Mice Bearing Lewis Lung Carcinoma Research Articles

Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology.

The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the beta-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-alpha) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3--5 microl plasmid DNA (1 microg DNA/microl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5--10 mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48 h and strong reporter gene expression 72 h and 96 h after jet injection. The simultaneous jet injection of the TNF-alpha and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24 h, 48 h, 72 h, 96 h and 120 h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA.

Cisplatin-loaded polymer-metal complex micelle with time-modulated decaying property as a novel drug delivery system.

The pharmacological activity and pharmacokinetics of cisplatin (CDDP)-loaded polymeric micelles were examined to reveal their usefulness as a novel tumor-directed drug carrier system of CDDP. In biodistribution assay, free CDDP or CDDP-loaded micelles were administered intravenously to Lewis lung carcinoma-bearing mice. Antitumor activity and nephrotoxicity were respectively evaluated by the measurement of tumor size and plasma blood urea nitrogen (BUN) after single bolus i.v. administration of each drug. The time profile of the plasma Pt level after the injection of the micelles exhibited a time-modulated disappearance as observed in saline in vitro. The micelles exhibited 5.2- and 4.6-fold higher AUC of Pt in the plasma and tumor, respectively, with minimal change in the kidney, in comparison with free CDDP, suggesting that prolonged circulation of Pt in circulation and specific accumulation in the tumor were achieved utilizing the micellar drug carrier system. Administration of the micelles at the dose exhibiting antitumor activity similar to free CDDP did not increase the plasma BUN, whereas free CDDP induced its remarkable increase. CDDP-loaded micelles restrained nephrotoxicity, which is the dose-limiting factor of CDDP, while exhibiting tumor-specific accumulation. Thus, CDDP-loaded micelles are expected to be a novel formulation of CDDP for clinical use.

Stress and chemotherapy. Combined effects on tumor progression and immunity in animal models.

In mice bearing Lewis lung carcinoma, rotational and restraint stress specifically increases the formation of lung metastasis, and restraint stress markedly attenuates the antitumor effects of cyclophosphamide. The aim of this investigation was therefore to examine the effects of restraint stress on tumor metastasis in mice bearing MCa mammary carcinoma, and on the effectiveness of CCNU and DTIC. Restraint stress increases MCa mammary carcinoma metastasis, causes a marked reduction in cyclophosphamide activity, and a minor attenuation of the effects of CCNU and DTIC. The possible occurrence of seasonal factors, observed for the increase by rotational stress of Lewis lung carcinoma metastasis, was also determined for cyclophosphamide effectiveness. The survival time of control mice is longer in February than in June, and is not appreciably modified by rotational stress. The effects of cyclophosphamide are similar in both seasonal periods, and are similarly attenuated by rotational stress. The seasonal effects of rotational stress, and the reduction of the effects of cyclophosphamide caused by rotational stress, are accompanied by corresponding variations in the number of CD3+ and CD4+ splenic T-lymphocyte subsets and in the CD4+/CD8+ ratio, respectively. The reported effects of stress on tumor progression and on the effectiveness of cyclophosphamide thus appear to occur via modulation of immune responses of the host directed against the tumor. These data appear of interest for their experimental implications, and suggest the opportunity to consider the role that the stress during treatment may play in determining the effectiveness of clinical antitumor chemotherapy.

Antitumor Components from an Actinomycete Strain 6011W

Three bioactive compounds that inhibited nucleoside transport were isolated from the cultured broth of Streptoverticillium sp. 6011W. The structures of those compounds were characterized as cinnamamide, N-(tetrahydro-2-oxo-3-thienyl)-acetamide and benzamide, respectively. They all inhibited radiolabeled thymidine transport into Ehrlich carcinoma cells, with IC50 values of 30.4, 97.2 and 85.4 μM, respectively. When administered i.p., cinnamamide not only inhibited the growth of transplanted tumors but also reduced the number of lung metastases in mice bearing Lewis lung carcinoma. The results suggest that nucleoside transport inhibition assay is a valuable model to search for antitumor agents of natural origin.

