Normal Renal Epithelial Cells Research Articles

Induction of Apoptosis and Growth-Inhibition by Thymoquinone in ACHN and GP-293 Cell Lines in Comparable with Cis-Platinum

ObjectiveIn the current work, we investigated the cytotoxic and apoptotic effects of Thymoquinone (TQ), an active compound of Nigella sativa (N. sativa) and Cis-platinum, on normal renal epithelial (GP-293) and human renal adenocarcinoma cell lines (ACHN).MethodsGP-293 and ACHN cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% penicillin plus streptomycin antibiotic. The MTT assay was used for cellular viability assessment. Viability of cells was observed using inverted light microscope 24, 48 and 72 h after exposure of the cells to various concentrations of TQ (1, 2.5, 5, 10, 50 and 100 μg/ml) and Cis-platinum (0.5, 1, 1.5, 2, 3, 6 and 12.5 μg/ml). Moreover, apoptosis was analyzed with a flow-cytometry method. The untreated cells were considered as control group.ResultsMorphological changes such as reduced cell number and increased intercellular distance and reduced cell viability in ACHN and GP-293cell lines were observed in both TQ and Cis-platinum groups; however, Cis-platinum had greater effect on ACHN cell line than GP-293 cell line. In addition, GP-293 cell line was more sensitive to TQ compared to ACHN cell line. Furthermore, TQ and Cis-platinum had apoptotic effects on both ACHN and GP-293 cell lines.ConclusionOur findings demonstrated that TQ and Cis-platinum had cytotoxic and apoptotic effects on both cell lines, However, GP-293 cell line was more sensitive to TQ. Additionally, Cis-platinum was more effective on ACHN cell line than on GP-293 cell line.

Abstract 3549: Elevated miR-141-3p inhibits renal cell carcinoma aggressiveness by targeting epithelial-to-mesenchymal transition pathway

Abstract Objective: Clear cell renal cell carcinoma (ccRCC), a common histological subtype of renal cell carcinoma (RCC) are categorized by their aggressive nature and comprise 90% of metastatic RCCs. Despite recent advances, management of the disease in the advanced metastatic phase is a significant challenge. Expression of miR-141-3p (miR-141) is low and function as tumor suppressor in various cancers. However, its association with long non-coding RNAs (lncRNAs) in RCC is not well understood. This study shows that miR-141 interacts with lncRNAs, and plays vital role in the regulation of stemness and epithelial-to-mesenchymal transition (EMT) in RCC. Experimental Design: Human renal cancer cell lines (ACHN and Caki-1), normal renal epithelial cells, RPTEC and tumor tissues were used for this study. We analyzed the expression of miR-141 in tissue samples and cell lines, and studied the function of miR-141 in kidney cancer progression using in vitro and in vivo models. Statistical analysis was performed to determine the clinical significance of miR-141 in kidney cancer patients. Results: Reduced expression of miR-141 was observed in ccRCC clinical specimens and cell lines. To elucidate the epigenetic role of miR-141 silencing in RCC, methylation status of CpG islands in its putative promoter region was analyzed. Result showed significant increase in miR-141 expression after 5-Aza-CdR treatment indicating that promoter hypermethylation is responsible for its inactivation. Ectopic expression of miR-141 reduced cell proliferation, clonogenicity, migration, invasion and induced apoptosis and cell cycle arrest compared to controls. An increase in cleaved PARP, cleaved caspase-3 and epithelial marker (CLDN1) was observed with a concomitant decrease in stemness (KLF4, Nanog) and EMT markers (FN1, VIM). We further investigated the biological significance of miR-141 in RCC. Loss of miR-141 function in RPTEC cells induced pro-cancerous characteristics. In addition, we examined lncRNAs (CDKN2B-As1, PCAT1 and PVT1) that bind to miR-141 and are overexpressed in RCC clinical samples compared to controls. Reduced expression of these lncRNAs was observed in RCC cells with overexpression of miR-141, supporting the notion that miR-141 interacts with these lncRNAs in RCC. Finally, in vivo experiment in nude mice revealed that intra-tumoral administration of miR-141 in the established tumors significantly suppressed tumor growth compared to controls. Furthermore, statistical analysis of patient samples showed that miR-141 may serve as a RCC diagnostic biomarker. Conclusion: Our results demonstrate that miR-141 overexpression inhibits RCC progression and inhibits epithelial-to-mesenchymal transition. These studies also show that miR-141 may be a useful RCC biomarker for early detection and monitoring RCC progression. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Nadeem S. Bhat, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. Elevated miR-141-3p inhibits renal cell carcinoma aggressiveness by targeting epithelial-to-mesenchymal transition pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3549.

