ACHN Cells Research Articles

Examination of the apoptotic effects of betulinic acid on renal cancer cell lines

Objective: Renal cancers are highly resistant to standard hormonal therapy, radiotherapy, and chemotherapy, and the survival rates are extremely low. Betulinic acid is a pentasilic triterpenoid saponin of lupine type obtained from various natural plants, especially from the shell of Betula plant. Betulinic acid was shown both in in vivo, and in vitro to have the ability to induce apoptotic pathways causing no toxicity for normal cells, and also has immunomodulatory effects. The aim of the present project is to investigate the anticancer effects of betulinic acid on CAKI-2 (ATCC® HTB-47™; clear cell renal carcinoma), ACHN (ATCC® CRL-1611™; renal cell adenocarcinoma) and MRC-5 (ATCC® CCL-171™: normal lung fibroblast) cell lines. Materials and Methods: The dose, and time-dependent cell viability was determined using the WST-1 test first in cell lines, and then apoptotic activity was determined with Annexin-V, apoptosis related nucleosomal enrichment factor levels, and Caspase 3 / BCA activity. Results: Betulinic acid reduced the CAKI-2, and ACHN cell viability in dose, and time-dependent manner inducing the apoptotic pathway. Conclusion: Researchers in the present study concluded in accordance with the results of Annexin-V, apoptosis-associated nucleosomal enrichment factor levels and Caspase 3 / BCA activity that betulinic acid triggered the apoptosis in both renal cancer cell lines, especially by the Caspase 3 activity.

Notch3 is involved in the proliferation of renal cancer cells via regulation of cell cycle progression and HIF-2α.

Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Although deregulation of the Notch signaling pathway is common in RCC and is involved in the tumorigenic process, the exact role of Notch3 and its underlying molecular mechanism in RCC, particularly in hypoxia, remain unknown. In the present study, RO4929097, a Notch3 inhibitor, was used to alter NICD3 expression. A Cell Counting Kit-8 assay, EdU incorporation assay, colony formation assay, flow cytometry and western blot analysis were used to investigate the effects of altered NICD3 expression on cell proliferation, cell cycle progression and HIF-2α protein expression. The results of western blot analysis showed that RO4929097 dose-dependently decreased the expression of Notch3 intracellular domain (NICD3) in 786-O and ACHN cells, which originate from clear cell RCC (ccRCC). The results of the Cell Counting Kit-8, EdU incorporation and colony formation assays demonstrated that downregulation of NICD3 significantly suppressed cell proliferation in both normoxia and hypoxia. In addition, flow cytometry and western blot analysis demonstrated that hypoxia (2% O2) promoted cell cycle progression in ccRCC cells with the increased expression of G1-S transition-associated proteins, namely cyclin-dependent kinase (CDK)4 and cyclin D1, while downregulation of NICD3 exerted negative effects on cell cycle progression, and the expression levels of CDK4 and cyclin D1. Furthermore, western blot analysis revealed that 2% O2-induced upregulated hypoxia-inducible factor-2α (HIF-2α) expression decreased following downregulation of NICD3 in 786-O and ACHN cells. Following transfection of the vector containing the NICD3 coding sequence, HIF-2α, CDK4, cyclin D1 and proliferating cell nuclear antigen expression, that were inhibited by RO4929097 in hypoxia, were rescued. Collectively, the results of the present study suggest that Notch3 is closely associated with the cell proliferation of ccRCC cells by regulating the cell cycle and HIF-2α.

Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

BackgroundKidney cancer is one of the most common cancers in the world. It is necessary to clarify its underlying mechanism and find its prognostic biomarkers. Current studies showed that SHMT2 may be participated in several kinds of cancer.MethodsOur studies investigated the expression of SHMT2 in kidney cancer by Oncomine, Human Protein Atlas database and ULCAN database. Meanwhile, we found its co-expression gene by cBioPortal online tool and validated their relationship in A498 and ACHN cells by cell transfection, western blot and qRT-PCR. Besides these, we also explored their prognostic values via the Kaplan–Meier plotter database in different types of kidney cancer patients.ResultsSHMT2 was found to be increased in 7 kidney cancer datasets, compared to normal renal tissues. For the cancer stages, ages and races, there existed significant difference in the expression of SHMT2 among different groups by mining of the UALCAN database. High SHMT2 expression is associated with poor overall survival in patients with kidney cancer. Among all co-expressed genes, NDUFA4L2 and SHMT2 had a high co-expression efficient. SHMT2 overexpression led to the increased expression of NDUFA4L2 at both mRNA and protein levels. Like SHMT2, overexpressed NDUFA4L2 also was associated with worse overall survival in patients with kidney cancer.ConclusionBased on above results, overexpressed SHMT2 and its co-expressed gene NDUFA4L2 were all correlated with the prognosis in kidney cancer. The present study might be benefit for better understanding the clinical significance of SHMT2 and provided a potential therapeutic target for kidney cancer in future.

Folliculin deficient renal cancer cells exhibit BRCA1 A complex expression impairment and sensitivity to PARP1 inhibitor olaparib.

Deficiency of folliculin (FLCN) may lead to renal cell carcinoma (RCC) in patients with Birt-Hogg-Dubé (BHD) disease. In this study, we investigated the cytotoxicity induced by PARP inhibitor olaparib in FLCN deficient RCC cells, and the interaction between FLCN and BRCA1 A complex-regulated DNA repair pathway. FLCN expressing (ACHN and UOK257-F) and FLCN deficient (ACHN-2 and UOK257) cell lines were used in this research. Cell viability was detected by clonogenic assay and MTT assay. Flow cytometry and TUNEL assay were used to detect apoptosis. Autophagy in cells was measured by MDC assay, western blot, and transmission electron microscopy. Co-immunoprecipitation, immunofluorescence and western blot experiments were performed to determine the interaction between FLCN protein and BRCA1 A complex. The in vivo experiments were performed in a xenograft model by inoculating UOK 257 in nude mice. RCC cells with FLCN protein deficiency were more sensitive to olaparib treatment than the cells with FLCN expression. Olaparib treatment led to more severe autophagy and apoptosis in FLCN deficient ACHN-2 and UOK257 cells compared to the FLCN expressing ACHN and UOK257-F cells. Decreased BRCA1 A complex expression and disruption of DNA repair ability were detected in FLCN-deficient cells, suggesting that FLCN deficiency impaired BRCA1 A complex expression and sensitized cells to PARP inhibitor olaparib. RCC cells deficient in FLCN are sensitive to olaparib treatment due to the impairment of BRCA1 A complex associated DNA repair ability. The results suggest that PARP inhibitor, such as olaparib, may be a potentially effective therapeutic approach for kidney tumors with deficiency of FLCN protein.

LncRNA NORAD stimulates proliferation and migration of renal cancer via activating the miR-144-3p/MYCN axis.

The purpose of this study was to clarify the potential role of long non-coding RNA (lncRNA) NORAD in the development of renal cancer. Expression levels of NORAD, miR-144-3p, and MYCN in renal cancer tissues and cell lines were detected. After overexpression of NORAD, proliferative and migratory changes in ACHN and A498 cells were evaluated by Cell Counting Kit-8 (CCK-8) and transwell assay, respectively. Thereafter, Luciferase assay was conducted to determine the interaction in the NORAD/miR-144-3p/MYCN axis. Besides, its biological function in influencing phenotype changes of renal cancer cells was finally demonstrated by rescue experiments. The results manifested that NORAD and MYCN were upregulated, while miR-144-3p was downregulated in renal cancer tissues. Overexpression of NORAD stimulated proliferative and migratory potentials in ACHN and A498 cells, which were partially abolished by co-overexpression of miR-144-3p. Moreover, NORAD/miR-144-3p/MYCN axis was found to be responsible for stimulating the malignant development of renal cancer. LncRNA NORAD stimulates proliferative and migratory potentials in renal cancer by sponging miR-144-3p to upregulate MYCN.

Chromodomain Helicase DNA-Binding Protein 5 Inhibits Renal Cell Carcinoma Tumorigenesis by Activation of the p53 and RB Pathways.

