Renal Cell Carcinoma Cell Lines ACHN Research Articles

Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 μM Pazopanib, respectively. Cellular senescence was measured using senescence-associated β-galactosidase (SA-β-Gal) staining. Western blot analysis and real-time polymerase chain reactionwere used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress by increasing the production of reactive oxygen species,reducing the levels of glutathione peroxidase,and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

CILP is a potential pan-cancer marker: combined silico study and in vitro analyses.

CILP (Cartilage intermediate layer protein), an ECM (extracellular matrix) glycoprotein, is found to be associated with intervertebral disc degeneration, chronic heart failure, obese and cardiac fibrosis. However, there are few reports on the role of CILP in tumors. Thus, in this study, we mainly explored the function of CILP in the occurrence and development of tumors and whether it could be a potential pan-cancer marker. Pan-cancer data in this study were obtained from UCSC Xena. Single-cell data were obtained from GSE152938. ROC (Receiver operating characteristic) curves were used to evaluate the accuracy of CILP in predicting the occurrence of different tumor types. The Kaplan-Meier plots were used to assess the relationship between CILP expression and survival prognosis in different tumor types by COX regression analysis. Pseudotime analysis and cell communication analysis were used to further explore the function of CILP at Single cell level. The human RCC (renal cell carcinoma) cell lines ACHN and 786-O were used for further experimental verification. Bulk RNA-seq showed differences in CILP expression in several tumors. ROC curves showed that 14 tumors have AUC > 0.7. Kaplan-Meier plots indicated that CILP is a risk factor for patients in 3 kinds of tumors. ScRNA-seq (Single cell RNA sequencing) suggested that CILP might influence tumors through fibroblasts and cell-cell communication. Finally, we verified the function of CILP at the cellular level by using RCC cell lines ACHN and 786-O and found that knockdown of CILP could significantly inhibit migration and invasion. This finding supports that CILP could be a risk factor as well as a pan-cancer predictor for patients.

MiR-30c-2-3p suppresses the proliferation of human renal cell carcinoma cells by targeting TOP2A.

The ambiguity of renal cell carcinoma (RCC) symptoms hinders early diagnosis, thereby contributing to high mortality rates. By attaching to the 3'-untranslated region (UTR) of the target gene, microRNAs (miRNAs) exert significant control over the expression of genes. To investigate the influence of miR-30c-2-3p and DNA topoisomerase II alpha (TOP2A) on RCC growth and the mechanisms underlying the regulation of its expression. The expression of miRNA-30c-2-3p and TOP2A in RCC cells was examined using quantitative real-time polymerase chain reaction (qRT-PCR). MiR-30c-2-3p mimics, its inhibitors, and controls, as well as TOP2A short hairpin RNA (shRNA) and controls, were used to transfect the human RCC cell lines 786-O, Caki-1, and ACHN. Additionally, the roles of miRNA-30c-2-3p and TOP2A in the growth of RCC were evaluated using the cell counting kit (CCK)-8 test, colony formation assay, apoptosis analysis, and Western blotting. Meanwhile, binding of miRNA-30c-2-3p and TOP2A was verified using dual-luciferase reporter assays and Western blotting. miR-30c-2-p is underexpressed in RCC cells. Overexpression of miR-30c-2-p promotes apoptosis and inhibits proliferation of ACHN, Caki-1, and 786-O cells. miR-30c-2-3p targets TOP2A, which is elevated in RCC tissues and cells, whereas TOP2A silencing inhibits the proliferation ability of RCC cells. The miRNA-30c-2-3p inhibitor compromises TOP2A shRNA-induced apoptosis of RCC. RCC cells cotransfected with miRNA-30c-2-3p inhibitors and TOP2A shRNAs have a higher proliferation rate than those transfected with only TOP2A shRNAs. Collectively, our results verify that miRNA-30c-2-3p has a tumor suppressor property. miRNA-30c-2-3p inhibits the proliferation of RCC through regulation of TOP2A. The data provide a viable therapeutic target for RCC.

