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1,5-Diazacyclooctane was prepared by a simple synthetic sequence and coupled to pentacyclic triterpenoic acids oleanolic acid, ursolic acid, betulinic acid, platanic acid, and asiatic acid; these amides were activated with oxalyl chloride and reacted with rhodamine B or rhodamine 101 to yield conjugates. The conjugates were screened in SRB assays with various human breast cancer cell lines (MDA-MB-231, HS578T, MCF-7, and T47D) and found to exert cytotoxic activity even at a low concentration. Therefore, for an asiatic acid rhodamine 101 conjugate (28), an IC50 = 0.60 nM was determined and found to induce apoptosis in MDA-MB-231 and HS578T cells. Extra experiments showed the compound to act as a mitocan and to induce inhibition of proliferation or growth arrest in MDA-MB-231 cells at lower doses followed by an induction of apoptosis at higher doses. Furthermore, differential responses to proliferation inhibition and apoptosis induction may explain differential sensitivity of mammary cell lines to compound 28.

Hypoxia-regulated protein carbonic anhydrase IX (CA IX) is up-regulated in different tumor entities and correlated with poor prognosis in breast cancer patients. Due to the radio- and chemotherapy resistance of solid hypoxic tumors, derivatives of betulinic acid (BA), a natural compound with anticancer properties, seem to be promising to benefit these cancer patients. We synthesized new betulin sulfonamides and determined their cytotoxicity in different breast cancer cell lines. Additionally, we investigated their effects on clonogenic survival, cell death, extracellular pH, HIF-1α, CA IX and CA XII protein levels and radiosensitivity. Our study revealed that cytotoxicity increased after treatment with the betulin sulfonamides compared to BA or their precursors, especially in triple-negative breast cancer (TNBC) cells. CA IX activity as well as CA IX and CA XII protein levels were reduced by the betulin sulfonamides. We observed elevated inhibitory efficiency against protumorigenic processes such as proliferation and clonogenic survival and the promotion of cell death and radiosensitivity compared to the precursor derivatives. In particular, TNBC cells showed benefit from the addition of sulfonamides onto BA and revealed that betulin sulfonamides are promising compounds to treat more aggressive breast cancers, or are at the same level against less aggressive breast cancer cells.

Many pentacyclic triterpenoids show anti-cancer and anti-inflammatory properties. Recently, we detected a pronounced cytotoxicity and radiosensitivity of two betulinyl sulfamates in human breast cancer cells. Besides betulinic acid scaffold (BSBA-S), we synthesized several new sulfamate-coupled scaffolds from oleanolic acid (OSBA-S), ursolic acid (USBA-S), platanic acid (PSBA-S) and maslinic acid (MSBA-S). Highest cytotoxicity was monitored in breast cancer cell lines after MSBA-S treatment showing in SRB assays IC50 values between 3.7 μM and 5.8 μM. Other sulfamate/triterpene conjugates, however, were less cytotoxic holding IC50 values between 6.6 μM and >50 µM, respectively. MSBA-S-treated breast cancer cells displayed significantly reduced clonogenic survival and an increased rate of apoptosis as compared to the other conjugates. In addition, MSBA-S in combination with irradiation resulted in effects on radiosensitivity in MDA-MB-231 cells (DMF10 = 1.14). In particular, ROS formation was strongly assessed in MSBA-S-treated breast cancer cells. Our findings suggest that the sulfamate derivative of maslinic acid MSBA-S might be a new option for the radiation therapy in breast cancer cells.

