The radiosensitivity of endothelial cells isolated from human breast cancer and normal tissue in vitro.

We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd-5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced suppressions of migration and capillary-like tube formations were greater for CECs than NECs from the same patients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients.

Hyperthermia improves therapeutic efficacy of doxorubicin carried by mesoporous silica nanocontainers in human lung cancer cells.

PURPOSE: We investigated the use of hyperthermia to improve the anti-cancer efficacy of doxorubicin (DOX)-loaded mesoporous silica nanocontainer Si-SS-CD-PEG. The hypothesis was that heat stimulates glutathione-mediated degradation of cyclodextrin gatekeeper, thereby causing the release of DOX from the carrier and DOX-induced cell death. MATERIALS AND METHODS: The release of DOX from DOX-loaded Si-SS-CD-PEG suspended in PBS containing glutathione (GSH) was studied by assessing the changes in DOX fluorescence intensity. The effect of heating at 42°C on the release of DOX from the intracellular carriers was determined with confocal microscopy. The extents of clonogenic and apoptotic cell death caused by DOX-loaded Si-SS-CD-PEG were determined. RESULTS: The release of DOX from DOX-loaded Si-SS-CD-PEG in PBS occurred only when GSH presented in the suspension, and heating at 42°C slightly increased the release of DOX from the carriers. Heating significantly elevated the GSH content in A549 cells and increased the release of DOX from the internalised carriers. Heating the cancer cells treated with the carriers at 42°C markedly increased the clonogenic death and apoptosis. The GSH content in A549 cells was greater than that in L-132 cells, and A549 cells were far more sensitive than L-132 cells to DOX-loaded Si-SS-CD-PEG at both 37°C and 42°C. CONCLUSIONS: Hyperthermia increased the GSH-mediated release of DOX from DOX-loaded Si-SS-CD-PEG. Furthermore, hyperthermia markedly elevated the GSH content in cancer cells, thereby increasing the release of DOX from the internalised carriers and potentiating the DOX-induced clonogenic and apoptotic cell death.

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.

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

Endoplasmic reticulum stress-induced JNK activation is a critical event leading to mitochondria-mediated cell death caused by ß-lapachone treatment.

BACKGROUND: ß-Lapachone (ß-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although ß-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which ß-lap causes apoptosis are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: ß-Lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in ß-lap-induced apoptosis were used to clarify the roles played by such factors in ß-lap-activated apoptotic signaling pathways. ß-Lap leads to clonogenic cell death and apoptosis in an NQO1-dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with ß-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate ß-lap-induced clonogenic and apoptotic cell death. Moreover, ß-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by ß-lap, and subsequent mitochondria-mediated cell death. In addition, ß-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition. CONCLUSIONS/SIGNIFICANCE: Our results indicate that ß-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies.

Aurora-a contributes to radioresistance by increasing NF-kappaB DNA binding.

Aurora-A, a serine/threonine kinase that is overexpressed in certain human cancer cell lines, plays an important role in mitotic progression. Aurora-A has also been reported to be involved in the activation of nuclear factor kappa B (NF-kappaB). The purpose of the present study was to identify the role of Aurora-A in the radiation-induced activation pathway of NF-kappaB. Wild-type and Aurora-A knockdown (Aurora-A(KD)) HeLa cells were irradiated with 4 Gy of gamma rays and the EMSA, luciferase reporter gene assay and immunoblot analysis were performed. The siRNA-based gene knockdown and overexpression system was adopted to elucidate the role of Aurora-A in radiation-induced NF-kappaB pathway activation. The clonogenic survival study indicated that Aurora-A(KD) cells and the wild-type cells transfected with Aurora-A siRNA or RelA/p65 siRNA were more radiosensitive than the wild-type cells. In both the wild-type and Aurora-A(KD) cells, radiation caused IkappaB kinase-mediated phosphorylation, degradation of IkappaBalpha and phosphorylation of RelA/p65. The nuclear translocation of RelA/p65 was also similar in the wild-type and Aurora-A(KD) cells. However, RelA/p65-DNA binding was markedly suppressed in Aurora-A(KD) cells compared to that in wild-type cells. It was concluded that Aurora-A enhances the binding of NF-kappaB to DNA, thereby increasing the gene transcription by NF-kappaB and decreasing the radiosensitivity of the cells.

