Our approach to examine the mechanism(s) of action for photodynamic therapy (PDT) has been via the generation of PDT-resistant cell lines. In this study we used three human cell lines, namely, human colon adenocarcinoma (HT29), human bladder carcinoma and human neuroblastoma. The three photosensitizers used were Photofrin, Nile Blue A and aluminum phthalocyanine tetrasulfonate. The protocol for inducing resistance consisted of repeated in vitro photodynamic treatments with a photosensitizer to the 1-10%-survival level followed by regrowth of single surviving colonies. Varying degrees of resistance were observed. The three induced variants of the HT29 cell line were the most extensively studied. Their ratios of increased survival at the LD90 level range between 1.5-and 2.62-fold more resistant.
Scrophularia ningpoensis has been used in China for centuries as a herbal tea to treat various diseases. Based on the numerous animal studies on its pharmaceutical effects and the long time clinical experiences, we studied the molecular and cellular mechanism underlying the bioactivity of aqueous extract of Scrophularia and its isolated compounds. Seven isolated compounds, unlike Scrophularia extract, failed to induce cytotoxicity on HaCaT cells, but their combination improved the effect of extract. Tumor cell line selectivity was not observed, when we studied its cytotoxic effect on melanoma cell lines. The apoptotic and anti-inflammatory effects of Scrophularia extract have been demonstrated on HaCaT cells. The extract induced those effects potentially through affecting the MAPK pathway and inhibition of the NF-κB pathway, Microarray-based bioinformatical analyses on the compound acetoside from Scrophularia revealed a gene expression profile which confirmed our findings with the extract on proliferation inhibition, anti-inflammation and apoptosis. With DNA alkylation as major proposed mechanism of action, we assume acetoside as one of the active compounds in Scrophularia.
Our approach to examine the mechanism(s) of action for photodynamic therapy (PDT) has been via the generation of PDT-resistant cell lines. In this study we used three human cell lines, namely, human colon adenocarcinoma (HT29), human bladder carcinoma and human neuroblastoma. The three photosensitizers used were Photofrin, Nile Blue A and aluminum phthalocyanine tetrasulfonate. The protocol for inducing resistance consisted of repeated in vitro photodynamic treatments with a photosensitizer to the 1-10%-survival level followed by regrowth of single surviving colonies. Varying degrees of resistance were observed. The three induced variants of the HT29 cell line were the most extensively studied. Their ratios of increased survival at the LD90 level range between 1.5- and 2.62-fold more resistant.
We are investigating treatments for cocaine abuse based on viral gene transfer of a cocaine hydrolase (CocH) derived from human butyrylcholinesterase, which can reduce cocaine-stimulated locomotion and cocaine-primed reinstatement of drug-seeking behavior in rats for many months. Here, in mice, we explored the possibility that anti-cocaine antibodies can complement the actions of CocH to reduce cocaine uptake in brain and block centrally-evoked locomotor stimulation. Direct injections of test proteins showed that CocH (0.3 or 1 mg/kg) was effective by itself in reducing drug levels in plasma and brain of mice given cocaine (10 mg/kg, s.c., or 20 mg/kg, i.p). Administration of cocaine antibody per se at a low dose (8 mg/kg, i.p.) exerted little effect on cocaine distribution. However, a higher dose of antibody (12 mg/kg) caused peripheral trapping (increased plasma drug levels), which led to increased cocaine metabolism by CocH, as evidenced by a 6-fold rise in plasma benzoic acid. Behavioral tests with small doses of CocH and antibody (1 and 8 mg/kg, respectively) showed that neither agent alone reduced mouse locomotor activity triggered by a very large cocaine dose (100 mg/kg, i.p.). However, dual treatment completely suppressed the locomotor stimulation. Altogether, we found cooperative and possibly synergistic actions that warrant further exploration of dual therapies for treatment of cocaine abuse.
Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.
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