O-GlcNAcylation regulation of RIPK1-dependent apoptosis dictates sensitivity to sunitinib in renal cell carcinoma.

Receptor interacting protein kinase 1 (RIPK1) has emerged as a key regulatory molecule that influences the balance between cell death and cell survival. Under external stress, RIPK1 determines whether a cell undergoes RIPK-dependent apoptosis (RDA) or survives by activating NF-κB signaling. However, the role and mechanisms of RIPK1 on sunitinib sensitivity in renal cell carcinoma (RCC) remain elusive. In this study, we demonstrated that the O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) of RIPK1 induces sunitinib resistance in RCC by inhibiting RDA. O-GlcNAc transferase (OGT) specifically interacts with RIPK1 through its tetratricopeptide repeats (TPR) domain and facilitates RIPK1 O-GlcNAcylation. The O-GlcNAcylation of RIPK1 at Ser331, Ser440 and Ser669 regulates RIPK1 ubiquitination and the formation of the RIPK1/FADD/Caspase-8 complex, thereby inhibiting sunitinib-induced RDA in RCC. Site-specific depletion of O-GlcNAcylation on RIPK1 affects the formation of the RIPK1/FADD/Caspase 8 complex, leading to increased sunitinib sensitivity in RCC. Our data highlight the significance of aberrant RIPK1 O-GlcNAcylation in the development of sunitinib resistance and indicate that targeting RIPK1 O-GlcNAcylation could be a promising therapeutic strategy for RCC.

CircPPAP2B controls metastasis of clear cell renal cell carcinoma via HNRNPC-dependent alternative splicing and targeting the miR-182-5p/CYP1B1 axis.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown. METHODS: High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis. RESULTS: In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC. CONCLUSIONS: Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.

[Treatment of premature ovarian failure patients by bushen tiaojing recipe combined hormone replacement therapy: a clinical observation].

OBJECTIVE: To observe the clinical effect on treatment of premature ovarian failure (POF) patients by Bushen Tiaojing Recipe (BTR) and hormone replacement therapy (HRT). METHODS: Totally 72 POF patients were randomly assigned to three groups by random digit table, i.e., the Chinese medicine group, the Western medicine group, and the integrative medicine group, 24 in each group. Those in the Chinese medicine group took BTR. Those in the Western medicine group were treated by HRT. Those in the integrative medicine group were treated by BTR + HRT. All were treated for three courses and followed-up for 3 months after treatment. The clinical efficacy, integrals of clinical symptoms, and serum sex hormones levels [follicular stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH)] were compared among the three groups before treatment, at the end of treatment, and 3 months after withdrawal. RESULTS: (1) The total effective rate was better in the integrative medicine group than in the Chinese medicine group and the Western medicine group (P < 0.05). (2) At the end of treatment, the integrals of clinical symptoms decreased in the 3 groups when compared with before treatment in the same group (P < 0.05, P < 0.01). The integrals of clinical symptoms were higher at 3 months after withdrawal than at the end of treatment in the Western medicine group (P < 0.05). But there was no statistical difference in changes of integrals between the Chinese medicine group and the integrative medicine group (P > 0.05). (3) By the end of treatment serum E2 increased (P < 0.01), FSH and LH decreased (P < 0.01) in the three groups, more significantly in the integrative medicine group and the Western medicine group (P < 0.05, P < 0.01). At 3 months after withdrawal serum E2 decreased, FSH and LH increased in the Western medicine group, showing statistical difference when compared with the other two groups (P < 0.05, P < 0.01). There was no statistical difference in changes of serum E2, FSH, or LH between the Chinese medicine group and the integrative medicine group (P > 0.05). CONCLUSIONS: BTR combined HRT had significant effect on treatment of POF, could significantly improve patients' clinical symptoms, menstrual states, and serum sex hormones levels. It had lower recurrence rate. Patients suffered from less adverse reactions.

Ozonated Water Inhibits Hepatocellular Carcinoma Invasion and Metastasis by Regulating the HMGB1/NF-κB/STAT3 Signaling Pathway.

