Heat shock protein 90 (Hsp90) chaperone complex inhibitor,Radicicol, potentiated radiation-induced cell killing in a hormone-sensitive prostate cancer cell line through degradation of the androgen receptor

Until now, there has not been enough information on how androgens or androgen deprivation may influence the response of cancer cells to radiation. In this study, the effect of dihydrotestosterone (DHT) on cellular proliferative activity and radiosensitivity was examined in a hormone-sensitive human prostate cancer cell line, LNCaP. In addition, the study also examined how a heat shock protein 90 (Hsp90) chaperone complex inhibitor modified the effect of DHT on the radiosensitivity of the cells, because binding of the androgen receptor (AR) to Hsp90 is required to maintain the stability and functioning of AR. The hormone-sensitive human prostate cancer cell line, LNCaP, was used. Radicicol was used as one of the known Hsp90 chaperone complex inhibitors, and the cells were incubated in the presence of this compound at a concentration of 500 nM. Cellular radiosensitivity was determined by the clonogenic assay; the changes in the protein expression were examined by Western blotting or immunofluorescence. DHT at a concentration of 1 nM caused enhancement of the proliferative activity and reduction of the radiosensitivity of the cells. Radicicol at a concentration of 500 nM abolished the DHT-induced decrease in cellular radiosensitivity and potentiated the radiation-induced cell killing synergistically. Consistent with the changes in the cellular radiosensitivity, radicicol degraded AR, Raf-1 and HER2/neu via reduced binding of AR to Hsp90, although selective degradation of HER2/neu caused by Herceptin, a monoclonal antibody against HER2, did not affect the cellular radiosensitivity. The results suggest that the Hsp90 chaperone complex may be a potential molecular target for potentiation of radiation-induced cell killing in a hormone-sensitive prostate cancer cell line.     

Radicicol potentiates heat-induced cell killing in a human oesophageal cancer cell line: the Hsp90 chaperone complex as a new molecular target for enhancement of thermosensitivity

PURPOSE: To examine the ability of a heat shock protein 90 (Hsp90) chaperone complex inhibitor, radicicol, to modify thermal response and heat-induced cell killing, and to clarify the underlining mechanisms. MATERIALS AND METHODS: A human oesophageal cancer cell line (TE-1), with a mutant p53 gene, was used. To examine the effect of radicicol on heat-induced cell killing, radicicol at a concentration of 100 nM was incubated with the cells for 7 h during heat treatment. Changes in the expression of proteins were examined by Western blot and immunofluorescence analysis. RESULTS: Radicicol in combination with heat synergistically potentiated heat-induced cellular killing despite an increase in the expression of Hsp72 and Hsp27 caused by radicicol. Heat alone activated Raf-1 and p42/p44 extracellular signal-regulated kinase (Erk), and heat in combination with radicicol inhibited the activation of Raf-1 and p42/p44 Erk through reduced binding of Raf-1 to Hsp90. Phosphorylation of Akt was also decreased by radicicol. CONCLUSIONS: The Hsp90 chaperone complex inhibitor, radicicol, potentiated heat-induced cellular killing, and inhibition of p42/p44 Erk and Akt activation rather than modification of Hsp expression might be involved in enhancing cellular thermosensitivity. Results suggest that the Hsp90 chaperone complex could be a new molecular target for the modification of the cellular response to heat.     

Androgen and the blocking of radiation-induced sensitization to Fas-mediated apoptosis through c-jun induction in prostate cancer cells