ALKBH1 contributes to renal cell carcinoma progression by reducing N6-methyladenine of GPR137.

Renal cell carcinoma (RCC) accounts for approximately 4% of all adult malignancies with high mortality worldwide. Although conventional chemotherapy and radiotherapy treatment has been applied for RCC in clinic, the mortality rate of patients is increasing each year, and patients with metastatic RCC are still suffering from poor prognosis. Thus, further investigation of the molecular mechanisms responsible for the development and progression of RCC is of particular importance. Total of 10 pairs of RCC tissues and adjacent nontumor tissues were collected for examination of ALKBH1 and GPR137 expression. The correlations between ALKBH1 and GPR137 expression in RCC patient were assessed by GEPIA online tool and were analyzed using auto best cutoff. The human RCC cell lines Caki-1, 786-O, ACHN, Osrc2, A498, and 769-P, were used for mechanistic investigation. Here, we report that the expression of AlkB homologue 1 (ALKBH1) is upregulated in RCC tissues, which is correlated with G-protein-coupled receptor 137 (GPR137) expression. The elevated expression of ALKBH1 is associated with RCC cell malignant characteristics, including cell proliferation and movement (migration and invasion). Mechanistic investigation further reveals that ALKBH1 reduces m6 A levels of GPR137 mRNA in RCC cells, which upregulates GPR137 mRNA levels, resulting in the increased GPR137 protein expression subsequently and the enhanced RCC cell biological actions consequently. In contrast, the suppression of GPR137 effectively alleviates the ALKBH1-induced malignancies of RCC cells. Our results indicate that ALKBH1-GPR137 axis might be used as a potential therapeutic target in RCC, contributing to finding new prognostic biomarkers for RCC at an early stage.

AKG induces cell apoptosis by inducing reactive oxygen species-mediated endoplasmic reticulum stress and by suppressing PI3K/AKT/mTOR-mediated autophagy in renal cell carcinoma.

Alpha-ketoglutarate (AKG) or 2-oxoglutarate is a key substance in the tricarboxylic acid cycle (TCA) and has been known to play an important role in cancerogenesis and tumor progression. Renal cell carcinoma (RCC) is the most common type of kidney cancer, and it has a high mortality rate. Autophagy is a phenomenon of self-digestion, and its significance in tumor genesis and progression remains debatable. However, the mechanisms underlying how AKG regulates autophagy in RCC remain unknown. Thus, the purpose of this study was to assess the therapeutic efficacy of AKG and its molecular mechanisms. RCC cell lines 786O and ACHN were treated with varying doses of AKG for 24 h. CCK-8, Transwell, and scratch wound healing assays were utilized to evaluate the role of AKG in RCC cells. Autophagy protein and PI3K/AKT/mTOR pathway protein levels were analyzed by Western blot. AKG inhibited the proliferation of RCC cells 786O and ACHN in a dose-dependent manner according to the CCK-8 assay. In addition, flow cytometry and Western blot analysis revealed that AKG dose-dependently triggered apoptosis and autophagy in RCC cells. By promoting cell apoptosis and autophagy, AKG dramatically suppressed tumor growth. Mechanistically, AKG induces autophagy by promoting ROS generation and inhibiting the PI3K/AKT/mTOR pathway. The anti-tumor effect of AKG promotes autophagy in renal cancer cells via mediating ROS-PI3K/Akt/mTOR, and may be used as a potential anticancer drug for kidney cancer.

SMOC2 promotes an epithelial-mesenchymal transition and a pro-metastatic phenotype in epithelial cells of renal cell carcinoma origin