Background: Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. High-mobility group box 1 (HMGB1), a highly conserved chromosome protein, is considered as a potential therapeutic target and novel biomarker because of its regulation in the proliferation and metastasis of HCC. Ozone has been shown to be beneficial in the treatment of cancer. The objective of this study was to explore the effects and molecular mechanism of ozonated water on the proliferation, migration, and invasion of BEL7402 HCC cells. Materials and Methods: We assessed cell proliferation using a CCK-8 assay kit and flow cytometry; we performed wound healing and transwell assays to evaluate the effects of ozonated water treatment on cell invasion and migration. We determined reactive oxygen species (ROS) values by flow cytometry and used ELISAs to detect cytokines HMGB1, IL-6, and TNF-α. In addition, we assessed mRNA and protein cytokine expressions using RT-qPCR and Western blot. Results: Ozonated water decreased the viability of the HCC cells; the IC50 of ozonated water at 24 h was approximately 1.5 µg/mL. Compared with control groups, ozone treated cells revealed reduced mobility on wound healing assays and decreased invasion in transwell assays. HMGB1, IL-6, and TNF-α cytokines were found at lower levels in ozone treated cells than in control cells. Ozonated water-induced ROS accumulation. Likewise, the expressions of phosphorylated nuclear factor Kappa B (NF-κB), p65, NF-κB, P-STAT3, IL-6, JAK2, Slug, Twist, vimentin, MMP-2, MMP-9, and HMGB1 were decreased in the treated cells. Conclusion: Our findings suggest that ozonated water inhibits the proliferation, invasion, and metastasis of HCC cells via regulation of the HMGB1/NF-κB/STAT3 signaling pathway.

Circulating plasma exosomal miRNA profiles serve as potential metastasis-related biomarkers for hepatocellular carcinoma.

Exosomes carry functional molecules that can regulate cancer progression. Understanding the function of exosomal markers may provide invaluable insights into the mechanism of metastasis in hepatocellular carcinoma (HCC). The aim of the present study was to identify metastasis-associated microRNAs (miRNAs/miRs) expressed in plasma exosomes. A miRNA microarray and reverse transcription-quantitative PCR were used to analyze the plasma exosome miRNA expression profiles of patients with metastatic or non-metastatic HCC. Receiver operating characteristic (ROC) curve and Kaplan-Meier analyses were used to evaluate the predictive performance and prognostic efficacy of candidate miRNAs identified in the Gene Expression Omnibus database (dataset accession no. GSE67140). Bioinformatics analysis was used to examine the role of exosomal miRNAs in HCC metastasis. A total of 32 miRNAs were differentially expressed in plasma exosomes of patients with metastatic HCC compared with in those of patients with non-metastatic HCC. Additionally, the expression levels of six miRNAs were consistent between plasma exosome samples and matched tissue samples. ROC analysis demonstrated that miR-18a, miR-27a and miR-20b could discriminate metastatic HCC from non-metastatic HCC. Furthermore, the prognostic efficacy of the combination of three miRNAs (miR-18a, miR-20b and miR-221) was superior to that of individual miRNAs. Survival analysis demonstrated that high expression levels of the candidate miRNAs were associated with poor prognosis. Bioinformatics analysis indicated that the potential target genes of these miRNAs were involved in biological processes, molecular functions and cellular components that were associated with metastasis. The present findings suggested that these exosomal miRNAs may serve important roles in HCC lung metastasis and could represent a complementary clinical tool for the assessment of HCC prognosis.

Ozone induces BEL7402 cell apoptosis by increasing reactive oxygen species production and activating JNK.

BACKGROUND: Oxidative stress is an important factor in the modulation of both tumorigenesis and anticancer responses. Ozone (O3) is a strong oxidant that causes redox reactions and exerts anticancer effects in various types of cancer cells. However, the pathways involved in O3-induced cell death are not well understood. METHODS: In vitro human hepatocellular carcinoma (HCC) BEL7402 cells were treated with various O3 concentrations to evaluate O3 cytotoxicity by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The regulatory mechanisms were analyzed by western blot analysis. In vivo, an HCC model was established to evaluate the inhibition of HCC with O3 treatment. RESULTS: In vitro cells treated with O3 exhibited a round and small morphology with nuclear shrinkage and fragmentation. The CCK-8 assay confirmed the potent cytotoxic activity of O3 against BEL7402 cells (IC50 value of 5 µg/mL). Acridine orange/ethidium bromide (AO/EB) staining revealed apoptosis of BEL7402 cells after O3 treatment. Flow cytometry analysis showed that S phase cell cycle arrest and apoptosis increased with O3 exposure. In addition, O3 exposure reduced the mitochondrial membrane potential (ΔΨm) and induced reactive oxygen species (ROS) accumulation. Western blot analysis showed that O3 exposure reduced B-cell lymphoma 2 (BCL-2) expression and increased cleaved poly ADP-ribose polymerase (PARP), cytochrome C (Cyt-C), caspase-3, caspase-9, and p-JNK expression. In vivo, treatment with intratumor injection O3 (20 µg/mL) inhibited HCC growth. CONCLUSIONS: Overall, our findings showed that O3 induces BEL7402 cell apoptosis via the intrinsic mitochondria-dependent pathway. Therefore, O3 has therapeutic potential for HCC.