PURPOSE: To clarify the key mechanism by which androgen makes prostate cancer cells highly resistant to Fas-mediated apoptosis. MATERIALS AND METHODS: The role of c-jun induction by 10 nM dihydrotestosterone (DHT) in 5 Gy radiation-induced up-regulation of Fas and sensitization to the apoptosis was studied by using the human prostate cancer cell line LNCaP. RESULTS: On exposure to 5 Gy radiation, LNCaP cells demonstrated high sensitization to Fas-mediated apoptosis through increased Fas expression, stabilized p53 expression and binding to p53 response elements within the promoter and first intronic region of the Fas gene. Following treatment with DHT, in vivo binding of p53 to its response elements was strongly inhibited. In addition, DHT significantly up-regulated c-jun expression through extracellular stress-regulated kinase (ERK) activation, and transfection of an antisense oligonucleotide for c-jun or ERK inhibition by PD98059 cancelled DHT-mediated suppression of radiation-induced transactivation of Fas gene and sensitization to Fas-mediated apoptosis. CONCLUSIONS: Radiation-induced Fas sensitization in prostate cancer cell was mediated through p53-dependent transactivation of the Fas gene, which can be blocked by androgen stimulation mainly through induction of c-jun.     

Radicicol potentiates heat‐induced cell killing in a human oesophageal cancer cell line: the Hsp90 chaperone complex as a new molecular target for enhancement of thermosensitivity

Purpose: To examine the ability of a heat shock protein 90 (Hsp90) chaperone complex inhibitor, radicicol, to modify thermal response and heat‐induced cell killing, and to clarify the underlining mechanisms.

Materials and methods: A human oesophageal cancer cell line (TE‐1), with a mutant p53 gene, was used. To examine the effect of radicicol on heat‐induced cell killing, radicicol at a concentration of 100?nM was incubated with the cells for 7?h during heat treatment. Changes in the expression of proteins were examined by Western blot and immunofluorescence analysis.

Results: Radicicol in combination with heat synergistically potentiated heat‐induced cellular killing despite an increase in the expression of Hsp72 and Hsp27 caused by radicicol. Heat alone activated Raf‐1 and p42/p44 extracellular signal‐regulated kinase (Erk), and heat in combination with radicicol inhibited the activation of Raf‐1 and p42/p44 Erk through reduced binding of Raf‐1 to Hsp90. Phosphorylation of Akt was also decreased by radicicol.

Conclusions: The Hsp90 chaperone complex inhibitor, radicicol, potentiated heat‐induced cellular killing, and inhibition of p42/p44 Erk and Akt activation rather than modification of Hsp expression might be involved in enhancing cellular thermosensitivity. Results suggest that the Hsp90 chaperone complex could be a new molecular target for the modification of the cellular response to heat.     

Androgen and the blocking of radiation‐induced sensitization to Fas‐mediated apoptosis through c‐jun induction in prostate cancer cells

Purpose: To clarify the key mechanism by which androgen makes prostate cancer cells highly resistant to Fas‐mediated apoptosis.

Materials and methods: The role of c‐jun induction by 10?nM dihydrotestosterone (DHT) in 5?Gy radiation‐induced up‐regulation of Fas and sensitization to the apoptosis was studied by using the human prostate cancer cell line LNCaP.

Results: On exposure to 5?Gy radiation, LNCaP cells demonstrated high sensitization to Fas‐mediated apoptosis through increased Fas expression, stabilized p53 expression and binding to p53 response elements within the promoter and first intronic region of the Fas gene. Following treatment with DHT, in vivo binding of p53 to its response elements was strongly inhibited. In addition, DHT significantly up‐regulated c‐jun expression through extracellular stress‐regulated kinase (ERK) activation, and transfection of an antisense oligonucleotide for c‐jun or ERK inhibition by PD98059 cancelled DHT‐mediated suppression of radiation‐induced transactivation of Fas gene and sensitization to Fas‐mediated apoptosis.

Conclusions: Radiation‐induced Fas sensitization in prostate cancer cell was mediated through p53‐dependent transactivation of the Fas gene, which can be blocked by androgen stimulation mainly through induction of c‐jun.     

