Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion

Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE. Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 mu mol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors. Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group. LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Mimosine and Cyclophosphamide: a Potential New Combination Therapy Used to Prevent Tumor Development

The effects of mimosine (MI), which is an amino acid that is derived from Leucaena leucocephala, were evaluated on the growth of ascitic Ehrlich tumors, and the effects of the combination treatment of MI and cyclophosphamide (CY) on tumor growth were also assessed. Mice were divided into groups that received the following treatments over the course of 20 days: phosphate buffer solution (CO), MI, Ehrlich cells (E), E plus CY (EC), E plus MI (EM) and E plus MI and CY (EMC). No signs of toxicity were detected in the mice from the MI group. The mice from the EMC group showed reductions in body weights when compared with those from the E group. The animals from the EC, EM and EMC groups showed reductions in ascitic volume compared with those from the E group. The mice from the EMC group showed reductions in total cell numbers of ascitic fluid compared with those from the E, EC and EM groups. The combination of MI and CY was the most effective treatment for Ehrlich tumor ascites.

Central Granular Cell Odontogenic Tumor of the Maxilla

The central granular cell odontogenic tumor (CGCOT) is a rare benign odontogenic neoplasm composed of varying amounts of large eosinophilic granular cells and apparently inactive odontogenic epithelium. It tends to occur as a small asymptomatic swelling in the posterior region of the mandible with nonaggressive appearance. We report an unusual case of CGCOT in the maxillary region with clinical features of malignancy. The patient underwent surgical treatment, and the 2-year follow-up revealed no signs of recurrence. Central granular cell odontogenic tumor is a very rare condition with few cases reported, especially in the maxillary region. This case highlights the possibility of aggressive behavior by these lesions.

Optimal Timing for Assessment of Tumor Response to Neoadjuvant Chemoradiation in Patients With Rectal Cancer: Do All Patients Benefit From Waiting Longer Than 6 Weeks?

Purpose: To estimate the metabolic activity of rectal cancers at 6 and 12 weeks after completion of chemoradiation therapy (CRT) by 2-[fluorine-18] fluoro-2-deoxy-D-glucose-labeled positron emission tomography/computed tomography ([18 FDG] PET/CT) imaging and correlate with response to CRT. Methods and Materials: Patients with cT2-4N0-2M0 distal rectal adenocarcinoma treated with long-course neoadjuvant CRT (54 Gy, 5-fluouracil-based) were prospectively studied (ClinicalTrials. org identifier NCT00254683). All patients underwent 3 PET/CT studies (at baseline and 6 and 12 weeks fromCRT completion). Clinical assessment was at 12 weeks. Maximal standard uptakevalue (SUVmax) of the primary tumor wasmeasured and recorded at eachPET/CTstudy after 1 h (early) and3 h (late) from 18 FDGinjection. Patientswith an increase in early SUVmax between 6 and 12 weeks were considered " bad" responders and the others as "good" responders. Results: Ninety-one patients were included; 46 patients (51%) were "bad" responders, whereas 45 (49%) patients were " good" responders. " Bad" responders were less likely to develop complete clinical response (6.5% vs. 37.8%, respectively; PZ. 001), less likely to develop significant histological tumor regression (complete or near-complete pathological response; 16% vs. 45%, respectively; PZ. 008) and exhibited greater final tumor dimension (4.3cmvs. 3.3cm; PZ. 03). Decrease between early (1 h) and late (3 h) SUVmax at 6-week PET/CTwas a significant predictor of " good" response (accuracy of 67%). Conclusions: Patients who developed an increase in SUVmax after 6 weeks were less likely to develop significant tumor downstaging. Early-late SUVmax variation at 6-week PET/CT may help identify these patients and allow tailored selection of CRT-surgery intervals for individual patients. (C) 2012 Elsevier Inc.

