Near-Infrared Fluorescence Imaging in Preclinical Models of Glioblastoma.

Cancer is a public health problem requiring ongoing research to improve current treatments and discover novel therapies. More accurate imaging would facilitate such research. Near-infrared fluorescence has been developed as a non-invasive imaging technique capable of visualizing and measuring biological processes at the molecular level in living subjects. In this work, we evaluate the tumor activity in two preclinical glioblastoma models by using fluorochrome (IRDye 800CW) coupled to different molecules: tripeptide Arg-Gly-Asp (RGD), 2-amino-2-deoxy-D-glucose (2-DG), and polyethylene glycol (PEG). These molecules interact with pathological conditions of tumors, including their overexpression of αvß3 integrins (RGD), elevated glucose uptake (2-DG), and enhanced permeability and retention effect (PEG). IRDye 800CW RGD gave the best in vivo fluorescence signal from the tumor area, which contrasted well with the low fluorescence intensity of healthy tissue. In the ex vivo imaging (dissected tumor), the accumulation of IRDye 800CW RGD could be appreciated at the tumor site. Glioblastoma tumors were presently detected with specificity and sensitivity by utilizing IRDye 800CW RGD, a near-infrared fluorophore combined with a marker of αvß3 integrin expression. Further research is needed on its capacity to monitor tumor growth in glioblastoma after chemotherapy.

Pharmacokinetics and Anti-Tumor Efficacy of PEGylated Liposomes Co-Loaded with Cisplatin and Mifepristone.

Although cisplatin is an effective chemotherapy drug used against many types of cancer, it has poor bioavailability, produces severe adverse effects, and frequently leads to tumor resistance. Consequently, more effective formulations are needed. The co-administration of cisplatin with mifepristone, which counters an efflux pump drug-resistance mechanism in tumor cells, has shown important synergism, but without resolving the problem of poor bioavailability. Specificity to tumor tissue and bioavailability have been improved by co-encapsulating cisplatin and mifepristone in a liposomal formulation (L-Cis/MF), which needs further research to complete pre-clinical requirements. The aim of this current contribution was to conduct a pharmacokinetic study of cisplatin and mifepristone in male Wistar rats after administration of L-Cis/MF and the conventional (unencapsulated) formulation. Additionally, the capacity of L-Cis/MF to reduce tumor growth in male nude mice was evaluated following the implantation of xenografts of non-small-cell lung cancer. The better pharmacokinetics (higher plasma concentration) of cisplatin and mifepristone when injected in the liposomal versus the conventional formulation correlated with greater efficacy in controlling tumor growth. Future research on L-Cis/MF will characterize its molecular mechanisms and apply it to other types of cancer affected by the synergism of cisplatin and mifepristone.

The Therapeutic Potential of Chemo/Thermotherapy with Magnetoliposomes for Cancer Treatment.

Cancer represents a very grave and quickly growing public health problem worldwide. Despite the breakthroughs in treatment and early detection of the disease, an increase is projected in the incidence rate and mortality during the next 30 years. Thus, it is important to develop new treatment strategies and diagnostic tools. One alternative is magnetic hyperthermia, a therapeutic approach that has shown promising results, both as monotherapy and in combination with chemo- and radiotherapy. However, there are still certain limitations and questions with respect to the safety of the systemic administration of magnetic nanoparticles. To deal with these issues, magnetoliposomes were conceived as a new generation of liposomes that incorporate superparamagnetic nanoparticles and oncological pharmaceuticals within their structure. They have the advantage of targeted and selective drug delivery to the diseased organs and tissues. Some of them can avoid the immune response of the host. When exposed to a magnetic field of alternating current, magnetoliposomes produce hyperthermia, which acts synergistically with the released drug. The aim of the present review is to describe the most recent advances in the use of magnetoliposomes and point out what research remains to be done for their application to chemo-thermal therapy in cancer patients.

Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells.

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20-30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinib-resistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 µM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.

