Biomedical subjects

Uma Raju

Publications and source records attributed to Uma Raju.

9 recordsLinked to original sources

PubMed2005

Inhibition of DNA repair as a mechanism of enhanced radioresponse of head and neck carcinoma cells by a selective cyclooxygenase-2 inhibitor, celecoxib.

PURPOSE: Previously, we reported that inhibitors of cyclooxygenase-2 (COX-2) enzyme enhanced murine and human tumor cell response to radiation in vitro and in vivo. However, the molecular mechanisms mediating the effects of COX-2 inhibitors are not clear. The present study was designed to investigate the ability of celecoxib, a selective COX-2 inhibitor, to sensitize human head-and-neck cancer cell line, HN5, to radiation, and examine its effects on DNA repair, which may be a potential mechanism of radiosensitization. METHODS AND MATERIALS: Cells were assessed for the effect of celecoxib (5-50 microM), by 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyltetrazolium bromide assay for growth inhibition and by clonogenic cell survival assay for the radiosensitizing effect. Kinase assay and Western analysis were conducted to assess the effect of celecoxib on DNA-dependent protein kinase catalytic subunit (PKcs) and Ku proteins. Electrophoretic mobility shift assays (EMSA) were performed to determine the DNA-binding activity of Ku/DNA-PKcs protein complex and nuclear factor kappa B (NFkappaB). RESULTS: Celecoxib (10 and 50 microM, for 2 days) inhibited the HN5 cell growth and significantly enhanced the cell radiosensitivity in a dose-dependent manner. It also reduced the shoulder region on the radiation-survival curve, suggesting that inhibition of DNA repair processes may have occurred. Western blot analysis demonstrated that celecoxib downregulated the expression of Ku70 protein and inhibited the kinase activity of DNA-PKcs, which are involved in the double-stranded DNA-break repair machinery. By EMSA, it was further shown that celecoxib reduced DNA-binding activity of Ku/DNA-PKcs protein complex. In addition, celecoxib inhibited the constitutively active NFkappaB and the radiation-induced NFkappaB in HN5 cells, suggesting that NFkappaB may play a role in mediating the effects of celecoxib. CONCLUSIONS: Celecoxib strongly enhanced the sensitivity of HN5 carcinoma cells to radiation, which, mechanistically, can be attributed to the inhibition of DNA repair processes in radiation-damaged cells.

Blotting, Western↗

PubMed2005

Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase.

Ionizing radiation, but not stimulation with epidermal growth factor (EGF), triggers EGF receptor (EGFR) import into the nucleus in a probably karyopherin alpha-linked manner. An increase in nuclear EGFR is also observed after treatment with H2O2, heat, or cisplatin. During, this process, the proteins Ku70/80 and the protein phosphatase 1 are transported into the nucleus. As a consequence, an increase in the nuclear kinase activity of DNA-dependent kinase (DNA-PK) and increased formation of the DNA end-binding protein complexes containing DNA-PK, essential for repair of DNA-strand breaks, occurred. Blockade of EGFR import by the anti-EGFR monoclonal antibody C225 abolished EGFR import into the nucleus and radiation-induced activation of DNA-PK, inhibited DNA repair, and increased radiosensitivity of treated cells. Our data implicate a novel function of the EGFR during DNA repair processes.

Active Transport, Cell Nucleus↗

PubMed2004

Flavopiridol increases therapeutic ratio of radiotherapy by preferentially enhancing tumor radioresponse.

PURPOSE: Recently we reported that inhibition of cyclin-dependent kinases (cdks) by flavopiridol enhanced the radiation response of murine ovarian carcinoma cells in culture. The purpose of this investigation was to extend these studies to in vivo tumor models and test whether flavopiridol increases the therapeutic ratio of radiotherapy. METHODS AND MATERIALS: Three transplantable syngeneic mouse tumors were used: mammary carcinoma (MCa-29), ovarian carcinoma (OCa-I), and a lymphoma (Ly-TH). Tumor treatment endpoints included growth delay, cure, and spontaneous lung metastases (OCa-I tumor). The normal tissue endpoint was survival of jejunal crypt cells quantified microscopically. A range of flavopiridol doses from 0.625 to 5.0 mg/kg were given systemically once or twice daily over 5, 10, or 20 days. Combined therapy flavopiridol treatments were initiated either several days before or shortly after the start of single dose or daily fractionated radiotherapy. RESULTS: The major findings of this study are that all three tumors treated with flavopiridol alone responded by tumor growth delay. Two of the tumors (MCa-29 and Ly-TH) responded in a schedule-dependent manner with larger radiation enhancement factors when flavopiridol treatment was started a few hours after irradiation (radioenhancement factors [EF] Ly-TH = 2.04, EF MCa-29 = 1.50 for single dose irradiation). When combined with fractionated irradiation (2.6 Gy daily for 10 or 20 days), flavopiridol enhanced the response of the MCa-29 tumor by a factor of 1.25-1.46. A fractional radiation dose of 6 Gy in combination with flavopiridol produced a 62.5% cure rate compared with 25% tumor cure for radiation alone. A novel finding of this study was the demonstration of antimetastatic activity of flavopiridol in addition to its effect on the local primary tumor. Both the incidence and absolute number of lung metastasis were reduced when flavopiridol followed surgical removal of the large (10 mm) primary leg tumor. The normal jejunum treated with flavopiridol and radiation responded in a schedule independent manner and the degree of radioenhancement (EF, 1.05-1.06) was much less than for any of the tumors studied. CONCLUSIONS: Therapeutic gain was achieved when flavopiridol treatment was initiated either before or after the start of radiotherapy. Flavopiridol shows promising clinical potential administered alone or in combination with other cytotoxic agents, including both chemotherapy and radiotherapy.

