, it did not affect the toxicity of the other antitumor drugs. Hypoxia or preincubation with antioxidant agents attenuated death induction, apoptotic and necrotic, by alkylating drugs. The generation of necrosis by cisplatin could not be mimicked by addition of exogenous H(2)O(2) instead of BSO and was not adequately explained by caspase inactivation nor by a selective fall in ATP content. Treatment with cisplatin and melphalan caused a late decrease in mitochondrial transmembrane potential (DeltaPsim), which was much greater during necrosis than during apoptosis. The administration of the antioxidant agents N-acetyl-l-cysteine and butylated hydroxyanisole after pulse treatment with cisplatin or melphalan did not affect apoptosis but attenuated necrosis. Under these conditions, both antioxidants attenuated the necrosis-associated DeltaPsim decrease. These results indicate that oxidation-mediated alterations in mitochondrial function regulate the selection between apoptosis and necrosis in alkylating drug-treated human promonocytic cells.

Topics: Adenosine Triphosphate; Alkylating Agents; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Buthionine Sulfoximine; Camptothecin; Cell Death; Cisplatin; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Doxorubicin; Enzyme Inhibitors; Etoposide; Flow Cytometry; Glutathione; Humans; Hydrogen Peroxide; Hypoxia; Immunoblotting; Melphalan; Membrane Potentials; Mitochondria; Monocytes; Necrosis; Nucleic Acid Synthesis Inhibitors; Oxygen; Radiation-Protective Agents; Reactive Oxygen Species; Spectrometry, Fluorescence; Time Factors; Topoisomerase I Inhibitors; U937 Cells

The aim of the present work was to investigate the origin and half-life of endogenous S100B protein reported by many investigators as a useful melanoma serum marker. Within cells, S100B protein exists in homo- or heterodimer form containing mainly Ca(++), having a substantial fraction bound to membranes. As such, S100B is believed to be involved in the regulation of cytoskeleton. Also, a role in the cell cycle progression has been suggested. Although S100B appears having important intracellular functions, proofs of its secretion, at least at concentrations such as the ones measured in melanoma patients, are still lacking. Consistent with this view is the fact that immunohistology for S100 protein reported by numerous authors clearly indicate an exclusive intracellular staining. For these reasons, it was of a major interest to investigate how and when S100B is shed to the blood. Knowing that significant S100B levels are seen only in stage IV patients, we hypothesized that cell death may be the major source of circulating S100B protein in these patients. This hypothesis was studied in an in vitro model simulating cell death and in vivo in melanoma and other cancer patients undergoing highly cytotoxic regional immunochemotherapy using isolated limb perfusion with tumor necrosis factor and melphalan, as well as in tumor exudates and pleural fluids. Our results strongly suggest melanoma and endothelial cell death and subsequent continuous drainage to the blood as the major mechanism behind S100B release to the blood circulation. We estimated the endogenous S100B protein half-life to be about 30 min.

Topics: Biomarkers, Tumor; Calcium; Calcium-Binding Proteins; Cell Death; Dimerization; Endothelium, Vascular; Hemangiosarcoma; Humans; Kinetics; Melanoma; Melphalan; Necrosis; Nerve Growth Factors; Perfusion; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Sarcoma; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Umbilical Veins

Amyloid beta protein ending at 42 (A beta 42) plays an important role in the pathology of Alzheimer's disease (AD). Here we show an increase in cellular A beta 42 in damaged neurons, with both ELISA and immunocytochemistry. The cellular A beta 42 increase was caused by 3-day treatments with H2O2, etoposide or melphalan, all of which induce genotoxic apoptosis, but not by treatment with sodium azide, which causes necrosis. Secreted A beta was similarly decreased with all these treatments. The cellular A beta 42 increase appeared even with minimal damage (ELISA) and A beta 42-positive cells were TUNEL negative (double staining), indicating that any early apoptosis mechanism may induce the cellular A beta 42 increase. Thus, neuronal apoptosis and cellular A beta 42 increase may be linked in a way that contributes importantly to AD pathology.

