Retracted: Connexin 43 upregulation by dioscin‐inhibited gastric cancer metastasis by suppressing PI3K/Akt pathway

Dioscin, a natural steroidal saponin isolated from traditional Chinese medicine, is re-ported to possess antitumor activity against various cancers including gastric cancer (GC). GC patients are commonly found with low expression of Connexin 43 (Cx43) and activated epithelial– mesenchymal transition (EMT). The aim of this study was to investigate the mechanism of dioscin in inhibiting the proliferation, invasion, and metastasis of GC. Cell migration was detected using wound healing analysis while the invasion was used in Transwell membrane chambers. Colony-forming assays were re-searched for cell potential capacity in proliferation. The data of 68 GC patients treated with radical gastrectomy were collected from 2017 to 2019. The clinicopathological data were recorded for retrospective analysis. Cx43 was measured by immunohistochemistry in GC tissues from patients. EMT-related proteins and PI3K/Akt pathway-associated proteins were observed by using Western blot analysis. Dioscin inhibited the proliferation, migration, and invasion of SGC-7901 cells. Dioscin increased the expression of E-cadherin but decreased the expression of N-cadherin, vimentin, and snail-1. Besides, lower-level Cx43 is expressed in GC tissues compared with adjacent normal tissues. Additionally, dioscin downregulated the levels of p-Akt, p-mTOR, and p-PI3K. This research indicated that dioscin may inhibit EMT via increasing the expression of Cx43 and suppress the phosphorylation of PI3K/Akt signaling pathway and then inhibit the proliferation, invasion, and metastasis of GC.

Epithelial-mesenchymal transformation (EMT) is considered to be the key mechanism in GC, in which the expression of E-cadherin and N-cadherin has been widely accepted as the key process of tumor cell invasion and metastasis (Mirzaei et al., 2022).In addition, matrix metalloproteinases (MMPs) are grouping enzymes, in which the increased expression of MMP2 and MMP9 is also related to the invasion and metastasis of GC (Dong et al., 2020).Previous studies have shown that PI3K/Akt pathway mediates the EMT process and serves as a new target for inhibition and prevention of GC (Kou et al., 2016;Li et al., 2021).Most importantly, the PI3K/Akt pathway may be related to the regulation of some key proteins by EMT and MMPs.
In recent years, gap junction (GJ) channels have frequently been researched in tumor promotion for over 30 years (Beckmann et al., 2019).GJ channels domain connexin family channel proteins, such as Connexin 43 (Cx43), Cx26, and Cx45, among which Cx43 is the most widely expressed connexin in the majority of tissues and intimately related to amounts of cancer development and metastatic process (Epifantseva & Shaw, 2018).Moreover, there was evidence that Wnt/ β-catenin signal transduction negatively regulates Cx43 expression (Won et al., 2021).Inhibition of Wnt/β-catenin and PI3K/ Akt signaling pathway can promote tumor cell apoptosis (Wang et al., 2022).It also had been reported that suppression of PI3K/ Akt could attenuate the promoting effects of Cx43 consumption on EMT and TAM resistance (Wu et al., 2021).Furthermore, Cx43 is described as a suppressor gene in tumors and its low expression and dysfunction are found in amounts of tumors (Poyet et al., 2015).
In GC, Cx43 was found to how low expression and positively enhanced the chemosensitivity of gastric tumors additionally (Yang et al., 2019).Hence, upregulation of Cx43 in GCs is a feasible method for therapeutics in GC treatment.Nevertheless, the underlying mechanism of Cx43 in GC remains ill-defined.Dioscin (Dio), a steroidal saponin, abundantly originated from many medicinal plants such as Polygonatum zanlanscianense, Dioscoreae rhizome, and Paridis rhizome (Li et al., 2017).It has been demonstrated that dioscin could exert significant effects on antiinflammation and antivirus, which also had activated actions on antitumor (Du et al., 2019;Tian et al., 2021).Recently, more and more studies reported that dioscin may possess potential functions against various cancers, for instance, melanoma, lung cancer, colorectal cancer, prostate cancer, and GC, through diverse molecular mechanisms including cell apoptosis induction, glycolysis, and cell cycle arrest which contributed to antitumor activities (Chae & Kim, 2021;He et al., 2020;Wu et al., 2020).Besides, there has been evidence that the key target of dioscin in preventing lung cancer is Akt1 kinase, which is the central protein of PI3K/Akt signaling pathway (Xi et al., 2022).However, the specific mechanism of diosgenininhibiting tumors is still not complete.More and more researches need to be carried out.
So far, the treatment of dioscin against GC and the possible mechanism still remain unclear.Therefore, in this study, we focally investigated the effect of dioscin on SGC-7901 cells and explored the expression of related proteins, so as to further study the related molecular mechanism.We also detected whether Cx43 was marked as a potential prognostic and predictive biomarker.
MTT was from Sigma.LY294002 was obtained from Calbiochem.
Primary and corresponding secondary antibodies were obtained from Cell Signaling Technology, Inc. (CST).SABC immunohistochemistry (IHC) kit was purchased from Boster Bioengineering Inc.

