Showing papers by "Bo Lu published in 2006"

Inhibition of Mammalian Target of Rapamycin or Apoptotic Pathway Induces Autophagy and Radiosensitizes PTEN Null Prostate Cancer Cells

Abstract: The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.

Targeting the Akt/mammalian target of rapamycin pathway for radiosensitization of breast cancer.

Abstract: The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be activated by radiation. The mammalian target of rapamycin (mTOR) is downstream of Akt, and we investigated the effects of radiation on Akt/mTOR signaling in breast cancer cell models. RAD001 (everolimus), a potent derivative of the mTOR inhibitor rapamycin, was used to study the effects of mTOR inhibition, as the role of mTOR inhibition in enhancing radiation remains unexplored. RAD001 decreased clonogenic cell survival in both breast cancer cell lines MDA-MB-231 and MCF-7, although the effect is greater in MDA-MB-231 cells. Irradiation induced Akt and mTOR signaling, and this signaling is attenuated by RAD001. The radiation-induced signaling activation is mediated by PI3K because inhibition of PI3K with LY294002 inhibited the increase in downstream mTOR signaling. Additionally, caspase-dependent apoptosis is an important mechanism of cell death when RAD001 is combined with 3 Gy radiation, as shown by induction of caspase-3 cleavage. An increase in G(2)-M cell cycle arrest was seen in the combination treatment group when compared with controls, suggesting that cell cycle arrest may have been a contributing factor in the increased radiosensitization seen in this study. We conclude that RAD001 attenuates radiation-induced prosurvival Akt/mTOR signaling and enhances the cytotoxic effects of radiation in breast cancer cell models, showing promise as a method of radiosensitization of breast cancer.

Integrin αvβ3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non–small-cell lung cancer models

Abstract: Purpose: Integrins α v β 3 and α v β 5 are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation. Methods and Materials: Levels of α v β 3 were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non–small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment. Results: Human umbilical vein endothelial cells had the highest α v β 3 level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of α v β 3 in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment. Conclusions: We conclude that radiation induces expression of α v β 3 integrin in endothelial and non–small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.

Radiosensitization of lung cancer by nutlin, an inhibitor of murine double minute 2

Abstract: p53 plays a critical role in cell cycle arrest and induction of apoptosis. Certain malignancies carry wild-type p53, which is frequently down-regulated by murine double minute 2 (MDM2) overexpression. Availability of a small-molecule inhibitor against MDM2, nutlin, has made it feasible to evaluate the anti-MDM2-based therapeutic strategies. The rationale for the current study is that functional p53 has been linked with improved responses to radiation treatment. Hence, this study evaluates the use of nutlin, a small-molecule inhibitor that blocks the interaction of p53 and MDM2, in sensitizing cancer cells to radiation. Expression of MDM2, p53, and p21 in both p53 wild-type and p53-defective lung cancer cell lines was examined. Clonogenic and 7-amino-actinomycin D studies were used to determine possible mechanisms of cell death. The combined effect of MDM2 inhibition and radiation on cell cycle was also studied. We found that radiosensitization by nutlin occurs in lung cancer cells with wild-type p53. There were increased apoptosis and cell cycle arrest following administration of nutlin and radiation. Furthermore, the combination of nutlin and radiation decreased the ability of endothelial cells to form vasculature, as shown by Matrigel assays. Our data suggest that nutlin is an effective radiosensitizer of p53 wild-type cells. The radiosensitizing effect seems to be at least partially due to induction of apoptosis and cell cycle arrest. In addition, nutlin may be an effective radiosensitizer of tumor vasculature.

Inhibition of signal transducer and activator of transcription 3 activity results in down-regulation of Survivin following irradiation

Abstract: Signal transducer and activator of transcription 3 (Stat3) and Survivin are constitutively up-regulated in various human tumor cells. We previously found Survivin to be significantly reduced in response to radiation in human umbilical vein endothelial cells (HUVEC) but not in tumor cell lines. In this study, we examined the effect of Stat3 on Survivin expression in irradiated HUVECs and breast cancer cells. We also studied how inhibition of Stat3 and Survivin activity affects cell survival and angiogenesis following irradiation. We determined that Survivin was significantly increased by overexpression of an active Stat3 (Stat3-C). Following irradiation, the level of phospho-Stat3 Tyr705, but not phospho-Stat3 Ser727, was reduced in HUVECs, whereas it remained unchanged in irradiated breast cancer cells. Correspondingly, Stat3 DNA-binding activity following irradiation was specifically down-regulated in HUVECs but not in breast cancer cells. Mutation of Tyr705 abolished radiation-induced down-regulation of Survivin. Clonogenic and endothelial cell morphogenesis assays suggested that DN-Stat3 and DN-Survivin together resulted in the greatest radiosensitization of MDA-MB-231, decreasing angiogenesis and cell survival. In summary, Stat3 modulates Survivin, and both are potential therapeutic targets for radiation sensitization in breast cancer. [Mol Cancer Ther 2006;5(11):2659–65]

Vascular endothelial growth factor tyrosine kinase inhibitor AZD2171 and fractionated radiotherapy in mouse models of lung cancer.

