Cancer stem cells and malignant gliomas. From pathophysiology to targeted molecular therapy.

High grade gliomas, the most frequent and most malignant brain cancers, grow rapidly and infiltrate the cerebrospinal axis causing deficits in cognition, mobility, balance or speech and are typically resistant to radiation and chemotherapy. Despite recent progress, WHO grade III and IV gliomas still represent a great challenge in oncology, with overall poor outcomes and inevitable lethality. While radiotherapy and temozolomide are considered the standard first-line approach for therapy of newly diagnosed malignant gliomas, the treatment protocols for recurrent tumors remain ill-defined. Increasing evidence suggests that tumors of the central nervous system are derived from proliferatively active neural stem cells residing in defined neuropoietic niches of the adult brain. These cancer stem cells, also identified in other tumors, provide a reservoir of cells with self-renewal capabilities, can maintain the tumor by generating differentiated non-stem tumor cells and are responsible for recurrences after ablative neurosurgical therapy and chemoradiotherapy. The only way to successfully control recurrent malignant gliomas and even hope for a cure in the future is by combining standard chemotherapy with immunotherapy. Despite the apparent improvements of current treatments, it should be realized that the characteristic brain tumor niche may provide recurrent gliomas an "escape mechanism" from anticancer treatments. Thus, the use of targeted molecular therapy drugs may effectively inhibit or at least slow down cancer stem cell proliferation and stop the brain microenvironment from allowing furtive invasion and proliferation of highly aggressive malignant gliomas.

Antidiabetic pharmacology: a link between metabolic syndrome and neuro-oncology?

One of the main topics of the annual meeting of the American Society for Clinical Oncology in 2011 were the results presented on breast cancer chemotherapy and concomitant administration of the oral antidiabetic metformin. The overall agreement was that current evidence is just enough to dramatically change the clinical practice of oncology, and in our case, brain cancer treatment, and that further research is needed to address the relationship between diabetes, metabolism, insulin analogues and neoplasia. Still, it is very interesting to explore the potentially beneficial effects of metformin in glioma chemo/immunotherapy and wait for results in the clinic. In the current paper we present the cell and molecular aspects of the metabolic syndrome, metformin administration and cancer chemotherapy, with a special emphasis in neuro-oncology, since brain tumors are usually devastating diseases with an extremely high mortality within two years of diagnosis even when surgical, radiotherapeutic and chemotherapeutic interventions are applied.

Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade III and IV malignant gliomas.

PURPOSE: Glioblastoma multiforme (GBM) remains one of the most devastating diseases known to man and affects more than 17,000 patients in the United States alone every year. This malignancy infiltrates the brain early in its course and makes complete neurosurgical resection almost impossible. Recent years have brought significant advances in tumor biology, including the discovery that many cancers, including gliomas, appear to be supported by cells with stem-like properties. In the current study we have investigated the effects of combining metformin with the standard treatment-of-care, as this drug, already used in the treatment of diabetes mellitus, has shown surprising results in the treatment of breast cancer, being also associated with lower mortality in several other malignancies. METHODS: The subjects of the current study were 8 patients with newly diagnosed high-grade gliomas, operated at the Department of Neurosurgery - Clinical University Emergency Hospital, Cluj Napoca. Tumor tissue cultures were established and characterized using immunofluorescence microscopy and PCR analysis and the sensitivity to metformin, epidermal growth factor (EGF) and temozolomide (TMZ) was tested. Microvascular density (MVD) assay was performed on the tumor samples. RESULTS: Seven of the 8 cases had a positive correlation between the number of endothelial cells, the phenotype of isolated tumor cells and the response to adjuvant chemoradiotherapy. The isolated tumor cells had a stem-like behavior, being resistant to conventional drugs. In most cases there was no statistical significant difference between TMZ alone and TMZ plus EGF arms, but there was a important difference between TMZ alone and TMZ plus metformin arms in 6 of the cases. CONCLUSION: New drugs and targeted molecular therapies are important for future therapeutics, but sometimes we must not exclude drugs already used in the clinic that might have remarkable results. Such is the case of metformin, a drug used for decades in the treatment of type 2 diabetes mellitus that has proven to enhance the effect of TMZ in the treatment of breast cancer and, starting with this paper, of brain cancer.

