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High-resolution measurements of stem radius variations provide information about the tree water status with changing climate conditions by swelling and shrinking due to the reduction of xylem water potential and to the exceedance of leaf transpiration over root water uptake. The aim of this study was to analyze daily stem radius variations of Norway spruce and European beech in intra- and interspecific neighborhood. The experimental plots are part of a rainfall exclusion experiment. These variations are species-specific, i.e. spruces have a higher phloem thickness and higher amplitudes during a day than beeches. The amplitudes were significantly higher at the rainfall exclusion plots, but the amplitudes of spruces decreased above 27°C with increasing drought due to reduced transpiration rates and exhausted soil water reserves. The shrinking amplitude was observed for spruces in intraspecific neighborhood from a soil volumetric water content of 0.21 m3 m−3. In interspecific neighborhood, a shrinkin...

Current and future climate change will be accompanied by more frequent and more severe drought events, with potential impacts on tree growth and forest stand productivity. Tree growth response may depend on its water status and on the competition or facilitation of the neighbouring trees. We analysed inter- and intra-annual diameter increments of European beech and Norway spruce trees within a mixed forest stand for two treatments, i.e., with and without rainfall exclusion and for two neighbourhood competition situations of the two climatically contrasting years 2014 and 2015. Rainfall exclusion by roofs at about 3 m height induced soil drought under 116 trees, particularly supported in the year 2015 by hot and dry weather conditions. The effects of extreme drought was examined at three levels, i.e. at two stem heights and at the main coarse root of 48 trees with inter- and intraspecific neighbourhood. We found species specific diameter growth performances during the year at all three levels. Compared to beech trees annual diameter increments of spruce trees were in most cases significantly higher in the year 2014, while in the drought year 2015 no significant differences between the two species were obvious. Under the heavy drought by the rainfall exclusion experiment and by the hot year 2015 diameter increments were significantly smaller for spruce trees at all three levels whereas for beech trees at all levels no significant differences were obvious. The diameter growth differences between inter- and intraspecific neighbourhood were in most cases small and not significant. In the drought year 2015 beech trees in intraspecific neighbourhood grew better at all levels compared to interspecific neighbourhood, with a significantly higher growth rate at the upper stem level. Reasons for the species specific reactions patterns on drought were discussed (phenology, water balance, species specific physiological reactions).

Although several studies suggest that tree species in mixed stands resist drought events better than in pure stands, little is known about the impact on growth and the tree water deficit (TWD) in different tree heights at heavy drought. With dendrometer data at the upper and lower stem and coarse roots, we calculated the TWD and growth (ZGmax) (referring to the stem/root basal area) to show (1) the relationship of TWD in different tree heights (50% tree height (H50), breast height (BH), and roots) and the corresponding leaf water potential and (2) how mixture and drought influence the partitioning of growth and tree water. The analyses were made in a mature temperate forest of Norway spruce (Picea abies (L.) Karst.) and European beech (Fagus sylvatica (L.)). Half of the plots were placed under conditions of extreme drought through automatic closing roof systems within the stand. We found a tight relationship of leaf water potentials and TWD at all tree compartments. Through this proven correlation at all tree heights we were also able to study the differences of TWD in all tree compartments next to the growth allocation. Whereas at the beginning of the growing period, trees prioritized growth of the upper stem, during the course of the year the growth of lower stem became a greater priority. Growth allocation of mixed spruces showed a tendency of a higher growth of the roots compared to the BH. However, spruces in interspecific neighborhoods exhibited a lesser TWD in the roots as spruces in intraspecific neighborhood. Beeches in intraspecific neighborhoods showed a higher TWD in BH compared to H50 as beeches in interspecific neighborhoods. Mixture seems to enhance the water supply of spruce trees, which should increase the stability of this species in a time of climatic warming.

Forest ecosystems in central Europe are predicted to face an increasing frequency and severity of summer droughts because of global climate change. European beech and Norway spruce often coexist in these forests with mostly positive effects on their growth. However, their different below-ground responses to drought may lead to differences in ectomycorrhizal (ECM) fungal community composition and functions which we examined at the individual root and ecosystem levels. We installed retractable roofs over plots in Kranzberg Forest (11 degrees 39'42 '' E, 48 degrees 25'12 '' N; 490 m a.s.l.) to impose repeated summer drought conditions and assigned zones within each plot where trees neighboured the same or different species to study mixed species effects. We found that ECM fungal community composition changed and the numbers of vital mycorrhizae decreased for both tree species over 3 drought years (2014-2016), with the ECM fungal community diversity of beech exhibiting a faster and of spruce a stronger decline. Mixed stands had a positive effect on the ECM fungal community diversity of both tree species after the third drought year. Ectomycorrhizae with long rhizomorphs increased in both species under drought, indicating long-distance water transport. However, there was a progressive decline in the number of vital fine roots during the experiment, resulting in a strong reduction in enzyme activity per unit volume of soil. Hydrolytic enzyme activities of the surviving ectomycorrhizae were stable or stimulated upon drought, but there was a large decline in ECM fungal species with laccase activity, indicating a decreased potential to exploit nutrients bound to phenolic compounds. Thus, the ectomycorrhizae responded to repeated drought by maintaining or increasing their functionality at the individual root level, but were unable to compensate for quantitative losses at the ecosystem level. These findings demonstrate a strong below-ground impact of recurrent drought events in forests.

