Renal Carcinoma Cell Lines Research Articles

Efficient anticarcinogenic activity of α-Fe2O3 nanoparticles: In-vitro and computational study on human renal carcinoma cells HEK-293

The present study has witnessed the synthesis of α-Fe2O3 NPs using polyethylene glycol (PEG) as a surfactant and l-ascorbic acid (LAA) as a stabilizer. The product has been characterized by UV–vis absorption spectroscopy, FTIR, Dynamic Light Scattering, particle size distribution analysis, X-ray diffraction analysis, TEM, FESEM, EDX and BET, which show formation of variable size and shape, mesoporous, PEG-coated α-Fe2O3 NPs (LAA@IONP-PEG) with β-FeOOH as an impurity. The present work emphasizes upon an anti-cancer study of LAA@IONP-PEG against renal carcinoma HEK-293 human embryonic kidney cell lines. The study suggests that LAA@IONP-PEG is a promising material against renal carcinoma HEK-293 human embryonic kidney cell lines. The docking study has confirmed anti-proliferative action of NPs through binding affinity with renal carcinoma molecular targets. The synthesized NPs show the synergistic effect with Axitinib as an anti-cancer drug effective against renal carcinoma cell lines. ROS and 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging assay have shown the antioxidant capability of synthesized NPs. The efficient biocatalytic activity of LAA@IONP-PEG prescribes its use as one of the best suited drugs for the future perspectives against fatal renal carcinoma and thus it is reckoned that synthesized NPs will have persistent utilization in different field of medical applications.

Differential sensitivity of renal carcinoma cells to doxorubicin and epigenetic therapeutics depends on the genetic background.

Differential sensitivity to chemotherapeutics is a limitation in chemotherapy of kidney cancer patients. Role of genetic background in chemotherapy is not fully understood. Therefore, this study evaluated the influence of genetic/epigenetic background of renal cancer cells on the sensitivity to chemotherapeutics. Two renal cell carcinoma (RCC) cell lines, Caki-1 and 786-0, with different genetic makeup of p53 and VHL were treated with doxorubicin either alone or in combination with epigenetic therapeutics 5-aza-2-dc and TSA. Sensitivity of RCC cells to these drugs was evaluated by cell viability and cell cycle analysis and was further confirmed by analysis of selected genes expression. Cell viability data revealed that 786-0 cells were more sensitive than Caki-1 to doxorubicin. Combination of doxorubicin with 5-aza-2-dc or TSA was more effective to inhibit growth of Caki-1 cells but not the 786-0. Data of cell cycle analysis and expression of representative genes for tumor suppressor, cell cycle and survival, drug transporter and DNA repair further provided the molecular basis for differential sensitivity of Caki-1 and 786-0 cell lines to doxorubicin. Important findings of this study suggest that doxorubicin is more cytotoxic to primary renal cancer 786-0 cells with mutant VHL and p53 than the metastatic Caki-1 cells with wild-type VHL and p53, and this differential response was independent of p53 expression level. This study suggests that combination of doxorubicin with epigenetic therapeutics could potentially be beneficial in clinical treatment of renal cancer patients with wild-type VHL and p53 but not in patients with mutant VHL and p53.

FAST1 Predicts Poor Survival of Renal Carcinoma and Promotes Its Progression Through the TGF-β/Smad Pathway.

