Studies on the Effect of Lantana camara Leaf Extract on the miRNA-mediated Regulation of Proliferation, Survival, and Migration of Breast Cancer Cells

Pal, Arundhaty (2026) Studies on the Effect of Lantana camara Leaf Extract on the miRNA-mediated Regulation of Proliferation, Survival, and Migration of Breast Cancer Cells. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Arundhaty Pal (20RS118)) 20RS118.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

Breast cancer is one of the most prevalent cancers among women and one of the major causes of cancer-related mortality globally. Among the different subtypes of breast cancers, luminal A is the most prevalent subtype and also has a better prognosis. However, the triple-negative breast cancer is the most invasive type of breast cancer and has the worst prognosis. There are different treatment options available for treating breast cancer, like surgery, radiation therapy, immunotherapy, and chemotherapy, but all these treatments come with various short-term and long-term side effects, and excessive use of these treatments has led to the development of drug resistance. Due to these reasons, exploration of newer or alternative treatment options, preferably from a natural source like plants, is crucial. Plants are a huge reservoir of bioactive compounds like flavonoids, alkaloids, terpenoids, saponins, etc., and all these compounds are known to have different medicinal properties, including anti-cancer activity. Many cancer drugs like vinblastine, vincristine, taxanes and camptothecin are plant-derived and are commercially available. However, the majority of the plant world is still unexplored. Lantana camara L., a flowering plant of the verbena family, has been known for its myriad medicinal values, including anti-tumour potential, for a long time. However, the knowledge on the effects of Lantana camara on breast cancer, specifically on triple-negative breast cancer, is limited. In this study, the anti-tumour potential of L. camara ethanolic leaf extract was explored on two different breast cancer cell lines: MCF-7 (luminal A) and MDA-MB-231 (triple negative breast cancer cell line), to delineate the effects of the leaf extract on the proliferation, survival and migration of breast cancer cells. Results of the present study revealed that L. camara leaf extract induced cytotoxicity in both MCF-7 and MDA-MB-231 cell lines, time and dose dependently. Analysis of cell cycle phase distribution through flow cytometer revealed that the leaf extract could induce G0/G1 and S-phase arrest in MCF-7 cells and G0/G1 phase arrest in MDA-MB-231 cells with a subsequent increase in the sub-G1 population. An increase in the sub-G1 population is an indication of DNA damage, and it was verified by DAPI staining. Further, AnnexinV-FITC/PI double staining confirmed that treatment with the ethanolic extract could result in significant cell death in both breast cancer cell lines, mainly through apoptosis. The molecular level expression of the regulators of cell cycle and cell death (Cyclin D1, Cyclin A2, p21, BCL-2, MCL-1, BAX, and BAK) also supported the above findings. To confirm whether the cytotoxicity of the extract was more specific for breast cancer cells, non-cancer human kidney epithelial-like HEK293T cells were used as a control and showed a higher IC50 value for L. camara and very little cell death compared to breast cancer cells. One of the phenomena of cancer cells is their ability to migrate to different parts of the body and form secondary tumours. Therefore, the ability to inhibit/reduce the migration of cancer cells can be regarded as one of the important requirements for any anti-tumour agent. MCF-7, a luminal A-type breast cancer cell, has lower migration potential, whereas MDA-MB-231, being a triple-negative breast cancer cell line, is highly invasive in nature. Treatment with L. camara leaf ethanolic extract impaired the migration of both cell lines, indicating the anti-migratory activity of the leaf extract. Expression of EMT-related genes also supported these findings. Epigenetic alteration is emerging as a crucial player in carcinogenesis. Among different epigenetic alterations, miRNAs are emerging as diagnostic and prognostic tools for cancer treatment, as several miRNAs are reported to be dysregulated in various cancers, including breast cancer. Different plant bioactive compounds are also known to regulate the expression of several miRNAs in cancer. However, to date, no report is available on the role of L. camara in miRNA regulations. Therefore, to have an extensive knowledge of the anti-cancer activity of the extract, the effect of the leaf extract on the regulation of miRNA-181a was observed. miR-181a-5p is known to play important roles in cancer progression and control in various cancers, including breast cancer. The present study revealed that L. camara leaf extract could upregulate the expression of miR-181a-5p in both MCF-7 and MDA-MB-231 cell lines, suggesting a possible tumour suppressor role of miR-181a-5p in breast cancer. To further investigate the role of miR-181a in breast cancer, miR-181a was ectopically overexpressed in both MCF-7 and MDA-MB-231 cells, and its effects on cell proliferation, survival and migration were observed. Through the cell cycle phase distribution assay, it was found that miR-181a was able to induce an increase in the sub-G1 population in both MCF-7 and MDA-MB-231 cell lines. Treatment with L. camara extract, along with miR-181a overexpression, further increased the sub-G1 population. AnnexinV-FITC/PI double staining revealed that miR-181a can induce significant cell death in both cell lines. Validation of miR-181a targets BCL-2 and MCL-1 also supported these observations. However, the ectopic overexpression of miR-181a alone could not impede the migration of MCF-7 and MDA-MB-231, but in the presence of L. camara extract in miR-181a overexpressed cells, cell migration was inhibited. Through bioinformatics analysis, CDKN3 was found to be one of the targets of miR-181a-5p, and it is also known to play an important role in migration and cell proliferation in various cancers, including breast cancer. However, no report is available to date confirming the interaction of miR-181a with CDKN3. Therefore, the present study explored CDNK3 as a target for miR-181a-5p in MCF-7 and MDA-MB-231 cells. Expression of CDKN3 was observed to be decreased at the mRNA level upon ectopic overexpression of miR-181a in breast cancer cells, indicating interaction of miR-181a with CDKN3. Reporter assay confirmed the interaction of miR-181a-5p with the 3ʹ untranslated region of CDKN3 mRNA. Overall, the findings revealed that the ethanolic leaf extract of Lantana camara could induce cytotoxicity, inhibit cell proliferation and induce cell death in both MCF-7 and MDA-MB-231 cells. The extract also impeded the migration potential of both breast cancer cell lines. Further investigation revealed that it could also upregulate the expression of miR-181a-5p in the MCF-7 and MDA-MB-231 cells, and ectopic overexpression of miR-181a induced cell death in both MCF-7 and MDA-MB-23 cells and inhibited cell migration in the presence of Lantana camara extract. Hence, Lantana camara can be regarded as a potential candidate for breast cancer management in future.

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