Preoperative treatment of rectal cancer with radiation, chemotherapy and hyperthermia: analysis of treatment efficacy and heat-shock response

Preoperative treatment of locally advanced rectal cancer with radiation, chemotherapy and hyperthermia is analyzed with regard to heat-shock response. In 23 patients with locally advanced rectal cancer (uT3/uT4), hyperthermia was administered in combination with radiotherapy and chemotherapy. In parallel, the effect of the treatment on levels of the heat-shock proteins HSP27 and inducible HSP70 in tumors and surrounding tissues was investigated by Western blotting. The patients' sera were also examined for autoantibodies against HSPs. HSP27 and inducible HSP70 were detected in most rectal tumors and surrounding tissues before and after treatment. HSP27 and inducible HSP70 levels had changed in 10 tumors after treatment. However, prior to treatment, there existed an unexpected diversity in HSP levels in the tumors and surrounding tissue. Hyperthermia doses in cumulative minutes for which 90% of the tumor is above the reference temperature (cum min T90 > or = 15 min) led to increased survival and response compared to that of a control group of patients treated without or with low-dose hyperthermia (cum min T90 < 15 min). However, there was no correlation to different expression of the HSPs. Hyperthermia as used in this setting does not lead to any sustained expression of HSPs in either the tumor or the surrounding tissue.     

Proteasome inhibition induces <Emphasis Type="Italic">hsp30</Emphasis> and <Emphasis Type="Italic">hsp70</Emphasis> gene expression as well as the acquisition of thermotolerance in <Emphasis Type="Italic">Xenopus laevis</Emphasis> A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.     

Conjugated linoleic acid isomers have differential effects on triglyceride secretion in Hep G2 cells

Treatment for 2 h with 200 microM cadmium chloride, followed by recovery, caused apoptosis and induced heat-shock protein 70 (HSP70) expression in U-937 promonocytic cells. However, pre-incubation with the GSH depleting agent L-buthionine-[S,R]-sulfoximine (BSO, 1 mM for 24 h) caused necrosis instead of apoptosis and failed to induce HSP70 expression. This failure was a consequence of necrosis instead of GSH depletion, since BSO allowed or even potentiated HSP70 induction when used in combination with heat shock (2 h at 42.5 degrees C) or with 50 microM cadmium, which caused apoptosis. The administration of N-acetyl-L-cysteine (NAC) at the beginning of recovery after BSO/200 microM cadmium treatment prevented the execution of necrosis and restored the execution of apoptosis, but did not restore HSP70 induction, indicating that the inhibition by BSO of HSP70 expression is an early regulated event. This contrasted with the capacity of NAC to prevent the alterations caused by BSO/200 microM cadmium in other proteins, namely the suppression of Bax expression and the increase in Bcl-2 and HSP-60 expression. Finally, it was observed that treatment with 200 microM cadmium rapidly increased the HSP70 mRNA level and stimulated heat-shock factor 1 (HSF1) trimerization and binding, and that these effects were prevented by pre-incubation with BSO. Taken together, these results indicate that the stress response is compatible with apoptosis but not with necrosis in cadmium-treated promonocytic cells. The suppression of the stress response is specifically due to the early inhibition of HSF1 activation.     

Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release

Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37 degrees C, 42 degrees C, or 45 degrees C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 +/- 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42 degrees C. We also observed a 7.7 +/- 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45 degrees C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42 degrees C. Heat shock in rats (core temperature: 42 degrees C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.     

Proteomic expression analysis of cardiomyocytes subjected to proteasome inhibition

We hypothesized that impaired proteasomal function affects gene expression in cardiomyocytes. To identify those genes, a proteomics-based analysis of neonatal rat cardiac myocytes treated with the proteasome inhibitor MG132 in comparison to vehicle treated control cells was performed. MG132 treatment induced reproducible changes in the protein expression profile, which was analyzed by two-dimensional difference gel electrophoresis followed by tryptic peptide mass fingerprinting for spot identification by MALDI-TOF mass spectrometry. The identified protein alterations could be grouped into three major categories: (1) induction of small heat shock proteins (HSPs) with chaperonic function, such as HSP27, alphaB-crystallin, and cardiovascular HSP, (2) altered expression of actin associated proteins, such as cofilin-1 and transgelin, and (3) induction of antioxidant proteins, such as peroxiredoxin-1, superoxide dismutase-1, and hemeoxygenase-1. Northern blotting revealed that expression was regulated at the mRNA level. Given that proteasomal activity is decreased in cardiovascular diseases, alterations in proteasome-dependent control of mRNA expression could provide a novel mechanism by which disease progression is modulated.     