Abstract 1901: Collagen remodeling in clear cell renal cell carcinoma: Influence of composition on growth and metabolism

Abstract The extracellular matrix (ECM) plays an important role in maintaining the structure and function of the kidney. During aggressive fibrotic disease of the kidney, such as chronic kidney disease (CKD), the kidney extracellular matrix is rapidly modified and degraded. Reports have shown that changes in the ECM composition and architecture can have profound influence on kidney function and recovery. The influence of ECM remodeling on disease progression and kidney function in Renal Cell Carcinoma (RCC), is still not well understood. By co-isolating primary cancer cell lines and the extracellular protein fraction from resected ccRCC tumors, we have determined that changes in collagen isoform composition are drastically altered compared to healthy normal kidney via Mass Spectrometry SWATH analysis (NvT COL1 p= 5.2x10-5; COLIII p=0.03; COLVI; COLIV p=0.004). RNA-seq analysis of TCGA data shows that altered collagen composition compared to normal correlates with patient outcomes (5 year survival COL1 HIGH=55%, LOW=72%, p=3.96e-4; COLIII HIGH=60%, LOW=73%, p=6.67e-2; COLIV HIGH=67%, LOW=56%, p=1.06e-2; COLVI HIGH=44%, LOW=71%, p=4.09e-9). Trichrome staining and IHC analysis of COLI, COLIII, and COLIV alongside cryptic-collagen epitopes XL313 and Hu177, suggests that subsets of tumor cell populations degrade surrounding collagen to drastically alter the ECM architecture. H&E scoring of regions of high XL313/Hu177 staining shows increased grade compared to low/no-staining. Isolated primary ccRCC cancer lines were analyzed for differential adherence to collagen isoforms, showing preferential binding preferences. In parallel, isolated normal kidney epithelia and renal proximal tubule epithelial cells (RPTECs) adherence preference was also investigated. Freshly dissociated tumor tissue cultured on a novel ECM cocktail coating in 3D scaffolds show changes in cell viability, growth, and purity compared to standard tissue culture conditions. In this study, we conclude that collagen composition analysis through IHC and Mass Spectrometry can be indicative of patient tumor aggressiveness. Preferential binding to collagen isoforms by tumor cells and the lack of adherence by normal kidney epithelia indicates a novel mechanism for tumor cells to promote local invasion. Additionally, we have generated a novel method by which to isolate human ccRCC cell lines using a collagen cocktail that will be useful for basic research and disease modeling. Note: This abstract was not presented at the meeting. Citation Format: Kyle H. Bond, Anna Deck, Jennifer Caron, Peter Brooks, Calvin Vary, Sunder Sims-Lucas, Leif Oxburgh. Collagen remodeling in clear cell renal cell carcinoma: Influence of composition on growth and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1901.

Abstract 2351: AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells

Abstract Purpose: The purpose of this research is to study the cross-talk between receptor tyrosine kinases (RTKs) c-MET and AXL in renal cancer cells; and to explore if the blockade of AXL-c-MET pathways can restrict PD-L1-mediated growth and immune escape of renal cancer cells. Background: c-MET and AXL are overexpressed in renal cell carcinoma (RCC). Upon binding with their respective ligands hepatocyte growth factor (HGF) and GAS6, c-MET and AXL activate a wide range of pathways, including tumor promoting events. Renal tumors show increased T cell infiltration, yet the T cells are rendered incapable of tumor rejection. The co-inhibitory molecules PD-1 and PD-L1 belong to cell surface glycoprotein B7 family that governs T cell function and proliferation. In RCC, tumor-infiltrating T cells express PD-1 and are associated with poor outcomes for patients. Similarly, PD-L1 expression on renal cancer cells hinders prognosis and indicates tumor aggressiveness. Therefore, PD-L1 in renal tumors may play a significant role in down-regulating host anti-tumor immune responses. Our recent report suggests that c-MET activation induces overexpression of PD-L1 in renal cancer cells; and the blockade of PD-L1 promotes immune-mediated killing of renal cancer cells. However, the cross talk between AXL and c-MET in the regulation of PD-L1 has not been studied in RCC. Results: We have confirmed that both AXL and c-MET are overexpressed in human renal cancer cells (786-O and ACHN) when compared with normal renal tubular epithelial cells. We performed co-immunoprecipitation assays with c-MET antibody and found that c-MET forms a complex with AXL in renal cancer cells. We utilized siRNA-mediated gene silencing approach and found that the knock-down of AXL increased the sensitivity of 786-O and ACHN cells to apoptosis following treatment with XL-184 (a small molecule inhibitor of c-MET, AXL and VGFRs, obtained from Exelixis) as measured by flow cytometry in Annexin V/PI stained cells. We also found that GAS6 treatment enhanced HGF-induced expression of cytoprotective molecule Heme oxygenase-1 (HO-1) and anti-apoptotic molecules Bcl-2 and Bcl-xL in renal cancer cells. In both 786-O and ACHN cells, c-MET activation increased the cell surface expression of PD-L1 as measured by flow cytometry. Interestingly, GAS6 treatment further increased the expression of PD-L1, suggesting a synergistic action of RTKs AXL and c-MET in renal cancer cells. We also utilized a PD-L1 promoter-luciferase plasmid to measure PD-L1 promoter activity and found that GAS6 treatment enhanced HGF-induced effects on PD-L1 transcription. In mouse renal cancer cells (RENCA), which express very high levels of PD-L1, we observed a significant decrease in the expression of PD-L1 when AXL was knocked down in cells. Conclusion: Together, our results demonstrate that AXL-mediated signaling enhances c MET-induced cytoprotection and PD-L1 expression on renal cancer cells. Citation Format: Murugabaskar Balan, Toni Choueiri, Soumitro Pal. AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2351.

