Poria acid inhibit the growth and metastasis of renal cell carcinoma by inhibiting the PI3K/akt/NF-κb signaling pathway.

Background: Poria acid (PAC) is a triterpene compound found in Poria cocos, a traditional Chinese medicine (TCM). The current study aims to explore the therapeutic effects and potential mechanisms of PAC on the migration and proliferation of human renal cell carcinoma (RCC) cells as well as tumor growth in animal model. Methods: Cell viability and proliferative capacity of normal renal cells and RCC cells were investigated by MTT assay. In addition, 786-O cells were divided into four groups and treated with different concentrations of PAC (0, 20, 40, and 60 µM) for 48 h. Cell scratch test and cell invasion assay were performed to evaluate the effects of PAC on the invasion and migration of RCC cells, respectively. The effects of PAC on apoptosis of RCC cells and expression levels of PI3K/Akt/NF-kB signaling pathway-related biomarkers were investigated using TUNEL staining and Western blotting methods, respectively. Effects of PAC on the inhibitory activity of RCC tumor in mice were evaluated in a 786-O CDX model. Results: The study found that PAC inhibited the viability of RCC cells in a dose-dependent manner, as demonstrated by in vitro cell assays (p < 0.05). However, PAC showed no significant inhibitory effect on normal renal cells (p > 0.05). PAC also significantly inhibited the migration and invasion of RCC via EMT/MMP signaling pathways (p < 0.05). Immunofluorescence and immunoblotting results showed that PAC induced the apoptosis of RCC, which was accompanied by changes in the expression levels of apoptosis-related proteins (p < 0.05). Moreover, PAC significantly downregulated the PI3K/Akt/NF-kB signaling pathway in a concentration-dependent manner (p < 0.05). The effect of PAC on RCC apoptosis was dramatically reversed by 740Y-P (PI3K agonist) (p < 0.05) but significantly enhanced in the presence of LY294002 (PI3K inhibitor) (p < 0.05). The results of in vivo experiment also demonstrated that the antitumor activity of PAC was achieved by affecting the PI3K/Akt/NF-kB signaling pathway. Conclusions: PAC can effectively suppress the proliferation, invasion and migration of RCC cells, and exhibit anti-tumor effects in RCC model by inhibiting the PI3K/Akt/NF-kB signaling pathway.

Antitumor and Protective Activity of TVLE against CdCl2-Induced Renal Damage in Rats.

<b>Background and Objective:</b> Cadmium is a heavy metal that has a wide range of applications in human existence. Cadmium may bind to the protein metallothionein and decrease kidney function once it enters the body. The purpose of this study was to investigate the renal protective activity of TVLE against CdCl₂-induced renal toxicity in rats. <b>Materials and Methods:</b> TVLE was prepared and characterized using instrumental analysis and spectral data. Furthermore, the IC₅₀ of TVLE against the Vero renal carcinoma cell line was calculated. Adult albino rats were used to assess the renal protective activity of TVLE (150 and 300 mg kg¹ b.wt.) in CdCl₂-treated rats. <b>Results:</b> IC₅₀of TVLE against Vero cell line equals 148.25 µg mL¹. The daily oral administration of TVLE at concentrations of 150 and 300 mg kg¹ b.wt. for 21 days to CdCl₂-treated rates resulted in a significant improvement in tumour volume and tumour weight, urea, creatinine, uric acid, TNF-α, NOx, TBARs, GSH, CAT, SOD, GPx and VEGF-C gene expression in CdCl₂-treated rats. Furthermore, TVLE almost normalized these effects in renal histoarchitecture. <b>Conclusion:</b> The biochemical, histological and MRI examinations of the current study suggested that TVLE have renal protective activity against CdCl₂-induced renal toxicity in rats.

lncRNAHIF1A-AS2 Promotes Renal Carcinoma Cell Proliferation and Migration via miR-130a-5p/ERBB2 Pathway.

