Matrine Activates PTEN to Induce Growth Inhibition and Apoptosis in V600EBRAF Harboring Melanoma Cells

Here, we report a natural chemical Matrine, which exhibits anti-melanoma potential with its PTEN activation mechanism. Matrine effectively inhibited proliferation of several carcinoma cell lines, including melanoma ^V600EBRAF harboring M21 cells. Flow cytometry analysis showed Matrine induced G₀/G₁ cell cycle arrest in M21 cells dose-dependently. Apoptosis in M21 cells induced by Matrine was identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis and Annexin-V/FITC staining. Molecular mechanistic study suggested that Matrine upregulated both mRNA level and protein expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), leading to inhibition of the PI3K/Akt pathway. Downregulation of phosphor-Akt^ser473 by Matrine activated p21 and Bax, which contributed to G₀/G₁ cell cycle and apoptosis. Besides, Matrine enhanced the PI3K/Akt inhibition effects to inhibit the cell proliferation with PI3K inhibitor, LY2940002. In summary, our findings suggest Matrine is a promising antitumor drug candidate with its possible PTEN activation mechanisms for treating cancer diseases, such as melanomas.     

Sulforaphane,a Dietary Isothiocyanate,Induces G2/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association

Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN) is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G₂/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa). We found that cytotoxicity is associated with an accumulation of cells in the G₂/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G₂/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins.     

Human Prostatic Acid Phosphatase: Structure,Function and Regulation

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.     

Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation,Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism,and Suppression of TGF-β and VEGF Production and Metastatic Potential of Cells

Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G₁ stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21^Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor β (TGF-β) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.     

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.     

Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

The β1-integrin receptor is broadly expressed on tumor and other cells in the tumor microenvironment (TME), and is an unfavorable prognostic factor for cancers. Nature-derived resveratrol has preventive and apoptotic effects on tumors, but whether resveratrol can exert its suppressive actions on TME-induced tumorigenesis through β1-integrin on the surface of CRC cells is still unknown. HCT116 or SW480 cells were exposed to inhibitory antibodies against β1-integrin, bacitracin (selective β1-integrin inhibitor), integrin-binding RGD (Arg-Gly-Asp) peptide, and/or resveratrol. We evaluated the anti-tumor actions and signaling impacts of resveratrol in colorectal cancer (CRC)-TME. We found that resveratrol completely altered the β1-integrin distribution pattern and expression on the surface of CRC cells in TME. Moreover, resveratrol down-regulated CRC cell proliferation, colony formation, viability, and up-regulated apoptosis in a concentration-dependent way. These actions of resveratrol were antagonized mainly by inhibitory antibodies against β1-integrin but not β5-integrin, and by an integrin-binding RGD peptide but not by RGE peptide, and by bacitracin in TME. Similarly, resveratrol-blocked TME-induced p65-NF-kB and its promoted gene markers linked to proliferation (cyclin D1), invasion (focal adhesion kinase, FAK), or apoptosis (caspase-3), were largely abrogated by anti-β1-integrin or RGD peptide, suggesting that β1-integrin is a potential transmission pathway for resveratrol/integrin down-stream signaling in CRC cells. The current results highlight, for the first time, the important gateway role of β1-integrins as signal carriers for resveratrol on the surfaces of HCT116 and SW480 cells, and their functional cooperation for the modulatory effects of resveratrol on TME-promoted tumorigenesis.     

Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T₁AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T₁AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates G_s signaling in vitro. Interestingly, 3-T₁AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T₁AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of G_s and/or G_i/o signaling. Activation of G-proteins G_q/11 and G_12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T₁AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal G_i/o signaling activity, a so far unknown signaling pathway for TAARs.     