Tree rings include retrospective information about the relationship between climate and growth, making it possible to predict growth reaction under changing climate. Previous studies examined species-specific reactions under different environmental conditions from the perspective of tree ring growth and 13C discrimination (Δ13C). This approach is extended to monospecific versus mixed stands in the present paper. We investigated the resistance and resilience of Norway spruce (Picea abies [L.] Karst) and European beech (Fagus sylvatica [L.]) in response to the drought event in 2003. The study was carried out along a precipitation gradient in southern Germany. Responses of basal area increment (BAI) and Δ13C were correlated with a Climate-Vegetation-Productivity-Index (CVPI). The species showed different strategies for coping with drought stress. During the summer drought of 2003, the BAI of spruces reveal a lower resistance to drought on dry sites than those of beech. For beech, we found an increasing resistance in BAI and Δ13C from dry to moist sites. In mixture with spruce, beech had higher resistance and resilience for Δ13C with increasing site moisture. The combination of Δ13C and tree ring growth proxies improves our knowledge of species-specific and mixture-specific reactions to drought for sites with different moisture conditions.

Tree rings include retrospective information about the relationship between climate and growth, making it possible to predict growth reaction under changing climate. Previous studies examined species-specific reactions under different environmental conditions from the perspective of tree ring growth and 13C discrimination (Δ13C). This approach is extended to monospecific versus mixed stands in the present paper. We investigated the resistance and resilience of Norway spruce (Picea abies [L.] Karst) and European beech (Fagus sylvatica [L.]) in response to the drought event in 2003. The study was carried out along a precipitation gradient in southern Germany. Responses of basal area increment (BAI) and Δ13C were correlated with a Climate-Vegetation-Productivity-Index (CVPI). The species showed different strategies for coping with drought stress. During the summer drought of 2003, the BAI of spruces reveal a lower resistance to drought on dry sites than those of beech. For beech, we found an increasing resistance in BAI and Δ13C from dry to moist sites. In mixture with spruce, beech had higher resistance and resilience for Δ13C with increasing site moisture. The combination of Δ13C and tree ring growth proxies improves our knowledge of species-specific and mixture-specific reactions to drought for sites with different moisture conditions.

Bone metastases are a frequent cause of morbidity in cancer patients. The present palliative therapeutic options are chemotherapy, hormone therapy, and the administration of bisphosphonates. The affinity between bisphosphonates and the apatite structure of bone metastases is strong. Thus, we designed two low-molecular-weight and water-soluble prodrugs which incorporate a bisphosphonate group as a bone targeting ligand, doxorubicin as the anticancer agent, and either an acid-sensitive bond (1) or a cathepsin B cleavable bond (3) for ensuring an effective release of doxorubicin at the site of action. Cleavage studies of both prodrugs showed a fast release of doxorubicin but sufficient stability over several hours in human plasma. Effective binding of prodrug 1 and 3 was demonstrated with hydroxyapatite and with native bone. In orientating toxicity studies in nude mice, the MTD of 1 was 3-fold higher compared to conventional doxorubicin, whereas 3 showed essentially the same MTD as doxorubicin.

PSA, which is overexpressed in prostate carcinoma, represents a molecular target for selectively releasing an anticancer agent from a prodrug formulation. In this study, we report on the in vivo antitumor efficacy of an efficacious albumin-binding prodrug of doxorubicin (PSA9) that incorporates p-aminobenzyloxycarbonyl (PABC) as a self-immolative spacer in addition to the heptapeptide, Arg-Ser-Ser-Tyr-Tyr-Ser-Leu, which serves as a substrate for PSA. The prodrug is cleaved very efficiently by PSA releasing H-Ser-Leu-PABC-doxorubicin and subsequently doxorubicin in PSA-positive cell lysates and prostate tumor homogenates as the final cleavage product. PSA9 at 3 × 6 mg kg(-1) doxorubicin equivalents (intravenous) was compared with conventional doxorubicin at equitoxic doses (at 3 × 3 mg kg(-1); intravenous) in an orthotopic mouse model of prostate cancer using LNCaP lentiviral luciferase-neomycin cells transduced with luciferase. Whereas doxorubicin did not show any efficacy against the primary tumor or metastases, the prodrug reduced the primary tumor by 30-50% and circulating PSA levels, and in addition, showed a pronounced reduction in lung and bone metastases by ∼77% and ∼96%, respectively, and a positive trend regarding the activity against liver and lymph-node metastases compared with control and doxorubicin-treated animals. The incorporation of PABC as a self-immolative spacer together with a PSA substrate demonstrates superior antitumor effects over doxorubicin attributed to an efficient cleavage by PSA releasing doxorubicin as the final active agent in prostate tumor homogenates. Using this approach for developing effective prodrugs against prostate cancer, is worthy of further preclinical optimization.

