Analysis of cell proliferation and cell death during in situ hyperthermic treatment of neoplastic cells: a case report of human non-Hodgkin lymphoma.

In this study we observed the effects in vivo of hyperthermic treatment on the cell kinetics (cell proliferation/cell death) in one case of human non-Hodgkin lymphoma, by analyzing the following morpho-cytochemical parameters: Acridine Orange fluorochromasia, mitotic index, proliferating cell nuclear antigen (PCNA) expression, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) labeling, and ultrastructure morphology. After two hyperthermic exposures there was a significant reduction of cell growth rate (e.g. mitotic and PCNA positive cells) and an increase in cell loss by death. The cell death occurred by the typical apoptotic cascade, namely DNA fragmentation, chromatin hypercondensation and margination, karyorrhexis, ribonucleoproteins segregation and cytoplasm cleavage; in addition some necrotic cells were found. The data indicates that the hyperthermic treatments limit the cell proliferation (e.g. arrest and/or deceleration of the cell cycle) by facilitating the trigger of programmed cell death. It was concluded that thermal injury can be considered an effective inducer of antiproliferative and apoptogenic associated effects on the growth of this kind of neoplasia.

Hyperthermia-induced cell death by apoptosis in myeloma cells.

We have analyzed by morphological (TEM) and histochemical (TUNEL reaction) criteria the type of cell death occurring in one case of human multiple myeloma after hyperthermia. Samples of cells examined immediately at the end of two treatments with a 15-day interval showed a significant degeneration, mostly demonstrating features of apoptosis (cell shrinkage, DNA fragmentation, karyorrhexis). The possible causes of the lag period between heating and apoptosis onset-expression are discussed.

Hyperthermic effects on the human metastatic liver: a TEM study.

Using ultrastructural analysis, we studied the effects of hyperthermic treatment of one case of human liver metastasis from colon carcinoma. The results indicate that the main hyperthermic response involves the neoplastic and the histiocytic cell population. The drastic decrease in metastatic cells was accompanied by the appearance of cell fragments and apoptotic bodies. Consequently, the histiocytic component (Kupffer cells) showed increased frequency, indicating an activated state. The data are consistent with a direct action of the heat on tumor cells with subsequent activation of Kupffer cells.

Adjuvant treatment of node-positive breast cancer with cyclophosphamide, doxorubicin, fluorouracil, and vincristine versus cyclophosphamide, methotrexate, and fluorouracil: final report after a 16-year median follow-up duration.

PURPOSE: To determine the long-term impact on disease-free survival (DFS) and overall survival (OS) of adjuvant anthracycline-based chemotherapy, when prospectively compared by random allocation with standard cyclophosphamide, methotrexate, and fluorouracil (CMF) in node-positive (N+) breast cancer patients. PATIENTS AND METHODS: Two hundred forty-nine patients with N+ breast cancer, recruited from eight French cancer centers, were randomized to receive 12 monthly cycles of adjuvant chemotherapy, either CMF (n = 112) or doxorubicin, vincristine, cyclophosphamide, and fluorouracil (AVCF) (n = 136). All had a negative metastatic work-up before inclusion, which was stratified by accrual center, tumor stage (International Union Against Cancer [UICC]), and menopausal status. RESULTS: No severe adverse effect related to grade 4 (World Health Organization [WHO]) toxicity was observed. There was no difference in second primary tumor incidence between the two arms. The treatment given was 88% of planned for AVCF and 75% for CMF in both premenopausal and menopausal patients. With a median follow-up time of 16 years (range, 13 to 17), the OS and DFS rates are significantly longer in the AVCF arm (56% v 41% [P = .01] for OS, and 53% v 36% [P = .006] for DFS). These differences are significant, irrespective of tumor stage (T1 to T2 v T3 to T4), and remain positive in patients with or without postoperative locoregional radiotherapy (55% of cohort). When analyzed according to menopausal status, the differences remain significant only for premenopausal patients. CONCLUSION: This set of mature controlled data confirms the added value of anthracycline-based combination adjuvant therapy for N+ breast cancer patients when compared with CMF, with both regimens given for 1 year.