| Patients and specimens
A total of 68 patients undergoing radical resection of GC in Luoyang Central Hospital from 2017 to 2019 were collected.All patients did not receive preoperative treatment and received postoperative adjuvant chemotherapy.This study was approved by the Institutional Review Committee and Human Ethics Committee of Luoyang Central Hospital affiliated with Zhengzhou University.Furthermore, all patients provided written and oral informed consent.The stage of GC was confirmed according to the AJCC/UICC TNM staging system, 8th edition.Clinical and pathologic dates, including age, gender, tumor size, and TNM stage, were obtained from hospital medical records.A total of 68 GC samples were used for IHC analysis.

| Cell culture
The human GC cell lines, SGC-7901 GC cells, were purchased from the China Cell Bank of Type Culture Collection, and cultured in RPMI 1640 medium with penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% fetal bovine serum (BI) incubated at 37°C with 5% CO 2 .

| Cell viability
MTT was performed to measure cell viability.The cells were inoculated in 96-well plate and cultured for 24, 48, and 72 hr, respectively.After adding MTT reagent (0.5 mg/ml) for 4 hr incubation, the formazan was dissolved with DMSO.The OD value was read by Victor X3 (PerkinElmer) at 490 nm.

| Transwell and wound healing assays
Cells (2 × 10 4 cells/well) were cultured in the upper chamber with a low-serum medium while a high-serum medium (20% FBS) was added to the lower chamber with the absence or presence of dioscin for 48 hr.After 48 hr of incubation at 37°C, cells were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 15 min.Then the crystal violet was dissolved in DMSO, and the OD value was detected using Victor X3 (PerkinElmer) at 600 nm.For wound healing assays, cells (5 × 10 5 cells/well) were seeded in six plate, scraped at the center of hole with pipe tips until they reached 80% of the area of six plate, and then photographed at 12, 24 and 48 hr by fluorescence inversion microscope (Olympus).

| Colony-forming assays
Logarithmic phase cells were digested with 0.25% tyrosine (Solarbio) first and then planted onto six-well plates at a density of 5 × 10 2 / well.After cultivation of 7 days, the cells were treated with dioscin.
When the cell colony was formed, it was fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet for 15 min.Finally, it was pictured with an inverted microscope (Olympus).

| Western blot analysis
Cells treated with dioscin for 48 hr were collected and the total protein was determined subsequently.According to the standard WB procedure, the blots were incubated with primary antibodies at 4°C overnight.Then, after incubating with the related secondary antibodies for 1 hr, the densities of bands were measured by ECL chemiluminescence detection (BD).

| Immunohistochemical staining
GC tissues and adjacent normal tissues were obtained from the hospital, the immunohistochemical staining was employed with anti-CX43 (CST, 1:500), and antibodies were incubated at 4°C overnight.The next day, the slides were incubated with the corresponding secondary antibody (Fcmacs Biotech Co., Ltd.) for 1 hr followed by 3,3-diaminobenzidine and hematoxylin staining.The slides were then examined and photographed using an Olympus BX53 fluorescence microscope.

