"Expand life , Comfort death is our minimum motto" 

This historical journey towards cancer treatment started along with the initiatives taken by victims of Cancer patients and their family members. This effort in the field of cancer treatment has been taken place from the March 2003. This program is based on the true story of person suffering from cancer disease, given the fact that to whom concerned hospitals, nursing homes and doctors said no more treatments possible actually got cured or relieved from the herbal treatments. It got started when all the related people gathered in a place and discussed about any additional possibilities of treatment of cancer through any means. Different people gave different ideas, and lastly a decision was made to use medicinal plants/herbals for cancer treatment. We have consulted with some doctors. Dr. Surendra is leading the team of doctors, and they analyze the patient reports. Each of them

belonging to different parts of the country, altogether made a decision to start study, research, and at the same time use the medicinal herbal plants practically in the treatments for different kinds of cancer under the direction of Mr. Devendra Basnet (traditional practitioner and experienced healer). The concerned people then committed to use whatever sources possible to find out herbalist and healer (Mr. Devendra Basnet) who have been working in this field from his generation. We have used our own sources to find out

traditional herbalists who are working in this field, and at the same time, we made a group first to search those traditional and experienced herbalists from different parts of the country, and we made gathering point in Kathmandu to know their practical knowledge about

the diseases. Under the leadership of Mr. Devendra Basnet, we collected more than 25 herbalists from different parts of the country and made a three days discussion program with them. Mr. Basnet is from Khotang district of Nepal, and he is an academic professional

herbalist. We also involved some cancer patients in checking them and starting treatment through those herbalists under the guideline and close supervision of Mr. Basnet.

 Research Group 

Principal Investigator

Dr. Bhupal Govinda Shrestha, the Principal Investigator for this project, has been an Associate Professor in the Department of Biotechnology at Kathmandu University since February 2009. He did his Ph.D. in Cancer Biology from the Tokyo University of Agriculture, Japan. During his Ph.D., he was also involved in a project in Ashwagandha, an Indian medicinal plant and cancer, where his team was able to give first molecular insights into the anti-cancer activity of the plant. Since then, he has worked as a postdoc fellow at the University of British Colombia, Vancouver, Canada, in zinc and breast cancer. After returning to Nepal and joining the Department of Biotechnology at Kathmandu University, he has been involved in medicinal plants and cancer research. He has received funds from the University Grants Commission, Nepal, the International Foundation for Science, Sweden, Nepal Ayurveda Research and Training Center, and the Third world Academy of Science, Italy, to research medicinal plants and cancer. Recently, he received a fellowship from the National Research Council of Thailand to study the isolation of bioactive components from medicinal plants. He has supervised several undergraduate and graduate students in this area and has over twenty publications. As Principal Investigator, he will be involved in all aspects of the project, including planning experiments, report writing, result analysis, finance, and human resource planning.

 Investigator

Rajib Kumar Shrestha works as Assistant Professor, in the Department of Chemical Science and Engineering, at Kathmandu University and will be an activity leader for the project. He has Master's and MPhil degrees in Chemistry and now doing Ph.D. in medicinal plants research. He has received a research grant from UGC and has numerous publications in peer-reviewed journals.

 Researcher

Prabal Singh Maharjan works as Researcher at the NTIC-funded project specializing in Baccatin III extraction from Taxus species of Nepal. He completed his Master of Technology in Biotechnology from Kathmandu University. He did his thesis on evaluating the medicinal plant's anticancer properties under Dr. Bhupal Govinda Shrestha.

 Research Process

 Collection of medicinal plants

Medicinal plants will be collected from different parts of Nepal with local people or healers' help. The herbarium will be prepared and identified by the National Herbarium & Plant Laboratories taxonomist.

 Sample processing

Plant sample collection will be placed in the dry and shaded storage space to let the plant material shade dry or put the plant sample into a plant dryer. Thus obtained plant small will be ground to a fine powder for the extraction process.

 Extraction process

The powdered sample is extracted with different solvents with high to low polarities, such as Methanol, chloroform, ethyl acetate, and hexane, respectively. The sample will be subjected to cooled maceration for three days, with three repetition of extraction. All the extracts will be pooled together and dried over rotary evapourator. Plant extract extracted in different solvent are subject to preliminary anticancer evaluation via MTT assay. Based on the cell death and cell inhibition, the extract with the best efficacy will be selected and subject to fractional separation. Each Fraction will be again tested against cancer cell lines.

