Lucas L. Colombo

Action of hexachlorobenzene on tumor growth and metastasis in different experimental models.

Hexachlorobenzene (HCB) is a widespread organochlorine pesticide, considered a possible human carcinogen. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). We have found that HCB activates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration, in an AhR-dependent manner in MDA-MB-231 breast cancer cells. The aim of this study was to investigate in vitro the effect of HCB (0.005, 0.05, 0.5, 5μM) on cell invasion and metalloproteases (MMPs) 2 and 9 activation in MDA-MB-231 cells. Furthermore, we examined in vivo the effect of HCB (0.3, 3, 30mg/kg b.w.) on tumor growth, MMP2 and MMP9 expression, and metastasis using MDA-MB-231 xenografts and two syngeneic mouse breast cancer models (spontaneous metastasis using C4-HI and lung experimental metastasis using LM3). Our results show that HCB (5μM) enhances MMP2 expression, as well as cell invasion, through AhR, c-Src/HER1 pathway and MMPs. Moreover, HCB increases MMP9 expression, secretion and activity through a HER1 and AhR-dependent mechanism, in MDA-MB-231 cells. HCB (0.3 and 3mg/kg b.w.) enhances subcutaneous tumor growth in MDA-MB-231 and C4-HI in vivo models. In vivo, using MDA-MB-231 model, the pesticide (0.3, 3 and 30mg/kg b.w.) activated c-Src, HER1, STAT5b, and ERK1/2 signaling pathways and increased MMP2 and MMP9 protein levels. Furthermore, we observed that HCB stimulated lung metastasis regardless the tumor hormone-receptor status. Our findings suggest that HCB may be a risk factor for human breast cancer progression.

Inhibition of Invasion and Metastasis by Glypican-3 in a Syngeneic Breast Cancer Model

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and β-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-β increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.

Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5β1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports a key role of HO-1 as a modulator of the angiogenic switch in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.

PKC delta (PKCδ) promotes tumoral progression of human ductal pancreatic cancer

Objective: Our objective was to study the role of protein kinase C delta (PKC&dgr;) in the progression of human pancreatic carcinoma. Methods: Protein kinase C delta expression in human ductal carcinoma (n = 22) was studied by immunohistochemistry. We analyzed the effect of PKC&dgr; overexpression on in vivo and in vitro properties of human ductal carcinoma cell line PANC1. Results: Human ductal carcinomas showed PKC&dgr; overexpression compared with normal counterparts. In addition, in vitro PKC&dgr;-PANC1 cells showed increased anchorage-independent growth and higher resistance to serum starvation and to treatment with cytotoxic drugs. Using pharmacological inhibitors, we determined that phosphatidylinositol-3-kinase and extracellular receptor kinase pathways were involved in the proliferation of PKC&dgr;-PANC1. Interestingly, PKC&dgr;-PANC1 cells showed a less in vitro invasive ability and an impairment in their ability to migrate and to secrete the proteolytic enzyme matrix metalloproteinase-2. In vivo experiments indicated that PKC&dgr;-PANC1 cells were more tumorigenic, as they developed tumors with a significantly lower latency and a higher growth rate with respect to the tumors generated with control cells. Besides, only PKC&dgr;-PANC1 cells developed lung metastasis. Conclusion: Our results showed that the overexpression of PKC&dgr; in PANC1 cells induced a more malignant phenotype in vivo, probably through the modulation of cell proliferation and survival, involving phosphatidylinositol-3-kinase and extracellular receptor kinase signaling pathways.

