New anti-proliferative agent, MK615, from Japanese apricot "Prunus mume" induces striking autophagy in colon cancer cells in vitro

Abstract

Aim: To investigate the anti-neoplastic effects of MK615, an extract from the Japanese apricot (Prunus mume), against colon cancer cells.

Methods: Three colon cancer cell lines, SW480, COLO, and WiDr, were cultured with MK615. Growth inhibition was evaluated by cell proliferation assay and killing activity was determined by lactate dehydrogenase assay. Induction of apoptosis was evaluated by annexin V flow cytometry. Morphological changes were studied by light and electron microscopy, and immunofluorescence staining with Atg8.

Results: MK615 inhibited growth and lysed SW480, COLO and WiDr cells in a dose-dependent manner. Annexin V flow cytometry showed that MK615 induced apoptosis after 6 h incubation, at which point the occurrence of apoptotic cells was 68.0%, 65.7% and 64.7% for SW480, COLO, and WiDr cells, respectively. Light and electron microscopy, and immunofluorescence staining with Atg8 revealed that MK615 induced massive cytoplasmic vacuoles (autophagosomes) in all three cell lines.

Conclusion: MK615 has an anti-neoplastic effect against colon cancer cells. The effect may be exerted by induction of apoptosis and autophagy.

Figure 1

Dose-dependent inhibition of colon cancer cell growth by MK615. Growth inhibition was evaluated by MTT assay. The percentage inhibition (Y axis) was calculated using the ratio of absorbance at each drug concentration relative to absorbance in absence of the drugs. ^aP < 0.05.

Figure 2

Dose-dependent lysis of colon cancer cells by MK615. Cells were challenged with 150, 300 or 600 μg/mL MK615. All three colon cancer cell lines were lysed effectively in a dose-dependent manner. ^aP < 0.05.

Figure 3

MK615-induced apoptosis in colon cancer cell lines. SW480, COLO and WiDr cells were cultured without (A, C and E) and with (B, D and F) MK615 at 300 mL, and harvested after 6 h incubation.

Figure 4

Massive induction of cytoplasmic vacuoles by MK615. MK615 (300 μg/mL) induced cytoplasmic vacuoles in SW480 (A), COLO (B) and WiDr (C) after 6 h incubation.

Figure 5

Electron micrographs of autophagy induced by MK615. A: MK615 induced typical features of apoptosis in SW480 cells; B-F: Autophagy induced by MK615. Cytoplasmic vacuoles (autophagosomes) induced by MK615 in SW480 B and C, COLO D and E and WiDr (F) cells. Degenerated mitochondria are evident (arrows in E and F).

Figure 6

Immunofluorescence staining with Atg8 (LC3). SW480 (A), COLO (B) and WiDr (C) cells were cultured with MK615 at 300 μg/mL for 6 h. Nuclei were stained with DAPI (A, C and E), and Atg8 (LC3) was localized in the cytoplasm (B, D and F; note the central blank area in the cells).