Renal Cell Carcinoma (RCC) is the most common form of all renal cancer cases, and well-known for its highly aggressive metastatic behavior. SMOC2 is a recently described non-structural component of the extracellular matrix (ECM) that is highly expressed during tissue remodeling processes with emerging roles in cancers, yet its role in RCC remains elusive. Using gene expression profiles from patient samples, we identified SMOC2 as being significantly expressed in RCC tissue compared to normal renal tissue, which correlated with shorter RCC patient survival. Specifically, de novo protein synthesis of SMOC2 was shown to be much higher in the tubular epithelial cells of patients with biopsy-proven RCC. More importantly, we provide evidence of SMOC2 triggering kidney epithelial cells into an epithelial-to-mesenchymal transition (EMT), a phenotype known to promote metastasis. We found that SMOC2 induced mesenchymal-like morphology and activities in both RCC and non-RCC kidney epithelial cell lines. Mechanistically, treatment of RCC cell lines ACHN and 786-O with SMOC2 (recombinant and enforced expression) caused a significant increase in EMT-markers, -matrix production, -proliferation, and -migration, which were inhibited by targeting SMOC2 by siRNA. We further characterized SMOC2 activation of EMT to occur through the integrin β3, FAK and paxillin pathway. The proliferation and metastatic potential of SMOC2 overexpressing ACHN and 786-O cell lines were validated in vivo by their significantly higher tumor growth in kidneys and systemic dissemination into other organs when compared to their respective controls. In principle, understanding the impact that SMOC2 has on EMT may lead to more evidence-based treatments and biomarkers for RCC metastasis.

Dysregulation of the Sirt5/IDH2 axis contributes to sunitinib resistance in human renal cancer cells.

Sunitinib (Sun), a tyrosine kinase inhibitor of vascular endothelial growth factor receptor, is the standard first‐line treatment against advanced clear cell renal cell carcinoma (RCC), but resistance to therapy is inevitable. Reactive oxygen species production is associated with sensitivity to chemotherapy, but the underlying mechanisms are not completely understood. Here, we investigated the mechanisms contributing to Sun resistance using the RCC cell lines ACHN and 786‐O. We report that Sun‐resistant cells exhibited reduced apoptosis, increased cell viability, increased reactive oxygen species production and disrupted mitochondrial function. Furthermore, chronic Sun treatment resulted in an up‐regulation of Sirt5/isocitrate dehydrogenase 2 (IDH2) expression levels. Knockdown of Sirt5/IDH2 impaired mitochondrial function and partially attenuated Sun resistance. Finally, up‐regulation of Sirt5 enhanced the expression of IDH2 via modulation of succinylation at K413 and promoted protein stability. In conclusion, dysregulation of Sirt5/IDH2 partially contributes to Sun resistance in RCC cells by affecting antioxidant capacity.

Long Non-Coding RNA LOC648987 Promotes Proliferation and Metastasis of Renal Cell Carcinoma by Regulating Epithelial-Mesenchymal Transition.

Renal cell carcinoma (RCC) is a type of urinary tumor with a high incidence and is often associated with tumor metastasis. Long non-coding RNA (lncRNA) regulates tumorigenesis, progression, and metastasis. However, the role and the predictive value of lncRNA in RCC progression and metastasis have not been elucidated. The purpose of this study was to evaluate the effect of a newly discovered lncRNA LOC648987 on RCC proliferation and metastasis. LOC648987 was identified by RT-PCR for high expression in human RCC tissues as well as in metastatic RCC tissues. In the cell experiments, we infected the RCC cell lines ACHN and 786-O cells with LOC648987-shRNA and its negative control (shNC). The results showed that the knockdown of LOC648987 inhibited the proliferation of ACHN and 786-O cells and colony formation. The cell cycle and the apoptosis progression of ACHN and 786-O cells were assessed using flow cytometry. The knockdown of LOC648987 significantly inhibited the progression of ACHN and 786-O cells from G0/G1 to S phase and promoted cell apoptosis. The metastasis promoting effects of LOC648987 on ACHN and 786-O cells were verified by transwell migration assays, which depended on vimentin and MMP-9 to regulate the epithelial–mesenchymal transition. Finally, the promotion of LOC648987 on RCC tumorigenesis was evaluated in BALb/c nude mice. These data confirmed that lncRNA LOC648987 promoted RCC cell proliferation and tumor metastasis and regulated the expression of EMT-related proteins in RCC cells.

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.

