Prostate cancer (PCA) is a common malignant tumor worldwide, and it is estimated to affect 1.4 million men by 2020.Although the mortality of prostate cancer began to decline in the mid -1990s, it is still the main cause of male cancer death. It is estimated that 375,000 male death was estimated in 2020.Among them, most advanced PCA patients eventually developed into metastatic dehuminating resistance PCA (MCRPC). This PCA responded to different treatments and eventually killed patients within two years.Therefore, it is imminent to develop drugs that are more effective in treating advanced prostate cancer.
Stronginoside is a type of steroid compound. It mainly inhibits NA+/K+-ATP enzymes and is widely used to treat various cardiac diseases, such as arrhythmia and hypotension.Recently, studies in vitro and in vivo show that some strong hearts can be resolved by killing aging cells, inducing apoptosis, or unlocking cyclic tumor cell clusters into a single cell to inhibit metastasis and use anti -cancer activity.Some strong percinoside also tested in prostate cancer. Different heartylins seem to have different mechanisms and have different effects.However, the anti -cancer effects in various cancers, including prostate cancer, have not been studied.In this study, it aims to evaluate whether Westland has anti -cancer activity in PCA cells.
1. Western ornament has an anti -tumor effect on PCA cells in the body and outside
In order to evaluate the influence of Westland on the late PCA, this study selected AR-positive 22RV1 and AR-negative PC-3 and DU 145 PCA cells to analyze cell vitality, falling formation and tumor occurrence.The CCK8 analysis shows that in 24 and 48 hours, Western Lanlan significantly reduced the live cells of each cell strain and presented in dosage (Figure 1A).When the Western Land dosage (22rv1 ≥ 5 nm, PC-3 ≥123 nm,; DU145 ≥123 nm), long-term treatment (48 h) can kill more cancer cells (Figure 1A).In 22RV1, PC-3, and DU 145, the IC50 of West Lan 48 H is 8410, 370, and 180 nm, respectively.This study also evaluated the cell vitality on the normal human prostic epithelial cell (RWPE-1) after the treatment of Westland.The results showed that Westland’s IC50 values on RWPE-1 cells 24 h and 48 h were 10.17 and 8.84 μm, respectively, indicating that its cytotoxic effects on non-tumor-1 cells were lower than prostate cancer cells.
Based on these IC50 values, 0, 60, and 120 nm westland in 22RV1 and PC -3 and DU -145 cell lines are used for forming experiments and other in vitro experiments.As shown in Figure 1b, all the number of settings is significantly reduced under all detection concentrations, and in the three cell lines, the effect of high doses is more obvious than that of low doses (Figure 1B).The setting scale of the West Lan Group is also less than the control group (Figure 1B, left).
Then study the treatment effect of Westland in the 22rv1 and PC-3 heterogeneous transplant models.First test the toxicity of mice with different doses of Westland (1, 5, 10, 20, 40, and 60 mg/kg BW).Westland’s MTD is less than 20 mg/kg weight. Choose 5 mg/kg BW dose for treatment test.Tumor cells are injected under the mice. When the size of the tumor increases to about 150mm3, each cell line is randomly divided into two groups.One group is given to West Lan 5 mg/kg daily IP for 3 consecutive weeks, 5 days/week, and the other group is given a control solution.Westland shows a significant tumor growth inhibitory effect in 22rv1 and PC-3 alien transplant tumor models.The right side of the tumor (Figure 1c, D, right) can be seen.
Compared with AR-negative PC-3 cells, AR-positive 22RV1 cells have stronger treatment effects. The tumor inhibition rates of 22RV1 and PC-3 cells are about 80%and 55%, respectively.Mouse weighs unchanged during the entire experiment (Figure S1A), and there are no obvious pathological changes in the main organs (heart, lung, spleen, and kidney) in each group (Figure S1B).We observe some liver fat degeneration in the liver of mice treated in Westland, which may be related to the impact of drugs on lipid metabolism, as shown in the analysis of biological information below (Figure 6A).
