Bis (3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine photodynamic therapy induces in vivo tumor ablation by triggering cytochrome c dependent mitochondria mediated apoptosis.

BACKGROUND: Despite findings that photodynamic treatment with bis (3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine initiated tumor regression in mice skin, queries regarding its mode of action - answers to which will be functional to design clinical trials on squaraine based photodynamic therapy - remain unanswered. Our investigation reveals the in vivo mechanism of action of the photosensitizer. METHODS: Skin tumor was induced in Swiss albino mice using 7,12-dimethyl benzanthacene. After the intraperitoneal administration of the dye in tumor induced mice, its concentration in subcellular fractions of the tumor tissue was determined fluorimetrically. Cytochrome c release from the mitochondrial membrane after the photodynamic treatment was analyzed. The observations stemming from this part lead to histopathological examination of tumor tissues. Apoptotic markers like caspase-3, Bcl-2 and Bax were also studied. RESULTS: Major portion of the dye accumulated in the mitochondria. Cytochrome c leakage from mitochondria after squaraine PDT suggests loss of mitochondrial membrane integrity, which was further confirmed by the results of histopathological analysis. The activity of caspase-3 was elevated, expression of Bcl-2 diminished and that of Bax increased - all these results show enhancement of apoptosis in the tumor region after the treatment. CONCLUSIONS: The results lead to the elucidation of mechanism of tumor destruction which proves to be mitochondria mediated apoptotic damage of tumor tissue. The study assumes significance since it defines the in vivo mode of action of a photosensitizer. Also, the query of how a squaraine based photosensitizer evokes tumor response is being dealt with here, for the first time.

Chemoprevention of two-stage skin cancer in vivo by Saraca asoca.

Saraca asoca (Family Caesalpiniaceae) has been widely used in traditional Indian medicine especially due to its wound-healing property. The present study investigates the chemopreventive property of flavonoids from Saraca asoca (flowers) on 2-stage skin carcinogenesis in mice models. Skin cancer was induced in Swiss albino mice by single topical application of 7,12-dimethyl benzanthracene (100 µg/50 µL of acetone) followed by thrice a week treatment of croton oil for 20 weeks. The topical pretreatment of the flavonoid fraction from S asoca (FF S asoca) was 30 minutes prior to the application of croton oil thrice weekly for 20 weeks. At the end of the experimental period the animals were sacrificed, and the tumor statistics and various marker parameters were studied (enzyme assays, Western blotting). The pretreatment of the FF of S asoca caused significant reduction in the number of tumors per mouse and the percentage of tumor-bearing mice. Also, the latency period for the appearance of the first tumor was delayed by S asoca pretreatment. In plant-treated animals there was a significant increase in the levels of reduced glutathione, catalase, and protein in skin when compared with the untreated animals. Conversely, there was a significant decrease in the lipid peroxidation levels. A significant reduction in the expression of ornithine decarboxylase, a key enzyme in the promotion stage of 2-stage skin cancer, in the plant-treated group was also observed. These findings suggest the chemopreventive activity of flavonoids from S asoca on 2-stage skin carcinogenesis.

Chemoprevention of skin cancer by the flavonoid fraction of Saraca asoka.

Saraca asoka (Family - Caesalpiniaceae) has been widely used in the Ayurvedic (traditional Indian) system of medicine especially due to its wound healing property. The present study investigated the chemopreventive property of flavonoids from the flowers of Saraca asoka on 7,12 dimethyl benz(a)anthracene (DMBA) induced skin cancer in mice models. A single topical application of DMBA (100 microg/50 microL of acetone) followed after 2 weeks by three times a week treatment with croton oil (1% in acetone), for 20 weeks resulted in tumor induction. The topical application of the flavonoid fraction of S. asoka (FF S. asoka), 30 min prior to the application of croton oil thrice weekly for 20 weeks, caused a significant reduction in the number of tumors per mouse and the percentage of tumor-bearing mice. Also the latency period for the appearance of the first tumor was delayed by S. asoka pretreatment. In the flavonoid fraction (5 mg and 10 mg/kg body weight) treated animals, the levels of biochemical markers - rhodanese, myeloperoxidase, beta-D-glucuronidase, sialic acid, hexokinase and caspase 3 were significantly restored to near normal levels. These findings suggest the chemopreventive activity of flavonoids from S. asoka on two stage skin carcinogenesis. Histological data also support the chemopreventive potential of S. asoka.

