G-Protein-coupled Estrogen Receptor 1 Agonist G-1 Perturbs Sunitinib Resistance-related Phosphoproteomic Signatures in Renal Cell Carcinoma.

BACKGROUND: Metastatic renal cell carcinoma (RCC) often develops resistance to first-line targeted therapy such as sunitinib. G-Protein-coupled estrogen receptor 1 (GPER1) agonist G-1 was recently reported to regulate RCC physiology but the role of G-1 in RCC tumorigenesis and sunitinib resistance remains largely unknown. MATERIALS AND METHODS: Parental and sunitinib-resistant 786-O cells were treated with GPER1 agonist G-1, and quantitative phosphoproteomics was performed. Bioinformatic analyses and validations, including immunoblotting, cell migration, and cell cycle distribution, were performed. RESULTS: G-1 repressed cell proliferation and migration in both parental and sunitinib-resistant 786-O cells. Phosphoproteomic signatures, including phosphoinositide 3-kinase and protein kinase B (PI3K-AKT) as well as other pathways, were up-regulated in sunitinib-resistant cells but application of G-1 reversed this effect. Among phosphoprotein candidates, activating transcription factor 2 (ATF2) Thr69/71 phosphorylation was antagonistically regulated by sunitinib resistance and G-1. CONCLUSION: Our results open up the possibility for managing RCC and sunitinib resistance by GPER1 agonist G-1 and its regulated pathways.

Gut butyrate-producing organisms correlate to Placenta Specific 8 protein: Importance to colorectal cancer progression.

Tumor metastasis or recurrence often occurs in patients with curative resection of colorectal cancer (CRC). Placental-specific 8 (PLAC8), which has increased expression in stool, may be associated with CRC recurrence. Insights into the role of PLAC8 in CRC recurrence and its clinical significance may support to develop strategies for preventing CRC recurrence and deterioration. Clinical tissues, cell and animal models were used to clarify the roles of PLAC8 in CRC tumorigenesis, invasion, and migration. Next-generation sequencing of 16S ribosomal DNA has been used to assess the gut microbiota in stool of CRC patients. We found that PLAC8 was upregulated in tissues from patients with late-stage CRC. In our in vitro studies, PLAC8 was dynamically regulated in mitotic cells. Overexpressed PLAC8 was nucleated at the centrosome during mitosis, and therefore, PLAC8 overexpression might increase cell growth and migration (all p < 0.05). The tumorigenic and invasive effects of PLAC8 on CRC cells were also confirmed in a xenograft mouse model. We further identified reduced levels of two butyrate-producing organisms, Butyricicoccus and Prevotella spp., in stools from CRC patients. We found that butyrate downregulated PLAC8 expression and induced apoptosis in PLAC8-overexpressing cells. Our data suggests that PLAC8 gene and protein expression and dysbiosis of gut microflora, especially in butyrate-producing microorganisms, may be indicators of CRC progression.

Dynamic expression of SMAD3 is critical in osteoblast differentiation of PDMCs.

Human pluripotent stem cells have the potential assist in the identification of genes involved in mammalian development. The human placenta is considered a repository of stem cells, termed placenta­derived multipotent cells (PDMCs), which are able to differentiate into cells with an osteoblastic phenotype. This plasticity of PDMCs maybe applied clinically to the understanding of osteogenesis and osteoporosis. In the presentstudy, osteoblasts were generated by culturing PDMCs in osteogenic medium. Reverse transcription quantitative polymerase chain reactionand the degree of osteoblast calcification were used to evaluate the efficacy of osteogenesis. The results suggestedthat the expression of mothers against decapentaplegic homolog 3 (SMAD3) increased in the initial stages of osteogenic differentiation but decreased in the later stages. However, osteogenesis was inhibitedwhen the PDMCs overexpressed SMAD3 throughout the differentiation period. In addition, the rate of osteogenic differentiation was decreased when SMAD3 signaling was impaired. In conclusion, SMAD3 serves an important role in osteoblast differentiation and bone formation in a time­dependent manner. The data from the present study indicate that arapid increase in SMAD3 expression is crucial for osteogenesis and suggest a role for PDMCs in the treatment of patients with osteoporosis.

Clinical significance of interleukin­6 and inducible nitric oxide synthase in ketamine­induced cystitis.