Hypoxia plays a key role in tumor progression and resistance to radiotherapy. Expression of the transmembrane-tethered enzyme carbonic anhydrase IX (CA IX) is strongly induced by hypoxia. High CA IX expression levels correlate with poor prognosis in cancer patients. Previously, we showed that the downregulation of CA IX expression by siRNA interference and the inhibition of CA IX activity results in increased cytotoxicity, inhibition of migration and radiosensitization of hypoxic cancer cells. Betulinic acid (BA) is a natural compound derived from birch bark. It has shown promising anti-tumor effects due to its cancer cell specific cytotoxic properties. We have shown that BA inhibits the HIF-1α pathway, resulting in apoptosis, inhibition of migration and enhanced cytotoxicity of breast cancer cells. In this study, we investigate the effects of the novel betulin derivative 3-O-acetylbetulin (3-AC) and carbonic anhydrase inhibitors (CAI) octyl disulfamate (OCT) or 4-(3-[4-fluorophenyl]ureido)benzenesulfonamide (SLC-0111), on cellular and radiobiological parameters in MDA-MB-231 and MCF-7 cells. Treatment with 3-AC or OCT alone only caused moderate cytotoxicity, reduction in cell migration, ROS production and DNA damage. However, the combined treatment with 3-AC and CAI strongly enhanced radiosensitivity, increased cytotoxicity, inhibited cell motility and enhanced DNA damage. Our findings suggest that the combination of two bioactive drugs 3-AC and a CAI, such as OCT or SLC-0111, could be a promising therapeutic approach for targeting hypoxic tumor cells.

Despite the existence of multimodal therapy concepts, glioblastoma remains a tumor type with one of the worst prognoses. In particular, the poor prognosis is due to the lack of therapeutic efficacy of chemical agents and irradiation in hypoxic tumor areas. New therapeutic strategies could improve the treatment of glioblastoma. In this study, we investigated the therapeutic efficacy of a conjugate of cisplatin (DDP), a widely used chemotherapeutic agent, and betulinic acid (BA), a natural product from plane tree bark, in glioblastoma cells under different oxygen conditions. We investigated the effects of the BA-DDP conjugate κN',N''-{3-acetyloxy-BA-28-[2-(2-aminoethyl)aminoethyl]amide} dichlorido platinum(II) (APC) and its precursor 3-acetyloxy-BA-28-[2-(2-aminoethyl)aminoethyl]amide (DE9B) on cytotoxicity, cell growth, apoptosis, migration and radiosensitivity compared to BA or DDP alone under different oxygen conditions. Based on the EC50 values, the precursor DE9B exhibited the strongest cytotoxic effects of the analyzed chemotherapeutic agents. The BA-DDP conjugate APC achieved a moderate cytotoxic effect in glioma cells. Both of the newly developed agents induced cell growth delay, apoptosis and inhibition of migration. Furthermore, additive effects could be achieved in combination with irradiation. In contrast to those of BA and DDP, the cell biological effects of APC and DE9B were not influenced by the oxygen concentration. In this study, the linking of BA and DDP did not produce a compound with additive therapeutic effects on glioblastoma cell lines in vitro. Nevertheless, the results of this study suggest that the precursor DE9B is an effective BA derivative for the treatment of glioblastoma in vitro.

The presence of an isocitrate dehydrogenase 1 (IDH1) mutation is associated with a less aggressive phenotype, increased sensitivity to radiation, and increased overall survival in patients with diffuse glioma. Based on in vitro experimentations in malignant glioma cell lines, the consequences on cellular processes of IDH1R132H expression were analyzed. The results revealed that IDH1R132H expression enhanced the radiation induced accumulation of residual &gamma, H2AX foci and decreased the amount of glutathione (GSH) independent of the oxygen status. In addition, expression of the mutant IDH1 caused a significant increase of cell stiffness and induced an altered organization of the cytoskeleton, which has been shown to reinforce cell stiffness. Furthermore, IDH1R132H expression decreased the expression of vimentin, an important component of the cytoskeleton and regulator of the cell stiffness. The results emphasize the important role of mutant IDH1 in treatment of patients with diffuse gliomas especially in response to radiation. Hence, detection of the genetic status of IDH1 before therapy massively expands the utility of immunohistochemistry to accurately distinguish patients with a less aggressive and radiosensitive IDH1-mutant diffuse glioma suitable for radiotherapy from those with a more aggressive IDH1-wildtype diffuse glioma who might benefit from an individually intensified therapy comprising radiotherapy and alternative medical treatments.

