Rat Colon Carcinoma Research Articles

A small mammal model of tumour implantation, dissemination and growth factor expression after partial hepatectomy

Surgical resection remains the most effective therapy for metastatic colorectal cancer confined to the liver, although the extrahepatic recurrence rate is high. To develop a mammal model in order to investigate by which mechanisms liver surgery affects distant tumour recurrence. In this animal study the effect of partial hepatectomy (phX) on the development of tumour noduli in the lungs was evaluated. CC531 rat colon carcinoma cells were inoculated i.v. 24h before, during or 24h after surgery. Rat serum was obtained at different time-points after phX and added to in vitro CC531 cell cultures. Finally, phX was compared with an ileum resection (ilX). phX leads to increased tumour noduli in the lungs, compared to Sham operation (p=0.002), but only when performed directly before the injection of tumour cells and not when performed 24h before or after the inoculation. Comparable results were obtained for ilX. No growth stimulation of tumour cells after incubation with rat serum, obtained at different time-points after phX, could be detected in vitro. Not only phX, but also surgery, in general promotes distant tumour recurrence exerting the effect during the early phase of tumour cell adhesion and not during tumour outgrowth.

γ-Glutamyltransferase is upregulated after oxidative stress through the Ras signal transduction pathway in rat colon carcinoma cells

γ-Glutamyltransferase (GGT) plays a central role in the homeostasis of the antioxidant glutathione (GSH). The expression of GGT has been shown to be upregulated after oxidative stress, but the signalling pathways implicated remain poorly characterized. The results here show that acute exposure of CC531 cells to oxidative stress resulted in activation of Ras and augmented GGT enzyme activity, both at the transcriptional and at the translation level. Moreover, an involvement of the GGT promoter II was detected after RT-PCR and transient transfection studies. Ectopic expression of activated Ras, but not dominant negative Ras, also resulted in increased GGT promoter II transcriptional activity, an effect that was attenuated by over-expression of dominant negative mutants of Akt, p38 MAPK and MEK1. Addition of specific inhibitors of these kinases during oxidative stress diminished the activation of GGT. In conclusion, oxidative stress-induced activation of GGT involves Ras and several downstream signalling pathways.

Decrease in size of azoxymethane induced colon carcinoma in F344 rats by 180-day fermented miso

The present study was designed to investigate the effects of fermented miso (fermented soybean paste) on the induction of colon tumors by azoxymethane (AOM) in male F344 rats. A total of 91 rats, 6 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. The animals were placed on diets one week before the first AOM dose: commercial normal control MF diet or a diet containing 10% 2-year, 180-day fermented, or 3-4-day fermented miso. There were no differences in body and organ weights, and no aberrant crypt foci (ACF) among carcinogen-treated groups at week 25. The rates of tumor incidence were 45%, 85%, 75% and 60% with the 2-year, 180-day, and 3-4-day fermented miso and MF, respectively, and those for colon tumors were 34%, 55%, 60% and 55%, respectively. The size of well-differentiated adenocarcinomas and total (well differentiated and signet ring cell) adenocarcinomas in the 180-day fermented miso group was significantly smaller than that in the 2-year fermented miso and MF+AOM groups. Nuclear staining of beta-catenin in colon tumors was increased for the 3-4-day fermented miso compared to the 180-day fermented miso. Cdx2 staining tendency was decreased in colon tumors and adenocarcinomas compared to normal mucosa and ACF, which stained in 100% of cases. In addition, the PCNA index was significantly reduced in the 180-day group compared with those groups receiving the 3-4-day fermented miso and MF diet. The germinal region was also decreased. The present results indicate that dietary supplementation with 180-day fermented dietary miso could act as a chemopreventive agent for colon carcinogenesis.

Combined Treatment with Octreotide, Galanin and Serotonin in Gastrointestinal Malignancies

Triple therapy with octreotide, galanin and serotonin reduces the volume and weight of both rat and human colon carcinoma in xenografts. The reduction in tumour volume and weight seems to be caused ...