Leaves of Cassia tora as a novel cancer therapeutic--an in vitro study.

Cassia tora Linn (Leguminacea) is a medicinal plant traditionally used as laxative, for the treatment of leprosy and various skin disorders. Preliminary phytochemical analysis of leaf showed the presence of polyphenols (3.7 mg gallic acid equivalent per gram dried leaves). The presence of phenolic compound prompted us to evaluate its antioxidant and antiproliferative potential. In the present study C. tora methanolic leaf extract (CTME) was evaluated for its nitric oxide scavenging activity and reducing power assays using Rutin and BHT as standards. The extract was studied for its lipid peroxidation inhibition assay using rat liver and brain. In all assays, a correlation existed between concentration of extract and percentage inhibition of free radical, reducing power and inhibition of lipid peroxidation. The antiproliferative activity of CTME with Cisplatin, anticancer drug was studied using human cervical cancer cells (HeLa). Proliferation of HeLa was measured by MTT assay, cell DNA content by modified diphenylamine method and apoptosis by Caspase 3 activity. The plant extract induced a marked concentration dependent inhibition on proliferation, reduced DNA content and apoptosis in HeLa. These results clearly indicate that C. tora is effective against free radical mediated diseases.

Bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine: a novel candidate in photodynamic therapy for skin cancer models in vivo.

Photodynamic therapy (PDT) is based on the light-induced activation of a photosensitizer generating highly reactive oxygen species that induce tissue destruction in malignant tissues. The present study was carried out to assess the photosensitizing potential of bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine in PDT trials in vivo. Male Swiss albino mice were divided into five groups. Skin tumor was induced using 7,12-dimethylbenz(a)anthracene - DMBA in the animals of Groups II, III, IV and V, while animals of Group I served as the control. At the completion of 20 weeks of induction, the tumor bearing mice from Group III, IV and V were given an intraperitoneal injection with the squaraine dye (12.5mg/kg body weight). After 24h, in the Group IV and V animals, the tumor area was exposed to visible light from a 1000W halogen lamp. The mice from groups I to IV were sacrificed two weeks after the PDT treatment and the marker enzymes (myeloperoxidase [MPO], beta-d-glucuronidase, rhodanese, lactate dehydrogenase [LDH], hexokinase, sialic acid and caspase) were assayed in tumor and normal tissues. Animals from Group V were sacrificed after 90 days of PDT treatment and the above parameters were recorded. Reduction in tumor volume and reversal of biochemical markers to near normal levels were observed in the treatment groups. The study assumes importance as it is the first report on PDT-a novel modality, using a squaraine dye for skin cancer therapy in vivo. The uniqueness of the mode of treatment lies in the selective uptake of squaraine dye by the cancer cells and their selective destruction using PDT without affecting the neighbouring normal cells, which is much advantageous over radiation therapy now frequently used. Also in skin cancer models, the progression/cure can be visualized by the naked eye which is another point of advantage, while seeking new modalities for the treatment of cancer.

Modulation of selenite cataract by the flavonoid fraction of Emilia sonchifolia in experimental animal models.

The purpose of the study was to investigate the role of flavonoids from Emilia sonchifolia (ES) on the progression of selenite-induced cataract. The antioxidant property of the flavonoids isolated from ES was assessed by measuring its capacity to inhibit superoxide production and serum oxidation in vitro in comparison with quercetin. Based on these experiments, an in vivo study was conducted to evaluate the modulatory effects of the flavonoids against selenite cataract. Cataract was induced by a single subcutaneous injection of sodium selenite (4 mg/kg body weight). The treatment group received flavonoids from ES (1 mg/kg) and this was compared with the quercetin treated group. Lens opacification was monitored by a slit lamp microscope and classified into six stages. Activity of the antioxidant enzymes - superoxide dismutase and catalase - and the level of lipid peroxidation products thiobarbituric acid reacting substances and reduced glutathione were studied. Slit lamp examination showed that the flavonoid fraction from ES could modulate the progression of cataract. Activities of superoxide dismutase, catalase and reduced glutathione were found to be increased in the ES treated groups, while thiobarbituric acid reacting substances were decreased compared with the selenite-induced group. The results suggest that flavonoids from ES can modulate lens opacification and oxidative stress in selenite-induced cataract.