Inhibition of cell growth induced by photosensitizer PP(Arg)2-mediated photodynamic therapy in human breast and prostate cell lines. Part I

Photodynamic therapy can become an effective alternative method to surgery. The experiments reveal that using low photosensitizer doses and relatively low energy doses allow us to obtain effective results after PDT (to limit formation of colonies by investigated cancer cells). The prostate and breast cancer cell lines were investigated: MCF-7, a human breast cancer responsive to androgen therapy; MDA-MB231, a more aggressive human breast cancer non-responsive to androgen therapy; LNCaP, a lymphonodal metastasis of prostate carcinoma responsive to androgen therapy; DU-145, a human prostate cancer non-responsive to androgen therapy. Clonogenic assay shows that certain PP(Arg)(2) and light energy low doses stimulate the researched colony-forming cancer cells growth. Some low energy doses used during PP(Arg)(2)-mediated PDT also cause the increase in the colony-forming tumor cells. Among investigated cancer lines, MCF-7 exhibited the biggest sensibility towards PP(Arg)(2) and LNCaP the smallest one. PP(Arg)(2) based PDT is an effective method in colony growth limitation of breast cancer cell lines: MCF-7, MDA-MB231 and prostate cancer cell lines: LNCaP, DU-145.     

Differences in radiosensitivity between three HER2 overexpressing cell lines

PURPOSE: HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin(R) treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. METHODS: The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from (211)At decays using the HER2-binding affibody molecule (211)At-(Z(HER2:4))(2) as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. RESULTS: SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of (211)At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from (211)At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. CONCLUSION: There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy.     

肌酸激酶B在前列腺细胞中的表达及临床意义

目的：探讨肌酸激酶B（脑型肌酸磷酸激酶,CKB）在前列腺良性及恶性细胞中的表达差异及临床意义。方法：采用实时定量PCR方法检测CKB在BPH1与LNCaP细胞中的表达差异,采用电泳仪法进行血标本CKB检验,最后采用半定量PCR方法研究CKB与雄激素的相关性。结果：CKB在LNCaP的表达量是BPH1细胞中的12．3倍,血检验发现,CKB在未经内分泌治疗前列腺癌组阳性率（5／10）显著高于前列腺增生组（0／10）,差异有统计学意义（P〈0．05）,内分泌治疗前列腺癌组CKB阳性率（4／37）与前列腺增生组阳性率（0／10）相比差异无统计学意义（P〉0．05）。CKB在比卡鲁胺（bicalutamide,Casodex）阻断组LNCaP细胞中的表达明显低于无比卡鲁胺阻断LNCaP细胞。结论：CKB在恶性细胞中高表达,对未经内分泌治疗前列腺癌患者有一定诊断价值,并且其表达受雄激素的调节,可能在前列腺癌的发生发展中起重要作用。     

Androgen sensitivity of rat prostate carcinoma studied by 31P NMR spectroscopy, 1H MR imaging, and 23Na MR imaging

31P NMR spectroscopy, 1H magnetic resonance (MR) imaging, and 23Na MR imaging were used to study the biochemical difference between nine hormone-sensitive and six hormone-resistant rat prostate cancers and to follow bioenergetic and morphologic changes subsequent to androgen deprivation in the hormone-sensitive model. Neither 1H nor 23Na MR image characteristics were useful in distinguishing androgen-sensitive from androgen-resistant prostate cancer nor in identifying androgen deprivation. 31P NMR spectroscopy did detect bioenergetic differences between the hormone-sensitive and hormone-resistant tumors. Baseline spectra showed a significantly higher PCr/ATP ratio (mean 0.86 +/- 0.09 SEM) for hormone-sensitive tumors than for hormone-resistant tumors (mean 0.26 +/- 0.07 SEM). By 3 days after androgen deprivation (orchiectomy (castration], PCr/ATP ratios had decreased noticeably; by 1 week, the decrease was statistically significant and remained so for the rest of the study (3 weeks). It appears that 31P NMR spectroscopy is useful in detecting androgen sensitivity of prostatic carcinoma.     

