Caspase Cascade Activation During Apoptotic Cell Death of Human Lung Carcinoma Cells A549 Induced by Marine Sponge Callyspongia aerizusa

Abstract

Introduction

In this study, Callyspongia aerizusa (CA), one of the most popular marine sponges for cancer therapy research, was investigated for its phytochemical compounds and evaluated for its anticancer activity in various cell lines. Since lung cancer is the most frequently diagnosed cancer, a solution from this marine source is a good choice to address the resistance to anticancer agents. Elucidation of the underlying mechanism of cell death elicited by a CA extract in human lung carcinoma cells A549 was undertaken.

Methods

The presence of secondary metabolites in CA methanol extract was revealed by gas chromatography-mass spectrometry (GC-MS) and evaluated on four cancerous cell lines and a non-cancerous cell line using Cell Counting Kit-8. Since the activity of CA extract in A549 cells was then evaluated through clonogenic assay, morphological detection of apoptosis, polymerase chain reaction (PCR) and Western blot assay, were also presented in this study.

Results

GC-MS analysis revealed the presence of two ergosteroids, ergost-22-en-3-one, (5β,22E), and ergost-7-en-3-ol, (35β) in the sponge extract that was suggested to suppress A549 cells (IC₅₀ 9.38 μg/mL), and another cancerous cell’s viability (IC₅₀ 3.12–10.72 μg/mL) in 24 h, but not in the non-cancerous cells. Moreover, CA extract was also able to reduce the colony-forming ability of A549 cells, and through A549 cells morphology seems that apoptosis is the underlying mechanism of cell death. Further, the treatment with CA extract induced the up-regulation of caspase-9, caspase-3, and PARP-1, and the down-regulation of BCL-2, in both mRNA and proteins expression level, promoting apoptotic cell death via caspase cascade.

Conclusion

These findings suggest that the compounds in CA extract possess the ability to induce apoptotic cell death in A549 cells and could become a promising candidate for future anticancer therapy.

Keywords:

• Callyspongia aerizusa
• ergosteroids
• lung cancer
• A549
• apoptosis

Acknowledgments

The authors would like to thank Professor Unang Supratman (Laboratorium Central, Universitas Padjadjaran West Java; Indonesia), for providing all the necessary facilities to carry out the present work. The authors would also like to thank Ms. Susianti and Ms. Kusmiati Sukmana for their excellent technical support and assistance. This research was funded by Universitas Padjadjaran Internal Grant 2017-2018, grant number 7511/UN6.O/PL/2018.

Abbreviations

CA, Callyspongia aerizusa; GC-MS, gas chromatography-mass spectrometry; CCK-8, cell counting Kit-8; CPI, cell proliferation inhibition; IC₅₀, half-maximal inhibitory concentration; PI, propidium iodide; RT-PCR, reverse transcription-polymerase chain reaction; BCL-2, B-cell lymphoma 2; PARP-1, Poly (ADP-ribose) polymerase 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBST, phosphate-buffered saline Tween-20.

Disclosure

All authors report no conflicts of interest in this work.