Chemical composition, antioxidant potential, macromolecule damage and neuroprotective activity of Convolvulus pluricaulis.

Herbal medicines are known to mitigate radical induced cell damage. Hence identification and scientific validation of herbal medicines contribute to better use in Ayurvedic/Unani research. In the present study, we investigated antioxidant and anti-apoptotic properties of Convolvulus pluricaulis (C. pluricaulis). C. pluricaulis exhibited antioxidant potential evident by free radical scavenging activities. C. pluricaulis pretreatment inhibited H2O2 induced macromolecule damage such as plasmid DNA damage and AAPH induced oxidation of bovine serum albumin and lipid peroxidation of rat hepatic tissues. Further to identify the neuroprotective properties of C. pluricaulis, SHSY5Y cells were treated with H2O2 with or without pretreatment of C. pluricaulis. The C. pluricaulis pretreatment at 50 µg/ml dose exhibited 50% cell survival against 100 µM H2O2 challenge for 24 h and it also decreased the lactate dehydrogenase leakage. Further C. pluricaulis pretreatment restored and regulated the antioxidant and apoptosis markers such as SOD, CAT, p53, and caspase-3 and inhibited, reactive oxygen species generation and depolarization of the mitochondrial membrane. C. pluricaulis possess a high content of flavonoids and polyphenols and GC-MS and FTIR analysis showed a wide variety of compounds which may contribute to the observed effects.

Anthrax lethal toxin (LeTx) neutralization by PA domain specific antisera.

Anthrax associated causalities in humans and animals are implicated mainly due to the action of two exotoxins that are secreted by the bacterium Bacillus antharcis during the infection. These exotoxins comprise of three protein components namely protective antigen (PA), lethal factor (LF) and edema factor (EF). The protective antigen is the common toxin component required to form both lethal toxin (LeTx) and edema toxin (EdTx). The LeTx is formed, when PA combines with LF and EdTx is formed when PA combines with EF. Therapeutic interventions aiming to neutralize these key effectors of anthrax pathology would therefore, provide an effective means to counter the toxicity imposed by the anthrax toxins on the host. The present work describes the lethal toxin neutralization potential of polyclonal antisera developed against the individual domains of the protective antigen component of the anthrax toxin. The individual domains were produced as recombinant proteins in E. coli and validated with peptide mass fingerprinting by MALDI-TOF analysis and corresponding mice polyclonal antisera by western blotting. Each domain specific antibody titre and isotype was ascertained by ELISA. The isotyping revealed the predominance of IgG1 isotype. The toxin neutralizing potential of these domain specific antisera were evaluated by in-vitro cell viability MTT assay, employing J774.1 mouse macrophage cell line against LeTx (0.25 µg ml-1 PA and 0.125 µg ml-1 LF concentrations). Among the four domain specific antisera, the antiserum against PA domain IV could neutralize LeTx with high efficiency. No significant neutralization of LeTx was observed with other domain specific antibodies. Results indicate that antibodies to r-PA domain IV could be explored further as therapeutic anti toxin molecule along with appropriate antibiotic regimens against anthrax.

Androgen receptor and microRNA-21 axis downregulates transforming growth factor beta receptor II (TGFBR2) expression in prostate cancer.

Prostate cancer cells escape growth inhibition from transforming growth factor ß (TGFß) by downregulating TGFß receptors. However, the mechanism by which cancer cells downregulate TGFß receptors in prostate is not clear. Here, we showed that coordinated action of miR-21 and androgen receptor (AR) signaling had a critical role in inhibiting TGFß receptor II (TGFBR2) expression in prostate cancer cells. Our results revealed that miR-21 suppresses TGFBR2 levels by binding to its 3'-UTR and AR signaling further potentiates this effect in both untransformed and transformed human prostate epithelial cells as well as in human prostate cancers. Analysis of primary prostate cancers showed that increased miR-21/AR expression parallel a significantly reduced expression of TGFBR2. Manipulation of androgen signaling or the expression levels of AR or miR-21 negatively altered TGFBR2 expression in untransformed and transformed human prostate epithelial cells, human prostate cancer xenografts and mouse prostate glands. Importantly, we demonstrated that miR-21 and AR regulated each other's expression resulting in a positive feedback loop. Our results indicated that miR-21/AR mediate its tumor-promoting function by attenuating TGFß-mediated Smad2/3 activation, cell growth inhibition, cell migration and apoptosis. Together, these results suggest that the AR and miR-21 axis exerts its oncogenic effects in prostate tumors by downregulating TGFBR2, hence inhibiting the tumor-suppressive activity of TGFß pathway. Targeting miR-21 alone or in combination with AR may restore the tumor inhibitory activity of TGFß in prostate cancer.

Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer.

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant.

Three haptens of the organophosphorus (OP) toxicant 'sarin' having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC(50) (inhibitory concentration) of free antigen was found to be 0.415 µg mL(-1) on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19-1.56 µg mL(-1) with a limit of detection (LOD) of 0.05 µg mL(-1). The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.

Characterization of native and denatured ricin using MALDI-TOF/MS.

Ricin is a toxic protein present in the seeds of castor bean plant. It can be inactivated by heat; therefore characterization of denatured ricin is essential to differentiate it from native ricin and to avoid any ambiguity in its identification. In this study, potential of mass spectrometry using MALDI—TOF/MS has been exploited to investigate the effects of heat treatment on ricin and spiked food matrices. The molecular weights of ricin, ricin A (A1 and A2) and B chain were found to be 62.8 kDa, 31.2 kDa, 32.5 kDa and 32 kDa respectively. The mass spectrum revealed a polypeptide chain of 11.1 kDa for denatured ricin. The peptide mass fingerprinting showed 24 peptides, six were common both in native and denatured ricin. The differentiating peptide at position 294—318 (m/z 934.533) was observed only in denatured ricin. The three selected marker peptides m/z 1013.6, 1310.7, 1728.9 are chosen for identification of ricin inactivated by heat in spiked apple juice and milk samples by immunocapture analysis. There is always a probability of denatured non— toxic ricin being confused with native (toxic) ricin to create unnecessary panic. Keeping this probability in mind, our study will be of immense value in minimising such risk.

MicroRNA-185 suppresses tumor growth and progression by targeting the Six1 oncogene in human cancers.

Homeobox genes encode transcription factors that are essential for normal development and are often dysregulated in cancers. The molecular mechanisms that cause their misregulation in cancers are largely unknown. In this study, we investigate the mechanism by which the Six1 homeobox protein, which has a crucial role during development, is frequently deregulated in several poor outcome, aggressive, metastatic adult human cancers, including breast cancer, ovarian cancer, hepatocellular carcinoma and pediatric malignancies such as rhabdomyosarcoma and Wilms' tumor. Our results reveal that miRNA-185 translationally represses Six1 by binding to its 3'-untranslated region. Analyses of ovarian cancers, pediatric renal tumors and multiple breast cancer cell lines showed decreased miR-185 expression, paralleling an increase in Six1 levels. Further investigation revealed that miR-185 impedes anchorage-independent growth and cell migration, in addition to suppressing tumor growth in vivo, implicating it to be a potent tumor suppressor. Our results indicate that miR-185 mediates its tumor suppressor function by regulating cell-cycle proteins and Six1 transcriptional targets c-myc and cyclin A1. Furthermore, we show that miR-185 sensitizes Six1-overexpressing resistant cancer cells to apoptosis in general and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in particular. Together, our findings suggest that the altered expression of the novel tumor suppressor miR-185 may be one of the central events that leads to dysregulation of oncogenic protein Six1 in human cancers.

Deep-notch, ultracompact long-period grating in a large-mode-area photonic crystal fiber.

An extremely short long-period grating (LPG) with strong resonance has been developed in a large-mode-area photonic crystal fiber (PCF) by use of the heat source of a CO2 laser. We believe that such a LPG in pure silica PCF is the first example to be obtained with the point-by-point technique. The fabrication method is simple and repeatable. The resulting LPG has 8 periods, written by a CO2 laser, within a 2.8-mm length of fiber, which yields a deep notch of core-cladding mode coupling of -31.5 dB at the telecommunication wavelength of 1529.2 nm, with a FWHM of approximately 0.7 nm. The principal advantages of this LPG are that it is practical, cost effective, and compact.

