Intratumoral gamma-linoleic acid therapy of human gliomas.

In vitro and in vivo studies have shown that gamma-linoleic acid (GLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) can selectively kill tumor cells. In a clinical trial, the effectiveness of intratumoral administration of GLA in patients with gliomas was studied. Of the 6 patients treated, all showed substantial response to GLA as documented by computerized tomography. There were no acute side-effects due to the therapy. This report demonstrates that intratumoral administration of GLA is a possible approach to the treatment of human glial tumors.     

From bench to the clinic: gamma-linolenic acid therapy of human gliomas

Malignant gliomas are among the most devastating of cancers and are a major cause of mortality in a young population with a median survival time of 9 months following cytoreductive surgery, radiotherapy and chemotherapy. Recent studies showed that polyunsaturated fatty acids especially gamma-linolenic acid (GLA) have selective tumoricidal action especially against malignant glioma cells both in vitro and in vivo. Limited open label clinical studies showed that intratumoral injection/infusion of GLA is safe and effective against malignant gliomas. In view of this, large-scale, double blind studies are needed to establish the usefulness of GLA in the treatment of malignant brain tumors.     

Intratumoral bacteria are an important “accomplice” in tumor development and metastasis

As emerging tumor components, intratumoral bacteria have been found in many solid tumors. Several studies have demonstrated that different cancer subtypes have distinct microbial compositions, and mechanistic studies have shown that intratumoral bacteria may promote cancer initiation and progression through DNA damage, epigenetic modification, inflammatory responses, modulation of host immunity and activation of oncogenes or oncogenic pathways. Moreover, intratumoral bacteria have been shown to modulate tumor metastasis and chemotherapy response. A better understanding of the tumor microenvironment and its associated microbiota will facilitate the design of new metabolically engineered species, opening up a new era of intratumoral bacteria-based cancer therapy. However, many questions remain to be resolved, such as where intratumoral bacteria originate and whether there is a direct causal relationship between intratumoral bacteria and tumor susceptibility. In addition, suitable preclinical models and more advanced detection techniques are crucial for studying the biological functions of intratumoral bacteria. In this review, we summarize the complicated role of intratumoral bacteria in the regulation of cancer development and metastasis and discuss their carcinogenic mechanisms and potential therapeutic aspects.     

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumours. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM) which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic-ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this point of view article we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.     

Glutamate and tumor-associated epilepsy: Glial cell dysfunction in the peritumoral environment

Seizures are a serious and debilitating co-morbidity of primary brain tumors that affect most patients, yet their etiology is poorly understood. In many CNS pathologies, including epilepsy and brain injury, high levels of extracellular glutamate have been implicated in seizure generation. It has been shown that gliomas release neurotoxic levels of glutamate through their high expression of system xc-. More recently it was shown that the surrounding peritumoral cortex is spontaneously hyperexcitable. In this review, we discuss how gliomas induce changes in the surrounding environment that may further contribute to elevated extracellular glutamate and tumor-associated seizures. Peritumoral astrocytes become reactive and lose their ability to remove glutamate, while microglia, in response to signals from glioma cells, may release glutamate. In addition, gliomas increase blood brain barrier permeability, allowing seizure-inducing serum components, including glutamate, into the peritumoral region. These factors, working together or alone, may influence the frequency and severity of tumor-associated epilepsy.     

Aromatase and its inhibitors

Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNA_arom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive, human breast carcinoma cells transfected with the human aromatase gene. Thus, the cells synthesized estrogen which stimulated tumor formation. Both aromatase inhibitors and antiestrogens were effective in suppressing tumor growth in this model. However, letrozole was more effective than tamoxifen. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. This suggests that sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.     

