Selectively enhanced radiation sensitivity in prostate cancer cells associated with proteasome inhibition

The purpose of this study is to evaluate the utility of MG-132, a broad spectrum proteasome inhibitor, to selectively enhance radiation sensitivity in prostate cancer without affecting normal surrounding urothelial tissue. PC3 prostate cancer cells and normal URO-tsa bladder epithelial cells were treated with or without MG-132 and exposed to 0, 2, 4, or 6 Gy radiation. Cell viability and clonogenic survival assays were performed, and nuclear factor kappa-B (NF-kappaB) activity was evaluated with electrophoretic mobility shift assay (EMSA). MG-132 was associated with decreased cell viability (between 24% and 33%) and clonogenic survival (between 71% and 88%) alone and in combination with radiation in PC3 cells. MG-132 had no effect on cell viability or clonogenic survival following radiation in URO-tsa cells. Constitutive and radiation-induced NF-kappaB binding activity was higher in PC3 cells compared with URO-tsa cells. Furthermore, MG-132 at concentrations associated with reductions in cell viability and clongenic survival inhibited NF-kappaB binding activity in PC3 cells with no effect in URO-tsa cells. These results provide strong evidence that proteasome inhibition and concomitant NF-kappaB inhibition can be used to selectively enhance tumor radiation sensitivity in prostate cancer without affecting normal surrounding bladder tissue.     

Resveratrol enhances radiation sensitivity in prostate cancer by inhibiting cell proliferation and promoting cell senescence and apoptosis

Radiation therapy (XRT) for treatment of localized prostate cancer (PCA) has outcomes similar to surgery and medical therapy. Toxicities of XRT and the relative radioresistance of PCA limit the effectiveness of this treatment method. Safe and effective radiosensitizing agents are lacking to enhance the effectiveness for XRT for PCA. In this study, the effect of XRT in combination with the radiosensitizing agent resveratrol (RSV) was investigated in a radioresistant PCA cell line, PC-3. Our results show the addition of RSV to XRT (XRT/RSV) synergistically enhanced XRT-induced apoptosis and inhibition of PC-3 proliferation. The antiproliferative effect of XRT/RSV treatment correlated with increased expression of p15, p21, and mutant p53 and decreased expression of cyclin B, cyclin D, and cdk2. Increased apoptosis correlated with increased expression of Fas and TRAILR1. Furthermore, XRT/RSV had little effect on the expression of p-AKT, whereas it increased the expression level of p-H2A.X, a marker for senescence. These data highlight the potential of RSV as a radiation sensitizer for PCA treatment and warrant further investigation.     

Enhanced radiosensitivity by inhibition of the anti-apoptotic gene clusterin using antisense oligodeoxynucleotide in a human bladder cancer model

Clusterin has been shown to be implicated in the acquisition of resistant phenotype to various kinds of apoptotic stimuli, including radiation. In bladder cancer, our previous study demonstrated that overexpression of clusterin is closely associated with disease progression and recurrence. The objective of this study was to investigate whether radiation sensitivity was enhanced by suppressing clusterin gene expression with antisense (AS) oligodeoxynucleotide (ODN) in the human bladder cancer KoTCC-1 model. Clusterin mRNA in KoTCC-1 cells after radiation was up-regulated in a dose-dependent manner; however, AS clusterin ODN treatment resulted in a marked inhibition of clusterin mRNA even after irradiation. Combined treatment of KoTCC-1 cells with radiation and AS clusterin ODN synergistically decreased plating efficacy and induced apoptotic cell death compared with either radiation or AS clusterin ODN treatment alone. In vivo systemic administration of AS clusterin ODN enhanced radiation sensitivity, significantly reducing subcutaneous KoTCC-1 tumor volume in nude mice, compared with that of mismatch control ODN. Moreover, additional administration of cisplatin to this combined regimen further achieved potential antitumor effects on subcutaneous KoTCC-1 tumor growth in nude mice. Collectively, these findings suggest that clusterin acts as a cell survival protein mediating radioresistance through the inhibition of apoptosis, and that inactivation of clusterin using AS technology might offer a novel strategy to improve the outcome of radiation therapy for patients with bladder cancer.     