BACKGROUND: Long non-coding RNAs (lncRNAs) are essential for tumorigenesis and progression of diverse cancers. This study aims to investigate the roles of lncRNAs on renal carcinoma. METHODS: The expression of lncRNA HIF1A-AS2 in clear cell renal cell carcinoma (ccRCC) and adjacent non-cancer tissues was identified by quantitative real-time PCR (qRT-PCR). Investigations were performed on biological function of lncRNA HIF1A-AS2 on cell proliferation, cell cycle, apoptosis and invasion of ccRCC by overexpression and knockdown experiments. Further, luciferase reporter assay and Western blot were constructed to explore molecular mechanisms underlying the function of lncRNA HIF1A-AS2. RESULTS: HIF1A-AS2 was highly expressed in kidney cancer tissues and ccRCC cells. Interference of HIF1A-AS2 in vivo hindered cell proliferation, invasion and migration while accelerated cell apoptosis. Overexpression of HIF1A-AS2 presented an opposite effect that repressed the expression of miR-130a-5p, and miR-130a-5p inhibited the expression of HIF1A-AS2. Additionally, rescue experiments exhibited that oncogenic function of HIF1A-AS2 was partially dependent on the suppression of miR-130a-5p. CONCLUSION: Our results indicated a critical role for the HIF1A-AS2-miR-130a-5p axis in renal carcinoma progression, which may act as a promising diagnostic biomarker and a pivotal therapeutic target for renal carcinoma cures.

Combined induction with anti-PD-1 and anti-CTLA-4 antibodies provides synergistic antitumor effects in DC-CIK cells in renal carcinoma cell lines.

Immune escape of cancer cells has become the main challenge in the immunocytotherapy field. In this study, we analyzed the cytotoxicity of DC-CIK cells induced by anti-PD-1 and anti-CTLA-4 antibodies in RCC cell lines. Flow cytometry analysis was performed to analyze the immune phenotypes of DC-CIK cells. Click-iT EdU assay was performed to analyze the proliferation of DC-CIK cells. ELISA analysis was performed to detect the expression of cytokines in DC-CIK cells. Compared with DC-CIK cells without any treatment, the growth inhibition rate was significantly higher in the other three groups. Moreover, combined induction with anti-PD-1 plus anti-CTLA-4 antibodies provides synergistic antitumor effects of DC-CIK cells in renal carcinoma cell lines. The combined treatment promoted DC-CIK cell proliferation and differentiation into CD3+CD56+ NKT cells and CD3+CD8+ CTL cells. Compared with the control group, combined treatment significantly up-regulated the secretion of immune-stimulatory cytokines, such as IFN-γ and TNF-α, and down-regulated the secretion of the immunosuppressive cytokine IL-10. Furthermore, the co-induction promoted the early activation of DC-CIK cells. These results indicated the co-induction with anti-PD-1 plus anti-CTLA-4 antibodies improved antitumor effects of DC-CIK cells by promoting proliferation, differentiation, and early activation and regulating the secretion of immune-stimulatory and suppressive cytokines in renal carcinoma cell lines.

Effects of autophage on the proliferation and apoptosis of clear cell renal carcinoma 786-O cells.

OBJECTIVE: Autophagy plays important roles in tumor occurrence and development. The present study aimed to investigate the association between autophagy and apoptosis in clear cell renal carcinoma cells (ccRCCs). METHODS: Atg7-overexpressing and -knockdown RCC 786-O cells (pLenti6.3-ATG7 and sh-ATG7-2 lv) were established using lentiviral transfection and interference shRNA. pLenti6.3-GFP and sh-scramb-con lv were used as controls. Cells were cultured in medium with or without the apoptosis inhibitor Z-VAD-FMK. Cell apoptosis were detected by flow cytometry. Cell proliferation was determined by MTT assay. Expression of apoptotic pathway proteins was measured by Western blot. RESULTS: The apoptosis rate of Z-VAD-FMK-treated cells was significantly decreased compared with untreated cells (P < 0.05). However, no significant difference in the apoptosis rate was detected among cell groups with different autophage level. The Z-VAD-FMK treatment induced significant changes in apoptosis rate in all cell groups, but only slightly changed the cell proliferation. When cell apoptosis were inhibited by Z-VAD-FMK, the cell viability in pLenti6.3-ATG7 group was significantly reduced compared with 786-O control (P < 0.05), whereas the cell viability in sh-ATG7-2 lv was significantly enhanced (P < 0.05) indicating that cell proliferation was closely associated with the level of autophage. The expression of caspase proteins in pLenti6.3-ATG7 was significantly higher compared with sh-ATG7-2 lv group (P < 0.05). CONCLUSION: autophagy and apoptosis are independent processes of PCD in human ccRCC 786-O cells. Autophagy is the main type of PCD and may be closely associated with apoptosis through the classical death receptor, mitochondria and endoplasmic reticulum apoptosis pathway.