Genetic diversity across natural populations of three montane plant species from the Western Ghats, India revealed by intersimple sequence repeats.

We analysed genetic diversity across the natural populations of three montane plant species in the Western Ghats, India; Symplocos laurina, Gaultheria fragrantissima and Eurya nitida using intersimple sequence repeat (ISSR) markers. These markers revealed genetic diversity within the populations of these plants from Nilgiri and also between two populations of S. laurina from Nilgiri and Amboli. Genetic variation within and between populations was analysed using various parameters such as total heterozygosity (HT), heterozygosity within population (HS), diversity between populations (DST), coefficient of population differentiation (GST), genetic distance (D) and gene flow (Nm). Total heterozygosity (HT) was higher for S. laurina (0.238) than for G. fragrantissima (0.172) and E. nitida (0.182). Two populations of S. laurina, separated by > 1000 km, showed a high within-population variation (53.7%) and a low gene flow (Nm = 0.447). upgma phenograms depicted a tendency of accessions to group according to their geographical locations in all the three plant species. The insight gained into the genetic structure of these plant populations might have implications in developing in situ and ex situ conservation strategies.

In vitro evaluation of 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-trifluoromethylquinoline hydrochloride (NLTQ-1), a new bioreductive agent as a hypoxia marker by 19F-magnetic resonance spectroscopy (19F-MRS).

19F-labeled bioreductive drugs bound to hypoxic cells in tumors could be detected by nuclear magnetic resonance, provided that they do not lose 19F during their metabolism. NLTQ-1, a 2-nitroimidazole-linked 7-trifluoromethylquinoline, has been synthesized to furnish this aim. NLTQ-1 demonstrated hypoxic selectivities of 7-10 in various cell-lines, in vitro. Uptake studies in V79 cells showed a 5 to 6 fold greater intracellular than extracellular concentration at a range of 100-300 microM input concentrations. A strong sharp peak, which was identified as the parent compound, was observed in the 19F-NMR spectrum of 90% MeCN extracts of V79 cells aerobically exposed to NLTQ-1, indicating that NLTQ-1 was not metabolized under aerobic conditions. Similarly, 19F NMR efflux studies in intact cells showed that the NLTQ-1 was bound to the cells predominantly under hypoxic conditions. 19F-NMR spectra of intact cells, exposed under hypoxic conditions to NLTQ-1, and of their lysates, after precipitation of various cellular components, indicated that possible covalent binding of NLTQ-1 had occurred with macromolecules such as proteins and nucleic acids. Therefore, NLTQ-1 might be suitable as a 19F-MRS/MRI hypoxia probe, although further in vivo work is necessary to verify this matter.

4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive agent as radiosensitizer in vitro and in vivo: comparison with tirapazamine.

The novel hypoxia-selective cytotoxin NLCQ-1, which is a weak DNA intercalator, was studied in conjunction with radiation against V79 cultured cells and EMT6 or SCCVII tumors in their syngeneic mice and compared with tirapazamine (TPZ). NLCQ-1 was a very potent and efficient radiosensitizer of hypoxic V79 cells, providing SER values of 2.27-2.56 at 20-80 microM concentration (measured at 10% survival level). Its C1.6 (concentration for an SER of 1.6 to be obtained) was 7.2+/-0.2 microM. Its in vitro therapeutic index (ThI, defined as CT50(Air),/C1.6) varied by the exposure time from 57 (1-h exposure) to 145 (4.5-h exposure). The corresponding C1.6 value for TPZ was 16.9 microM whereas its in vitro therapeutic index was 49 (3-h exposure). A schedule-dependent synergistic interaction was observed between NLCQ-1 or TPZ and 20 Gy of radiation in both tumor models examined, by using the in vivo-in vitro assay as endpoint. Optimal synergism (> 1 log) was observed in EMT6 tumors when each bioreductive drug was given between 45 and 60 min before irradiation. NLCQ-1 alone had no significant antitumor activity at 10 mg/kg (28% of its single LD50), whereas a 0.4 surviving fraction was obtained by TPZ at 30 mg/kg (38% of its single LD50). SER values of 1.52 and 1.25 were obtained with 10 mg/kg NLCQ-1 and 30 mg/kg TPZ, respectively, in EMT6 tumors. An SER value of 1.58 was obtained for both hypoxia-selective cytotoxins, at equitoxic doses, in SCCVII tumors, by using a fractionated regimen. These results suggest a possible use of NLCQ-1 or TPZ as adjuvants to radiotherapy.

