Studies on the effect of Lantana camara leaf extract on time dependent cytotoxicity, ROS generation and epigenetic regulations in MCF-7 breast cancer cell line

Chakraborty, Ahana (2025) Studies on the effect of Lantana camara leaf extract on time dependent cytotoxicity, ROS generation and epigenetic regulations in MCF-7 breast cancer cell line. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Ahana Chakraborty (20MS084)) 20MS084_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (4MB)

Abstract

Breast cancer, being the most common cancer type worldwide, serves as one of the most severe threats to human beings. Although females are the primary target of this fatal disease, it can also develop in males, in accordance to the World Health Organization (WHO). In order to treat breast cancer, there are various commercially available drugs which exhibit diverse side effects and toxicities like diarrhea, alopecia, skin infections etc. Hence, to circumvent these unwanted toxicities, plant-based treatment is a safer option. For our study, we have selected Lantana camara plant leaves, which is an important medicinal plant dispersed in various regions of the world including India. Along with numerous medicinal properties, this plant is also effective against cancer. Among the various cell-lines used for research studies on breast cancer, we have selected MCF-7 cell line as our in vitro model to study the effect of Lantana camara leaf extract (LCLE). MCF-7 is a luminal A subtype of breast cancer, i.e., it possesses Estrogen receptor (ER+) and Progesterone receptor (PR+) and does not possess the Human epidermal growth factor 2 receptor (HER2-). In this study, the cytotoxicity of our LCLE has been explored. By performing Trypan blue dye exclusion test and MTT assay and by morphological analysis, it has been reported here that LCLE induces cytotoxicity in MCF-7 cells in a dose-dependently as well as time-dependently. The half maximal Inhibition Concentration (IC50) of LCLE obtained at 12 hours and 48 hours are 98.46 μg/mL and 53.47 μg/mL, respectively. Furthermore, clonogenic assay was carried out at 12h, 24h and 48h to study the reproductive viability of MCF-7 cells after LCLE treatment. This indicated that LCLE also induces dose-dependent and time-dependent decrease in proliferative ability of MCF-7 cells. Hanging drop assay, performed with four different doses of LCLE, has inhibited spheroid formation in MCF-7 cells treated with LCLE for 24h. Additionally, ROS production was studied at six timepoints, 1h, 3h, 6h, 12h, 24h and 48h, with two selected LCLE doses, 40 μg/mL & 80 μg/mL. LCLE produces significant amount of ROS starting from 1h of treatment which time-dependently increases up to 48h. In objective 2, the MCF-7 viability was checked with antioxidant N-Acetyl Cysteine (NAC) to check if the LCLE cytotoxicity is ROS mediated. But, no significant increase in cell viability was observed, rather, NAC produced a cumulative cytotoxic effect on MCF-7 cells along with our plant extract. DCF-DA assay has revealed that both NAC has scavenged the ROS produced by LCLE. To further confirm this, another antioxidant Vitamin C or L-Ascorbic acid was used, which has produced similar results. Thus, it has been concluded that cytotoxicity of LCLE in MCF-7 cells is not ROS mediated, rather, through some other ROS associated pathway. Further, the DNA methylation status of LCLE treated MCF-7 was explored by checking the gene expression of DNA methyltransferases (DNMTs) in four different concentrations of LCLE. It has been observed that DNMT 1, 3A and 3B expression has reduced for higher LCLE doses which indicates that DNMTs act as oncogenes in MCF-7 cells.Breast cancer, being the most common cancer type worldwide, serves as one of the most severe threats to human beings. Although females are the primary target of this fatal disease, it can also develop in males, in accordance to the World Health Organization (WHO). In order to treat breast cancer, there are various commercially available drugs which exhibit diverse side effects and toxicities like diarrhea, alopecia, skin infections etc. Hence, to circumvent these unwanted toxicities, plant-based treatment is a safer option. For our study, we have selected Lantana camara plant leaves, which is an important medicinal plant dispersed in various regions of the world including India. Along with numerous medicinal properties, this plant is also effective against cancer. Among the various cell-lines used for research studies on breast cancer, we have selected MCF-7 cell line as our in vitro model to study the effect of Lantana camara leaf extract (LCLE). MCF-7 is a luminal A subtype of breast cancer, i.e., it possesses Estrogen receptor (ER+) and Progesterone receptor (PR+) and does not possess the Human epidermal growth factor 2 receptor (HER2-). In this study, the cytotoxicity of our LCLE has been explored. By performing Trypan blue dye exclusion test and MTT assay and by morphological analysis, it has been reported here that LCLE induces cytotoxicity in MCF-7 cells in a dose-dependently as well as time-dependently. The half maximal Inhibition Concentration (IC50) of LCLE obtained at 12 hours and 48 hours are 98.46 μg/mL and 53.47 μg/mL, respectively. Furthermore, clonogenic assay was carried out at 12h, 24h and 48h to study the reproductive viability of MCF-7 cells after LCLE treatment. This indicated that LCLE also induces dose-dependent and time-dependent decrease in proliferative ability of MCF-7 cells. Hanging drop assay, performed with four different doses of LCLE, has inhibited spheroid formation in MCF-7 cells treated with LCLE for 24h. Additionally, ROS production was studied at six timepoints, 1h, 3h, 6h, 12h, 24h and 48h, with two selected LCLE doses, 40 μg/mL & 80 μg/mL. LCLE produces significant amount of ROS starting from 1h of treatment which time-dependently increases up to 48h. In objective 2, the MCF-7 viability was checked with antioxidant N-Acetyl Cysteine (NAC) to check if the LCLE cytotoxicity is ROS mediated. But, no significant increase in cell viability was observed, rather, NAC produced a cumulative cytotoxic effect on MCF-7 cells along with our plant extract. DCF-DA assay has revealed that both NAC has scavenged the ROS produced by LCLE. To further confirm this, another antioxidant Vitamin C or L-Ascorbic acid was used, which has produced similar results. Thus, it has been concluded that cytotoxicity of LCLE in MCF-7 cells is not ROS mediated, rather, through some other ROS associated pathway. Further, the DNA methylation status of LCLE treated MCF-7 was explored by checking the gene expression of DNA methyltransferases (DNMTs) in four different concentrations of LCLE. It has been observed that DNMT 1, 3A and 3B expression has reduced for higher LCLE doses which indicates that DNMTs act as oncogenes in MCF-7 cells.

Actions (login required)

View Item