Naturally occurring hydroxytyrosol: synthesis and anticancer potential.

Several epidemiological and animal studies have suggested that polyphenols, a group of secondary plant metabolites occurring mainly in the plant kingdom, may have a protective effect against some chronic degenerative diseases such as cancer. Polyphenols are part of the human diet, being present in vegetal food and beverages. Among them, an olive biophenol named hydroxytyrosol [2-(3,4- dihydroxyphenyl)ethanol, HTyr] has recently received particular attention because of its antioxidant, antiproliferative, pro-apoptotic, and anti-inflammatory activities, which have the potential to specifically counteract all cancer hallmarks, thus representing the expectant biological activities underlying the anti-tumor properties of this polyphenol. After a description of the synthetic procedures to prepare pure HTyr, this review takes into consideration the chemopreventive and chemotherapeutic potential of HTyr as the result of its antioxidant, antiproliferative and anti-inflammatory activities. In particular, the review is focused on the current knowledge of the main cellular and molecular mechanisms used by HTyr to affect carcinogenesis, highlighting the specific oncogenic and inflammatory signaling pathways potentially targeted by HTyr.

Induction of apoptosis in human pancreatic cancer cells by docosahexaenoic acid.

Polyunsaturated fatty acids have been indicated to induce anti-proliferative and/or apoptotic effects in various tumor cells. We showed that, at a 200- micro M concentration, both alpha-linoleic (18:2 n-6; LA) or docosahexaenoic (22:6 n-3; DHA) acid inhibited cell growth, while only DHA induced apoptosis in the human Paca-44 pancreatic cancer cell line. Investigating the mechanism underlying DHA-induced apoptosis, we showed that DHA induced a rapid and dramatic (>60%) intracellular depletion of reduced glutathione (GSH), without affecting oxidized glutathione (GSSG). Moreover, using two specific inhibitors of carrier-mediated GSH extrusion, cystathionine or methionine, we observed that GSH depletion occurred via an active GSH extrusion, and that inhibition of GSH efflux completely reversed apoptosis. These results provide the first evidence for a possible causative role of GSH depletion in DHA-induced apoptosis.

Interleukin-2 gene transfer into human transitional cell carcinoma of the urinary bladder.

Transitional cell carcinoma of the bladder is one of the human cancers most responsive to immunotherapy, and local interleukin-2 (IL-2) production appears to be an important requirement for immunotherapy to be effective. In this study, we engineered two human bladder cancer cell lines (RT112 and EJ) to constitutively release human IL-2 by retroviral vector-mediated gene transfer. Following infection and selection, stable and consistent production of biologically active IL-2 was demonstrated at both the mRNA and the protein level. Morphology, in vitro growth rate and proliferation, as well as other cytokine gene mRNA or membrane adhesion receptor expression, were not altered in IL-2 transduced cells as compared to their parental or control vector-infected counterparts. Moreover, IL-2 engineered cells lost their tumorigenicity into nu/nu mice and the mechanism of rejection appeared to involve multiple host effector cell populations, among which a prominent role was played by neutrophils and radiosensitive cells. These findings may offer support to the development of an IL-2-based gene therapy approach to human bladder cancer.

Immune response following intravesical bacillus Calmette-Guerin instillations in superficial bladder cancer: a review.

Local immunotherapy with bacillus Calmette-Guerin (BCG) can prevent recurrences and progression of superficial bladder cancer, but the antitumoral mechanism of BCG is still unclear. The first event seems to be binding of BCG to urothelial cells via fibronectin, and processing of mycobacterial antigens by antigen-presenting cells. Experimental data suggest that bacterial antigens can also be processed by urothelial cells. CD4 lymphocytes subsequently recognize antigenic peptides presented by HLA class II molecules. The most common profile of urinary cytokines is interleukin-2 and interferon-gamma, suggesting the predominant involvement of the Th1 lymphocyte subpopulation. Natural killer cells, lymphocyte-activated killer cells, BCG-activated killer cells and macrophages are able to kill bladder tumor cells in vitro, but there is no evidence that a major histocompatibility complex (MHC)-restricted specific T cytotoxic response is involved in BCG antitumor activity.