Ketamine is an ionotropic glutamatergic N­methyl­D­aspartate receptor antagonist, which is widely used among recreational drug abusers. Ketamine abusers exhibit substantially reduced bladder capacity, which can lead to urinary frequency. The molecular pathogenesis of ketamine­induced cystitis has been scarcely reported. Given previous clinical findings, it may be hypothesized that pathological alterations in smooth muscle cells (SMCs) of the urinary bladder serve a crucial role in the mechanism underlying cystitis. In the present study, two lineages of SMCs, one from differentiated foreskin­derived fibroblast­like stromal cells and the other from cultured normal aortic SMCs, were used to study ketamine­induced molecular alterations. Polymerase chain reaction was used to study the effects of ketamine on oxidative stress. The effects of adjuvant chemotherapy with cyclophosphamide (CTX) were also investigated. The results indicated that the expression levels of interleukin­6 and inducible nitric oxide synthase (iNOS) were decreased, whereas collagen expression and deposition were increased in ketamine­treated SMCs. Conversely, treatment with CTX restored the expression of iNOS, which may prevent or limit oxidative damage. In conclusion, the present study demonstrated that ketamine may induce several molecular alterations in SMCs and these changes may be associated with the clinical symptoms observed in ketamine abusers. In addition, the specific chemotherapeutic agent CTX may reverse these ketamine­induced aberrations.

Ganoderma microsporum immunomodulatory protein induces apoptosis and potentiates mitomycin C-induced apoptosis in urinary bladder urothelial carcinoma cells.

Current chemotherapy and immunotherapy treatments followed by transurethral resection for urinary bladder urothelial carcinoma (UC) usually suffer from poor prognosis and high recurrence rate. Design and modification of current formulation with the novel adjuvants are needed. A recombinant protein derived from Ganoderma microsporum named as Ganoderma microsporum immunomodulatory protein (GMIP) was used to treat UC cells. We found GMIP elicits a dose-dependent and time-dependent anti-UC cell proliferation effect, with a half-maximal inhibition concentration (IC50 ) comparable to mitomycin C (MMC), a commonly used chemotherapy agent. After GMIP treatment, UC cells showed apoptotic phenomenon including cell cycle arrest in the G1 phase, elevated sub-G1 population, mitochondrial membrane potential loss, up-regulated p21 expression, p21 nuclear translocation, caspase activation, and PARP cleavage in a p53-independent but p21-mediated pathways. Unlike lung cancer cells, GMIP treated UC cells showed no autophagic scheme including Beclin-1, an autophagy to apoptosis switch marker, was not cleaved by caspase 3 and slight LC3B-II accumulation. Also, the classic autophagic inhibitor, chloroquine had no effect in GMIP-mediated cell death made us conclude that GMIP induced apoptosis through caspase activation but not autophagy in UC cells. Additionally, GMIP showed synergistic effects with MMC in killing UC cells and thus decreased the concentration of MMC usage to reach the comparable apoptotic effects. Our results delineate novel strategies for treatment of UC by GMIP alone or in combination with MMC application and provide a promising therapeutic cocktail for better treatment of urinary bladder urothelial carcinoma.

Cellular effects induced by 17-ß-estradiol to reduce the survival of renal cell carcinoma cells.

BACKGROUND: Renal cell carcinoma (RCC) is an adult malignancy with 2:1 men-to-women ratio, which implies the possible role of sex hormones in RCC carcinogenesis. One of the predominant sex hormones in women before menopause, 17-ß-estradiol (or E2), may regulate RCC growth by cellular mechanisms that are still not fully understood. METHODS: The expression levels of E2 receptors (ER1 and ER2) were determined in different RCC cell lines. The DNA damage response induced by E2 was determined by a DNA double-strand break marker γH2AX. To study the possible effect of E2 on oxidative stress response, RCC cells were stained with 2,7-dichlorofluorescein diacetate and analyzed by flow cytometry. Upregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) ser40 phosphorylation in response to oxidative stress was detected by immunoblotting. Finally, annexin V/propidium iodide (PI) double staining assay was used to determine E2-induced cellular apoptosis. RESULTS: Variable expression of ER1 and ER2 were found in the RCC cell lines studied (786-O, A498, and ACHN), in which ACHN and A498 showed highest and lowest ER expression, respectively. In A498 cells, E2 induced DNA double-strand breaks with positive staining of γH2AX. On the other hand, the level of reactive oxidative species were elevated in ACHN cells after E2 treatment. The E2-induced oxidative stress also induced the Ser40 phosphorylation and nuclear translocation of Nrf2. Finally, we also demonstrated that E2 induced apoptosis as revealed by annexin V/PI double staining. CONCLUSIONS: In this study, we demonstrated the cellular effects of E2 on DNA repair, ROS production as well as Nrf2 activation, and apoptosis in RCC cell lines. Together these cellular alterations may contribute to the reduced viability of RCC cells following E2 treatment.

Dual Roles of 17-ß Estradiol in Estrogen Receptor-dependent Growth Inhibition in Renal Cell Carcinoma.

