VHL-deficiency leads to reductive stress in renal cells.

Heritable renal cancer syndromes (RCS) are associated with numerous chromosomal alterations including inactivating mutations in von Hippel-Lindau (VHL) gene. Here we identify a novel aspect of the phenotype in VHL-deficient human renal cells. We call it reductive stress as it is characterised by increased NADH/NAD+ ratio that is associated with impaired cellular respiration, impaired CAC activity, upregulation of reductive carboxylation of glutamine and accumulation of lipid droplets in VHL-deficient cells. Reductive stress was mitigated by glucose depletion and supplementation with pyruvate or resazurin, a redox-reactive agent. This study demonstrates for the first time that reductive stress is a part of the phenotype associated with VHL-deficiency in renal cells and indicates that the reversal of reductive stress can augment respiratory activity and CAC activity, suggesting a strategy for altering the metabolic profile of VHL-deficient tumours.

Molecular Targets of Shenqi Dihuang, A Traditional Chinese Herbal Medicine, and Its Potential Role in Renal Cell Carcinoma Therapy.

Chinese herbal medicine (CHM), which includes herbal slices and proprietary products, is widely used in China. Shenqi Dihuang (SQDH) is a traditional Chinese medicine (TCM) formula with ingredients that affect tumor growth. Despite recent advances in prognosis, patients with renal cell carcinoma (RCC) cannot currently receive curative treatment. The present study aimed to explore the potential target genes closely associated with SQDH. The gene expression data for SQDH and RCC were obtained from the TCMSP and TCGA databases. The SQDH-based prognostic prediction model reveals a strong correlation between RCC and SQDH. In addition, the immune cell infiltration analysis indicated that SQDH might be associated with the immune response of RCC patients. Based on this, we successfully built the prognostic prediction model using SQDH-related genes. The results demonstrated that CCND1 and NR3C2 are closely associated with the prognosis of RCC patients. Finally, the pathways enrichment analysis revealed that response to oxidative stress, cyclin binding, programmed cell death, and immune response are the most enriched pathways in CCND1. Furthermore, transcription regulator activity, regulation of cell population proliferation, and cyclin binding are closely associated with the NR3C2.

Curcumin Inhibits Proliferation of Renal Cell Carcinoma in vitro and in vivo by Regulating miR-148/ADAMTS18 through Suppressing Autophagy.

OBJECTIVE: To explore the effect of curcumin on the proliferation of renal cell carcinoma and analyze its regulation mechanism. METHODS: In RCC cell lines of A498 and 786-O, the effects of curcumin (2.5, 5, 10 µ mo/L) on the proliferation were analyzed by Annexin V+PI staining. Besides, A498 was inoculated into nude mice to establish tumorigenic models, and the model mice were treated with different concentrations of curcumin (100, 200, and 400 mg/kg), once daily for 30 days. Then the tumor diameter was measured, the tumor cells were observed by hematoxylin-eosin staining, and the protein expressions of miR-148 and ADAMTS18 were detected by immunohistochemistry. In vitro, after transfection of miR-148 mimics, miR-148 inhibitor or si-ADAMTS18 in cell lines, the expression of ADAMTS18 was examined by Western blotting and the cell survival rate was analyzed using MTT. Subsequently, Western blot analysis was again used to examine the autophagy phenomenon by measuring the relative expression level of LC3-II/LC3-I; autophagy-associated genes, including those of Beclin-1 and ATG5, were also examined when miR-148 was silenced in both cell lines with curcumin treatment. RESULTS: Curcumin could inhibit the proliferation of RCC in cell lines and nude mice. The expression of miR-148 and ADAMTS18 was upregulated after curcumin treatment both in vitro and in vivo (P<0.05). The cell survival rate was dramatically declined upon miR-148 or ADAMTS18 upregulated. However, si-ADAMTS18 treatment or miR-148 inhibitor reversed these results, that is, both of them promoted the cell survival rate. CONCLUSION: Curcumin can inhibit the proliferation of renal cell carcinoma by regulating the miR-148/ ADAMTS18 axis through the suppression of autophagy in vitro and in vivo. There may exist a positive feedback loop between miR-148 and ADAMTS18 gene in RCC.