In various tumors, the hypoxia inducible factor-1α (HIF-1α) and the epidermal growth factor-receptor (EGFR) have an impact on survival. Nevertheless, the prognostic impact of both markers for soft tissue sarcoma (STS) is not well studied. We examined 114 frozen tumor samples from adult soft tissue sarcoma patients and 19 frozen normal tissue samples. The mRNA levels of HIF-1α, EGFR, and the reference gene hypoxanthine phosphoribosyltransferase (HPRT) were quantified using a multiplex qPCR technique. In addition, levels of EGFR or HIF-1α protein were determined from 74 corresponding protein samples using ELISA techniques. Our analysis showed that a low level of HIF-1α or EGFR mRNA (respectively, relative risk (RR) = 2.8; p = 0.001 and RR = 1.9; p = 0.04; multivariate Cox´s regression analysis) is significantly associated with a poor prognosis in STS patients. The combination of both mRNAs in a multivariate Cox’s regression analysis resulted in an increased risk of early tumor-specific death of patients (RR = 3.1, p = 0.003) when both mRNA levels in the tumors were low. The EGFR protein level had no association with the survival of the patient’s cohort studied, and a higher level of HIF-1α protein associated only with a trend to significance (multivariate Cox’s regression analysis) to a poor prognosis in STS patients (RR = 1.9, p = 0.09). However, patients with low levels of HIF-1α protein and a high content of EGFR protein in the tumor had a three-fold better survival compared to patients without such constellation regarding the protein level of HIF-1α and EGFR. In a bivariate two-sided Spearman’s rank correlation, a significant correlation between the expression of HIF-1α mRNA and expression of EGFR mRNA (p < 0.001) or EGFR protein (p = 0.001) was found, additionally, EGFR mRNA correlated with EGFR protein level (p < 0.001). Our results show that low levels of HIF-1α mRNA or EGFR mRNA are negative independent prognostic markers for STS patients, especially after combination of both parameters. The protein levels showed a different effect on the prognosis. In addition, our analysis suggests a possible association between HIF-1α and EGFR expression in STS.

Soft tissue sarcomas are a heterogeneous group of malignant neoplasms of mesenchymal origin. Partly due to hypoxia, an aggressive and radioresistant phenotype frequently develops, resulting in poorer patient outcome. microRNAs (miRNAs) are tiny, non-coding regulators of gene expression and in situations of cellular stress situations may predict clinical progression and patient outcome. In the present study, hypoxia-associated miR-199a-5p expression in 96 soft tissue sarcoma samples was analysed by reverse transcription-quantitative polymerase chain reaction and associations between miR-199a-5p expression and patient clinicopathological characteristics and survival were measured. Additionally, luciferase reporter assays analyzed the post-transcriptional regulation of hypoxia-associated genes hypoxia-inducible factor 1α (HIF-1α), oxidative stress induced growth inhibitor 2 (OSGIN2) and vascular endothelial growth factor (VEGF) by miR-199a-5p. Survival analyses indicated that low expression of miR-199a-5p was significantly correlated with poorer tumor-specific survival (univariate Cox's-Regression analyses; relative risk=1.92, P=0.029). Furthermore, it was demonstrated that the 3'UTR of HIF-1α and OSGIN2 genes were regulated by miR-199a-5p in-vitro, although the 3'UTR of VEGF was not. To the best of our knowledge, this is the first report demonstrating the regulation of the 3'untranslated region of the OSGIN2 gene by miR-199a-5p and a significant correlation between low miR-199a-5p expression and a poor outcome of patients with soft tissue sarcoma.