Abstract 1039: c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7

Abstract Purpose: The purpose of this study is to explore how the receptor tyrosine kinase (RTK) c-Met modulates the expression of the pro-tumorigenic molecule CXCR7, a chemokine receptor, in renal cancer cells; and to investigate the role of c-Met and CXCR7 in promoting the epithelial-mesenchymal transition (EMT) of renal cancer cells. Background: c-Met, a pro-oncogenic RTK, can play a major role in the survival and progression of cancer cells; and it is often overexpressed in both clear cell and papillary renal cell carcinoma (RCC). c-Met, upon activation by its specific ligand hepatocyte growth factor (HGF), can induce a broad spectrum of biological pathways involved in the growth of cancer cells. CXCR family of chemokine receptors and their chemokine ligands were shown to exhibit decisive roles in tumor initiation, promotion, progression, and metastasis. CXCR7 is upregulated in several types of cancers, including renal cancer, and found to be involved in tumor cell development, survival, and metastasis. A global process integral to the cancer metastasis is EMT, which results in the disruption of cell-to-cell junctions and endows epithelial cells with the ability to migrate and invade surrounding tissue. EMT process is associated with an increase of mesenchymal markers like vimentin and a decrease of epithelial markers like E-cadherin in cancer cells. Sustained activation of c-Met induces EMT in renal cancer cells, however, the cross-talk between CXCR7 and c-Met signaling in the promotion of renal cancer EMT remains unclear. Results: We assessed the expression levels of CXCR7 by Western blot analysis and found that CXCR7 is overexpressed in human renal cancer cells (both 786-O and ACHN) when compared to normal renal epithelial cells (RPTEC). Also, in both 786-O and ACHN cells, HGF-induced activation of c-Met further increased the expression of CXCR7 at both RNA and protein levels. In renal cancer cells (both 786-O and ACHN), siRNA-mediated knockdown of c-Met or treatment with XL-184 (a small molecule inhibitor of c-Met) inhibited c-Met-induced upregulation of CXCR7. c-Met activation increased the expression of pro-EMT markers such as vimentin, N-cadherin, and MMP-9 and suppressed the expression of anti-EMT molecule E-cadherin, as measured by Western blot and flow cytometry. On the other hand, siRNA-mediated silencing of CXCR7 attenuated the induction of EMT, suggesting that CXCR7 plays a major role in c-Met-induced EMT in renal cancer cells. Conclusion: Together, our findings demonstrate that the c-Met-CXCR7 axis can trigger the EMT, which can play an important role(s) in renal cancer proliferation and migration. Citation Format: Samik Chakraborty, Murugabaskar Balan, Soumitro Pal. c-Met activation promotes epithelial-to-mesenchymal transition (EMT) in renal cancer cells through the upregulation of the chemokine receptor CXCR7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1039.

MALAT1 accelerates the development and progression of renal cell carcinoma by decreasing the expression of miR-203 and promoting the expression of BIRC5.

