My Blog List

Saturday, May 01, 2010

Presented at Interdisciplinary Science Congress-2009 on Interface Between Chemistry and Biology, October 8, 2009, Jamia Millia Islamia, New Delhi.

Antiproliferative Effect of Cassia fistula Extracts on HeLa Cells.

Background:
Cancer is a dangerous disease of mankind which continuously requires the development of better therapeutic support for the treatment/management.Cervical cancer leads to 250,000 deaths per year world wide. Medicinal plants had been studied to serve as promising treatment strategies for cancer chemotherapy.Among several traditionally well-practiced herbal medicines from India,Cassia fistula had been shown to play some role as an anti-tumor agent.Cassia fistula Linn (Hindi-Amaltas, English-Golden shower, Indian labrum)is a member of Fabacea (alt. leguminasae) family.In Ayurvedic medicinal system, C. fistula was used against various disorders such asaematemesis, pruritus, leucoderma, diabetes and other ailments.The seed and pod of C. fistula have been evaluated as an important source of secondary metabolites such as alkalanoids, flavanoids, turpenoids. Gupta et al. (1998) had reported anti-tumor activity of seed in ascited mice model. However, anti-tumor potential of C. fistula in cervical cancer is yet to be explored.

Objective:
With these background information the present study is aimed to evaluate the anti-proliferative effect of Cassia fistula seeds and pod on Human Cervical Adenocarcinoma cell line (HeLa).

Materials and Methods:
Cell Lines and Culture Conditions: HeLa cell line was obtained from National Centre for Cell Sciences (NCCS), Pune. Cells were incubated in DMEM medium supplemented with 10% foetal bovine serum, penicillin (100U/ml) and streptomycin (100U/ml) at 37oC temperature and 5% CO2 atmosphere.
Extraction of C. fistula: Fresh ripe fruits of Cassia fistula (Linn) were collected from the campus of Delhi Institute of Pharmaceutical Science and Research (DIPSAR), New Delhi, India. A voucher specimen was stored in laboratory for future reference. Seeds and pod were separated and powdered. The aqueous extract of seed and pod were obtained and lyophilized.
Cell Proliferation Assay: Proliferation of cells was assessed by MTT assay. HeLa cells (2.5 × 105/ml) were briefly cultured in a final volume of 0.2 ml in a 96-well, flat-bottom plate for 48 h and then incubated with MTT (5 mg/ml, 20 μl/well) for 4 h. The medium was replaced with DMSO (150 μl/well) and after 10 min incubation the plate was read on the iMark Microplate Reader (BioRad, USA) with a wavelength of 570 nm. In these experiments, cells were treated with various concentrations (1, 2, 4, 6, 8, 10 mg/ml) of aqueous extract of C. fistula (Seed/Pod) for 48 h, and the cytotoxicity of C. fistula extract was expressed as IC50.
Detection of Apoptosis by DNA Fragmentation Assay: After being incubated with different concentrations of extract for 48 hr, approximately 107 cells were harvested and pelleted by centrifugation (Eppendorf 5804R, Germany). Cellular DNA was extracted by a standard method and the DNA samples were analyzed by 2% agarose gel electrophoresis and visulalized as a DNA Ladder with UV.

Results:
C.fistula extracts Inhibits HeLa Cell Proliferation: C. fistula extracts concentrations ranging from 1 to 10 mg/ml were monitored after 48hrs of cultivation in the cell line and the IC50 value for both C. fistula pod and seed extracts was calculated as 4.5 mg/ml and 3.8 mg/ml respectively. On the basis of the results, cell line treated with C. fistula extracts were incubated for 48 h in the next experiments.
Validation of Apoptosis Measurement by DNA Laddering: In our results, the cells were treated with C. fistula extracts, and the DNA was directly extracted and run on agarose gel. DNA fragmentation, if presented, was seen as a stepwise ladder of DNA fragments at about 200-basepair increments below 2.3 kb. The data shows that DNA laddering is pronounced for C. fistula extracts (Pod/ Seed) in cell line.

Conclusion:
The early trends revealed that the aqueous extract of C. fistula (seed/pod) has certain level of antiproliferative effects against HeLa cell line. Their therapeutic potential is yet to be explored.

Monday, January 18, 2010

Presented at International Symposium on Cancer Chemoprevention and Translational Research at Jawaharlal Nehru University, New Delhi on 21st Dec. 2009.