Betulinic acid (BA), a natural compound of birch bark, is cytotoxic for many tumors. Recently, a betulinyl sulfamate was described that inhibits carbonic anhydrases (CA), such as CAIX, an attractive target for tumor-selective therapy strategies in hypoxic cancer cells. Data on combined CAIX inhibition with radiotherapy are rare. In the human breast cancer cell lines MDA-MB231 and MCF7, the effects of BA and betulinyl sulfamates on cellular and radiobiological behavior under normoxia and hypoxia were evaluated. The two most effective betulinyl sulfamates CAI 1 and CAI 3 demonstrated a 1.8–2.8-fold higher cytotoxicity than BA under normoxia in breast cancer cells, with IC50 values between 11.1 and 18.1 µM. BA exhibits its strongest cytotoxicity with IC50 values of 8.2 and 16.4 µM under hypoxia. All three substances show a dose-dependent increase in apoptosis, inhibition of migration, and inhibition of hypoxia-induced gene expression. In combination with irradiation, betulinyl sulfamates act as radiosensitizers, with DMF10 values of 1.47 (CAI 1) and 1.75 (CAI 3) under hypoxia in MDA-MB231 cells. BA showed additive effects in combination with irradiation. Taken together, our results suggest that BA and betulinyl sulfamates seem to be attractive substances to combine with radiotherapy, particularly for hypoxic breast cancer.

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (rs = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients’ disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression.

Hypoxia‑induced carbonic anhydrase IX (CAIX) is involved in intracellular and extracellular pH regulation, which is critical for tumor growth and metastasis. CAIX is overexpressed in breast cancer and is associated with the poor survival of patients after radiotherapy. Therefore, we evaluated the cellular and radiobiological effects of CAIX inhibition in human breast cancer cells. We used CA9 siRNA and the CA inhibitor (CAI) U104, respectively, to inhibit CAIX expression and activity in basal triple‑negative MDA‑MB‑231 and luminal MCF‑7 cells under hypoxic conditions. We investigated the effects of CAIX inhibition on CA9 mRNA and CAIX protein level, as well as on CAIX activity, intracellular pH, proliferation, apoptosis, clonogenic survival, migration, cell cycle distribution and radiosensitivity. CA9 siRNA and CAI U104 decreased CA9 mRNA and CAIX protein level in MDA‑MB‑231 and MCF‑7 cells. Furthermore, incubation with CAI U104 significantly decreased carbonic anhydrase activity and reduced the intracellular pH. Additionally, CA9 siRNA or U104 reduced clonogenic survival, migration and the number of cells in the G0/G1 phase, induced apoptosis and demonstrated additive or synergistic effects in combination with irradiation. In conclusion, combination of CAIX inhibition and irradiation is a promising treatment strategy against breast cancer with hypoxia‑induced CAIX expression.

In malignant glioma the presence of the IDH1 mutation (IDH1(R132H)) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy.We determined the influence of IDH1(R132H) mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1(wt)) and mutated IDH1 (IDH1(R132H)).Overexpression of IDH1(R132H) in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1(R132H)-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p0.01 and 1.42, p0.01), U-343MG (DMF10: 1.78, p0.01 and 1.75, p0.01) and LN-229 (DMF10: 1.41, p0.05 and 1.68, p0.01) under normoxia and hypoxia, respectively.Our data indicate that IDH1(R132H) mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy.

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

In this report, we describe the synthesis, characterization, in vitro anticancer activity and Carbonic anhydrase IX (CAIX) inhibition of new sulfamate conjugates of Betulin and Betulinic acid (BA). The betulinyl sulfamates were subjected to inhibit carbonic anhydrases (CA), e.g. CAIX, an attractive target for tumor-selective therapy strategies in cancer cells. Data on combined in vitro antitumor activity with CAIX inhibition are very rare. The betulinyl sulfamates were tested against five tumor cell lines and normal human skin fibroblasts. The mode of cell death on MCF7 breast cancer cells induced by the most active compounds CAI1, CAI3 and CAI6 was investigated by Fluorescence Activated Cell Sorting (FACS) experiments. The compounds showed inhibitory activity towards CAIX, which was determined via in vitro enz-yme assay. Our preliminary investigations revealed that all compounds showed potent anticancer properties with IC50 values below 20 μM against all tumor cells. Interestingly, among the panel of sulfamate conjugates, CAI3 found to be highly cytotoxic (average IC50 = 5–10 μM) and possess high inhibitory activity (Ki = 1.25 nM) towards CAIX. Our results suggest that betulinyl sulfamates seem to be attractive substances, due to their possibility of targeted drug delivery they deserve to be proceeded for further pre-clinical (kinetic studies) and in vivo investigations.