A novel Salmonella Typhi-based immunotherapy promotes tumor killing via an antitumor Th1-type cellular immune response and neutrophil activation in a mouse model of breast cancer

We investigated the use of a live, attenuated Salmonella enterica serovar Typhi vaccine strain as an antitumor immunotherapy. Mice bearing a subcutaneous tumor (LM3 mammary adenocarcinoma) were immunized on three occasions with S. Typhi strain CVD 915 by injection into the tumor, the peritumoral tissue and the draining lymph node areas; this procedure was termed Salmonella multiple treatment (Salmonella MT). Tumor-bearing mice subjected to the Salmonella MT exhibited reduced tumor growth, prolonged survival and reduced incidence of lung metastases, compared to untreated mice. We examined the mechanisms mediating this effect and found that Salmonella MT promoted an antitumor Th1-type response characterized by increased frequencies of IFN-γ-secreting CD4(+) T and CD8(+) T cells with reduction of regulatory T cells in tumor draining lymph nodes. The main cells infiltrating bacteria-treated tumors were activated neutrophils, which can exert an antitumor effect through the secretion of TNF-α. These results demonstrate for the first time the efficacy of an attenuated S. Typhi vaccine strain as a cancer immunotherapeutic agent. By potentiating the host antitumor immune response, this approach could be a powerful adjunct tool for cancer therapy.

SLOWER GROWTH OF TUMOURS IN SYMPATHETICALLY DENERVATED MURINE SKIN

In order to examine tumour growth in sympathetically denervated murine skin, two breast cancer tumour lines were employed, i.e. M3 tumours, of a relatively high local growth and low metastatic capacity, and MM3-LN tumours, that grew locally at a slower rate but disseminated early to the lung. Mice subjected to unilateral superior cervical ganglionectomy or sham-operation 2 weeks earlier were used. M3 or MM3-LN tumours were implanted in the ipsilateral ear to the surgical procedure. Tumour size was assessed every 2-6 days, starting from the 7th day after tumour implantation. Growth of M3 and MM3-LN tumours was significantly slowed by a previous sympathetic denervation of the skin territory. There were no significant differences in the number or size of pulmonary metastases at autopsy between mice subjected to ganglionectomy or to sham-operation. Ganglionectomy increased significantly ipsilateral submaxillary lymph node ornithine decarboxylase activity by 62% and decreased noradrenaline content to 8% of the innervated contralateral lymph node. The present results indicate a local inhibitory modulation of tumour growth by the sympathetic nervous system.

Differential effects of raloxifene, tamoxifen and fulvestrant on a murine mammary carcinoma

The purpose of this study was to evaluate the effect of the selective estrogen receptor modulators raloxifene and tamoxifen and of the pure antiestrogen fulvestrant on tumor growth and progesterone receptor (PR) expression in an experimental model of breast cancer. The effects of these compounds on cell proliferation were studied in primary cultures of a progestin-dependent mammary carcinoma tumor line, in the presence of medroxyprogesterone acetate (MPA) or 17-β-estradiol (E2). In in vivo studies the tumor was inoculated subcutaneously in BALB/c female mice treated with 20 mg MPA depot. Raloxifene (12.5 mg/kg) or tamoxifen (5 mg/kg) were administered in daily doses or E2 silastic pellets (5 mg) were implanted. When the tumors reached about 25–50 mm2 MPA was removed in half of the animals. E2 induced complete tumor regressions, tamoxifen inhibited tumor growth in vivo while raloxifene disclosed proliferative effects in animals in which MPA had been removed. In vitro, E2 inhibited cell proliferation at concentrations higher than 10−14 M. Raloxifene and fulvestrant, but not tamoxifen, partially reverted E2-induced inhibition. Fulvestrant and tamoxifen inhibited MPA-induced cell proliferation while raloxifene had a stimulatory effect. Tamoxifen and E2 increased, raloxifene induced no effect, and fulvestrant significantly decreased PR expression. In this study we provide evidence for differential effects of tamoxifen and raloxifene on experimental mammary tumors. Since raloxifene is under evaluation for use in breast cancer prevention, these results may have important clinical implications.

