Claus Belka

Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-xL prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

Celecoxib activates a novel mitochondrial apoptosis signaling pathway

The cyclooxygenase (COX)‐2 inhibitor Celecoxib may inhibit cancer cell growth independently of its capacity to block the COX‐2 enzyme. The growth inhibitory effect had been attributed to its pro‐apoptotic effects. However, the molecular details of Celecoxib‐induced apoptosis have not been analyzed yet. To differentiate between death receptor and mitochondrial signaling pathways, induction of apoptosis upon treatment with Celecoxib was tested in Jurkat T‐ and BJAB B‐lymphoma cell lines with defects in either pathway. Celecoxib‐induced dose‐ and time‐dependent apoptosis in Jurkat and BJAB cells involving i) activation of caspases‐9, ‐8, and ‐3, ii) cleavage of poly(ADP‐ribose) polymerase and inhibitor of caspase‐activated DNAase, iii) breakdown of the mitochondrial membrane potential, and iv) release of cytochrome c. Lack of Fas‐associated death domain protein (FADD), overexpression of a dominant negative FADD, lack of caspase‐8, and treatment with caspase‐8‐specific inhibitors had no influence on Celecoxib‐induced apoptosis. In contrast, overexpression of a dominant negative caspase‐9 or pharmacological inhibition of caspase‐9 strongly interfered with Celecoxib‐induced cell death. Furthermore, expression of Apaf‐1 was required for Celecoxib‐induced apoptosis. Importantly, Bcl‐2 overexpression did not abrogate caspase activation, mitochondrial alterations, and apoptosis upon Celecoxib treatment while inhibiting radiation induced apoptosis. In conclusion, Celecoxib induces apoptosis via a novel apoptosome‐dependent but Bcl‐2‐independent mitochondrial pathway.

Irradiation and Bevacizumab in High-Grade Glioma Retreatment Settings

PURPOSE Reirradiation is a treatment option for recurrent high-grade glioma with proven but limited effectiveness. Therapies directed against vascular endothelial growth factor have been shown to exert certain efficacy in combination with chemotherapy and have been safely tested in combination with radiotherapy in a small cohort of patients. To study the feasibility of reirradiation combined with bevacizumab treatment, the toxicity and treatment outcomes of this approach were analyzed retrospectively. PATIENTS AND METHODS After previous treatment with standard radiotherapy (with or without temozolomide) patients with recurrent malignant glioma received bevacizumab (10 mg/kg intravenous) on Day 1 and Day 15 during radiotherapy. Maintenance therapy was selected based on individual considerations, and mainly bevacizumab-containing regimens were chosen. Patients received 36 Gy in 18 fractions. RESULTS The data of the medical charts of the 30 patients were analyzed retrospectively. All were irradiated in a single institution and received either bevacizumab (n = 20), no additional substance (n = 7), or temozolomide (n = 3). Reirradiation was tolerated well, regardless of the added drug. In 1 patient treated with bevacizumab, a wound dehiscence occurred. Overall survival was significantly better in patients receiving bevacizumab (p = 0.03, log-rank test). In a multivariate proportional hazards Cox model, bevacizumab, Karnovsky performance status, and World Health Organization grade at relapse turned out to be the most important predictors for overall survival. CONCLUSION Reirradiation with bevacizumab is a feasible and effective treatment for patients with recurrent high-grade gliomas. A randomized trial is warranted to finally answer the question whether bevacizumab adds substantial benefit to a radiotherapeutic retreatment setting.

FET-PET for malignant glioma treatment planning.