Estrogen Receptors as Molecular Targets of Endocrine Therapy for Glioblastoma.

Hormonal factors may participate in the development and progression of glioblastoma, the most aggressive primary tumor of the central nervous system. Many studies have been conducted on the possible involvement of estrogen receptors (ERs) in gliomas. Since there is a tendency for a reduced expression of ERs as the degree of malignancy of such tumors increases, it is important to understand the role of these receptors in the progression and treatment of this disease. ERs belong to the family of nuclear receptors, although they can also be in the plasmatic membrane, cytoplasm and mitochondria. They are classified as estrogen receptors alpha and beta (ER⍺ and ERß), each with different isoforms that have a distinct function in the organism. ERs regulate multiple physiological and pathological processes through the activation of genomic and nongenomic pathways in the cell. Nevertheless, the role of each isoform in the development and progression of glioblastoma is not completely clear. Diverse in vitro and in vivo studies have shown encouraging results for endocrine therapy as a treatment for gliomas. At the same time, many questions have arisen concerning the nature of ERs as well as the mechanism of action of the proposed drugs. Hence, the aim of the current review is to describe the drugs that could possibly be utilized in endocrine therapy for the treatment of high-grade gliomas, analyze their interaction with ERs, and explore the involvement of these drugs and receptors in resistance to standard chemotherapy.

Corrigendum: Mifepristone as a Potential Therapy to Reduce Angiogenesis and P-Glycoprotein Associated With Glioblastoma Resistance to Temozolomide.

[This corrects the article DOI: 10.3389/fonc.2020.581814.].

Mifepristone Repurposing in Treatment of High-Grade Gliomas.

Glioma is the most common and aggressive primary tumor of the central nervous system. The standard treatment for malignant gliomas is surgery followed by chemoradiotherapy. Unfortunately, this treatment has not produced an adequate patient response, resulting in a median survival time of 12-15 months and a 5-year overall survival of <5%. Although new strategies have been sought to enhance patient response, no significant increase in the global survival of glioma patients has been achieved. The option of developing new drugs implies a long and costly process, making drug repurposing a more practical alternative for improving glioma treatment. In the last few years, researchers seeking more effective cancer therapy have pursued the possibility of using anti-hormonal agents, such as mifepristone. The latter drug, an antagonist for progesterone and glucocorticoid receptors, has several attractive features: anti-tumor activity, low cytotoxicity to healthy cells, and modulation of the chemosensitivity of several cancer cell lines in vitro. Hence, the addition of mifepristone to temozolomide-based glioblastoma chemotherapy may lead to a better patient response. The mechanisms by which mifepristone enhances glioma treatment are not yet known. The current review aims to discuss the potential role of mifepristone as an adjuvant drug for the treatment of high-grade gliomas.

Statins as adjuvants in the treatment of ovarian cancer: Controversy and misunderstanding.

Ovarian cancer is frequently detected in advanced stages when the chances of survival are very low. Although chemotherapy is the treatment of choice, it is often rapidly compromised by the development of chemoresistance in patients. There are few pharmacological alternatives for managing chemoresistant ovarian cancer and statins have been suggested as an alternative, but their use is considered controversial. We present an overview of the most relevant epidemiological, in vitro and in vivo studies on the effects of statins in mono- or polytherapy for ovarian cancer. We conclude that the negative or inconclusive results of some epidemiological studies on statin-based cancer treatment are probably due, in large part, to the low doses given to patients, equivalent to those prescribed for hypercholesterolemia. Higher concentrations are well tolerated in animal models and by most patients in clinical trials. Future research is necessary to explore this possibility.

Mifepristone as a Potential Therapy to Reduce Angiogenesis and P-Glycoprotein Associated With Glioblastoma Resistance to Temozolomide.