Chromodomain helicase DNA-binding protein 5 (CHD5) plays a crucial tumor suppressor role in multiple types of tumors. For this study, we investigated its clinical significance and the molecular mechanism(s) underlying tumorigenesis in renal cell carcinoma (RCC). Initially, CHD5 expression was assessed in primary tumor tissue and in tissue array. Correlations among CHD5 expression and clinicopathological characteristics were analyzed. Next, lentivirus-mediated CHD5 overexpression in the ACHN and 769-P cells was used to assess effects on proliferation, migration, invasion ability, and the regulation of the p14ARF/p53 and p16INK4a/RB signaling pathways. Finally, a xenograft mouse model was used to verify its impact on tumor growth in vivo. Results demonstrated that CHD5 was downregulated in tumor tissues and that low CHD5 expression was correlated with advanced TNM stage, high Fuhrman grade, lymph node metastasis, and poor survival. Overexpression of CHD5 inhibited proliferation, migration, and invasion in vitro; prompted cell cycle G1 phase arrest; induced apoptosis; and suppressed tumor growth in vivo. Furthermore, we confirmed that CHD5 activates the p53 and RB pathways to inhibit tumorigenesis in RCC. In summary, CHD5 is involved in the initiation and progression of RCC and may serve as a diagnostic biomarker and a potential therapeutic target for RCC.

Cinnamaldehyde and Hyperthermia Co-Treatment Synergistically Induces ROS-Mediated Apoptosis in ACHN Renal Cell Carcinoma Cells.

Renal cell carcinoma (RCC) represents the most common form of kidney cancer, which accounts for 3–5% newly diagnosed cancer cases. Since limited therapies are available for RCC, a search for new options is required. Therefore, in this study, we evaluated the combination effect of cinnamaldehyde (CNM) and hyperthermia treatment. CNM treatment combined with 43 °C hyperthermia synergistically increased cytotoxicity in RCC cell line ACHN cells. Through Western blot assays, we observed increased apoptosis signaling and decreased proliferation/metastasis signaling, along with a repressed heat shock protein 70 level. In flow cytometry analyses, CNM and hyperthermia combination clearly induced apoptosis and mitochondrial potential of ACHN cells, while arresting the cell cycle. Investigation of reactive oxygen species (ROS) suggested a significant increase of ROS generation by CNM and 43 °C hyperthermia co-treatment. We could verify that ROS is crucial in the apoptotic action of combination treatment with CNM and hyperthermia through further experiments regarding an ROS scavenger. Overall, we suggest CNM and hyperthermia combination treatment as an alternative option of anticancer strategies for RCC.

USP22 promotes proliferation in renal cell carcinoma by stabilizing survivin.

Renal cell carcinoma (RCC) is one of the commonest urological tumors. The incidence of RCC ranks third among urological tumors, after prostate cancer and bladder tumors. However, the etiology of RCC remains unclear. Ubiquitin-specific protease 22 (USP22), a potential marker of cancer stem cells, is associated with the occurrence and progression of numerous tumors. However, the roles of USP22 in RCC have not yet been investigated. Survivin is a member of the inhibitor of apoptotic protein family involved in RCC progression. The present study first detected the expression of USP22 and survivin in RCC tissues using immunohistochemistry and western blotting. It was revealed that the protein levels of USP22 and survivin in RCC tissues were higher than those in adjacent normal renal tissue. Subsequently, it was demonstrated that USP22 knockdown inhibited the growth of an RCC cell line ACHN and downregulated the protein level of survivin, accompanied by an increased level of cleaved-caspase-3. By contrast, overexpression of USP22 promoted the growth of ACHN cells, upregulated the expression of survivin and decreased the level of cleaved-caspase-3. Notably, the changes in USP22 expression did not affect the SURVIVIN mRNA level. Finally, it was confirmed that USP22 interacted with survivin and stabilized it by downregulating its ubiquitination. The present results indicate that USP22 may regulate survivin via deubiquitination, thereby promoting the proliferation of RCC cells. The results of the current study suggest that USP22 may represent a novel therapeutic target for patients with RCC.

6-Gingerol suppresses tumor cell metastasis by increasing YAPser127 phosphorylation in renal cell carcinoma.