Antiproliferative Effect of Cassia fistula on U87 MG Cells

Objective:
Cancer is a complex disease which occurs as a result of a progressive accumulation of genetic and epigenetic changes that enable escape from normal cellular and environmental control.Glioblastomas are the most common tumor arising from the central nervous system. Approximately 74,000 glioblastomas are diagnosed worldwide each year and there are indications that the incidence of these types of tumors is increasing. Glioblastoma multiforme is a malignant neoplasm that accounts for approximately 55% of all gliomas.Recently, medicinal plants had been studied to serve as promising treatment strategies for cancer chemotherapy. Among several traditionally well-practiced herbal medicines from India, Cassia fistula had been shown to play some role as an anti-tumor agent.
Cassia fistula Linn (Hindi-Amaltas, English-Golden shower, Indian labrum)is a member of Fabacea (alt. leguminasae) family.In Ayurvedic medicinal system, C. fistula was used against various disorders such as haematemesis, pruritus, leucoderma, diabetes and other ailments.
The seed and pod of C. fistula have been evaluated as an important source of secondary metabolites such as alkalanoids, flavanoids, turpenoids. Gupta et al. (1998) had reported anti-tumor activity of seed in ascited mice model.However, anti-tumor potential of C. fistula in glioblastoma is yet to be explored.

Objective:
With these background information the present study is aimed to evaluate the anti-proliferative effect of Cassia fistula seeds and pod on Human Malignant Glioma cell line (U87 MG).

Materials and Methods:
Cell Lines and Culture Conditions: U87 MG cell line was obtained from National Centre for Cell Sciences (NCCS), Pune. Cells were incubated in DMEM medium supplemented with 10% foetal bovine serum, penicillin (100U/ml) and streptomycin (100U/ml) at 37oC temperature and 5% CO2 atmosphere.
Extraction of C. fistula: Fresh ripe fruits of Cassia fistula (Linn) were collected from the campus of Delhi Institute of Pharmaceutical Science and Research (DIPSAR), New Delhi, India. A voucher specimen was stored in laboratory for future reference. Seeds and pod were separated and powdered. The methanolic extract of seed and pod were obtained and lyophilized.
Cell Proliferation Assay: Proliferation of cells was assessed by MTT assay. U87 MG cells (2.5 × 105/ml) were briefly cultured in a final volume of 0.2 ml in a 96-well, flat-bottom plate for 48 h and then incubated with MTT (5 mg/ml, 20 μl/well) for 4 h. The medium was replaced with DMSO (150 μl/well) and after 10 min incubation the plate was read on the iMark Microplate Reader (BioRad, USA) with a wavelength of 570 nm. In these experiments, cells were treated with various concentrations (1, 2, 4, 6, 8, 10 mg/ml) of methanolic extract of C. fistula (Seed/Pod) for 48 h, and the cytotoxicity of C. fistula extract was expressed as IC50.
Detection of Apoptosis by DNA Fragmentation Assay: After being incubated with different concentrations of extract for 48 hr, approximately 107 cells were harvested and pelleted by centrifugation (Eppendorf 5804R, Germany). Cellular DNA was extracted by a standard method and the DNA samples were analyzed by 2% agarose gel electrophoresis and visulalized as a DNA Ladder with UV.
Phytochemical Tests: Phytochemicals were determined by standard chemical methods.

Results:
Preliminary phytochemical screening of methanolic extracts of C. fistula revealed the presence of phenols, flavanoids, tannins, alkaloids and glycosides in both the seed and pod.
C.fistula extracts Inhibits U87 MG Cell Proliferation
C. fistula extracts concentrations ranging from 1 to 10 mg/ml were monitored after 48 h of cultivation in the cell line and the IC50 value for both C. fistula pod and seed extracts was calculated as 6.1 mg/ml and 3.9 mg/ml respectively. On the basis of the results, cell line treated with C. fistula extracts were incubated for 48 h in the next experiments.
Validation of Apoptosis Measurement by DNA Laddering
In our results, the cells were treated with C. fistula extracts, and the DNA was directly extracted and run on agarose gel. DNA fragmentation, if presented, was seen as a stepwise ladder of DNA fragments at about 200-basepair increments below 2.3 Kb. The data shows that DNA laddering is pronounced for C. fistula extracts (Pod/ Seed) in cell line.

Conclusion:
The early trends revealed that the methanolic extract of C. fistula (seed/pod) has certain level of antiproliferative effects against U87 MG cell line. Their therapeutic potential is yet to be explored.

Wednesday, January 13, 2010

Presented at INTERNATIONAL CONFERENCE ON FRONTIERS IN PREVENTION, DIAGNOSIS & THERAPY OF CANCER (FPDTC – 2009) in Allahabad on 21st-22nd Nov. 2009.