Praeruptorin B Mitigates the Metastatic Ability of Human Renal Carcinoma Cells through Targeting CTSC and CTSV Expression.

Renal cell carcinoma (RCC) is the most common adult kidney cancer, and accounts for 85% of all cases of kidney cancers worldwide. Praeruptorin B (Pra-B) is a bioactive constituent of Peucedanum praeruptorum Dunn and exhibits several pharmacological activities, including potent antitumor effects. However, the anti-RCC effects of Pra-B and their underlying mechanisms are unclear; therefore, we explored the effects of Pra-B on RCC cells in this study. We found that Pra-B nonsignificantly influenced the cell viability of human RCC cell lines 786-O and ACHN at a dose of less than 30 μM for 24 h treatment. Further study revealed that Pra-B potently inhibited the migration and invasion of 786-O and ACHN cells, as well as downregulated the mRNA and protein expression of cathepsin C (CTSC) and cathepsin V (CTSV) of 786-O and ACHN cells. Mechanistically, Pra-B also reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), p-mitogen-activated protein kinase kinase (MEK), and p-extracellular signal-regulated kinases (ERK) in RCC cells. In addition, Pra-B treatment inhibited the effect of EGF on the upregulation of EGFR–MEK–ERK, CTSC and CTSV expression, cellular migration, and invasion of 786-O cells. Our findings are the first to demonstrate that Pra-B can reduce the migration and invasion ability of human RCC cells through suppressing the EGFR-MEK-ERK signaling pathway and subsequently downregulating CTSC and CTSV. This evidence suggests that Pra-B can be developed as an effective antimetastatic agent for the treatment of RCC.

LncRNA 00312 Attenuates Cell Proliferation and Invasion and Promotes Apoptosis in Renal Cell Carcinoma via miR-34a-5p/ASS1 Axis.

Background Previous studies have demonstrated that lncRNAs play functional roles in regulating cancer cell proliferation, invasion, and apoptosis. Recent studies confirmed that lncRNA 00312 has important biological functions in lung and colorectal cancer. However, the role of lncRNA 00312 in renal cell carcinoma (RCC) remains unclear. Our aim was to explore the function of lncRNA 00312 in RCC and its potential molecular mechanism. Methods RCC cell lines A498 and ACHN were used as in vitro models in this study. RT-PCR was performed to determine lncRNA 00312, miR-34a-5p, and ASS1 mRNA expression. Proliferation and invasion were examined by CCK-8 and Transwell assay to confirm the function role of lncRNA 00312. Western blot analysis was used to examine the expression of apoptotic proteins Bax and Bcl-2. Results lncRNA was significantly downregulated in RCC cells such as A498 and ACHN; the expression of lncRNA 00312 in RCC tissues was significantly lower than that in adjacent normal tissues. Patients with low expression of lncRNA 00312 have worse prognosis regarding pathological grade, tumor size, and TNM stage. Overexpression of lncRNA 00312 suppressed A498 and ACHN cell proliferation and invasion, while promoting apoptosis. Our study found that miR-34a-5p had the potential binding site with lncRNA 00312 and revealed the role of miR-34a-5p in RCC. Furthermore, we confirmed that lncRNA 00312 played its role with the participation of ASS1 and miR-34a-5p. Conclusion lncRNA 00312 can inhibit RCC proliferation and invasion and promote apoptosis in vitro by suppressing miR-34a-5p and overexpressing ASS1. Our study demonstrated that the lncRNA 00312/miR-34a-5p/ASS1 axis may play a functional role in the progression of RCC; lncRNA 00312 abundance is a prognostic factor candidate for RCC survival, which provides new insights for RCC clinical treatment.in vitro models in this study. RT-PCR was performed to determine lncRNA 00312, miR-34a-5p, and ASS1 mRNA expression. Proliferation and invasion were examined by CCK-8 and Transwell assay to confirm the function role of lncRNA 00312. Western blot analysis was used to examine the expression of apoptotic proteins Bax and Bcl-2.