Apoptotic Effects of Dietary and Synthetic Sphingolipids in Androgen-Independent (PC-3) Prostate Cancer Cells

Stress-induced activation and metabolism of plasma membrane sphingolipids results in intracellular ceramide accumulation and has been shown to induce apoptosis in human prostate cancer cells. This effect has been observed using synthetic ceramide analogs, such as C6-ceramide; however, the effects of naturally-occurring sphingolipids, such as C18-ceramide and sphingomyelin (CerPCho), on apoptosis and prostate cancer cell proliferation have not been examined. The results of the present study demonstrate that natural (CerPCho, C18-ceramide) and synthetic (C6-ceramide) sphingolipids reduced PC-3 cell proliferation by 15 ± 1.8, 17 ± 2.5, and 46 ± 2.1%, respectively (P < 0.05). These reductions in proliferation were due, in part, to increased cellular apoptosis. Treatment of PC-3 cells with CerPCho and C18-ceramide significantly increased apoptosis by 3.0 ± 0.8 and 3.6 ± 0.6%, respectively, compared to the untreated control, while the synthetic C6-ceramide significantly increased apoptosis by 55.7 ± 0.4%. C6-ceramide-induced apoptosis was associated with cell cycle arrest in the G₂/M phase, decreased extracellular signal-regulated kinase (ERK1/2) signaling and activation of the cell cycle regulatory protein, retinoblastoma (pRb). Treatment of PC-3 cells with C18-ceramide and CerPCho did not alter cell cycle distribution, pRb or ERK1/2 activation. Taken together, these results suggest that natural and synthetic sphingolipids induce apoptosis in PC-3 cells via distinct signaling mechanisms and potencies.     

Extracellular Prolidase (PEPD) Induces Anabolic Processes through EGFR, β1-integrin,and IGF-1R Signaling Pathways in an Experimental Model of Wounded Fibroblasts

The role of prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR) was studied in an experimental model of wound healing in cultured fibroblasts. The cells were treated with PEPD (1–100 nM) and analysis of cell viability, proliferation, migration, collagen biosynthesis, PEPD activity, and the expressions of EGFR, insulin-like growth factor 1 (IGF-1), and β₁-integrin receptor including downstream signaling proteins were performed. It has been found that PEPD stimulated proliferation and migration of fibroblasts via activation of the EGFR-downstream PI3K/Akt/mTOR signaling pathway. Simultaneously, PEPD stimulated the expression of β₁-integrin and IGF-1 receptors and proteins downstream to these receptors such as FAK, Grb2, and ERK1/2. Collagen biosynthesis was increased in control and “wounded” fibroblasts under PEPD treatment. The data suggest that PEPD-induced EGFR signaling may serve as a new attempt to therapy wound healing.     

BTG1 Expression Correlates with the Pathogenesis and Progression of Ovarian Carcinomas

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cell types. We aimed to clarify the role of BTG1 in ovarian carcinogenesis and progression. A BTG1-expressing plasmid was transfected into ovarian carcinoma cells and their phenotypes and related proteins were examined. BTG1 mRNA expression was detected in ovarian normal tissue (n = 17), ovarian benign tumors (n = 12), and ovarian carcinoma (n = 64) using real-time RT-PCR. Ectopic BTG1 expression resulted in lower growth rate, high cisplatin sensitivity, G₁ arrest, apoptosis, and decreased migration and invasion. Phosphoinositide 3-kinase, protein kinase B, Bcl-xL, survivin, vascular endothelial growth factor, and matrix metalloproteinase-2 mRNA and protein expression was reduced in transfectants as compared to control cells. There was higher expression of BTG1 mRNA in normal tissue than in carcinoma tissue (p = 0.001) and in benign tumors than in carcinoma tissue (p = 0.027). BTG1 mRNA expression in International Federation of Gynecology and Obstetrics (FIGO) stage I/II ovarian carcinomas was higher than that in FIGO stage III/IV ovarian carcinomas (p = 0.038). Altered BTG1 expression might play a role in the pathogenesis and progression of ovarian carcinoma by modulating proliferation, migration, invasion, the cell cycle, and apoptosis.     