PurposeRenal carcinoma (RC) originates in the renal tubular epithelial system, among which renal cell carcinoma (RCC) is the most frequent one. The forkhead activin signal transducer 1 (FAST1) has been shown to interfere with tumor progression as an oncogene, while its role in RC is limited. Therefore, this paper explored the prognostic significance, specific effects, and related mechanisms of FAST1 on RC.Patients and MethodsCell colony formation assay, cell counting kit-8 (CCK8) assay, flow cytometry and Transwell assay were used to test cell proliferation, viability, apoptosis, migration and invasion, respectively. Western blot (WB) was employed to determine the protein level of FAST1.ResultsOur study confirmed that FAST1 was up-regulated in RC tissues and cell lines, and its overexpression often represented a poor prognosis of RC patients. Meanwhile, the in vitro experiments showed that overexpressing FAST1 facilitated RC cell viability, proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), and repressed cell apoptosis. In addition, the in vivo experiments illustrated that the up-regulation of FAST1 strengthened tumor growth. On the contrary, knocking down FAST1 had the opposite effects. Mechanistically, The TGF-β/Smad pathway contributed to RC evolvement and was activated by FAST1 both in vitro and in vivo.ConclusionThis article suggests that FAST1 exerts a carcinogenic role in RC by regulating the TGF-β/Smad signaling.

Inhibition of Drp1 Sensitizes Cancer Cells to Cisplatin-Induced Apoptosis through Transcriptional Inhibition of c-FLIP Expression.

Mitochondrial fragmentation occurs during the apoptosis. Dynamin-related protein 1 (Drp1) acts as an important component in mitochondrial fission machinery and can regulate various biological processes including apoptosis, cell cycle, and proliferation. The present study demonstrates that dysfunction of mitochondrial dynamics plays a pivotal role in cisplatin-induced apoptosis. Inhibiting the mitochondrial fission with the specific inhibitor (Mdivi-1) did not affect apoptotic cell death in low concentrations (<10 μM). Interestingly, mdivi-1 enhanced cisplatin-induced apoptosis in cancer cells, but not in normal cells. Particularly in the presence of mdivi-1, several human cancer cell lines, including renal carcinoma cell line Caki-1, became vulnerable to cisplatin by demonstrating the traits of caspase 3-dependent apoptosis. Combined treatment induced downregulation of c-FLIP expression transcriptionally, and ectopic expression of c-FLIP attenuated combined treatment-induced apoptotic cell death with mdivi-1 plus cisplatin. Collectively, our data provide evidence that mdivi-1 might be a cisplatin sensitizer.

Cytotoxic and Apoptotic Effects of Ferulic Acid on Renal Carcinoma Cell Line (ACHN)

Background: Polyphenolic compounds have anti-proliferative effects and can trigger apoptosis in cancer cells. Ferulic acid is excessively found in various herbal products and fruits. Ferulic acid plays a role in the treatment of neurodegenerative diseases, cancer, diabetes, cardiovascular disease, inflammation, and bacterial and viral infections. Objectives: The purpose of this study was to evaluate the anticancer effect of ferulic acid on renal carcinoma cells (ACHN). Methods: To assess the anti-proliferative effect of ferulic acid, the renal carcinoma cell line (ACHN) was treated with different ferulic acid concentrations (10, 20, 40, 80, and 160 μM) for 24, 48, and 72 hours. Cell viability was measured using the MTT assay. The apoptosis of cancer cells was evaluated by flow cytometry and real-time PCR. Results: The IC50 of ferulic acid against ACHN cells was determined to be 30 μM at 72 hours with the MTT assay. The treatment of cells with ferulic acid concentrations of 30 and 60 μM caused a significant increase in the apoptosis index. The Bcl-2 gene expression level was significantly lower in the treated group than in the control group, and the Bax gene expression level was significantly higher in the treated group than in the control group (P &lt; 0.001). Conclusions: The results of this study showed the apoptotic activity of ferulic acid against ACHN cells. These results can be helpful in the better understanding of the anticancer mechanism of ferulic acid, suggesting this substance as an alternative drug or in combination with conventional chemical treatments for cancer treatment.

Different Role of Raptor and Rictor in Regulating Rasfonin-Induced Autophagy and Apoptosis in Renal Carcinoma Cells.