The cytotoxic effects of IL-2 combined with different dosages of sorafenib on renal cellular carcinoma / 实用医学杂志

Objective To investigate the cytotoxic effects of IL-2 combined with different dosages of sorafenib on renal cellular carcinoma cell line 786-0. Methods Renal carcinoma cell 786-0 was cultured. Then , IL-2 (20 μmol/L) combined with different dosages of sorafenib (6.9, 13.8, 20.8 μmol/L) were used to treat tumor cell 786-0. The inhibitory effect on cell proliferation was determined by MTT assay. Cell apoptosis was measured by Annexin V-FITC kit. The tumor-bearing mice models were established and divided into four groups. Results The tumor cell growth was inhibited with the time-course correlation in all groups. In the 48-hour high doses group, the inhibitory rate was up to (74.67±1.87) %. The rates of cell proliferation inhibition and cell apoptosis were higher in the high dosages group than those in the other groups. Conclusions Immunotherapy combined with target therapy could significantly inhibit the growth of renal cellular carcinoma. But we should find a proper dosage, which could improve the clinical effect and reduce the adverse effect.

Valproic acid upregulates NKG2D ligand expression and enhances susceptibility of human renal carcinoma cells to NK cell-mediated cytotoxicity.

INTRODUCTION: We aimed to investigate the effect of valproic acid (VPA) on NKG2D ligand expression in human renal carcinoma cell lines and to investigate the mechanisms. MATERIAL AND METHODS: Different concentrations of VPA from 0.5 mM to 8.0 mM were applied to 786-O and ACHN cell lines, respectively. Cell viability after treatment with VPA was determined by flow cytometry (FCM). Real-time PCR and FCM were used to detect the changes of mRNA and protein level of NKG2D ligands (MICA/B and ULBPs) in the two cell lines treated with 4 mM VPA. The cytotoxicity assay and CD107a mobilization assay were carried out to detect the cytotoxicity changes of NK cells against renal carcinoma cell lines after the same treatment. RESULTS: Valproic acid can efficiently upregulate MICA/B, ULBP1 and ULBP2 expression in the renal carcinoma cell lines at the mRNA and protein level (p < 0.05). 786-O and ACHN cells treated with VPA were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the pretreatment of NK cells with anti-NKG2D monoclonal antibodies (p < 0.05). CONCLUSIONS: Valproic acid can clearly induce the expression of NKG2D ligands of renal carcinoma cell lines, thereby enhancing the cytotoxicity of NK cells against renal carcinoma cell lines.

Regulation of Gene Expression in Murine Renal Cell Carcinoma Cells Growing in Ectopic or Orthotopic Organs of Syngenic Mice / 대한암학회지

PURPOSE: The biologic behavior of tumor cells is partially controlled by the microenvironment. We investigated the expression levels of several genes involved in metastasis and drug response in RENCA cells growing in ectopic (skin) and orthotopic (kidney) sites. MATERIALS AND METHODS: Murine renal carcinoma cells were injected into kidney (orthotopic) and subcutis (ectopic) of syngeneic mice. Mice were treated with doxorubicin (DXR) (8 mg/kg) on days 8 and 15 after tumor cell implantation. Drug response was measured both in vivo and ex vivo by measuring tumor size and MTT assay. We also performed an in situ mRNA hybridization to estimate the expression levels of mdr (multidrug resistance), EGFR (epidermal growth factor receptor) and type IV collagenase. RESULTS: RENCA cells growing in the kidney of syngeneic mice produced metastatic lesions in the lung (57% of mice), while the same cells growing in the subcutis did not. Tumors growing in the kidney were more resistant to DXR than tumors growing in the subcutis. MTT assays revealed that tumor cells derived from kidney were more resistant to DXR than those cells from subcutis. In situ hybridization analyses showed that transcripts of EGFR and type IV collagenase genes in kidney tumors were higher than those of subcutaneous tumors but mdr expression showed no difference between the two tumors. CONCLUSION: These results demonstrate that the organ environment influences the drug responsive ness and the expression of EGFR and type IV collagenase genes in murine renal cell carcinoma cells.