Isolated Flavonoids against Mammary Tumour Cells LM2

1 The purpose of the present study was to investigate antitumour and anti-inflammatory activities of flavonoids isolated from Byrsonima crassa, Davilla elliptica and Mouriri pusa. The antitumour activity was measured by the MTT assay in murine mammary tumour cells (LM2) and the IC50 values of the fl avonoids tested ranged from (31.5 ± 2.97) to (203.1 ± 5.9) μg/ml. The fl avonoids (myricetin-3-O-α-L-rhamnopyranoside) and 3 (quercetin-3-Ogalactopyranoside) from D. elliptica were the most active ones against the tumour cells. The same samples were tested to determine the inhibition of the release of nitric oxide (NO) and of the tumour necrosis factor-alpha (TNF-α) in murine macrophages by the Griess and ELISA sandwich assay, respectively. Almost all the samples showed inhibitory activity to the release of NO but not of TNF-α. Of all substances tested, flavonoids 2 (quercetin) and 6 (myricetin) may show promising activity in the treatment of murine breast cancer by immunomodulatory and antiproliferative activities.

Inhibition of nitric oxide is a good therapeutic target for bladder tumors that express iNOS.

Bladder cancer is the second cause of death for urological tumors in man. When the tumor is nonmuscle invasive, transurethral resection is curative. On the other hand, radical cystectomy is the treatment chosen for patients with invasive tumors, but still under treatment, these patients have high risk of dying, by the development of metastatic disease within 5 years. It is therefore important to identify a new therapeutic target to avoid tumor recurrences and tumor progression. Nitric oxide (NO) is an important biological messenger known to influence several types of cancers. In bladder cancer, production of NO and expression and activity of inducible NO synthase was associated to recurrence and progression. The objective of this work was to analyze if inhibition of nitric oxide production could be considered a therapeutic target for bladder tumors expressing iNOS. Using a bladder cancer murine model with different invasiveness grade we have demonstrated that NO inhibition was able to inhibit growth of bladder tumors expressing iNOS. Furthermore, invasive properties of MB49-I orthotopic growth was inhibited using NO inhibitors. This paper also shows that levels of NO in urine can be correlated with tumor size. In conclusion, inhibition of NO could be considered as a therapeutic target that prevents tumor growth and progression. Also, urine NO levels may be useful for measuring tumor growth.

Spleen and thymus cell subsets modified by long-term morphine administration and murine AIDS--II.

Intravenous heroin abusers suffer a great variety of infections, including AIDS (acquired immune deficiency syndrome). We developed an experimental mouse model to evaluate the long-term effect of in vivo morphine administration during retrovirus-induced immune dysfunction. Mice were treated daily for 11 weeks with increasing doses of morphine. Morphine treatment produced a decrease in body weight and spleen cell number. Murine retrovirus infection provoked an increase in body weight due to enlargement of lymphoid organs, and an increase in the percentage and absolute number of CD4+ and Mac 1+ cells. Interestingly, retrovirus-infected mice that were also morphine-treated did not show the increase in the relative proportion of Mac 1+ cells. Moreover, under the experimental conditions of protein-malnutrition and morphine treatment potentiation of immune dysfunction by murine retrovirus infection was investigated. Retrovirus infection-induced splenocyte proliferation was partially regulated by morphine treatment. Splenocytes from retrovirus-infected mice presented a higher percentage of IL-2R+ cells and, lower levels of sIL-2R in splenocyte supernatants. Mitogen-stimulated splenocytes had a lower production of interferon-gamma as well as an increase in the secretion of tumor necrosis factor-alpha. Thus morphine altered the immune system by down-regulating splenocyte proliferation, because retrovirus infection-induced splenocyte proliferation was partially regulated by morphine treatment. We also evaluated the effects of joint murine retrovirus infection and protein undernutrition on the thymus cell subsets. Retrovirus infection was associated with a decrease in the absolute number of Thy 1+, CD4+ and CD8+ cells per thymus with the CD8+ cell subset being the most affected. Moreover, retrovirus-infected mice presented a dramatic decrease in the percentage of double-positive (CD4+ CD8+) cells in the thymus as well as changes in its immunoarchitecture. While protein undernutrition alone did not produce further differences between infected versus non-infected, protein-undernourished, morphine treatment induced a greater decrease in thymocyte number than that seen in retrovirus- or morphine-treated animals alone.