Skewed differentiation of bone marrow CD34 + cells of tumor bearers from dendritic toward monocytic cells, and the redirection of differentiation toward dendritic cells by 1α,25-dihydroxyvitamin D 3

Tumor presence is detrimental to the development of antigen-presenting dendritic cells. Since dendritic cells can arise from CD34 + precursor cells, the present study assessed the capacity of bone marrow CD34 + cells from tumor bearers to develop into dendritic cells when cultured in the absence of either tumor cells or their products. Culturing bone marrow CD34 + cells from mice bearing Lewis lung carcinomas yielded a lower number of dendritic cells than arose from CD34 + cells of normal mice. This reduced yield of dendritic cells was associated with a shift to development of monocytic cells and a reduced antigen presenting capability by the cultures. When the CD34 + cell cultures from tumor bearers were supplemented with the differentiation-inducing hormone 1α,25-dihydroxyvitamin D 3, there was the restoration of dendritic cell development and antigen presenting ability. These results show that CD34 + cells from tumor bearers remain defective in their development into dendritic cells even when cultured outside the tumor environment, but development of dendritic cells can be restored with 1α,25-dihydroxyvitamin D 3.

Green Tea and Its Major Components Ameliorate Immune Dysfunction in Mice Bearing Lewis LungCarcinoma and Treated With the Carcinogen NNK

The protective effects of tea and/or its components on dysfunction of immune functions during tumor growth and carcinogenesis in mice were studied using two experimental models: C57/BL6J mice transplanted with Lewis lung carcinoma (LLC) and Kunming mice treated with a single dose of 4-(methylnitrosamino-)-1-(3-pyridyl)-1-butanone (NNK). In C57/BL6J mice bearing LLC, the weight of the thymus decreased, the proportion of CD4 +-positive T lymphocytes and the ratio of CD4 + to CD8 + decreased, luminol-enhanced chemiluminescence of white blood cells in peripheral blood stimulated by zymosan increased, and plaque-forming cells (PFC) decreased. However, in LLC-bearing mice given green tea as drinking water, all immune functions were improved, along with inhibition of tumor growth. In Kunming mice treated with NNK, during the four weeks of observation, their immunologic indicators, such as phagocytosis of macrophages in the abdominal cavity, luminol-enhanced chemiluminescence of white blood cells, plaque-forming cells, and delayed-type hypersensitivity, increased or decreased to various extents compared with normal controls. However, these changes were significantly prevented in the mice given green tea, mixed tea, or tea polyphenol as drinking water. In conclusion, tea and its components ameliorated immune dysfunction in mice bearing LLC or treated with the carcinogen NNK.

Inhibition of gelatinase A (MMP-2) by batimastat and captopril reduces tumor growth and lung metastases in mice bearing Lewis lung carcinoma.

We have examined the effects of the synthetic matrix metalloproteinase inhibitor, batimastat (BB-94) and the angiotensin-converting enzyme inhibitor, captopril, on metalloproteinase activity of murine Lewis-lung-carcinoma cells (3LL) in vitro, and on local growth and lung metastasis of the same tumor implanted intramuscularly in syngeneic C57BL/6 mice. The effect of BB-94 and captopril on the survival of the 3LL-tumor-bearing mice was also examined. Here we report that captopril treatment resulted in decreased transcription and protein levels of gelatinase A by 3LL cells. Both BB-94 and captopril also prevented substrate degradation by gelatinase A and B released in conditioned medium by cultured cells. Treatment of tumor-bearing animals with BB-94 (i.p.) or captopril (in drinking water) resulted in significant inhibition of the mean tumor volume (25 and 33% respectively) and of the mean lung metastasis number (26 and 29% respectively). When both agents were given, they acted in synergy, resulting in 51 and 80% inhibition of tumor growth and metastasis. The survival time of the mice treated with both BB-94 and captopril was also significantly longer compared with the groups treated with each agent alone or with the vehicle. Our data support the hypothesis of an essential role of metalloproteinase(s) in the metastatic process. Moreover, blockade of invasion, angiogenesis and other processes mediated by metalloproteinases may underlie the anti-tumor and anti-metastatic effect of BB-94 and captopril and their combination. It is conceivable that this combination could be tested in selected clinical conditions as an adjuvant modality to cytotoxic therapy.