| Dioscin suppressed the proliferation, invasion, and metastasis of GC by inhibiting EMT
First of all, MTT detection showed that the cell viability of dioscin (2 μM) treated for 48 hr was 51.76 ± 0.5%, in a time-and dosedependent manner, as shown in Figure 1a, in view of its toxicity, dioscin (0.5, 1, and 2 μM) that treated for 48 hr was chosen for subsequent assays (p < .001).In addition, a wound healing assay was performed to testify whether dioscin affects GC cell migration.As the results (Figure 1b, p < .001)demonstrated the migration capacity of SGC-7901 cells with the treatment of increased dioscin was inhibited compared with the control group.Moreover, dioscin inhibited SGC-7901 cell invasion in a dose-dependent manner that contrasted with the control group (Figure 1c, p < .001), in which cell invasion ratio was significantly reduced to 3.86 ± 1.3% at 48 hr.In addition, we measured the effects of dioscin on cell colony formation using anchorage-dependent growth assay.As demonstrated in Figure 1d (p < .001), the numbers of colonies with dioscin treatment compared with the control group were diminished.The results illustrated that dioscin may have a profound antitumor captivity in GC cells.
Moreover, the results displayed that dioscin (2 μM) decreased the expression levels of related proteins, for instance, N-cadherin, snail-1, and vimentin, but increased the expression level of E-cadherin (Figure 1f, p < .001).Moreover, MMPs are involved in the process of extracellular matrix.The results showed that MMP2 and MMP9 proteins' expression levels were decreased by dioscin (2 μM) treatment in a dose-dependent manner; the MMP2 protein levels decreased by 50.9 ± 2% as well as the MMP9 by approximately 63% (Figure 1e, p < .001).

| Correlation between low expression of Cx43 and prognosis
The IHC results demonstrated that the expression level of Cx43 in paracancer was significantly increased compared with cancer tissue (Figure 2a, p < .001).Moreover, the log-rank univariate analysis by SPSS showed that the GC patients with lower expression of Cx43 had prominently diminished the overall survival (OS) and disease-free survival (DFS) (Tables S1 and S2).The detailed clinicopathological characteristics of GC patients after resection were shown in Tables S3 and S4.The expression of Cx43 in GC tissues was associated with multiple clinicopathological factors (Table S5).

| Relationship between dioscin and PI3K/Akt signaling pathway in KEGG database
In the beginning, we displayed the relative pathway and mechanism of GC with the KEGG database (Figure 3, https://www.kegg.jp/).As shown in Figure 3, Wnt signaling pathway, TGFβ signaling pathway, and PI3K/Akt signaling pathway are mainly involved in the process of GC such as proliferation and DNA damage, whereas PI3K/Akt signaling pathway was more widely found in GC than other related signaling pathways.

| Dioscin upregulated Cx43 and inhibited PI3K/ Akt signal pathway
To validate whether Cx43 was performed as a novel target in GC, we detected the expression levels of Cx43 with Western blots.First, SGC-7901 was treated with different concentrations of dioscin (0.5, 1, and 2 μM) and RA (20 μM) acted as a positive control.The Western blots results showed that as the concentration of dioscin increased, the expression levels of Cx43 increased, and when SGC-7901 treated with 2 μM dioscin, Cx43 protein expression was notably upregulated by 41.65 ± 1.5% compared with the control group, while RA (20 μM) programmed as positive control significantly increased by 51.7 ± 5% (Figure 4a, p < .001).Next, we explored the protein expression levels of mTOR, p-mTOR, Akt, p-Akt, PI3K, and p-PI3K in SGC-7901 cells with the treatment of dioscin (0.5, 1, and 2 μM), and LY294002 (20 μM) acted as the PI3K/Akt signaling pathway inhibitor using Western blots assays to investigate whether dioscin exerted The results illuminated that dioscin did not affect the protein levels of total mTOR, Akt, and PI3K but significantly inhibited the phosphorylation of the three kinases in a dose-dependent manner.Two micrometers of dioscin notably suppressed the protein expression of p-mTOR with the declination of 49.6 ± 0.4% and p-Akt for 41.8 ± 0.4% as well as p-PI3K for 54.3 ± 1.5%.In addition, there was no significance with regard to total mTOR, Akt, and PI3K as shown in Figure 4b (p < .001).