2. Westland’s inhibitory effect on tumor growth involves G2/m cell cycle blocking
In order to understand the cell mechanism of Westland’s anti-cancer activity, immunohistochemical testing 22RV1 and PC-3 alien transplant tumors of proliferation cell marks KI67.Among 22rv1 and PC-3 tumors, Westland has significantly reduced the ratio of KI67-positive cells (Figure 2A), indicating that mushanoside inhibits cell proliferation to reduce tumor growth.
In order to further determine the impact of Westland on cell proliferation, the flow cytometer analysis 22RV1, PC-3, and DU 145 PCA cell lines are used to measure EDU income, which indicates DNA synthesis.Westland has significantly reduced the synthesis of DNA in each cell department, and the greater the dose, the more obvious the effect (Figure 2B and S2A), which provides direct evidence for Westland to suppress cell proliferation.The influence of Westland on the cell cycle was analyzed with fluid cells.Among the three PCA cell lines tested, Westland caused obvious cell accumulation in the G2/m stage, while decreased cells in stage of G1 and S (Figure 2C).We have also analyzed several cyclical regulatory proteins, including P21, CyClin D1, and the expression of CyClin B1 and CDK1 related proteins related to the G2/M checkpoints in PCA cells.At least two of the three cell lines have the expression of P21 to different degrees, and at the same time, the CYCLIN B1, CDK1 and CyClin D1 (Figure 2D) have been lowered, which further supports the inhibitory effect of Westland’s cycle process.
3. Westland can cause PCA cell apoptosis
In order to determine whether Western Lan or not caused the apoptosis of prostate cancer, Western Lan used 22RV1, PC-3, and DU 145 prostate cancer cells, and detected apoptotic cells with flow cells.After 48 h are treated, each cellular apoptosis percentage of the three cell lines increased significantly, and it was dosage dependent (Figure 3A).
We also analyzed the expression of apoptosis-related proteins through Western blotting, including Cleaved Caspase-3, Caspase-9 and PARP.West Lan adds three proteins in at least two cell lines (Figure 3B).Surprisingly, Cleaved -Caspase -3 in 22rv1 cells decreases (Figure 3B).
4. Westland inhibit the migration and invasion of prostate cancer cells
Transfer caused more than 90%of cancer -related deaths and often occurred in advanced PCA patients.The influence of Westland’s impact on three types of prostate cancer cell migration through TRANSWELL test.Westland treatment has led to a significant decrease in the number of migrants, indicating that Westland’s inhibitory effect on cell migration (Figure 4A).In order to evaluate the impact of Westland’s invasion of prostate cancer cells, Transwell data shows that compared with the control group, the Westland’s ability to deal with tumor cells is much lower (Figure 4B).These results show that Western Lan in vitro in vitro has inhibited the migration and invasion of prostate cancer cells.
5. Westland changes multiple biological processes and signal channels
In order to explore the molecular mechanism of Westland’s anti-cancer activity in PCA cells, 22RV1 and PC-3 cells were sequensed.Combined with correction P values <0.05 and | log2foldchange | <1, we identified a large number of differences of expression genes (DEGS).The volcanic map shows that there are 701 and 1085 DEGS (Figure 5A) in PC-3 and 22RV1 cells, respectively.There are 141 of these DEGs that are different in two cell lines, of which 84 expressions are raised and 46 expressions are lowered (Figure 5B).However, 11 of the 141 genes have the opposite expression of expression and changes between the two cell lines (Figure 5B) after the treatment of the two cell lines (Figure 5B). We eliminate these 11 genes for further analysis.
We carried out the Go Term enrichment analysis and KEGG Pathway analysis of 130 genes, showing the same expression of change in 22RV1 and PC-3 cells.GO enrichment analysis shows that the biological processes with more enrichment genes include “response to steroid hormone”, “lipid metabolic process”, “epithelial cell proliferation”, “cell migration”, “voltage door control calcium channel activity”,”DNA Copy”, “Periodic protein dependent linginine/soure apetease activity”, “apoptosis”, and et al. (Figure 6).