ObjectiveWe aimed to investigate the roles of the lncRNA MALAT1 in renal cell carcinoma (RCC) progression.MethodsqRT‐PCR was used for the assessment of BIRC5, miRNA‐203 and MALAT1 expression. Furthermore, the targeted relationships between miR‐203 and BIRC5, as well as MALAT1 and miR‐203, were predicted by the miRanda/starBase database and verified by dual‐luciferase reporter gene assay. The effects of MALAT1, miRNA‐203 and BIRC5 on cell proliferation, cell cycle, cell apoptosis, cell invasion and cell migration were studied by using CCK‐8, flow cytometry, transwell and wound healing assays, respectively. In addition, the effects of MALAT1 on RCC tumorigenesis were evaluated in vivo by nude mouse tumorigenesis.ResultsThe expression levels of BIRC5 and MALAT1 were higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. In contrast, the expression of miRNA‐203 in RCC tissues and cell lines was higher than that in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. BIRC5 and MALAT1 promoted cell proliferation yet decreased the percentage of RCC cells at G0/G1 phase.ConclusionsOur study demonstrated that MALAT1 functions as a miR‐203 decoy to increase BIRC5 expression in RCC.

Cross-linking of hyaluronic acid by curcumin analogue to construct nanomicelles for delivering anticancer drug

In this study, hyaluronic acid (HA) and curcumin (CCM) are used as functional molecular skeletons to prepare a reticular polymer HA-CCM* via the free radical polymerization reaction. The results showed that HA-CCM* formed nanomicelles through self-assembly in the aqueous medium. These nanomicelles were seen as star-shaped micelles and the particle diameter distribution range of 220–280 nm, which are used to carry doxorubicin (DOX) with a high drug loading and an entrapment efficiency. The experiment of drug release in vitro indicated that HA-CCM* micelles showed significant acid sensitivity. The anticancer activity of DOX-loaded by HA-CCM* micelles (HA-CCM* + DOX) was quantified by MTT assay, indicating that the cytotoxicity of HA-CCM* + DOX to human cervical cancer cell line (HeLa) and human renal cell adenocarcinoma cell line (786-O) was significantly improved and to normal human renal epithelial cell line (293A) was obviously reduced. DOX from HA-CCM* + DOX micelles can enter into the cells and resulted in cell death. The multifunctional HA-CCM* micelles have good prospects in the field of drug delivery. Overall, these results confirmed the potential of these nanomicelles for carrying DOX in the improved cancer chemotherapy.

Targeting the transcription factor receptor LXR to treat clear cell renal cell carcinoma: agonist or inverse agonist?

Growing evidence indicates that clear cell renal cell carcinoma (ccRCC) is a metabolism-related disease. Changes in fatty acid (FA) and cholesterol metabolism play important roles in ccRCC development. As a nuclear transcription factor receptor, Liver X receptor (LXR) regulates a variety of key molecules associated with FA synthesis and cholesterol transport. Therefore, targeting LXR may provide new therapeutic targets for ccRCC. However, the potential regulatory effect and molecular mechanisms of LXR in ccRCC remain unknown. In the present study, we found that both an LXR agonist and an XLR inverse agonist could inhibit proliferation and colony formation and induce apoptosis in ccRCC cells. We observed that the LXR agonist LXR623 downregulated the expression of the low-density lipoprotein receptor (LDLR) and upregulated the expression of ABCA1, which resulted in reduced intracellular cholesterol and apoptosis. The LXR inverse agonist SR9243 downregulated the FA synthesis proteins sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN) and stearoyl-coA desaturase 1 (SCD1), causing a decrease in intracellular FA content and inducing apoptosis in ccRCC cells. SR9243 and LXR623 induced apoptosis in ccRCC cells but had no killing effect on normal renal tubular epithelial HK2 cells. We also found that SRB1-mediated high-density lipoprotein (HDL) in cholesterol influx is the cause of high cholesterol in ccRCC cells. In conclusion, our data suggest that an LXR inverse agonist and LXR agonist decrease the intracellular FA and cholesterol contents in ccRCC to inhibit tumour cells but do not have cytotoxic effects on non-malignant cells. Thus, LXR may be a safe therapeutic target for treating ccRCC patients.

Effect of long-chain noncoding RNA NEAT1 on the proliferation and invasion of renal cell carcinoma