BACKGROUND AND PURPOSE The aim of this study was to compare MRI-based morphological gross tumour volumes (GTVs) to biological tumour volumes (BTVs), defined by the pathological radiotracer uptake in positron emission tomography (PET) imaging with (18)F-fluoroethyltyrosine (FET), subsequently clinical target volumes (CTVs) and finally planning target volumes (PTVs) for radiotherapy planning of glioblastoma. PATIENTS AND METHODS Seventeen patients with glioblastoma were included into a retrospective protocol. Treatment-planning was performed using clinical target volume (CTV=BTV+20mm or CTV=GTV+20mm+inclusion of the edema) and planning target volume (PTV=CTV+5mm). Image fusion and target volume delineation were performed with OTP-Masterplan®. Initial gross tumour volume (GTV) definition was based on MRI data only or FET-PET data only (BTV), secondarily both data sets were used to define a common CTV. RESULTS FET based BTVs (median 43.9 cm(3)) were larger than corresponding GTVs (median 34.1cm(3), p=0.028), in 11 of 17 cases there were major differences between GTV/BTV. To evaluate the conformity of both planning methods, the index (CTV(MRT)∩CTV(FET))/(CTV(MRT)∪CTV(FET)) was quantified which was significantly different from 1 (0.73 ± 0.03, p<0.001). CONCLUSION With FET-PET-CT planning, the size and geometrical location of GTVs/BTVs differed in a majority of patients. It remains open whether FET-PET-based target definition has a relevant clinical impact for treatment planning.

Counting colonies of clonogenic assays by using densitometric software

Clonogenic assays are a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumour cells. A colony is defined as a cluster of at least 50 cells which can often only be determined microscopically. The process of counting colonies is very extensive work and so we developed software that is able to count the colonies automatically from scanned flasks. This software is made freely available by us with a detailed description how to use and install the necessary features.

Type I and type II reactions in TRAIL-induced apoptosis – results from dose–response studies

Death receptor-induced apoptosis is paradigmatically mediated via the recruitment of FADD adapter molecule to the ligand/receptor complex and subsequent activation of caspase-8. However, several observations provided evidence that components of the mitochondrial apoptosis pathway are involved in death receptor-mediated apoptosis. In this regard, caspase-8-mediated activation of Bid induces the release of cytochrome c from the mitochondria, which, in turn, triggers the formation of the apoptosome protein complex, resulting in the activation of caspase-9. Whereas Bax or Bak were shown to be required for the proapoptotic effect of Bid, Bcl-2 was described to interfere with its action. Up to now, contradictory results regarding the role of Bcl-2 in TRAIL-induced apoptosis have been published. In order to study the influence of Bcl-2 on TRAIL-induced cell death more detailed, we utilized a tetracycline-regulated Bcl-2 expression system in Jurkat T cells. After having analysed the dose response for TRAIL-induced activation of caspase-8, -9, -3, breakdown of the mitochondrial membrane potential, and changes in the apoptotic morphology in cells expressing different Bcl-2 levels, we conclude that overexpression of Bcl-2 mediates a partial resistance towards lower doses of TRAIL that can be overcome when higher doses of TRAIL are applied. Thus, the requirement of the mitochondrial pathway for death receptor-induced apoptosis in type II cells should be reconsidered, since the protective effect of Bcl-2 is limited to lower TRAIL doses or early observation time points.

Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

Distribution of Prostate Sentinel Nodes: A SPECT-Derived Anatomic Atlas

PURPOSE The randomized Radiation Therapy Oncology Group 94-13 trial revealed that coverage of the pelvic lymph nodes in high-risk prostate cancer confers an advantage (progression-free survival and biochemical failure) in patients with ≥15% risk of lymph node involvement. To facilitate an improved definition of the adjuvant target volume, precise knowledge regarding the location of the relevant lymph nodes is necessary. Therefore, we generated a three-dimensional sentinel lymph node atlas. METHODS AND MATERIALS In 61 patients with high-risk prostate cancer, a three-dimensional visualization of sentinel lymph nodes was performed using a single photon emission computed tomography system after transrectal intraprostatic injection of 150 to 362 (median 295) mega becquerel (MBq) (99m)Technetium-nanocolloid (1.5-3 h after injection) followed by an anatomic functional image fusion. RESULTS In all, 324 sentinel nodes in 59 of 61 patients (96.7%) were detected, with 0 to 13 nodes per patient (median 5, mean 5.3). The anatomic distribution of the sentinel nodes was as follows: external iliac 34.3%, internal iliac 17.9%, common iliac 12.7%, sacral 8.6%, perirectal 6.2%, left paraaortic 5.3%, right paraaortic 5.3%, seminal vesicle lymphatic plexus 3.1%, deep inguinal 1.5%, superior rectal 1.2%, internal pudendal 1.2%, perivesical 0.9%, inferior rectal 0.9%, retroaortic 0.3%, superficial inguinal 0.3%, and periprostatic 0.3%. CONCLUSIONS The distribution of sentinel nodes as detected by single photon emission computed tomography imaging correlates well with the distribution determined by intraoperative gamma probe detection. A lower detection rate of sentinels in close proximity to the bladder and seminal vesicles is probably caused by the radionuclide accumulation in the bladder. In regard to intensity-modulated radiotherapy techniques, the presented anatomic atlas may allow optimized target volume definitions.