Glioblastoma, the most common primary central nervous system tumor, is characterized by extensive vascular neoformation and an area of necrosis generated by rapid proliferation. The standard treatment for this type of tumor is surgery followed by chemotherapy based on temozolomide and radiotherapy, resulting in poor patient survival. Glioblastoma is known for strong resistance to treatment, frequent recurrence and rapid progression. The aim of this study was to evaluate whether mifepristone, an antihormonal agent, can enhance the effect of temozolomide on C6 glioma cells orthotopically implanted in Wistar rats. The levels of the vascular endothelial growth factor (VEGF), and P-glycoprotein (P-gp) were examined, the former a promoter of angiogenesis that facilitates proliferation, and the latter an efflux pump transporter linked to drug resistance. After a 3-week treatment, the mifepristone/temozolomide regimen had decreased the level of VEGF and P-gp and significantly reduced tumor proliferation (detected by PET/CT images based on 18F-fluorothymidine uptake). Additionally, mifepristone proved to increase the intracerebral concentration of temozolomide. The lower level of O6-methylguanine-DNA-methyltransferase (MGMT) (related to DNA repair in tumors) previously reported for this combined treatment was herein confirmed. After the mifepristone/temozolomide treatment ended, however, the values of VEGF, P-gp, and MGMT increased and reached control levels by 14 weeks post-treatment. There was also tumor recurrence, as occurred when administering temozolomide alone. On the other hand, temozolomide led to 100% mortality within 26 days after beginning the drug treatment, while mifepristone/temozolomide enabled 70% survival 60-70 days and 30% survived over 100 days, suggesting that mifepristone could possibly act as a chemo-sensitizing agent for temozolomide.

Liposomes Co-Encapsulating Cisplatin/Mifepristone Improve the Effect on Cervical Cancer: In Vitro and In Vivo Assessment.

Cervical cancer is usually diagnosed in the later stages despite many campaigns for early detection and continues to be a major public health problem. The standard treatment is cisplatin-based chemotherapy plus radiotherapy, but patient response is far from ideal. In the research for new drugs that enhance the activity of cisplatin, different therapeutic agents have been tested, among them the antiprogestin mifepristone. Nevertheless, the efficacy of cisplatin is limited by its low specificity for tumor tissue, which causes severe side effects. Additionally, cervical tumors often become drug resistant. These problems could possibly be addressed by the use of liposome nanoparticles to encapsulate drugs and deliver them to the target. The aim of this study was to prepare liposome nanoparticles that co-encapsulate cisplatin and mifepristone, evaluate their cytotoxicity against HeLa cells and in vivo with subcutaneous inoculations of xenografts in nu/nu mice, and examine some plausible mechanisms of action. The liposomes were elaborated by the reverse-phase method and characterized by physicochemical tests. The nanoparticles had a mean particle size of 109 ± 5.4 nm and a Zeta potential of -38.7 ± 1.2 mV, the latter parameter indicating a stable formulation. These drug-loaded liposomes significantly decreased cell viability in vitro and tumor size in vivo, without generating systemic toxicity in the animals. There was evidence of cell cycle arrest and increased apoptosis. The promising results with the co-encapsulation of cisplatin/mifepristone warrant further research.

Preparation and Evaluation of Mebendazole Microemulsion for Intranasal Delivery: an Alternative Approach for Glioblastoma Treatment.

Although mebendazole (MBZ) has demonstrated antitumor activity in glioblastoma models, the drug has low aqueous solubility and therefore is poorly absorbed. Considering that other strategies are needed to improve its bioavailability, the current study was aimed to develop and evaluate novel microemulsions of MBZ (MBZ-NaH ME) for intranasal administration. MBZ raw materials were characterized by FTIR, DSC, and XDP. Subsequently, the raw material that contained mainly polymorph C was selected to prepare microemulsions. Two different oleic acid (OA) systems were selected. Formulation A was composed of OA and docosahexaenoic acid (3:1% w/w), while formulation B was composed of OA and Labrafil M2125 (1:1% w/w). Sodium hyaluronate (NaH) at 0.1% was selected as a mucoadhesive agent. MBZ MEs showed a particle size of 209 nm and 145 nm, respectively, and the pH was suitable for nasal formulations (4.5-6.5). Formulation B, which showed the best solubility and rheological behavior, was selected for intranasal evaluation. The nasal toxicity study revealed no damage in the epithelium. Furthermore, formulation B improved significantly the median survival time in the orthotopic C6 rat model compared to the control group. Moreover, NIRF signal intensity revealed a decrease in tumor growth in the treated group with MBZ-MaH ME, which was confirmed by histologic examinations. Results suggest that the intranasal administration of mebendazole-loaded microemulsion might be appropriated for glioblastoma treatment. Graphical abstract.