Mesenchymal stem cell irradiation in culture engages differential effect of hyper-fractionated radiotherapy for head and neck cancers.

PURPOSE: The purpose of this study was to challenge current knowledge on the potential therapeutic advantages of stem cells in radiotherapy by developing an in vitro model of the healthy tissue surrounding or replacing the widely resected tumor. After radical surgery, the start of radiotherapy is often delayed due to wound healing process, with potential loss of the opportunity for treating microscopic disease instead of macroscopic early recurrence. Hyperfractionated radiotherapy, contrary to the standard one, can extend the limits of radical surgery and shorten the gap before the onset of postoperative radiotherapy, with potential improvement in local control. METHODS: By using both mesenchymal stem cells and pre-differentiated osteoblasts, cultured in proper pro-osteogenic media after cell irradiation, we investigated both the differences in the response to DNA damage between lineages undergoing differentiation in culture and the intensity of the mineralization process. RESULTS: Ionizing radiation stimulated stem cell proliferation and differentiation at 0.5 Gy and 1 Gy, thus confirming in vitro the clinical results of hyperfractionated irradiation randomized trials in head and neck cancers -HNCs-. CONCLUSION: To our knowledge, this study is the first to investigate the biophysics of low dose gamma irradiation on stem cell culture, focusing on the potential applications in radiation oncology. For advanced oral cavity and oropharyngeal cancers, as radical surgery often implies major bone resection, the use of mesenchymal stem cells as bone reconstruction vectors might shorten the onset of adjuvant hyperfractionated radiotherapy which enhances the mineralization process. As postoperative radiotherapy has recently being revisited for osteosarcoma, this scenario could impact also on bone reconstruction process in this pathology.

Arsenic trioxide sensitizes cancer stem cells to chemoradiotherapy. A new approach in the treatment of inoperable glioblastoma multiforme.

PURPOSE: glioblastoma multiforme (GBM) still bears a very dismal prognosis even with complete resection followed by adjuvant chemoradiation. The aim of the current study was to evaluate in vitro the antitumor efficacy of arsenic trioxide (ATO) in combination with ionizing radiation plus temozolomide and bevacizumab against cultured glioblastoma stem-like cells, as possible way to increase the therapeutic index in patients diagnosed with recurrent, therapy-refractory GBM. METHODS: stem-like tumor cells isolated from a GBM biopsy were established by cell proliferation assays and upregulation of stem cell markers, as proven by reverse transcription - polymerase chain reaction (RT-PCR). Low concentrations of ATO were added prior to temozolomide, bevacizumab and ionizing irradiation. RESULTS: molecular analysis showed that cells expressed CXCR4, Oct-3/4 and GAPDH when compared to placental mesenchymal stem cells, as well as nestin, GFAP and neurofilament protein. Low concentrations of ATO led to morphologic differentiation, with fewer stem cells in Go state and differentiation-associated cytochemical features, like increased sensitivity to cytostatic drugs and radiotherapy. CONCLUSION: ATO exposure before conventional postoperative chemoradiotherapy for GBM might increase treatment efficacy. Further in vivo experiments on laboratory animals and analysis of absorption rate and side effects are required.

Oxaliplatin in patients with metastatic colorectal cancer: efficacy and pharmacokinetics parameters.

PURPOSE: The aim of this study was to investigate the efficiency of the FOLFOX-4 regimen and to evaluate the pharmacokinetics of oxaliplatin in untreated patients with metastatic colorectal cancer. METHODS: 43 patients were enrolled in the study. Patients received oxaliplatin 85 mg/m(2) as 2-h i.v. infusion, on day 1, and bolus 5-fluorouracil (5FU) 400 mg/m(2) plus leucovorin (LV) 200 mg/m(2) followed by 5FU 600 mg/m(2) as 22-h infusion on day 1 and 2, every 2 weeks. The pharmacokinetics of oxaliplatin evaluated in 4 patients was performed in blood, plasma and ultrafiltered plasma (UFT) by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). RESULTS: The overall response rate and the median time to progression (TTP) were 53.49% and 7.1 months, respectively. Grade 3-4 toxic effects were observed in 11 (25.5%) patients. Grade 3 neuropathy was observed in 13.95% of the cases. In univariate analysis only Eastern Cooperative Oncology Group (ECOG) performance status (PS) was correlated with response. No correlation was found between grade 3-4 adverse events and the patient characteristics. The area under the time-concentration curve (AUC) in UFT was 4.8 + or - 0.72 standard deviation (SD) microg h/ml and the total clearance 30.17 + or - 7.75 l/min. The values for volume of distribution and the maximum concentration were 567 + or - 20 liters and 0.38 + or - 0.17 ug/ml, respectively. CONCLUSION: FOLFOX-4 was an effective regimen with good tolerability in previously untreated metastatic colorectal cancer patients. The pharmacokinetics of oxaliplatin was triphasic with a short initial distribution phase and a long terminal elimination phase.

