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      Ganoderma lucidum put forth anti-tumor activity against PC-3 prostate cancer cells via inhibition of Jak-1/STAT-3 activity


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          Prostate cancer (PCA) is a frequent cancer that mainly affects the men. Studying growth feature pathways modified during PCA development may facilitate researchers to expand embattled therapeutic strategies for prostate cancer. In present study, we examined the anticancer potentials of Ganoderma lucidum against the prostate cancer (PC-3) cells by inflection of JAK-1/STAT-3 signalling pathway.


          The cytotoxicity of G. lucidum against the PC-3 cells was examined by MTT assay. The ROS production was monitored by using DCFH-DA staining. The apoptotic morphological alterations stimulatory potential of G. lucidum on PC-3 cells was inspected through the dual staining. The expression of Bcl-2, JAK-1, STAT3, Bax and CyclinD1 proteins were measured by western blotting. The caspase-3 and 9 functions were condensed by assay kit.


          Findings demonstrates the Ganoderma lucidum convince cytotoxicity, accretion of ROS, and apoptosis stimulation in PC-3 cells. In addition, signal transducer and activating transcription (STAT-3) is a successive oncogenic transcriptional factor that regularizes multiplication and apoptosis in cells. Discretion of STAT-3 transcription deliberated as original approach to hinder prostate cell growth. In present exploration, we ascertain that Ganoderma lucidum hold back STAT-3 translocation, in that way dropping the eminent expression of, BCL-2, cyclin-D1 and declined the Bax, caspase-3 and 9 expressions in PC-3 cells.


          In the end our finding concluded that Ganoderma lucidum hinder prostate cell development and convinces apoptosis via hampering the translocation STAT-3.

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          STAT3 as a target for inducing apoptosis in solid and hematological tumors.

          Studies in the past few years have provided compelling evidence for the critical role of aberrant Signal Transducer and Activator of Transcription 3 (STAT3) in malignant transformation and tumorigenesis. Thus, it is now generally accepted that STAT3 is one of the critical players in human cancer formation and represents a valid target for novel anticancer drug design. This review focuses on aberrant STAT3 and its role in promoting tumor cell survival and supporting the malignant phenotype. A brief evaluation of the current strategies targeting STAT3 for the development of novel anticancer agents against human tumors harboring constitutively active STAT3 will also be presented.
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            Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