Electromagnetic fields at mobile phone frequency induce apoptosis and inactivation of the multi-chaperone complex in human epidermoid cancer cells

The exposure to non-thermal microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004). On these basis we have evaluated the in vivo effect of MW-EMF in human epidermoid cancer KB cells. We have found that MW-EMF induces time-dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5-fold decrease of the expression of ras and Raf-1 and of the activity of ras and Erk-1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5-fold increase of the ubiquitination of ras and Raf-1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 microM caused an accumulation of the ubiquitinated isoforms of ras and Raf-1 and counteracted the effects of MW-EMF on ras and Raf-1 expression suggesting an increased proteasome-dependent degradation induced by MW-EMF. The exposure of KB cells to MW-EMF induced a differential activation of stress-dependent pathway with an increase of JNK-1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras --> Erk-dependent survival signal induced by MW-EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek-1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW-EMF. In conclusion, these results demonstrate for the first time that MW-EMF induces apoptosis through the inactivation of the ras --> Erk survival signaling due to enhanced degradation of ras and Raf-1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.     

Maximal eccentric exercise induces a rapid accumulation of small heat shock proteins on myofibrils and a delayed HSP70 response in humans

In this study the stress protein response to unaccustomed maximal eccentric exercise in humans was investigated. Eleven healthy males performed 300 maximal eccentric actions with the quadriceps muscle. Biopsies from vastus lateralis were collected at 30 min and 4, 8, 24, 96, and 168 h after exercise. Cellular regulation and localization of heat shock protein (HSP) 27, alpha B-crystallin, and HSP70 were analyzed by immunohistochemistry, ELISA technique, and Western blotting. Additionally, mRNA levels of HSP27, alpha B-crystallin, and HSP70 were quantified by Northern blotting. After exercise (30 min), 81 +/- 8% of the myofibers showed strong HSP27 staining (P < 0.01) that gradually decreased during the following week. alpha B-Crystallin mimicked the changes observed in HSP27. After exercise (30 min), the ELISA analysis showed a 49 +/- 13% reduction of the HSP27 level in the cytosolic fraction (P < 0.01), whereas Western blotting revealed a 15-fold increase of the HSP27 level in the myofibrillar fraction (P < 0.01). The cytosolic HSP70 level increased to 203 +/- 37% of the control level 24 h after exercise (P < 0.05). After 4 days, myofibrillar-bound HSP70 had increased approximately 10-fold (P < 0.01) and was accompanied by strong staining on cross sections. mRNA levels of HSP27, alpha B-crystallin, and HSP70 were all elevated the first day after exercise (P < 0.01); HSP70 mRNA showed the largest increase (20-fold at 8 h). HSP27 and alpha B-crystallin seemed to respond immediately to maximal eccentric exercise by binding to cytoskeletal/myofibrillar proteins, probably to function as stabilizers of disrupted myofibrillar structures. Later, mRNA and total HSP protein levels, especially HSP70, increased, indicating that HSPs play a role in skeletal muscle recovery and remodeling/adaptation processes to high-force exercise.     

Regulation of the levels of small heat-shock proteins during differentiation of C2C12 cells

Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.     