The urokinase plasminogen activator system (uPA, uPAR, PAI‑1) is upregulated in cancer and high plasma levels are associated with poor prognosis. Their interaction with hypoxia-related osteopontin (OPN) which is also overexpressed in malignant tumors suggests potential clinical relevance. However, the prognostic role of the uPA system in the radiotherapy (RT) of non-small-cell lung cancer (NSCLC), particularly in combination with OPN, has not been investigated so far. uPA, uPAR, PAI‑1 and OPN plasma levels of 81 patients with locally advanced or metastasized NSCLC were prospectively analyzed by ELISA before RT and were correlated to clinical patient/tumor data and prognosis after RT. uPAR plasma levels were higher in M1; uPA and PAI‑1 levels were higher in M0 NSCLC patients. uPAR correlated with uPA (p

Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

Betulinic acid (BA), a pentacyclic triterpene, represents a new therapeutic substance that has potential benefits for treating glioblastoma. Recently, new strategies for producing BA derivatives with improved properties have evolved. However, few studies have examined the combination of BA or BA derivatives using radiotherapy. The effects of two BA derivatives, NVX-207 and B10, on cellular and radiobiological behavior were analyzed using glioblastoma cell lines (U251MG, U343MG and LN229). Based on IC50 values under normoxic conditions, we detected a 1.3–2.9-fold higher cytotoxicity of the BA derivatives B10 and NVX-207, respectively, compared to BA. Incubation using both BA derivatives led to decreased cell migration, cleavage of PARP and decreased protein expression levels of Survivin. Weak radiation sensitivity enhancement was observed in U251MG cells after treatment with both BA derivatives. The enhancement factors at an irradiation dose of 6 Gy after treatment with 5 µM NVX-207 and 5 µM B10 were 1.32 (p = 0.029) and 1.55 (p = 0.002), respectively. In contrast to BA, neither NVX-207 nor B10 had additional effects under hypoxic conditions. Our results suggest that the BA derivatives NVX-207 and B10 improve the effects of radiotherapy on human malignant glioma cells, particularly under normoxic conditions.

The epidermal growth factor receptors, EGFR (HER1) and HER2, have proven prognostic relevance in a variety of human malignancies and both are functionally involved in the molecular pathogenesis of malignant gliomas.We selectively inhibited EGFR and HER2 in glioblastoma cell lines via EGFR- and HER2-specific siRNAs and through the binding of the therapeutic antibodies cetuximab and trastuzumab. The expression of EGFR and HER2 was verified by real-time PCR and western blot analyses. We examined the growth rate, cell cycle distribution, cell migration, clonogenic survival, and radiosensitivity of U251MG and LN-229 glioblastoma cell lines to determine the physiological and cell biological effects of EGFR and HER2 targeting.EGFR and HER2 targeting using the therapeutic antibodies cetuximab and trastuzumab had no effect on cellular growth rate, cell cycle distribution, cell migration, clonogenic survival, and radiosensitivity in the cell lines U251 and LN-229. In contrast, siRNA knock-down of EGFR and HER2, reduced the growth rate by 40-65 %. The knock-down of EGFR did not change the cell migration rate in the cell lines U251 and LN-229. However, knock-down of HER2 reduced the cell migration rate by 50 %. Radiobiological analysis revealed that EGFR knock-down induced no radiosensitization in U251MG and LN-229 cells. However, the knock-down of HER2 induced radiosensitization in U251MG cells.The epidermal growth factor receptor HER2 is a promising anti-tumor target for the therapy of glioblastoma. HER2 targeting may represent a promising strategy to induce cell physiological and radiobiological anti-tumor effects in glioblastoma.

