Thaiz F. Borin

Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho‐associated kinase protein (ROCK‐1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK‐1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti‐angiogenic effects and can modulate ROCK‐1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK‐1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK‐1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X‐ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK‐1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK‐1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of ‘hot’ spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK‐1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK‐1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

HET0016, a selective inhibitor of 20-HETE synthesis, decreases pro-angiogenic factors and inhibits growth of triple negative breast cancer in mice.

A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and in vitro cell line. MDA-MB-231 tumor cells were implanted in animals’ right flank and randomly assigned to early (1 and 2), starting treatments on day 0, or delayed groups (3 and 4) on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg) treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05). Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found in vitro at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05) compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day’s data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.

Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines.

Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p < .05). This treatment also decreased both gene and protein expression of HIF-1α and VEGF-A under hypoxic conditions (p < .05). Among the proteins evaluated by protein array, melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p < .05). Taken together, these results showed that melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions.

Vascular mimicry in glioblastoma following anti-angiogenic and anti-20-HETE therapies

Glioblastoma (GBM) is one hypervascular and hypoxic tumor known among solid tumors. Antiangiogenic therapeutics (AATs) have been tested as an adjuvant to normalize blood vessels and control abnormal vasculature. Evidence of relapse exemplified in the progressive tumor growth following AAT reflects development of resistance to AATs. Here, we identified that GBM following AAT (Vatalanib) acquired an alternate mechanism to support tumor growth, called vascular mimicry (VM). We observed that Vatalanib induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer-side. The PAS+ matrix is positive for basal laminae (laminin) indicating vascular structures. Vatalanib treated GBM displayed various stages of VM such as initiation (mosaic), sustenance, and full-blown VM. Mature VM structures contain red blood cells (RBC) and bear semblance to the functional blood vessel-like structures, which provide all growth factors to favor tumor growth. Vatalanib treatment significantly increased VM especially in the core of the tumor, where HIF-1α was highly expressed in tumor cells. VM vessels correlate with hypoxia and are characterized by co-localized MHC-1+ tumor and HIF-1α expression. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at periphery of the tumors. In summary, AAT induced resistance characterized by VM is an alternative mechanism adopted by tumors to make functional vessels by transdifferentiation of tumor cells into endothelial-like cells to supply nutrients in the event of hypoxia. AAT induced VM is a potential therapeutic target of the novel formulation of HET0016. Our present study suggests that HET0016 has a potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.

Melatonin Regulates Angiogenic and Inflammatory Proteins in MDA-MB-231 Cell Line and in Co-culture with Cancer-associated Fibroblasts.

BACKGROUND Cancer-associated fibroblast (CAFs) are the most abundant cells in the tumor microenvironment, able to secrete growth factors and act on tumor progression. Melatonin is associated with several mechanisms of action with oncostatics and oncoprotectors effects, and also participate in the reduction of synthesis of surrounding fibroblasts and endothelial cells in breast cancer. OBJECTIVE The objectives of this study were to determine the effectiveness of melatonin in cell viability and expression of proteins involved in angiogenesis and inflammation in triplenegative mammary tumor cell line (MDA-MB-231) and in co-culture with CAFs. METHOD Cell viability was measured by MTT assay and the protein expression was evaluated by Membrane Antibody Array after melatonin treatment. RESULTS Melatonin treatment (1 mM) for 48 hours reduced the cell viability of MDA-MB-231, CAFs and co-culture (p < 0.05). The semi-quantitative protein analysis showed that when monoculture of tumor cells were compared with co-culture of CAFs, there was a regulation of angiogenic and inflammatory proteins (p < 0.05). Melatonin treatment also leads a differential expression of angiogenic and inflammatory proteins in both monoculture and co-culture of tumor cells and CAFs (p < 0.05). CONCLUSION The influence of CAFs under the tumor microenvironment was confirmed, increasing the malignancy of the tumor. In addition, melatonin is effective in both monoculture and co-culture, regulating angiogenic and inflammatory proteins that contribute to tumor progression. This study show an overview of melatonin ability in regulating angiogenic and inflammatory proteins, and opens the way for exploration of each individual protein in further studies.

Differential biodistribution of intravenously administered endothelial progenitor and cytotoxic T-cells in rat bearing orthotopic human glioma.

