Polyphyllin I attenuates the invasion and metastasis via downregulating GRP78 in drug-resistant hepatocellular carcinoma cells.

Drug resistance to chemotherapy agents presents a major obstacle to the effective treatment of hepatocellular carcinoma (HCC), a common type of liver cancer. Increasing evidence indicates a link between drug resistance and the recurrence of HCC. Polyphyllin I (PPI), a promising pharmaceutical candidate, has shown potential therapeutic advantages in the treatment of sorafenib-resistant hepatocellular carcinoma (SR-HCC cells). In this study, we sought to investigate the mechanism underlying the inhibitory effect of PPI on the invasion and metastasis of SR-HCC cells. Our in vitro studies included scratch wound-healing migration assays and transwell assays to examine PPI's effect on HCC cell migration and invasion. Flow cytometry was employed to analyze the accumulation or efflux of chemotherapy drugs. The results of these experiments demonstrated that PPI increased the susceptibility of HCC to sorafenib while inhibiting SR-HCC cell growth, migration, and invasion. Molecular docking analysis revealed that PPI exhibited a higher binding affinity with GRP78. Western blot analysis and immunofluorescence experiments showed that PPI reduced the expression of GRP78, E-cadherin, N-cadherin, Vimentin, and ABCG2 in SR-HCC cells. Interference with and overproduction of GRP78 in vitro impacted the proliferation, migration, invasion, and metastasis of HCC cells. Further examination revealed that PPI hindered the expression of GRP78 protein, resulting in a suppressive effect on SR-HCC cell migration and invasion. Histological examination of tumor tissue substantiated that administering PPI via gavage to HepG2/S xenograft nude mice inhibited tumor growth and significantly reduced tumor size, as evidenced by xenograft experiments involving nude mice. Hematoxylin and eosin (HE) staining of tumor tissue specimens, along with immunohistochemistry (IHC), were conducted to evaluate the expression levels of Ki67, GRP78, N-cadherin, Vimentin, and ABCG2. The results indicated that PPI administration decreased the levels of proteins associated with metastasis and markers of drug resistance in tumor tissues, impeding tumor growth and spread. Overall, our findings demonstrated that PPI effectively suppressed the viability, proliferation, invasion, and metastasis of SR-HCC cells both in vitro and in vivo by modulating GRP78 activity. These findings provide new insights into the mechanism of PPI inhibition of SR-HCC cell invasion and metastasis, highlighting PPI as a potential treatment option for sorafenib-resistant HCC.

Mouse promyelocytic leukemia zinc finger protein (PLZF) regulates hepatic lipid and glucose homeostasis dependent on SIRT1.

Previous studies have demonstrated that promyelocytic leukemia zinc finger protein (PLZF) promotes the expression of gluconeogenic genes and hepatic glucose output, which leads to hyperglycemia. However, the role played by PLZF in regulating lipid metabolism is not known. In this study, we aimed to examine the function of PLZF in regulating hepatic lipid and glucose homeostasis and the underlying mechanisms. The expression of PLZF was determined in different mouse models with regard to non-alcoholic fatty liver disease (NAFLD). In the next step, adenoviruses that express PLZF (Ad-PLZF) or PLZF-specific shRNA (Ad-shPLZF) were utilized to alter PLZF expression in mouse livers and in primary hepatocytes. For the phenotype of the fatty liver, histologic and biochemical analyses of hepatic triglyceride (TG), serum TG and cholesterol levels were carried out. The underlying molecular mechanism for the regulation of lipid metabolism by PLZF was further explored using luciferase reporter gene assay and ChIP analysis. The results demonstrated that PLZF expression was upregulated in livers derived from ob/ob, db/db and diet-induced obesity (DIO) mice. Liver PLZF-overexpressing C57BL/6J mice showed fatty liver phenotype, liver inflammation, impaired glucose tolerance and insulin sensitivity. On the other hand, hepatic PLZF knockdown in db/db and DIO mice alleviated hepatic steatosis. Of note, we found that PLZF activates SREBP-1c gene transcription through binding directly to the promoter fragment of this gene, which would induce a repressor-to-activator conversion depending on its interaction with SIRT1 in the role played by PLZF in the transcription process through deacetylation. Thus, PLZF is identified as an essential regulator of hepatic lipid and glucose metabolism, where the modulation of its liver expression could open up a therapeutic path for treating NAFLD.