BACKGROUND: It has been proposed that 17-ß-estradiol (E2) activates estrogen receptor and inhibits renal cell carcinoma (RCC) growth. In the present study we explored the role of E2 and ER in the regulation of RCC growth. MATERIALS AND METHODS: The RCC cell line ACHN was treated by E2 combining with E2 antagonist Fulvestrant or ER knockdown, and cell growth was monitored. Quantitative phosphoproteomics was applied to study the E2 regulated non-genomic phosphorylation changes. Western blotting, immunofluorescence microscopy, and apoptosis assays were used for validation. RESULTS: E2 induced ER-dependent growth inhibition in RCC cell lines. Quantitative phosphoproteomics revealed that E2 induced both apoptosis and autophagy. Cellular apoptosis was confirmed by altered mitochondrial membrane potential, and ER-dependent autophagosome formation was also found. CONCLUSION: Our data revealed the potential dual roles of E2 in regulating RCC growth via autophagy and apoptosis pathways.

Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

BACKGROUND: Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. RESULTS: After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. CONCLUSION: Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

Hydrostatic pressure enhances mitomycin C induced apoptosis in urothelial carcinoma cells.

OBJECTIVES: Urothelial carcinoma (UC) of the bladder is the second most common cancer of the genitourinary system. Clinical UC treatment usually involves transurethral resection of the bladder tumor followed by adjuvant intravesical immunotherapy or chemotherapy to prevent recurrence. Intravesical chemotherapy induces fewer side effects than immunotherapy but is less effective at preventing tumor recurrence. Improvement to intravesical chemotherapy is, therefore, needed. METHODS AND MATERIALS: Cellular effects of mitomycin C (MMC) and hydrostatic pressure on UC BFTC905 cells were assessed. The viability of the UC cells was determined using cellular proliferation assay. Changes in apoptotic function were evaluated by caspase 3/7 activities, expression of FasL, and loss of mitochondrial membrane potential. RESULTS: Reduced cell viability was associated with increasing hydrostatic pressure. Caspase 3/7 activities were increased following treatment of the UC cells with MMC or hydrostatic pressure. In combination with 10 kPa hydrostatic pressure, MMC treatment induced increasing FasL expression. The mitochondria of UC cells displayed increasingly impaired membrane potentials following a combined treatment with 10 µg/ml MMC and 10 kPa hydrostatic pressure. CONCLUSIONS: Both MMC and hydrostatic pressure can induce apoptosis in UC cells through an extrinsic pathway. Hydrostatic pressure specifically increases MMC-induced apoptosis and might minimize the side effects of the chemotherapy by reducing the concentration of the chemical agent. This study provides a new and alternative approach for treatment of patients with UC following transurethral resection of the bladder tumor.

Human foreskin fibroblast-like stromal cells can differentiate into functional hepatocytic cells.

Foreskin fibroblast-like stromal cells (FDSCs) are progenitors isolated from human tissue that can differentiate into diverse cell types. Many types of stem cells can differentiate into hepatocyte-like cells, which could be used for drug testing or in liver regeneration therapy, but whether FDSCs can be converted into functional hepatocytes is unknown. FDSCs show divergent properties when cultured in distinct media, forming spheres in Dulbecco's modified Eagle's medium (DMEM) containing F12, epidermal growth factor (EGF), and basic fibroblast growth factor (b-FGF), but have fibroblast-like morphology when cultured in DMEM-based growth medium. Both cell populations express the typical mesenchymal stem cell markers CD90, CD105, and CD73, but the p75 neurotrophin receptor (p75NTR) was detected only in FDSC spheres. Both types of FDSCs can differentiate into hepatocyte-like cells, which express typical liver markers, including albumin and hepatocyte paraffin 1 (Hep Par1), along with liver-specific biological activities. When plasmids containing the human hepatitis B virus (HBV) genome were transfected transiently into FDSCs, differentiated hepatocyte-like cells secrete large amounts of HBe and HBs antigens. FDSCs could be used for clinical hepatic therapy and/or serve as a model of HBV.

Multilineage differentiation potential of fibroblast-like stromal cells derived from human skin.

Adult stem cells that reside in adult tissues have been deemed to possess great potential for clinical application because of their capabilities of self-renewal and differentiation. However, the limitations such as infection risks and low isolation rate make the search for appropriate source to be continued. Here, we demonstrate isolation of progenitors from human foreskin tissue samples, which have fibroblast-like morphology and could be easily propagated for more than 50 passages. These foreskin-derived fibroblast-like stromal cells (FDSCs) expressed CD90, CD105, CD166, CD73, SH3, and SH4, which is similar to the immunophenotypes of human bone marrow-derived mesenchymal stem cells. In comparison with Hs68, the human fibroblast cell line, FDSCs are positive for CD105 and absent for CD54 expression. Further, FDSCs could be induced to differentiate into osteocytes, adipocytes, neural cells, smooth muscle cells, Schwann-like cells, and hepatocyte-like cells. Interestingly, when cultured in Dulbecco's modified Eagle's medium/F12 medium, FDSCs can form spheres with increased expression levels of fibronectin and vimentin. In conclusion, foreskin can serve as a valuable source of multipotent cells with the capabilities for endodermal, mesodermal, and ectodermal cells. Coupled with the advantages of their easy access, isolation, and propagation, these foreskin-derived stromal cells might be of potential use in future studies in stem cell differentiation and therapeutic application.