Natural and chemotherapy-induced clonal evolution of tumors

Evolution and natural selection of tumoral clones in the process of transformation and the following carcinogenesis can be called natural clonal evolution. Its main driving factors are internal: genetic instability initiated by driver mutations and microenvironment, which enables selective pressure while forming the environment for cell transformation and their survival. We present our overview of contemporary research dealing with mechanisms of carcinogenesis in different localizations from precancerous pathologies to metastasis and relapse. It shows that natural clonal evolution establishes intratumoral heterogeneity and enables tumor progression. Tumors of monoclonal origin are of low-level intratumoral heterogeneity in the initial stages, and this increases with the size of the tumor. Tumors of polyclonal origin are of extremely high-level intratumoral heterogeneity in the initial stages and become more homogeneous when larger due to clonal expansion. In cases of chemotherapy-induced clonal evolution of a tumor, chemotherapy becomes the leading factor in treatment. The latest research shows that the impact of chemotherapy can radically increase the speed of clonal evolution and lead to new malignant and resistant clones that cause tumor metastasis. Another option of chemotherapy-induced clonal evolution is formation of a new dominant clone from a clone that was minor in the initial tumor and obtained free space due to elimination of sensitive clones by chemotherapy. As a result, in ~20% of cases, chemotherapy can stimulate metastasis and relapse of tumors due to clonal evolution. The conclusion of the overview formulates approaches to tumor treatment based on clonal evolution: in particular, precision therapy, prediction of metastasis stimulation in patients treated with chemotherapy, methods of genetic evaluation of chemotherapy efficiency and clonal-oriented treatment, and approaches to manipulating the clonal evolution of tumors are presented.     

Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion

Despite temozolomide (TMZ) treatment, the prognosis for patients with glioblastoma multiforme is still dismal. As dose escalation of TMZ is limited by systemic toxicity, intratumoral delivery emerges as an attractive treatment modality, which may sustain cytotoxic drug concentrations intratumorally and induce immunogenic cell death. Both clinical and experimental gliomas have responded to immunotherapy, but the benefit of simultaneous chemo- and immunotherapy is inadequately studied. Here, we monitored survival of GL261-bearing C57BL/6 mice following a 3-day treatment with either intratumoral TMZ (micro-osmotic pump, 4.2 mg/kg/day) or systemic TMZ (i.p. injections, 50 mg/kg/day) alone, or combined with immunization using GM-CSF secreting GL261 cells. Peripheral and intratumoral leukocytes were analyzed by flow cytometry and immunohistochemistry. Intratumoral TMZ induced higher survival rate than systemic TMZ (45 vs. 8 %). When T cells were depleted following intratumoral TMZ, the therapeutic effect was completely abrogated (0 % survival). Intratumoral TMZ synergistically increased survival rate of immunized mice (from 25 to 83 %), while systemic TMZ failed (0 %). While systemic TMZ induced a transient leukopenia, intratumoral TMZ and immunotherapy sustained the proliferation of CD8⁺ T cells and decreased the number of intratumoral immunosuppressive cells. In conclusion, intratumoral TMZ alone or in combination with immunotherapy could cure glioma-bearing mice, due to attenuation of local immunosuppression and increase in potential effector immune cells.     

SIRT2 activity is required for the survival of C6 glioma cells

SIRT2 is a tubulin deacetylase, which can play either detrimental or beneficial roles in cell survival under different conditions. While it has been suggested that reduced SIRT2 expression in human gliomas may contribute to development of gliomas, there has been no study that directly determines the effects of decreased SIRT2 activity on the survival of glioma cells. In this study we applied both pharmacological and molecular approaches to determine the roles of SIRT2 in the survival of glioma cells. Our studies, by conducting such assays as flow cytometry-based Annexin V assay and caspase-3 immunostaining, have indicated that decreased SIRT2 activity leads to apoptosis of C6 glioma cells by caspase-3-dependent pathway. Our experiments have further shown that reduced SIRT2 activity produces necrosis of C6 glioma cells. Moreover, our study applying SIRT2 siRNA has also shown that decreased SIRT2 leads to both necrosis and apoptotic changes of C6 glioma cells. Collectively, our study has provided novel evidence indicating that SIRT2 activity plays a key role in maintaining the survival of glioma cells, and that reduced SIRT2 activity can induce both necrosis and caspase-3-dependent apoptosis of C6 glioma cells. These results have also suggested that inhibition of SIRT2 might become a novel therapeutic strategy for gliomas.     