MicroRNA-143-3p Inhibits Wilms' Tumor Cell Growth By Targeting the Ras/Raf/MEK/ERK Pathway.

Background: A previous study found that microRNA-143-3p (miR-143-3p) is a tumor suppressor in various types of human cancer. However, the roles and molecular mechanisms of miR-143-3p in the progression of Wilms' tumor (WT) remain to be clarified. The aim of the present study was to determine the expression and biological functions of miR-143-3p in WT. Material and Methods: The expression levels of miR-143-3p in primary WT tissues and adjacent tissues were determined using quantitative-reverse transcription polymerase chain reaction (qRT-PCR), and the association of the miR-143-3p expression level with various clinicopathological features of WT was investigated. Western blotting was used to evaluate the protein expression of the related signaling pathway. Results: The expression of miR-143-3p was significantly downregulated in WT tissues and its expression levels were closely associated with tumor stage and lymph node metastasis. Overexpression of miR-143-3p in SK-NEP-1 and G401 cell lines inhibited cell proliferation by G0/G1 cell cycle phase arrest and induction of apoptosis. Moreover, k-Ras, a unique oncogene, was confirmed as a direct target of miR-143-3p, and k-Ras messenger RNA (mRNA) expression was increased in WT tissues and inversely correlated with miR-143-3p. Knockdown of k-Ras by si-k-Ras could inhibit, whereas overexpression of k-Ras could promote. cell proliferation in WT cells. Meanwhile, overexpression of k-Ras reversed the inhibitory effects on WT cells induced by miR-143-3p mimics. Conclusion: Our findings indicate that miR-143-3p may be a potential novel prognostic biomarker and therapeutic target for WT.

Pueraria montana (Kudzu vine) Ameliorate the Inflammation and Oxidative Stress against Fe-NTA Induced Renal Cancer.

Renal tissue plays a crucial function in maintaining homeostasis, making it vulnerable to xenobiotic toxicity. Pueraria montana has more beneficial potential against the various diseases and has long history used as a traditional Chinese medicine. But its effect against the renal cancer not scrutinize. The goal of this study is to see if Pueraria montana can protect rats from developing kidney tumors caused by diethylnitrosamine (DEN) and ferric nitrite (Fe-NTA). Wistar rats was selected for the current study and DEN (use as an inducer) and Fe-NTA (promoter) for induction the renal cancer. For 22 weeks, the rats were given orally Pueraria montana (12.5, 25, and 50 mg/kg) treatment. At regular intervals, the body weight and food intake were calculated. The rats were macroscopically evaluated for identification of cancer in the renal tissue. The renal tumor makers, renal parameters, antioxidant enzymes, phase I and II enzymes, inflammatory cytokines and mediators were estimated at end of the experimental study. Pueraria montana treated rats displayed the suppression of renal tumors, incidence of the tumors along with suppression of tumor percentage. Pueraria montana treated rats significantly (p < 0.001) increased body weight and suppressed the renal weight and food intake. It also reduced the level of renal tumor marker ornithine decarboxylase (ODC) and [3H] thymidine incorporation along with suppression of renal parameter such as uric acid, blood urea nitrogen (BUN), urea and creatinine. Pueraria montana treatment significantly (p < 0.001) altered the level of phase enzymes and antioxidant. Pueraria montana treatment significantly (p < 0.001) repressed the level of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and improved the level of interleukin-10 (IL-10). Pueraria montana treatment suppressed the level of prostaglandin (PGE2), cyclooxygenase-2 (COX-2), nuclear kappa B factor (NF-κB) and transforming growth factor beta 1 (TGF-ß1). Pueraria montana suppressed the inflammatory necrosis, size the bowman capsules in the renal histopathology. Pueraria montana exhibited the chemoprotective effect via dual mechanism such as suppression of inflammatory reaction and oxidative stress.

YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) such as sunitinib are multitarget antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). They are widely used in the treatment of advanced/metastatic renal cancer. However, resistance to TKIs is common in the clinic, particularly after long-term treatment. YTHDC1 is the main nuclear reader protein that binds with m6A to regulate the splicing, export and stability of mRNA. However, the specific role and corresponding mechanism of YTHDC1 in renal cancer cells are still unclear. METHODS: The Cancer Genome Atlas (TCGA) dataset was used to study the expression of YTHDC1 in ccRCC. Cell counting kit-8 (CCK-8), wound healing, Transwell and xenograft assays were applied to explore the biological function of YTHDC1 in ccRCC. Western blot, quantitative real time PCR (RT‒qPCR), RNA immunoprecipitation PCR (RIP-qPCR), methylated RIP-qPCR (MeRIP-qPCR) and RNA sequencing (RNA-seq) analyses were applied to study the YY1/HDAC2/YTHDC1/ANXA1 axis in renal cancer cells. The CCK-8 assay and xenograft assay were used to study the role of YTHDC1 in determining the sensitivity of ccRCC to sunitinib. RESULTS: Our results demonstrated that YTHDC1 is downregulated in ccRCC tissues compared with normal tissues. Low expression of YTHDC1 is associated with a poor prognosis in patients with ccRCC. Subsequently, we showed that YTHDC1 inhibits the progression of renal cancer cells via downregulation of the ANXA1/MAPK pathways. Moreover, we also showed that the YTHDC1/ANXA1 axis modulates the sensitivity of tyrosine kinase inhibitors. We then revealed that HDAC2 inhibitors resensitize ccRCC to tyrosine kinase inhibitors through the YY1/HDAC2 complex. We have identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC. CONCLUSION: We identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC.

Icariside II induces ferroptosis in renal cell carcinoma cells by regulating the miR-324-3p/GPX4 axis.

Icariside II (ICS II) is an active flavonoid having anti-tumor properties. However, the role of ICS II in renal cell carcinoma (RCC) and its underlying mechanisms have not been investigated to date. In this study, we demonstrated that ICS II inhibited proliferation, migration, and invasion of RCC cells. Furthermore, ferroptosis, a novel form of cell death, induced in RCC cells by ICS II, accompanied by accumulation of Fe2+, MDA (lipid peroxidation), and ROS (reactive oxygen species), and reduced GSH levels. The underlying mechanism was found to be the downregulation of GPX4, independent of p53, that occurs during ICS II-induced ferroptosis. Overexpression of GPX4 reversed the ferroptosis induced by ICS II. Moreover, ICS II treatment resulted in the upregulation of miR-324-3p, which directly targets GPX4. Overall, our results suggested that ICS II-induced ferroptosis via the miR-324-3p/GPX4 axis in RCC cells could be a promising therapeutic agent for RCC.

Asiatic acid from centella asiatica exert anti-invasive ability in human renal cancer cells by modulation of ERK/p38MAPK-mediated MMP15 expression.