Significance and prognostic role of human epidermal growth factor receptor 2 and RAB1A expression in gastric cancer.

Human epidermal growth factor receptor 2 (HER-2) has an important clinical role in various cancers. However, the prognostic impact of HER-2 in gastric cancer (GC) is controversial. RAB1A is an important small molecule in the mechanistic target of rapamycin signalling pathway, which is one of the downstream signalling pathways of the epidermal growth factor receptor family. In recent years, the aberrant expression of RAB1A has been reported in a number of tumours, but its regulation in GC has not been extensively examined. Therefore, the present study investigated the expression pattern and prognostic significance of HER-2 and RAB1A in gastric adenocarcinoma (CAG). A comprehensive analysis was performed to examine the expression level of HER-2 and RAB1A in 280 cases of paired paraffin-embedded GAC tissues and an additional 120 archived GAC tissue samples. HER-2 and RAB1A protein expression was assessed by immunohistochemistry and cases with a 2+ score for HER-2 expression levels were subjected to fluorescence in situ hybridization to determine the HER-2 amplification status. Furthermore, HER-2 and RAB1A mRNA expression was quantified by reverse transcription-quantitative polymerase chain reaction. The comparison of continuous data between two groups was performed using a paired-samples t-test. Clinical correlations were determined using Pearson's Chi-square and Fisher's exact tests. Kaplan-Meier survival curves were used to estimate overall survival (OS). Cox proportional hazards models were used to determine associations between HER-2 and RAB1A expression and outcomes. Regression analyses were performed to detect the correlation between the mRNA levels of HER-2 and RAB1A in GAC tissues. It was observed that RAB1A was significantly overexpressed in GAC tissues compared with normal tissues (P<0.001). Approximately 12.86% of the 280 GAC patients had HER-2 amplification. Additionally, RAB1A expression was significantly associated with a short OS (P<0.001) but there were no significant differences in survival between the HER-2 high-expression group and the HER-2 low-expression group. Additionally, the co-expression of HER-2 and RAB1A indicated poorer OS than the overexpression of each protein (P=0.001), and the two factors were significantly positively correlated in GAC (P=0.012). These findings may be used to further explore the molecular mechanisms and regulatory associations among signalling pathways in GC.

Aberrant amino acid signaling promotes growth and metastasis of hepatocellular carcinomas through Rab1A-dependent activation of mTORC1 by Rab1A.

mTORC1 is a master regulator of cell growth and proliferation, and an established anticancer drug target. Aberrant mTORC1 signaling is common in hepatocellular carcinoma (HCC), but the underlying mechanism remains elusive. Rab1A is a newly identified mTORC1 activator that mediates an alternative amino acid (AA) signaling branch to Rag GTPases. Because liver is a physiological hub for nutrient sensing and metabolic homeostasis, we investigated the possible role of Rab1A in HCC. We found that Rab1A is frequently overexpressed in HCC, which enhances hyperactive AA-mTORC1 signaling, promoting malignant growth and metastasis of HCC in vitro and in vivo. Moreover, aberrant Rab1A expression is closely associated with poor prognosis. Strikingly, aberrant Rab1A overexpression leads to increased rapamycin sensitivity, indicating that inappropriate activation of AA signaling is a cancer-driving event in HCC. Our findings further suggest that Rab1A is a valuable biomarker for prognosis and personalized mTORC1-targeted therapy in liver cancer.