Objective To observe the expression of long non-coding RNA (LncRNA-NEAT1) in renal cell carcinoma and cell lines, and to explore its influence on the malignant biological behavior of renal cell carcinoma and its possible molecular mechanism. Methods The expression of NEAT1 was detected by quantitative polymerase chain reaction (qPCR) in 10 cases of renal cell carcinoma and paracancerous tissues, and in 4 types of renal cell carcinoma cells and normal renal tubular epithelial cells. ACHN cells infected with negative control lentivirus (LV-NC) were used as control group, and ACHN cells infected with recombinant lentivirus carrying NEAT1 gene (LV-NEAT1) were used as experimental group. Bioinformatics predicts targeted miRNA and downstream genes of NEAT1. The effect of overexpression of NEAT1 on the mRNA expression of miR-342-3p and cell adhesion molecule 1 (CADM1) was detected by qPCR. The protein expression of CADM1 was detected by Western blot. The proliferation and invasion of ACHN cells were detected by methyl thiazolyl tetrazolium (MTT) method and Transwell invasion assay. Results The expression level of NEAT1 in renal cell carcinoma was lower than that in paracancerous tissue (P<0.01). The expression of NEAT1 in renal carcinoma cell lines was lower than that in renal tubular epithelial cells (P<0.01). After LV-NEAT1 infection, the expression of NEAT1 and CADM1 gene were significantly increased (P<0.01), while the expression of miR-342-3p decreased (P<0.01). Overexpression of NEAT1 significantly inhibited cell proliferation and invasion of ACHN cells (P<0.05). Conclusions LncRNA NEAT1 was down-regulated in renal cell carcinoma and cell lines. Overexpression of NEAT1 inhibited the proliferation and invasion of ACHN cells. The molecular mechanism may be that NEAT1 up-regulates the expression of CADM1 gene through complementary binding to miR-342-3p. Key words: RNA, long noncoding; Kidney neoplasms; Cell proliferation; Neoplasm invasiveness; In vitro

Long non-coding RNA HOTTIP affects renal cell carcinoma progression by regulating autophagy via the PI3K/Akt/Atg13 signaling pathway.

Renal cell carcinoma (RCC) is the most common malignancy of the urinary system, and it is a serious threat to human health. HOXA transcript at the distal tip (HOTTIP), located at the 5' end of the HOXA locus, is a long non-coding RNA that has been newly discovered in recent years. It has been reported to promote the development of several types of tumors. Moreover, accumulating evidence has indicated that autophagy plays an important role in tumor cell survival or death. However, whether HOTTIP affects RCC development by regulating autophagy remains unknown. In this study, we first measured HOTTIP expression in 42 paired RCC and adjacent non-tumor tissues, as well as in 4 RCC cell lines and 1 normal renal tubular epithelial cell line. Then, we selected 2 RCC cell lines to silence HOTTIP expression and 1 RCC cell line to overexpress HOTTIP, and we measured their proliferation, migration and invasion, as well as autophagy, after pretreatment with an autophagy inhibitor or inducer. In addition, we assessed the growth, metastasis and autophagy of tumors in nude mice and explored the mechanism involved. The results showed that HOTTIP expression was significantly upregulated in the RCC tissues and cell lines, and it was closely associated with TNM stage, histological grade, lymph node metastasis and patient prognosis. The in vitro and in vivo assays indicated that HOTTIP silencing inhibited RCC cell proliferation, migration and invasion and induced autophagy, and 3-MA (an autophagy inhibitor) reversed these effects. In contrast, HOTTIP overexpression and rapamycin (an autophagy inducer) yielded the opposite results. Further research revealed that HOTTIP modification could affect RCC cell autophagy via the PI3K/Akt/Atg13 signaling pathway. Our study will help in finding a potential marker for RCC diagnosis and supply a target molecule for RCC treatment.

Engineering of pectin-dopamine nano-conjugates for carrying ruthenium complex: A potential tool for biomedical applications

In this work, we designed polysaccharide-metal complex of dopamine-modified pectin linking ruthenium compound, which exhibited a certain inhibition specificity to human renal cell adenocarcinoma cell line 786-O. The chemical structure and physical properties of the polysaccharide-metal complex were well characterized by multiple analysis methods. The multi-spectral results revealed that pectin-dopamine have been successfully coordinated by ruthenium complex to form nano-conjugates, which self-assembled into relatively regular nanospheres of approximately 200 nm. The polysaccharide-metal complex has more amorphous and less viscosity than pectin, and cannot withstand as much strain as the pectin and pectin-dopamine systems. Pectin-dopamine can decrease the toxicity effect of Ru complex in normal cell line such as human normal renal epithelial cell line 293A.

Polydatin exerts anti-tumor effects against renal cell carcinoma cells via induction of caspase-dependent apoptosis and inhibition of the PI3K/Akt pathway.

