PI3K/Akt/GSK-3β signal pathway is involved in P2X7 receptor-induced proliferation and EMT of colorectal cancer cells

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Abstract

P2X7 receptor (P2X7R) plays an important role in regulating the growth of tumor cells. However, the role of P2X7R in colorectal cancer (CRC) has remained poorly understood. Therefore, in this study, in vivo and in vitro experiments were performed to investigate the effect of P2X7R on the proliferation of CRC. The results showed that P2X7R was expressed in CRC cell lines (SW620 and HCT116). ATP and BzATP increased the expression of P2X7R in CRC cells, while the application of P2X7R antagonist A438079 and AZD9056 decreased the P2X7R expression induced by BzATP. Moreover, ATP and BzATP induced the activation of P2X7R to promote the proliferation, migration and invasion of CRC cells. Conversely, A438079, AZD9056 or siRNA transfection targeting P2X7R (siP2X7R) knockdown P2X7R expression inhibited the proliferation and migration of CRC cells. TGF-β1 promoted the migration and invasion of CRC cells, while the application of P2X7R antagonist could inhibit TGF-β1 induced migration of CRC cells. Furthermore, activation of P2X7R increased the expression of Vimentin, Snail, Fibronectin and decreased the expression of E-cadherin. While reducing the expression of P2X7R could inhibit these genes expression. In addition, ATP and BzATP increased the expression of p-Akt, p-GSK-3beta and β-catenin via P2X7R. P13/Akt pathway inhibitor LY294002 inhibited the proliferation of CRC cells, and the P13/Akt signaling was required for BzATP induced the proliferation of CRC cells. Our conclusion is that P2X7R mediated the PI3K/Akt/GSK-3beta signaling to promote the proliferation and EMT of CRC, indicating that P2X7R may be used as a potential therapeutic target for CRC.

Introduction

Colorectal cancer (CRC) is a common tumor of the digestive system, which seriously threatens human health (Piawah and Venook, 2019). CRC invades the mucosa at an early stage, most patients have no obvious clinical symptoms, and the early screening rate and detection rate are low, leading to the fact that most patients have entered the advanced stage of diagnosis and lost the best time for treatment (Eslami et al., 2019). Clinically, the treatment of CRC is still mainly surgery, and multiple methods combined with adjuvant therapy have improved the overall survival rate of patients. However, the proliferation and metastasis of tumors are still the main cause of death (Huang et al., 2019b, Huang et al., 2019a). Therefore, exploring the new molecular basis of tumor pathological mechanism and inhibiting its proliferation and metastasis are expected to become a new target for treatment of CRC. ATP is not only an important energy information substance, but also has an important effect on regulating the life activities of cells (such as cell adhesion and migration) (Subauste, 2019). Various studies have shown that ATP plays an important role in regulating tumor development (Yang et al., 2019; Jones et al., 2019). P2X purine receptors are dependent on ATP cation channel receptors and currently can be divided into 7 subtypes (1–7). P2X7R is an important member of the P2X family (Jimenez-Mateos et al., 2019). Currently, P2X7R has been found to be expressed in almost all body tissues and cells, such as blood, dental, exocrine, endothelial, muscle, renal and intestinal mucosal epithelial cells (Sluyter, 2017; Zhang et al., 2020a, Zhang et al., 2020b, Zhang et al., 2020c; Uekawa et al., 2018). It has been identified that P2X7R is expressed in most tumors (such as lung cancer, liver cancer, pancreatic cancer, and prostate cancer), and affects tumor progression (Zhang et al., 2020a, Zhang et al., 2020b, Zhang et al., 2020c). ATP is a natural activator of P2X7R in vitro. When the body is in a pathological state, such as tumors, ATP is released in large amounts, activates P2X7R, opens the ion channels on the cell membrane (sodium and calcium ions influx, potassium ions outflow), and plays an important role in regulating the progression of tumor cells (Zhang et al., 2020a, Zhang et al., 2020b, Zhang et al., 2020c; Bergamin et al., 2019). BzATP, an ATP analogue, has a high affinity for P2X7R, and its activity of activating P2X7R is higher than that of ATP (Zhang et al., 2019). Studies have found that BzATP (10 μM) significantly promotes the proliferation of human gliomas, while BzATP promotes cell proliferation to a peak value of 100 μM (Ji et al., 2018). Studies have found that BzATP promotes tumor cell invasiveness through ERK1/2, AKT and NFKB signals by activating P2X7R (Tafani et al., 2011). Studies have found that ATP and P2X7R activation increase TGF-β1 induced the migration of lung cancer cells, but down-regulating the expression of P2X7R can inhibit TGF-β1 induced cell migration and actin remodeling (Takai et al., 2012). Previous studies have reported that P2X7R can be used as a potential predictor of survival and prognosis of CRC (Zhang et al., 2019; Calik et al., 2020). However, these studies are limited to the correlation between P2X7R and clinicopathological features, and there is a lack of understanding of the specific role and molecular basis of P2X7R on CRC. Therefore, in this study, we investigated the effect and molecular mechanism of P2X7R on the progression of CRC through in vitro and in vivo experiments, providing new theoretical basis and data support for the treatment of CRC.