Melatonin administration in tumor-bearing mice (intact and pinealectomized) in relation to stress, zinc, thymulin and IL-2

Melatonin (MEL) may counteract tumors through a direct oncostatic role. MEL is also an antistress agent with immunoenhancing properties against tumors due to a suppressive role of MEL on corticosterone release. Rotational stress (RS) (spatial disorientation) facilitates metastasis progression in mice. Also, MEL counteracts tumors because of its influence on immune responses via the metabolic zinc pool, which, is reduced in tumors and stress. Zinc is required for normal thymic endocrine activity (i.e. thymulin) and interleukin-2 (IL-2) production. Because in vivo data is still controversial, exogenous MEL treatment (22 days in drinking water) in both intact and pinealectomized (px) mice bearing Lewis lung carcinoma leads to significant decrements of metastasis volume, restoration of the negative crude zinc balance, recovery of thymulin activity and increment of IL-2 exclusively in intact and px tumor bearing mice subjected to RS. Significant inverse correlations are found in both stressed intact and px tumor bearing mice after MEL treatment between zinc and corticosterone (r = 0.78, P < 0.01; r = 0.80, P < 0.01, respectively). Positive correlations between zinc and IL-2 (r = 0.75, P < 0.01; r = 0.73, P < 0.01, respectively) or thymulin (r = 0.75, P < 0.01; r = 0.82, P < 0.01, respectively) are observed in same models of mice. These findings suggest a MEL action to decrease metastasis mediated by a possible interplay between zinc and MEL, via corticosterone, with consequent restoration of thymic efficiency and IL-2 production. MEL as an antistress agent with immunoenhancing properties in cancer deserves further consideration.

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Seasonal effects of rotational stress on Lewis lung carcinoma metastasis and T-lymphocyte subsets in mice

Rotational stress specifically increases the formation of spontaneous lung metastasis in mice bearing Lewis lung carcinoma, without significantly modifying the growth of primary tumor. The increase in metastasis number and volume caused by rotational stress varies in magnitude with a highly significant circannual rhythm; the acrophase approximately coincides with summer solstice. Rotational stress causes a significant reduction in the number of CD3+ and CD4+ T-lymphocyte subsets in summer, whereas in winter the number of CD3+ subset is significantly increased; the CD4+ CD8+ ratio and the number of NK 1.1 antigen positive cells are not significantly modified by rotational stress in both periods considered. The increase in metastasis formation by rotational stress thus appears to negatively correlate with the number of splenic CD3+ and CD4+ T-lymphocyte subsets. This seasonal behavior occurs in spite of the control of light cycle, temperature and humidity in the animal housing, suggesting the existence in the host of an endogenous oscillator with a circannual period. These data indicate the opportunity to consider endogenous rhythms within the host, as well as seasonal factors, in studies on stress and neuro-immunomodulation in experimental oncology.

Restraint Stress Reduces the Antitumor Efficacy of Cyclophosphamide in Tumor-Bearing Mice