PurposePolydatin, a stilbenoid glucoside of a resveratrol derivative, has many biological functions, including antitumor effects. However, the antitumor effects of polydatin in renal cell carcinoma (RCC) have not been investigated.Materials and methodsIn the current study, MTT assays, transwell invasion assays and wound healing assays were performed to examine cell proliferation, invasion and migration. An apoptosis nucleosome ELISA was used to measure apoptosis. Caspase activity assays were applied to measure the activities of caspase-3/9. A Western blot assay was used to measure the change in protein levels.ResultsOur data demonstrated that polydatin inhibited the proliferation of RCC cells but not normal renal epithelial cells in a time- and dose-dependent manner. Polydatin also triggered apoptosis in a caspase-dependent manner. Moreover, polydatin treatment also led to the downregulation of Bcl-2 and Mcl-1 and to activation of Bax. Ectopic expression of Bcl-2 and Mcl-1 or silencing of Bax could repress the apoptosis that was induced by polydatin. Moreover, incubation with polydatin also suppressed the PI3K/Akt signaling pathway in RCC cells.ConclusionTaken together, our data indicated that polydatin may be applied as a potent agent against RCC.

PBN11-8, a Cytotoxic Polypeptide Purified from Marine Bacillus, Suppresses Invasion and Migration of Human Hepatocellular Carcinoma Cells by Targeting Focal Adhesion Kinase Pathways.

The development of antitumor drugs has attracted cancer researchers and the identification of novel antitumor lead compounds is certainly of great interest. The fermentation broth of Bacillus sp. N11-8, which was isolated from the Antarctic waters, showed cytotoxicity towards different cells. A cytotoxic polypeptide, PBN11-8, was purified from the fermentation broth of Bacillus sp. N11-8 using ultrafiltration, ammonium sulfate precipitation, anion exchange liquid chromatography and high performance liquid chromatography (HPLC). Cloning and sequence analysis showed that PBN11-8 polypeptide (MW: ~19 kDa by the electrospray-ionization (ESI)) displayed high similarity with peptidase M84 from Bacillus pumilus. PBN11-8 possessed moderate cytotoxicity towards several cancer cell lines with IC50 values of 1.56, 1.80, 1.57, and 1.73 µg/mL against human hepatocellular carcinoma cell line BEL-7402, human renal clear cell adenocarcinoma cell line 786-0, human hepatocellular carcinoma cell line HepG2, and human pancreatic cancer cell line Panc-28, respectively. Moreover, the polypeptide displayed weak cytotoxicity towards normal cell line renal tubular epithelial cell line HK2 and human normal liver cell line L02 cells. Wound healing migration and Transwell experiments demonstrate that PBN11-8 could inhibit the migration and invasion of BEL-7402. Further investigation revealed that PBN11-8 suppresses focal adhesion kinase (FAK)-mediated adhesion, migration, and invasion by disturbing FAK/extracellular regulated protein kinases (ERK) signaling and matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) in BEL-7402 cells. Thus, PBN11-8 represents a potential novel anti-cancer lead compound.

Knockdown of LETM1 inhibits proliferation and metastasis of human renal cell carcinoma cells.

The leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) has been reported to serve an important role in a number of human malignancies and is correlated with poor prognosis. However, little is known about the role of LETM1 in renal cell carcinoma (RCC). In the present study, the expression levels of LETM1 were investigated in RCC cell lines (Caki-1, 786-O, OS-RC-2, A498 and ACHN) and the HK-2 normal human renal tubular epithelial cell line. Short interfering RNA (siRNA) was used to knock down the expression of LETM1 in 786-O and A498 cells. The results indicated that the constitutive expression of LETM1 was notably upregulated in RCC cell lines. Knockdown of LETM1 significantly decreased cell proliferation, migration and invasion. Mechanistically, it was revealed that the knockdown of LETM1 expression sharply downregulated the protein expression of β-Catenin, Cyclin D1 and c-Myc in 786-O and A498 cells. In conclusion, these results suggest that knockdown of LETM1 exhibits tumor suppressive effects, at least in part by controlling the downstream Wnt/β-Catenin signaling pathway. Therefore, LETM1 may act as a novel therapeutic target for the treatment of RCC.

Development of highly sensitive and specific in vitro renal solute carrier (SLC) uptake cell models using normal human adult renal proximal tubule epithelial cells for drug transporter interaction studies

Development of highly sensitive and specific in vitro renal solute carrier (SLC) uptake cell models using normal human adult renal proximal tubule epithelial cells for drug transporter interaction studies

Decreased expression of BRAF-activated long non-coding RNA is associated with the proliferation of clear cell renal cell carcinoma