SFRP1 increases TMPRSS2-ERG expression promoting neoplastic features in prostate cancer in vitro and in vivo.

BACKGROUND: Prostate cancer (PCa) is the second cause of cancer related death in North American men. Androgens play an important role in its progression by regulating the expression of several genes including fusion ones that results from structural chromosome rearrangements. TMPRSS2-ERG is a fusion gene commonly observed in over 50% of PCa tumors, and its expression can be transcriptionally regulated by the androgen receptor (AR) given its androgen responsive elements. TMPRSS2-ERG could be involved in epithelial-mesenchymal transition (EMT) during tumor development. ERG has been reported as a key transcriptional factor in the AR-ERG-WNT network where five SFRP proteins, structurally similar to WNT ligands and considered to be WNT pathway antagonists, can regulate signaling in the extracellular space  by binding to WNT proteins or Frizzled receptors. It has been shown that over-expression of SFRP1 protein can regulate the transcriptional activity of AR and inhibits the formation of colonies in LNCaP cells. However, the effect of SFRP1 has been controversial since differential effects have been observed depending on its concentration and tissue location. In this study, we explored the role of exogenous SFRP1 protein in cells expressing the TMPRSS2-ERG fusion. METHODS: To evaluate the effect of exogenous SFRP1 protein on PCa cells expressing TMPRSS2-ERG, we performed in silico analysis from TCGA cohort, expression assays by RT-qPCR and Western blot, cell viability and cell cycle measurements by cytometry, migration and invasion assays by xCELLigance system and murine xenografts. RESULTS: We demonstrated that SFRP1 protein increased ERG expression by promoting cellular migration in vitro and increasing tumor growth in vivo in PCa cells with the TMPRSS2-ERG fusion. CONCLUSIONS: These results suggest the possible role of exogenous SFRP1 protein as a modulator of AR-ERG-WNT signaling network in cells positive to TMPRSS2-ERG. Further, investigation is needed to determine if SFRP1 protein could be a target in against this type of PCa.

Gadolinium-containing carbon nanomaterials for magnetic resonance imaging: Trends and challenges.

Gadolinium-containing carbon nanomaterials are a new class of contrast agent for magnetic resonance imaging. They are characterized by a superior proton relaxivity to any current commercial gadolinium contrast agent and offer the possibility to design multifunctional contrasts. Intense efforts have been made to develop these nanomaterials because of their potential for better results than the available gadolinium contrast agents. The aim of the present work is to provide a review of the advances in research on gadolinium-containing carbon nanomaterials and their advantages over conventional gadolinium contrast agents. Due to their enhanced proton relaxivity, they can provide a reliable imaging contrast for cells, tissues or organs with much smaller doses than currently used in clinical practice, thus leading to reduced toxicity (as shown by cytotoxicity and biodistribution studies). Their active targeting capability allows for improved MRI of molecular or cellular targets, overcoming the limited labelling capability of available contrast agents (restricted to physiological irregularities during pathological conditions). Their potential of multifunctionality encompasses multimodal imaging and the combination of imaging and therapy.

Mesenchymal Stromal Cells Derived from Normal Cervix and Cervical Cancer Tumors Increase CD73 Expression in Cervical Cancer Cells Through TGF-ß1 Production.

