Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells.

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.

Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation.

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr 78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50 values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity.

Hypersensitivity to DNA cross-linking agents associated with up-regulation of glucose-regulated stress protein GRP78.

We have shown previously that NAD/poly(ADP-ribose) polymerase-deficient cells that overexpress Mr 78,000 glucose-regulated stress protein (GRP78) are resistant to topoisomerase II inhibitors, such as etoposide, m-amsacrine, and doxorubicin. However, these cells have been found to be hypersensitive to DNA cross-linking agents, including melphalan, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These observations prompted us to examine whether overexpression of GRP78 is associated with modulation of cytotoxicity of clinically useful DNA-cross-linking agents such as melphalan, BCNU, and cisplatin. We up-regulated GRP78 in V79 Chinese hamster cells by 2-5-fold using two independent approaches that include exposure to 6-aminonicotinamide, or 2-deoxyglucose. Subsequently, these GRP78-overexpressing cells were trypsinized, plated in regular medium without GRP78-inducing agents, and allowed a 5-h attachment time before being treated with melphalan, BCNU, or cisplatin for 1 h to determine clonogenic survivals. In addition, repair of DNA cross-links induced by those agents were determined by alkaline elution assay. Our results show that the GRP78-overexpressing V79 cells are hypersensitive to DNA cross-linking agents compared to the control V79 cells. Furthermore, repair of drug-induced DNA cross-links appears to be considerably slower in these cells relative to that found in control V79 cells. Thus, our results suggest that (a) up-regulation of GRP78 is associated with an impairment of DNA cross-link repair, (b) up-regulation of GRP78 is associated with potentiation of cytotoxicity induced by alkylating and platinating agents, and (c) up-regulation of GRP78 can be considered as a potentially useful tool to modulate the cytotoxicity of clinically useful alkylating and platinating agents.

Insulin and insulin-like growth factor-1 (IGF-1) inhibit repair of potentially lethal radiation damage and chromosome aberrations and alter DNA repair kinetics in plateau-phase A549 cells.

Plateau-phase A549 cells exhibit a high capacity for repair of potentially lethal radiation damage (PLD) when allowed to recover in their own spent medium. Addition of either insulin or insulin-like growth factor-1 (IGF-1) to the spent medium 60 to 120 min before irradiation significantly inhibits PLD repair. The 9-h recovery factor (survival with holding/survival without holding) is reduced from 10.8 +/- 0.7 to 3.4 +/- 0.3 by insulin and to 3.0 +/- 0.4 by IGF-1. Neither growth factor alters the cell age distribution of the plateau-phase cells, increases the rate of incorporation of 5-bromo-2'-deoxyuridine into DNA, or alters the extent of radiation-induced mitotic delay in cells subcultured immediately after irradiation. Both insulin and IGF-1 alter the kinetics for rejoining of DNA double-strand breaks (DSBs), slowing the fast component of rejoining significantly. However, these growth factors have no effect on the initial level of DSBs or on the percentage of residual unrejoined breaks at 120 min postirradiation. Both growth factors affect repair of lesions leading to dicentric, but not to acentric, chromosome aberrations significantly. In control cells (treated with phosphate-buffered saline, 90 min prior to irradiation), the half-time for disappearance of dicentrics was 4.1 h (3.4 to 5.1 h), and 47.1 +/- 3.7% of the residual damage remained at 24 h postirradiation. Insulin and IGF-1 increased the half-time for disappearance of dicentrics to 5.2 h (3.9 to 7.7 h) and 5.7 h (5.5 to 5.9 h), respectively, and increased residual damage to 56.1 +/- 5.9% and 60.8 +/- 6.0%, respectively. Overall, these data show that insulin and IGF-1 inhibit PLD repair in A549 cells by mechanisms which are independent of changes in cell cycle parameters. The data suggest that the growth factors act by inducing changes in chromatin conformation which promote misrepair of radiation-damaged DNA.

Comparison of the effects of hydralazine on tumor and normal tissue blood perfusion by MRI.

PURPOSE: The differential effects on blood perfusion of the vasodilator hydralazine (HYD) between tumor and normal muscle have been measured using the dynamic enhanced-magnetic resonance imaging (DE-MRI) technique. METHODS AND MATERIALS: DE-MRI is a noninvasive method of determining blood perfusion in tumors and normal tissues using the MR contrast agent Gd-DTPA. Hydralazine is currently being used in an attempt to increase tumor response to bioreductive agents and to hyperthermia. RESULTS: We show that a dose of 1.2 mg/kg HYD causes an increase in tumor perfusion while doses > or = 2.5 mg/kg cause a decrease in tumor perfusion. The latter was accompanied by a dose-dependent increase in normal muscle perfusion consistent with the "steal effect." CONCLUSION: This study demonstrates the sensitivity of the DE-MRI technique and its capability of providing estimates of blood perfusion in normal and tumor tissue as well as in smaller regions of a solid tumor. Such features would make it clinically useful in the study of tumor response to radiation therapy and chemotherapy in patients.

Determination of changes in tumor blood perfusion after hydralazine treatment by dynamic paramagnetic-enhanced magnetic resonance imaging.