Allelic loss of 6q25-27, the PARKIN tumor suppressor gene locus, in cervical carcinoma from northern Indian population

Background:
Frequent loss of heterozygosity (LOH) within genetically defined chromosomal regions is considered an indication of the presence of a putative TSG. Several loss of heterozygosity (LOH) studies have indicated that the chromosome 6q25-q27 region is frequently altered in a variety of human cancers.
Recently, Parkin, a gene implicated in autosomal recessive juvenile Parkinsonism, was found to be a target of LOH at chromosome 6q25-q27 in breast, ovarian and lung tumorigenesis. Although various deletions and point mutations have been described in patients with early onset of Parkinsonism, mutation analysis failed to identify somatic point mutations in any of the breast or ovarian tumors with LOH at the Parkin/FRA6E locus examined.However, truncating and homozygous deletions were identified in the lung adenocarcinoma cell lines Calu-3 and H-1573. RT–PCR of Parkin on a panel of cell lines derived from breast, kidney, cervical and prostate cancers revealed that Parkin is also genetically altered in cancer types other than ovarian cancer.

Objective:
With these background information the present study is aimed to define regions of allelic loss at human chromosome 6q25–q27 in cervical cancer patients from northern India.

Materials and Methods:
Tissue samples: One hundred and five cervical tissue biopsies of carcinoma patients and their matched control samples (blood/precancerous lesions) were collected from Maulana Azad Medical College and associated LNJP Hospital, New Delhi and was immediately stored in -80oC.
DNA extraction: DNA was extracted from the above frozen cervical tissue biopsies and their matched control samples by standard phenol-chloroform method, then dissolved and stored in TE buffer. Finally, purity and concentration of extracted DNA were analyzed by gel electrophoresis and ultraviolet spectrophotometry.
Microsatellite Marker selection and PCR amplification: Three microsatellite marker sites: D6S1599, D6S305 and D6S1008 in chromosome 6q25-27 were selected to detect LOH of Parkin gene. D6S1599 and D6S305 are intragenic markers which are present in Parkin introns 2 and 7, respectively where as D6S1008 is present in the flanking region at telomeric end. Primer sequences are available at the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov/). PCR amplification was carried out in a final volume of 25 μL, containing 50 ng DNA, 2.5 μL of 10X PCR Buffer, 1.5 mmol/L MgCl2, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, and 1 U Taq DNA polymerase. The amplification conditions were as follows: an initial incubation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, at 58°C , 62°C and 57°C for 1 min for above mentioned primers respectively, at 72°C for 1 min, and a final extension at 72°C for 5 min.
LOH analysis: The amplified PCR products were denatured at 95°C for 5 min and run on 8% denaturation polyacrylamide gel (1xTBE buffer, circulatory water) at a voltage of 500 V, and power of 45 W for 3.5 h. Silver staining was performed as previously described in Laboratory manual by Sambrook.
Determination of Parkin LOH: The heterozygous genomic allele was targeted for LOH information analysis. LOH was defined as a complete loss or up to 40% decreased relative density of silver staining bands of PCR products in cervical cancer samples compared to their matched control samples (blood/precancerous lesions.
Statistical Analysis: LOH found in two intragenic markers (D6S1599, D6S305) was compared with the marker of flanking region (D6S1008) using the Chi-Square test. P<0.05>Parkin gene in 3 microsatellite sites are shown in Table 1. 59 of 105 (56%) samples showed LOH in at least one locus in the region examined. The percentage of LOH for each of the three markers ranged from 25% (D6S1008) to 48% (D6S305). The most frequent site was D6S305, accounting for 48%. Presence of LOH was significantly higher in both the intragenic markers (D6S1599 and D6S305) as compared with the locus of flanking region (D6S1008) with their p value 0.04 and 0.003, respectively.
Results:
59 of 105 (56%) samples showed LOH in at least one locus in the region examined. The percentage of LOH for each of the three markers ranged from 25% (D6S1008) to 48% (D6S305). The most frequent site was D6S305, accounting for 48%. Presence of LOH was significantly higher in both the intragenic markers (D6S1599 and D6S305) as compared with the locus of flanking region (D6S1008) with their P value 0.04 and 0.003, respectively.

Conclusion:
Chromosomal region 6q25–q27 has frequently undergone deletions in a wide spectrum of human neoplasms, such as melanoma, ovarian cancer, breast cancer, non-Hodgkin’s B cell lymphoma, and several other types of cancer.
Our findings suggest that Parkin is a strong candidate TSG located at human chromosome 6q25–q27 and that its reduced expression and inactivation may play a major role in the progression of cervix carcinoma.