According to the World Health Organization, the incidence and mortality rates of renal cell carcinoma (RCC) are rapidly increasing worldwide. Serious side effects caused by immune therapy and resistance to targeted drug therapy are urgent clinical problems facing kidney treatment. There is increasing global interest in developing natural products with a reduced number of side effects as adjunctive therapeutic options for RCC. Ginger is a spice and herbal remedy used worldwide, and 6-gingerol is a major pharmacologically active ingredient in ginger. In our study, we found that 6-gingerol suppressed RCC cell migration and metastasis in vitro and in vivo. Moreover, reduction in MMP2, Slug, and Vimentin protein levels was observed following 6-gingerol treatment of 786-O and ACHN cells. Furthermore, we revealed the mechanisms underlying the ability of 6-gingerol to inhibit RCC cell migration and metastasis. 6-Gingerol increased yes-associated protein (YAP)ser127 phosphorylation and reduced YAP levels in cell nuclei. We also used a series of loss-of-function and gain-of-function experiments to support our results. Western blot results showed that MMP2, Slug, and Vimentin protein expression was downregulated in YAP-silenced cells and upregulated in YAP-overexpressing cells. Transwell data demonstrated that YAP suppressed RCC migration ability. Immunofluorescence images showed that 6-gingerol decreased YAP levels, leading to disordered F-actin and a reduction in cell lamellipodia. Overall, our results indicated that 6-gingerol is a potential antimetastatic compound for use in kidney therapy.

TRIM33 Overexpression Inhibits the Progression of Clear Cell Renal Cell Carcinoma In Vivo and In Vitro.

Purpose To evaluate the expression of tripartite motif-containing 33 (TRIM33) in ccRCC tissues and explore the biological effect of TRIM33 on the progress of ccRCC. Method The Cancer Genome Atlas (TCGA) database was used to examine the mRNA expression levels of TRIM33 in ccRCC tissues and its clinical relevance. Immunohistochemistry (IHC) was performed to evaluate its expression in ccRCC tissues obtained from our hospital. The correlation between TRIM33 expression and clinicopathological features of the patients was also investigated. The effects of TRIM33 on the proliferation of ccRCC cells were examined using the CCK-8 and colony formation assays. The effects of TRIM33 on the migration and invasion of ccRCC cells were explored through wound healing and transwell assays, along with the use of Wnt signaling pathway agonists in rescue experiments. Western blotting was used to explore the potential mechanism of TRIM33 in renal cancer cells. A xenograft model was used to explore the effect of TRIM33 on tumor growth. Result Bioinformatics analysis showed that TRIM33 mRNA expression in ccRCC tissues was downregulated, and low TRIM33 expression was related to poor prognosis in ccRCC patients. In agreement with this, low TRIM33 expression was detected in human ccRCC tissues. TRIM33 expression levels were correlated with clinical characteristics, including tumor size and Furman's grade. Furthermore, TRIM33 overexpression inhibited proliferation, migration, and invasion of 786-O and ACHN cell lines. The rescue experiment showed that the originally inhibited migration and invasion capabilities were restored. TRIM33 overexpression reduced the expression levels of β-catenin, cyclin D1, and c-myc, and inhibited tumor growth in ccRCC cells in vivo. Conclusion TRIM33 exhibits an abnormally low expression in human ccRCC tissues. TRIM33 may serve as a potential therapeutic target and prognostic marker for ccRCC.

Abstract 6439: CapioCyte™ technology for efficient capture and downstream analysis of circulating tumor cells from renal cell carcinoma