Animals↗

PubMed2004

Predicting radiosensitivity using DNA end-binding complex analysis.

Previous reports have suggested that measuring radiosensitivity of normal and tumor cells would have significant clinical relevance for the practice of radiation oncology. We hypothesized that radiosensitivity might be predicted by analyzing DNA end-binding complexes (DNA-EBCs), which form at DNA double-strand breaks, the most important cytotoxic lesion caused by radiation. To test this hypothesis, the DNA-EBC pattern of 21 primary human fibroblast cultures and 15 tumor cell lines were studied. DNA-EBC patterns were determined using a modified electrophoretic mobility shift assay and were correlated with radiosensitivity, as measured by SF2. DNA-EBC analysis identified a rapidly migrating ATM-containing band (identified as "band-A") of which the density correlated with SF2 (0.02 </= SF2 </= 0.41) in primary fibroblasts (r(2) = 0.77). The DNA-EBC pattern of peripheral blood lymphocytes was identical to that of fibroblasts. In addition, band-A density correlated with SF2 (0.35 </= SF2 </= 0.80) in 15 human tumor cell lines (r(2) = 0.91). Densitometry of other bands, or total DNA-EBC binding, correlated more poorly with SF2 (r(2) < 0.45). These data indicate that DNA-EBC analysis may be a practical, clinically relevant predictor of tumor and primary cell radiosensitivity.

Cell Line↗

PubMed2004

Potentiation of tumor response to radiation or chemoradiation by selective cyclooxygenase-2 enzyme inhibitors.

Cyclooxygenase-2 (COX-2) is an enzyme expressed primarily in pathologic states, such as inflammatory disorders and cancer, where it mediates prostaglandin production. Its overexpression is associated with more aggressive biologic tumor behavior and adverse patient outcome. Increasing evidence shows that agents that selectively inhibit COX-2 enhance tumor response to radiation or chemotherapeutic agents. This article gives an overview of some of this evidence. In addition, we describe new results showing that celecoxib, a selective COX-2 inhibitor, enhanced response of A431 human tumor xenografts in nude mice to radiation by an enhancement factor (EF) of 1.43 and to the chemotherapeutic agent docetaxel by an EF of 2.07. Celecoxib also enhanced tumor response when added to the combined docetaxel plus radiation treatment (EF = 2.13). Further experiments showed that selective COX-2 inhibitors enhanced tumor cell sensitivity to ionizing radiation, involving inhibition of cellular repair from radiation damage and cell cycle redistribution as mechanisms for some cell types. The results show that selective COX-2 inhibitors have the potential to improve tumor radiotherapy or radiochemotherapy, and this therapeutic strategy is currently under clinical testing.

Animals↗

PubMed2003

Flavopiridol, a cyclin-dependent kinase inhibitor, enhances radiosensitivity of ovarian carcinoma cells.

Flavopiridol, a cyclin-dependent kinase (cdk) inhibitor, can cause cell cycle arrest, induce apoptosis in cancer cells, and inhibit tumor cell growth in vivo. The present study investigated the in vitro radiosensitizing effect of flavopiridol and the underlying molecular mechanisms in a murine ovarian cancer cell line, OCA-I. Flavopiridol inhibited cell growth in a dose-dependent manner and enhanced cell radiosensitivity assessed by the clonogenic cell survival assay. A flavopiridol dose of 300 nM, given for 1 day, enhanced radiosensitivity by a factor of 2.1. Clonogenic cell survival after split-dose radiation showed that flavopiridol inhibited repair from radiation damage. In addition, flavopiridol treatment (300 nM, 1 day) resulted in decreased levels of Ku70 and Ku86 proteins that play a role in DNA repair processes, suggesting that DNA repair processes may have been disrupted by this agent. Flow cytometry analysis showed that flavopiridol (300 nM, 1 day) accumulated the cells in G(1) and G(2) phases, with a significant reduction in the S phase component. This cell cycle redistribution is likely another mechanism underlying flavopiridol-induced cell radiosensitivity. Flavopiridol down-regulated cyclin D1 and cyclin E protein levels and also inhibited phosphorylation of retinoblastoma protein, which is inconsistent with the observed cell cycle arrest. Among the cdks tested, cdk-9, the catalytic subunit of positive transcription elongation factor b, was significantly down-regulated by flavopiridol, suggesting that flavopiridol may modulate cellular transcription processes. Furthermore, flavopiridol on its own induced apoptosis in the OCA-I cells, whereas in combination with radiation, exerted no additional increase in apoptosis. Taken together, our data show that flavopiridol strongly augmented the response of ovarian carcinoma cells to radiation and that the underlying mechanisms included inhibition of sublethal DNA damage repair and cell cycle redistribution. At the molecular level, transcriptional regulation by flavopiridol may have been involved.