BACKGROUND: Asiatic acid (AA) is a naturally pentacyclic triterpenoids extracted from traditional medicine Centella asiatica l. that has demonstrated possesses potential health benefits and antitumor ability. However, the precise anticancer effects and mechanisms by which AA impact RCC cells remains unclear. METHODS: Cell proliferation and cell cycle distribution were detected by MTT, colony formation assay and PI stain by flow cytometry, respectively. Cell mobility and invasiveness were determined by in vitro migration and invasion assay. The secretory MMP15 was detected by ELISA assay. Quantitative RT-PCR, siRNA, and immunoblot were used to determine gene expression/regulation and protein expression, respectively. Antimetastatic effect of AA were performed to lung nodule numbers in vivo metastasis mice model. MMP15, pERK1/2 and p-p38MAPK expressions were determined by immunohistochemistry. RESULTS: Our findings indicated cell proliferation and cell cycle distribution of RCC cells were not significantly influenced by AA treatment. AA suppressed cell migration, invasion and significantly down-regulated mRNA and protein expression of MMP-15 (Matrix Metallopeptidase-15). Activation of ERK1/2 and p38MAPK were inhibited with AA, whereas combined AA with siRNA-ERK or siRNA-p38MAPK markedly reduced the metastatic effect and decreased MMP-15 expression in 786-O and A498 cells. Finally, AA significantly reduced the lung metastasis formation and metastasis-related proteins of human 786-O cells in vivo metastasis mice model. CONCLUSION: AA inhibits the metastatic properties of RCC cells via inhibition of the p-ERK/p-p38MAPK axis and the subsequent down-regulation of MMP-15 in vitro and in vivo. Further study of AA as a potential anti-metastatic agent for RCC is warranted.

LINC02532 Contributes to Radiosensitivity in Clear Cell Renal Cell Carcinoma through the miR-654-5p/YY1 Axis.

BACKGROUND: Studies have shown that long non-coding RNAs (lncRNAs) play essential roles in tumor progression and can affect the response to radiotherapy, including in clear cell renal cell carcinoma (ccRCC). LINC02532 has been found to be upregulated in ccRCC. However, not much is known about this lncRNA. Hence, this study aimed to investigate the role of LINC02532 in ccRCC, especially in terms of radioresistance. METHODS: Quantitative real-time PCR was used to detect the expression of LINC02532, miR-654-5p, and YY1 in ccRCC cells. Protein levels of YY1, cleaved PARP, and cleaved-Caspase-3 were detected by Western blotting. Cell survival fractions, viability, and apoptosis were determined by clonogenic survival assays, CCK-8 assays, and flow cytometry, respectively. The interplay among LINC02532, miR-654-5p, and YY1 was detected by chromatin immunoprecipitation and dual-luciferase reporter assays. In addition, in vivo xenograft models were established to investigate the effect of LINC02532 on ccRCC radioresistance in 10 nude mice. RESULTS: LINC02532 was highly expressed in ccRCC cells and was upregulated in the cells after irradiation. Moreover, LINC02532 knockdown enhanced cell radiosensitivity both in vitro and in vivo. Furthermore, YY1 activated LINC02532 in ccRCC cells, and LINC02532 acted as a competing endogenous RNA that sponged miR-654-5p to regulate YY1 expression. Rescue experiments indicated that miR-654-5p overexpression or YY1 inhibition recovered ccRCC cell functions that had been previously impaired by LINC02532 overexpression. CONCLUSIONS: Our results revealed a positive feedback loop of LINC02532/miR-654-5p/YY1 in regulating the radiosensitivity of ccRCC, suggesting that LINC02532 might be a potential target for ccRCC radiotherapy. This study could serve as a foundation for further research on the role of LINC02532 in ccRCC and other cancers.

Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

BACKGROUND/AIMS: Maintenance of whole-body ascorbate levels and distribution is mediated via sodium-dependent vitamin C transporters (SVCTs). The kidney is one of a few organs that express both SVCT1 and SVCT2. Recent evidence suggests that accumulation of ascorbate may be different in tumour compared to normal tissue, but data on SVCT levels in tumours is sparse. METHODS: The role of the two SVCT isoforms in ascorbate uptake in renal cell carcinoma (RCC) was investigated in vitro and in clinical samples. In three human RCC cell lines, we investigated SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal. In clinical RCC samples (n=114), SVCT patterns of staining and protein levels were analysed and compared to ascorbate levels. RESULTS: In cell culture, transporter levels and cellular location were not modified by ascorbate availability at any time up to 8h, although basal SVCT2 levels governed maximal ascorbate accumulation. In clinical samples, SVCT1 protein levels in papillary RCC (pRCC) were similar to matched normal renal cortex, but were increased in clear-cell RCC (ccRCC). Native SVCT2 (72 kDa) was significantly decreased in both pRCC and ccRCC tissues compared to cortex (p<0.01), whereas a modified form of SVCT2 (100 kDa) was significantly increased (p<0.001). There was no association between the transporters (SVCT1, native or modified SVCT2) and ascorbate concentrations in either normal or tumour tissues. SVCT1 and SVCT2 displayed diffuse cytoplasmic staining in both pRCC and ccRCC tumour cells, with cortex showing distinct membrane staining for SVCT1. CONCLUSION: We observed a re-distribution of ascorbate transporters in tumour tissue compared to normal cortex and a shift from native to modified SVCT2 in cell culture and clinical samples. Data presented here show that SVCT protein levels do not appear to predict intracellular ascorbate accumulation in RCC.