Abstract Objective: The objective of this work was to obtain highly sensitive purification and post-capture analysis of renal cell carcinoma (RCC) circulating tumor cells (CTCs) using CapioCyte™ technology. Background: Circulating tumor cells (CTCs) may be a highly valuable source of actionable information for the practice of precision medicine. However, CTCs are exceptionally rare in peripheral blood. The CapioCyte™ chip purifies live CTCs through a combination of biomimetic cell rolling and dendrimer-mediated multivalent immunorecognition. The CapioCyte™ platform has been previously shown to isolate CTCs with high sensitivity from peripheral blood collected from patients with head and neck cancer using a mixture of antibodies against epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) [Myung et al., Clinical Cancer Research 2018, 24(11):2539-2547]. However, this antibody cocktail is not universally applicable to all types of cancer. In particular, renal cell carcinoma (RCC), having no clinically actionable circulating biomarkers, often do not express HER2. To address this, we have employed a new antibody mixture targeting EpCAM, EGFR, carbonic anhydrase IX (CAIX), and hepatocyte growth factor receptor (c-MET). Our study presents enhanced capture of CTCs from renal cancer, demonstrating the modular nature of our CapioCyte™ technology. Methods: Antibody-coated beads were used to determine the functional recognition of ACHN and 786-O RCC cell lines. Flow channel experiments and atomic force microscopy (AFM) force spectroscopy measurements were used to quantify off-rate kinetics. A clinical pilot study of 7 RCC patients (pre-treatment) and 5 healthy controls was performed to validate the CapioCyte™ surface tuned for RCC capture. In recent work, CTCs recovered from the CapioCyte™ surface were sequenced using 10X Chromium system with v3 chemistry and analyzed using RaceID clustering and Gene Set Enrichment Analysis (GSEA). Results: The optimized mixture of EpCAM, CAIX, EGFR, and c-MET showed 80% enhancement in the capture of RCC cell lines compared to the mixture of EpCAM, EGFR, and HER2. Force spectroscopy measurements were used to show that dendrimers contributed to an order of magnitude enhancement in off-rate kinetics. In patient samples, the RCC capture surface isolated a total of 9.8 +/- 5.1 CTC ml-1 whole blood compared to just 1.8 +/- 2.0 CTC ml-1 with the conventional three-antibody mixture. Finally, preliminary results show the detection of oncogenic signatures within two additional peripheral blood samples. Conclusions: The prototype CapioCyte™ platform is a flexible liquid biopsy that can be tuned for various disease indications. Here, enhanced capture of RCC CTCs was demonstrated in clinical blood samples with high sensitivity. Proof-of-concept RNA sequencing shows the promise of CapioCyte™ in providing further clinically-actionable information from CTCs. Citation Format: Jiyoon Bu, Michael J. Poellmann, Marco Reyes-Martinez, Andrew Armstrong, Daniel George, Tian Zhang, Andrew Z. Wang, seungpyo hong. CapioCyte™ technology for efficient capture and downstream analysis of circulating tumor cells from renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6439.

Curcumin Conjugates of Non-steroidal Anti-Inflammatory Drugs: Synthesis, Structures, Anti-proliferative Assays, Computational Docking, and Inflammatory Response.

In an effort to combine the anti‐proliferative effect of CUR‐BF2 and CUR compounds with anti‐inflammatory benefits of non‐steroidal anti‐inflammatory drugs (NSAIDs), a library of the bis‐ and mono‐NSAID/CUR‐BF2 and NSAID/CUR conjugates were synthesized by coupling flufenamic acid, flurbiprofen, naproxen, indomethacin, and ibuprofen to diversely substituted hydroxy‐benzaldehydes via an ester linkage, and by subsequent reaction with acetylacetone‐BF2 to form the bis‐ and the mono‐NSAID/CUR‐BF2 adducts. Since conversion to NSAID/CUR by the previously developed decomplexation protocol showed limited success, a set of NSAID/CUR conjugates were independently prepared by directly coupling the NSAIDs with parent curcumin. The bis‐NSAID/CUR‐BF2 and bis‐NSAID‐CUR hybrids exhibited low cytotoxicity in NCI‐60 assay, and in independent cell viability assay on colorectal cancer (CRC) cells (HCT116, HT29, DLD‐1, RKO, SW837, CaCo2) and in normal CR cells (CCD841CoN). By contrast, the mono‐naproxin and mono‐flurbiprofen CUR‐BF2 adducts exhibited remarkable anti‐proliferative and apoptopic activity in NCI‐60 assay most notably against HCT‐116 (colon), OVCAR‐3 (ovarian), and ACHN (renal) cells. Computational molecular docking calculations showed favorable binding energies to HER2, VEGFR2, BRAF, and Bcl‐2 as well as to COX‐1 and COX‐2, which in several cases exceeded known inhibitors. The main interactions between the ligands and the proteins were hydrophobic, although several hydrogen bonds were also observed. A sub‐set of six compounds that had exhibited little or no cytotoxicity were tested for their anti‐inflammatory response with THP‐1 human macrophages in comparison to parent NSAIDs or parent curcumin.