| DISCUSS ION
Based on the high incidence and mortality of GC, research on effective treatment of GC is also increasing.There were many studies that proved dioscin exerted effects on various solid tumors, however, studies on dioscin in the treatment of GC are still few.This research described that dioscin possessed antitumor activity and increased the expression level of Cx43 in GC cells.
Dioscin is a kind of steroidal saponins of Chinese herbal medicine, which has a protective effect on many kinds of chronic injury and antitumor effects (Liu et al., 2022;Shang et al., 2022).Previous evidence had demonstrated that Dioscin upregulates the expression of Cx43 and improves gap junction communication in B16 cells (Kou et al., 2017;Zhang et al., 2022).Cx43, an important component of cell-to-cell communication, in recent years, has been reported to be involved in cancer progression (Solan et al., 2021).Although it also exerted a complex dichotomous activity in cancer, it has been widely accepted that Cx43 was programmed as a tumor suppressor (Choudhary et al., 2015;Uzu et al., 2017).In our experiment, the expression level of Cx43 was detected by Western blotting.The results showed that the expression level of Cx43 was upregulated in a dose-dependent manner after treatment with different concentrations of dioscin (Figure 4a).In addition, our results showed that the expression level of Cx43 in cancer tissues is significantly lower than that in paracancerous tissues in patients with GC (Figure 2a).
As shown in Tables S1 and S2, Cx43 protein expression is not only positively correlated with prognosis but also OS and DFS.This evidence may suggest that there was a low expression level of Cx43 in GC with low expression levels being associated with poor prognosis.In the basement of inhibiting cancer growth, migration, and invasion by dioscin treatment, which elevated Cx43 spontaneously suggested Cx43 could serve as a potential prognostic and predictive biomarker.

F I G U R E 3
The main regulatory pathway of PI3K/Akt in gastric cancer in the KEGG database Previous evidence has shown that Cx43 was closely linked with EMT, which was considered as the main approach to be involved in the original process of tumor invasion, angiogenesis, and micrometastases formation (Babaei et al., 2021;Diepenbruck & Christofori, 2016;Suarez-Carmona et al., 2017).Cx26 and Cx43 reduced cell migration, decreased the level of vimentin, and increased the expression of cytokeratin 18, indicating a phenotypic transition from stroma to epithelium (McLachlan et al., 2006).
Besides, the downregulation of Cx43 leads to the expression of mesenchymal marker N-cadherin, while the overexpression of Cx43 promotes the expression of epithelial marker E-cadherin (Kazan et al., 2019).Furthermore, MMPs expression frequently increased in cells that underwent EMT (Hwang et al., 2021).As shown in Figure 1b-d, dioscin exerted antitumor activities on SGC-7901 cells that dioscin-inhibited colony formation ability and the capacities of migration and invasiveness in vitro.Additionally, the results also demonstrated that dioscin suppressed the expression of MMP2 and MMP9 simultaneously.Furthermore, the study also found upregulated expression of E-cadherin and downregulated expression of N-cadherin, which revealed that dioscin decreased the potentialities of EMT in vitro (Figure 1f).However, the molecular mechanism has yet to be determined and far more experiments are needed.
Emerging evidence displayed that PI3K/Akt pathway was involved in the process of metastasis via regulating autophagy, apoptosis, and EMT (Ma et al., 2020;Rong et al., 2020;Zhang et al., 2019).Previous studies have shown that diosgenin has strong medicinal properties and can significantly reduce the protein expression level of Akt signaling pathway in vivo and in vitro (Liu et al., 2018;Mao et al., 2019).Network pharmacological studies detected that dioscin was associated with PI3K/Akt signaling pathway in GC (Figure 3).It was widely accepted that PI3K/Akt signaling pathway facilitated E-cadherin switch to N-cadherin (Georgakopoulos-Soares et al., 2020).The results demonstrated that dioscin suppressed the phosphorylation of PI3K/Akt signaling pathway while reversing EMT in SGC-7901 cells, in addition, the expression level of MMP9 and MMP2 was also decreased (Figures 1e,f and 4b).Previous research showed that Cx43 expression was downregulated and PI3K/Akt signaling pathway was activated in cardiomyocytes in vitro (Liu et al., 2021), however, Cx43 expression was upregulated and PI3K/Akt signaling pathway was inhibited in SGC-7901 (Figure 4a,b).This suggested that Cx43 may closely relate to PI3K/Akt signaling pathway.Moreover, LY294002, which acted as PI3K/Akt pathway inhibitor, was used to confirm that the inhibited effects on dioscin in the process of EMT on SGC-7901 cells involved the PI3K/Akt pathway regulation.Therefore, we speculated that dioscin regulated PI3K/Akt signaling pathway leading to the inhibition of the process of EMT.
In conclusion, our research revealed that dioscin inhibited the proliferation, invasion, and metastasis of GC, as well as EMT.
Statistical analysis was performed using SPSS 19.0 and GraphPad Prism 5.0 (GraphPad Software) software.Data from the individual experiments were expressed as the mean ± SD in triplicate.Pearson chi-square test and Fisher's exact test were used to analyze the correlation between Cx43 and clinicopathological parameters.Survival difference and prognosis factors were established by the Kaplan-Meier method with the log-rank test.The Kaplan-Meier method was used to draw the survival curve of the relationship between various factors and survival time.In all statistical analyses, p < .05 is considered to be statistically significant.