Topics: Amyloid beta-Peptides; Animals; Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Apoptosis; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Etoposide; Guinea Pigs; Humans; Hydrogen Peroxide; Immunohistochemistry; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Melphalan; Necrosis; Peptide Fragments; Reactive Oxygen Species; Sodium Azide

Hyperthermic isolated limb perfusion with tumor necrosis factor-alpha and melphalan was used as induction treatment in locally advanced extremity soft-tissue sarcomas for limb sparing surgery. The typical histopathological changes that occur in these tumoral masses are described in a series of 30 patients.. Fresh tumor specimens of 27 high grade extensive soft-tissue sarcomas and 3 recurrent desmoid tumors of the extremities were collected 6 to 8 weeks after hyperthermic isolated limb perfusion with tumor necrosis factor-alpha plus melphalan. The specimens were studied for surgical margins, extent and type of tumor necrosis, lymph node involvement, perineural and vascular invasion, and the effects on adjacent normal tissues such as nerves, muscles, and blood vessels.. The typical histological changes were central cystic hemorrhagic necrosis with pericystic extensive fibrosis. Some nonspecific changes were noted in the soft tissues around the mass. In eight cases, more than 90% necrosis was found. In 17 cases, the extent of necrosis ranged between 60% and 90% (80%-90% in 4 of 17 cases). In five cases, less than 60% necrosis was noted. The best responses (>90% necrosis) were observed in distally located tumors. The responsive types were malignant fibrous histiocytoma, followed by myxoid liposarcoma and synovial sarcoma. Desmoid tumors showed less necrosis than high grade sarcomas. Vascular invasion was observed in two cases and intralesional venous thrombosis in one case. No perineural invasion or lymph nodes involvement were observed. The soft tissues adjacent to the tumor bed did not show major morphological changes. No correlation was found between the histological changes and each of the following: the anatomical (upper vs. lower limb) or compartmental location of the tumor; whether the tumor was primary or recurrent; and the types of previous treatment (systemic chemotherapy or radiotherapy) and tumor size.. This is the first serial histological description of the effects of tumor necrosis factor-alpha and melphalan administered via hyperthermic isolated limb perfusion on the tumoral masses of limb soft-tissue sarcomas. The small number of specimens and, especially, the variability of tumors preclude definite conclusions. Larger numbers and more homogeneity are needed in future studies.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents, Alkylating; Chemotherapy, Cancer, Regional Perfusion; Extremities; Female; Fibrosis; Humans; Hyperthermia, Induced; Male; Melphalan; Middle Aged; Necrosis; Sarcoma; Soft Tissue Neoplasms; Tumor Necrosis Factor-alpha

Isolated limb perfusion (ILP) with TNFalpha in combination with melphalan and IFNgamma has resulted in an immediate and dramatic tumor response in patients. Such an effect was also noted following ILP in a rat sarcoma model. This model enables us to investigate several factors responsible for the TNFalpha-induced tumor responses. We applied total body irradiation (TBI) to reduce white blood cell count, to investigate the contribution of leukocytes to the anti-tumor effect of TNFalpha.. Small fragments of the nonimmunogenic BN 175 sarcoma were implanted sc in the lower hind leg. A 5 Gy TBI was performed before ILP at a tumor diameter of approximately 15 mm. The hind limbs of 63 rats were perfused and were divided into 6 groups: group 1, sham perfusion, n = 9; group 2, TBI + sham perfusion, n = 6; group 3, TNFalpha 50 microgram, n = 9; group 4, melphalan 40 microgram, n = 9; group 5, TNFalpha 50 microgram + melphalan 40 microgram, n = 22; group 6, TBI + TNFalpha + melphalan ILP, n = 8. In addition, 10 rats were perfused for histological analysis at 24 h post-ILP.. We observed in Group 1: 9/9 progressive disease (PD); Group 2: 6/6 PD; Group 3: 9/9 PD; Group 4: 9/9 no change (NC) of tumor diameter for at least 4 days; Group 5: 6/22 NC, 16/22 complete remission (CR), 12/16 of which showed skin necrosis at the tumor site; and Group 6: 7/8 NC and 1/8 CR (without skin necrosis). After TBI, WBC reduction of 80-95% was observed, while the number of platelets was not significantly reduced and platelet aggregation was maintained at 72 %. Histological analysis revealed decreased hemorrhagic necrosis associated with the absence of PMN infiltration at the tumor margins in the TBI rats.. TBI and the associated reduction in WBC count decreased the tumor response by TNFalpha and melphalan significantly and abrogated the immediate response of skin necrosis at the tumor site, as found in rats treated with TNFalpha and melphalan without TBI. These data strongly suggest that leukocytes play an important role in the hemorrhagic effects of TNFalpha.