Cure, cell killing, growth delay and fragmentation of X-irradiated human melanoma HMV-I multicellular spheroids

Human melanoma, HMV-I, multicellular spheroids were irradiated and cure was determined by the absence of cellular outgrowth. Their cellular radiosensitivity was measured by the colony-forming ability of cells dispersed from the spheroid. Analysis of radiocurability of spheroids in terms of their cellular radiosensitivity predicted three necessary conditions: a linearity of dose versus the double-minus logarithm of curability; constancy of a critical cell number; and constancy of cellular radiosensitivity. These conditions were found to exist in the observed data for each of three size classes of spheroids. Analysis suggests that cellular radiosensitivity in multicellular spheroids with diameters of 250 and 400 microns was different from that of monolayers, and that the increase of spheroid-control doses was found to be a function of cellular radiosensitivity, total cell number per spheroid and a critical cell number. The critical cell number increased from 0.8 in a 150 microns spheroid to 4 in a 250 microns spheroid and to 57 in 400 microns spheroid. This number is a unique characteristic of multicellular systems and is one important factor in determining their radiocurability. X-ray-induced growth delay of spheroid size was increased with increasing dose. At high doses a sharp increase in delay time was seen, sometimes accompanying fragmentation of spheroids at late postirradiation times. The clonogenic activity of these fragments may serve as a model of exfoliation, the first step of radiation-induced metastasis.     

Geldanamycin, an inhibitor of Hsp90, sensitizes human tumour cells to radiation

PURPOSE: The effects of the heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) were examined on the radiosensitivity and signal transduction pathways in human tumour cell lines. MATERIALS AND METHODS: Two human cell lines, SQ-5 and DLD-1, derived from lung carcinoma and colon adenocarcinoma, respectively, were incubated for 16 h at 37 degrees C in medium containing 0.2 microM GA. The cells were then irradiated with X-rays and incubated with GA for a further 8 h. Radiation sensitivity was determined by clonogenic assays and protein levels were examined by Western blotting. RESULTS: GA radiosensitized both cell lines, but potentiated X-ray sensitivity more in SQ-5 than in DLD-1 cells. It was found that GA depleted EGFR and ErbB-2 in DLD-1 cells and depleted only ErbB-2 in SQ-5 cells. GA also reduced the expression of Akt and phosphorylated Akt (pAkt) expression in SQ-5 cells. In addition, the ratio (%) of apoptotic cells and poly [ADP-ribose] polymerase cleavage increased in SQ-5 but not in DLD-1 cells after exposure to GA and X-ray irradiation. The findings suggest that GA enhances the radiation sensitivity of human tumour cells by inhibiting the EGFR signal transduction system and the Akt signalling pathway. CONCLUSION: Targeting Hsp90 with GA provides a promising experimental strategy for radiosensitization of carcinoma.     

Physical exercise on the rat ventral prostate: Steroid hormone receptors,apoptosis and cell proliferation

Studies have investigated the effect of exercise on prostate cancer risk. However, there are still doubts regarding the correlation between physical activity and the steroid hormones with respect to the reduction of the risk for prostatic lesions. We evaluated the levels of corticosterone, dihydrotestosterone (DHT), testosterone, estradiol, and steroid hormone receptors, and investigated the relationship between apoptosis and cell proliferation in the rat ventral prostate after training. Two groups were included in this study: control and trained. The trained group was submitted to training for 13 weeks (1 week of adaptation). Two days after the last training session, all animals were euthanized, and the intermediate and distal regions of the ventral prostate were collected and processed for immunohistochemistry, Western blotting and hormonal analyses. Physical exercise increased the corticosterone plasma, DHT and testosterone. In addition, androgen receptor expression was lower and estrogen receptor (ER) α and ER β expression were higher in the trained group. However, the trained group showed disruption of the ratio of apoptotic to proliferating cells, indicating a predominance of apoptosis. We conclude that physical exercise alters the sex hormones and their receptors and is associated with the disruption of the balance between apoptosis and cell proliferation in the rat ventral prostate.     