Pancreatic cancer cell radiation survival and prenyltransferase inhibition: the role of K-Ras

Activating K-ras mutations are found in approximately 90% of pancreatic carcinomas and may contribute to the poor prognosis of these tumors. Because radiotherapy is frequently used in pancreatic cancer treatment, we assessed the contribution of oncogenic K-ras signaling to pancreatic cancer radiosensitivity. Seven human pancreatic carcinoma lines with activated K-ras and two cell lines with wild-type ras were used to examine clonogenic cell survival after Ras inhibition. Ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression and by blocking Ras processing using a panel of prenyltransferase inhibitors of differing specificity for the two prenyltransferases that modify K-Ras. K-ras knockdown by siRNA or inhibition of prenyltransferase activity resulted in radiation sensitization in vitro and in vivo in tumors with oncogenic K-ras mutations. Inhibition of farnesyltransferase alone was sufficient to radiosensitize most K-ras mutant tumors, although K-Ras prenylation was not blocked. These results show that inhibition of activated K-Ras can promote radiation killing of pancreatic carcinoma in a superadditive manner. The finding that farnesyltransferase inhibition alone radiosensitizes tumors with K-ras mutations implies that a farnesyltransferase inhibitor-sensitive protein other than K-Ras may contribute to survival in the context of mutant K-ras. Farnesyltransferase inhibitors could therefore be of use as sensitizers for pancreatic carcinoma radiotherapy.     

Enhanced radiation late effects and cellular radiation sensitivity in an ATM heterozygous breast cancer patient.

We observed severe late effects in a patient treated with radiation therapy for breast cancer. Radiation survival studies of patient fibroblasts show an enhanced cellular radiation sensitivity (Do = 0.95 Gy). Genetic analysis reveals that the patient is heterozygous for a mutated ATM gene. Protein truncation test (PTT) and sequence analysis identified a truncation within the leucine zipper domain, corresponding to a fragment previously reported to exhibit dominant negative function. These findings demonstrate that ATM heterozygosity may be associated with enhanced clinical radiation sensitivity and suggest a clinical relevance to this truncation that results in a dominant negative-acting protein.     

Enhanced sensitivity to irinotecan by Cdk1 inhibition in the p53-deficient HT29 human colon cancer cell line

Mutations in the tumor-suppressor gene p53 have been associated with advanced colorectal cancer (CRC). Irinotecan (CPT-11), a DNA topoisomerase 1 inhibitor, has been recently incorporated to the adjuvant therapy. Since the DNA-damage checkpoint depends on p53 activation, the status of p53 might critically influence the response to CPT-11. We analysed the sensitivity to CPT-11 in the human colon cancer cell line HT29 (mut p53) and its wild-type (wt)-p53 stably transfected subclone HT29-A4. Cell-cycle analysis in synchronised cells demonstrated the activation of transfected wt-p53 and a p21(WAF1/CIP1)-dependent cell-cycle blockage in the S phase. Activated wt-p53 increased apoptosis and enhanced sensitivity to CPT-11. In p53-deficient cells, cDNA-macroarray analysis and western blotting showed an accumulation of the cyclin-dependent kinase (cdk)1/cyclin B complex. Subsequent p53-independent activation of the cdk-inhibitor (cdk-I) p21(WAF1/CIP1) prevented cell-cycle progression. Cdk1 induction was exploited in vivo to improve the sensitivity to CPT-11 by additional treatment with the cdk-I CYC-202. We demonstrate a gain of sensitivity to CPT-11 in a p53-mutated colon cancer model either by restoring wild-type p53 function or by sequential treatment with cdk-Is. Considering that mutations in p53 are among the most common genetic alterations in CRC, a therapeutic approach specifically targeting p53-deficient tumors could greatly improve the treatment outcomes.     

Determinants of prostate cancer-specific survival after radiation therapy for patients with clinically localized prostate cancer.

PURPOSE: Identifying pretreatment and posttreatment predictors of time to prostate cancer-specific death (PCSD) after external-beam radiation therapy (RT) was the subject of this study. PATIENTS AND METHODS: A Cox regression analysis was used to evaluate the ability of the pretreatment risk group to predict time to PCSD for 381 patients who underwent RT for clinically localized prostate cancer. Posttreatment factors analyzed for the 94 patients who experienced prostate-specific antigen (PSA) failure included the time to PSA failure, the posttreatment PSA doubling time (DT), and the timing of salvage hormonal therapy. RESULTS: Despite the median age of 73 years at diagnosis, 45% of patients with high-risk disease were estimated to die from prostate cancer within 10 years after RT compared with 0% (P =.004) and 6% (P =.05) for patients with low- or intermediate-risk disease, respectively. Predictors of time to PCSD after PSA failure included PSA DT (P =.01) and delayed use of hormonal therapy (P 相似文献   

Enhanced chemosensitivity of bladder cancer cells to cisplatin by suppression of clusterin in vitro