Both Raptor and Rictor are the key components in the complexes of mammalian target of rapamycin (mTOR), which play a vital role in mediating autophagy. Unlike mTOR, the regulatory role of either Raptor or Rictor in the regulation of autophagic process is relatively less explored. In present study, we found that rasfonin, which isolated from Talaromyces sp. 3656-A1 and was a fungal natural product, activated both caspase-dependent apoptosis and autophagy in ACHN, a renal carcinoma cell line. Knockdown of Raptor decreased both rasfonin-induced autophagic flux and PARP-1 cleavage, and in contrast, Rictor silencing increased apoptosis concomitantly enhancing rasfonin-induced autophagy. Unexpectedly, API-2, which was widely used as an inhibitor of Akt, promoted rasfonin-dependent autophagy in Raptor-depleted but not Rictor-deprived cells. Collectively, these results demonstrated that Raptor and Rictor could play a distinctly regulatory role in rasfonin-enhanced autophagy and apoptosis.

Inducing regulated necrosis and shifting macrophage polarization with anti-EMMPRIN antibody (161-pAb) and complement factors.

Treatment of solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME) that prevents effector immune cells from eradicating tumor cells and promotes tumor progression, angiogenesis, and metastasis. Therefore, targeting components of the TME to restore the ability of immune cells to drive anti‐tumoral responses has become an important goal. One option is to induce an immunogenic cell death (ICD) of tumor cells that would trigger an adaptive anti‐tumoral immune response. Here we show that incubating mouse renal cell carcinoma (RENCA) and colon carcinoma cell lines with an anti‐extracellular matrix metalloproteinase inducer polyclonal antibody (161‐pAb) together with complement factors can induce cell death that inhibits caspase‐8 activity and enhances the phosphorylation of receptor‐interacting protein kinase 3 (RIPK3) and mixed‐lineage kinase‐like domain (MLKL). This regulated necrotic death releases high levels of dsRNA molecules to the conditioned medium (CM) relative to the necrotic death of tumor cells induced by H2O2 or the apoptotic death induced by etoposide. RAW 264.7 macrophages incubated with the CM derived from these dying cells markedly enhanced the secretion of IFNβ, and enhanced their cytotoxicity. Furthermore, degradation of the dsRNA in the CM abolished the ability of RAW 264.7 macrophages to secrete IFNβ, IFNγ‐induced protein 10 (IP‐10), and TRAIL. When mice bearing RENCA tumors were immunized with the 161‐pAb, their lysates displayed elevated levels of phosphorylated RIPK3 and MLKL, as well as increased concentrations of dsRNA, IFNβ, IP‐10, and TRAIL. This shows that an antigen‐targeted therapy using an antibody and complement factors that triggers ICD can shift the mode of macrophage activation by triggering regulated necrotic death of tumor cells.

Polysaccharides extract from Vaccaria segetalis seeds inhibits kidney infection by regulating cathelicidin expression

Ethnopharmacological relevanceAccording to the Chinese Pharmacopoeia, the seeds of Vaccaria segetalis, a traditional medicinal herb, can be used for treating urinary diseases. The polysaccharides extract from V. segetalis seeds (VSP) has been shown to prevent urinary tract infections (UTIs). Aim of the studyInvestigate the effects of VSP on treating kidney infection induced by uropathogenic Escherichia coli (UPEC) and the underlying mechanisms. Materials and methodsBoth in vivo and in vitro infection models were established with the UPEC strain CFT073. After oral administration of VSP, the levels of bacterial load, cathelicidin (CRAMP), Toll-like receptors (TLRs) in the kidney were evaluated. The expression of cathelicidin (LL-37) in human renal cell carcinoma cell line (A498) was tested after the treatment of VSP. ResultsIn the kidneys of infection models, high-titer bacteria was detected. In the kidney of rat model, the expression of CRAMP was down-regulated, no significant change was observed in the levels of TLRs. After oral administration of VSP, the bacterial load was significantly decreased in rat and mouse models, and the levels of CRAMP and TLRs were significantly up-regulated in rat model. In vitro, the expression of LL-37 was significantly inhibited by CFT073. VSP up-regulated the expression of LL-37 in A498 cells. ConclusionsThe up-regulation of cathelicidin expression may contribute to the therapeutic effects of VSP on kidney infection.