Lysosomal exocytosis induced by hyperthermia: a new model of cancer death. III. Effect on liver metastasis.

The purpose of the present study was to evaluate by morphological approaches (light and electron microscopy), the effect of hyperthermic treatment in one case of human liver metastasis. The results demonstrate that hyperthermia causes a significant reduction of the metastatic cells circulating into sinusoids and the "normalization" of the hepatocytes substructure. The data are consistent with a direct and/or indirect action of the temperature on the presence of infiltrating tumor cells. Particular importance is attributed to a general activation of lysosomes present in neoplastic cells, Kupffer cells and hepatocytes.

Lysosomal exocytosis induced by hyperthermia: a new model of cancer cell death. II. Effect on peritoneal macrophages.

Recently we described the "macrophagic lysosomal exocytosis" (MLE) as a possible new mode of cancer cell death induced by hyperthermia (HT) [1]. In order to confirm this first report, HT was applied at the peritoneum level with perfusional procedure, after catheter insertion under local anesthesia. We evaluated the subcellular changes of peritoneal macrophages in human gastric tumor, before and after hyperthermic treatment at 42 degrees C for 90 minutes. Transmission electron microscopy showed that treatment produced the disappearance of the cytoplasmic granules, with a consequent extracellular scavenger action of the phagocytic cells, the proliferation of some organelles such as mitochondria, endoplasmic reticulum and Golgi complex that may be addressed to a subsequent restoration of the granular pool in the degranulated macrophages. This phenomenon can enhance the already documented effect of hyperthermic perfusional chemotherapy in peritoneal tumors.

Can neo-adjuvant chemotherapy prevent residual tumors?

MA 16/C is a spontaneous mouse mammary adenocarcinoma. It is hormone-dependent and was injected s.c. into C3H/He female mice on day 0. Tumors were excised on day 15. Neo-adjuvant treatments were applied from day 1 to day 21 for hormonotherapy and immunotherapy and on days 1, 5 and 9 for chemotherapy. Adjuvant treatments were applied from day 21 to day 42 for hormonotherapy and immunotherapy, and on days 21, 25 and 29 for chemotherapy. Mixed (neo-adjuvant and adjuvant) treatments combined the two patterns. Chemotherapy consisted of an oxalato-platinum complex of trans-l-dach (l-OHP) at a dose of 5 mg/kg i.p. Hormonotherapy consisted of the LH-RH agonist (D-Trp6) LH-RH, at a dose of 100 micrograms/kg i.p. Zinc gluconate (6mg/kg per os) and bestatin (6mg/kg per os) were administered as immunoregulators. Under present experimental conditions, surgery alone did not increase the life span. Both neo-adjuvant and adjuvant chemotherapy and neo-adjuvant hormonotherapy, however, when added to surgery, increased survival significantly (p less than 0.02-p less than 0.03).

Do tuftsin and bestatin constitute a biopharmacological immunoregulatory system?

Tuftsin is the tetrapeptide Thr-Lys-Pro-Arg. It is spontaneously released from the Fc fragment of IgG by two specific enzymes. One 25-micrograms dose administered to mice in good immunologic status stimulated phagocytosis, macrophage killing of tumor cells, delayed hypersensitivity, cytolytic T-cell activity, antibody production, antibody-dependent cell-mediated cytotoxicity (ADCC), and natural killer (NK) cell activity. Administered for 6 months at the dose of 10 micrograms once a week to old, immunodepressed mice, tuftsin restored macrophage and T-cell cytotoxic activities. At this dosage, tuftsin prevented spontaneous tumor development. Tuftsin was also well tolerated in phase I studies in humans in increased polymorphonuclear leukocytes and OKT4-positive lymphocytes. Bestatin is extracted from Streptomyces olivoreticuli. One 100-micrograms dose of bestatin injected in young mice with normal immunologic status increased macrophage cytotoxicity, antibody production, ADCC, and NK cell activities. Long-term administration of bestatin (100 micrograms once a week) corrected macrophage and T-cell cytotoxicity and prevented age-related spontaneous tumors. Bestatin inhibited lymphocyte membrane aminopeptidase, which degrades tuftsin into a tripeptide that is an antagonist competing with it for receptors. Tuftsin and bestatin constitute a biopharmacologic system that can be developed as other aminopeptidase inhibitors are available for study.