Relationships between clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine human cervix carcinoma cell lines

PURPOSE: To compare clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine cervix carcinoma cell lines. MATERIALS AND METHODS: Initial and residual (after 24h repair) radiation-induced DNA damage was evaluated using pulsed field gel electrophoresis. Chromosome damage was measured by micronucleus (MN) induction in cytochalasin-B-induced binucleate cells. RESULTS: Significant differences between the cell lines were obtained in the induced levels of initial damage, residual damage and MN. Values for SF2 for the nine cell lines ranged from 0.36 to 0.92. No correlation was found between clonogenic measurements of radiosensitivity and initial DNA damage dose response slopes. However, borderline significant correlations were seen between clonogenic radiosensitivity data and the levels of residual DNA damage. There was no correlation between clonogenic radiosensitivity and the levels of radiation-induced MN. Cell lines with high levels of initial damage had high yields of MN induced by radiation and the correlation seen was significant. CONCLUSIONS: The poor correlation between the different endpoints precludes their use in a clinical setting on primary tumour samples in vitro. It may be that tumour cell lines in vitro are a poor model for tumours in vivo. Studies aimed at assessing assays for measuring tumour radiosensitivity therefore should employ clinical samples. In vitro cell line work should concentrate on unravelling the complex mechanisms involved in determining a radiosensitive or radioresistant phenotype.     

Relationships between clonogenic cell survival,DNA damage and chromosomal radiosensitivity in nine human cervix carcinoma cell lines

Purpose : To compare clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine cervix carcinoma cell lines. Materials and methods : Initial and residual (after 24h repair) radiation-induced DNA damage was evaluated using pulsed field gel electrophoresis. Chromosome damage was measured by micronucleus (MN) induction in cytochalasin-B-induced binucleate cells. Results : Significant differences between the cell lines were obtained in the induced levels of initial damage, residual damage and MN. Values for SF2 for the nine cell lines ranged from 0.36 to 0.92. No correlation was found between clonogenic measurements of radiosensitivity and initial DNA damage dose-response slopes. However, borderline significant correlations were seen between clonogenic radiosensitivity data and the levels of residual DNA damage. There was no correlation between clonogenic radiosensitivity and the levels of radiation-induced MN. Cell lines with high levels of initial damage had high yields of MN induced by radiation and the correlation seen was significant. Conclusions : The poor correlation between the different endpoints precludes their use in a clinical setting on primary tumour samples in vitro. It may be that tumour cell lines in vitro are a poor model for tumours in vivo. Studies aimed at assessing assays for measuring tumour radiosensitivity therefore should employ clinical samples. In vitro cell line work should concentrate on unravelling the complex mechanisms involved in determining a radiosensitive or radioresistant phenotype.     

Chiral dimethylamine flutamide derivatives--modeling, synthesis, androgen receptor affinities and carbon-11 labeling

Most prostate cancers are androgen dependent upon initial diagnosis. On the other hand, some very aggressive forms of prostate cancer were shown to have lost the expression of the androgen receptor (AR). Although the AR is routinely targeted in endocrine treatment, the clinical outcome remains suboptimal. Therefore, it is crucial to demonstrate the presence and activity of the AR in each case of prostate cancer, before and after treatment. While noninvasive positron emission tomography (PET) has the potential to determine AR expression of tumor cells in vivo, fully optimized PET imaging agents are not yet available. Based on molecular modeling, three novel derivatives of hydroxyflutamide (Compounds 1-3) were designed and synthesized. They contain an electron-rich group (dimethylamine) located on the methyl moiety, which may confer a better stability to the molecule in vivo. Compounds 1-3 have AR binding that is similar or higher than that of the currently used commercial drugs. An automated carbon-11 radiolabeling route was developed, and the compounds were successfully labeled with a 10-15% decay-corrected radiochemical yield, 99% radiochemical purity and a specific activity of 4Ci/mumol end of bombardment (n=15). These labeled biomarkers may facilitate the future quantitative molecular imaging of AR-positive prostate cancer using PET and may also allow for image-guided treatment of prostate cancer.     