Dying cell clearance and its impact on the outcome of tumor radiotherapy

The induction of tumor cell death is one of the major goals of radiotherapy and has been considered to be the central determinant of its therapeutic outcome for a long time. However, accumulating evidence suggests that the success of radiotherapy does not only derive from direct cytotoxic effects on the tumor cells alone, but instead might also depend – at least in part – on innate as well as adaptive immune responses, which can particularly target tumor cells that survive local irradiation. The clearance of dying tumor cells by phagocytic cells of the innate immune system represents a crucial step in this scenario. Dendritic cells and macrophages, which engulf, process and present dying tumor cell material to adaptive immune cells, can trigger, skew, or inhibit adaptive immune responses, respectively. In this review we summarize the current knowledge of different forms of cell death induced by ionizing radiation, the multi-step process of dying cell clearance, and its immunological consequences with special regard toward the potential exploitation of these mechanisms for the improvement of tumor radiotherapy.

Phase III Study of Surgery Versus Definitive Concurrent Chemoradiotherapy Boost in Patients With Resectable Stage IIIA(N2) and Selected IIIB Non-Small-Cell Lung Cancer After Induction Chemotherapy and Concurrent Chemoradiotherapy (ESPATUE)

PURPOSE Concurrent chemoradiotherapy with or without surgery are options for stage IIIA(N2) non-small-cell lung cancer. Our previous phase II study had shown the efficacy of induction chemotherapy followed by chemoradiotherapy and surgery in patients with IIIA(N2) disease and with selected IIIB disease. Here, we compared surgery with definitive chemoradiotherapy in resectable stage III disease after induction. PATIENTS AND METHODS Patients with pathologically proven IIIA(N2) and selected patients with IIIB disease that had medical/functional operability received induction chemotherapy, which consisted of three cycles of cisplatin 50 mg/m(2) on days 1 and 8 and paclitaxel 175 mg/m(2) on day 1 every 21 days, as well as concurrent chemoradiotherapy to 45 Gy given as 1.5 Gy twice daily, concurrent cisplatin 50 mg/m(2) on days 2 and 9, and concurrent vinorelbine 20 mg/m(2) on days 2 and 9. Those patients whose tumors were reevaluated and deemed resectable in the last week of radiotherapy were randomly assigned to receive a chemoradiotherapy boost that was risk adapted to between 65 and 71 Gy in arm A or to undergo surgery (arm B). The primary end point was overall survival (OS). RESULTS After 246 of 500 planned patients were enrolled, the trial was closed after the second scheduled interim analysis because of slow accrual and the end of funding, which left the study underpowered relative to its primary study end point. Seventy-five patients had stage IIIA disease and 171 had stage IIIB disease according to the Union for International Cancer Control TNM classification, sixth edition. The median age was 59 years (range, 33 to 74 years). After induction, 161 (65.4%) of 246 patients with resectable tumors were randomly assigned; strata were tumor-node group, prophylactic cranial irradiation policy, and region. Patient characteristics were balanced between arms, in which 81 were assigned to surgery and 80 were assigned to a chemoradiotherapy boost. In arm B, 81% underwent R0 resection. With a median follow-up after random assignment of 78 months, 5-year OS and progression-free survival (PFS) did not differ between arms. Results were OS rates of 44% for arm B and 40% for arm A (log-rank P = .34) and PFS rates of 32% for arm B and 35% for arm A (log-rank P = .75). OS at 5 years was 34.1% (95% CI, 27.6% to 40.8%) in all 246 patients, and 216 patients (87.8%) received definitive local treatment. CONCLUSION The 5-year OS and PFS rates in randomly assigned patients with resectable stage III non-small-cell lung cancer were excellent with both treatments. Both are acceptable strategies for this good-prognosis group.