Efficient infection of tumor endothelial cells by a capsid-modified adenovirus

Targeted antiangiogenic gene therapy is an attractive approach to treat metastatic cancer. However, the relative paucity of the receptors of the commonly used adenovirus serotype 5 in endothelial cells as compared with liver cells undermines the use of this vector for targeting the endothelial cells in tumors. To overcome this problem, we analyzed the ability of a hybrid Ad5/35 virus, where the serotype 5 fiber has been replaced with the fiber from serotype 35, to target tumor vasculature. Infection of human umbilical vein endothelial cells (HUVECs) with Ad5/35 at MOI 120 infected 100% of cells. In contrast, infection with Ad5 at the same MOI infected only 10% HUVECs. Ad5/35 was even more effective in transducing human aortic endothelial cells (HAECs), as infection with Ad5/35 at MOI 3.6 was sufficient to transduce 95% of cells. Gene expression analyses demonstrated that infection of HUVECs and HAECs with Ad5/35 resulted in between 1 and 3 orders of magnitude higher gene expression than infection with Ad5. Furthermore, various liver-derived cells were less infectable with Ad5/35 than Ad5, indicating a favorable toxicity profile for this virus. In a rat colon carcinoma tumor model, Ad5 was located mainly in the liver parenchyma after hepatic artery administration. In contrast, Ad5/35 was found only in the angiogenesis-rich border region of the tumor. Double immunostaining revealed that Ad5/35 colocalized with CD31 and Flk-1 positive endothelial cells. These results indicate that Ad5/35 may be useful in anticancer strategies targeting tumor endothelial cells.

Triple Treatment with Octreotide, Galanin and Serotonin is a Promising Therapy for Colorectal Cancer

In patients with colorectal cancer, low levels of colonic somatostatin, galanin and serotonin have been found. Based on these findings, the effects of triple treatment with octreotide (a somatostatin analogue), galanin and serotonin on colorectal cancer has been studied. Triple therapy was found to reduce the volume and weight of both rat and human colon carcinoma in xenografts, apparently by necrosis, but also by reducing proliferation and expression of epidermal growth factor of cancer cells, and also by inducing apoptosis. It has been suggested that tumour necrosis results from ischemia in the tumour caused by a reduction in the tumour blood flow, a consequence of reduced number of tumour-feeding blood vessels and by constricting of tumour feeding arterioles. The effects of treating rat colorectal cancer using single, double and triple therapy with octreotide, galanin and serotonin were studied. Of these substances, galanin alone achieved a significant reduction in tumour-feeding blood vessels. Single and double regimes had some effect, but were not nearly so successful as triple treatment. The optimum treatment dose of triple therapy lies between 40 and 80 microg/kg/day, smaller doses had no effect on the tumours at all, while larger doses had no additional effect. The optimal administration route is continuous i.p. infusion, for 14 days. Triple therapy gave no obvious side effects, and had equivalent anti-tumour and therapeutic efficacy as standard treatment with 5-fluorouracil/leucovorin. Although this treatment appears to be a promising option, clinical trials need be conducted to establish whether it can be beneficial in clinical use.

Evaluation of the Interaction Between TGF β and Nitric Oxide in the Mechanisms of Progression of Colon Carcinoma

It is recognised that stromal cells determine cancer progression. We have previously shown that active TGFbeta produced by rat colon carcinoma cells modulated NO production in rat endothelial cells. To elucidate the role of TGFbeta and NO in the mechanisms of interaction of colon carcinoma cells with stromal cells and in cancer progression, we transfected REGb cells, a regressive colon carcinoma clone secreting latent TGFbeta, with a cDNA encoding for a constitutively-secreted active TGFbeta. Out of 20 injected rats only one tumour progressed, which was resected and sub-cultured (ReBeta cells). ReBeta cells secreted high levels of active TGFbeta. The adhesive properties of REGb and Rebeta cells to endothelial cells were similar, showing that the secretion of active TGFbeta is not involved in tumour cell adhesion to endothelial cells. ReBeta, but not REGb, cell culture supernatants inhibited cytokine-dependent NO secretion by endothelial cells, but inhibition of NO production was similar in co-cultures of REGb or ReBeta cells with endothelial cells. Therefore, secretion of active TGFbeta regulated endothelial NO synthase activity when tumour cells were distant from, but not in direct contact with, endothelial cells. However, only ReBeta cells inhibited cytokine-dependent secretion of NO in coculture with macrophages, indicating that the active-TGFbeta-NO axis confers an advantage for tumour cells in their interaction with macrophages rather than endothelial cells in cancer progression.