Whey Protein Hydrolysate Enhances HSP90 but Does Not Alter HSP60 and HSP25 in Skeletal Muscle of Rats

Whey protein hydrolysate (WPH) intake has shown to increase HSP70 expression. The aim of the present study was to investigate whether WPH intake would also influences HSP90, HSP60 and HSP25 expression, as well as associated parameters. Forty-eight male Wistar rats were divided into sedentary (unstressed) and exercised (stressed) groups, and were fed with three different sources of protein: whey protein (WP), whey protein hydrolysate (WPH) and casein (CAS) as a control, based on the AIN93G diet for 3 weeks. WPH intake increased HSP90 expression in both sedentary and exercised animals compared to WP or CAS, however no alteration was found from exercise or diet to HSP60 or HSP25. Co-chaperone Aha1 and p-HSF1 were also increased in the exercised animals fed with WPH in comparison with WP or CAS, consistent with enhanced HSP90 expression. VEGF and p-AKT were increased in the WPH exercised group. No alteration was found in BCKDH, PI3-Kinase (p85), GFAT, OGT or PGC for diet or exercise. The antioxidant system GPx, catalase and SOD showed different responses to diet and exercise. The data indicate that WPH intake enhanced factors related to cell survival, such as HSP90 and VEGF, but does not alter HSP60 or HSP25 in rat skeletal muscle.     

Heat-shock treatment-mediated increase in transduction by recombinant adeno-associated virus 2 vectors is independent of the cellular heat-shock protein 90

Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in approximately 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.     

Inactivation of DNA–Dependent Protein Kinase Promotes Heat–Induced Apoptosis Independently of Heat–Shock Protein Induction in Human Cancer Cell Lines

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.     

Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings

Tomato (Lycopersicon esculentum cv. 'Moneymaker') was transformed with a codA gene, from Arthrobacter globiformis, for choline oxidase that had been modified to allow targeting to both chloroplasts and the cytosol. Glycinebetaine (GB) accumulated in seeds of transformed plants up to 1 μmol g(-1) dry weight (DW), while no detectable GB was found in wild-type (WT) seeds. The codA-transgenic seeds germinated faster and at higher frequency than WT seeds with high temperature treatment. After heat stress, levels of expression of a mitochondrial small heat-shock protein (MT-sHSP), heat-shock protein 70 (HSP70) and heat-shock cognate 70 (HSC70) were higher in transgenic seeds than in WT seeds during heat stress, and the accumulation of HSP70 was more prominent in codA-transgenic seeds than in WT seeds. Addition of GB to the germination medium or imbibition of seeds in a solution of GB enhanced the tolerance of WT seeds to high temperatures. WT seeds treated with exogenous GB also expressed heat-shock genes at elevated levels and accumulated more HSP70 than controls. Our results suggest that GB, either applied exogenously or accumulated in vivo in codA-transgenic seeds, enhanced the expression of heat-shock genes in and improved the tolerance to high temperature of tomato seeds during germination.     

Proteasome inhibitors lactacystin and MG132 inhibit the dephosphorylation of HSF1 after heat shock and suppress thermal induction of heat shock gene expression.

Recently, we have shown that two proteasome inhibitors, MG132 and lactacystin, induce hyperphosphorylation and trimerization of HSF1, and transactivate heat shock genes at 37 degrees C. Here, we examined the effects of these proteasome inhibitors and, in addition, a phosphatase inhibitor calyculin A (CCA) on the activation of HSF1 upon heat shock and during post-heat-shock recovery, with emphasis on HSF1 hyperphosphorylation and the ability of HSF1 to transactivate heat shock genes. When lactacystin, MG132, or CCA was present after heat shock, HSF1 remained hyperphosphorylated during post-heat-shock recovery at 37 degrees C. Failure of HSF1 to recover to its preheated dephosphorylated state correlated well with the suppression of the heat-induced hsp70 expression. In vitro, HSF1 from heat-shocked cells, when dephosphorylated, showed an increase in HSE-binding affinity. Taken together, these data suggest that phosphorylation of HSF1 plays an important role in the negative regulation of heat-shock response. Specifically, during post-heat-shock recovery phase, prolonged hyperphosphorylation of HSF1 suppresses heat-induced expression of heat shock genes.     

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri4.5-fold higher steady-state level of the respective mRNA. Also animals taken from stressed locations in the field showed an increased expression. The amount of HSP90 protein in D. klunzingeri was found to be strongly increased under thermal stress, or exposure to polychlorinated biphenyl (congener 118), but not after treatment with cadmium. Field studies revealed that samples taken from a nonstressed area have a low level of HSP90, but those collected from locations at which the corals are under physical stress (sedimentation through landfilling) show a high expression of HSP90. It is concluded that the chaperone HSP90 might become a suitable biomarker to monitor environmental stress on corals.