Effects suramin, tumor necrosis factor-alpha(TNF-alpha, interferon-alpha(IFN-alpha on human renal carcinoma cell line growth / 대한비뇨기과학회지

Recently much interest has been expressed for the use of biological response modifiers and growth factor antagonists in the treatment or radiation and chemotherapeutic drug-refractory renal cell carcinoma. Herein antiproliferative effects of Suramin, human recombinanl tumor necrosis factor alpha (TNF-alpha) and human recombinant interferon alpha (IFN-alpha) as a single and in combination were studied in vito on human renal carcinoma cell line (Caki-1). Antiproliferative effect was evaluated by trypan blue dye exclusion assay after 3 days exposure to Suramin at 10, 30, 100, 300, 1,000 mcg/ ml. TNF-alpha at 1, 10, 50, 100, 500, 1,000 units/ml and IFN-alpha at 10, 100, 1,000. 10,000 units/ml concentration, respectively. In addition, effects of combined administration in tolerable peak plasma level or suramin (300 mcg/ml). TNF-alpha (500 units/ml) and IFN-alpha (1000 units/ml) were comparatively analyzed to those of single drug administration. The results were as follows: 1. Significant dose dependent antiproliferative effects were shown by Suramin at above 100 mcg/ml. TNF-alpha at above 500 units/ml and IFN-alpha at above 10 units/ml, respectively (p<0.05). 2. At peak plasma level, suramin, TNF-alpha and IFN-alpha showed less than 50% inhibition of proliferation. 3. On combined administration, suramin plus TNF-alpha (61.0%), Suramin plus IFN-alpha(71.7%) and TNF-alpha plus IFN-alpha (57.2%) induced significantly greater inhibition of proliferation (p<0.005). These results suggest that further in vivo study using combination of Suramin plus TNF-alpha Suramin plus IFN-alpha and TNF-alpha plus IFN-alpha is necessary and that these combination trials may become one of the treatment options for renal cell carcinoma.

The study of killing effect by T-lymphocytes after activiated by dendritic cells loaded with RCC 786-0 cell line freeze thawing antigen in vitro / 重庆医科大学学报

Objective: To study the immune responses of cytotoxic T-lymphocytes against Renal carcinoma cell 786-0(RCC) after activiated by dendritic cells(DCs) loaded with RCC freeze thawing antigen.Methods:DCs were obtained by cultured plastic-adherent monocytes isolated from health human peripheral blood with granulocyte-monocyte colony-stimulating factor(GM-CSF) and interleukin-4(IL-4) and Tumor necrosis factor(TNF) for 9 days.Host lymphocytes were stimulated with freeze thawing antigen of RCC 786-0 under the culture medium containing interleukin-2(IL-2) for 5 day.Killing activity and cytokine release were measured by MTT assay and ELISA.Results:The immune response of CTL activiated by DCs loaded with tumor soluble antigen was demonstrated by the following facts:(1)DCs loaded with tumor antigen could induce the growth of CTL.(2)The cyotoxicity of obtained specific CTL against RCC-7860 was highly enhanced with a significant difference from that of non-specific CTL.(3)The interleukin-12 release/secretion was increased by tumor antigen,which suggest an improved anti-tumor effect.(4)Apoptosis was observed in the RCCs after treated with CTL obtained.Conclution: These findings indicate that specific CTL induced by DCs sensibilized by RCC freeze thawing antigen exerts a remarkable killing activity on RCC 786-0.It is suggestd that DCs antitumor vaccines poses a clinically useful prospect with RCC.