Chemoinducible gene therapy: A strategy to enhance doxorubicin antitumor activity

A replication-defective adenoviral vector, Ad.Egr-TNF.11D, was engineered by ligating the CArG (CC(A/T)6GG) elements of the Egr-1 gene promoter upstream to a cDNA encoding human tumor necrosis factor-alpha. We report here that Ad.Egr-TNF.11D is activated by the clinically important anticancer agents cisplatin, cyclophosphamide, doxorubicin, 5-fluorouracil, gemcitabine, and paclitaxel. N-acetylcysteine, a free radical scavenger, blocked induction of tumor necrosis factor-alpha by anticancer agents, supporting a role for reactive oxygen intermediates in activation of the CArG sequences. Importantly, resistance of PC-3 human prostate carcinoma and PROb rat colon carcinoma tumors to doxorubicin in vivo was reversed by combining doxorubicin with Ad.Egr-TNF and resulted in significant antitumor effects. Treatment with Ad.Egr-TNF.11D has been associated with inhibition of tumor angiogenesis. In this context, a significant decrease in tumor microvessel density was observed following combined treatment with doxorubicin and Ad.Egr-TNF.11D as compared with either agent alone. These data show that Ad.Egr-TNF.11D is activated by diverse anticancer drugs.

Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis.

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.

965. A Novel Approach for Gene Transduction with Adenovirus/Fibrin Glue System

Introduction: The major cause of local recurrence after initial surgery for hepatic malignancy is residual carcinoma macroscopically or microscopically at the surgical site. Although many surgical devices and skills have developed, the recurrence rate of hepatic malignancy is still high. At present, for those patients with recurrent disease, adjuvant therapies such as chemotherapy, radiation therapy and immunotherapy are combined. However, in such cases, severe side-effects make the patient's QOL (quality of life) considerably worse. Therefore, a novel approach for residual carcinoma at the time of surgery is urgently needed. In this study we proposed the use of a novel gene therapy for residual carcinoma of the liver. Purpose: The fibrin glue system (FGS) consists of liquid forms of fibrinogen and thrombin and is used widely in surgical operation as it has a sealing effect and promotes tissue adhesion at the surgically resected site. In this study, we proposed the efficacy and safety of loco-regional gene transfer using adenoviral vector mixed with FGS to prevent local recurrence of liver carcinoma. We demonstrated that the adenoviral vector containing the E.coli LacZ gene (AxCALacZ) was delivered to the liver of rats by FGS. Materials and Methods: Experiment 1 in vitro Rat colon carcinoma cell line, RCN-9 was incubated on a membrane containing multiple 0.02μm-sized holes. Various concentrations of the adenoviral vector mixed with FGS (AxCALacZ/FGS) were applied on the RCN-9 and incubated for 48 hours. The optimum efficiency of transduction was evaluated by X-Gal staining and NIH imaging. Experiment 2 in vitro To examine the survival period of adenoviral vector in FGS, AxCALacZ/FGS put on the monolayer cultured RCN9 after being left for various periods. Transgene expression was evaluated by β-gal assay. The β-gal activity was measured photometrically using the Promega β-gal enzyme assay kit according to the manufacture's instructions (Promega, Madison, USA). Experiment 3 in vivo 8-week-old male F344 rats underwent a partial hepatectomy. AxCALacZ/FGS was applied on the cut surface of the dissected liver and the rats were kept for 48 hours under normal conditions. AxLacZ/PBS was treated as the control study. The transducted and non-transducted parts of the liver were extirpated, stained with X-gal liquid and β-gal was measured to compare the efficacy of transduction in each organ. Results: Experiment 1 significantly more transgene expression was shown by the X-gal staining and NIH imaging than 1MOI. Experiment 2 LacZ expression revealed infected RCN9 when AxCALacZ/FGS was left for a period of less than 96 hours. Experiment 3 AxCALacZ/FGS transgene expression was shown at the treated and non-treated part of the liver more than AxCALacZ/PBS expression. Discussion and conclusion: It was demonstrated that adenoviral vector survives and being stable for enough time to transduct in FGS. AxCALacZ/FGS can transduct the target gene. Furthermore, the transduction efficiency was comparable to those of existing methods. In the future, FGS will be studied with an adenoviral vector containing the gene which enhances antitumor immunity for preventing recurrence loco-regionally site as well as its original tissue adhesive purpose.