Human epidermal growth factor receptor 2 (ErbB2) is overexpressed in a variety of human malignancies. Moreover, ErbB2 has been reported to influence cancer patient survival and progression of different tumor entities. However, information regarding the prognostic impact of ErbB2 in soft tissue sarcoma (STS) patients is limited and conflicting.ErbB2 mRNA and protein levels were defined by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), and the prognostic impact of ErbB2 mRNA and protein levels in tumor tissue of 124 soft tissue sarcoma patients were investigated.The median ErbB2 mRNA expression level in tumor tissue was decreased 3.9-fold compared to non-neoplastic surrounding tissue (p = 0.001). Furthermore, an increased ErbB2 mRNA expression level was associated with an improved tumor-specific survival (p = 0.01, log rank test). Multivariate Cox's proportional hazard regression analyses revealed an increased ErbB2 mRNA expression level as an independent favorable prognostic factor for tumor-specific survival of STS patients (n = 124; RR = 3.0; 95 % CI = 1.6-5.7; p 0.001). In addition, multivariate Cox's proportional hazard regression analyses showed that an increased ErbB2 protein expression level correlated with poorer recurrence-free survival of STS patients (n = 47; RR = 9.9; 95 % CI = 1.7-59.7; p = 0.012), in particular for STS patients who received postoperative radiotherapy (n = 27; RR = 17.9; 95 % CI = 1.3-247.7; p = 0.031).This study suggests an inverse prognostic value of ErbB2 mRNA and protein expression level.

Chemokines are involved in both the negative and positive regulation of inflammatory processes, angiogenesis and cancer/cancer stem cell proliferation as well as the chemoattraction of tumor cells to metastatic sites. The aim of this study was to measure the mRNA expression levels of three chemokines, CCL2, CCL7 and CX3CL1, in soft tissue sarcomas (STSs) and to assess the correlations between these levels as well as their correlations with clinicopathological data and the disease-specific survival of STS patients. The mRNA levels of CCL2, CCL7 and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death as compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) as compared to those with high expression of both parameters in female patients. In conclusion, this is the first study to show a significant correlation between combined low expression of CCL2 and CX3CL1 and a poor prognosis for STS patients, particularly in female patients.

Hypoxic radioresistance plays a critical role in the radiotherapy of cancer and adversely impacts prognosis and treatment response. This prospective study investigated the interrelationship and the prognostic significance of several hypoxia-related proteins in non-small cell lung cancer (NSCLC) patients treated by radiotherapy ± chemotherapy. Pretreatment osteopontin (OPN), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) plasma levels were determined by ELISA in 55 NSCLC (M0) patients receiving 66 Gy curative-intent radiotherapy or chemoradiation. Marker correlation, association with clinicopathological parameters and the prognostic value of a biomarker combination was evaluated. All biomarkers were linearly correlated and linked to different clinical parameters including lung function, weight loss (OPN), gross tumor volume (VEGF) and T stage (CA IX). High OPN (p = 0.03), VEGF (p = 0.02) and CA IX (p = 0.04) values were significantly associated with poor survival. Double marker combination additively increased the risk of death by a factor of 2 and high plasma levels of the triple combination OPN/VEGF/CA IX yielded a 5.9-fold risk of death (p = 0.009). The combined assessment of OPN/VEGF/CA IX correlated independently with prognosis (p = 0.03) in a multivariate Cox regression model including N stage, T stage and GTV. This pilot study suggests that a co-detection augments the prognostic value of single markers and that the integration of OPN, VEGF and CA IX into a hypoxic biomarker profile for the identification of patients with largely hypoxic and radioresistant tumors should be further evaluated.

