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Purpose: Effectiveness of a new device inserted within the common cellulose acetate cigarette filter (named hypobaric chamber tar-removing system, HCTRS) to remove tar and its carcinogenic compounds from mainstream cigarette smoke (MCS)., Methods: Eighty HCTRS prototypes were mounted inside the cellulose acetate filter of commercial-brand cigarettes (13 mg tar) and smoked by eighty smoker volunteers. Tar retained by HCTRS prototypes was determined by weighing them before and after smoking. Subsequently, an aliquot (3-5 mg) of the tar retained by twenty randomly chosen HCTRS prototypes was analysed by high-performance liquid chromatography (HPLC) for the detection of polycyclic aromatic hydrocarbons (PAH)., Results: The mean value of tar retained was 12.80 mg/HCTRS prototype (S.D. = 5.31), thus showing that this simple device is capable of removing 98.5% of tar present in MCS. Minimal and maximal amounts of retained tar were 4.15 and 31.47 mg/HCTRS prototype, respectively. Moreover, these tar samples contained, although in differing amounts, each of the 16 priority pollutant PAH. A mean value of 259.42 ng/mg of tar (S.E.M. = 44.37) of the 16 main PAH was found in the tar of the 20 HCTRS prototypes examined. These data cogently demonstrate that the use of the HCTRS prototype can effectively eliminate about 100% of tar from MCS, thus reducing the inhalation of PAH (considered the most obvious carcinogenic tar components)., Conclusions: The application of this device could be a suitable tool for effectively improving human health through the prevention of smoking-associated cancer.

Adriamycin (ADR), one of the major antitumor agents used for the clinical treatment of a wide variety of human cancers and its glutathione(GSH)-conjugated adduct, ADRIGLU, induced apoptosis in K562 erythroleukemia and TVM-A12 clone 2 melanoma human cell lines. We have previously reported that ADR has nuclear localization and that ADRIGLU localizes exclusively in the cytoplasm. During ADR or ADRIGLU treatment, significant depletion of the cell energy state, demonstrated by a reduction in high-energy phosphates (ATP and GTP) and a decrease in energy charge potential (ECP), were recorded between 2 hours and 24 hours, by HPLC analysis. Transmission electron microscopy also revealed that between 2 hours and 24 hours of ADR or ADRIGLU treatment, mitochondria underwent evident morphological changes, from an initial "high amplitude swelling state" to a "shrinkage state" and finally, in early apoptotic cells, to an "abnormal shrinkage state", in which a marked accumulation of pycnotic mitochondria was also observed. Confocal microscopic analysis, using the potential-sensitive dye JC-1, showed that inhibition of cell energy metabolism was preceded by a rapid decrease in mitochondrial transmembrane potential (delta psi m). With the progression of exposure time, the early depolarization of the mitochondrial membrane was followed by a transient reversion to normal delta psi m until, in apoptotic cells, almost all mitochondrial subpopulations appeared to be hyperpolarized. Our results indicated that mitochondria are actively involved in anthracycline-induced programmed cell death, suggesting a novel mechanism that may be common to all forms of apoptosis.

In spite of extensive investigation, the mechanism for cell cytotoxicity of the anthracycline antitumor drug adriamycin (ADR) has not yet been completely understood but the nature of the cytotoxic effects of this drug is generally related to its interaction with nuclear components, such as DNA and topoisomerase II. In a previous paper, we studied, using Confocal Laser Scanning Microscopy (CLSM), the localization of ADR and its glutathione (GSH)-conjugate (ADRIGLU), obtained by the anaerobic reaction of the parent anthracycline with reduced GSH, in drug-sensitive and in multidrug resistant (MDR) cells. In all drug-sensitive lines used, ADR was mostly located in the nuclei, while its GSH-conjugate was found only in the cytoplasm, predominantly in the Golgi region. In this study we examined the morphological changes induced by ADR or its GSH-conjugated adduct (ADRIGLU) treatments in TVM-A12 (clone 2) melanoma and K562 erythroleukemia human cell lines, correlated to programmed cell death (apoptosis). We observed that ADR-induced apoptosis in both cell lines tested after 5 h treatment: CLSM and Scanning Electron Microscopy (SEM) showed cell shrinkage, fragmentation and condensation of nuclear chromatin, cell surface blebbing and cytoplasmic vacuolization. On the contrary, ADRIGLU-induced fragmentation and condensation of nuclear chromatin, typical of apoptosis, only after 48-72 h treatment. Cytoflourimetric assay by propidium iodide staining confirmed the data obtained by CLSM and SEM. Our data suggest that apoptosis activation by anthracycline antitumor drugs is induced not only by direct interaction with nuclear components but also with cytoplasmic compartments.