Glucocorticoid modulation of protein phosphorylation and sarcoplasmic reticulum function in rat myocardium.

To decipher the mechanism(s) underlying glucocorticoid action on cardiac contractile function, this study investigated the effects of adrenalectomy and dexamethasone treatment on the contents of sarcoplasmic reticulum (SR) Ca(2+)-cycling proteins, their phosphorylation by endogenous Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), and SR Ca(2+) sequestration in the rat myocardium. Cardiac SR vesicles from adrenalectomized rats displayed significantly diminished rates of ATP-energized Ca(2+) uptake in vitro compared with cardiac SR vesicles from control rats; in vivo administration of dexamethasone to adrenalectomized rats prevented the decline in SR function. Western immunoblotting analysis showed that the relative protein amounts of ryanodine receptor/Ca(2+)-release channel, Ca(2+)-ATPase, calsequestrin, and phospholamban were neither diminished significantly by adrenalectomy nor elevated by dexamethasone treatment. However, the relative amount of SR-associated CaM kinase II protein was increased 2.5- to 4-fold in dexamethasone-treated rats compared with control and adrenalectomized rats. Endogenous CaM kinase II activity, as judged from phosphorylation of ryanodine receptor, Ca(2+)-ATPase, and phospholamban protein, was also significantly higher (50--80% increase) in the dexamethasone-treated rats. The stimulatory effect of CaM kinase II activation on Ca(2+) uptake activity of SR was significantly depressed after adrenalectomy and greatly enhanced after dexamethasone treatment. These findings identify the SR as a major target for glucocorticoid actions in the heart and implicate modification of the SR CaM kinase II system as a component of the mechanisms by which dexamethasone influences SR Ca(2+)-cycling and myocardial contraction.

4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive compound as a hypoxia-selective cytotoxin.

A novel weakly DNA-intercalative bioreductive compound. 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1). has been synthesized and studied as a hypoxia-selective cytotoxin in vitro. NLCQ-1, which shares a similar structure with the DNA-intercalative antimalarial drug chloroquine, bound more strongly to DNA than the nonchlorinated analog NLQ-1 (4-[3-(2-nitro-1-imidazolyl)propylamino]-quinaldine hydrochloride). Thus, NLCQ-1 exhibited a C50 [concentration for 50% displacement of the ethidium bromide (EB) from a DNA-EB complex] of 44 microM, whereas a C50 value could not be reached for NLQ-1 up to 225 microM. NLCQ-1 demonstrated significant hypoxic selectivity in several rodent (V79, EMT6, SCCVII) or human (A549, OVCAR-3) tumor cell lines. Its potency as a hypoxic cytotoxin (expressed as the product of exposure time and concentration for 50% survival) ranged between 10 and 136 microM x h, for the cell lines tested, at 30 microM input concentration. Because uptake in all cell lines was similar, the differences in potency may reflect differences in the enzymatic profile or damage repair processes among the cell lines. In addition, however, the most striking feature of NLCQ-1 was that hypoxic selectivity increased with exposure time, a common feature normally found in only bis-bioreductive agents carrying two moieties with different redox potentials. Thus, hypoxic selectivity of NLCQ-1 in V79 cells at 50% survival was increased from fivefold up to 388-fold by increasing exposure time from 1 to 4.5 h, as the result of a concomitant increase and decrease in its hypoxic and aerobic potency, respectively, over time. Because the nonchlorinated analog NLQ-1 did not demonstrate similar behavior, we hypothesized that the C-7 chlorine of NLCQ-1 might play a significant role in this phenomenon.