Topics: Animals; Extremities; Leukocyte Count; Male; Melphalan; Necrosis; Neovascularization, Pathologic; Neutrophils; Organ Culture Techniques; Platelet Aggregation; Rats; Rats, Inbred BN; Sarcoma, Experimental; Tumor Necrosis Factor-alpha

Treatment of stage IIIA/B melanoma patients by isolated limb perfusion (ILP) with a combination of tumor necrosis factor-alpha (TNF-alpha) and melphalan induces a complete response in 80-90% of the cases. The mechanism of tumor regression induced by the combination of TNF-alpha and melphalan is not precisely understood. Previous studies focused on the immediate (ie., within a few days) clinico-pathological changes after perfusion involving hemorrhagic necrosis. However, clinical data clearly indicate that complete tumor remission frequently requires a period of a few weeks to as much as months after ILP. Because the mechanism underlying this delayed-type reaction is completely unknown, we studied the clinico-pathological events in patients with such slowly regressing melanoma lesions. For this purpose, 94 biopsies of in-transit melanoma metastasis that were taken sequentially from 11 patients between 1 week and 9 months after ILP were analyzed by light and electron microscopy and immunohistochemistry. Clinical data included patient sex, age, anatomical localization and size of the tumor, and follow-up. All of the 11 patients ultimately responded to perfusion treatment (9 complete, 1 partial, 1 stable disease). Serial biopsies showed scattered individual tumor cell necrosis without hemorrhage. Most of the lesions with this delayed-type reaction pattern were less than 0.5 cm in diameter. They contained varying amounts of histologically viable-looking tumor cells and tumor-infiltrating melanophages. In addition, a marked but transient infiltrate of peritumoral eosinophils and moderate interstitial edema and dermal fibrosis were encountered. Only small numbers of lymphocytes were present. In comparison with the reaction pattern after treatment with melphalan alone, the delayed-type reaction pattern was similar but more intense. The scattered tumor cell necrosis in the latter type may be explained by a TNF-alpha-induced increase in permeability of the tumor vascular bed, which results in higher intratumoral concentrations of melphalan or in a prolongation of its effect. Subsequently, degenerated tumor cells are cleared by macrophages, and, finally, repair by fibrosis occurs. Because the immediate reaction type is evoked by hyperpermeability of the tumor vessels as well, quantitative differences seem to determine which reaction type ensues. We suggest that the extent of tumor vasculature that is sensitive to TNF-alpha determines the onset and histopathological

Topics: Aged; Aged, 80 and over; Chemotherapy, Cancer, Regional Perfusion; Female; Humans; Male; Melanoma; Melphalan; Middle Aged; Necrosis; Tumor Necrosis Factor-alpha

