Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected... more Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected to cisplatin or hyperthermia treatment. In an ovarian carcinoma line (A2780s) and its derivative cisplatin resistant line (A2780cp) the variation in response was expressed for both the colony survival endpoint and the apoptosis endpoint. Apoptosis was measured by the number of floating cells, DNA agarose gels, and electron microscopy. In fact, cisplatin resistance was expressed to a higher level for apoptosis, than colony survival in the A2780cp cell line compared to the A2780s line. The melanoma cell line (Sk Mel-3) also showed induced apoptosis by cisplatin treatment while the glioma line (U87MG) showed little to no apoptosis in response to cisplatin treatment. Hyperthermia (43 degrees C for 1 hour) induced apoptosis in the human melanoma cell line but not in the glioma cell line. These data indicate that, while both cisplatin and hyperthermia can induce apoptosis in human tumour cell...
Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainl... more Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainly restricted by uptake and ODN release into cytoplasm, which is difficult to evaluate in cell culture studies. Well-designed models of cellular membranes, aim of the present study, might facilitate investigation of such processes. In this investigation, a phosphorothioate ODN was actively encapsulated in a DODAP-containing cationic liposome by ethanol injection with 73% efficiency. ODN release was determined by fluorescence dequenching of FITC-ODN upon incubation of liposomes with early endosomal (EE), late endosomal (LE) and plasma membranes (PM) models. LE provided the highest release (up to 76%) in a temperature-dependent manner. Release by EE (<16%), total PM (<11%) and PM external layer ( approximately 0) were not temperature sensitive. These differences are attributed to lipid charge, chain mobility, critical packing parameter and cholesterol content of the models. Intracellular distribution of FITC-ODN, determined by fluorescence microscopy and flowcytometry in the presence and absence of sodium azide, confirmed that liposomes were internalized mainly via endocytosis; hence inability of our PL models to simulate such active processes. Instead, release of ODN from endosomes into cytoplasm was pH-sensitive and in good agreement with model membrane studies in terms of amount and mechanism.
Introduction. 4-Aminopyridine (4-AP) is a potassium channel blocker used to increase muscle stren... more Introduction. 4-Aminopyridine (4-AP) is a potassium channel blocker used to increase muscle strength in the treatment of demyelinating diseases such as multiple sclerosis. We describe a case of ingestion by an 8-month-old child that resulted in severe but transient symptoms.
N-(phosphonacetyl)-L-aspartate (PALA) modulates the activity of 5-fluorouracil (5-FU) by inhibiti... more N-(phosphonacetyl)-L-aspartate (PALA) modulates the activity of 5-fluorouracil (5-FU) by inhibiting pyrimidine biosynthesis. A cross-over study was conducted to determine whether PALA affects the pharmacokinetic parameters of 5-FU in patients given 5-FU/folinic acid (FA). Six patients (3 males, 3 females) aged 63 4.3 (mean SD) years (body surface area of 1.84 18 m2) with metastatic colorectal carcinoma were given two courses of treatment. The treatment consisted of 250 mg/m2 of PALA on day 1 followed by 20 mg/m2 FA and 400 mg/m2 5-FU (5 min i.v. bolus injection) on days 2-5 in one cycle of treatment (PALA+). In another treatment cycle, these doses of 5-FU and FA were given for all 5 days without PALA (PALA-). The two courses were given four weeks apart. It was determined by random selection whether the course with PALA was given before or after the course without PALA. Blood samples were collected over a period of three hours, starting from the beginning of 5-FU infusion on days 2 and 5 of both courses. Plasma concentrations of 5-FU were determined by an HPLC technique. Pharmacokinetic parameters were calculated using a non-compartmental model. While there were no significant differences between pharmacokinetic parameters in the PALA+ vs PALA- courses, there was a trend towards a decreasing area under the curve (AUC) and increasing clearance (Cl) in PALA+ courses of treatment.
the reasons that account for the noncytotoxic ability of HP-RNase is its inhibition by the protei... more the reasons that account for the noncytotoxic ability of HP-RNase is its inhibition by the proteic ribonuclease inhibitor present in the cytosol of mammalian cells. We have reasoned that directing this enzyme to a cellular compartment devoid of this inhibitor would endow it with the desired property. Thus, we have constructed an HP-RNase variant, named PE5, which presents a nonclassical nuclear localization sequence (NLS) responsible for its import to the nucleus. Accordingly, unlike ONC, this variant degrades mainly nuclear RNA. Its cytotoxic properties have been tested on different tumor cell lines being one of the most sensible ovarian cell line (NCI-ADR/RES) that presents multiple drug resistance phenotype at least in part associated with the overexpression of P-glycoprotein (P-gp). The cytotoxic mechanism of PE5 was investigated on this cell line. The results showed that cellular death is produced by apoptosis. The apoptotic process was evidenced by the visualization of cell morphology using confocal microscopy, cell staining with annexin V-Alexa Fluor 488 and propidium iodide followed by FACS and by assaying caspase-3, -8 and -9 activation. Unlike ONC, the cytotoxic effect of PE5 is produced mainly in phase G0/G1 of the cell cycle. Selectivity studies carried out comparing the cytoxicity of PE5 on tumor and on normal human cell lines show similar results to those found for ONC. Finally, PE5 presents synergy with doxorubicin on NCI-ADR-RES cell line. This effect is explained by a specific inhibition of P-gp expression.