Animals↗

PubMed2002

Enhancement of radiotherapy by oleandrin is a caspase-3 dependent process.

Cardiac glycosides such as digitoxin and ouabain have previously been shown to be selectively cytotoxic to tumor as opposed to normal cells. Moreover, this class of agents has also been shown to act as potent radiosensitizers. In the present study we explored the relative radiosensitization potential of oleandrin, a cardiac glycoside contained within the plant extract known as Anvirzel that recently underwent a Phase I trial as a novel drug for anticancer therapy. The data show that oleandrin produces an enhancement of sensitivity of PC-3 human prostate cells to radiation; at a cell survival of 0.1, the enhancement factor was 1.32. The magnitude of radiosensitization depended on duration of exposure of cells to drug prior to radiation treatment. While a radiosensitizing effect of oleandrin was evident with only 1h of cell exposure to drug, the effect greatly increased with 24h oleandrin pretreatment. Susceptibility of PC-3 cells to oleandrin and radiation-induced apoptosis was dependent on activation of caspase-3. Activation was greatest when cells were exposed simultaneously to oleandrin and radiation. Inhibition of caspase-3 activation with Z-DEVD-FMK abrogated the oleandrin-induced enhancement of radiation response suggesting that both oleandrin and radiation share a caspase-3 dependent mechanism of apoptosis in the PC-3 cell line.

Adenocarcinoma↗

PubMed2002

In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme: mechanistic considerations.

PURPOSE: Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor response to radiation in vivo, but the cellular mechanisms underlying the radiosensitizing effect are not understood. In the present study, we investigated several possible mechanisms using a murine sarcoma cell culture system. METHODS AND MATERIALS: Cells derived from a murine sarcoma, designated NFSA, were cultured in vitro and exposed to different (either single or split) doses of radiation with and without a pretreatment of SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-l-yl] benzene sulfonamide), a selective cyclooxygenase-2 (COX-2) inhibitor. The cells were assayed for clonogenic survival to determine the radiosensitizing effect of SC-236. In addition, MTT assay and TUNEL assay were performed to determine the effects of SC-236 and radiation on the cell survival and cell cycle distribution. RNase protection assay was performed on the total RNA extract using probes that encoded for selected cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases. To monitor the extent of COX-2 activity and its role in radiosensitization, the cellular content of prostaglandin E2, a major metabolite of COX-2 activity on arachidonic acid, was also determined. RESULTS: The cell clonogenic survival assay showed that SC-236 significantly enhanced tumor cell radiosensitivity: 50 microM SC-236 increased it by a factor of 1.51 at the 0.1 cell survival level. Treatment with SC-236 (50 microM, 3 days) removed the "shoulder" region on the radiation survival curve, suggesting that the drug inhibited repair of sublethal radiation damage. The inhibition was confirmed by split-dose experiments where two doses (3 Gy each) of radiation were given 4 h apart. The cells exposed to radiation only repaired the damage by a factor of 1.44, whereas those treated with SC-236 plus radiation repaired it by a factor of 1.1 only. Whereas SC-236 induced apoptosis in these NFSA cells, radiation did not. No further increase in apoptosis was observed when the cells were exposed to both SC-236 and radiation, suggesting that SC-236 did not render tumor cells more susceptible to radiation-induced apoptosis. The RNase protection assay showed that SC-236 (50 microM, 3 days) inhibited the expression of cyclins A and B, as well as cyclin-dependent kinase-1. Inhibition of these cell cycle regulatory elements by SC-236 was associated with the arrest of cells in the radiosensitive G2-M phase (67%), determined by flow cytometry. CONCLUSIONS: SC-236 significantly enhanced radiosensitivity of tumor cells; the magnitude of sensitivity was dependent on the drug's concentration. The likely mechanisms involve accumulation of cells in the radiosensitive G2-M phase of the cell cycle and inhibition of repair from sublethal radiation damage.

Animals↗

PubMed2002

Inhibition of radiation-induced nuclear factor-kappaB activation by an anti-Ras single-chain antibody fragment: lack of involvement in radiosensitization.

We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-kappaB (NF-kappaB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-kappaB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IkappaB kinase activity and IkappaBalpha phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-kappaB. Whereas AV1Y28 inhibited NF-kappaB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-alpha-induced NF-kappaB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-kappaB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-kappaB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-kappaB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-kappaB activation. However, inhibition of NF-kappaB activation via the expression of a dominant-negative form of IkappaBalpha in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-kappaB activation, this process does not appear to play a direct role in its radiosensitizing actions.

DNA-Binding Proteins↗