Radiation-induced effects on telomerase in gynecological cancer cell lines with different radiosensitivity and repair capacity

PURPOSE: Telomerase activation in response to irradiation might enhance the radioresistance of cells. Thus, we have investigated radiation-induced effects on telomerase in six gynecological cancer cell lines, with different intrinsic radiosensitivity and capacity for sublethal damage repair (SLDR). MATERIALS AND METHODS: Three endometrial adenocarcinoma (UM-EC-1, UT-EC-2B and UT-EC-3) and three vulvar squamous cell carcinoma (A431, UM-SCV-2 and UM-SCV-7) cell lines were irradiated with doses of 5, 10 and 25 Gy and the effects on telomerase were evaluated at 0.5, 6, 24 and 48 h post-irradiation. Telomerase activity was quantitatively measured by SYBR Green real-time telomeric repeat amplification protocol. RESULTS: The most radioresistant cell line A431 had the strongest stimulatory effects (approximately 2.0 - 2.5-fold) on telomerase activity 24 and 48 h post-irradiation with the highest radiation doses. In contrast to that, telomerase activities in the highly radiosensitive cell line UT-EC-2B remained below the basal level throughout the 48-h period of post-irradiation with the highest doses, and even a decline to approximately 50% of the basal level was found 24 h after exposure. In other cell lines being either moderately or highly radiation resistant, telomerase activity levels in response to irradiation remained mainly at the basal level or gradually increased. CONCLUSIONS: The present findings indicate that there might be a connection between the radiation-induced telomerase response and radiosensitivity. However, no correlation was found between the radiation-induced effects on telomerase and the sublethal damage repair capacity of the cells.     

Geldanamycin,an inhibitor of Hsp90, sensitizes human tumour cells to radiation

Purpose: The effects of the heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) were examined on the radiosensitivity and signal transduction pathways in human tumour cell lines.

Materials and methods: Two human cell lines, SQ‐5 and DLD‐1, derived from lung carcinoma and colon adenocarcinoma, respectively, were incubated for 16?h at 37°C in medium containing 0.2?µM GA. The cells were then irradiated with X‐rays and incubated with GA for a further 8?h. Radiation sensitivity was determined by clonogenic assays and protein levels were examined by Western blotting.

Results: GA radiosensitized both cell lines, but potentiated X‐ray sensitivity more in SQ‐5 than in DLD‐1 cells. It was found that GA depleted EGFR and ErbB‐2 in DLD‐1 cells and depleted only ErbB‐2 in SQ‐5 cells. GA also reduced the expression of Akt and phosphorylated Akt (pAkt) expression in SQ‐5 cells. In addition, the ratio (%) of apoptotic cells and poly [ADP‐ribose] polymerase cleavage increased in SQ‐5 but not in DLD‐1 cells after exposure to GA and X‐ray irradiation. The findings suggest that GA enhances the radiation sensitivity of human tumour cells by inhibiting the EGFR signal transduction system and the Akt signalling pathway.

Conclusion: Targeting Hsp90 with GA provides a promising experimental strategy for radiosensitization of carcinoma.     

Evaluation of the Hsp90 inhibitor NVP-AUY922 in multicellular tumour spheroids with respect to effects on growth and PET tracer uptake