Low-grade gliomas: clinical and pathobiological aspects

The optimal management of patients with low-grade gliomas remains a challenge for the treating physician. The natural history of the disease shows a large variety, and there is a substantial controversy about many of everyday treatment recommendations. However, new developments in clinical and basic research in neuro-oncology have occurred during the last years. In this review some of these new insights into clinical and biological aspects of low-grade gliomas are discussed, with focus on the translation of new knowledge from basic research into clinical practice. For example, molecular genetic profiling of tumour material has started to guide treatment recommendations and clinical management of some patients with oligodendrogliomas. Experimental studies of the different molecular pathways in tumour cells and in their normal counterparts involved in cell-cycle check-point control have elucidated some of the underlying mechanisms of resistance of gliomas to radiotherapy and chemotherapy. Finally, improved classification of the different subtypes of low-grade gliomas may be achieved in the near future by characterization of the genetic heterogeneity within the tumour and by identification of a putative stem cell as the origin of the tumour cells.     

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumors. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM), which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic- ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this Commentary & View, we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.Key words: brain tumor, blood coagulation, hypoxia, MAP kinase, cancer stem cells, tumor invasion     

The role of radiation therapy for carcinoma of the lung

A large proportion of patients with carcinoma of the lung may benefit from the use of radiation therapy. Operable patients have not been shown to benefit from preoperative irradiation, but postoperative irradiation has improved survival in those found to have involvement of hilar or mediastinal lymph nodes. Radiation therapy is the only potentially curative treatment for patients who are inoperable, but do not have distant metastasis. Control of the local tumor is very dependent upon dose-fractionation-time relationships. Patients who are relatively asymptomatic, i.e., they have a high performance status, are curable if treated promptly with radiation therapy. Small cell carcinoma requires both radiation therapy and chemotherapy. The optimal method of combining the two modalities is yet to be determined, but prophylactic cranial irradiation is necessary to control microscopic metastases that are not affected by systemic chemotherapy, and thoracic irradiation is necessary to give the highest probability of control of the primary tumor. Prophylactic cranial irradiation has also been shown to reduce the frequency of brain metastasis in patients with squamous carcinoma, large cell carcinoma, and adenocarcinoma; it may become more important in these cell types when more effective chemotherapy is developed.     

The characterisation of gliomas using human monoclonal antibodies. Minireview on cancer research

Malignant gliomas contain large numbers of invading lymphocytes. The function of these lymphocytes is unknown. It is likely that they are involved in host defence against the developing tumour. By isolating these cells and fusing them with a suitable myeloma system, hybrids can be established and their antibody activity analysed. Human monoclonal antibodies reactive to glioma cells have now been isolated and their specificity has been determined by radioimmunoassay, binding assays and immunoprecipitation. Furthermore, such antibodies can be radiolabelled with 131I and administered intravenously to localise tumours in patients. These antibodies have therapeutic potential as selective targeting agents for chemotherapy and other cytotoxic agents.     

5-Aminolevulinic Acid Induced Fluorescence Is a Powerful Intraoperative Marker for Precise Histopathological Grading of Gliomas with Non-Significant Contrast-Enhancement

Background

Intraoperative identification of anaplastic foci in diffusely infiltrating gliomas (DIG) with non-significant contrast-enhancement on MRI is indispensible to avoid histopathological undergrading and subsequent treatment failure. Recently, we found that 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence can visualize areas with increased proliferative and metabolic activity in such gliomas intraoperatively. As treatment of DIG is predominantely based on histopathological World Health Organisation (WHO) parameters, we analyzed whether PpIX fluorescence can detect anaplastic foci according to these criteria.

Methods

We prospectively included DIG patients with non-significant contrast-enhancement that received 5-ALA prior to resection. Intraoperatively, multiple samples from PpIX positive and negative intratumoral areas were collected using a modified neurosurgical microscope. In all samples, histopathological WHO criteria and proliferation rate were assessed and correlated to the PpIX fluorescence status.

Results

A total of 215 tumor specimens were collected in 59 patients. Of 26 WHO grade III gliomas, 23 cases (85%) showed focal PpIX fluorescence, whereas 29 (91%) of 33 WHO grade II gliomas were PpIX negative. In intratumoral areas with focal PpIX fluorescence, mitotic rate, cell density, nuclear pleomorphism, and proliferation rate were significantly higher than in non-fluorescing areas. The positive predictive value of focal PpIX fluorescence for WHO grade III histology was 85%.

Conclusions

Our study indicates that 5-ALA induced PpIX fluorescence is a powerful marker for intraoperative identification of anaplastic foci according to the histopathological WHO criteria in DIG with non-significant contrast-enhancement. Therefore, application of 5-ALA optimizes tissue sampling for precise histopathological diagnosis independent of brain-shift.     