Clonality of tumor-infiltrating lymphocytes in human urinary bladder carcinoma.

The immune system has been implicated in the control of bladder tumor growth. To evaluate the clonality of bladder tumor-infiltrating T lymphocytes (TILs) in vivo, we studied the T-cell antigen receptor (TCR) repertoire in tumor biopsy specimens from 10 patients with transitional-cell carcinoma (TCC) of the bladder. Nine patients had a primary tumor, and one had a multifocal disease, consisting of two bladder tumors and three bilateral upper urinary tract sites of involvement. The following specimens from the nine patients with a primary tumor also were analyzed: a recurrent tumor from four patients, a metastatic lymph node from one patient, and peripheral blood from five patients. We used a high-resolution polymerase chain reaction (PCR) method to determine CDR3 (complementarity-determining region 3) size lengths of TCR beta-chain transcripts. Oligoclonal T-cell expansion was identified in all specimens, with a larger number of expanded clones in the tumors than in peripheral blood. Expanded clones were identified in several beta-chain variable region (BV) subfamilies and varied from one patient to the next and also in different specimens from the same patient. However, a number of clones with the same VJ combination and the same CDR3 size were identified in a given patient (in specimens collected either simultaneously or at different times), suggesting homogeneity in the immunogenic environment. Clonal T-cell expansion in patients with bladder cancer may reflect prolonged exposure of T lymphocytes to tumor antigens. Our findings provide a basis for functional studies to elucidate T lymphocyte-bladder tumor cell interactions.

Local immunotherapy of superficial bladder cancer by intravesical instillation of recombinant interleukin-2.

Local immunotherapy of superficial bladder cancer by endovesical administration of recombinant interleukin-2 (rIL-2) was investigated in a phase I-II study. Twenty-five patients with Ta-T1, N0, M0, G1-G2 transitional cell carcinoma of the bladder received an induction course of rIL-2 (10 daily instillations) with the tumor in place using an interpatient dose escalation scheme from 3 to 18 x 10(6) IU/day. Seven to fourteen days after the end of the induction course, the objective clinical response was evaluated and transurethral resection of the bladder tumor was carried out. Four maintenance courses (10 daily instillations) were started 1 month after surgery and carried out every 4 months at a dose of 6 x 10(6) IU/day in all patients. Follow-up visits were scheduled bimonthly during treatment and then every 6 months. No evidence of laboratory, local or systemic toxicity was observed even at the highest rIL-2 dosages. Induction of a local inflammatory response could be demonstrated at the tumor site after intravesical rIL-2 treatment. The significant reduction in tumor diameters observed in some patients may be interpreted as a sign of the biological activity of this rIL-2 regimen. Further exploratory work is required to evaluate the clinical efficacy of this immunotherapeutic approach.

A phase II study of advanced colorectal cancer patients treated with combination 5-fluorouracil plus leucovorin and subcutaneous interleukin-2 plus alpha interferon.

Twenty-one patients with advanced, pretreated colorectal cancer in disease progression were entered in a phase II study to investigate the use of 5-fluorouracil (5FU) + leucovorin with subcutaneous Interleukin-2 + alpha interferon (alpha-IFN). Eighteen of these patients were evaluable for response to treatment: 1 partial response (PR) (duration 8 months), 9 stable disease (SD) (median duration of 6.5 months, range 2-15) and 8 progressive disease (PD). The PR patient survived for 15 months, the SD patients for a median of 11 months and 8 months for PD patients. Toxicity evaluated in the 21 patients reached grade 4 for mucositis in two cases. Grade 3 toxicity was observed more frequently for fever (52.3%) and diarrhea (33.3%) and was most probably the result of the combined side-effect of chemotherapy and the biological response modifiers (BRMs). Treatment was, for the most part, carried out on an out-patient basis as originally planned. In 15 patients tests were carried out to verify whether any immuno-activation had taken place. Significant increases were found during the course of therapy regarding cluster of differentiation activation (HLA-DR, CD71, CS25). Different curves were observed during the course of treatment with respect to the CD8 value, which proved higher in SD patients than in PD patients. Our study would seem to suggest that the addition of BRMs to 5FU + leucovorin could increase survival. The next step, however, must be to determine lower doses of IL-2 for subcutaneous administration in order to reduce toxicity but maintain the same immunostimulation.

Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine-activated killer cells with NK phenotype (LAK-NK).

Granzymes A-G are a family of serine proteases localized in the cytoplasmic granules of cytotoxic T lymphocytes (Masson, D. et al. Cell 1987. 49: 679) and granzyme A is secreted by T lymphocytes in response to antigenic stimulation. Granzyme A is also expressed by natural killer (NK) and lymphokine-activated killer (LAK) cells. Here we show that fresh rat NK cells constitutively express granzyme B and that granzyme A and granzyme B are differentially regulated in unstimulated NK cells vs. LAK cells with NK phenotype (LAK-NK cells). We also show that both granzymes A and B are secreted in a calcium-dependent manner, by NK and LAK-NK cells in response to stimuli which trigger NK cell functions.

Interleukin-2 lengthens extrajunctional acetylcholine receptor channel open time in mammalian muscle cells.

The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000-3,000 units/ml). Cell-attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07 +/- 0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system.

Continuous intra-arterial administration of recombinant interleukin-2 in low-stage bladder cancer. A phase IB study.

Toxicity and clinical effects of intra-arterial (IA) continuous infusion of recombinant interleukin-2 (rIL-2) were evaluated in twelve patients with low-stage transitional cell carcinoma (TCC) of the bladder (T1NOMO; G1 to G2). rIL-2 dosages were escalated from 18 x 10(3) to 18 x 10(6) IU/m2/d in four groups of three patients. After two 5-day courses, separated by a 48-hour interval, evaluation of clinical response and transurethral resection (TUR) were carried out. World Health Organization (WHO) Grade 3 toxicity occurred in 2 of 12 patients (hypotension/mental confusion and fever, respectively); all side effects rapidly disappeared after infusion was abandoned. No laboratory toxicity developed in any patient. Two pathologically proven complete responses (CR) were achieved using 18 x 10(4) IU/m2/d, and three partial responses (PR) were achieved using 18 x 10(5) IU/m2/d in two patients and 18 x 10(6) IU/m2/d in one patient, giving an overall response rate of 42%. All objective responses are still ongoing after a mean follow-up time of 23 months (range, 12 to 32 months). Local relapses occurred 3 months after TUR only in two nonresponders.

Local activation of immune response in bladder cancer patients treated with intraarterial infusion of recombinant interleukin-2.

Tumor regression induced in cancer patients by i.v. infusion of interleukin-2 (IL-2) is often accompanied by severe side effects. To investigate whether local administration would affect immune response without the side effects, two 5-day cycles of continuous intraarterial [internal iliac artery] infusion of recombinant interleukin-2 (rIL-2) were performed in 12 patients with transitional cell carcinoma (tumor stage 1, node stage 0, metastasis stage 0, and grade 1-2) of the bladder. Four groups of 3 patients were treated at each of 4 escalating doses of rIL-2 (18 x 10(3), 18 x 10(4), 18 x 10(5), and 18 x 10(6) IU/m2/day) throughout the course of the two IL-2 cycles. This treatment was effective in inducing a marked intratumor inflammatory response, consisting mainly of T-lymphocytes and macrophages. A remarkable dose-dependent increase in the levels of soluble CD25 was observed in the urine of all patients, which was associated constantly with an enhanced number of intratumor CD25+ cells. Intratumor macrophages were often immunoreactive for interleukin-1 and/or tumor necrosis factor, suggesting an activated status. Increased levels of soluble CD25 and CD25+ lymphocytes were observed in peripheral blood only at the two highest doses of rIL-2, while increased percentages of circulating HLA-DR+ and CD71+ lymphoid cells and enhancement of CD3+/CD16+ T-lymphocytes were found at lower doses. Peripheral blood eosinophils were augmented in almost all patients but were rarely increased in situ. We provide evidence that continuous intraarterial infusion of rIL-2 activates host immune response, acting preferentially at the tissue level.