Vitexin suppresses renal cell carcinoma by regulating mTOR pathways.

BackgroundRenal cell carcinoma (RCC) is one of the most common malignant tumors in the world. Vitexin (apigenin-8-C-D-glucopyranoside), a bioactive compound isolated from a variety of plants, has multiple protective effects on human health. The purpose of this study was to investigate the role of vitexin in RC and the related molecular mechanism.MethodsProliferation was tested with Cell Counting Kit-8 and Edu staining. Apoptosis was studied with flow cytometry. Immunofluorescent was applied to show LC3 spots. BALB/c nude mice bearing ACHN cells were established and immunohistochemical staining was applied to validate the in vivo effects of vitexin. All the effects and possible signaling pathways involved were validated with western blotting.ResultsSeventy micromole of vitexin started to show significant effect on the growth of normal renal tubular epithelial cells (HK-2), so 0, 10, 20 and 40 µM of vitexin were used in later experiments. Vitexin inhibited growth and induced apoptosis of ACHN and OS-RC-2 cells in a dose-dependent manner, and promoted excessive autophagy by reducing p62 levels and increasing Beclin1 and LC3II levels. Western blotting revealed that vitexin significantly increased the phosphorylation levels of Adenosine Monophosphate Activated Protein Kinase (AMPK) and c-Jun N-terminal kinase (JNK) in ACHN and OS-RC-2 cells, while decreasing the phosphorylation levels of phosphatidylinositol 3-kinase/activates protein kinase/mammalian target of rapamycin (PI3K/AKT/mTOR). In BALB/c nude mice bearing ACHN cells, vitexin inhibited tumor growth, reduced Ki67 and increased caspase-3 levels in the tumor tissues.ConclusionsThe results indicated that the tumor suppressive role of vitexin in ACHN and OS-RC-2 cells involved AMPK/mTOR, PI3K/AKT/mTOR, and JNK pathways. Therefore, vitexin may be a promising drug for the treatment of RCC.

Impacts of the Staphylococcal Enterotoxin H on the Apoptosis and lncRNAs in PC3 and ACHN.

Cancer is considered as the most lethal disease for human beings, and up to now many attempts were failed for prevention and treatment of this tremendous health problem. Consequently, this study purpose was to investigate novel therapeutic methods for cancer. The bacterial toxin can result in cell death throughout the induction of apoptosis in cancer cell lines. We evaluated apoptosis and the expression levels of long non-coding RNAs (lncRNAs) in PC3, ACHN and HDF cell lines that were transfected with pCDNA3.1(+)-seh and empty plasmid. pCDNA3.1(+)-seh treatment showed overexpression of GAS5 (p = 0.0033 and p = 0.0033) in PC3 and ACHN cells, down regulation of PCA3 and NEAT1 (p = 0.0092 and p = 0.0097) in the PC3 cells, and down regulation of PVT1 and MALAT1 (p = 0.0239 and p = 0.0133) in the ACHN cells in comparison with the empty plasmid, but there was no significant effect on HDF normal cells. Additionally, this study data demonstrated that the cell adhesion was down regulated. The flow cytometry data showed transfection by pCDNA3.1 (+)-seh could elevate PC3 and ACHN cell apoptosis levels in comparison with empty plasmid. This study findings propose that SEH toxin of S. aureus could be a useful candidate for therapeutic researches in cancer vaccine development.

Brain-Derived Neurotrophic Factor Up-Regulation by the Methanol Extract of Foxtail Millet in Human Peripheral Cells.