Cytostatic action of two nitrosoureas derived from cysteamine.

2-Chloroethyl nitrosocarbamoylcystamine or ICIG-1325 (CNCC) is a lipid-soluble isomeric mixture of nitrosoureas. Its dose-effect relationship on L1210 leukaemia is characterized by a large maximally efficient dose-range (MEDR), greater than that of other nitrosoureas. CNCC also demonstrated significant therapeutic activity on intracerebrally (i.c.) transplanted L1210 leukaemia and on six transplanted solid tumours, TM2 mammary carcinoma, M555 ovarian carcinoma, B16 melanoma, glioma 26, 3LL, Lewis lung carcinoma and colon 26 carcinoma. It was inactive on fibrosarcoma ICIG-Ci4. Its antitumour activity spectrum is wider than that of the related compounds 2-[3-(2-chloroethyl) 3-nitrosoureido]D-glucopyranose (CZT), (chloro-2-ethyl)-1(ribofuranosyl-isopropylidene-2'-3' paranitrobenzoate-5')-3 nitrosourea (RFCNU), and (chloro-2-ethyl)-1 (ribopyranosyl triacetate-2'-3'-4')-3 nitrosourea (RPCNU). A study of its metabolic disposition in animals has shown that CNCC undergoes extensive first-pass metabolism leading to the formation of four main plasma metabolites. These metabolites are water-soluble nitrosoureas that arose from the bioreduction of the disulphide bridge followed by the methylation and the oxidation of the thiol groups. Experimental screening was performed with these chemically synthesized metabolites. Both N'-(2-chloroethyl)-N-[2-(methylsulphinyl)ethyl]-N'-nitrosourea (CMSOEN2) and N'-(2-chloroethyl)-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea (CMSO2EN2) are very active on L1210 leukaemia grafted intraperitoneally (i.p.) and i.c., L40 leukaemia, B16 melanoma, glioma 26 and Lewis lung carcinoma. Their effectiveness is better than that of the parent compound CNCC. In addition,the percentage of mice cured after CMSOEN2 or CMSO2EN2 treatment is increased especially on B16 melanoma and glioma 26. 6 Haematological toxicity of both active metabolites is lower than that of CNCC, particularly on platelets which is the main toxicity location due to nitrosoureas.

In vivo immunopharmacological properties of tuftsin (Thr-Lys-Pro-Arg) and some analogues.

Tuftsin (Thr-Lys-Pro-Arg) is part of the Fc fragment of a leukophilic IgG and is a stimulator of the phagocytic activity of macrophages and polymorphonuclear cells (PMN) when cleaved from its carrier molecule. Tuftsin was shown to stimulate in vitro all PMN and macrophage functions examined through binding to specific cell surface receptors. In the present work, we provide further evidence that synthetic tuftsin administered to mice may act as an immunomodulator and that its effects on immune functions may result from a primary action on macrophages. After i.v. injection at a dosage of 25 micrograms/mouse, tuftsin stimulated effector (phagocytosis) and regulatory (IL1 production) functions of macrophages and potentiated DTH reaction. Lymphocyte functions (proliferative response to mitogens, T cell-mediated cytotoxicity, IL2 and gamma IFN production) were depressed at times at which macrophage activities were maximally enhanced, suggesting that negative regulatory functions of these latter cells were also stimulated. Tuftsin analogues were synthetized representing substitution or derivatization of the threonyl residue. The relative potencies of these analogues in augmenting phagocytosis-induced chemiluminescence of macrophages were tuftsin greater than or equal to (Gly1)-tuftsin greater than for-tuftsin greater than (for-Met1)-tuftsin greater than (Met1)-tuftsin. Concerning potentiation of DTH reaction the order was (Gly1)-tuftsin greater than or equal to (for-Met1)tuftsin greater than tuftsin greater than (Met1)-tuftsin greater than for-tuftsin. In contrast to tuftsin, none of the analogues induced depression of spleen cell reactivity to mitogens. In addition, (for Met1)-tuftsin administration resulted in an increased production of IL2 and IFN by ConA-stimulated spleen cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of three new nitrosourea analogs (CNCC, RFCNU and chlorozotocin) on in vivo destruction of L 1210 leukemia cells and on the immune response.