Coupling the near-infrared fluorescent dye IR-780 with cabazitaxel makes renal cell carcinoma chemotherapy possible

Renal cell carcinoma (RCC) has always been considered resistant to chemotherapy. IR-780 is a near-infrared fluorescent (NIRF) dye that can be efficiently taken up by RCC cells. Cabazitaxel is a cytotoxic drug that interferes with mitosis by acting on tubulin. We chemically fused IR-780 and cabazitaxel into a new drug, Caba-780, which is expected to increase the sensitivity of RCC to chemotherapy. Infrared spectrum, nuclear magnetic resonance spectra, high-resolution mass spectra, and IR spectra were used for detecting structural characterization of the new synthetic drug Caba-780. The RCC cells lines ACHN and 786-O, as well as the non-cancerous human embryonic kidney cell line HEK293, were used to assess the cytotoxicity and tumor-efficient uptake of Caba-780 in vitro. The xenograft tumor-bearing mice and C57 mice were used to estimate the tumor-efficient imaging of Caba-780 as well as the safety and efficacy of its anti-tumor effects in vivo. The new synthetic drug Caba-780 retains the NIRF properties of IR-780. In vitro, Caba-780 was efficiently absorbed by the RCC cell lines ACHN and 786-O, and had an inhibitory effect on their growth, clonogenicity migration, and invasion. At the same time, Caba-780 retained the anti-tumor effect of cabazitaxel, which can inhibit the growth of tumor cells and promote apoptosis by inhibiting mitosis. In vivo experiments showed that Caba-780 can be taken up and imaged in tumor tissue, whereby it inhibits tumor growth. The novel fused molecule Caba-780 has application prospects in the diagnosis and treatment of RCC and makes RCC chemotherapy possible.

Timosaponin AIII inhibits metastasis of renal carcinoma cells through suppressing cathepsin C expression by AKT/miR-129-5p axis.

Timosaponin AIII (TSAIII) is a steroidal saponin that exerts anticancer activity on various cancer cells. In this study, we explore the effects of TSAIII on renal cell carcinoma (RCC) cells. Our findings show that TSAIII treatment (<8 μM) insignificantly influenced cell viability and cell cycle distribution of human RCC cell lines 786-O, A-498, and ACHN. Further observations revealed that TSAIII inhibited migration and invasion of 786-O and A-498 cells, as well as significantly decreased the production and expression of cathepsin C (CTSC) in both the cell types. Kinase cascade analysis exhibited that PI3K/AKT activation was inhibited, but PTEN expression was increased, in response to TSAIII treatments. Combining TSAIII and PI3K inhibitors, LY294002 synergically reduced the migration and invasion of 786-O and A-498 cells, as well as decreased the CTSC expression in both the cell types. We also observed that miR-129-5p bound to CTSC gene and suppressed the expression of CTSC and demonstrated that the miR-129-5p expression was synergically enhanced by TSAIII and LY294002. In addition, pretreatment with antago-miR-129-5p significantly restored the CTSC expression and the migration and invasion of TSAIII-treated 786-O cells. In conclusion, our findings reveal that TSAIII inhibits the metastatic properties of RCC cells, contributing to the inhibition of PI3K/AKT and the increase of miR-129-5p and the subsequent downregulation of CTSC. This suggests that TSAIII has significant antimetastatic activity against RCC cells and may be beneficial to RCC treatments.

Long noncoding RNA ATB participates in the development of renal cell carcinoma by downregulating p53 via binding to DNMT1.

Long noncoding RNA (lncRNA) exerts an essential role in the pathological processes of many diseases. Our previous study found that lncRNA ATB was highly expressed in renal cell carcinoma (RCC). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and migration-related assays were conducted to access the regulatory effects of lncRNA ATB on proliferative and migratory capacities of RCC cells. Flow cytometry was carried out to determine cell cycle and apoptosis influenced by lncRNA ATB. The interaction among lncRNA ATB, DNMT1, and p53 was evaluated through RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and western blot analyses. The results showed that lncRNA ATB knockdown in RCC cell line ACHN inhibited proliferative and migratory capacities and promoted apoptosis. Meanwhile, overexpression of lncRNA ATB in RCC cell line A-498 promoted proliferative and migratory capacities but inhibited apoptosis. RIP and ChIP assays confirmed that lncRNA ATB can bind to DNMT1 and stabilize its expression; meanwhile, it can promote the binding of DNMT1 to p53. Overexpression of p53 partially reversed the proliferative and migratory changes caused by lncRNA ATB. To sum up, our study revealed that high expression of lncRNA ATB could accelerate the proliferative and migratory rates of RCC cells and inhibit cell apoptosis through downregulating p53 via binding to DNMT1.

