Silibinin Induces Both Apoptosis and Necroptosis with Potential Anti-tumor Efficacy in Lung Cancer
Authors: Zhang Guoqing , Wang Li , Zhao Limei , Yang Fang , Lu Chunhua , Yan Jianhua , Zhang Song , Wang Haiping , Li Yixiang 
Published: July 2024

Background:
The incidence of lung cancer is steadily on the rise, posing a growing threat to human
health. The search for therapeutic drugs from natural active substances and elucidating their mechanism have
been the focus of anti-tumor research.

Objective:
Silibinin (SiL) has been shown to be a natural product with a wide range of pharmacological activities,
including anti-tumour activity. In our work, SiL was chosen as a possible substance that could inhibit lung cancer.
Moreover, its effects on inducing tumor cell death were also studied.

Methods:
CCK-8 analysis and morphological observation were used to assess the cytotoxic impacts of SiL on
lung cancer cells in vitro. The alterations in mitochondrial membrane potential (MMP) and apoptosis rate of cells
were detected by flow cytometry. The level of lactate dehydrogenase (LDH) release out of cells was measured.
The expression changes of apoptosis or necroptosis-related proteins were detected using western blotting. Protein
interactions among RIPK1, RIPK3, and MLKL were analyzed using the co-immunoprecipitation (co-IP) technique.
Necrosulfonamide (Nec, an MLKL inhibitor) was used to carry out experiments to assess the changes in
apoptosis following the blockade of cell necroptosis. In vivo, SiL was evaluated for its antitumor effects using
LLC tumor-bearing mice with mouse lung cancer.

Results:
With an increased dose of SiL, the proliferation ability of A549 cells was considerably inhibited, and the
accompanying cell morphology changed. The results of flow cytometry showed that after SiL treatment, MMP
levels decreased, and the proportion of cells undergoing apoptosis increased. There was an increase in cleaved
caspase-9, caspase-3, and PARP, with a down-regulation of Bcl-2 and an up-regulation of Bax. In addition, the
amount of LDH released from the cells increased following SiL treatment, accompanied by augmented expression
and phosphorylation levels of necroptosis-related proteins (MLKL, RIPK1, and RIPK3), and the co-IP assay
further confirmed the interactions among these three proteins, indicating the necrosome formation induced by
SiL. Furthermore, Nec increased the apoptotic rate of SiL-treated cells and aggravated the cytotoxic effect of SiL,
indicating that necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL
on A549 cells. In LLC-bearing mice, gastric administration of SiL significantly inhibited tumor growth, and H&E
staining showed significant damage to the tumour tissue. The results of the IHC showed that the expression of
RIPK1, RIPK3, and MLKL was more pronounced in the tumor tissue.

Conclusion:
This study confirmed the dual effect of SiL, as it can induce both biological processes, apoptosis and
necroptosis, in lung cancer. SiL-induced apoptosis involved the mitochondrial pathway, as indicated by changes
in caspase-9, Bcl-2, and Bax. Necroptosis may be activated due to the changes in the expression of associated
proteins in tumour cells and tissues. It has been observed that blocking necroptosis by SiL increased cell death
efficiency. This study helps clarify the anti-tumor mechanism of SiL against lung cancer, elucidating its role in
the dual induction of apoptosis and necroptosis. Our work provides an experimental basis for the research on cell
death induced by SiL and reveals its possible applications for improving the management of lung cancer.