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. 2020 Oct;9(10):5994-6004.
doi: 10.21037/tcr-20-1512.

Paris polyphylla ethanol extract induces G2/M arrest and suppresses migration and invasion in bladder cancer

Zhiyong Liu  1 , Zhonghua Sun  2 , Denglu Zhang  1 , Chenchen Ma  1 , Yuehua Jiang  1 , Guangshang Cao  3 , Chao Sun  4 , Kailin Li  4 , Dawei Xu  5 , Jiang Liu  6 , Shengtian Zhao  7
Affiliations

Paris polyphylla ethanol extract induces G2/M arrest and suppresses migration and invasion in bladder cancer

Zhiyong Liu et al. Transl Cancer Res. 2020 Oct.

Abstract

Background: Paris polyphylla is a traditional Chinese medicinal herb with multiple antitumor activities, but the role of P. polyphylla in bladder cancer (BC) is under investigation. This study aims to examine the antitumor activities of P. polyphylla ethanol extract (PPE) on BC cells and elucidate the underlying mechanisms.

Methods: Viable cells were counted using the trypan blue exclusion assay. The cell cycle was analyzed using flow cytometry, and scratch wound-healing and transwell assays were used to evaluate cell migration and invasion abilities, respectively. The protein expression levels were determined by western blotting. A xenograft model was used to assess the in vivo inhibitory effect of PPE on BC tumor growth.

Results: Our results showed that PPE inhibited the growth of BC cells in vivo and in vitro. Mechanistically, PPE regulated the levels of cell cycle-associated proteins, with PPE-induced G2/M phase arrest occurring through cyclin-dependent kinase inhibitor 1 (CDKN1A) accumulation and cyclin B1 (CCNB1)/cyclin-dependent kinase 1 (CDK1) inhibition. BC tumor growth was also inhibited by PPE treatment. Moreover, the migration and invasion abilities of J82 cells were suppressed through modulating epithelial-mesenchymal transition (EMT) regulatory factors with upregulation of cadherin-1 (CDH1) and downregulation of cadherin-2 (CDH2), snail family transcriptional repressor 2 (SNAI2), and twist family bHLH transcription factor 1 (TWIST1).

Conclusions: PPE inhibited cell growth, induced G2/M arrest, and suppressed the migration and invasion of J82 cells. BC tumor growth in vivo was also inhibited by PPE. Our results lay the foundation for further studies on the antitumor mechanisms of PPE.

Keywords: Urinary bladder neoplasm; cell cycle; epithelial-mesenchymal transition (EMT); medicine, Chinese traditional.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr-20-1512). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
PPE inhibits J82 cell growth. (A) J82 cells were treated with PPE or cisplatin for 24 hours. Subsequently, viable cells were enumerated using the trypan blue exclusion assay. (B) Different concentrations of PPE were used to treat J82 cells for 24 hours. Cell morphologies and cell growth were recorded, and representative images are shown. Scale bar =100 µm. (C) J82 cells were treated with PPE as indicated, and representative images of colony formation are shown. (D) The number of colonies was calculated and presented as the means ± SD of three independent experiments (* indicates P<0.05; *** indicates P<0.001). PPE, P. polyphylla ethanol extract.
Figure 2
Figure 2
PPE induces G2/M phase arrest in J82 cells. J82 cells were seeded into 6-well plates and starved for 12 hours. Different concentrations of PPE (as indicated) were used to treat J82 cells for 24 hours, and cell cycle analyses were then performed. Cell cycle distributions are shown in (A and B). G2/M cell cycle arrest was statistically analyzed, and the results are presented as the means ± SD of three independent experiments (NS represents not significant; * P<0.05; *** P<0.001). PPE, P. polyphylla ethanol extract.
Figure 3
Figure 3
PPE inhibits xenograft tumor growth in vivo. (A) After 21 days of PPE treatment, the mice were sacrificed, and tumor tissues were obtained (scale bar =1 cm). (B) Tumor weights were calculated, and the results are presented as the means ± SD. The tumor sizes and weight curves of the three groups are shown in (C and D), respectively (NS represents not significant; * indicates P<0.05). PPE, P. polyphylla ethanol extract.
Figure 4
Figure 4
PPE inhibits J82 cell migration. (A) J82 cells were treated with PPE at the indicated concentrations for 48 hours, and a CCK-8 kit was used to evaluate cell viability. The error bars represent the SD (NS, not significant; *** P<0.001). (B) Confluent cells were scratched and then treated with various concentrations of PPE. Images show cells at 0 and 48 hours for each group (magnification, 100×). (C) Differences in migration rates between groups were evaluated (** indicates P<0.01; *** indicates P<0.001). Data are representative of at least three experiments. PPE, P. polyphylla ethanol extract.
Figure 5
Figure 5
PPE inhibits J82 cell invasion. (A) J82 cells were treated with PPE at the indicated concentrations for 48 hours. Representative images show invaded cells in each PPE treatment group (magnification, 100×). (B) Differences in invading cell numbers between groups were analyzed (** indicates P<0.01; *** indicates P<0.001). Data are representative of three individual experiments. PPE, P. polyphylla ethanol extract.
Figure 6
Figure 6
PPE modulates cell cycle signaling pathway and EMT-associated biomarkers. (A) J82 cells were starved for 12 hours. Subsequently, cells were treated with different concentrations of PPE for 24 hours, and the levels of cell cycle arrest-associated proteins were detected by western blotting. (B) J82 cells were treated with PPE at the indicated concentrations for 48 hours. Epithelial marker (CDH1), mesenchymal marker (CDH2), and transcription inhibitors of CDH1 (SNAI2 and TWIST1) were evaluated by western blotting. PPE, P. polyphylla ethanol extract; EMT, epithelial-mesenchymal transition.
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