Hypoxia contributes to galectin-3 expression in renal carcinoma cells

Galectin-3 is supposed as a prognostic factor and therapeutic target for many cancers. In a previous study, we have reported that galectin-3 was related to the development of renal cell cancer and served a therapeutic target for renal cell carcinoma (RCC). However, the mechanisms underlying the regulation of galectin-3 in RCC are still not known. In this study, we detected the expression of galectin-3 and hypoxia-inducible factor 1 (HIF-1) α in RCC using immunohistochemistry, and then conducted in vitro experiments to verify the regulation of galectin-3 by hypoxia in RCC. Our results showed that the expression of galectin-3 and HIF-1α were remarkably high in RCC tissues compared with those in the paracancerous tissues. Interestingly, hypoxia significantly promoted cytoplasmic and nuclear HIF-1α and galectin-3 expression in renal carcinoma cell lines, but not in renal tubular epithelial cell (HK-2). Renal carcinoma cell line (Caki-1), but not HK-2 showed significant increase of luciferase reporter activity of galectin-3 encoding the fragment from the site of −845 to +50 upon hypoxic insult. Moreover, HIF-1α overexpression vector promoted, while HIF-1α silencing vector reduced luciferase reporter activity of galectin-3 in Caki-1 and HK-2 cells in both normal and hypoxia conditions. A direct interaction of HIF-1α with Gal-3 promoter was also verified by electrophoretic mobility shift assay and chromatin immunoprecipitation. Together, our data indicated that hypoxia was critical for galectin-3 expression in RCC in a HIF-1α-dependent manner.

Abstract B13: Targeting mTOR/JUN/AXL pathway in TSC-related tumors

Abstract Renal angiomyolipoma (AML) and cysts are seen in 70-80% of patients with the human tumor suppressor gene syndrome tuberous sclerosis complex (TSC). Renal cell carcinoma (RCC) also is relatively common in TSC. Although two-hit loss of TSC1/2 is established in AML and TSC-RCC, the mechanisms and signaling pathways underlying TSC tumor development are not well understood. TSC-related tumor models were generated through i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 in Tsc2f/fROSA26-CreERT2 (n=24), Tsc2f/f CAG-CreERT2 mice (n=2) and Tsc1f/f CAG-CreERT2 mice (n=19). Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney of these mice at age of 6 months, confirmed by IHC. A phospho-RTK immunoblot screen was performed using lysates from kidney tumors in comparison to normal kidneys. pAXL levels were ~2.8-fold higher versus normal kidney, whereas other pRTKs showed no apparent change. Immunoblotting confirmed the upregulated levels of both p-AXL (Tyr 702) and total AXL in kidney tumor lysates vs. normal kidneys. Q-RT-PCR of mRNA also showed 2-fold increase in AXL mRNA. Bioinformatic analysis suggested that AXL is a probable target of the transcription factor JUN. RNA-seq data showed that AMLs had the highest level of JUN expression in comparison to 28 different kinds of cancer (from TCGA). JUN was highly expressed in the mouse kidney tumors compared to normal kidneys in both protein and mRNA levels. JUN levels were also assessed using TSC-null cell lines (TSC2-/- angiomyolipoma cells (h), Tsc2-/- renal cell carcinoma cells (m), TSC1-/- bladder cancer cells (h), Tsc1-/- lung cancer cells (m)) versus their TSC add-back counterparts. All TSC-null cells showed a higher level of JUN and AXL compared to their TSC add-back counterparts. Both rapamycin (20nM) and Torin (500nM) treatment of these cell lines for 24h led to a reduction in JUN expression, with a stronger effect from Torin than rapamycin. To identify connections between JUN and AXL, JUN and AXL CRISPR KO were performed in 2 Tsc2-/- renal cell carcinoma cell lines. JUN KO led to a major decrease in AXL expression, and AXL KO led to a decrease of JNK, ERK and JUN. A positive feedback loop may exist between JUN and AXL through JNK and ERK pathway. R428, an inhibitor of AXL, was used to treat our Tsc2f/f ROSA26-CreERT2 mice and Tsc2+/- AJ mice (12.5mg/kg, twice daily, oral gavage, 3 weeks). Kidney tumor volume decreased dramatically compared with vehicle-treated mice in both cohorts (31.38 vs 4.11 mm³, p=0.001; 1.84 vs 0.05 mm³, p=0.02). These findings suggest that JUN and AXL are both upregulated in response to TSC1/TSC2 loss and mTORC1 activation and contribute to tumorigenesis, and that there is potential therapeutic value to targeting these events. Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Targeting mTOR/JUN/AXL pathway in TSC-related tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B13.