Functional and molecular characterization of glioblastoma multiforme-derived cancer stem cells.

PURPOSE: Brain tumors are the leading cause of cancer mortality in children and remain incurable despite advances in surgery and adjuvant therapies. The failure of malignant gliomas to respond to conventional treatment reflects the unique biology of these tumors, linked to a small population of stem-like precursors. This study describes the characteristics of stem cells isolated from glioblastoma multiforme (GM) and gives insight into the mechanism of brain tumorigenesis. METHODS: Tumor stem-like precursors were identified from primary human GM-derived cell culture using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). Cells were cultured in vitro in stem cell medium supplemented with growth factors and then the capacity of the surviving stem-like precursors to form tumor spheres and to continue to proliferate after chemoradiotherapy were tested. RESULTS: The tumor cells expressed the cellular markers CD133, CD105, CD90, Nanog, Oct 3/4, CXCR4, nestin, glial fibrillary acidic protein (GFAP), neurofilament protein (NF) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells also displayed a high proliferative potential despite chemotherapy and irradiation and also had the ability to form spheroids in suspension. CONCLUSION: High grade gliomas contain stem-like precursors, which exhibit neural stem cell properties with tumorigenicity, establishing a novel developmental paradigm in the study of brain carcinogenesis and providing a powerful tool to develop patient-tailored therapy for this devastating disease.

Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.

BACKGROUND AND PURPOSE: Ovarian cancers are composed of heterogeneous cell populations, including highly proliferative immature precursors and differentiated cells that may belong to different lineages. The main reason why epithelial ovarian cancer is difficult to treat is the unusual mechanism of dissemination that involves local invasion of pelvic and abdominal organs. But, unlike many other carcinomas, initial dissemination rarely requires blood or lymph vessels. Because it has been proven that aggregates of malignant cells within the ascites of patients diagnosed with ovarian cancer represent an impediment to cure such cancers, in the present study we adopted suspension culture combined with anti-cancer regimens as a laboratory strategy for research of the initial process of peritoneal micrometastasis. EXPERIMENTAL DESIGN: MLS human ovarian cancer cells were cultured in serum-free medium. Cells of passage eight were treated in combination with the anticancer agent doxorubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The acquired increased aggressiveness properties was confirmed by multidrug resistance assays and by their ability to grow in an anchorage-independent manner in vitro as tumor spheroids. RESULTS: Cells selected after chemotherapy had a increased proliferative potential, eliminated Rhodamine 123 in culture and also formed spheroids in suspension. CONCLUSIONS: Here we present direct evidence that the metastasis of human ovarian cancer may be a result of transformation and dysfunction of immature precursor cells in the ovary. Also, spheroid formation may represent a key component of chemotherapy recurrence and a better understanding of these 3D structures can contribute to the development of new treatments for metastatic carcinoma.

Adjuvant partial breast radiotherapy for early breast cancer--ready for a change?

Partial breast radiotherapy (PBRT) has recently gained an optional place as local adjuvant treatment after breast-conserving surgery (BCS) for selected early-stage breast cancer. With high-dose-rate (HDR) interstitial multicatheter brachytherapy (BT) performed under strict quality assurance criteria, there is level B evidence derived from one single randomized study with follow-up longer than 5 years and level C from several mature prospective non-randomized studies. For intra-lumpectomy cavity Mammosite HDR-BT and for external beam RT, as well as 3-dimensional (3D)-conformal or intra-operative therapy by electrons or photons, there is only level D evidence with no mature enough prospective data. Large phase III trials are ongoing for each PBRT technique, but their results might offer a level A evidence no earlier than 5 years.

Adiponectin predicts cardiovascular events in diabetes dialysis patients.