Salinomycin suppresses cancer cell stemness and attenuates TGF-β-induced epithelial-mesenchymal transition of renal cell carcinoma cells

Metastatic Renal cell carcinoma (RCC) remains a difficult oncologic challenge. Salinomycin is a monocarboxylic polyether antibiotic, which has been proved to possess anti-tumor activities in multiple types of cancer cells. However, its effects on RCC cells remains unclear. In our study, salinomycin could inhibit the proliferation and viability of RCC cell lines 786-O and ACHN. The TUNEL assay revealed that treatment with salinomycin induced DNA breaking in RCC cells. Consistently, Western blotting showed up-regulation of pro-apoptotic biomarkers (cleaved caspase3/9 and cleaved PARP1) and down-regulation of anti-apoptotic biomarker (survivin) in RCC cells after salinomycin treatment, suggesting that salinomycin could induce RCC cell apoptosis. salinomycin treatment also suppressed the sphere formation ability of RCC cells and decreased the expressions of CD105, ALDH1 and CD44, biomarkers for reflecting the stemness of RCC cells. salinomycin treatment effectively down-regulated SMO and Gli1, two key proteins in Hedghog signaling pathway, in a dose-dependent manner. Moreover, salinomycin could suppress the invasion and migration of RCC cells in the presence of TGF-β1, as observed in wound-healing and Transwell assays. salinomycin treatment attenuated TGF-β1-induced epithelial-mesenchymal transition (EMT), as evidenced by its ability to increase E-cadherin expression and decrease N-cadherin, Snail and MMP-2 expressions in RCC cells. Finally, salinomycin inhibited the tumorigenecity of RCC cells in vivo. Our study provides the evidence that salinomycin possess multiple anti-tumor activities against RCC, as it, in particular, suppressed the cancer stem cell properties and attenuated TGF-β-induced EMT. Therefore, it may serve as a potentially therapeutic candidate for metastatic RCC and improve the prognosis of RCC patients.

VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

The majority of molecular targets of anticancer agents are limited to a subset of patients, and therefore identification of more specific biomarkers that can be used to improve clinical outcomes is of increasing interest. The present study showed that von Hippel‑Lindau tumor suppressor (VHL) tumor‑suppressor activity may influence the therapeutic response to Aurora kinase A (AURKA) inhibitors in human renal cell carcinoma (RCC). VHL protein (pVHL) expression was evaluated by immunoblotting in the human RCC cell lines CAKI, ACHN, 786‑O, 769‑P and A498. The anti‑tumor activities of alisertib, an AURKA‑specific chemical inhibitor, were detected by Cell Counting Kit‑8 assay invitro and mouse xenograft model invivo. Additionally, the VHL‑mediated anti‑tumor activity was assessed in 769‑P and CAKI cells via the loss or gain of VHL. The results revealed that VHL‑deficient 786‑O, 769‑P and A498 cells were sensitive to alisertib. By contrast, alisertib‑resistant CAKI and ACHN cells expressed the wild type VHL gene. In addition, rescue or knockdown of VHL was observed to increase or decrease alisertib anti‑proliferation activity, respectively, in RCC cells. The inverse correlation between the VHL gene expression profile and alisertib sensitivity was further confirmed in human cancer xenografts models. Taken together, these results suggested that VHL loss could potentially serve as a biomarker for predicting the efficacy of AURKA inhibitors.

The Mediator complex subunit MED15, a promoter of tumour progression and metastatic spread in renal cell carcinoma.