Integrating Phenotypic Search and Phosphoproteomic Profiling of Active Kinases for Optimization of Drug Mixtures for RCC Treatment.

Simple SummaryRenal cell carcinoma (RCC) cancer is among the ten most common malignancies, and frequently presents as metastatic disease (mRCC). For these patients, systemic treatment is in order, but mRCC is often highly heterogeneous, and resistant to conventional therapies, or acquires resistance over time. Application of a combination of targeted therapeutic agents can tackle these problems, however, experimental optimization is not feasible given the enormous number of possible drug- and dose-combinations. We used a phenotypic approach, the streamlined-feedback system control (s-FSC) technique, which does not use a priori information on the mechanism of action of drugs. Using a number of searches, this method selects for optimized drug combinations (ODC) given at low doses (ED5-25), that can act synergistically. This way, we selected effective ODC for different RCC cell lines. Analysis of kinase activity was performed to provide mechanistic insight into the ODC action, and to further improve the found drug combinations. Combined application of multiple therapeutic agents presents the possibility of enhanced efficacy and reduced development of resistance. Definition of the most appropriate combination for any given disease phenotype is challenged by the vast number of theoretically possible combinations of drugs and doses, making extensive empirical testing a virtually impossible task. We have used the streamlined-feedback system control (s-FSC) technique, a phenotypic approach, which converges to optimized drug combinations (ODC) within a few experimental steps. Phosphoproteomics analysis coupled to kinase activity analysis using the novel INKA (integrative inferred kinase activity) pipeline was performed to evaluate ODC mechanisms in a panel of renal cell carcinoma (RCC) cell lines. We identified different ODC with up to 95% effectivity for each RCC cell line, with low doses (ED5–25) of individual drugs. Global phosphoproteomics analysis demonstrated inhibition of relevant kinases, and targeting remaining active kinases with additional compounds improved efficacy. In addition, we identified a common RCC ODC, based on kinase activity data, to be effective in all RCC cell lines under study. Combining s-FSC with a phosphoproteomic profiling approach provides valuable insight in targetable kinase activity and allows for the identification of superior drug combinations for the treatment of RCC.

Effect of murine double-minute 2 inhibitors in preclinical models of advanced clear cell carcinomas originating from ovaries and kidneys.