195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cispl... more 195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cisplatin in water and plasma ultrafiltrate (PUF). Cisplatin was found to be stable for at least 2, but no longer than 5 months in a reconstituted clinical formulation, as determined by 195Pt NMR. In aqueous solution, the cis-PtCl2(NH3)2 195Pt and 15N NMR signal intensities decreased with time and the formation of [PtCl(H2O)(NH3)2]+ at PH values of 3.0, 6.5, 7.5 and 9.5 was observed within 24 h of sample preparation. In addition, [Pt(H2O)2(NH3)2]++ was observed at pH 3.0, and [PtCl(OH)(NH3)2] and [Pt(OH)2(NH3)2] were observed at pHs 7.5 and 9.5. During incubation of PUF with cisplatin for 35 h, 15N NMR signals for at least eight cisplatin derivatives appeared at different times, whereas only four were observed by 195Pt NMR. With our NMR protocols, the detection limit for quantifiable cisplatin derivatives is estimated at 500 microM using 195Pt NMR and < or = 200 microM using 15N NMR. In addition to providing useful information about the chemical stability of cisplatin and derivatives formed in aqueous solution, these magnetic resonance techniques, particularly 15N NMR, can provide useful information about the metabolism of cisplatin in biological regimes.
Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected... more Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected to cisplatin or hyperthermia treatment. In an ovarian carcinoma line (A2780s) and its derivative cisplatin resistant line (A2780cp) the variation in response was expressed for both the colony survival endpoint and the apoptosis endpoint. Apoptosis was measured by the number of floating cells, DNA agarose gels, and electron microscopy. In fact, cisplatin resistance was expressed to a higher level for apoptosis, than colony survival in the A2780cp cell line compared to the A2780s line. The melanoma cell line (Sk Mel-3) also showed induced apoptosis by cisplatin treatment while the glioma line (U87MG) showed little to no apoptosis in response to cisplatin treatment. Hyperthermia (43 degrees C for 1 hour) induced apoptosis in the human melanoma cell line but not in the glioma cell line. These data indicate that, while both cisplatin and hyperthermia can induce apoptosis in human tumour cell...
Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected... more Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected to cisplatin or hyperthermia treatment. In an ovarian carcinoma line (A2780s) and its derivative cisplatin resistant line (A2780cp) the variation in response was expressed for both the colony survival endpoint and the apoptosis endpoint. Apoptosis was measured by the number of floating cells, DNA agarose gels, and electron microscopy. In fact, cisplatin resistance was expressed to a higher level for apoptosis, than colony survival in the A2780cp cell line compared to the A2780s line. The melanoma cell line (Sk Mel-3) also showed induced apoptosis by cisplatin treatment while the glioma line (U87MG) showed little to no apoptosis in response to cisplatin treatment. Hyperthermia (43 degrees C for 1 hour) induced apoptosis in the human melanoma cell line but not in the glioma cell line. These data indicate that, while both cisplatin and hyperthermia can induce apoptosis in human tumour cell...
Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainl... more Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainly restricted by uptake and ODN release into cytoplasm, which is difficult to evaluate in cell culture studies. Well-designed models of cellular membranes, aim of the present study, might facilitate investigation of such processes. In this investigation, a phosphorothioate ODN was actively encapsulated in a DODAP-containing cationic liposome by ethanol injection with 73% efficiency. ODN release was determined by fluorescence dequenching of FITC-ODN upon incubation of liposomes with early endosomal (EE), late endosomal (LE) and plasma membranes (PM) models. LE provided the highest release (up to 76%) in a temperature-dependent manner. Release by EE (<16%), total PM (<11%) and PM external layer ( approximately 0) were not temperature sensitive. These differences are attributed to lipid charge, chain mobility, critical packing parameter and cholesterol content of the models. Intracellular distribution of FITC-ODN, determined by fluorescence microscopy and flowcytometry in the presence and absence of sodium azide, confirmed that liposomes were internalized mainly via endocytosis; hence inability of our PL models to simulate such active processes. Instead, release of ODN from endosomes into cytoplasm was pH-sensitive and in good agreement with model membrane studies in terms of amount and mechanism.
Introduction. 4-Aminopyridine (4-AP) is a potassium channel blocker used to increase muscle stren... more Introduction. 4-Aminopyridine (4-AP) is a potassium channel blocker used to increase muscle strength in the treatment of demyelinating diseases such as multiple sclerosis. We describe a case of ingestion by an 8-month-old child that resulted in severe but transient symptoms.