Tackling of Renal Carcinogenesis in Wistar Rats by Silybum marianum Total Extract, Silymarin, and Silibinin via Modulation of Oxidative Stress, Apoptosis, Nrf2, PPARγ, NF-κB, and PI3K/Akt Signaling Pathways.

The present work was designed to assess the efficacy of Silybum marianum total extract (STE), silymarin (Sm), and silibinin (Sb) against experimentally induced renal carcinogenesis in male Wistar rats and their roles in regulating oxidative stress, inflammation, apoptosis, and carcinogenesis. The diethylnitrosamine (DEN)/2-acetylaminofluorene (AAF)/carbon tetrachloride (CCl4)-administered rats were orally treated with STE (200 mg/kg b.w.), Sm (150 mg/kg b.w.), and Sb (5 mg/kg b.w.) every other day either from the 1st week or from the 16th week of carcinogen administration to the end of 25th week. The treatments with STE, Sm, and Sb attenuated markers of toxicity in serum, decreased kidney lipid peroxidation (LPO), and significantly reinforced the renal antioxidant armory. The biochemical results were further confirmed by the histopathological alterations. The treatments also led to suppression of proinflammatory mediators such as NF-κß, p65, Iκßα, and IL-6 in association with inhibition of the PI3K/Akt pathway. Furthermore, they activated the expressions of PPARs, Nrf2, and IL-4 in addition to downregulation of apoptotic proteins p53 and caspase-3 and upregulation of antiapoptotic mediator Bcl-2. The obtained data supply potent proof for the efficacy of STE, Sm, and Sb to counteract renal carcinogenesis via alteration of varied molecular pathways.

Cyclovirobuxine inhibits the progression of clear cell renal cell carcinoma by suppressing the IGFBP3-AKT/STAT3/MAPK-Snail signalling pathway.

Of all pathological types of renal cell cancer (RCC), clear cell renal cell carcinoma (ccRCC) has the highest incidence. Cyclovirobuxine (CVB), a triterpenoid alkaloid isolated from Buxus microphylla, exhibits antitumour activity against gastric cancer and breast cancer; however, the mechanism by which CVB inhibits ccRCC remains unclear. The aim of our study was to explore the antitumour effects of CVB on ccRCC and to elucidate its exact mechanism. Cell viability, proliferation, cell cycle distribution, apoptosis, wound healing and invasion were evaluated. Furthermore, Western blotting, immunofluorescence staining, immunohistochemical staining, and bioinformatics analyses were utilized to comprehensively probe the molecular mechanisms. The in vivo curative effect of CVB was explored using a 786-O xenograft model established in nude mice. CVB reduced cell viability, proliferation, angiogenesis, the epithelial-mesenchymal transition (EMT), migration and invasion. In addition, CVB induced cell cycle arrest in S phase and promoted apoptosis. The expression of the EMT-related transcription factor Snail was significantly downregulated by CVB via the inhibition of the AKT, STAT3 and MAPK pathways. We revealed that insulin-like growth factor binding protein 3 (IGFBP3) was the true therapeutic target of CVB. CVB exerted anti-ccRCC effects by blocking the IGFBP3-AKT/STAT3/MAPK-Snail pathway. Targeted inhibition of IGFBP3 with CVB treatment may become a promising therapeutic regimen for ccRCC.