Anticancer Effect of Polyphyllin I in Suppressing Stem Cell-Like Properties of Hepatocellular Carcinoma via the AKT/GSK-3ß/ß-Catenin Signaling Pathway.

Polyphyllin I (PPI), also called Chong Lou saponin I, is a steroidal saponin isolated from the rhizome of Paris polyphylla. PPI has been demonstrated to have strong anticancer activity. However, its effect on the stemness of liver cancer stem cells (LCSCs) is not completely understood. Herein, we aimed to investigate the effect of PPI on the stem cell-like features of LCSCs and hepatocellular carcinoma (HCC). LCSCs were enriched in a serum-free medium and treated with PPI, sorafenib (Sora), or PPI and Sora. Several endpoints, including spheroid formation and differentiation, cell proliferation, surface markers of LCSCs, PPI binding targets, and stemness-associated protein expression, were evaluated. Immunofluorescence staining, quantitative real-time polymerase chain reaction, siRNA transfection, and coimmunoprecipitation ubiquitination assays were conducted for in-depth mechanistic studies. Evaluation of in vivo antitumor efficacy demonstrated that PPI effectively inhibited the proliferation of liver cancer cells and the self-renewal and differentiation of LCSCs. Flow cytometry indicated that PPI suppressed the expression of the stem cell surface markers EpCAM and CD13. Molecular docking showed a high affinity between PPI and proteins of the Wnt/ß-catenin signaling pathway, including AKT, GSK-3ß, and ß-catenin, with the binding energies of -5.51, -5.32, and -5.40 kcal/mol, respectively, which suggested that PPI might regulate the Wnt/ß-catenin signaling pathway to affect the stem cell-like properties of HCC. Further ex vivo experiments implied that PPI activated the AKT/GSK-3ß-mediated ubiquitin proteasomal degradation of ß-catenin and subsequently attenuated the prooncogenic effect of LCSCs. Finally, the anticancer property of PPI was confirmed in vivo. It was found that PPI inhibited the tumor growth in an HCC cell line xenograft model. Taken together, molecular docking analysis and experimental data highlighted the novel function of PPI in suppressing the stem cell-like characteristics of LCSCs via the AKT/GSK-3ß/ß-catenin signaling pathway.

Corrigendum: Autophagy Blockade by Ai Du Qing Formula Promotes Chemosensitivity of Breast Cancer Stem Cells Via GRP78/ß-Catenin/ABCG2 Axis.

[This corrects the article DOI: 10.3389/fphar.2021.659297.].

Construction of a genome instability-derived lncRNA-based risk scoring system for the prognosis of hepatocellular carcinoma.

Emerging evidence revealed the critical roles of long non-coding RNAs (lncRNAs) in maintaining genomic instability. However, genome instability-associated lncRNAs (GILncRNAs) and their performance in clinical prognostic significance in hepatocellular carcinoma (HCC) are rarely reported. Our study constructed a computational framework integrating somatic mutation information and lncRNA expression profiles of HCC genome and we identified 88 GILncRNAs of HCC. Function enrichment analysis revealed that GILncRNAs were involved in various metabolism processes and genome instability of cancer. A genome instability-derived lncRNA-based gene signature (GILncSig) was constructed using training set data. The performance of GILncSig for outcome prediction was validated in testing set and The Cancer Genome Atlas (TCGA) set. The multivariate cox regression analysis and stratification analysis demonstrated GILncSig could serve as an independent prognostic factor for the overall survival of HCC patients. The time-dependent Receiver Operating Characteristic (ROC) curve illustrated GILncSig outperformed two recently published lncRNA signatures for overall survival prediction. The combination of GILncSig and tumor protein p53 (TP53) mutation status exhibited better prognostic performance in survival evaluation compared to TP53 mutation status alone. AC145343.1 was further validated to be a risk factor for HCC in vitro among GILncSig. Overall, our study provided a novel approach for identification of genome instability-associated lncRNAs and established an independent risk score system for outcome prediction of HCC patients, which provided a new insight for exploring in-depth mechanism and potential therapy strategy.