OBJECTIVES: Adiponectin is an insulin-sensitizing, anti-inflammatory adipokine with anti-atherogenic actions in the general population. In dialysis patients it is unclear whether adiponectin conserves its protective value or is, on the contrary, associated to worse prognosis. We assessed the predictive value of adiponectin for atherosclerosis related cardiovascular events in type 2 diabetic dialysis patients. DESIGN AND METHODS: Prevalent diabetic dialysis patients from three dialysis units (n=77) were enrolled in a 3years' prospective observational study. Serum adiponectin, clinical and laboratory parameters were determined at baseline; new occurrence of atherosclerosis related events (coronary events, atherosclerosis obliterans, and stroke) was recorded. RESULTS: Baseline adiponectin was 17.25(9.53-31.97) µg/mL and significantly correlated to HDL cholesterol (r=0.29, p=0.01), triglycerides (r=-0.40, p=0.0004), ferritin (r=-0.29, p=0.02), transferrin (r=-0.28, p=0.02), and uric acid (r=-0.24, p=0.04). In multivariate analysis association to triglycerides (p=0.001), HDL cholesterol (p=0.01) and ferritin (p=0.04) remained significant. 36 new fatal and non-fatal new cardiovascular events occurred, 29 patient died. Cox proportional regression analysis showed that adiponectin below or above a ROC-derived cut-off of 27.33µg/mL significantly influenced event-free survival: hazard ratio (HR) 2.48, 95% confidence interval (CI) (1.09-5.66), p=0.031 along with fasting glucose HR 1.01, 95%CI(1.00-1.02), p=0.01 and history of cardiovascular events at inclusion HR 3.16, 95%CI(1.36-7.32), p=0.007. In multivariate analysis baseline adiponectin HR 5.02, 95%CI(0.98-25.06), p=0.05 and glycemia HR 1.01, 95%CI(1.00-1.02), p=0.01 influenced event-free survival. Adiponectin also predicted cardiovascular events in patients without cardiovascular disease at inclusion but was not associated to overall mortality. CONCLUSIONS: In diabetes dialysis patients low adiponectin favors occurrence of atherosclerosis related cardiovascular events.

Acetylsalicylic acid and acetaminophen.

Aspirin is the most extensively prescribed analgesic, antipyretic, and anti-inflammatory agent. It is effective against the mild-to-moderate pain of inflammation. The best single dose of aspirin is that which is adequate to relieve pain; the proper dosage interval is that which sustains relief without causing toxicity. It would be unfortunate to withhold aspirin from a patient who desperately requires the relief provided by this most useful analgesic because there have been some adverse effects when it is used improperly. Acetaminophen is a suitable substitute for aspirin for its analgesic or antipyretic uses in patients in whom aspirin is contraindicated (e.g., prepartum patients, children with febrile conditions, patients with asthma, peptic ulcer, gouty arthritis, hyperuricemia, hemophilia or other bleeding disorders, and those taking anticoagulants). However, the general substitution of acetaminophen for aspirin as an analgesic is not recommended.

Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma.

OBJECTIVES: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence of a role of metformin as an anticancer drug, the mechanisms by which metformin exerts its oncostatic actions are not fully understood yet. Therefore, we tried to bring new insights by analyzing the anti-neoplastic effect of metformin for hepatocellular carcinoma-derived stem-like cells treated with conventional combination chemotherapy. METHODS: Cancer stem-like cells previusly isolated from a hepatocellular carcinoma biopsy were treated with metformin, PIAF chemotherapy regimen and the combination of these two protocols. Measurements of lipid peroxidation, reduced glutathione, fluorescein diacetate and proliferation rates were determined, apart from the autophagy assay and apoptosis determination by chip flow cytometry. RESULTS: Metformin alone and especially metformin in association with PIAF increases oxidative stress within the cells by increasing the levels of lipid peroxids as well as decreasing the levels of reduced glutathione. The MTT cell proliferation assay showed decreased prolife-ration rates for the arm treated with metformin and with the combination of drugs in comparison with the control arm, proving high correlation with the oxidative stress results. The autophagy assay and determination of apoptosis by chip flow cytometry confirmed the results obtained in the previous assays. CONCLUSION: Metformin could be used in chemotherapy treatments to induce reactive oxygen species and increase the cytostatics effects within the tumor cell. Still, further experiments must be carried out on murine models before we can move on and use this drugs in the adjuvant setting for unresectable primary liver cancer.