Dose-intensive chemotherapy with stem cell support as a treatment strategy for bone and soft-tissue sarcomas

Whether high-dose chemotherapy with stem cell support improves the long-term outcome for patients with bone and soft-tissue sarcoma is debatable and controversial. Prognosis of patients with unresectable or advanced metastatic sarcoma remains poor with a disease-free survival at 5 years less than 10%; treatment is generally considered to be palliative. Doxorubicin, epirubicin and ifosfamide are the most active single agents with response rates above 20%. Although drug combinations result in higher response rates, superiority against single agent chemotherapy in terms of survival could not have been demonstrated yet. As a dose-response relationship has been shown for the anthracyclines and especially for ifosfamide, high-dose chemotherapy with stem cell support has been evaluated by several investigators. However, all studies were not randomized, comprised small patient numbers and included heterogeneous histological subtypes of soft-tissue sarcomas. Nevertheless, higher doses of chemotherapy result in higher remission rates, which could correlate with longer survival. Well-designed randomized trials should be performed. In this review article, we overview the literature and on the basis of our own data we emphasize the value of high-dose chemotherapy as a treatment option for younger patients with a good performance status in complete or partial remission prior to high-dose chemotherapy.     

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220. © 2012 Wiley Periodicals, Inc.     

Targeting siRNA in colorectal cancer therapy: Nanotechnology comes into view

Colorectal cancer (CRC) is known as one of the most important causes of death and mortality worldwide. Although several efforts have been made for finding new therapies, no achievements have been made in this area. Multidrug resistance (MDR) mechanisms are one of the key factors that could lead to the failure of chemotherapy. Moreover, it has been shown that various chemotherapy drugs are associated with several side effects. Hence, it seems that finding new drugs or new therapeutic platforms is required. Among different therapeutic approaches, utilization of nanoparticles (NPs) for targeting a variety of molecules such as siRNAs are associated with good results for the treatment of CRC. Targeting siRNA-mediated NPs could turn off the effects of oncogenes and MDR-related genes. In the current study, we summarized various siRNAs targeted by NPs which could be used for the treatment of CRC. Moreover, we highlighted other routes such as liposome for targeting siRNAs in CRC therapy.     

Controversies of aromatase localization in human breast cancer--stromal versus parenchymal cells

Aromatase is a key enzyme of estrogen production through conversion from serum androgens in estrogen-dependent postmenopausal breast cancer. Aromatase has been reported to be predominantly located in intratumoral stromal cells and adipocytes but not in parenchymal or carcinoma cells in breast cancer tissue. It is, however, true that there have been controversies regarding intratumoral localization of aromatase in human breast carcinoma, especially whether intratumoral production of estrogens through aromatase occurs in parenchymal or stromal cells. Results of several studies suggested that aromatase present in parenchymal carcinoma cells plays more important roles in the growth and invasion of breast carcinomas than that in stromal cells through providing higher levels of estrogens to carcinoma cells. Aromatase inhibitors are increasingly being used in place of tamoxifen after results of various clinical trials demonstrated that aromatase inhibitors are more effective in increasing survival and recurrence of estrogen-dependent breast cancer patients. Therefore, it is important to clarify the estrogen supplying pathway by aromatase inside of breast carcinoma tissues in order to evaluate the possible efficacy of aromatase inhibitor treatment. In this review, the controversies regarding these intratumoral localization patterns in human breast carcinoma will be briefly summarized.     

Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation

Delta9-Tetrahydrocannabinol, the main active component of marijuana, induces apoptosis of transformed neural cells in culture. Here, we show that intratumoral administration of Delta9-tetrahydrocannabinol and the synthetic cannabinoid agonist WIN-55,212-2 induced a considerable regression of malignant gliomas in Wistar rats and in mice deficient in recombination activating gene 2. Cannabinoid treatment did not produce any substantial neurotoxic effect in the conditions used. Experiments with two subclones of C6 glioma cells in culture showed that cannabinoids signal apoptosis by a pathway involving cannabinoid receptors, sustained ceramide accumulation and Raf1/extracellular signal-regulated kinase activation. These results may provide the basis for a new therapeutic approach for the treatment of malignant gliomas.