N-(phosphonacetyl)-L-aspartate (PALA) modulates the activity of 5-fluorouracil (5-FU) by inhibiti... more N-(phosphonacetyl)-L-aspartate (PALA) modulates the activity of 5-fluorouracil (5-FU) by inhibiting pyrimidine biosynthesis. A cross-over study was conducted to determine whether PALA affects the pharmacokinetic parameters of 5-FU in patients given 5-FU/folinic acid (FA). Six patients (3 males, 3 females) aged 63 4.3 (mean SD) years (body surface area of 1.84 18 m2) with metastatic colorectal carcinoma were given two courses of treatment. The treatment consisted of 250 mg/m2 of PALA on day 1 followed by 20 mg/m2 FA and 400 mg/m2 5-FU (5 min i.v. bolus injection) on days 2-5 in one cycle of treatment (PALA+). In another treatment cycle, these doses of 5-FU and FA were given for all 5 days without PALA (PALA-). The two courses were given four weeks apart. It was determined by random selection whether the course with PALA was given before or after the course without PALA. Blood samples were collected over a period of three hours, starting from the beginning of 5-FU infusion on days 2 and 5 of both courses. Plasma concentrations of 5-FU were determined by an HPLC technique. Pharmacokinetic parameters were calculated using a non-compartmental model. While there were no significant differences between pharmacokinetic parameters in the PALA+ vs PALA- courses, there was a trend towards a decreasing area under the curve (AUC) and increasing clearance (Cl) in PALA+ courses of treatment.
the reasons that account for the noncytotoxic ability of HP-RNase is its inhibition by the protei... more the reasons that account for the noncytotoxic ability of HP-RNase is its inhibition by the proteic ribonuclease inhibitor present in the cytosol of mammalian cells. We have reasoned that directing this enzyme to a cellular compartment devoid of this inhibitor would endow it with the desired property. Thus, we have constructed an HP-RNase variant, named PE5, which presents a nonclassical nuclear localization sequence (NLS) responsible for its import to the nucleus. Accordingly, unlike ONC, this variant degrades mainly nuclear RNA. Its cytotoxic properties have been tested on different tumor cell lines being one of the most sensible ovarian cell line (NCI-ADR/RES) that presents multiple drug resistance phenotype at least in part associated with the overexpression of P-glycoprotein (P-gp). The cytotoxic mechanism of PE5 was investigated on this cell line. The results showed that cellular death is produced by apoptosis. The apoptotic process was evidenced by the visualization of cell morphology using confocal microscopy, cell staining with annexin V-Alexa Fluor 488 and propidium iodide followed by FACS and by assaying caspase-3, -8 and -9 activation. Unlike ONC, the cytotoxic effect of PE5 is produced mainly in phase G0/G1 of the cell cycle. Selectivity studies carried out comparing the cytoxicity of PE5 on tumor and on normal human cell lines show similar results to those found for ONC. Finally, PE5 presents synergy with doxorubicin on NCI-ADR-RES cell line. This effect is explained by a specific inhibition of P-gp expression.
195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cispl... more 195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cisplatin in water and plasma ultrafiltrate (PUF). Cisplatin was found to be stable for at least 2, but no longer than 5 months in a reconstituted clinical formulation, as determined by 195Pt NMR. In aqueous solution, the cis-PtCl2(NH3)2 195Pt and 15N NMR signal intensities decreased with time and the formation of [PtCl(H2O)(NH3)2]+ at PH values of 3.0, 6.5, 7.5 and 9.5 was observed within 24 h of sample preparation. In addition, [Pt(H2O)2(NH3)2]++ was observed at pH 3.0, and [PtCl(OH)(NH3)2] and [Pt(OH)2(NH3)2] were observed at pHs 7.5 and 9.5. During incubation of PUF with cisplatin for 35 h, 15N NMR signals for at least eight cisplatin derivatives appeared at different times, whereas only four were observed by 195Pt NMR. With our NMR protocols, the detection limit for quantifiable cisplatin derivatives is estimated at 500 microM using 195Pt NMR and < or = 200 microM using 15N NMR. In addition to providing useful information about the chemical stability of cisplatin and derivatives formed in aqueous solution, these magnetic resonance techniques, particularly 15N NMR, can provide useful information about the metabolism of cisplatin in biological regimes.
Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected... more Four human tumour cell lines were evaluated for their ability to undergo apoptosis when subjected to cisplatin or hyperthermia treatment. In an ovarian carcinoma line (A2780s) and its derivative cisplatin resistant line (A2780cp) the variation in response was expressed for both the colony survival endpoint and the apoptosis endpoint. Apoptosis was measured by the number of floating cells, DNA agarose gels, and electron microscopy. In fact, cisplatin resistance was expressed to a higher level for apoptosis, than colony survival in the A2780cp cell line compared to the A2780s line. The melanoma cell line (Sk Mel-3) also showed induced apoptosis by cisplatin treatment while the glioma line (U87MG) showed little to no apoptosis in response to cisplatin treatment. Hyperthermia (43 degrees C for 1 hour) induced apoptosis in the human melanoma cell line but not in the glioma cell line. These data indicate that, while both cisplatin and hyperthermia can induce apoptosis in human tumour cell...
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