Low plasma adiponectin levels predict increased urinary albumin/creatinine ratio in type 2 diabetes patients.

BACKGROUND: Experimental studies have shown that adiponectin has antiproteinuric and nephroprotective effects. The purpose of the study was to assess the value of plasma adiponectin as a predictor of proteinuria in type 2 diabetes (T2D) patients. METHODS: In this one-year prospective follow-up study, we included T2D patients with positive visual test for microalbuminuria (Micral) and negative visual test for proteinuria. Exclusion criteria were: glomerular filtration ratio (GFR) < 30 ml/min, acute infection/inflammation, uncontrolled hypertension, and atherosclerotic complications. The main outcome measure was the change in urinary albumin/creatinine ratio (UACR) after 1 year follow-up (Δ UACR). RESULTS: Fifty-six patients (66% males) completed the study. Their initial mean UACR was 81.58 ± 26.42 mg/g and mean GFR was 81.15 ± 3.96 ml/min. At baseline, simple regression disclosed significant correlations between UACR and plasma adiponectin (r = 0.54, P = 0.00002) and GFR (r = -0.28, P = 0.03); in multiple regression analysis, plasma adiponectin remained the only predictor of UACR (P = 0.00007). Baseline plasma adiponectin was significantly correlated to body mass index (r = -0.28, P = 0.04), waist circumference (r = -0.27, P = 0.05), HDL cholesterol (r = 0.35, P = 0.01), and LDL cholesterol (r = 0.27, P = 0.04). Baseline plasma adiponectin significantly correlated in simple (r = -0.38, P = 0.004) and multiple regression (P = 0.04) to Δ UACR. When patients were divided according to Δ UACR in nonprogressors (Δ UACR < 0) and progressors (Δ UACR > 0), logistic regression showed that baseline GFR (OR = 1.04, CI95%: 1.00-1.09, P = 0.04) and plasma adiponectin (OR = 1.16, CI95%: 1.02-1.32, P = 0.02) were the only factors that predicted whether the patient would be a progressor or not. CONCLUSION: In T2D patients, lower plasma adiponectin levels seem to be predictive of increased UACR.

The axis of evil in the fight against cancer.

Over the past years medicine has undergone intensive changes, evolving from classical semiology and internal medicine to individualized treatments, based on recent breakthroughs in immunology and genetics. This concept has had a profound impact in all medical specialties and as a consequence pharmacology and various treatment plans will be based on monoclonal antibodies and targeted cell therapies. One such target is the SDF-1-CXCR4 axis bacause it plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis and neuronal development to immune cell trafficking. The chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1alpha are also implicated in various pathological conditions, including metastatic spread and HIV infection. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells, based on the growing evidence that it plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells and their homing to the bone marrow. Because most malignancies originate in the progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and migrate to organs that highly express SDF-1. Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, which may be regulated by several small molecules related to inflammation. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications in both tissue engineering and in clinical hematology and oncology to inhibit metastasis of cancer stem cells.

Stem-like cells in colorectal oncology.

Although the treatment for colorectal cancer has seen considerable progress during the past few years, the mortality associated with this type of tumor remains high. This article presents the existing methods of treatment, focusing on the new treatments made possible by the advances in the field of normal and tumor stem cells. Starting from the normal architecture of the colon and the properties of the cells identified in it, we sought to present a few notions concerning these cells which have a direct relevance for both pathology and treatment. The manner in which they divide (symmetrically or asymmetrically) as well as the molecules which control their circulation through the body are just a few examples which are likely to influence the treatment of colorectal cancer in the future.

Cellular interactions in prostate cancer genesis and dissemination. Looking beyond the obvious.

Similar to normal organs arising from normal stem cells, cancers can be viewed as organs composed of heterogeneous cellular populations arising from cancer cells with indefinite proliferation abilities. The continuous malignant progression is maintained by the proliferation of cancer stem cells and not the progeny that undergo limited proliferation before terminally differentiating. Effective therapy must eradicate malignant cells with unlimited clonogenic expansion within the primary tumor bulk. Thus, resolving both the specific cell of origin for prostate cancer and the interactions between the cells and the surrounding microenvironment within the cancer stem cell niche are crucial to appropriately define rational targets for therapeutic intervention and cure prostate cancer.