BackgroundA major challenge in the development of cell based therapies for glioma is to deliver optimal number of cells (therapeutic dose) to the tumor. Imaging tools such as magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) and single-photon emission computed tomography (SPECT) has been used in cell tracking and/or biodistribution studies. In this study, we evaluate the dynamic biodistribution of systemic injected labeled cells [human cord blood derived endothelial progenitor cells (EPCs) and cytotoxic T-cells (CTLs)] in rat glioma model with in vivo SPECT imaging.MethodsHuman cord blood EPCs, T-cells and CD14+ cells (monocytes/dendritic cells) were isolated using the MidiMACS system. CD14+ cells were converted to dendritic cells (DC) and also primed with U251 tumor cell line lysate. T-cells were co-cultured with irradiated primed DCs at 10:1 ratio to make CTLs. Both EPCs and CTLs were labeled with In-111-oxine at 37°C in serum free DMEM media. Glioma bearing animals were randomly assigned into three groups. In-111 labeled cells or In-111 oxine alone were injected through tail vein and SPECT imaging was performed on day 0, 1, and 3. In-111 oxine activity in various organs and tumor area was determined. Histochemical analysis was performed to further confirm the migration and homing of injected cells at the tumor site.ResultsEPCs and CTLs showed an In-111 labeling efficiency of 87.06 ± 7.75% and 70.8 ± 12.9% respectively. Initially cell migration was observed in lung following inravenous administration of In-111 labeled cells and decreased on day 1 and 3, which indicate re-distribution of labeled cells from lung to other organs. Relatively higher In-111 oxine activity was observed in tumor areas at 24 hours in animals received In-111 labeled cells (EPCs or CTLs). Histiological analysis revealed iron positive cells in and around the tumor area in animals that received labeled cells (CTLs and EPCs).ConclusionWe observed differential biodistribution of In-111-oxine labeled EPCs and CTLs in different organs and intracranial glioma. This study indicates In-111 oxine based SPECT imaging is an effective tool to study the biodistribution of therapeutically important cells.

Vascular Mimicry: A Novel Neovascularization Mechanism Driving Anti-Angiogenic Therapy (AAT) Resistance in Glioblastoma

Glioblastoma (GBM) is a hypervascular neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT therapy resistance. Alternative neovascularization mechanisms were shown to be at work in these resilient tumors to counter the AAT therapy insult. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties and lay down vascular patterned networks reminiscent of host endothelial blood vessels. The VM channels served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands of the tumor in the event of the ensuing hypoxia resulting from AAT. In our previous studies, we have demonstrated that AAT accelerates VM in GBM. In this review, we will focus on the origins of VM, visualizing VM in AAT-treated tumors and the development of VM as a resistance mechanism to AAT.

Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.

Efficacy of melatonin, IL-25 and siIL-17B in tumorigenesis-associated properties of breast cancer cell lines

ABSTRACT Mammary tumorigenesis can be modulated by melatonin, which has oncostatic action mediated by multiple mechanisms, including the inhibition of the activity of transcription factors such as NF‐&kgr;B and modulation of interleukins (ILs) expression. IL‐25 is an active cytokine that induces apoptosis in tumor cells due to differential expression of its receptor (IL‐17RB). IL‐17B competes with IL‐25 for binding to IL‐17RB in tumor cells, promoting tumorigenesis. This study purpose is to address the possibility of engaging IL‐25/IL‐17RB signaling to enhance the effect of melatonin on breast cancer cells. Breast cancer cell lines were cultured monolayers and 3D structures and treated with melatonin, IL‐25, siIL‐17B, each alone or in combination. Cell viability, gene and protein expression of caspase‐3, cleaved caspase‐3 and VEGF‐A were performed by qPCR and immunofluorescence. In addition, an apoptosis membrane array was performed in metastatic cells. Treatments with melatonin and IL‐25 significantly reduced tumor cells viability at 1 mM and 1 ng/mL, respectively, but did not alter cell viability of a non‐tumorigenic epithelial cell line (MCF‐10A). All treatments, alone and combined, significantly increased cleaved caspase‐3 in tumor cells grown as monolayers and 3D structures (p < 0.05). Semi‐quantitative analysis of apoptosis pathway proteins showed an increase of CYTO‐C, DR6, IGFBP‐3, IGFBP‐5, IGFPB‐6, IGF‐1, IGF‐1R, Livin, P21, P53, TNFRII, XIAP and hTRA proteins and reduction of caspase‐3 (p < 0.05) after melatonin treatment. All treatments reduced VEGF‐A protein expression in tumor cells (p < 0.05). Our results suggest therapeutic potential, with oncostatic effectiveness, pro‐apoptotic and anti‐angiogenic properties for melatonin and IL‐25‐driven signaling in breast cancer cells.