TF-RBP-AS Triplet Analysis Reveals the Mechanisms of Aberrant Alternative Splicing Events in Kidney Cancer: Implications for Their Possible Clinical Use as Prognostic and Therapeutic Biomarkers.

Aberrant alternative splicing (AS) is increasingly linked to cancer; however, how AS contributes to cancer development still remains largely unknown. AS events (ASEs) are largely regulated by RNA-binding proteins (RBPs) whose ability can be modulated by a variety of genetic and epigenetic mechanisms. In this study, we used a computational framework to investigate the roles of transcription factors (TFs) on regulating RBP-AS interactions. A total of 6519 TF-RBP-AS triplets were identified, including 290 TFs, 175 RBPs, and 16 ASEs from TCGA-KIRC RNA sequencing data. TF function categories were defined according to correlation changes between RBP expression and their targeted ASEs. The results suggested that most TFs affected multiple targets, and six different classes of TF-mediated transcriptional dysregulations were identified. Then, regulatory networks were constructed for TF-RBP-AS triplets. Further pathway-enrichment analysis showed that these TFs and RBPs involved in triplets were enriched in a variety of pathways that were associated with cancer development and progression. Survival analysis showed that some triplets were highly associated with survival rates. These findings demonstrated that the integration of TFs into alternative splicing regulatory networks can help us in understanding the roles of alternative splicing in cancer.

Curcumin induces apoptosis and autophagy inhuman renal cell carcinoma cells via Akt/mTOR suppression.

Renal cell carcinoma (RCC) is a highly aggressive cancer leading to high economic and social burden, and has increasing annual cases. Curcumin is a traditional Chinese medicine widely used as anti-inflammatory, anti-viral and anti-cancer agent, thus can be applicable in RCC therapy. The work assessed the effects of RCC treatment with Curcumin, Curcumin+3-MA, Curcumin+ CQ or curcumin+ Z-VAD in vitro and in vivo, and the mechanisms involved in inhibition of tumor cells proliferation. The study used ACHN tumor cells and C57BL/6 nude mice for results validation. Cell proliferation was determined through MTT assays while apoptosis was investigated using Annexin V-FITC/PI kit and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect IL-6, IL-8, and TNF-α cytokines expressions. AKT/mTOR and autophagy proteins expressions were investigated through western blot and immunofluorescence. The results indicated significantly inhibited cell viability following ACHN tumor cells treatments with curcumin alone, or with the various combinations, as compared to the control. Apoptosis was significantly increased following curcumin treatment, but was significantly reversed after treatment with curcumin+ 3-MA. Likewise, AKT/mTOR proteins expression were significantly reduced while the autophagy-related proteins were significantly elevated following curcumin treatment. The tumor size, weight and volumes were also significantly suppressed following treatment with curcumin. In conclusion, the investigation demonstrated that curcumin suppressed ACHN cell viability, induced apoptosis and autophagy, through the suppression of AKT/mTOR pathway. Use of curcumin to target AKT/mTOR pathway could be an effective treatment alternative for renal cell carcinoma.

Garlic (Allium sativum)-derived SEVs inhibit cancer cell proliferation and induce caspase mediated apoptosis.

As a key component of the cell-to-cell communication, small extracellular vesicles (SEVs) released from various sources are known to be affecting the physiological conditions of the target cells. Although it has been suggested that edible plant-derived nanoparticles contributes to the cross kingdom communication with the mammalian cells, the effect of these particles on cancer cell progression still needs a further exploration. Here, we isolated and then characterized garlic derived SEVs by nanoparticle tracking analysis, electron microscopy and SEV surface antibodies. In order to investigate anti-cancer property of garlic SEVs A498 human kidney carcinoma, A549 human lung carcinoma were used as cell models along with the normal human dermal fibroblast cell lines. Annexin V/pI staining and analysis of apoptotic mRNA and protein expression levels suggested that garlic SEVs induced apoptosis through activation of intrinsic pathway. Furthermore, angiogenic VEGF protein expression levels significantly decreased in response to SEVs treatment in cancer cells. Our results support that garlic derived SEVs could cause apoptotic cell death among cancer cells while normal cells remain unaffected with the treatment. This study revealed for the first time that plant SEVs possess anti-cancer affects by inducing caspase mediated apoptosis and provided a new alternative for cancer treatment.