MED15 is a part of the multiprotein Mediator complex which is involved in the transcription of polymerase (Pol) II-dependent genes. Several studies in this field have reported altered expressions of distinct subunits in human malignancy. However, the role of MED15 in renal cell carcinoma (RCC) has not be investigated yet. First, we performed an RNA expression and survival analysis of MED15 in RCC by using the database cBioPortal. To confirm these data on the protein level, we executed immunohistochemical (IHC) staining against MED15 on a tissue microarray containing 184 samples of the most common subtypes of the tumour at the various stages. Further, we performed functional analysis including proliferation, migration, and invasion assays on the RCC cell lines A-498 and ACHN following the siRNA-mediated MED15 knockdown. On the mRNA level, higher expression of MED15 was associated with worse patient survival rates. IHC staining validated this tendency, unfortunately the results were not significant. However, supporting this tendency, in vitro-assays showed a significant decrease in proliferation, migration, and invasion after knockdown of MED15. The research concludes that MED15 does seem to play a tumour promoting role in the progression and metastatic spread of renal cell carcinoma.

Down-regulation of transient receptor potential melastatin member 7 prevents migration and invasion of renal cell carcinoma cells via inactivation of the Src and Akt pathway.

PurposeTransient receptor potential melastatin member 7 (TRPM7), an ion channel and serine/threonine protein kinase, has been linked with distinct human malignancies. However, the role of TRPM7 in renal cell carcinoma (RCC) has not been investigated. The aim of this study is to determine whether TRPM7 regulates the migration and invasion of RCC cells. Its relationship with signal transduction pathways was also studied.Materials and MethodsThe human RCC cell lines ACHN and SN12C were chosen for this study. The molecular mechanisms of TRPM7 action were studied using Western blot analysis and small interfering RNA (siRNA)-based knockdown. The effect of TRPM7 knockdown on RCC cells was measured by using Transwell invasion and wound healing migration assays.ResultssiRNA-induced silencing of TRPM7 notably decreased the migration and invasion of ACHN and SN12C RCC cells. The phosphorylation levels of Src in both cells were obviously reduced after TRPM7 silencing compared with that of the control ACHN and SN12C cells. Furthermore, the phosphorylation levels of Akt were greatly decreased in ACHN cells after siRNA-induced knockdown of TRPM7. Additionally, the treatment of cells with Src and Akt inhibitors clearly limited the migration and invasion of RCC cells.ConclusionsOur data show that TRPM7 regulated ACHN and SN12C RCC cell invasion via the Src/Akt signaling pathway. Therefore, targeting the Src/Akt signaling pathway and/or the expression or function of TRPM7 could be a potential beneficial treatment for patients with RCC.

CMTM5 inhibits renal cancer cell growth through inducing cell-cycle arrest and apoptosis.

CKLF-like MARVEL transmembrane domain-containing 5 (CMTM5) has been reported to function as a potential tumor suppressor in several human cancers. However, the involvement of CMTM5 in human renal cell carcinoma (RCC) remains unclear. The current study aimed to detect its expression pattern in RCC tissues and cells, and to determine its anti-proliferative functions in this malignancy. The mRNA and protein expression levels of CMTM5 in RCC tissues and cells were detected by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. Following the transfection with CMTM5 lentivirus or control lenti-EGFP lentivirus into the RCC cell line ACHN, the viability, migration, apoptosis and cell cycle of these cells were detected by Cell Counting kit-8 assay, Transwell assay and flow cytometry, respectively. Compared with the adjacent non-malignant kidney tissue samples, CMTM5 expression was significantly downregulated in RCC tissues (P<0.05). In addition, enforced expression of CMTM5 could efficiently inhibit the cell growth of ACHN cells, which were arrested in G0/G1 phase. Furthermore, the migration and invasion of ACHN cells were also inhibited by restoration of CMTM5 expression. The present data suggest that CMTM5 may function as a tumor suppressor in human RCC by suppressing the viability of RCC cells, implying its potential as a therapeutic target for this malignancy.