A vitronectin M381T polymorphism increases risk of hemangioblastoma in patients with VHL gene defect.

Hemangioblastomas, highly vascular tumors, occur sporadically or associated with von Hippel-Lindau (VHL) disease. Diverse mutations in the VHL gene inactivate the VHL protein and constitute the molecular etiology of the disease. Changes in VHL gene were analyzed in patients with multiplex ligation-dependent probe amplification and single-strand conformation polymorphism analyses. We report here that other angiogenesis-related changes in vitronectin were identified with 2D electrophoresis of plasma samples and restriction fragment length polymorphisms. Our findings revealed that most patients (80.0%) with a familial VHL deletion carried the threonine (T) allele at vitronectin codon 381. Adults simultaneously carrying a VHL defect and the T allele were 5.0-fold more likely to be affected by VHL disease than were methionine/methionine (M/M) homozygotes carrying a VHL defect. Patients with sporadic hemangioblastoma, C-terminally truncated VHL protein or a large deletion in the VHL gene, and the T allele were 18.0-fold more likely to develop recurrent disease. Taken together, individuals with mutated VHL are more likely to be affected by familial or recurrent sporadic hemangioblastoma when carrying the M/T or T/T genotype at codon 381 of vitronectin.

(-)-Epicatechin-3-gallate, a green tea polyphenol is a potent agent against UVB-induced damage in HaCaT keratinocytes.

(-)-Epicatechin-3-gallate (ECG) is a polyphenolic compound similar to (-)-epigallocatechin-3-gallate (EGCG) which is abundant in green tea. Numerous workers have proposed that EGCG protects epidermal cells against UVB-induced damage. However, little has been known about whether ECG protects keratinocytes against UVB-induced damage. We decided to investigate the protective effects and underlying mechanisms of ECG on UVB-induced damage. Cell viability was determined by the MTT assay. Activation of ERK1/2, p38 and JNK was analyzed by Western blotting. Intracellular H2O2 production and DNA content was analyzed by flow cytometry. Lipid peroxidation was assayed by colorimetry. In our study, we found that ECG dose-dependently attenuated UVB-induced keratinocyte death. Moreover, ECG markedly inhibited UVB-induced cell membrane lipid peroxidation and H2O2 generation in keratinocytes, suggesting that ECG can act as a free radical scavenger when keratinocytes were photodamaged. In parallel, H2O2-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by ECG. UVB-induced pre-G1 arrest leading to apoptotic changes of keratinocytes were blocked by ECG. Taken together, we provide here evidence that ECG protects keratinocytes from UVB-induced photodamage and H2O2-induced oxidative stress, possibly through inhibition of the activation of ERK1/2, p38 and JNK and/or scavenging of free radicals.

(+)-Catechin prevents ultraviolet B-induced human keratinocyte death via inhibition of JNK phosphorylation.

High levels of (+)-catechin are found in the skin and seed of many fruits such as apples and grapes. Dietary supplementation with (+)-catechin has been demonstrated to protect epidermal cells against damage induced by ultraviolet B (UVB) radiation. However, the underlying mechanisms are not well understood yet. To determine whether (+)-catechin protects keratinocytes from UVB-induced damage, the viability of UVB- and H2O2-treated cells was determined by cell viability assay. Intracellular H2O2 level was measured by flow cytometry. UVB- or H2O2-induced signaling pathways were detected by Western blotting. The results indicated that (+)-catechin inhibited UVB- and H2O2-induced keratinocyte death. In parallel, intracellular H2O2 generation in keratinocytes irradiated by UVB was inhibited by (+)-catechin in a concentration-dependent manner. (+)-Catechin also inhibited UVB- and H2O2-induced JNK activation in keratinocytes. However, it had little inhibitory effect on UVB- and H2O2-induced ERK and p38 activation even at a higher concentration, suggesting indirectly that JNK activation is required for the induction of apoptosis in keratinocytes exposed to UVB. Finally, we compared the cytotoxicity of (+)-catechin and (-)-epigallocatechin-3-gallate (EGCG) on keratinocytes. Cell viability assay showed that (+)-catechin was relatively nontoxic at higher doses. Taken together, our results demonstrate that (+)-catechin inhibits UVB- and oxidative stress-induced H2O2 production and JNK activation and enhances human keratinocyte survival. However, although it seems that (+)-catechin and EGCG are equally effective in preventing keratinocyte death, (+)-catechin is relatively nontoxic and thus is suitable for developing as an anti-ageing agent for skin care.