Elsevier

Life Sciences

Volume 257, 15 September 2020, 118034
Life Sciences

Levamisole enhances DR4-independent apoptosis induced by TRAIL through inhibiting the activation of JNK in lung cancer

https://doi.org/10.1016/j.lfs.2020.118034Get rights and content

Highlights

  • •

    Levamisole exhibited antitumor activity in lung cancer cells in vitro and in vivo.

  • •

    Levamisole enhanced DR4-independent TRAIL-induced apoptosis by inhibiting JNK.

  • •

    LC3B-DR4/Erk was firstly disclosed as a new cellular protective pathway.

Abstract

The headings aims

Levamisole has anti-parasite and antitumor activities, but the anti-lung cancer mechanism has not been studied. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a promising drug because of the ability to selectively target cancer cells. However, the tolerance of cancer cells to TRAIL limits its antitumor activity. Other drugs combined with TRAIL need to be explored to enhance its antitumor activity. Based on the adjuvant anticancer effect of levamisole on anticancer drugs activity, the antitumor activity of levamisole combined with TRAIL will be investigated.

Materials and methods

In vitro and in vivo experiments were employed to investigate the anti-tumor activity. Flow-cytometry analysis, western blotting and siRNA transfection were used to explore the molecular mechanism.

Key findings

Levamisole decreased the proliferation of lung cancer cells in vitro and in vivo and induced cell cycle arrest in G0/G1 phase. Besides, levamisole also enhanced TRAIL-induced DR4-independent apoptosis by inhibiting the phosphorylation of cJUN. A new cellular protective pathway LC3B-DR4/Erk was also disclosed, in which levamisole only increased the expression of LC3B and then activated the phosphorylation of Erk and increased the expression of DR4, while p-Erk and DR4 inter-regulated.

Significance

Levamisole may be used as an adjuvant of TRAIL in treating lung cancer. The discovery of LC3B-DR4/Erk as a new protective pathway provides a new direction for sensitizing lung cancer cells to TRAIL.

Section snippets

Instruction

Levamisole is originally recognized as a broad-spectrum anti-helminthic drug, mainly used to eliminate roundworm and hook worm [1,2]. In recent years, with the in-depth study of levamisole, more functions of it have been discovered, such as antitumor activity [3,4], immune modulation [[5], [6], [7]], good inhibitory effect on alkaline phosphatase (APase) activity [1,8], anti-angiogenic effect [8,9] and an effective reduction in the risk of recurrence of steroid-sensitive nephrotic syndrome [10,

Cell culture

All of the five human lung cancer cell lines (HCC827, H1975, H157, H460 and A549) were kept in our laboratory and cultured in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) foetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (National China Pharmaceutical Inc., Beijing, China) at 37 °C in a 5% CO2 incubator.

Reagents

Levamisole was purchased from Sigma (St. Louis, MO, USA) and dissolved in PBS. Recombinant TRAIL was constructed in our

Levamisole exerts antitumor activity in lung cancer in vitro and in vivo

Levamisole is an imidazole derivative used as the form of hydrochloride [19] and the structural formula of levamisole hydrochloride was shown in Fig. 1A. SRB method was applied to detect the antitumor activity of levamisole on five kinds of lung cancer cells (HCC827, H1975, H157, A549 and H460) in vitro. As shown in Fig. 1B, levamisole displayed an inhibitory effect on the proliferation of lung cancer cells in a dose-dependent manner, especially on A549 and H460 cells. The half-inhibitory

Discussion

Levamisole, an imidazole derivative, mainly has anti-parasite, antitumor, immune-regulating and other effects [20]. For its antitumor effect, levamisole has been used in adjuvant chemotherapy for colorectal cancer [21], but few studies have been explored its specific anti-tumor mechanisms and target proteins, especially on the treatment of lung cancer. The current study is the first to find that levamisole possesses an antitumor effect on lung cancer cells both in vitro and in vivo. Some

Conclusions

We found for the first time that levamisole exhibited antitumor activity in lung cancer cells in vitro and in vivo by blocking cell cycle in G0/G1 phase and inhibiting the phosphorylation of cJUN. Meanwhile, levamisole facilitated the proliferation inhibition of TRAIL on lung cancer cells, and levamisole enhanced DR4-independent apoptosis induced by TRAIL through inhibiting the phosphorylation of cJUN. Besides, a new cellular protective pathway was also disclosed, which was that levamisole only

Abbreviations

DR4/5 death receptor 4/5

LC3B microtubule associated protein 1 light chain 3 beta

SRB sulforhodamine B

TRAIL tumor necrosis factor-related apoptosis-inducing ligand

Funding

The manuscript was supported by grants from the National Natural Science Foundation of China (81621064, 81702934), CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-02-002, 2019-I2M-1-003), and “Significant New Drug Development” Major Science and Technology Development Projects of China (no. 2018ZX09711001-007-002).

Author contributions

Shuzhen Chen designed all experiments and revised the manuscript. Xinran Qiao and Chen Wang did all experiments, and Xinran Qiao wrote the manuscript. Wendie Wang, Yue Shang and Yi Li participated in the animal experiments. Jun Ni help to do the purification processes of TRAIL. All authors have read and agreed with the final submitted version.

Competing interests

The authors declare no conflicts of interest.

Availability of data and materials

Not applicable.

Consent for publication

Not applicable.

Ethical approval

All animal experiments were performed in the Experimental Animal Center of our institute, which was an organization approved by Beijing Municipal Science & Technology Commission, and in accordance with Regulation on the Administration of Experimental Animals (2013 Revision), which was promulgated by the National Scientific and Technological Committee of People's Republic of China. All animal experiments procedures were approved by the Ethical Committee.

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