New frontiers against sorafenib resistance in renal cell carcinoma: From molecular mechanisms to predictive biomarkers.

Renal cell carcinoma (RCC) is a highly vascularized tumor and prone to distant metastasis. Sorafenib is the first targeted multikinase inhibitor and first-line chemical drug approved for RCC therapy. In fact, only a small number of RCC patients benefit significantly from sorafenib treatment, while the growing prevalence of sorafenib resistance has become a major obstacle for drug therapy effectivity of sorafenib. The molecular mechanisms of sorafenib resistance in RCC are not completely understood by now. Herein, we comprehensively summarize the underlying mechanisms of sorafenib resistance and molecular biomarkers for predicting sorafenib responsiveness. Moreover, we outline strategies suitable for overcoming sorafenib resistance and prospect potential approaches for identifying biomarkers associated with sorafenib resistance in RCC, which contributes to guide individualized and precision drug therapy.

Traditional Chinese medicine Bu-Shen-Jian-Pi-Fang attenuates glycolysis and immune escape in clear cell renal cell carcinoma: results based on network pharmacology.

Clear cell renal cell carcinoma (ccRCC) is the most common malignant type of kidney cancer. The present study aims to explore the underlying mechanism and potential targets of the traditional Chinese medicine Bu-Shen-Jian-Pi-Fang (BSJPF) in the treatment of ccRCC based on network pharmacology. After obtaining the complete composition information for BSJPF from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, we analyzed its chemical composition and molecular targets and then established a pharmacological interaction network. Twenty-four significantly differentially expressed genes and nine pathways mainly related to tumor proliferation were identified and screened. Functional enrichment analysis indicated that the potential targets might be significantly involved in glycolysis and the HIF-1 signaling pathway. To further confirm the effect of BSJPF on ccRCC cell proliferation, a BALB/c xenograft mouse model was constructed. Potential targets involved in regulating glycolysis and the tumor immune microenvironment were evaluated using RT-qPCR. VEGF-A expression levels were markedly decreased, and heparin binding-EGF expression was increased in the BSJPF group. BSJPF also inhibited tumor proliferation by enhancing GLUT1- and LDHA-related glycolysis and the expression of the immune checkpoint molecules PD-L1 and CTLA-4, thereby altering the immune-rejection status of the tumor microenvironment. In summary, the present study demonstrated that the mechanism of BSJPF involves multiple targets and signaling pathways related to tumorigenesis and glycolysis metabolism in ccRCC. Our research provides a novel theoretical basis for the treatment of tumors with traditional Chinese medicine and new strategies for immunotherapy in ccRCC patients.

Raloxifene, identified as a novel LSD1 inhibitor, suppresses the migration of renal cell carcinoma.

Aim: As an important epigenetic modulator, histone lysine-specific demethylase 1 (LSD1) has been proved to be associated with the progression of renal cell carcinoma (RCC). Discovering novel LSD1 inhibitors offers therapeutic potential for RCC treatment. Methods & Results: We identified raloxifene as a novel LSD1 inhibitor (IC50 = 2.08 µM) through small compound library screening. Molecular docking indicated raloxifene might bind LSD1 in the flavin adenine dinucleotide (FAD) binding cavity in a reversible manner. Cell viability and migration assays showed raloxifene could suppress the proliferation and migration of RCC cells bearing overexpressed LSD1. Conclusion: Our findings indicated that LSD1 might be a promising therapeutic target for RCC and that raloxifene could serve as a lead compound for further anti-RCC metastasis drug discovery.