Anti-cancer effect of bio-reductive drug beta-lapachon is enhanced by activating NQO1 with heat shock.

PURPOSE: Bio-reduction/activation of anti-cancer drug beta-lapachone (beta-lap) is mediated by NAD(P)H: Quinone oxidoreductase (NQO1). We investigated the feasibility of using mild temperature hyperthermia to increase the anti-cancer effect of beta-lap by up-regulating NQO1 expression. METHODS: NQO1 expression in FSaII fibrosarcoma of C3H mice and A549 human lung cancer cells was evaluated with Western blot analysis and immunostaining of cells at different times after water-bath heating. Clonogenic cell survival method was used to determine the sensitivity of cells to heating, beta-lap, and in combination. The growth of FSaII tumors in the right hind legs of C3H mice was studied after heating the tumors at 42 degrees C for 1 h with water bath, an i.p. injection of beta-lap to host mice or an i.p. injection of beta-lap 24 h after heating the tumors. RESULTS: Heating at 42 degrees C for 1 h significantly increased the expression of NQO1 in the cancer cells with a maximum increase occurring 8-24 h after heating. The sensitivity of cancer cells to beta-lap treatment progressively increased until 24 h after heating most likely due to the increase in NQO1 expression. Heating the FSaII tumors at 42 degrees C for 1 h and treating the host mice with an i.p. injection of 50 mg/kg beta-lap 24 h after the tumor heating was far more effective than heating alone or beta-lap treatment alone to suppress the tumor growth. CONCLUSION: Mild temperature heat shock elevates the NQO1 expression in cancer cells, which in turn markedly increases the sensitivity of the cells to the bioreductive drug beta-lap in vitro and in vivo.

Synergistic effect of ionizing radiation and beta-Lapachone against RKO human colon adenocarcinoma cells.

PURPOSE: To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-Lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation-induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-Lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against cancer cells.

Characterization of monoclonal antibody reacting exclusively against intracellular Orientia tsutsugamushi.

Intracellular bacteria often change the expression of their genes in order to adapt to new environmental conditions. Here we describe a monoclonal antibody (MAb) that reacts exclusively against intracellular Orientia tsutsugamushi. Although MAb applied to the 56-kDa protein, a major outer membrane protein, reacted against a large number of bacteria that had attached to host cells at the early stage of infection, M686-13 reacted against only a minor portion of the attached bacteria. In the later stage of the intracellular growth cycle, both antibodies showed identical staining patterns by double immunofluorescent staining. These results suggest that M686-13 reacted to an epitope or a protein that had probably been expressed during the intracellular growth cycle and rapidly diluted or degraded upon release into the extracellular environment. Although its molecular characteristics remain unknown, the reactive antigen may prove to be a novel developmental antigen and this MAb could be used as reagent for the staining of viable O. tsutsugamushi.

Application of monoclonal antibody, specific for intracellular Orientia tsutsugamushi, to immunofluorescent antibody test for determining antibiotic susceptibility.

The simple quantification of viable intracellular bacteria is important for the study of an obligate intracellular bacterium, Orientia tsutsugamushi. We applied a novel monoclonal antibody (M686-13)--specific for intracellular Orientia--to an immunofluorescent antibody (IFA) test for determining antibiotic susceptibility of O. tsutsugamushi. M686-13 did not react with Orientia that was inhibited by doxycycline, although bacterial particles still remained in the cells. This preferential staining of proliferating bacteria made the IFA test rapid and precise. Using this method, we could successfully measure the minimal inhibitory concentration (MIC) of a Korean strain of O. tsutsugamushi to doxycycline and clindamycin. This method may be used in other procedures to evaluate the growth of Orientia.

Orientia tsutsugamushi inhibits apoptosis of macrophages by retarding intracellular calcium release.

Orientia tsutsugamushi shows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited by O. tsutsugamushi infection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected with O. tsutsugamushi. Antiapoptotic activities of O. tsutsugamushi in infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.