In vivo bioluminescence imaging of locally disseminated colon carcinoma in rats.

Animal tumour models using orthotopic tumours for the evaluation of cancer therapies are of greater clinical relevance than subcutaneous models, but they also pose greater difficulties for measuring tumour size and quantifying response to treatment. In this study, we used noninvasive bioluminescence imaging to monitor the intraperitoneal growth of luciferase-transfected CC531 colorectal cells in adult WAG/RIJ rats. The bioluminescence signal correlated well with post-mortem assessment of tumour load by visual inspection of the peritoneal cavity at specific follow-up times. Using bioluminescence imaging, we were able to monitor peritoneal tumour growth sequentially in time and to calculate a tumour growth rate for each animal; this is not possible with invasive methods of evaluating tumour load. Bioluminescence imaging of rats treated with a single dose of cisplatin (4 mg kg−1, i.p.) demonstrated a significant delay in peritoneal tumour growth relative to saline controls (mean 45.0±s.d. 13.0 vs 28.2±10.3 days; P=0.04). Similar protocols evaluated by visual scoring of tumour load at 40 days after inoculation supported these findings, although no quantitative assessment of treatment-induced growth delay could be made by this method. This study shows that in vivo imaging of luciferase-transfected tumour cells is a useful tool to investigate the dynamics of disseminated tumour growth and efficacy of anticancer treatment in orthotopic models of peritoneal cancer in rats. It offers an attractive alternative to invasive methods, and requires fewer animals for measuring tumour response to therapy.

CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative.

We investigated the mechanisms of immune tolerance raised by tumors by comparing immunogenic and tolerogenic tumor cell clones isolated from a rat colon carcinoma. When injected into syngeneichosts, the immunogenic REGb cells yield tumors that are rejected, while the tolerogenic PROb cells yield progressive tumors and inhibit the regression of REGb tumors. We show here that PROb tumor volume is correlated with an expansion of CD4(+)CD25(+) regulatory T lymphocytes in lymphoid tissues. These cells delay in vivo the rejection of REGb tumors and inhibit in vitro T cell-mediated immune responses against REGb cells through a mechanism that requires cell contact between effector and regulatory T cells and involves TGF-beta. While total T cells fromPROb tumor-bearing rats yield no apparent anti-tumor immune response, depletion of CD25(+) T cells restores this reactivity. A single administration of cyclophosphamide depletes CD4(+)CD25(+) T cells in PROb tumor-bearing animals, delays the growth of PROb tumors, and cures rats bearing established PROb tumors when followed by an immunotherapy which has no curative effect when administered alone. These results demonstrate the role of CD4(+)CD25(+) regulatory T cells in tumor-induced immune tolerance and the interest of regulatory T cell depletion to sensitize established tumors to immunotherapy.

Cytotoxic reactions of CC531s towards liver sinusoidal endothelial cells: a microscopical study

Colorectal cancer cells can induce apoptosis in cells of various tissues [1]. Apoptosis can be induced by a number of factors such as Fas inducing apoptosis through the Fas/FasL pathway; other factors involve the TRAIL pathway and TNF. It is known that metastasizing colon cancer cells express more FasL then primary carcinoma cells [2]. We investigated whether a rat colon carcinoma cell line CC531s could induce apoptosis in liver sinusoidal endothelial cells (LSECs). LSECs and CC531s were co-cultured for 18 hrs and cells were visualized by SEM and TEM. Apoptosis was visualized by markers such as Hoechst and Propidium iodide. Furthermore, cells were recorded by time lapse video microscopy with and without an antagonistic antibody for FasL.