We examined the functional role of clusterin in chemotherapy-induced apoptosis and tested whether anti-sense transfection targeted against clusterin enhances the chemosensitivity in human bladder cancer cells in vitro. Clusterin mRNA and protein expression of 253J cells, a human bladder carcinoma cell line, after treatment with cisplatin were measured by RT-PCR and Western blot analysis. Clusterin expression and cell growth were compared between 253J cells transfected with constructed a clusterin anti-sense plasmid vector (pCR-CLU-AS) and controls. Tumor cell viability was measured with MTT assay after cisplatin treatment. DNA fragmentation and CPP32 assay were performed. Clusterin expression was increased after treatment with cisplatin and highest at 8 h in 253J cells. Clusterin anti-sense transfectants were highly sensitive to apoptotic cell death induced by cisplatin compared with parental 253J cells or control transfectants. Collectively, our results showed that expression of clusterin was increased in the acute phase of cell death caused by cisplatin and that suppressing the expression of clusterin enhanced the susceptibility of apoptosis caused by cisplatin in human bladder cancer cells. These results suggest that lowering the expression of clusterin might increase the sensitivity of bladder cancer cells to chemotherapeutic agents.     

Enhanced radiation killing by 5-fluorouracil of biliary tract cancer cell lines

Despite the frequent clinical use of 5-fluorouracil (5-FU) in combination with radiotherapy for patients with biliary tract cancers, data remain scarce concerning specifically the influence of 5-FU on the sensitivity of these cancer cells to radiation. The present study was carried out to evaluate the effects of concomitant treatment with 5-FU on radiation-induced cell killing in two established human biliary tract cancer cell lines (Mz-ChA-2 and SK-ChA-1 cells). These lines were chosen as we have previously shown that SK-ChA-1 cells are significantly more resistant to both radiation and 5-FU than Mz-ChA-2 cells. Clonogenic survival was employed as the end-point for cell killing. Administration of 5-FU at LD50 doses to each cell line significantly enhanced radiation-induced cell killing. The enhancement ratio (ER) was obtained by dividing the radiation dose required to decrease the cell survival fraction to 37% (D0) by the dose to decrease cell survival to the same level when the cells were also treated with 5-FU. The ER in each of the cell lines was greater when they were incubated with 5-FU after radiation rather than prior to radiation. Longer exposure times with 5-FU resulted in enhanced radiation killing. The ER was significantly higher in the radioresistant cell line than in the radiosensitive line. These findings suggest that therapy with radiation and 5-FU may be of value as components of multidisciplinary treatment for biliary tract cancer. Protracted low dose exposure to 5-FU may prove to be most efficacious in enhancing the effects of radiation therapy.     

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.     

Cytoplasmic clusterin expression is associated with longer survival in patients with resected non small cell lung cancer.

BACKGROUND: Clusterin is a glycoprotein that has been implicated in many processes, including apoptosis, cell cycle regulation, and DNA repair. Previous studies have examined the prognostic value of clusterin expression in various malignancies. In the present study, we examined clusterin staining in tumors resected from patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Tumor specimens were obtained for 113 patients with completely resected NSCLC from paraffin-embedded tissue microarrays and stained with an antibody specific for clusterin. Staining patterns were observed and graded based on intensity and then correlated with clinical data. RESULTS: Positive cytoplasmic clusterin staining was observed in 44 patients, and weak/negative staining was observed in 62 patients. Patients who had tumors that stained positive for cytoplasmic clusterin had significantly longer survival in multivariate analysis (hazard ratio 0.487, 95% confidence interval 0.27-0.89). A correlation was also observed for recurrence-free survival, which approached statistical significance (hazard ratio 0.345, 95% confidence interval 0.12-1.02). In univariate analysis, patients with clusterin-positive tumors had a 63% 3-year survival, whereas patients with clusterin-negative tumors had a 42% 3-year survival (P = 0.0108); clusterin-positive tumors also had significantly less recurrence (P = 0.0231). CONCLUSIONS: Cytoplasmic clusterin staining is present in a substantial number of NSCLC tumors and may be a biomarker for longer survival in patients with surgically resected NSCLC.     

Functional p53 increases prostate cancer cell survival after exposure to fractionated doses of ionizing radiation