Treatment with the cytotoxic antitumor drug cyclophosphamide is highly effective in mice bearing Lewis lung carcinoma, causing the absence of macroscopically detectable tumors at necroscopy after sacrifice. When the effects of the treatment on survival are determined, a significant increase in survival time and in the proportion of long-term survivors is observed. When restraint stress is further applied, tumors develop in all of the mice treated with cyclophosphamide, and survival time and the fraction of long-term survivors are significantly reduced. Flow cytometry of splenic T-lymphocyte subsets in normal mice indicates a significant decrease in the number of CD3+, CD4+, and CD8+subsets after treatment with cyclophosphamide and after application of restraint stress; the interaction of the two treatments is significant for CD3+and marginally significant for CD4+subsets. The attenuation by restraint stress which was observed for the effects of cyclophosphamide on the presence of tumors at necroscopy and for the survival of the treated mice might thus be interpreted as follows: restraint stress attenuates the immune functions of the host directed toward the weakly immunogenic tumor, an effect which, in the absence of restraint stress, interacts effectively with the cytotoxic action of cyclophosphamide toward tumor cells. The results obtained using this animal model thus indicate that experimental stress reduces the therapeutic efficacy of a cytotoxic antitumor drug; experimental and clinical implications are discussed.

Specific Uptake Ratio of Octa-α-butyloxy-zinc Phthalocyanine in Lewis Lung Carcinoma

Summary Octa-α-butyloxy-zinc phthalocyanine (8-α-bo-ZnPc) is a newly synthesized photosensitizer characterized by a high absorption coefficient at the far red wavelength 735 nm and a good singlet oxygen quantum yield. The pharmacokinetics of 8-α-bo-ZnPc was studied in mice bearing lewis lung carcinoma after i.v. administration of 7.8 μmole/kg body weight at 2 h, 6 h, 12 h, 24 h, 48 h, and 168 h incubation intervals. The accumulated photosensitizer was chemically extracted from selected tissues and the concentrations were measured by absorption spectroscopy. The ratio of the accumulated photosensitizer concentration to its injected dose is named the specific uptake ratio (SUR). In this study, the biodistributions of 8-α-bo-ZnPc in different tissues and organs was recorded in the form of SUR. The parenchymatous organs, liver and spleen, showed a maximum 8-μ-bo-ZnPc SUR after 6 h incubation. An extensive uptake was detected in the lung at 6 h, 12 h, 24 h, and 48 h extracts. 8-α-bo-ZnPc SUR in malignant tissue was low during the entire investigation period. The photosensitizer uptake by muscle and skin were even lower. Because of the low uptake by tumour, 8-α-bo-ZnPc is suggested to be administered topically or in combination with a suitable carrier system in order to increase the photosensitizer uptake in the target tissue, as well as to decrease the loss of dye to other tissues.

132-hydroxy-bacteriopheophorbidea-methylester pharmacokinetics in mice bearing Lewis lung carcinoma

The photosensitiser 132-hydroxy bacteriopheophorbide a-methylester (132-OH-BPME) is characterised by a high absorption coefficient at the far red wavelength 750 nm and a good singlet oxygen quantum yield. The pharmacokinetics of 132-OH-BPME was studied on the Lewis lung carcinoma in mice after i.v. administration of 5 mg/kg body weight at different incubation intervals. The accumulation dye was chemically extracted from selected tissues and the concentrations were measured by absorption spectroscopy. The parenchymatous organs, liver, spleen and kidney, showed maximum 132-OH-BPME concentrations after 2 h incubation (liver, spleen) and 4 h postinjection (kidney). A high uptake was detected in the lung with the maximum concentration at 2 h. The malignant tissue accumulated high 132-OH-BPME concentrations at 2–12 h postinjection with peaking at 8 h. The 132-OH-BPME concentrations in muscle tissue, representing the normal tumour surroundings, and in the skin were very low. The results of our study suggest that photodynamic therapy (PDT) using 132-OH-BPME could be effective at an incubation time of about 8 h postsensitiser injection, when the tumour 132-OH-BPME concentration has reached its maximum and the muscle and skin concentrations are at a minimum.

Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.

