The usefulness of a continuous administration of tirapazamine combined with reduced dose-rate irradiation using {gamma}-rays or reactor thermal neutrons.

We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using gamma-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using gamma-rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.

The usefulness of continuous administration of hypoxic cytotoxin combined with mild temperature hyperthermia, with reference to effects on quiescent tumour cell populations.

To evaluate the usefulness of continuous administration of hypoxic cytotoxins in terms of targeting acute hypoxia in solid tumours and the significance of combination with mild temperature hyperthermia (MTH) (40 degrees C, 60 min), the cytotoxic effects of singly or continuously administered tirapazamine (TPZ) and TX-402 were examined in combination with or without MTH in vivo. Further, the effects were also analysed on total (=proliferating (P)+quiescent (Q)) and Q cell populations in solid tumours with the method for selectively detecting the Q cell response. C3H/He mice bearing SCC VII tumours received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all P cells. The tumour-bearing mice then received a single intra-peritoneal injection or 24 h continuous subcutaneous infusion of hypoxic cytotoxin, TPZ or TX-402, with or without MTH. On the other hand, to detect the changes in the hypoxic fraction (HF) in the tumours by MTH, another group of mice with or without MTH received a series of test doses of gamma-rays while alive or after tumour clamping. After each treatment, the tumour cells were isolated and incubated with a cytokinesis blocker (=cytochalasin-B) and the micronucleus (MN) frequency in cells without BrdU labelling (=Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total tumour cells was determined from the tumours that were not pre-treated with BrdU. The sensitivity to TX-402 was slightly higher than that to TPZ in both total and Q tumour cells. Continuous administration elevated the sensitivity of both total and Q cells, especially total cells. MTH raised the sensitivity of Q cells more remarkably than that of total cells in both single and continuous administrations. It was thought to be probably because of the higher dose distribution of hypoxic cytotoxin in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. From the viewpoint of tumour control as a whole including both total and Q tumour cells, the continuous administration of hypoxic cytotoxin combined with MTH may be useful for sensitizing tumour cells in vivo.

Evaluation of the potential of p-boronophenylalaninol as a boron carrier in boron neutron capture therapy, referring to the effect on intratumor quiescent cells.

C57BL mice bearing EL4 tumors and C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p-boronophenylalanine-(10)B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-(10)B (BSH) or l-p-boronophenylalaninol (BPA-ol), a newly developed (10)B-containing alpha-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and / or tirapazamine (TPZ), the tumors were heated at 40 degrees C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the (10)B uptake of all (10)B-compounds into both tumor cells. BPA-ol has good potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

Combined effects of tirapazamine and mild hyperthermia on anti-angiogenic agent (TNP-470) treated tumors-reference to the effect on intratumor quiescent cells.

PURPOSE: To evaluate the efficacy of the use of tirapazamine (TPZ), especially combined with mild hyperthermia (40 degrees C, 60 min), in the treatment of solid tumors following an anti-angiogenic treatment with TNP-470. In addition, we assessed the effect of TPZ and/or mild hyperthermia (MHT) combined with conventional radiotherapy or chemotherapy on TNP-470 treated tumors. MATERIALS AND METHODS: C3H/He mice bearing SCC VII tumors subcutaneously received TNP-470 at two doses of 100 mg/kg after tumor cell inoculation. At the same time, the tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received TPZ administration combined with or without MHT, gamma-ray irradiation combined with or without TPZ and/or MHT, or cisplatin injection with or without TPZ and/or MHT. Another group of mice received a series of test doses of gamma-rays while alive or after being killed to obtain hypoxic fractions (HFs) in the tumors at various time points after the above-mentioned cytotoxic treatment point. After each treatment, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (or quiescent [Q] cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. For the measurement of the HFs, the MN frequency of BrdU-unlabeled cells was then used to calculate the surviving fraction of the unlabeled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumor cells. RESULTS: TPZ administration combined with TNP-470 treatment and MHT increased the MN frequency more markedly than treatment with TPZ alone, and this tendency was more remarkable in Q cells than total cells. In both total and Q cells, combined treatment with TPZ and MHT produced significant increases in MN frequencies whether gamma-rays were delivered to TNP-470 treated tumors or cisplatin was injected into the TNP-470 administered mice. Although not significantly, the HFs of total and Q cell populations within solid tumors increased after TNP-470 treatment. CONCLUSION: Combined treatment with TPZ and MHT, whether other cytotoxic treatments such as gamma-ray irradiation or chemotherapy using cisplatin were combined or not, was useful for sensitizing tumor cells in vivo including Q cells even after TNP-470 treatment.

