Effect of Secoisolariciresinol Diglucoside Rich Extract of Linum Usitatissimum L. on Skin Cancer and Determination of Its Mechanism of Action

Abstract

INTRODUCTION AND OBJECTIVES: Occurrence and mortality rates due to skin cancer are increasing worldwide. Regardless of different treatments available against skin cancer, there are many limitations like recurrence of the disease, low effectiveness, side-effects and cost of the treatments with its compliance. These limitations accentuate the need of newer molecules against skin cancer. Many herbal molecules have been screened against the effect of different cancer cells, amongst which one is lignans. One of the richest sources of lignan is flaxseed, which consist of ecoisolariciresinol as major lignan present. Earlier reports have shown great potential of this lignan against the breast cancer, colon cancer and prostate cancer. Despite this, till date the no reports are available on its effect against the skin cancer. Hence in light to these facts, the objectives of the present study are as follows: 1. To study effect of different extracts of L. usitatissimum invitro on skin cancer cell lines. 2. To study the effect of L. usitatissimumon two stage skin cancer model of mice using dimethylbenz (a) anthracene and croton oil. 3. To determine the mechanism of action of extract of L. usitatissimum. 4. To develop the therapeutic strategy for skin cancer. MATERIALS AND METHOD: 1. Phytochemical studies: We prepared four different types of extracts/preparations namely flaxseed oil, ethanolic extract of flaxseed, secoisolariciresinol diglucoside (SDG) rich extract and secoisolariciresinol(SECO) rich extract. Flaxseeds were crushed and were subjected to defattening process with n-hexane. The filterate obtained consisted of flaxseed oil dissolved in n-hexane which was further subjected to distillation to remove solvent from the oil. The residue was collected and was subjected to two different treatments, for making SDG rich extract and ethanolic extract. Further SDG rich extract was used as prerequisite in preparing SECO rich extract. 2. Cell viability studies To evaluate the potency of different extracts in inhibiting the cancer cells, (MTT) assay was carried out. Inhibitory action of different extracts was tested using A-375 human melanoma cell line. The extract with the IC 50 value nearest to the IC 50 value of the standard 5- Fluorouracil was selected for the further study. 3. Phytochemical analysis Preliminary identification test of SDG rich extract was carried out to be check the presence of flavonoids, carbohydrate, proteins, cyanogenic glycoside, tannins, steroids, saponins and alkaloids. Qualitative test of the SDG rich extract was carried out using TLC and UV spectrophotometry. 4. Pharmacological studies Female Balb/C mice were used for the present study. Skin cancer was induced using Dimethlybenz (a) anthracene (DMBA) and croton oil. 50ul of DMBA (1mg/ml in acetone) was sprayed directly on the caudal region of the mice at 1st week. Application was made twice weekly for period of one week. On 3rd week from the day of DMBA application, 50ul of croton oil (1% in acetone) was sprayed on the caudal region of the mice twice weekly for period of 16 weeks. From 7th week the application of 5-FU, 2% and 5% SDG rich suspension was applied to the tumor affected areas to the respective groups of mice. From 7th to 16th week, we measured the change in tumor volume and tumor burden levels. After 16th week of dosing the blood samples along with the skin tissue samples were collected from the mice back. Blood samples collected were used to calculate the levels of nonspecific biomarkers of cancer like LDH, GGT, and C-reactive protein. Skin samples were used to determine collagen levels, oxidative stress markers, histopathological studies and mRNA expression of p53 and CDK4.