Brain-derived neurotrophic factor (BDNF) plays important roles in synaptic plasticity and neuronal differentiation. The neurotrophic hypothesis of depression, which suggests that reduced BDNF in the hippocampus underlies depression, has attracted increasing attention. Stress, a major cause of depression, leads to decreased BDNF levels, and administration of BDNF into the hippocampus shows an antidepressant effect. BDNF is synthesized in peripheral tissues as well as in the brain. Since BDNF crosses the blood-brain barrier, intake of food ingredients that elevate BDNF in peripheral tissues may be useful for the prevention and treatment of depression. However, no screening method for BDNF up-regulators in peripheral tissues has been reported. In this study, we revealed that ACHN human kidney adenocarcinoma cells secreted BDNF. In addition, we demonstrated that the methanol extract of foxtail millet up-regulated BDNF levels in ACHN cells. Our results indicate that ACHN cells could be useful in the screening for peripheral-BDNF up-regulators, and that foxtail millet may have the potential to elevate BDNF levels in peripheral tissues.

Resveratrol Inhibition of Renal Cancer Stem Cell Characteristics and Modulation of the Sonic Hedgehog Pathway

Aims Renal cell cancers typically exhibit high metastasis and recurrence, and this is thought to be due to renal cancer stem cells (CSCs). Meanwhile, aberrant activation of Sonic hedgehog (Shh) signaling is linked with CSCs. Resveratrol has direct or indirect impacts on the pathological activities of CSCs. However, the effects of resveratrol on renal CSCs remain to be elucidated. Methods We cultured renal CSCs in serum-free medium. Western blotting was used to analyze the expression levels of related proteins. The mRNA changes were detected by qRT-PCR after resveratrol treatment. The CD133+ cells were quantified by flow cytometry analysis. Immunofluorescence staining images showed the changes in CD44 and Smoothened expression in the cell spheres. Results Renal CSCs were enriched by tumorsphere formation assays of ACHN and 786-O cells. Resveratrol treatments markedly decreased the size and number of cell spheres and downregulated the expression of the Shh pathway-related proteins and CSCs markers. Moreover, we observed that resveratrol inhibited cell proliferation and induced cell apoptosis, while purmorphamine upregulated the Shh pathway and weakened the effects of resveratrol on renal CSCs. Conclusions These results suggested that resveratrol is a potential novel therapeutic agent that targets inactivation of renal CSCs by affecting the function of the Shh pathway.

CircHIPK3 promotes proliferation and metastasis and inhibits apoptosis of renal cancer cells by inhibiting MiR-485-3p

BackgroundThe intervention of circHIPK3 in renal carcinoma (RC) has not been reported, and thus, the current study investigated the intervention and mechanism of circHIPK3 in RC.MethodsThe expression of circHIPK3 in RC tissues and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Ribonuclease R (RNase R) resistance and distribution of circHIPK3 and HIPK3 were analyzed by RNase R digestion experiments and cytoplasm/nucleus separation experiments. CircHIPK3 was knocked down in ACHN and 769-P cells. Cell counting kit-8 (CCK-8), colony formation assay, scratch assay, and Transwell assay were performed to detect cell proliferation and metastasis. CircInteractome, qRT-PCR and dual-luciferase reporter assay were used to predict the target miRNAs of circHIPK3. Furthermore, a series of rescue experiments were performed to analyze the regulatory relationship between circHIPK3 and miR-485-3p. Epithelial-mesenchymal transition (EMT) and the expressions of apoptosis-associated markers were detected by Western blot and qRT-PCR. The regulatory relationship between circHIPK3 and miR-485-3p in vivo was explored by xenograft experiments, Western blot, qRT-PCR and immunohistochemistry (Ki-67).ResultsCircHIPK3 was mainly overexpressed in the cytoplasm of RC tissues and cells. Knocking down circHIPK3 inhibited the proliferation, migration, and invasion of RC cells. The expression of circHIPK3 was negatively related to that of its target gene miR-485-3p. Results of the rescue experiments showed that circHIPK3 overexpression could partially reverse the anti-carcinoma effect of miR-485-3p mimic. The specific mechanism of circHIPK3 was related to the effect of miR-485-3p on partially reversing the up-regulated expressions of Clever caspase-3, Bax, E-Cadherin and down-regulated expressions of Bcl-2, N-Cadherin and Vimentin. The results of in vivo experiments demonstrated that circHIPK3 promoted tumor growth and the expression of Ki-67 by down-regulating miR-485-3p.ConclusionCircHIPK3 promotes the proliferation and metastasis and inhibits the apoptosis of RC cells through competitively binding to miR-485-3p.