Advanced clear cell carcinomas originating from both ovaries and kidneys with cancerous peritonitis have poor prognoses. Murine double‐minute 2 (MDM2) is a potential therapeutic target for clear cell ovarian carcinomas with WT TP53. Herein, we characterized the antiangiogenic and antitumor effects of the MDM2 inhibitors DS‐3032b and DS‐5272 in 6 clear cell ovarian carcinoma cell lines and 2 clear cell renal carcinoma cell lines, as well as in clear cell ovarian carcinomas s.c. xenograft and ID8 (murine ovarian cancer cells with WT TP53) cancer peritonitis mouse models. In clear cell ovarian carcinoma s.c. xenograft mouse models, DS‐3032b significantly reduced WT TP53 clear cell ovarian carcinoma‐ and clear cell renal carcinoma‐derived tumor volumes. In ID8 mouse models, DS‐5272 significantly inhibited ascites production, reduced body weight, and significantly improved overall survival. Additionally, DS‐5272 reduced the tumor burden of peritoneal dissemination and decreased CD31+ cells in a dose‐dependent manner. Furthermore, DS‐5272 significantly decreased vascular endothelial growth factor concentrations in both sera and ascites. Combined therapy with MDM2 inhibitors and everolimus showed synergistic, and dose‐reduction potential, for clear cell carcinoma treatment. Our findings suggest that MDM2 inhibitors represent promising molecular targeted therapy for clear cell carcinomas, thereby warranting further studies to evaluate the efficacy and safety of dual MDM2/mTOR inhibitors in clear cell carcinoma patients.

Development of an artificial antibody specific for HLA/peptide complex derived from cancer stem-like cell/cancer-initiating cell antigen DNAJB8

BackgroundPeptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8.MethodsWe screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed.ResultsscFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10−9 M and 5.04 × 10−9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8).ConclusionWe isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

Anterior gradient 2 promotes tumorigenesis through upregulation of CCAAT-enhancer binding protein beta and hypoxia-inducible factor-2α and subsequent secretion of interleukin-6, interleukin-8, and vascular endothelial growth factor in the Caki-1 clear cell renal cell carcinoma cell line.

It has been previously established that hypoxia leads to tumor development, treatment resistance, and a poor prognosis. Under oxygen deprivation, hypoxia-inducible factors (HIFs) are stimulated to activate the genes necessary for tumor development in a low-oxygen environment. These genes encode regulators of angiogenesis, epithelial-mesenchymal transition, and cellular metabolism. A disulfide isomerase, anterior gradient 2 (AGR2), has been shown to increase hypoxia-inducible factor 1, alpha subunit (HIF-1α) stability in breast cancer. Our goal was to determine if AGR2 affects the level of transcription factor hypoxia-inducible factor 2, alpha subunit (HIF-2α). As a model, we used the clear cell renal cell carcinoma (ccRCC) cell line Caki-1. The cells were transduced with lentiviral vector (Tet-On) encoding AGR2. After induction of AGR2 expression, cells were grown under either hypoxic (0.5% O2 ) or normoxic (21% O2 ) conditions. Our data showed that AGR2 upregulated both HIF-1α and HIF-2α expression in Caki-1 cells increasing the expression of HIF-activated genes (glucose transporter 1, phosphoglycerate kinase 1, vascular endothelial growth factor A, and transforming growth factor-alpha) under the hypoxic conditions. Under the normoxic conditions, AGR2 strongly activated CCAAT-enhancer binding protein beta (C/EBPβ). Upregulation of C/EBPβ correlated with increased expression and secretion of the interleukin-6 and interleukin-8, inducing angiogenesis and inflammation in Caki-1 cells. In summary, our studies revealed that AGR2 has essential functions in ccRCC progression through upregulation of C/EBPβ and HIF-2α expressions, which affects cell signaling and metabolism.

Acetate promotes SNAI1 expression by ACSS2-mediated histone acetylation under glucose limitation in renal cell carcinoma cell.

Metastasis is the main cause of cancer-associated deaths, yet this complex process is still not well understood. Many studies have shown that acetate is involved in cancer metastasis, but the molecular mechanisms remain to be elucidated. In the present study, we first measured the effect of acetate on zinc finger transcriptional repressor SNAI1 and acetyl-CoA synthetase 2 (ACSS2) under glucose limitation in renal cell carcinoma cell lines, 786-O and ACHN. Then, RNA interference and overexpression of ACSS2 were used to detect the role of acetate on SNAI1 expression and cell migration. Finally, chromatin immunoprecipitation assay (ChIP) was used to investigate the regulatory mechanism of acetate on SNAI1 expression. The results showed that acetate increased the expressions of SNAI1 and ACSS2 under glucose limitation. ACSS2 knockdown significantly decreased acetate-induced SNAI1 expression and cell migration, whereas overexpression of ACSS2 increased SNAI1 level and histone H3K27 acetylation (H3K27ac). ChIP results revealed that acetate increased H3K27ac levels in regulatory region of SNAI1, but did not increase ACSS2-binding ability. Our study identified a novel inducer, acetate, which can promote SNAI1 expression by ACSS2-mediated histone acetylation in partly. This finding has important implication in treatment of metastatic cancers.

