Curcumin‐Induced Apoptosis Pathways in Positive and Negative Human Epidermal Growth Factor Receptor Breast Cancer Cells
Authors: Ali Mohammed Showkat , Smiley Rebecca 
Published: April 2020

Aim

Curcumin, have several advantages and potential health benefits, such as low cytotoxicity and minimal development of resistance, as compared to chemically‐based therapies, and have anti‐cancerous efficacies, including induction of apoptosis, in cancer cells of various tissue origins. The aim of this study was toelucidate the mechanisms of curcumin‐induced apoptosis in human epidermal growth factor receptor positive (BT474) and negative (MCF‐7) breast cancer cell lines.

Methods

Cytotoxic activity of curcumin was measured by the sulphorhodamine B assay. We studied the induction of apoptosis by flow cytometry and detection of an apoptosis‐associated PARP cleavage product via Western blotting. Westernblotting was also used to measure Cytochrome C translocation, which determines mitochondrial involvement and caspase‐7 activation. The Caspase cascade was measured by activity levels of caspase‐3, caspase‐8, and caspase‐9 using chromogenic substrates.

Results

Curcumin CC50 (concentration that kills 50% of the cells) was 25.92 ±5.13μM for MCF‐7, and 49.96 ± 5.98 μM for BT474 cells. Flow cytometry results indicated a 13.36 ±5.96% expression increase and a 6.87 ± 1.42% expression increase in early stage apoptosis (Annexin V positively stained cells) in MCF‐7 and BT474 cells, respectively. Late stage apoptosis (dual stained Annexin V/Propidium Iodide (PI)cells) in curcumin exposed MCF‐7 cells was increased by 3.45 ± 0.94% expression, but no increase was seen in BT474 cells. PARP cleavage product (89kD) was detected at similar levels in both curcumin exposed MCF‐7 and BT474 cells and in a dose dependent manner. Curcumin‐exposedMCF‐7 cells, but not BT474 cells, showed substantial release of Cytochrome C from the mitochondria into the cytoplasm. Curcumin significantly upregulatedcaspase‐8 activity without modulating the activity of caspase‐9 in both BT474 and MCF‐7 cells. However, Caspase‐3 activity was upregulated only in BT474 cells. MCF‐7 cells reportedly have a gene deletion for Caspase‐3 and therefore do not express it. Caspase‐7 was then tested in MCF‐7 cells, but no upregulation was measured.

Conclusion

Cytotoxicity of Curcumin is low for both MCF‐7 and BT474 cells and concentrations are easily achievable. Apoptosis induction by Curcumin exposure was detected by both PARP cleavage and flow cytometry. Flow cytometry showed that curcumin induced predominantly early stage apoptosis (Annexin V stained) in both BT474 and MCF‐7 cells, with a late‐stage apoptosis increase seen only in MCF‐7. Caspase studies determined an extrinsic pathway (Caspase‐8 and Caspase‐3 positive, Caspase‐9 negative) in BT474 cells. MCF‐7 cells were also Caspase‐8 positive, Caspase‐9 negative, but Caspase‐3 was not detected. In the absence of Caspase‐3, Caspase‐7 might serve as an effector caspase. However, Curcumin exposure did not upregulate its expression in MCF‐7 cells. While curcumin activated the mitochondria‐independent extrinsic pathway in BT474 cells (no translocation of Cytochrome C into the cytoplasm), a mitochondria‐dependent (translocation of Cytochrome C into the cytoplasm), extrinsic with possible intrinsic pathway involvement was activated in MCF‐7 cells. Further studies are needed to elucidate the altered apoptotic pathway utilized in MCF‐7 cells.