Mesenchymal stromal cells (MSCs) in the tumor microenvironment (TME) participate together with tumor cells to suppress antitumor effector cells through the production of immunosuppressive factors, such as transforming growth factor-beta 1 (TGF-ß1). Furthermore, TGF-ß1 can induce 5'-nucleotidase (CD73) expression in various cell types; this functional activity is associated with the production of adenosine (Ado), which is an immunosuppressive nucleoside. In this study, we provide evidence that coculture of MSCs derived from cervical tumors (CeCa-MSC) with CeCa tumor cells increases CD73 expression in tumor cells and the capacity of these cells to generate Ado in a MSC ratio-dependent manner. Interestingly, the increase in CD73 in the CeCa cell membrane corresponded to an increase in the TGF-ß1 expression level in the tumor cells and the TGF-ß1 content in the supernatants of the CeCa/CeCa-MSC cocultures. The addition of anti-hTGF-ß neutralizing antibodies strongly reversed CD73 expression in the tumor cells. This phenomenon was not exclusive to CeCa-MSCs; coculture of MSCs derived from the normal cervix with CeCa cells produced similar results. These results suggest that the interaction of MSCs with CeCa tumor cells in the TME may condition higher TGF-ß1 production to maintain an immunosuppressive status not only through the activity of this cytokine per se but also through its ability to induce CD73 expression in tumor cells and generate an immunosuppressive microenvironment rich in Ado.

Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma.

The standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemo/radiotherapy. A major limitation on patient improvement is the high resistance of tumors to drug treatment, likely responsible for their subsequent recurrence and rapid progression. Therefore, alternatives to the standard therapy are necessary. The aim of the present study was to evaluate whether mifepristone, an antihormonal agent, has a synergistic effect with temozolomide (used in standard therapy for gliomas). Whereas the mechanism of temozolomide involves damage to tumor DNA leading to apoptosis, tumor resistance is associated with DNA damage repair through the O6-methylguanine-DNA-methyltransferase (MGMT) enzyme. Temozolomide/mifepristone treatment, herein examined in Wistar rats after orthotopically implanting C6 glioma cells, markedly reduced proliferation. This was evidenced by a decreased level of the following parameters: a proliferation marker (Ki-67), a tumor growth marker (18F-fluorothymidine uptake, determined by PET/CT images), and the MGMT enzyme. Increased apoptosis was detected by the relative expression of related proteins, (e.g. Bcl-2 (B-cell lymphoma 2), Bax (bcl-2-like protein 4) and caspase-3). Thus, greater apoptosis of tumor cells caused by their diminished capacity to repair DNA probably contributed significantly to the enhanced activity of temozolomide. The results suggest that mifepristone could possibly act as a chemo-sensitizing agent for temozolomide during chemotherapy for GBM.

Liposomes Loaded with Cisplatin and Magnetic Nanoparticles: Physicochemical Characterization, Pharmacokinetics, and In-Vitro Efficacy.

With the aim improving drug delivery, liposomes have been employed as carriers for chemotherapeutics achieving promising results; their co-encapsulation with magnetic nanoparticles is evaluated in this work. The objective of this study was to examine the physicochemical characteristics, the pharmacokinetic behaviour, and the efficacy of pegylated liposomes loaded with cisplatin and magnetic nanoparticles (magnetite) (Cis-MLs). Cis-MLs were prepared by a modified reverse-phase evaporation method. To characterize their physicochemical properties, an evaluation was made of particle size, ζ-potential, phospholipid and cholesterol concentration, phase transition temperature (Tm), the encapsulation efficiency of cisplatin and magnetite, and drug release profiles. Additionally, pharmacokinetic studies were conducted on normal Wistar rats, while apoptosis and the cytotoxic effect were assessed with HeLa cells. We present a method for simultaneously encapsulating cisplatin at the core and also embedding magnetite nanoparticles on the membrane of liposomes with a mean vesicular size of 104.4 ± 11.5 nm and a ζ-potential of -40.5 ± 0.8 mV, affording a stable formulation with a safe pharmacokinetic profile. These liposomes elicited a significant effect on cell viability and triggered apoptosis in HeLa cells.