Serum response factor increases renal cell carcinoma migration and invasion through promoting epithelial-mesenchymal transition.

To explore the regulation and function of serum response factor in epithelial-mesenchymal transition in renal cell carcinoma. First, bioinformatics analysis of human renal cell carcinoma tissues was carried out. Then, the expression of serum response factor, mesenchymal markers (N-cadherin, vimentin and fibronectin) and epithelial markers (zonula occludens-1 and epithelial cadherin) was examined in 786-O cells (a human renal cell carcinoma cell line). Serum response factor was overexpressed with pcDNA-serum response factor plasmid, and suppressed by CCG-1423 (a small molecule inhibitor of serum response factor) to study how serum response factor affects epithelial-mesenchymal transition in renal cell carcinoma. A xenograft nude mouse model was established to explore whether serum response factor affected the tumorigenic ability of renal cell carcinoma cells. Serum response factor interacted with several important differentially expressed genes in renal cell carcinoma. In 786-O cells, serum response factor, N-cadherin, vimentin and fibronectin were upregulated, whereas zonula occludens-1 and epithelial cadherin were downregulated. Serum response factor upregulation in 786-O cells increased the Snail expression. Serum response factor suppression reduced Snail induction, and migration and invasion in renal cell carcinoma, which decreased the xenograft tumor volume. Serum response factor is a critical transcription factor in human renal cell carcinoma. Increased serum response factor activity induces epithelial-mesenchymal transition, migration and invasion in 786-O cells, and facilitates the progression of renal cell carcinoma. Targeting serum response factor with CCG-1423 might be an attractive therapeutic strategy for renal cell carcinoma.

Identification of low-dose multidrug combinations for sunitinib-naive and pre-treated renal cell carcinoma

BackgroundCombinations of drugs can improve the efficacy of cancer treatment, enable the reduction of side effects and the occurrence of acquired drug resistance.MethodsWe approached this challenge mathematically by using the validated technology called the Therapeutically Guided Multidrug Optimization (TGMO) method. In a set of genetically distinct human renal cell carcinoma (RCC) cell lines, either treated chronically with sunitinib (−ST) or sunitinib-naive, we identified cell line-specific low-dose-optimised drug combinations (ODC).ResultsSix cell-type-specific low-dose drug combinations for three sunitinib-naive as well as three sunitinib pre-treated cells were established. These ODCs effectively inhibited the RCC cell metabolic activity while being ineffective in non-cancerous cells. Based on a single screening test and three searches, starting with ten drugs, we identified highly efficacious drug mixtures containing four drugs. All ODCs contained AZD4547 (FGFR signalling pathway inhibitor) and pictilisib (pan-phosphatidylinositol 3-kinase inhibitor), but varied in the third and fourth drug. ODC treatment significantly decreased cell metabolic activity (up to 70%) and induced apoptosis, independent of the pretreatment with sunitinib. The ODCs outperformed sunitinib, the standard care for RCC. Moreover, short-term starvation potentiated the ODC activity. The translation of the 2D-based results to 3D heterotypic co-culture models revealed significant inhibition of the spheroid growth (up to 95%).ConclusionWe demonstrate a promising low-dose drug combination development to obtain drug combinations effective in naive as well as resistant tumours. Nevertheless, we emphasise the need for further mechanistic investigation and preclinical development.