Magnetic resonance imaging, using the paramagnetic chelate gadopentetate dimeglumine as a perfusing agent, was used to investigate the effect of the vasoactive drug hydralazine on tumor blood perfusion. The method requires measurements of the magnetic resonance image intensity changes with time on a pre-selected region of interest in the tumor image, immediately following intravenous injection of gadopentetate dimeglumine. The present study showed that the initial slope of the intensity-time curve can be used, to a first approximation, to infer tumor blood perfusion. With the dynamic imaging technique, it was demonstrated that, in the KHT sarcoma implanted intramuscularly in the hind leg of C3H/HeN mice, intraperitoneal administration of hydralazine reduced the volume-averaged tumor blood perfusion in a dose-dependent manner. The intrinsically high spatial resolution of magnetic resonance imaging allows a detailed study of the heterogeneous nature of tumor blood perfusion. The potential applications of this imaging technique to study the differential effects of hydralazine on perfusion between tumor and normal tissues will be discussed. The clinical utility of the technique should be promising because of its non-invasive nature.

The dependence of proton longitudinal and transverse relaxation times on cell-cycle phase: mouse MCA-transformed 10T1/2 TCL-15 cells.

Attempts to determine proton NMR longitudinal relaxation times (T1) as a function of cell-cycle stage using cells synchronized by chemical methods have yielded conflicting results (P. T. Beall, C. F. Hazlewood, and P. N. Rao, Science 192, 904 (1976); R. N. Muller et al., FEBS Lett. 114, 231 (1980); D. N. Wheatley, et al., J. Cell Sci. 88, 13 (1987]. This has raised the question whether a true dependence of T1 on cell-cycle phase exists. In the present study, the centrifugal elutriation technique was used to obtain relatively pure, synchronized cell populations of TCL-15 cells (a methylcholanthrene-transformed line of mouse 10T1/2 cells) for measurement of proton NMR relaxation rates. This technique provides a means to procure synchronized cell populations without the use of chemical agents as in the above-cited investigations and therefore avoid possible effects caused by the chemical agents of the NMR relaxation processes. Both T1 and the transverse relaxation time, T2, of water protons in synchronized-cell pellets obtained in this study, exhibited a dependence on cell-cycle phase at least for the first half of the cell cycle (G1 to S). Cells in G1 phase exhibited quantitatively higher T1 and T2 relaxation times compared to those measured for cells in mid S phase. Such changes were found to correlate with changes in water content. The distribution of cell-cycle phases of each cell population was determined by the DNA histogram using flow cytometric methods. Possible relaxation mechanisms which may contribute to the cell-cycle-specific phenomena of the intracellular T1 and T2 times are discussed.

The response of the KHT sarcoma to radiotherapy as measured by water proton NMR relaxation times: relationships with tumor volume and water content.

The potential application of magnetic resonance imaging (MRI) to predict tumor response to radiotherapy is investigated. The water proton spin-lattice and spin-spin relaxation times (T2 and T2, respectively) of murine sarcomas (designated KHT) were measured shortly after excision. This study has demonstrated significantly different responses in T1 and T2 between the control and the irradiated tumors at various times following single doses of X rays. Quite generally, the changes in relaxation times correlated with the changes in tumor water content, indicating that the MR relaxation-time probes are fairly sensitive to radiation-induced edema and dehydration. The possible relationships between the T1 and T2 responses and radiobiological effects such as those on tumor blood flow, vascular permeability, physiological state of cells, and cell death are discussed. It is conceivable that the findings obtained from this investigation could be extended to in situ studies for potential applications in clinical radiotherapy.

Radiation damage to dinucleoside monophosphates: mediated versus direct damage.

The mediation of radiation-induced damage to dinucleoside monophosphate by oxygen and by glutathione was studied. The sequence isomers d(TpA) and d(ApT) were X-irradiated in aqueous solutions and the products isolated by reverse-phase high-performance liquid chromatography. The main products were characterized by proton NMR spectroscopy. In the presence of oxygen the principal products are the formamido derivative formed by breakdown of thymine and the aldehyde derivative formed at the 5' end of the dinucleoside monophosphate, both nucleoside monophosphates and free bases. In the presence of glutathione, the two stereoisomers of the 5,6-dihydrothymine derivatives are prominent. Radiation-induced damage to d(TpA) and d(ApT) in the solid state was also studied.

Radiation chemistry of a dinucleoside monophosphate and its sequence isomer.

A combination of high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy was used to analyze the products of X-irradiated aqueous solutions of the dinucleoside monophosphate thymidylyl(3'-5')-2'-deoxyadenosine, d(TpA), and its sequence isomer 2'-deoxyadenylyl(3'-5')thymidine, d(ApT). The products of d(TpA) include both bases and nucleotides and a variety of thymine modifications of d(TpA) including the two cis and two trans glycol stereoisomers, two cis monohydroxy derivatives, an N-formamide derivative, and the hydroxymethyl derivative. Attention is focused on using NMR spectral features to distinguish among the various stereoisomers. The radiation chemistry of d(ApT) is also explored and differences in product formation compared with d(TpA) are described, particularly the formation of two products involving modification of adenine base. The potential of the HPLC-NMR approach to the study of radiation chemistry in DNA model compounds is discussed.

HPLC-n.m.r. studies of radiation damage to d(TpA).

Nine of the products isolated from X-irradiated aqueous solutions of d(TpA) by HPLC are identified from their proton n.m.r. spectra.