Abstract
Background
Novel chemotherapeutic drugs with good anti-tumor activity are of pressing need for bladder cancer treatment. In this study, plumbagin (PL), a natural plant-derived drug extracted from Chinese herbals, was identified as a promising candidate for human bladder cancer (BCa) chemotherapy.
Methods
The anti-tumor activity of PL was evaluated using a series of in vitro experiments, such as MTT, transwell assay, flow cytometry, quantitative real-time PCR (qRT-PCR) and western blotting. We established xenograft tumors in nude mice by subcutaneous injection with the human bladder cancer T24 cells.
Results
The results showed that PL could inhibit the proliferation, migration and survival of BCa cells (T24 and UMUC3 cells) in a time- and dose-dependent way. We found PL promotes the cell cycle arrest and apoptosis by inhibiting PI3K/AKT/mTOR signaling pathway, which inhibits cell proliferation. In vivo, anti-tumor activity of PL was further investigated using a BCa cell xenograft mice model. To simulate clinical chemotherapy, the PL were intravenously injected with a dose of 10 mg/kg for 10 times. Compared with the blank control, the tumor weight in PL treated group decreased significantly from 0.57 ± 0.04 g to 0.21 ± 0.06 g (P < 0.001).
Conclusions
In our study. We found PL inhibits the proliferation of T24 and UMUC3 cells in vivo and in vitro, which may play a role through several downstream effectors of PI3K/AKT/mTOR signaling pathway to promote the cell cycle arrest and apoptosis. Meanwhile, we consider that PL may inhibit the migration of bladder cancer cells via EMT suppression and induce ROS generation to make cell apoptosis. This work screened out a novel chemotherapeutic drug (plumbagin) with relatively good anti-tumor activity, which possessed great potential in BCa chemotherapy.
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Introduction
Bladder cancer (BCa) is the most common urological carcinoma with more than 4.3 × 105 new cases per year [1, 2]. BCa is complex in histopathological characteristics and is usually divided into two independent groups including muscle invasive and non-muscle invasive BCa [3]. The natural storage of urine in the bladder makes intravesical administration the most effective route of medication and treatment besides surgical resection, which has led to researchers have never stopped exploring new drugs for bladder cancer. A distinctive biological reagent named as Bacille Calmette-Guérin (BCG) vaccine also received great success in bladder cancer treatment. European Association of Urology (EAU) has recommended BCG bladder irrigation as one of the preferred adjuvant treatments for non-muscle invasive bladder cancer in 2018 [4]. Despite extensive research achievements have been successfully applied, the survival of BCa patients is still not fully satisfactory. The major factor is toxic side effects on off-target organs with the treatment of disease. It has been reported that the 5-year mortality rate of the muscle invasive BCa patients with lymph node (LN) metastasis is more than 77% [5]. Thus, finding a new antineoplastic drug with higher bioavailability is currently urgent.
Plant-derived drugs (PDDs) have been used to anti-cancer applications for thousands of years [6,7,8]. Our group has screened out a series of BCa chemotherapeutic PDDs, and their anti-tumor mechanisms were also investigated. For instance, capsaicin could suppress the tumorigenesis of BCa xenograft in vivo via FOXO3a mediated pathways [ In our study, we found PL inhibits the proliferation of T24 and UMUC3 cells in vivo and in vitro, which may play a role through several downstream effectors of PI3K/AKT/mTOR signaling pathway promoting the cell cycle arrest and apoptosis. Meanwhile, we consider that PL may inhibit the migration of bladder cancer cells via EMT suppression and induce ROS generation to make cell apoptosis. This work screened out PL with relatively good anti-tumor activity in human bladder cancer. It provides a reference for future clinical trials and new drug research and development, adds a choice for multi-drug combination to prevent drug resistance.Conclusions
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional file 1.
Abbreviations
- PL:
-
Plumbagin
- DCFH-DA:
-
The fluorescent probe 2′,7′-dichlorofluorescin diacetate
- qRT-PCR:
-
Quantitative real-time PCR
- BCa:
-
Bladder cancer
- PDD:
-
Plant-derived drug
- BCG:
-
Bacille Calmette-Guérin
- EAU:
-
European Association of Urology
- LN:
-
Lymph node
- FDA:
-
Food and Drug Administration
- PI3K:
-
Phosphatidylinositol 3-kinase
- PIP3:
-
Phosphatidyl inositol triphosphate
- PDK1:
-
Phosphoinositide dependent kinase-1
- mTOR:
-
Mammalian target of rapamycin
- EMT:
-
Epithelial–mesenchymal transition
- FBS:
-
Fetal bovine serum
- DMSO:
-
Dimethylsulfoxide
- PBS:
-
Phosphate buffer saline
- DCFH-DA:
-
2,7-Dichlorofluorescin diacetate
- MDM2:
-
Murine Double Minute 2
- GADD:
-
Growth arrest and DNA damage
- PVDF:
-
Polyvinylidene fluoride
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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Acknowledgements
We would like to thank other members of Department of Urology for their assistance and invaluable support.
Funding
This work was financially supported by the Medical Science Advancement Program (Clinical Medicine) of Wuhan University (TFLC2018002) and Improvement Project for Theranostic Ability on Difficulty Miscellaneous Disease (Tumor) from National Health Commission of China (ZLYNXM202006).
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All authors read and approved the final manuscript. RZ designed the study. ZW and WY analyzed the experimental data. FZ and ZG performed in the statistical analysis. KQ, YX and XW contributed funding for the project. RZ and ZW prepared the manuscript. KQ and YX edited the manuscript and provided comments. All authors read and approved the final manuscript.
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Supplementary information
Additional file 1: Figure S1.
PL inhibits proliferation and migration in T24 and UMUC3 cells. (a) Representative images of clonogenic BCa cells (T24 and UMUC3) after plumbagin treatment; (b) Quantitative analysis of clonogenic survival assay for T24 and UMUC3 cells; (c) The anti-migration activity of plumbagin for T24 cells was further evaluated using wound healing assay; (d) Quantitative analysis of wound healing assay for T24 cells. Table S1. Primer sequences used for qRT-PCR. Table S2. List of primary antibodies and secondary antibodies.
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Zhang, R., Wang, Z., You, W. et al. Suppressive effects of plumbagin on the growth of human bladder cancer cells via PI3K/AKT/mTOR signaling pathways and EMT. Cancer Cell Int 20, 520 (2020). https://doi.org/10.1186/s12935-020-01607-y
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DOI: https://doi.org/10.1186/s12935-020-01607-y