Three new nitrosourea analogs (CNCC, RFCNU, and chlorozotocin) had comparable activities in vivo against L1210 leukemia cells. In addition to the antileukemia effect, these drugs also decreased both the humoral immune response to sheep red blood cells and the delayed hypersensitivity reaction to oxazolone. The immunodepression induced by these agents lasted at least 25 days, and could not be reversed by the transplantation of normal syngenic bone marrow cells into treated animals.

From experimental to clinical attempts in immunorestoration with bestatin and zinc.

Severe impairment of the lymphopoietic cell renewal system is an important etiological factor of cancer development and it may be the consequence of massive radio and/or chemotherapeutic regimens. In a comparative study, we analysed the potential, systemic immunorestoratory capacity of bestatin, a microbial leucil-aminopeptidase inhibitor and of the ubiquitous trace element zinc. In vivo administration of bestatin in mice stimulated both Interleukin 1 and Interleukin 2 production, and enhanced T cell, B cell as well as macrophage mediated immunoreactions. In a phase II clinical trial on 41 patients with non-Hodgkin lymphoma, Hodgkin disease and solid tumors, bestatin treatment corrected the pathological frequency of both OKT4 and OKT8 lymphocyte subpopulations. Zinc-saturated transferrin had a significative stimulatory effect on the ongoing DNA synthesis of antigen activated human lymphocytes in culture. Oral administration of zinc-gluconate to patients who manifested a severe T cell subpopulation defect corrected preferentially the OKT8 suppressor/cytotoxic T cell unbalances. The clinical results obtained by both bestatin and zinc were observed only on a short-term, so further studies are needed to elaborate long lasting regiments and to establish whether these treatments have determinant influence on the underlying disease.

Enhancement of NK-cell activity in normal and tumor-bearing mice after administration of aclacinomycin.

Aclacinomycin (ACM) a new cytotoxic antibiotic employed in cancer chemotherapy, can either enhance or inhibit the NK-cell activity of the immune system, depending on the dose administered. A single intraperitoneal injection of 2-4 mg/kg of ACM augments the cytolytic activity by spleen and peritoneal exudate cells of normal mice and spleen cells depleted of nylon-adherent cells and peritoneal exudate cells of tumor-bearing mice. In contrast to the stimulatory effect of NK-cell activity by low doses of ACM, a single injection of 8 mg/kg of this agent leads to depression in the level of NK-cell activity in both normal and tumor-bearing animals. We suggest that the mechanism through which the ACM enhances NK-cell activity may be through the deletion of a suppressor cell population acting on the NK cells.

Adriamycin, vincristine, cyclophosphamide and 5-fluorouracil (AVCF) compared with cyclophosphamide, methotrexate and 5-fluorouracil (CMF) in premenopausal breast carcinoma. Personal results.

Adjuvant treatment of breast cancer with AVCF gave significantly longer disease-free survival than CMF in the group of patients taken as a whole, in the subgroup with lymph node involvement and in the premenopausal subgroup. No significant difference was found regarding overall survival.