External beam radiation therapy is an effective therapy for localized prostate cancer, although failures occur at high rates. One variable that may affect the radiosensitivity of prostate tumor cells is their p53 status because this gene controls radiation-induced cell cycle arrest, apoptosis, and the repair of DNA damage. Using a system in which p53 function was conditionally restored to p53-null PC3 prostate cancer cells by stable transfection with a human temperature-sensitive p53 mutant allele, we tested the hypothesis that functional p53 increases cell cycle arrest and contributes to increased clonogenic survival after ionizing radiation (IR) of prostate carcinoma cells. Cell cycle arrest and clonogenic survival in response to single and multiple daily exposures to clinically relevant 2-Gy doses of IR were examined. Whereas the temperature-sensitive p53 protein was activated by phosphorylation after IR exposure at both the restrictive and permissive temperatures, Cdkn1/p21 was only induced by functional p53 (at the permissive temperature). In the presence of functional p53, the maintenance of G(2) arrest was significantly longer (P < 0.01), and a small increase in cell survival measured by clonogenic assay was seen after exposure to a single 2-Gy dose of IR. However, functional p53 significantly increased clonogenic survival (P < 0.01) after exposure to daily doses of 2 Gy of IR and contributed to a more sustained G(2) arrest and increased G(1) arrest in response to the multifraction regimen. These studies implicate the presence of wild-type p53 with increased survival of prostate carcinoma cells after fractionated exposure to radiation. Additionally, the data provide evidence that wild-type p53 in prostate tumor cells may reduce the effectiveness of radiation therapy.     

Role of fibroblast growth factor receptor signaling in prostate cancer cell survival.

BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies.     

DNA-dependent protein kinase activity correlates with Ku70 expression and radiation sensitivity in esophageal cancer cell lines.

We investigated the relationship between DNA-dependent protein kinase (DNA-PK) activity and radiation sensitivity using 14 esophageal cancer cell lines, TE 1-14. DNA-PK activities differed significantly among the cell lines. The highest DNA-PK activity observed in TE-8 was more than two times higher than the lowest DNA-PK activity observed in TE-5. Significant correlation was observed between DNA-PK activity and D0 (r = 0.766; P = 0.0008). Western blots analysis revealed a significant correlation between DNA-PK activity and Ku70 expression, suggesting that the regulation in DNA-PK activity was associated with Ku70 expression. The data suggest that the measurement of DNA-PK activity and/or Ku70 expression may provide a useful way to predict radiation sensitivity.     

Differential expression of protein kinase C epsilon protein in lung cancer cell lines by ionising radiation.

The effect of ionising radiation on the regulation of gene and protein expression is complex. This study focuses on the translational regulational of the epsilon isoform of protein kinase C by ionising radiation. We found that protein kinase C epsilon is rapidly increased in the human lung adenocarcinoma cell line A549 following irradiation. Western blots showed increased accumulation of this protein at doses as low as 75 cGy after 15 min post irradiation. Maximal induction (11-fold over unirradiated cells) of PKC epsilon occurred at 150 cGy within 1 h after treatment by X-rays in A549 cells. The increased levels of PKC epsilon protein after X-rays does not require de novo protein or RNA synthesis, suggesting that this increase is post-translationally controlled. In contrast to A549 cells PKC epsilon levels in the large cell lung carcinoma cell line NCI H661 were not induced by radiation. In the small cell lung carcinoma cell line NCI N417, PKC epsilon was also not induced but a higher molecular weight PKC epsilon protein, suggestive of phosphorylation, appeared at 2 h after irradiation. The variation in induction or phosphorylation of PKC epsilon by ionising radiation in the cell lines tested in this study suggested that no clear correlation existed between intrinsic radiation sensitivity and PKC epsilon induction. To determine whether PKC epsilon does play a role in cell survival to irradiation, we used the protein kinase inhibitor staurosporin to decrease PKC activity and found that staurosporin sensitised cells to killing by ionising radiation. Pulsed field gel electrophoresis, however, indicated that DNA double-strand break repair was not decreased, suggesting that PKC epsilon is modifying the fidelity of rejoining and not the overall magnitude of repair. The regulation of PKC by ionising radiation will be discussed with respect to the biological consequences of gene induction by DNA damage agents.     

Molecular mechanisms of the antimetastatic activity of nuclear clusterin in prostate cancer cells

The proapoptotic activity of nuclear clusterin (nCLU) in cancer cells is now well established. We previously showed that nCLU decreases the motility of prostate cancer cells by triggering a dramatic dismantling of the actin cytoskeleton. Here, we sought to unravel the molecular mechanisms of the antimetastatic activity of nCLU. We found that nCLU: i) decreases LIMK1 expression, thus increasing the levels of the active (unphosphorylated) form of cofilin, the well known actin depolymerizing factor; ii) binds to vimentin, sequestering the protein from its adhesion sites at the cell periphery, thus interfering with its role in cell motility and adhesion; iii) affects the intracellular distribution of E-cadherin (the major component of epithelial adherens junctions) which appears to be diffusely distributed in the cells. Through these mechanisms nCLU reduces the migratory/invasive behavior of PC3 cells; this effect is further demonstrated by a decreased secretion of active MMP-2 from the cells. Thus, in addition to its proapoptotic function, nCLU also exerts a strong anti-migratory/anti-invasive activity in prostate cancer cells, by interfering with the cytoskeletal components and by decreasing MMP-2 activity.