In developed countries, prostate cancer is the third most common cause of death from cancer in men. Unfortunately, whilst accumulating clinical data have suggested that taxanes may prolong the survival in a subset of men with prostate carcinoma, the dose and duration of administration of these drugs are limited by their significant systemic toxicities due to a lack of tumour selectivity. In an attempt to improve both the water solubility and tumour-targeting properties of paclitaxel (Taxol®), we set out to develop a water soluble paclitaxel prodrug that is activated specifically by prostate-specific antigen (PSA) which is almost exclusively expressed in prostate tissue and prostate carcinoma making it an ideal molecular target for prodrug strategies. Using albumin as a drug carrier, we describe a novel albumin-binding prodrug of paclitaxel, EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-Leu-PABC-paclitaxel [EMC: e-maleimidocaproyl; PABC: p-aminobenzyloxycarbonyl] that was synthesised by reacting EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-OH with H-Leu-PABC-paclitaxel. This prodrug was water soluble and was bound to endogenous and exogenous albumin. Moreover, incubation studies with PSA demonstrated that the albumin-bound form of the prodrug was cleaved rapidly at the P1-P1' scissile bond releasing the paclitaxel-dipeptide H-Ser-Leu-PABC-paclitaxel. Last but not least, due to the incorporation of a PABC self-eliminating linker, this dipeptide was rapidly degraded to liberate paclitaxel as a final cleavage product within a few hours in prostate tumour tissue homogenates. Of note was that the albumin-bound form of the prodrug was approximately three-fold more active in killing PSA-positive LNCaP cells than paclitaxel. In addition, orientating toxicity studies in mice showed that the maximum tolerated dose of the novel paclitaxel prodrug was twice that of conventional paclitaxel. When tested in vivo in an orthotopic mouse model of human prostate cancer using luciferase-transduced LNCaP LLN cells, both paclitaxel and the new paclitaxel prodrug showed distinct antitumour efficacy on the primary tumour and metastases that was significantly better than the effect of doxorubicin which was used as a comparison and showed no antitumour efficacy. The new paclitaxel prodrug (3 × 24 mg paclitaxel equivalents) showed comparable antitumour activity on the primary tumour to paclitaxel at its maximum-tolerated dose (3 × 12mg/kg), reduced circulating PSA levels and demonstrated a better antitumour effect on lung metastases but not on bone metastases.

Bone metastases occur in up to 80 % of patients with advanced breast or prostate cancer and significantly affect the quality of life. Most frequently, bone metastases cause bone fractures, severe pain and hypercalcaemia. Current options for treating of bone metastases are surgery, radiotherapy, chemotherapy, hormone therapy, and the administration of bisphosphonates. These treatments are primarily palliative and can improve the quality of life in some cases, however without increasing the survival time of the patients. Bisphosphonates are presently the most active inhibitors of bone degradation, and they can reduce bone pain, bone destruction and tumor growth. Their therapeutic effects are due to the high affinity between bisphosphonates and the apatite structure of bone and bone metastases. In order to improve the outcome of treating bone metastases, new therapeutic approaches are urgently required. As bone-seeking agents, bisphosphonates show an uptake in the bone of 20-60% of the applied dose, and in general the uptake in bone metastases is 10-20-fold higher than in healthy bone tissue. In addition, bone metastases are characterized by an acidic pH and the over-expression of several proteases including cathepsins, matrix metalloproteases, plasmin and uPA. Surprisingly, no prodrug approaches have been reported to date that exploit the bone targeting effect of low-molecular weight bisphosphonates with a concomitant release of a bound drug in bone metastases. To this purpose, we developed water-soluble bisphosphonate prodrugs of doxorubicin for targeting bone metastases by conjugating a thiol-bearing aliphatic bisphosphonate with the 6-maleimidocaproyl hydrazone derivative of doxorubicin (1) that is cleaved pH dependently, or by attaching a maleimide derivative of the dipeptide Val-Ala-PABC (PABC = p-benzyloxycarbonyl) to the aliphatic bisphosphonate (2). Incubation studies with cathepsin B and 2 at 37 °C showed a cleavage of doxorubicin after two hours with a half time of ∼ 45 min and 1 released doxorubicin quantitatively at pH 5.0 after 2 h. The plasma stabilities (human and mouse) of both prodrugs were in the range of 3-6 h at 37 °C. Binding studies of 1 and 2 with a major component of bone, i.e. hydroxyapatite, demonstrated binding of 80-90 % of both prodrugs after 4 h at 37 °C. Finally, in an orientating toxicity study in nude mice the maximum tolerated dose (MTD) of 1 was three-fold higher compared to conventional doxorubicin whereas 2 showed essentially the same MTD as doxorubicin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1760. doi:1538-7445.AM2012-1760