F
Detection of SGC-7901 cells migration, invasion, growth, and EMT-related protein.(a) Cell viability analysis of SGC-7901 cells cultivated with the treatment of dioscin for 24, 48, and 72 hr by MTT assay.(b) Cell migration of SGC-7901 cells in the presence of dioscin (0.5, 1, and 2 μM) and its control was detected using wound-healing assays.(c) Cell invasion of SGC-7901 cells in the presence of dioscin (0.5, 1, and 2 μM) and its control was detected with Transwell membrane chambers.(d) Cell growth of SGC-7901 cells in the presence of dioscin (0.5, 1, and 2 μM) and its control was detected using colony formation assay.(e) Expression levels of MMP2 and MMP9 and (f) EMT markers E-cadherin, N-cadherin, snail 1, and vimentin in SGC-7901 cells treated in the presence or absence of dioscin (0.5, 1, and 2 μM) were measured by Western blotting.The bar graphs (mean ± SD) and representative images are shown.*p < .05,**p < .01,and ***p < .001were compared with the Ctrl group.EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase the prognostic impact of Cx43, we compared DFS and OS in GC patients with different expressions of Cx43, which determined by cut-point (5.43) of IHC score, found that there was a remarkably influence between high Cx43 expression and longer DFS and OS.As shown on Figure 2b, we found that the depth of tumor invasion (p = .012)and tumor size (p = .004)had positive correlation with Cx43 expression levels.Kaplan-Meier survival analysis showed that there was a significant correlation between high level of Cx43 expression and shorter DFS (Figure 2, p < .001).DFS was reduced in patients with low expression of Cx43, whereas it was prolonged in patients with high Cx43 expression.Meanwhile, there was also a strong association between low Cx43 expression and shorter OS (Figure 2d, p < .001).

F
The expression levels of Cx43 and prognosis in GC samples.(a) Expression levels of Cx43 detected by immunohistochemical staining in GC cancer and adjacent to cancer tissues.Scale bar, 100 μM.(b) The expression of Cx43 in adjacent nontumorous tissues, GC tissues with different sizes of tumors, and T stage were detected by IHC.(c, d) High expression of Cx43 is positively correlated with a positive prognosis in human gastric cancer patients.GC, gastric cancer; IHC, immunohistochemistry an important role in inhibiting GC developments via PI3K/Akt signaling pathway.