Luffa echinata Roxb. Induced Apoptosis in Human Colon Cancer Cell (SW-480) in the Caspase-dependent Manner and Through a Mitochondrial Apoptosis Pathway

Abstract

Background: Luffa echinata Roxb. (LER) (Cucurbitaceae) showed tremendous medicinal importance and are being used for the treatment of different ailments.

Objective: In this study, the antiproliferative properties and cell death mechanism induced by the extract of the fruits of LER were investigated.

Materials and methods: MTT and LDH assay were used to test the antiproliferative and cytotoxicity of LER extract, respectively. The intracellular ROS were measured by a fluorometric assay. The expression of several apoptotic-related proteins in SW-480 cells treated by LER was evaluated by Western blot analysis.

Results: The methanolic extract of LER fruits inhibited the proliferation of human colon cancer cells (SW-480) in both dose- and time-dependent manners. The LER-treated cells showed obvious characteristics of cell apoptosis, including cell shrinkage, destruction of the monolayer, and condensed chromatin. In addition, treatments of various concentrations of LER extracts caused the release of lactate dehydrogenase as a dose-dependent manner via stimulation of the intracellular metabolic system. LER induced apoptosis, DNA fragmentation, and cellular ROS accumulation in SW-480 cells. Treatment of LER on SW-480 cells promoted the expression of caspases, Bax, Bad, and p53 proteins and decreased the levels of Bcl-2 and Bcl-XL.

Conclusions: These results indicated that treatment with LER-induced cell death in mitochondrial apoptosis pathway by regulating pro-apoptotic proteins via the up regulation of the p53 protein. These findings highlight the potentials of LER in the treatment of human colon cancer.

Summary: LER induced apoptosis, DNA fragmentation, and cellular ROS accumulation in SW-480 cells. Treatment of LER on SW-480 cells promoted the expression of caspases, Bax, Bad, and p53 proteins and decreased the levels of Bcl-2 and Bcl-XL.

Keywords: Apoptosis; Luffa echinata Roxb For the treated cells, viability was calculated; caspases; colon cancer cells; p53.

Figure 1

Anti-proliferative activity of Luffa echinata Roxb. Cultured SW-480 cells were treated with LER extract (25, 50, 100, and 200 μg/ml) for 6, 12, 24, 48, and 72 h, respectively. The cell viability of SW-480 cells treated by Luffa echinata Roxb. extract was measured by MTT assay

Figure 2

Morphological changes of SW-480 cells under a light microscope. SW-480 cells were incubated for 24 h with Luffa echinata Roxb. extracts. The medium was removed and the cells were rinsed and visualized under light microscope in control (a), 50 µg/ml (b), 100 µg/ml, (c) and 200 µg/ml (d) of methanolic extract of Luffa echinata Roxb. fruits

Figure 3

Cytotoxicity assays of SW-480 cells exposed to 25, 50, 100, and 200 µg/ml of Luffa echinata Roxb. extracts for 24 h. The cytotoxicity of Luffa echinata Roxb. extract was analyzed by LDH assays (Triton X-100 as the positive control)

Figure 4

Effect of Luffa echinata Roxb. extracts on chromosomal DNA fragmentation. Samples were prepared from SW-480 cells incubated with or without 200 µg/ml of Luffa echinata Roxb. extract for 24 h. The DNA samples were analyzed by agarose gel electrophoresis. Lanes M is marker

Figure 5

Reactive oxygen species content of SW-480 cells exposed to 50, 100, and 200 µg/ml of Luffa echinata Roxb. extracts for 24 h

Figure 6

Effects of Luffa echinata Roxb. extracts on the expression of apoptosis-related proteins. Cells were treated with 25, 50, 100, and 200 µg/ml of Luffa echinata Roxb. extracts for 24 h, and aliquots containing 30–50 g of protein were subjected to sodium dodecyl sulfate-polyacrylamide gels followed by immunoblot analysis with specific antibodies. β-actin was used as an internal control for integrity and an equal amount of protein used in each lane

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Prof. Xiao-Long Zou