Abstract: The vascular endothelial growth factor receptor (VEGFR) tyrosine kinases are being explored as targets for antiangiogenic cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We investigated the combination of the potent VEGFR tyrosine kinase inhibitor AZD2171 and ionizing radiation in cell culture and mouse models of lung cancer. We show that ionizing radiation induces expression of phosphorylated VEGFR-2 (Flk-1) in endothelial cells and that this phosphorylation is inhibited by AZD2171. Human umbilical vascular endothelial cells become more sensitive to radiation after treatment with AZD2171 as determined by clonogenic assay. Matrigel assay showed a decrease in in vitro endothelial tubule formation with AZD2171/radiation combination treatment. When similar combination was applied to the H460 lung cancer xenograft model in nude mice, loss of radiation-induced phosphorylated Flk-1 was observed in the combination treatment group, which also showed a large decrease in tumor vascular density by staining of the von Willebrand factor. H460 tumor growth delay was enhanced in the combination treatment group compared with the groups treated with AZD2171 or radiation alone. Additionally, after therapy, Ki67 index showed >4-fold reduction of tumor proliferation in the combination therapy group, which also showed increased intratumoral apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. In conclusion, AZD2171 sensitizes lung tumor xenografts to radiation and inhibits angiogenesis both in vitro and in vivo. When used as a radiation enhancer, AZD2171 has the potential to improve tumor growth delay by inhibiting tumor proliferation and promoting apoptosis. Clinical trials are needed to determine the potential of this combination therapy in patients with locally advanced lung cancer.

E-cadherin promoter polymorphisms are not associated with the aggressiveness of prostate cancer in Caucasian patients.

Abstract: Background −160C→A and −347G→GA polymorphisms in the promoter region decrease E-cadherin gene transcription. Decreased E-cadherin expression predicts poor outcome among patients with cancer. We sought to investigate whether −160C→A and/or −347G→GA polymorphisms were associated with the aggressiveness of prostate cancer. Methods TaqMan single nucleotide polymorphism genotyping assay (Applied Biosystems, Foster City, CA) was used to detect −160C→A and −347G→GA polymorphisms in deoxyribonucleic acid from the paraffin-embedded prostate tissues of 98 Caucasian patients. Results The genotype frequencies were −160C/C: 48% (47 of 98); −160C/A: 44% (43 of 98); −160A/A: 8% (8 of 98); −347G/G: 68% (67 of 98); −347G/GA: 28% (27 of 98); and −347GA/GA: 4% (4 of 98). Using the chi-square test, we found that the polymorphisms −160C→A and −347G→GA were not related to other clinical and pathologic parameters (i.e., age, prostate-specific antigen level, Gleason grade, and clinical stage) ( P > 0.05). In combination analysis, there was no significant relationship between patients with both −160C/C and −347G/G, and these same parameters ( P > 0.05). Using the log-rank test, we found no significant difference in relapse-free survival and overall survival between patients with −160C/C and those with −160A/C or −160A/A ( P = 0.0764 and 0.2746, respectively), and also no significant difference between patients with −347G/G and those with −347GA/G or −347GA/GA ( P = 0.9416 and 0.7367, respectively). There was also no significant difference in relapse-free survival and overall survival between patients with homozygosities of −160C/-347G and patients with other genotypes ( P = 0.1418 and 0.2434, respectively). Conclusion We conclude that E-cadherin −160C→A and/or −347G→GA polymorphisms are not associated with the aggressiveness of prostate cancer in Caucasian patients.

Radiation-sensitization in the absence of Bax/Bak

Abstract: 4953 Abstract Bax and Bak act as a mitochondrial gateway for various apoptotic signals. However, cells lacking both Bax and Bak are resistant to apoptosis induced by various apoptotic stimuli. Instead, they undergo autophagic cell death. Resistance to apoptosis has been associated with radiation resistance, although little is known regarding the relationship between Bax/Bak -/- and irradiation-induced autophagy. In the present study, we investigated radiation-induced autophagy in the presence or absence of Bax and Bak. We found that mouse embryonic fibroblasts (MEFs) lacking both Bax and Bak are much more sensitive to radiation than the wild-type (WT) MEFs as demonstrated by clonogenic assay. Following radiation, increased levels of cleaved caspase 3 and higher apoptotic index were detected in the WT cells, but not in the Bax/Bak -/- double knockout (DKO) cells. Punctate fluorescence of GFP-LC3, characteristic of autophagy, was significantly increased in the irradiated DKO cells. Electron microscopy revealed that these cells had numerous autophagosomes and lysosomes. Inhibition of autophagy by 3-methyl adenine (3MA) increased the radiation resistance of Bax/Bak-/- MEF cells. In addition, expression of the pro-autophagic proteins APG5 and Beclin 1 was induced by radiation in the DKO cells. These results indicate that deficiency of Bax and Bak sensitized MEF cells to radiation and upregulated non-apoptotic cell death in response to radiation.