Acquired drug resistance is a major problem in the treatment of cancer. Of the more than 500,000 annual deaths from cancer in the United States, many follow the development of resistance to chemotherapy. The emergence of resistance depends in part on the genetic instability, heterogeneity and high mutational rate of tumour cells. In contrast, endothelial cells are genetically stable, homogeneous and have a low mutational rate. Therefore, antiangiogenic therapy directed against a tumour's endothelial cells should, in principle, induce little or no drug resistance. Endostatin, a potent angiogenesis inhibitor, was administered to mice bearing Lewis lung carcinoma, T241 fibrosarcoma or B16F10 melanoma. Treatment was stopped when tumours had regressed. Tumours were then allowed to re-grow and endostatin therapy was resumed. After 6, 4 or 2 treatment cycles, respectively, no tumours recurred after discontinuation of therapy. These experiments show that drug resistance does not develop in three tumour types treated with a potent angiogenesis inhibitor. An unexpected finding is that repeated cycles of antiangiogenic therapy are followed by prolonged tumour dormancy without further therapy.

Echistatin inhibits Lewis lung carcinoma cell-matrix adhesion in vitro and experimental metastasis in vivo

Echistatin, a low molecular weight, RGD-containing protein isolated from the venom of Echis carinatus, inhibited Lewis lung carcinoma cell (3LL) adhesion to immobilized fibronectin and laminin. The inhibition was specific, noncytotoxic, dose-dependent and fully reversible. Echistatin showed a stronger activity in inhibiting cell adhesion to fibronectin rather than to laminin and it resulted about 3-fold more effective than kistrin, an other ROD-snake venom protein, in inhibiting 3LL cell attachment to both substrates. The ability of echistatin to modulate experimental metastasis formation in vivo was also evaluated. A 20% inhibition of the lung metastasis spread with respect to controls was observed when 3LL cells and echistatin were coinjected i.m. into male C57BL/6NCr1BR mice. When echistatin was administered i.p. 1 mu g/g of body weight/72 h x 4 doses into mice bearing Lewis lung carcinoma, it promoted only a 15% inhibition of tumor growth but inhibited by 45% lung metastasis formation. These results demonstrate that echistatin is able to inhibit metastasis attachment and spreading in experimental system in vivo independently by its effect on the primary tumor.

821 The effect of experimental stressor and its immuno-modulation on tumor metastasis in mice bearing lewis lung carcinoma

821 The effect of experimental stressor and its immuno-modulation on tumor metastasis in mice bearing lewis lung carcinoma

132-hydroxy bacteriopheophorbide a methylester specific uptake ratio in mice bearing lewis lung carcinoma

Summary The ratio of the accumulated photosensitizer concentration to its injected dose is named the specific uptake ratio (SUR). Investigations on a photosensitizer's biodistribution using the parameter SUR offer a better understanding of the pharmacokinetic behaviours of different photosensitizers and render the comparison of different photosensitizers possible by overcoming the problem of using different sensitizer doses in different studies. The SUR of 13 2 -OH-BPME was recorded in mice bearing Lewis lung carcinoma after i.v administration of 7.8 αmole/kg body weight at different incubation time intervals. The photosensitizer was chemically extracted from selected tissues and concentrations were measured by absorption spectroscopy. The parenchymatous organs liver, spleen, and kidney as well as the lung showed maximum 13 2 -OH-BPME uptake ratios of 1.5 up 5.4 between 2 h and 4 h incubation times. The malignant tissue showed high 13 2 -OHBPME SUR between 2 h and 12 h post injection with a maximum SUR of 1.2 at 8 h incubation time. The 13 2 -OH-BPME SUR in muscle tissue, which represented the normal tumour surrounding tissue, and in the skin were very low and never exceeded 0.09 and 0.3 in muscle and skin, respectively. These results suggest that PDT using 13 2 -OH-BPME could be effective at a photosensitizer incubation time of about 8 h post injection, when the tumour 13 2 -OH-BPME SUR is maximum value, while muscle and skin exhibit minimum 13 2 -OH-BPME uptake. Therefore, phototoxic side effects on normal tissues in the tumour surrondings should not be induced.