NK and LAK susceptibility varies inversely with target cell MHC class I antigen expression in a rat epithelial tumour system.

Several cell clones derived from cell lines obtained from a rat thyroid carcinoma, induced by in vivo injection of the Kirsten murine sarcoma virus into thyroid gland, and from its spontaneous lung metastases were analysed for their major histocompatibility complex (MHC) class I antigen expression. The susceptibility to natural killer (NK) cell lysis of these clones, differing in their levels of MHC class I antigen expression, was determined and found to vary inversely with the target cell MHC level, confirming numerous reports of the literature. We then tried to localize the step of the multistage natural cytotoxic process, in which class I antigens could interfere, and tested first whether lymphokine (IL-2) activation of the killer (LAK) cells could overcome the differences in MHC class I expression of target cells. As this did not appear to be the case, we studied the binding step by either a cold target inhibition assay and a target binding assay and found that target cells expressing class I antigens show a lower competitive capacity for effector cells than targets not expressing such antigens, indicating that this interference may occur, at least in our system, in the binding step of the cytotoxic process.

Interleukin-2 suppresses established long-term potentiation and inhibits its induction in the rat hippocampus.

The effects of recombinant interleukin-2 (rIL-2) on the potentiation of the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. The application of rIL-2 inhibited the induction of both short-term (STP) and long-term potentiation (LTP) in a dose-dependent manner. In addition, rIL-2 (1000 U/ml) reduced both post-tetanic potentiation (PTP) and LTP maintenance phase. The possible involvement of rIL-2 action on the synaptic potentiation with the enzymatic activity of protein kinase systems is discussed.

Local/regional recombinant interleukin 2 (rIL-2) immunotherapy of tumors. Intra-arterial continuous infusion of rIL-2 in bladder cancer patients: a phase I study.

We have investigated clinical and immunological effects of two 5-day cycle continuous infusion of four escalating doses (3,000; 30,000; 300,000; 3,000,000 U/m2/day) of intra-arterial (i.a.) rIL-2, in ten patients with superficial (Ta-T1, N0, M0) bladder TCC. Tumor TUR was performed four days after the end of the second IL-2 cycle. No clinical or laboratory toxicity was detected in any but one patient, who developed grade III hypotension and grade III neurological toxicity. Two CR and two PR were observed in patients treated with 30,000 and 300,000 U/m2/day of rIL-2, respectively. Clinical responses lasted 8+, 8+, 4+ and 4+ months, respectively. Two out of ten patients developed recurrences two months after tumor resection. In the peripheral blood, no changes in the percentage of T (CD3+) lymphocytes were observed, while a significant increase of CD3+ CD16+ T cells was found in 6/9 patients. In contrast, NK (CD3- CD16+) cells were augmented only in 2/9 patients. An increase of B lymphocytes (CD19+ cells) and monocytes (CD14+ cells) was also observed in 3/9 and 7/9 patients, respectively. Lymphocyte activation marker expression (CD25, CD71, HLA-DR) increased mainly on T lymphocytes. NK and LAK activities were enhanced in 4/10 patients, while ADCC activity augmented in 2/10 patients. No detectable increased levels of plasmatic lymphokines (gamma-IFN and alpha-TNF) were found. Enhanced CD8+ and CD4+ tumor infiltrating lymphocytes were demonstrated after IL-2 treatment. Moreover, at the tumor site, lymphocytes expressing CD25 and HLA-DR antigens were noted. Tumor infiltrating macrophages were positive for IL-1 alpha, IL-1 beta and alpha-TNF.