Autophagy Blockade by Ai Du Qing Formula Promotes Chemosensitivity of Breast Cancer Stem Cells Via GRP78/ß-Catenin/ABCG2 Axis.

Accumulating evidence suggests that the root of drug chemoresistance in breast cancer is tightly associated with subpopulations of cancer stem cells (CSCs), whose activation is largely dependent on taxol-promoting autophagy. Our pilot study identified GRP78 as a specific marker for chemoresistance potential of breast CSCs by regulating Wnt/ß-catenin signaling. Ai Du Qing (ADQ) is a traditional Chinese medicine formula that has been utilized in the treatment cancer, particularly during the consolidation phase. In the present study, we investigated the regulatory effects and molecular mechanisms of ADQ in promoting autophagy-related breast cancer chemosensitivity. ADQ with taxol decreasing the cell proliferation and colony formation of breast cancer cells, which was accompanied by suppressed breast CSC ratio, limited self-renewal capability, as well as attenuated multi-differentiation. Furthermore, autophagy in ADQ-treated breast CSCs was blocked by taxol via regulation of ß-catenin/ABCG2 signaling. We also validated that autophagy suppression and chemosensitizing activity of this formula was GRP78-dependent. In addition, GRP78 overexpression promoted autophagy-inducing chemoresistance in breast cancer cells by stabilizing ß-catenin, while ADQ treatment downregulated GRP78, activated the Akt/GSK3ß-mediated proteasome degradation of ß-catenin via ubiquitination activation, and consequently attenuated the chemoresistance-promoted effect of GRP78. In addition, both mouse breast cancer xenograft and zebrafish xenotransplantation models demonstrated that ADQ inhibited mammary tumor growth, and the breast CSC subpopulation showed obscure adverse effects. Collectively, this study not only reveals the chemosensitizating mechanism of ADQ in breast CSCs, but also highlights the importance of GRP78 in mediating autophagy-promoting drug resistance via ß-catenin/ABCG2 signaling.

Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of ß-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.

Compound Phyllanthus urinaria L. (CP) is a traditional Chinese medicine (TCM) formula for cancer treatment in the clinic, particularly during progression of hepatitis B-associated hepatocellular carcinoma (HBV-associated HCC). Nevertheless, its anti-metastatic action and mechanisms are not well elucidated. In this study, CP was found to exert remarkable inhibitory effects on the proliferation, migration and invasion of HBV-associated HCC cells. The following network and biological analyses predicted that CP mainly targeted Caveolin-1 (Cav-1) to induce anti-metastatic effects, and Wnt/ß-catenin pathway was one of the core mechanisms of CP action against HBV-associated HCC. Further experimental validation implied that Cav-1 overexpression promoted metastasis of HBV-associated HCC by stabilizing ß-catenin, while CP administration induced autophagic degradation of Cav-1, activated the Akt/GSK3ß-mediated proteasome degradation of ß-catenin via ubiquitination activation, and subsequently attenuated the metastasis-promoting effect of Cav-1. In addition, the anti-cancer and anti-metastatic action of CP was further confirmed by in vivo and ex vivo experiments. It was found that CP inhibited the tumor growth and metastasis of HBV-associated HCC in both mice liver cancer xenograft and zebrafish xenotransplantation models. Taken together, our study not only highlights the novel function of CP formula in suppressing metastasis of HBV-associated HCC, but it also addresses the critical role of Cav-1 in mediating Akt/GSK3ß/ß-catenin axis to control the late-phase of cancer progression.