Section snippets

Cell culture

CRC cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) (SW620 and HCT116 cell lines are highly invasive and poorly differentiated, with high tumorigenicity. Both cell lines have been identified by STR, and no mycoplasma contamination was detected by PCR), and cells were placed in a CO2 incubator (37 °C 5%CO2) for cultivation. The next day, the culture dish was removed from the incubator, the culture medium was discarded in the culture dish, and

Expression of P2X7R in CRC cell lines

SW620 and HCT116 were treated or untreated with P2X7R agonists ATP (100 μM) and BzATP (100 μM) or BzATP + P2X7R antagonist A438079 (10 μM) for 24 h. The results showed that P2X7R was expressed in the two CRC cells. ATP increased the expression level of P2X7R protein in SW620 and HCT116 cells, especially BzATP significantly increased P2X7R protein expression. On the contrary, A438079 significantly reduced BzATP-induced P2X7R expression in SW620 and HCT116 cells (Fig. 1A and B). Similarly,

Discussion

Tumor proliferation and metastasis are still the main cause of death in patients with CRC (Li et al., 2019a, Li et al., 2019b). Therefore, more detailed molecular studies are needed to reveal the mechanism involved in CRC proliferation and metastasis. Currently, there is a lack of detailed understanding of the role of P2X7R in CRC. Previous studies have shown that P2X7R is highly expressed in CRC tissues, which is closely related to clinicopathological characteristics (Zhang et al., 2019; Calik

Ethical approval and consent to participate

Ethical approval has been exempted by the Ethics Committee of the Second Affiliated Hospital of Nanchang University (No. 2017 [028]). All protocols were approved by the Animal Care and Ethics Committee, China.

Consent for publication

Not applicable.

Availability of supporting data

All data generated or analyzed during this study are included in this article. And we have not used other data that has already been published. All the data presented in this article are original results derived from this study.

Funding

This study was supported by grants from the Natural Science Foundation of Jiangxi Province (20202BABL206163 and 20202BABL206091).

Author contributions

Wen-jun Zhang carried out the study of the whole experiment and drafted the manuscript. Chen Luo, Chao Huang, Fan-qin Pu and Jin-feng Zhu carried out western-blotting analysis and collected relevant data. Zheng-ming Zhu carried out the design of the study and helped revise this paper. All authors read and approved the final manuscript.

Author agreement

We confirmed that the paper has been seen and admitted by co-authors, approved the submission to the journal. We confirmed that there are no conflicts of interest in this paper.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

We thank Wen-jun Zhang for writing this article, the help of Jin-feng Zhu, Chen Luo, Fan-qin Pu and Chao Huang in the experiment, Zheng-ming Zhu for his guidance and support, and the molecular key laboratory and staff of the Second Affiliated Hospital of Nanchang University.

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