            Inflammatory hepatocellular adenomas (IHCA) are benign liver tumors that predominantly develop in women and are frequently associated with obesity and alcohol abuse (Zucman-Rossi et al., 2006; Bioulac-Sage et al., 2007). These tumors are characterized by overexpression of the acute-phase inflammatory response in tumor hepatocytes and highly polymorphous inflammatory infiltrates. We recently showed that 60% of IHCA activate signal transducer and activator of transcription 3 (STAT3) via somatic heterozygous activating mutations in IL6ST (IL-6 signal transducer) that encodes the gp130 co-receptor and signal transducer of IL-6 family cytokines (Rebouissou et al., 2009). Inflammatory adenoma is a homogenous disease in which gp130-mutated and nonmutated IHCAs are similar in their expression profiles, particularly in regard to the expression of acute phase inflammatory response target genes (Rebouissou et al., 2009). To identify genetic alterations causing the inflammatory phenotype, defined as an acute-phase inflammatory response with elevated levels of serum amyloid A and C-reactive protein (CRP) observed in nonmutated gp130 IHCA, we used a candidate gene strategy. We focused on STAT3, as it encodes a key nuclear transcription factor that induces the acute-phase inflammatory response in hepatocytes. Moreover, STAT3 has oncogenic roles in epithelial cell tumorigenesis (Grivennikov et al., 2009, 2010; Yu et al., 2009). STAT proteins are latent transcription factors activated by phosphorylation, typically after the binding of extracellular ligands to cytokine receptors that activate associated JAK kinases via growth factor receptors having intrinsic tyrosine kinase activity (e.g., epidermal growth factor receptor, platelet-derived growth factor receptor, and colony stimulating factor 1 receptor), as well as by nonreceptor tyrosine kinases such as Src family kinases (Levy and Darnell, 2002). STAT3, one of seven STAT family members, is activated by gp130 in response to IL-6 (Zhong et al., 1994; Inoue et al., 1997). STAT3 is persistently phosphorylated in many human cancer cell lines and in epithelial cells and primary tumors, including hepatocellular carcinoma, breast, prostate, and head and neck cancers, and also in several hematological malignancies. STAT3 mutations have never been described in human tumors, yet we reasoned that alterations activating its function would explain the inflammatory phenotype of IHCA lacking mutations in gp130. RESULTS AND DISCUSSION Identification of STAT3 mutations in IHCA Among 114 screened hepatocellular adenomas, we identified 7 STAT3 mutations in 6 tumors (Table I and Fig. S1, A and B). All of these adenomas were inflammatory (6 out of 75 IHCA), and no additional somatic mutations were identified in hepatocyte nuclear factor 1α (HNF1A), catenin (cadherin-associated protein), β1 (CTNNB1), or IL6ST, genes that are recurrently altered in IHCA and HCA (Bluteau et al., 2002; Zucman-Rossi et al., 2006; Rebouissou et al., 2009). Thus, STAT3 IHCA mutations are exclusive from all other known genetic alterations in hepatocellular tumors. STAT3-mutated IHCA were associated with obesity (n = 4) and/or alcohol abuse (n = 3), and multiple (≥4) nodules were present in half of these cases. All STAT3 mutations were somatic, as they were not observed in adjacent nontumor liver tissues that were steatotic in 5/6 cases. All mutations were also monoallelic and led to amino acid substitutions in 5 cases or to in-frame insertions of 1–4 aa in the remaining 2 cases. In one case (#379), we identified 3 nucleotide mutations that led to amino acid substitutions at codons 502 and 658. Sequencing cloned STAT3 cDNA from this tumor showed that the three different nucleotide mutations were carried by the same allele. We also showed in three different cases (#379, #966, and #1351) that the mutations were harbored by all of the isoforms of STAT3, including splicing of nucleotides 2099–2101 and 2145–2194 (STAT3β) that lead to p.Ser701del and p.Thr716PhefsX8, respectively (Fig. S1 C; Schaefer et al., 1995). Sequencing STAT3 RT-PCR products in mutated tumors showed that all cases expressed the normal and mutated STAT3 alleles at comparable levels (Fig. S1, A and B). Thus, the expression of one mutated STAT3 allele is a rare but recurrent genetic event in IHCA that is selected for during benign hepatocellular tumorigenesis. Table I. STAT3 somatic mutations identified in inflammatory hepatocellular tumors Tumors Nucleotide change Amino acid change Mutant ID CHC341T T233>G L78>R L78 CHC379T G1504>T D502>Y D502 A1972>T; G1974>T K658>Y K658 CHC574T G496>C E166>Q E166 CHC966T A1919>T Y640>F Y640 CHC1021T T1969_G1980dup Y657_M660dup Y657 CHC1351T C1968>T; 1969_1970insTTT G656_Y657insF G656 Codons and mutated nucleotides are numbered according to STAT3 cDNA open reading frame. Four of the seven identified mutations were located in the domain of STAT3 (residues 585–688) that shares homology with Src homology 2 (SH2) domains (Fig. 1 A). The STAT3 SH2 domain mediates STAT3 dimerization via binding of phosphotyrosine residue Y705 (Shuai et al., 1994; Wen et al., 1995). Moreover, three of these mutations clustered in a hotspot in the SH2 domain at codons 657 and 658. Interestingly, introduction of cysteine at residues 662 and 664 promotes STAT3 dimerization and creates a constitutively active transcription factor (Stat3-C; Bromberg et al., 1999). The fourth mutation, Y640F, was in the PYTK motif conserved in STAT1, STAT2, and STAT3. Interestingly, substitution of the corresponding Y631 to phenylalanine in STAT2 promotes type I IFN signaling (Scarzello et al., 2007; Constantinescu et al., 2008). The three other mutations found in IHCA were distributed throughout the protein: the altered leucine-78, which contributes to dimerization (Chen et al., 2003); glutamate-166, which is part of helix α1 involved in the interaction with gp130 (Zhang et al., 2000a); and aspartate-502, which is located in the α-helical “connector” domain. Remarkably, none of the mutations identified in IHCA were similar to the 150 germline-inactivating STAT3 heterozygous mutations described in patients with Job’s syndrome, which causes hyper-IgE syndrome (Holland et al., 2007; Minegishi et al., 2007). Figure 1. Gain-of-function mutations in STAT3 in