Butyrate reduces liver metastasis of rat colon carcinoma cells in vivo and resistance to oxidative stress in vitro

Injection of the rat colon carcinoma cell line CC531 into spleen of syngeneic rats results in considerable amounts of liver metastases within 14 days. We investigated whether preincubation of the cells with butyrate reduced their metastatic ability in vivo and whether this was accompanied by reduction in related properties such as secretion of metalloproteinases and their ability to withstand oxidative stress. Butyrate incubation reduced cell growth rate and initiated apoptosis in a dose- and time-related manner, but proliferation was retrieved when cultivation was continued in medium without butyrate. Splenic injection of butyrate treated, proliferating cells resulted in significantly reduced amounts of tumor mass compared to untreated cells. The butyrate treated cells were more susceptible to oxidative stress than control cells, as demonstrated by increased number of apoptotic cells and reduced cell growth after exposure to menadione. A reduction in cellular glutathione was found after prolonged incubation with butyrate. Butyrate appeared not to alter the secretion of active metalloproteinases from the cells although an apparent increase in proforms was demonstrated. Neither did butyrate alter the synthesis of metalloproteinase inhibitors. Lastly, a reduced adhesion of the tumor cells to collagen coated matrix was found after butyrate treatment. Thus, the inhibitory effects of butyrate on tumor malignancy are caused by a diversity of mechanisms.

Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix

A preformed gelatin matrix containing adherent rat colon carcinoma cells was transplanted subcutaneously into rats to analyze the outgrowth of the tumor and the inflammatory response. The gelatin matrix simplifies the precise localization of the tumor cells early after implantation and allows the gelatin piece with a growing tumor to be dissected for analysis in vitro, after various times in vivo. The immortalized mesenchymal progenitor cell line MPC1cE was cocultured with rat colon carcinoma cells in vivo in gelatin matrix. The mesenchymal progenitor cells inhibited the outgrowth of the rat colon carcinoma and a complete inhibition was seen if the number of mesenchymal progenitor cells were at least equal to the number of tumor cells. The mixture of tumor cells and mesenchymal progenitor cells induced more infiltration of monocytes and granulocytes than tumor cells or mesenchymal progenitor cells alone. Infiltration of T cells and CD31 + endothelial cells correlated to the presence of tumor cells and not to mesenchymal progenitor cells. These findings suggest that tumor cell culture in vivo in a gelatin matrix is effective for early localization of tumor cells in vivo and that mesenchymal progenitor cells effectively inhibit the growth of the tumor cells in vivo.

Hyaluronan content in experimental carcinoma is not correlated to interstitial fluid pressure

Mechanism(s) for generation of the high tumor interstitial fluid pressure (TIFP) that is characteristic of carcinoma is not known. We investigated the role of hyaluronan, the major water-binding polysaccharide of the extracellular matrix, for the generation of a high TIFP. A human anaplastic thyroid carcinoma (KAT-4) xenografted to athymic mice and a syngeneic rat colon carcinoma (PROb) were used. Neither KAT-4 nor PROb cells produced hyaluronan (HA) in culture, however, both cell lines produced factors that stimulated HA-synthesis by cultured fibroblasts. Modulating hyaluronan levels by transfection of PROb carcinoma cells with hyaluronan synthase-2 revealed no correlation between hyaluronan content and TIFP. Furthermore, lowering of TIFP by treating KAT-4 tumors with a specific inhibitor of TGF-β1 and -β3 did not change the concentration of hyaluronan in the tumors. In summary, our results suggest that a modulation of hyaluronan content is not a major pathogenetic mechanism for the generation of the characteristically high TIFP in malignant carcinomas.