BackgroundBRAF-activated long non-coding RNA (BANCR) has been associated with various types of cancer. Nevertheless, the role of BANCR in clear cell renal cell carcinoma (ccRCC) is still not fully understood. This study aims to investigate the relationship between ccRCC and BANCR.MethodsExpression of BANCR in TCGA renal cancer data sets was analyzed. The expression pattern of BANCR in Immortalized normal human proximal tubule epithelial cell line HK-2 and ccRCC cell lines (ACHN, CAKI-1, A498 and 786-O) was detected by real-time quantitative RT-PCR (qRT-PCR). ccRCC tissues with adjacent normal renal tissues diagnosed by pathological methods from 62 patients were used to detect the expression of BANCR, and its correlation with prognosis of ccRCC patients was assessed by Kaplan-Meier method. The LV-BANCR vector was used to examine the influence of BANCR on the proliferation, migration, invasion, apoptosis and cell cycle distribution of ccRCC cells in vitro.ResultsBANCR was downregulated in renal cancer according to TCGA data sets. Compared with adjacent normal renal tissues and normal human proximal tubule epithelial cell line HK-2, BANCR expression was significantly decreased in ccRCC tissues and ccRCC cell lines, and its low expression was associated with poor prognosis. Moreover, in the condition of BANCR overexpression by LV-BANCR vector, the proliferation, migration, invasion capacity of ccRCC cells was inhibited, while the apoptosis was increased and the G1 cell cycle arrest was induced in vitro.ConclusionsBANCR is downregulated in ccRCC tissues and cell lines, and is associated with ccRCC progression. Thus, BANCR may represent a novel prognostic biomarker and a potential therapeutic target for ccRCC patients.

Aberrant DNA Damage Response and DNA Repair Pathway in High Glucose Conditions

Higher cancer rates and more aggressive behavior of certain cancers have been reported in populations with diabetes mellitus. This association has been attributed in part to the excessive reactive oxygen species generated in diabetic conditions and to the resulting oxidative DNA damage. It is not known, however, whether oxidative stress is the only contributing factor to genomic instability in patients with diabetes or whether high glucose directly also affects DNA damage and repair pathways. Normal renal epithelial cells and renal cell carcinoma cells are more chemo- and radiation resistant when cultured in high concentrations of glucose. In high glucose conditions, the CHK1-mediated DNA damage response is not activated properly. Cells in high glucose also have slower DNA repair rates and accumulate more mutations than cells grown in normal glucose concentrations. Ultimately, these cells develop a transforming phenotype. In high glucose conditions, defective DNA damage responses most likely contribute to the higher mutation rate in renal epithelial cells, in addition to oxidative DNA damage. The DNA damage and repair are normal enzyme dependent mechanisms requiring euglycemic environments. Aberrant DNA damage response and repair in cells grown in high glucose conditions underscore the importance of maintaining good glycemic control in patients with diabetes mellitus and cancer.

Abstract 2457: Epithelial-to-mesenchymal transition and stemness: Key targets of HOTAIR-miR-203 interaction in renal cell carcinoma

Abstract Background: Tumor metastasis and recurrence are major obstacles in renal cell carcinoma (RCC) treatment. RCC aggressiveness is highly associated with stemness and epithelial-to-mesenchymal transition (EMT) which involves increased cellular migration, invasion, inhibition of apoptosis and senescence. HOX transcript antisense RNA (HOTAIR), a lncRNA, is reported to be over-expressed, whereas miR-203 has low expression and is a tumor suppressor in various cancers. However, their association and role in RCC is not well understood. The aim of the present study was to examine the interaction of HOTAIR and miR-203 in the regulation of stemness and EMT in RCC. Experimental Procedure: ACHN, Caki-1 (human RCC cell lines), normal renal epithelial cells HK-2 and clinical specimens were used for this study. Profiling of HOTAIR and miR203 expression were done by quantitative real-time PCR and luciferase assay was performed to confirm their interaction. Attenuation of HOTAIR (25nM siRNA) and overexpression of miR-203 (10nM mimic) for 72 hours were used for functional studies. The biological role of miR-203 and its interaction with HOTAIR was also investigated using nude mouse models. Results: HOTAIR was observed to be overexpressed in RCC cell-lines and clinical specimens whereas, miR-203 was significantly under-expressed when compared to normal cells and tissues. Overexpression of miR-203 altered the cell cycle, inhibited epithelial-to-mesenchymal transition (EMT) and decreased cell proliferation along with induction of epithelial marker proteins and decrease in mesenchymal and stemness marker proteins. Conversely, knockdown of miR-203 in non-malignant HK-2 cells induced pro-cancerous characteristics. Direct binding of miR-203 to HOTAIR was shown by RNA immunoprecipitation and luciferase assay. Attenuation of HOTAIR expression reduced cell migration, invasion, and induced apoptosis, mimicking the effects of miR-203 overexpression. Administration of miR-203 mimic to established tumors in nude mice significantly suppressed tumor growth compared to controls. Conclusion: Our results show that HOTAIR-miR-203 interaction inhibits EMT and stemness to regulate RCC progression. Statistical analysis of the clinicopathological data from kidney cancer patients suggests that HOTAIR and miR-203 may be useful in RCC diagnostics and therapeutics. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Yutaka Hashimoto, Nadeem S. Bhat, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura Yamamura, Yuichiro Tanaka, Rajvir Dahiya. Epithelial-to-mesenchymal transition and stemness: Key targets of HOTAIR-miR-203 interaction in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2457.