IHCA. (A) Distribution of STAT3 mutations identified in IHCA is represented according to the different protein domains. Asterisk indicates the two mutations that are found on the same allele in tumor #379. Stat3-C location is indicated in gray. (B) STAT3 mutants L78R (L78), E166Q (E166), Y640F (Y640), D502Y K658Y (D502/K658), G656_Y657insF (G656), Y657_M660dup (Y657), and Stat3-C (SC) or control STAT3 WT (WT) and empty plasmid (EP) were transfected in Hep3B, HepG2, and Huh7 cells expressing a STAT3-driven luciferase (Luc) reporter construct. STAT3 activation was measured after 6 h of serum starvation and, when indicated, was treated for 3 h with 100 ng/ml IL-6. Shown is the Luc activity (mean) determined from triplicate co-transfections (± SD) relative to pSIEM-luc alone (EP) without IL-6. (C) Graphs are qRT–PCR results showing the induction of endogenous CRP, STAT3, and SOCS3 mRNA after overexpression of mutant STAT3 relative to unstimulated EP-transfected Hep3B cells (EP; mean ± SD). (D) Endogenous CRP mRNA expression in Hep3B cells transfected with increasing amounts of expression plasmids encoding WT or E166 STAT3 mutant (dotted line: mock-transfected cells). (E) Activity of the STAT3 mutants, including D502Y (D502), K658Y (K658), and the double mutant D502Y K658Y (D502/K658) were evaluated using STAT3-driven Luc in Hep3B cell line. Data are from triplicate transfections (± SD) relative to pSIEM-luc alone (EP). *, P 80%. JAK1 siRNA (S7646), JAK2 siRNA (S7651), and IL6ST (gp130) siRNA (S229006) were purchased from Applied Biosystems; siRNA control (Block-IT Alexa Fluor Red) was purchased from Invitrogen. Pharmacological treatments. Cells were exposed, in serum-free medium, for 6 h to Tyrphostin AG490 (Sigma-Aldrich), 9 h to Src inhibitor-1 (Sigma-Aldrich), Src inhibitor-5 (JS Res Chemical Trading), or PP2 (Sigma-Aldrich); for 18 h to Ruxolitinib (INCB-018424; JS Res Chemical Trading); and for 24 h to Curcumin (Sigma-Aldrich). In the last 3 h of treatment, cells were stimulated with 100 ng/ml IL-6, or not, as indicated. Western blot analysis. Western blot analyses were performed as previously described (Rebouissou et al., 2009) using antibodies specific for STAT3 (Cell Signaling Technology; 1:500) and phospho-STAT3 Tyr705, (Cell Signaling Technology; 1:200). For dimerization assays, cell lysates were incubated with Immobilized Protein G agarose (Thermo Fisher Scientific) and anti-flag (Cell Signaling Technology; 1:50) or anti-myc (Cell Signaling Technology; 1:200) antibody, at 4°C overnight, before Western blot analysis. Immunohistochemistry. Immunohistochemistry was performed using a Dako autostainer, on paraffin sections of 10% fixed tumor tissue using a monoclonal anti–phospho-STAT3 Tyr705 (Cell Signaling Technology; 1:50). For each immunohistochemical procedure, antigen retrieval was performed in citrate buffer; detection was amplified by the Dako Envision system. STAT3 DNA-binding assays. 1 d after transfection, cells were starved in a serum-free medium overnight, and the TransFactor Extraction kit (Takara Bio, Inc.) was used to prepare nuclear extracts following the manufacturer’s protocol. Nuclear extracts were analyzed for STAT3 DNA-binding activity using the TransFactor Universal STAT3-specific kits (Takara Bio, Inc.), which is an ELISA-based method. Immunofluorescence. 24 h after transfection, cells were fixed in 4% formaldehyde for 15 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 15 min. Cells were saturated with 2% BSA for 30 min, washed with PBS, and then incubated with primary antibody overnight at 4°C. After 3 washes with PBS, cells were incubated with secondary antibodies for 1 h. The slides were washed, and then mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Immunofluorescence images were obtained using an HBO 100 optic microscope (Carl Zeiss, Inc.). All data from one experiment were collected at the same intensity and background level. The following monoclonal antibodies were used: rabbit anti–phospho-STAT3 Tyr705 (Cell Signaling Technology; 1:200), mouse anti-total STAT3 (Cell Signaling Technology; 1:100). The secondary antibodies were anti–mouse (Invitrogen; 1:200) and anti–rabbit (Invitrogen; 1:200). Online supplemental material. Fig. S1 shows the somatic DNA alterations identified on STAT3 gene, and distribution of mutations according to the different isoforms. Fig. S2 shows serine 727 phosphorylation of STAT3 mutants. Fig. S3 shows the effect of inhibitors on STAT3 IHCA mutant activation. Table S1 lists the TaqMan predesigned gene expression assays used for qRT-PCR analyses. Table S2 lists the primers used in PCR and sequencing. Table S2 lists the primers used in in site-directed mutagenesis. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20110283/DC1.
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              Resveratrol inhibits the proliferation and induces the apoptosis in ovarian cancer cells via inhibiting glycolysis and targeting AMPK/mTOR signaling pathway


                Author and article information

                Saudi J Biol Sci
                Saudi J Biol Sci
                Saudi Journal of Biological Sciences
                03 June 2020
                October 2020
                03 June 2020
                : 27
                : 10
                : 2632-2637
                [a ]Department of Urology, The Second Affiliated Hospital of Guangxi Medical University, Nanning City, Guangxi Province 530007, China
                [b ]Department of Biochemistry, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai 600 077, India
                [c ]Department of Biochemistry, Panimalar Medical College Hospital & Research Institute, Varadharajapuram, Poonamallee, Chennai 600 123, India
                [d ]Department of Urology, The Second People's Hospital of Lianyungang, Jiangsu Province 222006, China
                Author notes
                [* ]Corresponding author. xinfeng225588@ 123456sina.com
                © 2020 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                : 23 April 2020
                : 22 May 2020
                : 27 May 2020
                Original Article

                ganoderma lucidum,pc-3 cells,apoptosis,prostate cancer,cyclin-d1,stat-3 pathway


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