 Fractional purification

The crude extract contains hundreds of phytochemicals. However, only some will have significant medicinal properties. To isolate and purify these phytochemicals, we perform fractional purification. The crude extract will be separated into a fraction in column chromatography using suitable solvent. The Fraction separation is observed using the thin layer chromatography method. The fraction will be tested against cancer cell line. If it shows a good result, it will be further separated in column chromatography using suitable solvent. Individual phytochemicals obtained after separation will be studied for the mechanism of action and sent for Mass spectroscopy.

 Anticancer activity and study of the mechanism of its action

Human normal cell lines TIG-1, WI-38 and cancer lines osteogenic sarcomaU2OS, breast carcinomaMCF-7, lung carcinoma PC14, faibrsarcoma HT 1080 and colon carcinoma HCT116, cell lines will be purchased from ATCC(American Type Culture Collection). The cells will be cultured in respective medium supplemented with 10% fetal bovine serum in a humidified incubator, 37C, and 5%CO2. All cells will be counted by hemocytometer and plated and harvested at around 80-90% confluency. For lysate making, cells will be washed with PBS (Phosphate Saline Buffer), trypsinized, and cell pellet collected. The harvested cell pellets will be washed with PBS twice, and lysis will be done over ice for 30 mins with vortexing every 10 minutes, using NP-40 lysis buffer. The clear supernatant will be used for further assays.

 For this the plants will be tested for their selective anti cancer activity in cancer and

normal cell lines at different concentrations, and MTT assay will be done to find the IC 50 value. MTT assay is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. Equal number of cells, cancer and normal cell lines, will be plated in 96 well plate and allowed to grow for a day. Our DMSO solubilized extract will be added to each well and allowed to grow for 48 hours. MTT reagent will then be added, and the change in colour will be observed colorimetrically after 4-5 hours of addition of the MTT reagent. The control consists of DMSO in equal amount available in the extract. All experiments will be done in triplicates.The mechanism of action will be studied using molecular biology tools like apoptosis assay, western blotting, immunostaining, TRAP assay.

 For western blotting, protein sample (20ug) will be separated on a SDS polyacrylamide gel which will be electroblotted onto a nitrocellulose membrane. Proteins will be visualized with horseradish peroxidase conjugated secondary antibody and ECL Plus using a X-ray developer. Immunoassays will be done using p53, pRb, p21, Bax, caspase-3, ATM, ATR and actin anti body, among others. We know p53, a tumor suppressor gene, has central role in cancer initiation. The p53 protein acts as a checkpoint in the cell cycle, either preventing or initiating programmed cell death. Since cancer is the unchecked proliferation of cells, p53’s role is critical. Mutation in p53 gene, causing it to loose its activity, is implicated for 50-60% of all cancers. So, first we begin by looking if the addtion of the extract to the cells increases the p53 protein level by western blotting. Increase in p53 protein level leads to increased transcription of apoptotic/senescence proteins like p21, Bax, BH3 only proteins. These will also be quantitated along with other proteins.

For immunostaining, cells are grown on coverslips, and after treatment with our extract, are fixed on the coverslips using 4% formaldehyde or cold aceton-methanol.

 For Immunostaining, we permeabilized the cells with 0.2-0.5% Triton-X 100 in TBS for 20min and incubated with first antibody for an hour and then with fluorescence conjugated secondary antibody for 30 mins. Cells are washed and fixed in a plate using glycerol and observed under fluorescent microscope. This experiment gives us the localization of proteins, like if we use p53 antibody and we see that p53 in untreated sample is cytoplasmic and that in our treated sample is nuclear ie in the nucleus, we can says that p53 is transcriptionally active and that it acts as an transcriptional activator of its down stream genes.

 TRAP(Telomere Repeat Amplification Protocol) assay

To proliferate indefinitely, cancer cells must maintain the length of their telomeres. By interfering with the telomere elongation mechanism within these cells, it may be possible to force cell proliferation to stop, thus halting tumour progression. Telomere elongation process in cancer cells is mediated by an enzyme called telomerase. Reactivation of telomerase is the most frequent mechanism found by cancer cells to acquire virtually unlimited growth capacity.
We will use the TRAP assay, Gel based and ELISA based, to detect the telomerase activity to ascertain whether, the mechanism of action of our extract is by its ability to inhibit telomease.