Tumour growth modifies intravascular polyamine transport by plasma lipoproteins in the mouse

Polyamines are polycationic compounds which are implicated in cell division and tumor growth. We have evaluated the potential role of plasma lipoproteins in the transport of major polyamines, spermine, spermidine and putrescine, and the effect of tumor growth on such transport. Plasmas of healthy male BL6/DBA2 mice and of mice bearing Lewis lung carcinoma (3LL) were fractionated by isopycnic density gradient ultracentrifugation, and polyamine content determined in lipoprotein fractions. Spermidine was the most abundant polyamine in the lipoproteins of both control and tumor-bearing mice and was principally associated with HDL (d: 1.046–1.136 g/ml); approx. 40% of total plasma polyamines was lipoprotein-associated in control mice and 60% in cancerous mice. Only minor amounts were transported by LDL (<10% of total lipoprotein-associated polyamines), while VLDL were devoid of these substances. Marked elevations of circulating levels of LDL were found in 3LL grafted mice: in these particles however, the contents of spermidine and spermine were significantly reduced. A preferential uptake of polyamines by red blood cells could in part explain this marked reduction of LDL polyamine content, but the consequence of this reduction on the net electrical charge and biochemical function of LDL remains unknown. Elevations of plasma LDL and HDL levels in 3LL-grafted mice underlie the finding that only minor modification was detected in the putrescine content of these particles. However, it is evident that elevated total amounts of putrescine were present in the plasma of such animals. Finally, the density profile of polyamines was modified in cancerous mice in which a shift to transport in lighter apo.AI-containing HDL particles was observed for spermidine; an even more marked shift was found for spermine. In conclusion, our data demonstrate that HDL particles constitute the major plasma vehicle for polyamine transport in both control and in tumor-bearing mice. © 1997 Elsevier Science B.V. All rights reserved.

Pharmacological control of lung metastases of solid tumours by a novel ruthenium complex.

Imidazolium trans-imidazoledimethylsulphoxidetetrachlororuthenate ImH[trans-RuCl4(DMSO)Im] (NAMI-A), a ruthenium compound that replaces Na+ with ImH+ in the molecule of Na[trans-RuCl4(DMSO)Im] (NAMI), was studied for the anti-metastasis effects in models of solid metastasizing tumours of the mouse. NAMI-A, given i.p. at 35 mg/kg/day for six consecutive days, a dose equimolar to that of NAMI, to mice bearing Lewis lung carcinoma and MCa mammary carcinoma, markedly reduces lung metastasis weight by 80-90%, with an effect equal or even superior to that of NAMI, depending on the experimental system adopted. Correspondingly, NAMI-A increases the content of connective tissue in the tumour matrix, around blood vessels, and in the tumour capsule, augments the percentage of tumour cells in G2/M phase and reduces the amount of CD45+ cells infiltrating the tumour parenchyma. The effects of the same doses on spleen lymphocytes correspond to an increase of CD8+ subset without any change of the distribution of cells in G0/G1, S and G2/M phases. The study shows that NAMI-A behaves similarly to NAMI on the several parameters examined in comparison experiments and therefore we suggest to credit NAMI-A with all the biological actions already described for NAMI during the last 3 years. The replacement of Na+ with ImH+ therefore, besides the better chemical stability of the molecule, confers to [trans-RuCl4(DMSO)Im]- a closer similarity with a true drug to be used in humans, and suggests this molecule for future studies of preclinical toxicology and phase I and II clinical trials.

Antitumor activity of Hypsizigus marmoreus. II. Preventive effect against lung metastasis of Lewis lung carcinoma

Antitumor activity of Hypsizigus marmoreus, an edible mushroom, was investigated by in vivo bioassay. The aqueous extract, hereinafter referred to as YH, was tested against syngeneic tumor, Lewis lung carcinoma. YH was found to give a significant increase in life span when assayed using a solid tumor, Lewis lung carcinoma, by intraperitoneal administration, but not as much by oral administration. It was also found that YH have an inhibitory activity of spontaneous tumor metastasis in mice bearing Lewis lung carcinoma by intraperitoneal administration. YH significantly decreased the number of metastasized nodules. It was suggested by Winn test that antitumor and antimetasatic activities shown by YH was effective in increasing activity on immunological by competent cells.