An Intravital Model to Monitor Steps of Metastatic Tumor Cell Adhesion Within the Hepatic Microcirculation

Organ-specific tumor cell adhesion within the microcirculation of host organs is an important step in the metastatic cascade. Circulating tumor cells have to adhere within the microcirculatory vessels, quickly stabilize their adhesion and probably leave the circulation to avoid toxic effects of hydrodynamic shear forces of circulating blood. Using intravital fluorescence microscopy we established a new model for the intravital observation of colon carcinoma cell adhesion within the hepatic microcirculation. HT-29 (human) and CC531 (rat) colon carcinoma cells were fluorescence labeled using CalceinAM. Single cell suspensions were injected intraarterially in Sprague-Dawley rats. Using intravital fluorescence microscopy adhesive interactions of circulating tumor cells within the hepatic microcirculation were observed at the liver surface. These interactions were analyzed regarding their time course and the localization within the vascular tree. Autofluorescence of liver parenchyma was sufficient for distinction of hepatic sinusoids. Intravital microscopy enabled the differentiation of early events in adhesion formation within hepatic sinosoids, adhesion stabilization, and extravasation of the tumor cells into the liver parenchyma. Tumor cell adhesion occurred almost exclusively within sinusoidal capillaries; however, the diameter of these vessels was usually larger than that of the tumor cells leaving remaining perfused lumen of the capillaries. Colon carcinoma cells rapidly migrated into the liver parenchyma after successful adhesion within the sinusoids. In contrast to common endpoint assays of the metastatic cascade, this in vivo model allows investigations of metastatic colon carcinoma cell adhesion within the liver microcirculation as specific steps during the formation of hematogenous metastasis and their underlying mechanisms. ( J Gastrointest Surg 2003;7:507–515).

Triple therapy with octreotide, galanin, and serotonin reduces the size and blood vessel density and increases apoptosis of a rat colon carcinoma

A rat colonic adenocarcinoma was implanted subcutaneously in female nude (C57BL/6JBom-nu) mice. After 7 days, the animals were divided into different groups. One group received triple therapy with octreotide, galanin, and serotonin, 10 μg/kg body weight of each, twice daily. The second group served as controls and received only saline solution. Three groups received 10 μg/kg body weight twice daily of octreotide, galanin, or serotonin. The last group consisted of controls that received only saline solution. The treatment lasted for 5 days. The tumour volume, wet weight, and relative volume density of blood vessels were significantly decreased after the triple treatment, as compared to controls. Apoptotic index was significantly increased, but the proliferation index was not affected in the group of mice that received triple therapy. There was no significant difference between controls and mice treated with octreotide, galanin, or serotonin regarding tumour volume or weight. The relative volume density of blood vessels was decreased in tumours treated with galanin, but not with octreotide or serotonin. There was no statistical difference in the proliferation index between controls and animals treated with octreotide, galanin, or serotonin, as compared with controls. Tumour necrosis and increased apoptosis may be responsible for the reduction in the volume and weight of the tumour after triple therapy. Tumour necrosis may be caused by the induction of tumour ischemia due to a reduction in tumour blood flow, which is caused by decreased incidence of tumour-feeding blood vessels, and by constriction of tumour-feeding arterioles. These results are promising and may offer treatment for colon cancer.

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.

Immunological aspects of photodynamic therapy of liver tumors in a rat model for colorectal cancer.

We have investigated tumor immunological effects of photodynamic therapy (PDT) of liver metastases. Livers of Wag/Rij rats were inoculated with three tumors of a syngeneic rat colon carcinoma cell line, CC531. One tumor in each rat was illuminated, with or without previous administration of the photosensitizer metatetrahydroxyphenylchlorin (mTHPC). PDT was effective in causing necrosis of tumors, but it did not affect the growth rate of nearby, nonilluminated tumors in the liver. Immunological staining of tumors showed natural killer (NK) cells to be significantly lower in PDT-treated tumors than in control tumors (P < 0.05). T cells in PDT-treated tumors and in their margins were lower than in tumors that received only sensitizer or only illumination (P = 0.015) at day 2 after treatment but reappeared at the tumor margins from day 7 after treatment. For macrophages, a similar pattern was found. NK cells, T cells or macrophages in nonilluminated tumors in mTHPC-treated rats did not increase significantly when compared with tumors in rats without mTHPC treatment. These findings indicated that no antitumor effect of a systemic immune response was present, as measured by the effect of PDT on growth of distant tumors and the number of T lymphocytes, NK cells and macrophages in these tumors.