Zinc transporter ZIP7 is a novel determinant of ferroptosis.

Ferroptosis is a newly described form of regulated cell death triggered by oxidative stresses and characterized by extensive lipid peroxidation and membrane damages. The name of ferroptosis indicates that the ferroptotic death process depends on iron, but not other metals, as one of its canonical features. Here, we reported that zinc is also essential for ferroptosis in breast and renal cancer cells. Zinc chelator suppressed ferroptosis, and zinc addition promoted ferroptosis, even during iron chelation. By interrogating zinc-related genes in a genome-wide RNAi screen of ferroptosis, we identified SLC39A7, encoding ZIP7 that controls zinc transport from endoplasmic reticulum (ER) to cytosol, as a novel genetic determinant of ferroptosis. Genetic and chemical inhibition of the ZIP7 protected cells against ferroptosis, and the ferroptosis protection upon ZIP7 knockdown can be abolished by zinc supplementation. We found that the genetic and chemical inhibition of ZIP7 triggered ER stresses, including the induction of the expression of HERPUD1 and ATF3. Importantly, the knockdown of HERPUD1 abolished the ferroptosis protection phenotypes of ZIP7 inhibition. Together, we have uncovered an unexpected role of ZIP7 in ferroptosis by maintaining ER homeostasis. These findings may have therapeutic implications for human diseases involving ferroptosis and zinc dysregulations.

Mechanism of gypenosides of Gynostemma pentaphyllum inducing apoptosis of renal cell carcinoma by PI3K/AKT/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynostemma pentaphyllum (Thunb.) Makino is a traditional medicine commonly used in China, East Asia and Southeast Asia. In clinic, it is mainly used for hyperlipidemia and antitumor. Its antitumor activity was first recorded in "Illustrated Catalogue of Plants". Gypenosides were the main active ingredients of G. pentaphyllum. The anticancer activity of gypenosides in vivo and in vitro had been widely reported. However, the mechanism of gypenosides in renal cell carcinoma (RCC) still unclear. AIM OF THE STUDY: In this study, we tried to investigate the active constituents from G. pentaphyllum and potential mechanisms in RCC treatment through network pharmacology and in vitro experiments. MATERIAL/METHODS: Active compounds and their targets were evaluated and screened through TCMSP and Swiss Target Prediction database. Notably, nine preliminary screened components obtained from database were identified by LC-MS and LC-MS/MS. The targets associated with RCC were obtained from OMIM, TTD and GeneCards database. The PPI network and active component/target/pathway networks were constructed to identify the potential drug targets using String database and Cytoscape software. The functions and pathways of targets were analyzed through DAVID database. Finally, AutoDockTools 1.5.6 was used for molecular docking to assess the binding ability between compounds and targets. To support our prediction, we then explore the antitumor effect and mechanism of gypenosides by vitro experiments. CCK8 and flow cytometry were performed to evaluate cell death treated with gypenosides. Quantitative real-time PCR and Western blot were conducted to detect the changes of PI3K/AKT/mTOR signaling pathway. RESULTS: Nine saponins and 68 targets have been screened. The hub targets covered PIK3CA, VEGFA, STAT3, JAK2, CCND1 and MAPK3. Enrichment analysis showed that the pathways mainly contained PI3K/Akt/mTOR, HIF-1, TNF, JAK-STAT and MAPK signaling pathways. Gypenosides extracted from G. pentaphyllum showed strong activity against 786-O and Caki-1 cells, and cell apoptosis were detected through Annexin V/PI dual staining assay. RT-qPCR showed that gypenosides downregulated the levels of PIK3CA, Akt and mTOR in Caki-1 and 786-O cells. Mechanistically, gypenosides induced apoptosis of RCC cells through regulating PI3K/Akt/mTOR signaling pathway which was implemented though decreasing the phosphorylation level of Akt and mTOR. CONCLUSIONS: Gypenosides induced apoptosis of RCC cells by modulating PI3K/Akt/mTOR signaling pathway.