ABSTRACT
We have designed an amphiphilic prodrug of the anticancer agent gemcitabine (dFdC), by covalent coupling to squalene. This bioconjugate, which self-assembled into nanoparticles (NPs) in water, was previously found to display an impressive anticancer activity both in vitro and in vivo. The present study aims to investigate the impact of SQdFdC nanoparticles on cellular membranes. MTT assays showed that, in the nanomolar range, squalenoyl gemcitabine (SQdFdC) was slightly less active than dFdC on a panel of human cancer cell lines, in vitro. However, above 10 µmol L(-1) SQdFdC was considerably more cytotoxic than dFdC. Contrarily to its parent drug, SQdFdC also induced cell lysis in a few hours, as evidenced by LDH release assays. Erythrocytes were used as an experimental model insensitive to the antimetabolic activity of dFdC to further investigate the putative membrane-related cytotoxic activity of SQdFdC. The bioconjugate also induced hemolysis in a time- and dose-dependent fashion, unlike squalene or dFdC, which clearly proved that SQdFdC could permeabilize cellular membranes. Structural X-ray diffraction and calorimetry studies were conducted in order to elucidate the mechanism accounting for these observations. They confirmed that SQdFdC could be transferred from NPs to phospholipid bilayers and that the insertion of the prodrug within model membranes resulted in the formation of nonlamellar structures, which are known to promote membrane leakage. As a whole, our results suggested that due to its amphiphilic nature, the cell uptake of SQdFdC resulted in its insertion into cellular membranes, which could lead to the formation of nonlamellar structures and to membrane permeation. Whether this mechanism could be the source of toxicity in vivo, however, remains to be established, since preclinical studies have clearly proven that squalenoyl gemcitabine displayed a good toxicity profile.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cell Membrane/drug effects , Deoxycytidine/analogs & derivatives , Prodrugs/pharmacology , Squalene/analogs & derivatives , Surface-Active Agents/pharmacology , Animals , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/chemistry , Antimetabolites, Antineoplastic/pharmacokinetics , Calorimetry, Differential Scanning , Cell Culture Techniques , Cell Line, Tumor , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Survival/drug effects , Deoxycytidine/administration & dosage , Deoxycytidine/chemistry , Deoxycytidine/pharmacokinetics , Deoxycytidine/pharmacology , Erythrocytes/drug effects , Female , Hemolysis/drug effects , Humans , Mice , Mice, Inbred Strains , Phospholipids/chemistry , Prodrugs/administration & dosage , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Squalene/administration & dosage , Squalene/chemistry , Squalene/pharmacokinetics , Squalene/pharmacology , Surface-Active Agents/administration & dosage , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacokinetics , X-Ray DiffractionABSTRACT
This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.
Subject(s)
Chlorophyll/analogs & derivatives , Chlorophyll/pharmacokinetics , Colonic Neoplasms/drug therapy , Dihematoporphyrin Ether/pharmacokinetics , Photochemotherapy/methods , Animals , Chlorophyll/administration & dosage , Chlorophyll/pharmacology , Colonic Neoplasms/pathology , Dihematoporphyrin Ether/administration & dosage , Dihematoporphyrin Ether/pharmacology , Humans , Male , Mice , Mice, Nude , Photosensitizing Agents/administration & dosage , Photosensitizing Agents/pharmacokinetics , Photosensitizing Agents/pharmacology , Porphyrins/administration & dosage , Porphyrins/pharmacokinetics , Porphyrins/pharmacology , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Laparoscopic influence on cell-mediated immunity and tumour evolution is controversial. The objective of the present study was to assess tumour growth and immune patterns after laparoscopy on an experimental study. Lewis rats, bearing an intrapancreatic ductal carcinoma randomly underwent one of the following 2-hour procedures: anaesthesia, laparotomy or CO(2) pneumoperitoneum. Cell-mediated immunity was investigated through determination of serum IL1beta concentrations by ELISA and TNFalpha, IL6 and iNOS gene transcriptions in blood white cells and peritoneal cells by RT-PCR 1 day after operation. Tumour growth and spread patterns were assessed on anatomopathological examination 2 weeks after surgery. Tumour growth and spread were unaffected no matter what procedure was applied, but port-site seeding occurred in half of the cases undergoing laparoscopy. No significant change in acute-phase protein response, represented by IL1beta serum concentration, was found after laparoscopy. TNFalpha, IL6 and inducible NO synthase gene transcriptions were enhanced in blood white cells and depressed in peritoneal immune cells after laparoscopy. In our experimental conditions, cell-mediated immune response to CO(2) pneumoperitoneum seems to be a good systemic immune activation and a less acute peritoneal immune response as opposed to control laparoscopy. This early impairment of peritoneal macrophage immune activity, observed after a long-lasting CO(2) pneumoperitoneum, might be responsible for the high rate of port site recurrence.
Subject(s)
Immunity, Cellular , Laparoscopy , Animals , Interleukin-1/blood , Interleukin-6/genetics , Neoplasms, Experimental/immunology , Neoplasms, Experimental/surgery , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Osmolar Concentration , Pancreatic Ducts , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/secondary , Pancreatic Neoplasms/surgery , Rats , Rats, Inbred Lew , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Tests of diadochokinesia are an inherent part of a neurological examination. Various quantifying methods have been proposed to increase the objectivity, sensitivity, and reliability of such examinations. The methods used and analyses performed, however, differ substantially between tasks. We used a three-dimensional, ultrasound-based recording device to continuously record joint angles during three diadochokinetic movements, avoiding any external constraints of the movements. Alternate pronation and supination of the forearm, tapping with the whole hand and with the index finger in isolation were analyzed in a sample of 63 healthy control subjects. The most sensitive measure for capturing effects of gender, sex, and active hand was frequency. The right hand was faster than the left in all tasks, tapping performance declined with increasing age, and male subjects were faster than females in forearm diadochokinesia. Other measures that characterize speed of movement such as maximum angular velocities and accelerations did not yield comparable sensitivity in detecting the same statistical effects. However, angular velocity achieved the highest test-retest reliability for forearm diadochokinesia, while frequency was reproduced in the tapping tasks. Additional measures characterizing symmetry of the angular velocity profiles and intraindividual variability were shown to be largely independent of movement speed. Examples in neurological patients showed that the data define a valuable standard against which pathological performance can be precisely evaluated. In addition, the different measures captured dissociable aspects of motor performance that may further help to characterize the deficit and adjust therapy.
Subject(s)
Fingers/physiology , Forearm/physiology , Hand/physiology , Movement/physiology , Neurologic Examination , Adult , Aged , Female , Functional Laterality , Humans , Male , Middle Aged , Nervous System Diseases/physiopathology , Pronation/physiology , Reproducibility of Results , Supination/physiology , UltrasonicsABSTRACT
Activities of the NADP+-dependent isocitrate dehydrogenase were measured along the entire sinusoidal path (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the liver of male Wistar rats using a Lowry technique. The measured activities show a slight increase from the periportal to the perivenous end, whereas no such septal-) perivenous gradient could be established. These profiles of enzyme activity give further support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.