Head and neck squamous cell carcinoma is the 6th most malignant tumor entity worldwide and has exhibited a 5-year mortality of approximately 50% for the last fifty years. For the therapy monitoring and successful management of this tumor entity new and easily accessible biomarkers are greatly needed. The aim of the study was to determine whether and to what extent microRNAs, a class of small regulatory RNAs, are detectable in saliva post-radiation therapy. The expression and feasibility as therapy monitoring marker of the microRNAs were analyzed by RT-qPCR in 83 saliva samples from 33 patients collected at several time points pre-, during and post-radiotherapy treatment. Ten head and neck squamous cell carcinoma- or radiation-associated microRNAs (miR-93, miR-125a, miR-142-3p, miR-200a, miR-203, miR-213, let-7a, let-7b, let-7g and let-7i) were analyzed. All were detectable to a different extent in the saliva of the patients. miR-93 and miR-200a were significantly higher expressed 12 months post-radiotherapy than at baseline (p=0.047 and p=0.036). These results point towards miR-93 and miR-200a as biomarkers for the treatment monitoring post-radiation of head and neck squamous cell carcinoma.

The prognostic quality of increased osteopontin (OPN) plasma levels has been demonstrated for the chemotherapy and surgery of lung cancer. There is also evidence in the literature that tumor volume impacts prognosis in definitive radiotherapy (RT) of (lung) cancer. We previously demonstrated that elevated plasma levels of OPN before, and increasing OPN plasma levels after RT significantly correlate with survival and outcome after curative-intent RT of non-small-cell lung cancer (NSCLC). Tumor volume was also associated with prognosis. The present prospective clinical study investigated the prognostic interrelation of OPN plasma levels and tumor volume and their changes in the radical RT of NSCLC. We evaluated a subset of patients (n=27) with inoperable, non-metastasized NSCLC of the previously published patient collective. Patients were treated with radical radiochemotherapy (2 Gy ad 66 Gy). OPN plasma concentrations were determined by ELISA before (t0), at the end (t1), and 4 weeks after RT (t2). GTV was delineated PET- and CT-correlated before RT (GTV1) and after 40 Gy (GTV2). The course of OPN during and after RT and the change of GTV during RT was monitored over time and correlated with prognosis. Median GTV2 after 40 Gy (63 ml) was significantly lower than pre-RT GTV1 (90 ml, P

The following information is missing from the Funding section: “This work was supported by a grant of the von Behring-Roentgen-Stiftung to SPG.”

The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1α (HIF1α) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1α has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood. We investigated the conditions for normoxic HIF1α stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR. Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1α in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1α. Additionally, the normoxic HIF1α protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1α was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1α to the same extent as glutamine. Our study suggests that HIF1α is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1α and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions. Therefore, the inhibition of HIF1α (and/or HIF1α target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.

Expression of microRNAs can affect age of tumor onset and prognosis of cancer patients. However, nothing is known about the effects of microRNAs on altered age of cancer onset and disease-specific survival of soft-tissue sarcoma (STS) patients. The levels of miR-210, also known as hypoxia-regulated microRNA, were analyzed by quantitative real-time (RT)-PCR in the tumors of 78 STS patients. The patients were stratified according to their microRNA levels with low, intermediate and high expression levels and the association of microRNA expression and patients' survival was analyzed using multivariate Cox's regression hazard analyses. A significant correlation between an intermediate miR-210 expression and disease-specific death of STS patients [relative risk (RR) = 3.19; p = 0.018] was observed compared with patients with high expression levels in their tumors. Interestingly, the association between an intermediate expression of miR-210 and a poor prognosis was only significant in female STS patients (RR = 11.28; p = 0.010), but not observed in male individuals. Furthermore, the expression of miR-210 showed a significant association with the age of tumor onset in a gender-specific manner. Specifically, male patients with an intermediate expression of miR-210 associated with a 9.6-year later age of tumor onset (p = 0.017) compared with males with a low expression of miR-210 in their tumors. However, no significant differences in the female patients were observed. This study provides the first evidence of a correlation of expression levels of a single microRNA (miR-210) with the prognosis and age of tumor onset in a gender-specific manner in STS patients.