Growth differentiation factor 15 (GDF-15) inhibition to increase muscle mass and function in cancer cachexia.

e15633 Background: Almost half of cancer deaths are attributed to cancers most frequently associated with cachexia. Cachexia is a complex metabolic disease characterized by anorexia and unintentional weight loss. Skeletal muscle depletion has been recognized as a key feature of the disease, however muscle anabolic therapies have not been successful, suggesting that treatments that target multiple aspects of the disease will be most effective. Growth differentiation factor 15 (GDF-15) is a cytokine that induces anorexia and weight loss and is associated with cachexia in cancer patients. In preclinical cancer cachexia models, GDF-15 inhibition is sufficient to normalize food intake and body weight, including skeletal muscle mass. However, it remains to be determined whether the increased skeletal muscle mass also results in restoration of muscle function. Therefore, we examined the effect of GDF-15 inhibition on muscle mass and function in mouse models of cancer cachexia in comparison with myostatin inhibition, an established muscle anabolic pathway. Methods: Cachectic mouse tumor models were established with subcutaneous implantation of tumor cell lines reported to be GDF-15-dependent; mouse renal cell carcinoma (RENCA) and human ovarian cancer (TOV-21G) cell lines. Mice were treated with anti-GDF-15 (mAB2) or anti-myostatin (RK35) monoclonal antibodies and skeletal muscle function was assessed in vivo via maximum force, maximum rate of contraction and half relax time. In the RENCA tumor model, GDF-15 inhibition fully restored body weight and skeletal muscle mass whereas myostatin inhibition showed only a modest effect. Results: Consistent with the muscle mass improvement, GDF-15 inhibition dramatically increased functional muscle endpoints compared to the partial effect of myostatin inhibition. Interestingly, in the TOV-21G tumor model GDF-15 inhibition only partially restored body weight, however skeletal muscle mass and muscle function were completely normalized. Consistent with the functional assessment, GDF-15 inhibition in the RENCA tumor model decreased the expression of several catabolic genes (i.e. Trim63, Fbxo32, Myh7 and Myh2). The GDF-15 effect is likely to be secondary to the reversal of anorexia since wildtype mice pair-fed to Fc-GDF-15-treated mice demonstrated equivalent muscle mass loss. Conclusions: Taken together these data suggest that GDF-15 inhibition holds potential as an effective therapeutic approach to alleviate multiple aspects of cachexia.

CRISPR-mediated ablation of overexpressed EGFR in combination with sunitinib significantly suppresses renal cell carcinoma proliferation.

Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma.

Design of Fluorescent Probes for Bioorthogonal Labeling of Carbonylation in Live Cells

With the rapid development of chemical biology, many diagnostic fluorophore-based tools were introduced to specific biomolecules by covalent binding. Bioorthogonal reactions have been widely utilized to manage challenges faced in clinical practice for early diagnosis and treatment of several tumor samples. Herein, we designed a small molecule fluorescent-based biosensor, 2Hydrazine-5nitrophenol (2Hzin5NP), which reacts with the carbonyl moiety of biomolecules through bioorthogonal reaction, therefore can be utilized for the detection of biomolecule carbonylation in various cancer cell lines. Our almost non-fluorescent chemical probe has a fast covalent binding with carbonyl moieties at neutral pH to form a stable fluorescent hydrazone product leading to a spectroscopic alteration in live cells. Microscopic and fluorometric analyses were used to distinguish the exogenous and endogenous ROS induced carbonylation profile in human dermal fibroblasts along with A498 primary site and ACHN metastatic site renal cell carcinoma (RRC) cell lines. Our results showed that carbonylation level that differs in response to exogenous and endogenous stress in healthy and cancer cells can be detected by the newly synthesized bioorthogonal fluorescent probe. Our results provide new insights into the development of novel bioorthogonal probes that can be utilized in site-specific carbonylation labeling to enhance new diagnostic approaches in cancer.