Apoptotic Cells Contribute to Melanoma Progression and This Effect is Partially Mediated by the Platelet-Activating Factor Receptor

There is evidence that the platelet-activating factor receptor (PAFR) is involved in the clearance of apoptotic cells by macrophages, and that this is associated with anti-inflammatory phenotype. Our group has previously shown that coinjection of a large number of apoptotic cells can promote tumor growth from a subtumorigenic dose of melanoma cells. Here, we studied the involvement of the PAFR in the tumor growth promoting effect of apoptotic cells. A sub-tumorigenic dose of melanoma cells (Tm1) was coinjected with apoptotic Tm1 cells, subcutaneously in the flank of C57Bl/6 mice, and the volume was monitored for 30 days. Animals received the PAFR antagonists, WEB2170 or PCA4248 (5 mg/kg body weight) or vehicle, by peritumoral daily injection for 5 days. Results showed that PAFR antagonists significantly inhibited the tumor growth induced by the coinjection of a subtumorigenic dose of melanoma cells together with apoptotic cells. This was accompanied by inhibition of early neutrophil and macrophage infiltration. Addition of (platelet-activating factor) to this system has no significant effect. PAFR antagonists did not affect the promoting effect of carrageenan. We suggest that the recognition of apoptotic cells by phagocytes leads to activation of PAFR pathways, resulting in a microenvironment response favorable to melanoma growth.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Synthetic phosphoethanolamine a precursor of membrane phospholipids reduce tumor growth in mice bearing melanoma B16-F10 and in vitro induce apoptosis and arrest in G2/M phase

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes, especially phosphatidylcholine. We recently reported that synthetic Pho-s has potent effects on a wide variety of tumor cells. To determine if Pho-s has a potential antitumor activity, in this study we evaluated the activity of Pho-s against the B16-F10 melanoma both in vitro and in mice bearing a dorsal tumor. The treatment of B16F10 cells with Pho-s resulted in a dose-dependent inhibition of cell proliferation. At low concentrations, this activity appears to be involved in the arrest of the cell cycle at G2/M, while at high concentrations Pho-s induces apoptosis. In accordance with these results, the loss of mitochondrial potential and increased caspase-3 activity suggest that Phos has dual antitumor effects; i.e. it induces apoptosis at high concentrations and modulates the cell cycle at lower concentrations. In vivo, we evaluated the effect of Pho-s in mice bearing B16-F10 melanoma. The results show that Pho-s reduces the tumoral volume increasing survival rate. Furthermore, the tumor doubling time and tumor delays were substantially reduced when compared with untreated mice. Histological analyses reveal that Pho-s induces changes in cell morphology, typical characteristics of apoptosis, in addition the large areas of necrosis correlating with a reduction of tumor size. The results presented here support the hypothesis that Pho-s has antitumor effects by the induction of apoptosis as well as the inhibition of cell proliferation by arrest at G2/M. Thus, Pho-s can be regarded as a promising agent for the treatment of melanoma. Published by Elsevier Masson SAS.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Anticancer Effects of Synthetic Phosphoethanolamine on Ehrlich Ascites Tumor: An Experimental Study

Background: Antineoplastic phospholipids (ALPs) represent a promising class of drugs with a novel mode of action undergoes rapid turnover in the cell membrane of tumors, interfering with lipid signal transduction, inducing cell death. The aim of this study was to investigate the synthetic phosphoethanolamine (Pho-s) as a new anticancer agent. Materials and Methods: Cell viability and morphology were assessed by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst and rhodamine staining. Apoptosis was assessed by Annexin V and propidium iodide (PI) staining, caspase-3 activity, mitochondrial membrane potential (Delta m psi) and cell cycle analysis, combined with evaluation of tumor growth in Ehrlich Ascites Tumor (EAT) bearing mice. Results: We found that Pho-s 2.30 mg/ml induced cytotoxicity in all tumor cell lines studied without affecting normal cells. In vitro studies with EAT cells indicated that Pho-s induced apoptosis, demonstrated by an increase in Annexin-V positive cells, loss of mitochondrial potential (Delta m psi) and increased caspase-3 activity. It was also shown to increase the sub-G(1) apoptotic fraction and inhibit progression to the S phase of the cell cycle. Additionally, antitumor effects on the EAT-bearing mice showed that Pho-s, at a concentration of 35 and 70 mg/kg, inhibited tumor growth and increased the lifespan of animals without causing liver toxicity. Conclusion: These findings suggest that Pho-s is a potential anticancer candidate drug.

The Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

Differential Gene Expression Profiles May Differentiate Responder and Nonresponder Patients with Rheumatoid Arthritis for Methotrexate (MTX) Monotherapy and MTX plus Tumor Necrosis Factor Inhibitor Combined Therapy

Objective. We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-alpha (anti-TNF) combined therapy. Methods. We evaluated 25 patients with RA taking MTX 15-20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received intliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. Results. Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF nonresponders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. Conclusion. Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1 CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study. (First Release July 1 2012; J Rheumatol 2012;39:1524-32; doi:10.3899/jrheum.120092)

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

Analysis by MRI of Residual Tumor After Radiofrequency Ablation for Early Stage Breast Cancer

OBJECTIVE. The objective of our study was to evaluate the effectiveness of MRI in the detection of possible residual lesions after radiofrequency ablation (RFA) in the treatment of breast cancer. SUBJECTS AND METHODS. We prospectively evaluated 14 patients who had undergone ultrasound-guided core biopsies diagnostic of invasive ductal carcinoma (IDC; range of diameters, 1.0-3.0 cm) and then ultrasound-guided percutaneous RFA with sentinel node biopsy as the primary treatment. Breast MRI was performed 1 week before RFA to evaluate tumor extension and again 3 weeks after RFA to verify the presence of possible residual lesions. Conventional surgical resection of the tumors was performed 1 week after RFA. The MRI findings were compared with histopathologic analyses to confirm the presence or absence of residual tumor. RESULTS. There was no residual enhancement in seven lesions on the postablation breast MRI scans. These findings were confirmed by negative histopathologic findings in the surgical specimens. The MRI scans of five patients showed small areas of irregular enhancement that corresponded to residual lesions. In the two remaining patients, we observed enhancement of almost the entire lesion, indicating that RFA had failed. CONCLUSION. Breast MRI is effective in detecting residual lesions after RFA in patients with IDC.

Accuracy of positron emission tomography/computed tomography and clinical assessment in the detection of complete rectal tumor regression after neoadjuvant chemoradiation Long-Term Results of a Prospective Trial (National Clinical Trial 00254683)

BACKGROUND: Neoadjuvant chemoradiation (CRT) therapy may result in significant tumor regression in patients with rectal cancer. Patients who develop complete tumor regression have been managed by treatment strategies that are alternatives to standard total mesorectal excision. Therefore, assessment of tumor response with positron emission tomography/computed tomography (PET/CT) after neoadjuvant treatment may offer relevant information for the selection of patients to receive alternative treatment strategies. METHODS: Patients with clinical T2 (cT2) through cT4NxM0 rectal adenocarcinoma were included prospectively. Neoadjuvant therapy consisted of 54 grays of radiation and 5-fluorouracil-based chemotherapy. Baseline PET/CT studies were obtained before CRT followed by PET/CT studies at 6 weeks and 12 weeks after the completion of CRT. Clinical assessment was performed at 12 weeks after CRT completion. PET/CT results were compared with clinical and pathologic data. RESULTS: In total, 99 patients were included in the study. Twenty-three patients were complete responders (16 had a complete clinical response, and 7 had a complete pathologic response). The PET/CT response evaluation at 12 weeks indicated that 18 patients had a complete response, and 81 patients had an incomplete response. There were 5 false-negative and 10 false-positive PET/CT results. PET/CT for the detection of residual cancer had 93% sensitivity, 53% specificity, a 73% negative predictive value, an 87% positive predictive value, and 85% accuracy. Clinical assessment alone resulted in an accuracy of 91%. PET/CT information may have detected misdiagnoses made by clinical assessment alone, improving overall accuracy to 96%. CONCLUSIONS: Assessment of tumor response at 12 weeks after CRT completion with PET/CT imaging may provide a useful additional tool with good overall accuracy for the selection of patients who may avoid unnecessary radical resection after achieving a complete clinical response. Cancer 2012;35013511. (C) 2011 American Cancer Society.