Isolated limb perfusion (ILP) with tumor necrosis factor alpha (TNF alpha) +/-interferon gamma (IFN gamma) and melphalan in patients with primarily irresectable soft tissue sarcoma is promising in terms of tumor regression and limb salvage. However, the feasibility of radiotherapy in combination with this treatment modality has not been established.. Fifteen patients with irresectable soft tissue tumors of the limb underwent ILP with TNF alpha, +/-IFN gamma, and melphalan. Three groups could be distinguished with respect to the role of radiotherapy. In nine patients, the residual tumor could be resected after ILP, and this was followed by radiotherapy with a total dose of 50-70 Gy (2 Gy/day). In one patient with aggressive fibromatosis, ILP was followed by radiotherapy without tumor resection (Group I). In two patients who underwent ILP for recurrent sarcoma, the primary tumor had been treated before by resection and radiotherapy (60 Gy) (Group II). In three patients whose tumors remained irresectable after ILP, radiotherapy was applied later in the course of disease for local palliation (Group III).. In Group I, healing of the resection wound was markedly delayed in four patients, with soft tissue necrosis and infection necessitating amputation in two of them. Following completion of radiotherapy, persistent lymphoceles were encountered in two patients. Radiotherapy-induced fibrosis was encountered in five patients, resulting in a mild limb malfunction in two. Three-patients developed mild edema during radiotherapy. Tumor-associated neuropathy was aggravated by ILP in three patients causing severely disabling motor deficits and limb contractures in two of them. In Group II, ILP did not cause any local problem in the heavily irradiated areas. In Group III, pre-existing limb edema was increased after a total palliative dose of 20 Gy in one patient. Another patient, who had been re-operated for arterial thrombosis immediately after ILP, developed occlusion of the brachial artery 4 months after completion of palliative radiotherapy (36 Gy in 6 Gy fractions).. In patients with irresectable soft tissue tumors, multimodality treatment using ILP with TNF alpha +/- IFN gamma and melphalan, tumor resection, and postoperative high-dose radiotherapy is associated with a considerable risk of tissue necrosis and impaired healing. This risk should be weighed against a possible benefit from radiotherapy in local tumor control.

Topics: Adult; Aged; Antineoplastic Agents; Arm; Chemotherapy, Cancer, Regional Perfusion; Combined Modality Therapy; Edema; Female; Humans; Interferon-gamma; Leg; Liposarcoma; Male; Melphalan; Middle Aged; Necrosis; Radiation Injuries; Radiotherapy Dosage; Retrospective Studies; Risk; Sarcoma; Skin; Soft Tissue Neoplasms; Tumor Necrosis Factor-alpha

A 57-year-old woman presented with the second recurrence of a high-grade malignant fibrous histiocytoma of the right foot, following previous local resection plus curative adjuvant radiotherapy. The first recurrence of the lesion was treated by isolated limb perfusion with cisplatin; the second recurrence was treated by isolated limb perfusion with tumor necrosis factor, interferon, and melphalan. The tumor and the area that had been irradiated showed a bluish color a few hours after tumor necrosis factor perfusion. Amputation of the right foot and leg below the knee had to be performed because of severe necrosis.

Topics: Amputation, Surgical; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Cisplatin; Combined Modality Therapy; Female; Foot Diseases; Histiocytoma, Benign Fibrous; Humans; Interferons; Melphalan; Middle Aged; Necrosis; Neoplasm Recurrence, Local; Perfusion; Radiotherapy Dosage; Radiotherapy, Adjuvant; Time Factors; Tumor Necrosis Factor-alpha

Isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan has shown impressive results in patients with irresectable soft tissue sarcomas and stage III melanoma of the extremities. The mechanisms of the reported in vivo synergistic anti-tumour effects of TNF-alpha and melphalan are not precisely understood. We have developed an ILP model in the rat using a non-immunogenic sarcoma in which similar in vivo synergy is observed. The aim of this present study was to analyse the morphological substrate for this synergistic response of TNF-alpha in combination with melphalan to shed more light on the pathomechanisms involved. Histology of the tumours from saline- (n = 14) and melphalan-treated (n = 11) rats revealed apparently vital tumour cells in over 80% of the cross-sections. Interstitial oedema and coagulation necrosis were observed in the remaining part of the tumour. Haemorrhage was virtually absent. TNF-alpha (n = 22) induced marked oedema, hyperaemia, vascular congestion, extravasation of erythrocytes and haemorrhagic necrosis (20-60% of the cross-sections). Oedema and haemorrhage suggested drastic alterations of permeability and integrity of the microvasculature. Using light and electron-microscopy, we observed that haemorrhage preceded generalised platelet aggregation. Therefore, we suggest that the observed platelet aggregation was the result of the microvascular damage rather than its initiator. Remarkably, these events hardly influenced tumour growth. However, perfusion with the combination of TNF-alpha and melphalan (n = 24) showed more extensive haemorrhagic necrosis (80-90% of the cross-sections) and revealed a prolonged remission (mean 11 days) in comparison with the other groups of rats. Electron microscopical analysis revealed similar findings as described after TNF-alpha alone, although the effects were more prominent at all time points after perfusion. In conclusion, our findings suggest that the enhanced anti-tumour effect after the combination of TNF-alpha with melphalan results from potentiation of the TNF-alpha-induced vascular changes accompanied by increased vascular permeability and platelet aggregation. This may result in additive cytotoxicity or inhibition of growth of residual tumour cells.

Topics: Animals; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Blood Platelets; Chemotherapy, Cancer, Regional Perfusion; Cytoplasm; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Endothelium, Vascular; Immunohistochemistry; Male; Melphalan; Microscopy, Electron; Necrosis; Rats; Sarcoma, Experimental; Tumor Necrosis Factor-alpha

The effect of lowering intracellular glutathione (GSH) concentrations on the toxicity of alkylating agents, and RNA synthesis inhibitor and topoisomerase 1 and 2 inhibitors to a number of human leukaemic cell lines were evaluated. By using the GSH synthesis inhibitor DL-buthionine-(S,R)-sulfoximine (BSO), GSH levels were artificially reduced. Cells with low GSH concentrations were exposed to a number of cytotoxic agents and the resultant mode of cell death was analysed using morphological and biochemical criteria. It was found that untreated cells exposed to the above drugs underwent apoptosis to varying extents. However, the toxicity of alkylating agents was dramatically increased to all cell lines on lowering GSH levels, with the mode of cell death switching from apoptosis to necrosis. The reduction of GSH levels had no effect on the toxicity of actinomycin-D, camptothecin or etoposide, nor did it affect the mode of cell death induced by these agents. These observations suggest that modulation of GSH levels effect the toxicity of alkylating agents and that GSH influences the mode of cell death induced by alkylating agents.

Topics: Alkylating Agents; Apoptosis; Camptothecin; Cell Death; Cell Line; Chlorambucil; Dactinomycin; DNA; Glutathione; Humans; Melphalan; Necrosis

Topics: Antistreptolysin; Arthritis; Clone Cells; Electrophoresis; Female; Humans; Immunoelectrophoresis; Melphalan; Middle Aged; Multiple Myeloma; Myeloma Proteins; Necrosis; Radiography; Remission, Spontaneous; Skull; Spine; Tonsillitis

Topics: Amputation, Surgical; Bone Marrow Diseases; Chemotherapy, Cancer, Regional Perfusion; Extracorporeal Circulation; Extremities; Fascia; Female; Hepatitis B; Humans; Lymph Node Excision; Lymphatic Metastasis; Male; Melanoma; Melphalan; Methods; Necrosis; Neoplasm Recurrence, Local; Neuritis; Prognosis; Thrombophlebitis

Topics: Female; Humans; Leg Ulcer; Melphalan; Middle Aged; Multiple Myeloma; Necrosis; Prednisone; Purpura; Waldenstrom Macroglobulinemia