BackgroundMolecular targeting has become a prominent concept in cancer treatment and heat shock protein 90 (Hsp90) inhibitors are suggested as promising anticancer drugs. The Hsp90 complex is one of the chaperones that facilitate the refolding of unfolded or misfolded proteins and plays a role for key oncogenic proteins such as Her2, Raf-1, Akt/PKB, and mutant p53. NVP-AUY922 is a novel low-molecular Hsp90 inhibitor, currently under clinical development as an anticancer drug. Disruption of the Hsp90-client protein complexes leads to proteasome-mediated degradation of client proteins and cell death.The aim of the current study was to use a combination of the multicellular tumour spheroid (MTS) model and positron emission tomography (PET) to investigate the effects of NVP-AUY922 on tumour growth and its relation to PET tracer uptake for the selection of appropriate PET tracer. A further aim was to evaluate the concentration and time dependence in the relation between growth inhibition and PET tracer uptake as part of translational imaging activities.MethodsMTS of two breast cancer cell lines (MCF-7 and BT474), one glioblastoma cell line (U87MG) and one colon carcinoma cell line (HCT116) were prepared.Initially, we investigated MTS growth pattern and ³H-thymidine incorporation in MTS after continuous exposure to NVP-AUY922 in order to determine dose response. Then the short-term effect of the drug on the four PET tracers 2-[¹⁸F] fluoro-2-deoxyglucose (FDG), 3′-deoxy-3′-fluorothymidine (FLT), methionine and choline was correlated to the long-term effect (changes in growth pattern) to determine the adequate PET tracer with high predictability.Next, the growth inhibitory effect of different dose schedules was evaluated to determine the optimal dose and time. Finally, the effect of a 2-h exposure to the drug on growth pattern and FDG/FLT uptake was evaluated.ResultsA dose-dependent inhibition of growth and decrease of ³H-thymidine uptake was observed with 100% growth cessation in the dose range 7–52 nM and 50% ³H-thymidine reduction in the range of 10–23 nM, with the most pronounced effect on BT474 cells.The effect of the drug was best detected by FLT. The results suggested that a complete cessation of growth of the viable cell volume was achieved with about 50% inhibition of FLT uptake 3 days after continuous treatment.Significant growth inhibition was observed at all doses and all exposure time spans. Two-hour exposure to NVP-AUY922 generated a growth inhibition which persisted dose dependently up to 10 days. The uptake of FDG per viable tumour volume was reduced by just 25% with 300 nM treatment of the drug, whereas the FLT uptake decreased up to 75% in correlation with the growth inhibition and recovery.ConclusionsOur results indicate a prolonged action of NVP-AUY922 in this cell culture, FLT is a suitable tracer for the monitoring of the effect and a FLT PET study within 3 days after treatment can predict the treatment outcome in this model. If relevant in vivo, this information can be used for efficient planning of animal PET studies and later human PET trial.     

Radiation-induced effects on telomerase in gynecological cancer cell lines with different radiosensitivity and repair capacity

Purpose: Telomerase activation in response to irradiation might enhance the radioresistance of cells. Thus, we have investigated radiation-induced effects on telomerase in six gynecological cancer cell lines, with different intrinsic radiosensitivity and capacity for sublethal damage repair (SLDR).

Materials and methods: Three endometrial adenocarcinoma (UM-EC-1, UT-EC-2B and UT-EC-3) and three vulvar squamous cell carcinoma (A431, UM-SCV-2 and UM-SCV-7) cell lines were irradiated with doses of 5, 10 and 25 Gy and the effects on telomerase were evaluated at 0.5, 6, 24 and 48 h post-irradiation. Telomerase activity was quantitatively measured by SYBR Green real-time telomeric repeat amplification protocol.

Results: The most radioresistant cell line A431 had the strongest stimulatory effects (～2.0 – 2.5-fold) on telomerase activity 24 and 48 h post-irradiation with the highest radiation doses. In contrast to that, telomerase activities in the highly radiosensitive cell line UT-EC-2B remained below the basal level throughout the 48-h period of post-irradiation with the highest doses, and even a decline to ～50% of the basal level was found 24 h after exposure. In other cell lines being either moderately or highly radiation resistant, telomerase activity levels in response to irradiation remained mainly at the basal level or gradually increased.

Conclusions: The present findings indicate that there might be a connection between the radiation-induced telomerase response and radiosensitivity. However, no correlation was found between the radiation-induced effects on telomerase and the sublethal damage repair capacity of the cells.     

Radiosensitization of tumor cells by modulation of ATM kinase

PURPOSE: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization. METHOD: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied. RESULTS: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts. CONCLUSIONS: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.