Knockdown of Myoferlin Suppresses Migration and Invasion in Clear-Cell Renal-Cell Carcinoma

Myoferlin (MYOF) has emerged as an oncogenic protein in various human cancer types. This study was conducted to investigate comprehensively the expression and functional properties of MYOF in clear-cell renal-cell carcinoma (ccRCC) with respect to its value as diagnostic biomarker and therapeutic target. mRNA and protein expression of MYOF were assessed by quantitative polymerase chain reaction and immunohistochemistry. siRNA-mediated knockdown of MYOF was performed in the RCC cell line ACHN followed by proliferation, migration and invasion assays. MYOF mRNA and protein expression were significantly up-regulated in ccRCC. Higher mRNA levels were measured in advanced tumors. MYOF protein expression was increased in tumors with higher histological grades, and those with positive lymph node and surgical margin status. MYOF knockdown led to reduction of migration and invasion in ACHN cells, whereas expression of angiogenesis-associated genes tyrosine-protein kinase receptor-2 (TIE2), angiopoietin 2 (ANG2) and caveolin-1 (CAV1) was up-regulated following knockdown. MYOF may serve as a diagnostic biomarker of tumor progression and a potential therapeutic target in ccRCC.

Circular RNA circ_0001368 inhibited growth and invasion in renal cell carcinoma by sponging miR-492 and targeting LATS2

Circular RNAs (circRNAs) act as essential regulators in the tumorigenesis of renal cell carcinoma. Our study aims to investigate the underlying function and the molecular mechanisms of circ_0001368 in renal cell carcinoma. The qRT-PCR results indicate that the circ_0001368 expression level was downregulated in the carcinoma cells and tissues of the renal cell. circ_0001368 weakened cell proliferation and invasion in the ACHN and 786-O cells. The luciferase reporter assay showed that circ_0001368 functioned as an endogenous sponge for miR-492. The transwell and CCK8 assays showed that circ_0001368 suppressed cell proliferation and invasion in the ACHN and 786-O cells. Large tumor suppressor kinase 2 (LATS2) was confirmed by Targetscan as a target gene of miR-492. The overexpression of LATS2 repressed the growth and invasion of ACHN and 786-O cells. circ_0001368 upregulated the LATS2 expression and suppressed ACHN and 786-O cell growth and invasion by sponging miR-492. circ_0001368 suppressed the proliferation ability of 786-O in vivo. In conclusion, circ_0001368 was identified in this study as a novel anti-tumor RNA in renal cell carcinoma and can function as a potential therapeutic target for renal cell carcinoma treatment.

Knockdown of HMGB2 inhibits proliferation and invasion of renal tumor cells via the p-38MAPK pathway.

To investigate the function of HMGB2 in renal tumor ACHN cells in vitro and in vivo and to study the underlying molecular mechanisms. Kaplan-Meier analysis was used to study the relationship between expression of HMGB2 and prognosis of renal tumor. MTT assay was employed to examine cell proliferation and flow cytometry analysis was used to study the role of HMGB2 in cell apoptosis in ACHN cells. Transwell assays were used to explore the migration and invasion of ACHN cells. The effect of HMGB2 on tumor growth was investigated in vivo. Western blot was performed to evaluate the expression levels of p-JNK, p-ERK and p-p38MAPK. HMGB2 was upregulated in renal tumor and correlated with worse overall survival in renal tumor patients. Down-regulation of HMGB2 suppressed ACHN cells proliferation, invasion and migration in vitro. Moreover, down-regulation of HMGB2 inhibited tumor growth in vivo and HMGB2 exerts the oncogene function partly via the inhibition of p-p38MAPK activation. Our results provide novel insights into neuropathic pain and help to explore therapeutic targets in the treatment.