Usefulness of tirapazamine as a combined agent in chemoradiation and thermo-chemoradiation therapy at mild temperatures: reference to the effect on intratumor quiescent cells.

C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received one of six different DNA-damaging agents with or without mild temperature hyperthermia (40 degrees C, 30 min, MTH). These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ). After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of gamma-rays. Immediately after irradiation, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that had not been pretreated with BrdU. MTH significantly increased the MN frequency of total cells in tumors irradiated with gamma-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with gamma-rays combined with BLM or TPZ. The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ. TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.

DNA sensing on a DNA probe-modified electrode using ferrocenylnaphthalene diimide as the electrochemically active ligand.

Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.

Apoptosis induction in HCT116 cells by cytochalasins isolated from the fungus Daldinia vernicosa.

We examined the induction of apoptosis by cytochalasin (cc) derivatives (1-14) isolated from the Japanese fungus Daldinia vernicosa to HCT116 human colon cancer cell line based on their cytotoxicity, DNA ladder and DNA fragmentation ratio in agarose gel electrophoresis, and morphological changes. Most cc derivatives tested here induced apoptosis. Particularly cytochalasin 1 (cc1), monoacetate of 1 (cc1Ac), and cc14 were the most potent apoptosis inducers. These apoptotic activities were stronger than that of cytochalasin D as a known apoptosis inducer in HCT116 cell. However, cc4 and cc12 induced necrosis. The structure-activity relationship including their cytotoxicity will be discussed.

Modification of tirapazamine-induced cytotoxicity in combination with mild hyperthermia and/or nicotinamide: reference to effect on quiescent tumour cells.

C3H/He and Balb/c mice bearing SCC VII or EMT6/KU tumours received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all proliferating (P) cells. The tumours were locally heated at 40 degrees C for 60 min and/or the tumour-bearing mice received intraperitoneal injection of nicotinamide, and then tirapazamine (TPZ) was injected intraperitoneally. Sixty minutes after TPZ injection, the tumours were excised, minced and trypsinized. The tumour cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labelling (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumour cells was determined from the tumours that were not pretreated with BrdU. The cytotoxicity of TPZ was evaluated in terms of the frequency of induced micronuclei in binuclear tumour cells (= MN frequency). In both tumour systems, the MN frequencies of Q cells were greater than those of total tumour cell populations. Mild heat treatment elevated the MN frequency in total and Q cells in both tumour systems, but the effect was more marked in Q cells. In total cells, mild heat treatment increased the MN frequency in EMT6/KU tumour cells more markedly than in SCC VII tumour cells. In contrast, in both tumour systems, nicotinamide decreased the MN frequency in both cell populations, with a greater influence on the total cells. The combination of TPZ and mild heat treatment may be useful for sensitizing tumour cells in vivo, including Q cells.

Enhancement of cisplatin sensitivity of quiescent cells in solid tumors by combined treatment with tirapazamine and low-temperature hyperthermia.

We examined the enhanced chemosensitivity of quiescent (Q) cells in solid tumors to cis-diamminedichloroplatinum (II) (cisplatin) by combined treatment with tirapazamine (TPZ) and mild heating. C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. TPZ was administered intraperitoneally 2 h before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection. Sixty minutes after cisplatin injection, the tumors were excised, minced and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). Other groups of tumor-bearing C3H/He and Balb/c mice not given BrdU were injected with 195mPt-radiolabeled cisplatin. In both tumor systems, the MN frequency in Q cells was lower than that in the total cells. TPZ and mild heat treatment elevated the MN frequency in total and Q cells in both tumor systems, and to a higher extent in Q cells. The combination of TPZ and mild heat treatment increased the MN frequency more markedly than treatment with either TPZ or mild heating alone. In total tumor cells, TPZ and mild heat treatment increased the MN frequency in EMT6/KU tumor cells more markedly than in SCC VII tumor cells. 195mPt-labeled cisplatin uptake into total tumor cells was increased by mild heat treatment but not by TPZ. The cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems. TPZ was thought to sensitize Q cells by killing the hypoxic cells without influencing tumor blood flow, and mild hyperthermia appeared to sensitize Q cells by distributing more cisplatin with an increase in blood flow in solid tumors.