Prostate Infiltration by Treg and Th17 Cells as an Immune Response to Propionibacterium acnes Infection in the Course of Benign Prostatic Hyperplasia and Prostate Cancer

Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) belong to the most frequent diseases in ageing men. It has been proposed that prostate chronic inflammation is a risk factor for the development of both BPH and PCa. However, potential stimuli that cause or maintain inflammation in the prostate gland are still poorly characterized. Bacterial infections seems to be one of the potential sources of prostatitis. Recent studies show that Propionibacterium acnes (P. acnes) is the most prevalent microorganism in the prostate gland and may be a predisposing factor for inflammation of prostatic tissue. It indicates that P. acnes may contribute to cancer development by enhancing proinflammatory responses, as well as by modifying the prostate extracellular environment. In this review, we discuss the potential role of P. acnes in the development of BPH and PCa and highlight the importance of regulatory T CD4(+)FoxP3(+) (Treg) and Th17 cells in response to P. acnes infection in the context of both prostate diseases.     

Yohimbine,an α2-Adrenoceptor Antagonist,Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.     

Fargesin Inhibits EGF-Induced Cell Transformation and Colon Cancer Cell Growth by Suppression of CDK2/Cyclin E Signaling Pathway

Although the lignan compound fargesin is a major ingredient in Shin-Yi, the roles of fargesin in carcinogenesis and cancer cell growth have not been elucidated. In this study, we observed that fargesin inhibited cell proliferation and transformation by suppression of epidermal growth factor (EGF)-stimulated G₁/S-phase cell cycle transition in premalignant JB6 Cl41 and HaCaT cells. Unexpectedly, we found that signaling pathway analyses showed different regulation patterns in which fargesin inhibited phosphatidylinositol 3-kinase/AKT signaling without an alteration of or increase in mitogen activated protein kinase (MAPK) in JB6 Cl41 and HaCaT cells, while both signaling pathways were abrogated by fargesin treatment in colon cancer cells. We further found that fargesin-induced colony growth inhibition of colon cancer cells was mediated by suppression of the cyclin dependent kinase 2 (CDK2)/cyclin E signaling axis by upregulation of p21^WAF1/Cip1, resulting in G₁-phase cell cycle accumulation in a dose-dependent manner. Simultaneously, the suppression of CDK2/cyclin E and induction of p21^WAF1/Cip1 were correlated with Rb phosphorylation and c-Myc suppression. Taken together, we conclude that fargesin-mediated c-Myc suppression inhibits EGF-induced cell transformation and colon cancer cell colony growth by the suppression of retinoblastoma (Rb)-E2F and CDK/cyclin signaling pathways, which are mainly regulated by MAPK and PKB signaling pathways.     

Genomic Features and Clinical Implications of Intraductal Carcinoma of the Prostate

Intraductal carcinoma of the prostate (IDC-P) is a rare and unique form of aggressive prostate carcinoma, which is characterized by an expansile proliferation of malignant prostatic epithelial cells within prostatic ducts or acini and the preservation of basal cell layers around the involved glands. The vast majority of IDC-P tumors result from adjacent high-grade invasive cancer via the retrograde spreading of tumor cells into normal prostatic ducts or acini. A subset of IDC-P tumors is rarely derived from the de novo intraductal proliferation of premalignant cells. The presence of IDC-P in biopsy or surgical specimens is significantly associated with aggressive pathologic features, such as high Gleason grade, large tumor volume, and advanced tumor stage, and with poor clinical courses, including earlier biochemical recurrence, distant metastasis, and worse survival outcomes. These architectural and behavioral features of IDC-P may be driven by specific molecular properties. Notably, IDC-P possesses distinct genomic profiles, including higher rates of TMPRSS2–ERG gene fusions and PTEN loss, increased percentage of genomic instability, and higher prevalence of germline BRCA2 mutations. Considering that IDC-P tumors are usually resistant to conventional therapies for prostate cancer, further studies should be performed to develop optimal therapeutic strategies based on distinct genomic features, such as treatment with immune checkpoint blockades or poly (adenosine diphosphate–ribose) polymerase inhibitors for patients harboring increased genomic instability or BRCA2 mutations, as well as genetic counseling with genetic testing. Patient-derived xenografts and tumor organoid models can be the promising in vitro platforms for investigating the molecular features of IDC-P tumor.