Association of interleukin-6 and other biologic variables with depression in older people living in the community.

OBJECTIVES: The prevalence of depression increases with age, as does the prevalence of higher levels of the cytokine interleukin-6 (IL-6). This analysis was performed to determine the association between increased levels of this cytokine and depression in a population-based sample. DESIGN: Cross-sectional cohort study. SETTING: Rural and urban counties in North Carolina. PARTICIPANTS: Community-dwelling older people. MEASUREMENTS: The association between IL-6 and other biologic variables with self-report depression was examined in 1686 persons aged 70 years and older in the third in-person survey wave (1991) of the Duke Established Population for Epidemiologic Studies of the Elderly (EPESE). Bivariate associations were established by the Spearman correlation, adjusted for age. A stepwise linear logistic regression model was used to derive a final model to assess multivariable effects on CES-D scores. RESULTS: Depression was correlated with IL-6 (P = .011), D-Dimer (P = .017), alpha-1-globulin (P = .023), alpha-2-globulin (P = .002), and beta globulin (P = .012). After controlling for age, race, and gender, IL-6 levels remained the only biologic variable significantly associated with depression (P = .035). CONCLUSION: These data suggest that the inflammatory marker, IL-6, is associated with depression in older people in this cross-sectional study. These results are compatible with the hypothesis of cytokine (IL-6) stimulation in geriatric depression as part of an overall immunoendocrine dysregulation.

Disulfiram augments oxidative stress in rat brain following bilateral carotid artery occlusion.

We examined the brain oxidative stress which accompanies 30 min of bilateral carotid artery ligation (BCAL) in terms of changes in brain levels of glutathione; reduced (GSH) and oxidized (GSSG) forms and the exacerbation of oxidative stress by disulfiram (DSF). These results indicate that BCAL alone decreases GSH content and limits glutathione reductase (GR) activity, and these changes were enhanced by DSF pretreatment. Similar observations were recorded with DSF alone. GR activity (74.3 +/- 4.0 micromol min(-1) mg(-1) tissue; p < 0.001) and GSH content (1.23 +/- 0.06 micromol min(-1) g(-1) tissue; p < 0.001) was attenuated in rats subjected to synergistic effect of BCAL and DSF with a concomitant increase of GSSG (0.006 +/- 0.006 micromol min(-1) g(-1) tissue; p < 0.001). Recovery of GSH/GSSG level and GR activity during reperfusion following 30 min BCAL was considerably delayed (96 h) in the BCAL and DSF group as compared to the recovery time of 24 h in the group subjected to BCAL-reperfusion alone. Perturbation of GSH/GSSG homeostasis as a result of BCAL was augmented by DSF. These findings clearly demonstrate central nervous system oxidative stress due to a BCAL-DSF synergistic effect. Based on the results obtained with this model, we conclude that DSF increases brain oxidative stress and this may be detrimental to alcoholics who might drink and develop an acetaldehyde-induced hypotension while taking DSF.

Racial differences in the prevalence of monoclonal gammopathy in a community-based sample of the elderly.

PURPOSE: To determine if there is an increased prevalence of monoclonal gammopathy in elderly blacks compared with whites, analogous to the difference in incidence of multiple myeloma reported for the two racial groups and to confirm age and gender relationships. PATIENTS AND METHODS: Subjects were from the Duke Established Populations for the Epidemiologic Study of the Elderly, selected on the basis of stratified random household sampling. Blacks were oversampled to allow for increased statistical precision in racial comparisons. In all, 1,732 subjects (aged > 70 years) consented to blood drawing and constitute the sample for this study. Monoclonal immunoglobulins were determined by agarose gel electrophoresis and immunofixation. RESULTS: One hundred six subjects (6.1%) had a monoclonal gammopathy. There was a greater than twofold difference in prevalence between blacks (8.4%) and whites (3.8%) (P < 0.001); monoclonal gammopathy prevalence increased with age, and was greater in men than women. Those with monoclonal gammopathy did not differ from those without in socioeconomic status, urban/rural residence, or education. The presence of monoclonal gammopathy was not associated with any specific diseases nor with impaired functional status. There was a slight increase in serum creatinine levels and decrease in hemoglobin and albumin levels in patients with monoclonal gammopathy, but no difference in interleukin-6 (IL-6) levels. Moreover, IL-6 levels were not correlated significantly with the level of monoclonal protein. CONCLUSION: Prevalence of monoclonal gammopathy is significantly greater among blacks than whites in a community-based sample, in approximately the same ratio that multiple myeloma has been reported in the two groups. Given the absence of correlation with environmental factors, there may be a biological racial difference in susceptibility to an early event in the carcinogenic process leading to multiple myeloma.