Plasticizer Di-(2-Ethylhexyl)Phthalate Induces Epithelial-to-Mesenchymal Transition and Renal Fibrosis In Vitro and In Vivo.

Plasticizer di-(2-ethylhexyl)phthalate (DEHP) is known as an endocrine disruptor and a peroxisome proliferator. A currently epidemiological study has suggested that daily high DEHP intake from phthalate-tainted foods in children may be a risk factor for renal dysfunction. DEHP can leach from medical devices such as blood storage bags and the tubing. Long-term exposure to DEHP is associated with nephropathy and exacerbates chronic kidney diseases (CKDs) progression. However, the detailed effects and molecular mechanisms remain unclear. Here, we hypothesized that DEHP and its major metabolite mono-(2-ethylhexyl)phthalate (MEHP) incited epithelial-to-mesenchymal transition (EMT) and lead to aggravate renal fibrosis progression. Treatment with low-cytotoxic concentration DEHP, but not MEHP, for 72 h obviously induced the morphological and phenotypic changes and EMT markers induction in normal rat renal tubular epithelial cells (NRK-52E). AKT inhibitor MK-2206 inhibited DEHP-induced EMT features and signals of AKT phosphorylation and downstream NF-κB and GSK3β. DEHP did not affect the expression of transforming growth factor-β1 mRNA. DEHP down-regulated the peroxisome proliferator-activated receptor (PPAR)α and PPARγ protein expressions. PPARγ agonist pioglitazone partially and significantly inhibited DEHP-induced EMT induction. In vivo DEHP exposure for 6 weeks enhanced the renal dysfunction and renal fibrosis and mortality rate, but decreased the PPARα and PPARγ protein expressions, in a folic acid-induced kidney fibrosis mouse model. Taken together, these results demonstrate for the first time that DEHP arouses EMT induction and renal fibrosis progression in renal tubular cells and is associated with PPARs downregulation. DEHP exposure potentially exacerbated renal fibrosis/nephropathy in a kidney disease condition.

PAX2 regulates NRP-1 expression in renal tubular epithelial cellsto promote epithelial-to-mesenchymal transition and renal fibrosis

To observe the expression of neuropilin-1 (NRP-1) induced by paired-box gene 2 (PAX2) during the process of epithelial-to-mesenchymal transition (EMT) and renal fibrosis in unilateral ureteral obstruction (UUO) model in rats, and explore the mechanism of EMT induced by PAX2. Methods: The recombinant lentivirus expression vector for PAX2 was constructed and transfected into rat normal renal tubular epithelial cell line (NRK52E). The experimental cells were divided into three groups: transfection group, empty vector group, and normal group. E-cadherin and α-SMA were detected by western blot and real-time PCR. Expression of NRP-1 was detected by western blot, real-time PCR, and immunofluorescence. Sixty male Wistar rats were randomly divided into two groups: the sham-operation group (n=30) underwent left ureteral dissection, the UUO group (n=30) underwent left ureteral ligation. Post-operation on days 3, 7, 14, 21 and 28, 6 rats from each of the groups were sacrificed and the obstructed kidneys were dissected out. The histopathological changes were observed by hematoxylin-eosin and Masson staining. E-cadherin and α-SMA were detected by western blot and immunohistochemistry. Expression of NRP-1 and PAX2 were determined by western blot, immunohistochemistry, and real-time PCR. Results: Expression of NRP-1 mRNA and protein and α-SMA protein increased (P&lt;0.05) while E-cadherin protein expression decreased (P&lt;0.05) in the transfection group as compared to the empty vector group in vitro. In the UUO group, fibrosis was obvious, and there was decreased expression of E-cadherin protein (P&lt;0.05) and increased expression of α-SMA protein and NRP-1 mRNA and protein (P&lt;0.05) in comparison to the sham group. Conclusion: NRP-1 maybe mediate PAX2-induced EMT in renal tubular epithelial cells and renal fibrosis.