Background: The urokinase plasminogen activator (uPA) system is one of the best-investigated protease systems, both under physiological and pathological conditions, including various types of cancer. However, effects of co-expression of members of the uPA system in soft-tissue sarcoma (STS) patients at the protein level in both tumour tissue and serum have not been investigated yet. Methods: We examined 82 STS patients for protein levels of uPA, PAI-1and uPAR in tumour tissue and serum by ELISA. Results: A significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumour tissue, and of uPAR in serum, with poor outcome of STS patients was found for the first time. Most strikingly, we observed an additive effect of combined uPA, PAI-1 or uPAR levels in tumour tissue extracts with uPAR levels in serum on patients' prognosis. High uPA/uPAR, PAI-1/uPAR and uPAR/uPAR antigen levels in tumour tissue/serum were associated with a 5.9-fold, 5.8-fold and 6.2-fold increased risk of tumour-related death (P=0.003, 0.001 and 0.002, respectively) compared with those patients who displayed low levels of the respective marker combination. Conclusion: As expression of members of the uPA system in tumour tissue and serum is additively correlated with prognosis of STS patients, our results suggest that combinations of these biomarkers can identify STS patients with a higher risk of tumour-related death.

The p53 stress response is crucial for the prevention of tumor formation. The oncogene HDM2 is one of the key negative regulators of p53 and is a central node in the p53 pathway. P53 and HDM2 form an oscillating feedback loop. HDM2 expression is regulated by different promoters. To evaluate its clinical relevance, we determined the levels of HDM2 transcripts originating from the constitutive P1 and p53-sensitive P2 promoter in 133 soft tissue sarcomas and correlated the results with the age of diagnosis and the patients' outcome. We show that only high levels of the HDM2-P1 transcript but not the P2 transcript are associated with an 11-year earlier age of onset (50.5 years) compared with low P1 levels (61.5 years; P < 0.0001, t test). In addition, low P1 and P2 mRNA expression levels were independent predictors of poor outcome for patients with soft tissue sarcomas (low P1: relative risk, 3.7; P < 0.0001; low P2: relative risk, 2.5; P = 0.001). A change in the expression levels of the HDM2 transcripts originating from the two HDM2 promoters could disrupt the oscillating P53-HDM2 feedback loop in a way that elevated levels of HDM2-P1 transcript are associated with an earlier age of tumor onset and that reduced levels of HDM2-P1 or HDM2-P2 transcripts are correlated with poor prognosis of patients with soft tissue sarcomas. (Mol Cancer Res 2008;6(10):1575–81)

Poor oxygenation of solid tumors is a major indicator of adverse prognosis after standard treatment, e.g. radiotherapy. This observation founded on intratumoral pO(2) electrode measurements has been supported more recently by studies of injected hypoxia markers (pimonidazole, EF5) or hypoxia-related proteins (hypoxia-inducible factor-1alpha, carbonic anhydrase IX) detected immunohistochemically. Alternative approaches include imaging of tumor hypoxia by nuclear medicine studies and the measurement of hypoxia-related proteins (osteopontin) in patient plasma. Low oxygen levels as found in tumors are rarely observed in normal tissues. The presence of hypoxic tumor cells is therefore regarded not only as an adverse prognostic factor but as an opportunity for tumor-specific treatment. Classic approaches to normalize tumor oxygenation involve the breathing of modified gas mixtures and pharmacologic modification of blood flow as in the "accelerated radiotherapy, carbogen, nicotinamide" (ARCON) scheme. Specific killing of hypoxic tumor cells can potentially be achieved by hypoxia-selective cytotoxins (model substance tirapazamine), which has shown promise in head and neck cancer. Direct targeting of hypoxia-related molecules such as hypoxia-inducible factor-1alpha, the central regulator of the hypoxic response in tumor cells, is an attractive approach currently tested in preclinical models. For clinical applications, the appropriate combination of hypoxia detection for patient selection with a hypoxia-specific treatment is essential. A therapeutic benefit has been suggested for the selection of patients by plasma osteopontin level and treatment with the hypoxic radiosensitizer nimorazole in addition to radiotherapy, for selection by F-misonidazole positron-emission tomography (PET) and treatment with tirapazamine in addition to chemoradiation and for selection by pimonidazole immunohistochemistry and ARCON treatment, all in head and neck cancer.