Enhancement of lymphocyte proliferation and IL-2 receptor expression by a processed form (GM-1/P) of monosialoganglioside GM-1.

In this study we investigated the ability of GM-1/P, a calcium mediated processed form of monosialoganglioside GM-1, of in vivo augmenting mouse T and B-lymphocyte blastogenesis induced by mitogens. We have also determined its effect on IL-2 responsiveness by analyzing the induction of the expression of IL-2 receptor (IL-2r) on mouse spleen cells. Lymphocyte blastogenesis was evaluated by 3H-TdR incorporation of spleen cells from untreated or GM-1/P (1mg/Kg, i.v., day-1) treated mice cultured in the presence of T (PHA, ConA) B (LPS) cell specific mitogens. The stimulatory effects appeared to be due to a direct action on T and B lymphocytes, since proliferative response was not abolished by removal of macrophages. Splenocytes from GM-1/P treated mice showed increased proliferation in response to various concentrations of HrIL-2; moreover under these conditions an increased generation of LAK activity was found. A direct evidence for enhanced expression of IL-2r was obtained by immunofluorescence and FACS analysis using a monoclonal antibody (PC.61) directed against the p55 subunit of murine IL-2r. 29% PC.61+ cells were found in IL-2 cultures from treated spleen cells.

Granzyme A expression by normal rat natural killer (NK) cells in vivo and by interleukin 2-activated NK cells in vitro.

Granzyme A (TSP-1) is a serine esterase expressed in cytotoxic T and natural killer (NK) cell lines. Its presence in in vivo primed T cells is disputed at present. Here we show that normal rat NK cells contain granzyme A in vivo and that its expression is augmented by their short-term in vitro treatment with IL 2. Granzyme A expression in a NK cell population with LAK activity has also been examined.

Overnight incubation of mouse spleen cells in recombinant IL-2 generates cytotoxic cells with NK characteristics from precursors enriched with or devoid of LGL.

Interleukin-2 (IL-2) augments natural killer (NK) activity as well as generating effector cells named lymphokine activated killer cells (LAK) which are capable of lysing a wide spectrum of target cells. A large body of evidence has been accumulated to evaluate the relationship between NK and LAK cells and conflicting results have been reported. Our study was addressed to further analyse this relationship and in particular to investigate whether in a short incubation IL-2 is merely capable of augmenting the activity of pre-existing killer cells, or whether it can also promote the differentiation of precursor cells. Eighteen-hour culture of mouse spleen cells in human recombinant IL-2 induced a DNA-synthesis-independent generation of cytotoxic cells bearing an NK phenotype (aGM-1+, Thy1.2+/-, CD8-, CD4-). These were generated from precursor cells also bearing an NK phenotype, recovered either from low density Percoll fractions enriched in lytic cells with LGL morphology as well as from high density fractions devoid of LGL and cytotoxic activity.

Appearance of granule-associated molecules during activation of cytolytic T-lymphocyte precursors by defined stimuli.

Lysis of target cells by cytolytic T lymphocytes (CTL) is associated with the exocytosis of cytoplasmic granules. Purified granules from CTL cell lines contain a pore-forming protein (perforin), tree serine esterases, granzyme A (60,000 MW), granzyme B (29,000 MW), and granzyme C (27,000 MW). We have compared the kinetics of appearance of cytolytic activity with that of perforin and granzyme A activity during activation of lymphocytes from normal animals with leukoagglutinin (LA) and recombinant interleukin-2 (rIL-2). Unstimulated lymph node cells do not express any of these activities, which appear between Day 3 and Day 4 of stimulation and increase rapidly to reach a pronounced peak on Day 6. On Day 7 all the activities are considerably lower, even though the cells still proliferate exponentially. There is a good correlation between the kinetics of appearance of all of these activities. Using antisera against perforin and against granzyme C, one can detect positive cytoplasmic granules in a small fraction of cells on Day 3; by Day 5, 80-90% of the cells are stained. This proportion decreases again on Day 7.