Costus pictus extracts stimulate insulin secretion from mouse and human islets of Langerhans in vitro.

Plant-derived extracts have been used as folk remedies for Type 2 diabetes mellitus (T2DM) for many centuries, and offer the potential of cheap and readily available alternatives to conventional pharmaceuticals in developing countries. Extracts of Costus pictus (CP), a plant belonging to the Costaceae family, are reported to have antidiabetic activity in vivo. The exact molecular mode of action(s) of CP is unclear but the antihyperglycemic effect seen in animal studies was associated with dramatic increases in insulin secretion so in our study we have measured the effect of aqueous CP extract on insulin secretion in vitro from the MIN6 ß-cell line and isolated mouse and human islets. Our data demonstrate that CP has a direct stimulatory effect on insulin secretion at basal but not stimulatory glucose concentrations which was not associated with compromised membrane integrity or decrease ß-cell viability. Single cell calcium microfluorimetry measurements showed that CP caused elevations in ß-cell intracellular Ca(2+) concentrations ([Ca(2+)](i)), an effect which was completely abolished by the removal of extracellular Ca(2+) or blockade of voltage-gated Ca(2+) channels (VGCC). These in vitro observations suggest that one mode of action of CP is through stimulating insulin secretion which may be mediated, in part, by the ability of CP to increase [Ca(2+)](i) levels through VGCC. CP extracts may provide an affordable and inexpensive alternative for treating patients with T2DM.

Low-dose radiation hypersensitivity in human tumor cell lines: effects of cell-cell contact and nutritional deprivation.

The hyper-radiosensitivity at low doses recently observed in vitro in a number of cell lines is thought to have important implications for improving tumor radiotherapy. However, cell-cell contact and the cellular environment influence cellular radiosensitivity at higher doses, and they may alter hyper-radiosensitivity in vivo. To confirm this supposition, we investigated the effects of cell density, multiplicity and nutritional deprivation on low-dose hypersensitivity in vitro. Cell survival in the low-dose range (3 cGy to 2 Gy) was studied in cells of two human glioma (BMG-1 and U-87) and two human oral squamous carcinoma (PECA-4451 and PECA-4197) lines using a conventional macrocolony assay. The effects of cell density, multiplicity and nutritional deprivation on hyper-radiosensitivity/induced radioresistance were studied in cells of the BMG-1 cell line, which showed prominent hypersensitivity and induced radioresistance. The induction of growth inhibition, cell cycle delay, micronuclei and apoptosis was also studied at the hyper-radiosensitivity-inducing low doses. Hyper-radiosensitivity/induced radioresistance was evident in the cells of all four cell lines to varying extents, with maximum sensitivity at 10-30 cGy, followed by an increase in survival up to 50 cGy-1 Gy. Both the glioma cell lines had more prominent hyper-radiosensitivity than the two squamous carcinoma cell lines. Low doses inducing maximum hyper-radiosensitivity did not cause significant growth inhibition, micronucleation or apoptosis in BMG-1 cells, but a transient G(1)/S-phase block was evident. Irradiating and incubating BMG-1 cells at high density for 0 or 4 h before plating, as well as irradiating cells as microcolonies, reduced hyper-radiosensitivity significantly, indicating the role of cell-cell contact-mediated processes. Liquid holding of BMG-1 cells in HBSS + 1% serum during and after irradiation for 4 h significantly reduced hyper-radiosensitivity, suggesting that hyper-radiosensitivity may be due partly to active damage fixation processes at low doses. Therefore, our findings suggest that the damage-induced signaling mechanisms influenced by (or mediated through) cell-cell contact or the cellular environment, as well as the lesion fixation processes, play an important role in hyper-radiosensitivity. Further studies are required to determine the exact nature of the damage that triggers these responses as well as for evaluating the potential of low-dose therapy.

DNA ligand Hoechst-33342 enhances UV induced cytotoxicity in human glioma cell lines.

The effects of minor groove binding ligand bisbenzimidazole derivative Hoechst-33342 on the cellular response to UV damage have been studied in two human glioma cell lines BMG-1 and U-87 grown as monolayer cultures. Treatment induced cell death (macro-colony assay) and growth inhibition, potential lethal damage recovery, cytogenetic damage (micronuclei formation) and proliferation kinetics were studied as parameters for cellular response. Pre and post-irradiation treatment with Hoechst-33342 (1-20 microM) enhanced the UV-induced growth inhibition and cell death in a concentration dependent manner in both cell lines. At higher Hoechst-33342 concentrations (>5 microM), the cytotoxic effects of the combination (Hoechst-33342+UV) were highly synergistic and mainly mediated through apoptosis implying the possible interactions of lesions caused by both the agents. The enhanced cell death due to Hoechst-33342 was accompanied by a significant increase (2-3 folds at 5 microM) in UV-induced micronuclei formation in BMG-1 cells. Under these conditions, Hoechst-33342 also enhanced the UV-induced cell cycle delay, mainly due to S and G(2) blocks. The increase in UV-induced micronuclei formation observed after treatment with Hoechst-33342 indicates that the DNA bound Hoechst-33342 may interfere with the rejoining of DNA strand breaks. Since the treatment of cells with the replication inhibitor aphidicolin reduced the enhancement of UV induced cytotoxicity by Hoechst-33342, ongoing DNA replication appears to stimulate Hoechst-33342 and UV-induced cytotoxicity.

Preparation and comparative evaluation of 99mTc-labeled 2-iminothiolane modified antibodies and CITC-DTPA immunoconjugates of anti-EGF-receptor antibodies.