Background: We have reported previously that combined chemo-immunotherapy with cyclophosphamide (CY), thymosin alpha 1 (T alpha 1) and low dose interferon alpha,beta (IFN alpha beta) has significant anti-tumour effects. Here, using mouse B16 melanoma as a model, we tested whether increasing the dose of T alpha 1 could increase the anti-tumour activity of triple combination chemo-immunotherapy., Materials and Methods: C57BL/6 mice were challenged subcutaneously with B16 melanoma cells and injected intraperitoneally with saline, CY (200 mg/kg, day 7), or CY with T alpha 1 (200, 600 or 6000 micrograms/kg/day, days 10-13) and IFN alpha beta (30,000 I.U., day 13)., Results: Chemo-immunotherapy with high dose (HD)-T alpha 1 caused complete tumour regression for 27.5 days after tumour cell injection (3.9 times longer than untreated controls) and delayed tumour relapse compared to all other groups. Moreover, it significantly increased the median survival time of treated mice, and cured an average of 23% of animals, while none was cured in any other group. Splenocytes from HD-T alpha 1-treated mice showed markedly increased cytotoxic activities against both YAC-l and autologous B16 tumour cells. HD-T alpha 1 treatment reversed the tumour-induced reduction in percentages of CD3 and CD4-positive splenocytes to non-tumour levels, and it increased percentages of CD8, B220 and IL-2R beta-positive cells to well beyond non-tumour controls., Conclusions: High doses of T alpha 1 improve anti-tumour efficacy of tnple chemo-immunotherapy against B16 melanoma. These effects appear to be mediated by stimulation of the host immune response.

The accumulation of adriamycin (ADR), daunomycin (DAUNO) and their glutathione (GSH)-conjugates, recently obtained by anaerobic reaction of the parent anthracyclines with reduced GSH, was examined in drug-sensitive and multidrug resistant (MDR) cell lines using confocal laser scanning microscopy. In all drug-sensitive lines used (TVM-A12 and TVM-A197 human melanoma cells, K562 lymphoblastoid cells and MCF-7 human breast cancer cells) ADR and DAUNO were mostly located in the nuclei, while their GSH-conjugates were found only in the cytoplasm, predominantly in the Golgi region. On the contrary, in MDR MCF-7/Dox cells, both conjugated and non conjugated anthracyclines gave fluorescence only in the cytoplasm, mostly in the Golgi region, the intensity of the fluorescence being stronger in cells pretreated with verapamil. Viability assay showed that the GSH-conjugate are significantly less cytotoxic than the parent anthracyclines in sensitive cells and showed the same scarce cytotoxicity in MDR MCF-7/Dox cells. These results demonstrate that conjugation of anthracycline antitumor drugs with GSH prevents their access to the nucleus and decreases their cytotoxicity. Furthermore, the observations on MCF-7/Dox suggest that GSH-conjugation of anthracycline might occur in resistant cells and can be in part responsible for the MDR in breast cancer cells.

TLP (Tumour Liberated Proteins) is a 214 kDa protein, isolated from lung cancer tissue and synthetic nonapeptide CSH-275 is a major epitope identified on a 100 kDa TLP fragment and used to create antibodies in rabbit (antiserum termed CSH-419). CSH-419 antiserum, labelled or conjugated as necessary, was used to detect TLP on sera from NSCLC patients by a new ELISA test set up as a 1 step sandwich format test. This ELISA was performed on sera from 534 individuals. TLP was detected in 53.1% of NSCLC patients, with a 0% response in patients with cancers other than NSCLC, 7.6% response in unknown blood donors, and 17.4% response in patients with chronic lung diseases correlated with an elevated risk for lung cancer. TLP was particularly present in early stages of disease: 75% in stage I, 56% in stage II and III and 45% in stage IV. The presence of TLP antigen in sera from NSCLC patients indicates that TLP could represent an useful tumour marker.

22 patients with advanced non-small-cell lung cancer were randomized to receive chemotherapy (ifosfamide) or chemotherapy followed by thymosin alpha1 + low-dose IFNalpha. Chemo-immunotherapy induced an enhanced response rate compared with chemotherapy alone (33% and 10% respectively). Although these differences were not significant, the difference in time to progression was (p=0.0059). CD4+, CD8+ and NK cell counts were significantly depressed after two cycles of chemotherapy, while no difference in cell count were seen in chemo-immunotherapy treated patients. Hematologic toxicity was reduced by the immunotherapy, with no grade 3/4 toxicity seen compared to 50% in patients treated with chemotherapy alone.