ECM receptor interaction, choline metabolism in cancer, and linoleic acid metabolism (Figure 6B).Most of these signal pathways are related to cancer, which provides mechanism of Westland’s anti -cancer activity.
6. The expression level of 15 Western Lanlan regulatory genes is related to the survival of patients with prostate cancer
In order to further determine the importance of the 130 Westland regulating genes in human prostate cancer (Figure 5), the TCGA PCA database and single -variable COX regression analysis evaluated a genetic expression level and disease -free survival period (DFS) and total survival of survival.Period (OS) correlation.In general, the high -expression level of 17 genes is significantly related to the poor RFS, including ITGA2B, C1QTNF4, CDKN2B, CNIH2, FBF1, PABPC1L, MMP11, DUSP9, COL8A1, SLC6A6, CMPK2, GPNMB, Cyp2e1111,, MAMDC4 and Rem2 (Table 1).
At the same time, there are two genes with high expression levels related to better DFS, including RASD1 and RBP7 (Table 1).Among these 17 genes, 10 genes have been lowered by West Lanlan, including ITG2B, CNIH2, FBF1, PABPC1L, MMP11, DUSP9, TMEM121, SOX18, CMPK2 and MAMDC4 (Figure 5C and 7A).EssenceAt the same time, the expression of RASD1, one of the two genes related to better survival rates, was raised by Westland, indicating that RASD1 has a cancer suppression function (Figure 5C and 7A).Kaplan-Meier analysis confirms the results of the single variable Cox regression analysis (Figure 7, Table 1).We also performed a single variable COX regression analysis in the GSE16560 data set with OS information.The higher the level of ENG and GEM, the worse the OS, and the ENG is lowered by West Lanlan (Table 1, Figure 7B), indicating that ENG has carcinogenic function in PCA cells.High -level Jun, MXD1, AQP3 are related to better OS (Table 1, Figure 7B), and these three proteins are raised by Western Lanlan (Figure 5C), indicating that they have a tumor suppression in PCA.Kaplan-Meier analysis once again confirmed the results of the single variable Cox regression analysis (Figure 7, Table 1).
In summary, this study uses three PCA cell lines to discover that Westland has reduced the formation of collision in vitro and weakened the growth of tumor growth of nude rats.At the cell level, Westland blocked the cycle of cells at G2/m, causing apoptosis and a consistent molecular change.Genome analysis shows that Westland has changed the expression of 130 genes, including many cancer -related cell processes and signal pathways.The change in the level of 15 Westernland regulating gene expression is significantly related to the disease -free survival or overall survival period of PCA patients.
In summary, this study proves that another kind of strong heartylidis incense can inhibit the growth of prostate cancer cells by inducing cell cycle blocking and apoptosis.In terms of mechanism, Westland regulates gene expression under a variety of biological processes and signal pathways.There are 15 genes related to DFS or OS, including AQP3, CMPK2, CNIH2, DUSP9, ENG, FBF1, ITGA2B, Jun, MAMDC4, MMP11, MXD1, PABPC1L, RASD1, SOX18 and TMEM121.Cancer activity is consistent.Although most of these 15 genes play a role in the progress of cancer, four of them have not involved cancer before, which provides new candidate genes for understanding how Westland inhibits the growth of prostate cancer cells.These discoveries also show that, like many other strong heartyls, Westland uses a wide range of signal pathways to play an anti -cancer activity in PCA cells and provides another candidate for anti -cancer drugs to be converted into anticancer drugs.
Mingcheng liu, et al.The Cardiac Glycoside DesiSide Exrts Anticancer Activity in Prostate Cancer Cells by Multiple Signaling Pathways 2021, 13, 5809.