Regulatory role of cortisone and dietary carbohydrate energy in prepubertal rat mammary gland growth and development.

Role of cortisone and regulated carbohydrate energy in mammary gland growth, based on studies of morphometric and certain growth and energy related biochemical parameters has been evaluated in the adrenalectomized prepubertal female rat. Results showed improvement or restoration of mammary features like fat pad weight, area, duct system and sprouting of end and lateral buds by cortisone treatment from adrenalectomy induced suppressed state. Similarly the hormone rectified the gland nucleic acids, glycogen and rate of tissue oxidation of glucose levels, reduced under adrenalectomy. Adrenalectomy caused loss of appetite (33.5% less than normal intake). This amount of diet restriction to normal intact did not affect the gland morphology but reduced nucleic acids, protein, glycogen and glucose oxidation rate. Provision of dextrose mixed diet to the adrenalectomized rats showed variable improvements of these biochemical parameters except the gland protein level in association with increase in appetite. However, cortisone therapy under provision of dextrose supply, restored gland protein level and increased further in nucleic acids, glycogen and glucose oxidation rate. The morphological growth parameters with dextrose and cortisone also showed further improvements. Results suggested that glucocorticoid was essential mammary growth factor during prepubertal ages. The hormone appeared to operate via tissue metabolism and stimulating energy intake through appetite stimulation.

9-[3-(2-Nitro-1-imidazolyl)propylamino]-cyclopenteno[b]quinoline hydrochloride (NLCPQ-1): a novel DNA-affinic bioreductive agent as chemosensitizer. I.

9-(3-(2-Nitro-1-imidazolyl)propylamino]-cyclopentano[b]quinoline hydrochloride (NLCPQ-1) is one member of a limited series of 2-nitroimidazole-linked derivatized quinolines we have synthesized to be weak DNA binding compounds. On a concentration basis, NLCPQ-1 is the most potent analogue of the series as a radiosensitizer and cytotoxin of hypoxic cells in vitro and in vivo. This improved efficacy compared to untargeted nitroimidazolic bioreductive compounds has been mainly attributed to its weak DNA binding. In the present study, we investigated the ability of NLCPQ-1 to act synergistically with 4-[bis(2-chloroethyl)amino]-L-phenylalanine (L-PAM, melphalan) or cis-diamminedichloroplatinum (cis-DDP, cisplatin) against tumor cells in vitro and in vivo. We demonstrated that 7 microM NLCPQ-1 potentiated the toxic effect of cis-DDP and L-PAM against V79 cells under hypoxic pretreatment conditions with dose modification factors (DMF) of 2.6 and 2.4, respectively, measured at 0.1 survival. Potentiation was dependent on the concentrations of both the chemotherapeutic agent and NLCPQ-1 as well as on the duration of the hypoxic pretreatment with NLCPQ-1. No potentiation was observed under aerobic cotreatment conditions in vitro. Significant synergism was observed when 15 mg/kg NLCPQ-1 was administered IP at various time intervals before a single dose of L-PAM (5 mg/kg) or cis-DDP (5 mg/kg) in Balb/c mice bearing EMT6 tumors. The in vivo/in vitro assay was used as the investigational endpoint and either the fractional product or isobologramic analysis was used to determine synergistic interactions.