Effect of Pinocembrin Isolated from Mexican Brown Propolis on Diabetic Nephropathy.

Propolis is a resinous beehive product that has been used worldwide in traditional medicine to prevent and treat colds, wounds, rheumatism, heart disease and diabetes. Diabetic nephropathy is the final stage of renal complications caused by diabetes and for its treatment there are few alternatives. The present study aimed to determine the chemical composition of three propolis samples collected in Chihuahua, Durango and Zacatecas and to evaluate the effect of pinocembrin in a model of diabetic nephropathy in vivo. Previous research demonstrated that propolis of Chihuahua possesses hypoglycemic and antioxidant activities. Two different schemes were assessed, preventive (before renal damage) and corrective (once renal damage is established). In the preventive scheme, pinocembrin treatment avoids death of the rats, improves lipid profile, glomerular filtration rate, urinary protein, avoid increases in urinary biomarkers, oxidative stress and glomerular basement membrane thickness. Whereas, in the corrective scheme, pinocembrin only improves lipid profile without showing improvement in any other parameters, even pinocembrin exacerbated the damage. In conclusion, pinocembrin ameliorates diabetic nephropathy when there is no kidney damage but when it is already present, pinocembrin accelerates kidney damage.

The Effect of Gabapentin and Tramadol in Cancer Pain Induced by Glioma Cell in Rat Femur.

Preclinical Research The presence of pain as part of the cancer process is variable. Glioblastoma multiform (GBM) can produce bone metastasis, a condition that involves other pathological phenotypes including neuropathic and inflammatory pain. Tramadol and gabapentin are drugs used in the treatment of neuropathic pain. However, there are no studies evaluating their analgesic effects in bone metastasis. We produced a pain model induced by the inoculation of glioma cells (105 ) into the rat femur, by perforating the intercodiloid fossa. Painful behavior was evaluated by measuring mechanical allodynia using the Von Frey test while thermal hyperalgesia was assessed in the plantar test. Histopathological features were evaluated and antinociceptive responses were compared using tramadol and gabapentin. The inoculation of cells inside the right femur produced nociceptive behaviors. Tramadol and gabapentin produced an anti-allodynic effect in this condition, but tramadol did not produce an anti-hyperalgesic response. The development of this model will allow us to perform tests to elucidate the pathology of bone metastasis, cancer pain, and in particular the pain produced by glioma. Drug Dev Res 78 : 173-183, 2017. © 2017 Wiley Periodicals, Inc.

NF-kappaΒ-inducing kinase regulates stem cell phenotype in breast cancer.

Breast cancer stem cells (BCSCs) overexpress components of the Nuclear factor-kappa B (NF-κB) signaling cascade and consequently display high NF-κB activity levels. Breast cancer cell lines with high proportion of CSCs exhibit high NF-κB-inducing kinase (NIK) expression. The role of NIK in the phenotype of cancer stem cell regulation is poorly understood. Expression of NIK was analyzed by quantitative RT-PCR in BCSCs. NIK levels were manipulated through transfection of specific shRNAs or an expression vector. The effect of NIK in the cancer stem cell properties was assessed by mammosphere formation, mice xenografts and stem markers expression. BCSCs expressed higher levels of NIK and its inhibition through small hairpin (shRNA), reduced the expression of CSC markers and impaired clonogenicity and tumorigenesis. Genome-wide expression analyses suggested that NIK acts on ERK1/2 pathway to exert its activity. In addition, forced expression of NIK increased the BCSC population and enhanced breast cancer cell tumorigenicity. The in vivo relevance of these results is further supported by a tissue microarray of breast cancer samples in which we observed correlated expression of Aldehyde dehydrogenase (ALDH) and NIK protein. Our results support the essential involvement of NIK in BCSC phenotypic regulation via ERK1/2 and NF-κB.