The use of antibodies as targeting agents for the delivery of radioisotopes to tumors is a promising concept that has received widespread attention since the advent of monoclonal antibody (mAb) technology. The following studies are described in this article: the 99mTc-randiolabeling of 2-iminothiolane (2-IT) modified antibodies and 6-p-isothiocyanatobenzyl- diethylene-triamine penta-acetic acid (CITC-DTPA) immunoconjugates of anti-EGF-receptor antibodies murine ior egf/r3 and humanized h-R3; the analytical methods for quality control of the radiopharmaceutical such as instant thin layer chromatography-silica gel (ITLC-SG); the biological assessment of the radiolabeled molecule using flow cytometry analysis; in vitro stability studies with cysteine and DTPA challenge and the biodistribution studies in 4NMRI xenografted nude mice with U-87 human glioblastoma multiforme and MDA-MB-468 breast cancer cell lines. Labeling efficency of (96.48 +/- 0.70%) (98.42 +/- 0.38%), (94.8 +/- 1.25%) and (96.41 +/- 0.89%) was achieved for 99mTC-2-IT ior efg/r3, 99mTc-CITC-DTPA- ior egf/r3, 99mTc-CITC-DTPA- h-R3 and 99mTc-DIACIM h-R3, respectively. Radiocolloids were less than 2.0% in all cases. The biological activity measured by flow cytometry analysis using the MDA-MB-468 breast cancer cell line showed an immunoreactivity fraction greater than 85% in all concentrations of each immunoconjugate. Challenge studies demonstrated no evidence of transcomplexation of 99mTc to 1.0 mM DTPA for 2-IT modified antibody ior egf/r3 and CITC-DTPA immunoconjugates and only 8.7%, 4.9% and 5.0% of the 99mTc-radiolabeled was transcomplexed to 1.0 mM cysteine after 1 h incubation at 37 degrees C for 2-IT modified antibody ior egf/r3, CITC-DTPA ior egf/r3 and CITC-DTPA h-R3, respectively. Biodistribution studies with 2-IT modified antibodies and CITC-DTPA immunoconjugates indicated high tumor uptake in both cell lines with both immunoconjugates and no accumulation of the radiolabeled antibodies in normal organs.

Suicidal oxidative stress induced by certain antioxidants.

Well known antioxidants-coumarins (7,8-dihydroxy-4-methyl coumarin-DHMC and 7,8-diacetoxy-4-methyl coumarin-DAMC) and flavonoids (quercetin-Q and quercetin penta-acetate-QPA) were investigated for their pro-oxidant effects in two human tumor cell lines. The breast carcinoma cell line (MDA-MB-468) was found to be more sensitive to treatment by the drugs-DAMC, Q and QPA at 10 microM than the glioma cell line (U-87MG), while DHMC was non toxic in both cell lines at this concentration. In MDA-MB-468 distinct growth inhibition was observed by 48 hr post treatment. Paradoxically, an increase in the formazan production was revealed by MTT assay at this time indicating an increase in the production of free radicals. An increase in the levels of reactive oxygen species (ROS) was also confirmed by DCFH-DA assay. In cells treated with DAMC, Q and QPA an increase in the percentage of cells with the hypodiploid DNA content was suggestive of apoptotic cell death. Taken together, these results suggest that an increase in oxidative stress caused by the pro-oxidant action of these drugs is responsible for cell death.

Optimizing cancer radiotherapy with 2-deoxy-d-glucose dose escalation studies in patients with glioblastoma multiforme.

BACKGROUND AND PURPOSE: Higher rates of glucose utilization and glycolysis generally correlate with poor prognosis in several types of malignant tumors. Own earlier studies on model systems demonstrated that the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) could enhance the efficacy of radiotherapy in a dose-dependent manner by selectively sensitizing cancer cells while protecting normal cells. Phase I/II clinical trials indicated that the combination of 2-DG, at an oral dose of 200 mg/kg body weight (BW), with large fractions of gamma-radiation was well tolerated in cerebral glioma patients. Since higher 2-DG doses are expected to improve the therapeutic gain, present studies were undertaken to examine the tolerance and safety of escalating 2-DG dose during combined treatment (2-DG + radiotherapy) in glioblastoma multiforme patients. PATIENTS AND METHODS: Untreated patients with histologically proven glioblastoma multiforme (WHO criteria) were included in the study. Seven weekly fractions of (60)Co gamma-rays (5 Gy/fraction) were delivered to the tumor volume (presurgical CT/MRI evaluation) plus 3 cm margin. Escalating 2-DG doses (200-250-300 mg/kg BW) were administered orally 30 min before irradiation after overnight fasting. Acute toxicity and tolerance were studied by monitoring the vital parameters and side effects. Late radiation damage and treatment responses were studied radiologically and clinically in surviving patients. RESULTS: Transient side effects similar to hypoglycemia were observed in most of the patients. Tolerance and patient compliance to the combined treatment were very good up to a 2-DG dose of 250 mg/kg BW. However, at the higher dose of 300 mg/kg BW, two out of six patients were very restless and could not complete treatment, though significant changes in the vital parameters were not observed even at this dose. No significant damage to the normal brain tissue was observed during follow-up in seven out of ten patients who received complete treatment and survived between 11 and 46 months after treatment. CONCLUSION: Oral administration of 2-DG combined with large fractions of radiation (5 Gy/fraction/week) is safe and could be tolerated in glioblastoma patients without any acute toxicity and late radiation damage to the normal brain. Further clinical studies to evaluate the efficacy of the combined treatment are warranted.