P388 Cells Research Articles

PEMISAHAN SENYAWA-SENYAWA YANG BERSIFAT SITOTOKSIK TERHADAP SEL MURIN LEUKEMIA P388 DARI EKSTRAK METANOL KULIT BATANG DIPTEROCARPUS CONFERTUS SLOOT (DIPTEROCARPACEAE)

Six compounds, KP-1 up to KP-6 were isolated from the methanol extract of the tree bark of Dipterocarpus confertus Sloot (Dipterocarpaceae). The cytotoxic activities of these compounds were evaluated against murine leukemia P388 cells. Result of these examination indicated that KP-2 compound and KP-1 were very active with each IC 50 value of these compounds were 2.25 and 5.1 μg/mL, respectively. While the others were not active against murine leukemia P388 cells.Keywords: Dipterocarpaceae, Dipterocarpus confertus Sloot, isolated compounds, cytotoxic, and leukemia P-388 cell

Nazumazoles A-C, Cyclic Pentapeptides Dimerized through a Disulfide Bond from the Marine Sponge Theonella swinhoei.

A mixture of nazumazoles A-C (1-3) was purified from the extract of the marine sponge Theonella swinhoei. The mixture was eluted as an extraordinarily broad peak in the reversed-phase HPLC. The structures of nazumazoles were determined by interpretation of the NMR data and chemical degradations. Nazumazoles contain one residue each of alanine-derived oxazole and α-keto-β-amino acid residue. Nazumazoles exhibited cytotoxicity against P388 cells.

Cyclohexene long-chain fatty acid esters from Uvaria dulcis (Dunal)

Two new cyclohexene long-chain fatty acid esters, namely Dulcisenes A and B, were isolated from the twigs of the Uvaria dulcis together with seven known compounds, uvarigranol E, (−)-zeylenol, ellipeiopsol B, 5,7-dihydroxyflavone, 8-hydroxy-5,7-dimethoxyflavanone, lupeol, and benzyl benzoate. The structures of the isolated compounds were elucidated by spectroscopic and mass-spectrometric analyses, including 1D, 2D NMR and HR TOF MS. Several of these metabolites were tested for cytotoxicity against HepG2, A549, S102, HuCCA-1, HeLa, MDA-MB-231, T47D, HL-60, and P388 cell lines.

QSAR based docking studies of marine algal anticancer compounds as inhibitors of protein kinase B (PKBβ)

Marine algae are prolific source of bioactive secondary metabolites and are found to be active against different cancer cell lines. QSAR studies will explicate the significance of a particular class of descriptor in eliciting anticancer activity against a cancer type. Marine algal compounds showing anticancer activity against six different cancer cell lines namely MCF-7, A431, HeLa, HT-29, P388 and A549 taken from Seaweed metabolite database were subjected to comprehensive QSAR modeling studies. A hybrid-GA (genetic algorithm) optimization technique for descriptor space reduction and multiple linear regression analysis (MLR) approach was used as fitness functions. Cell lines HeLa and MCF-7 showed good statistical quality (R2∼0.75, Q2∼0.65) followed by A431, HT29 and P388 cell lines with reasonable statistical values (R2∼0.70, Q2∼0.60). The models developed were interpretable, with good statistical and predictive significance. Molecular descriptor analyses revealed that Baumann’s alignment-independent topological descriptors had a major role in variation of activity along with other descriptors. Incidentally, earlier QSAR analysis on a variety of chemically diverse PKBα inhibitors revealed Baumann’s alignment-independent topological descriptors that differentiated the molecules binding to Protein kinase B (PKBα) kinase or PH domain, hence a docking study of two crystal structures of PKBβ was performed for identification of novel ATP-competitive inhibitors of PKBβ. Five compounds had a good docking score and Callophycin A showed better ligand efficiency than other PKBβ inhibitors. Furthermore in silico pharmacokinetic and toxicity studies also showed that Callophycin A had a high drug score (0.85) compared to the other inhibitors. These results encourages discovering novel inhibitors for cancer therapeutic targets by screening metabolites from marine algae.

Microbial Metabolite Butyrate Suppresses Murine Mast Cell Activation

BackgroundMast cell is known for its roles in mediating food allergy and regulating gut inflammation. Butyrate, a major short chain fatty acid produced by gut microbiota, exerts beneficial effects on intestinal homeostasis. However, its role in modulating mast cell function remain undefined. Methods: Murine mastocytoma P815 cells (P815) or murine bone marrow derived mast cells (BMMC) were treated with sodium butyrate (NaB). The proliferation, cell cycle, apoptosis, degranulation and cytokine secretion were assessed.ResultsIn P815 cells, NaB inhibited cell proliferation, increased acetylation of both histone 3 and α‐tubulin. The growth inhibition effect of NaB is probably through reducing c‐kit expression and inducing cell cycle arrest and apoptosis, which is indicated by enhanced annexin V/PI staining, caspase‐3 and PARP cleavage. In BMMC, NaB treatment dramatically suppressed FcεRI dependent TNFα and IL‐6 release without affecting degranulation. Furthermore, NaB enhanced histone 3 acetylation but diminished mRNA expression of Tnf ‐α and Il‐6, possibly through inhibiting the transcription initiation as revealed by reduced RNA polymerase II binding to the promoters of Tnf‐α and Il‐6.ConclusionsNaB modulates murine mast cell function possibly through enhancing histone acetylation, which is of importance in developing therapeutics for diseases involving mast cells (NIH R15HD073864).

Immunoregulatory effects of 1, 25(OH)2D3 on Der p stimulating the expression of TLR4 and IL-4 in P815 mast cells

Objective To investigate the effects of Dermatophagoides pteronyssinus (Der p) on the expression of TLR4 and IL-4 in P815 mast cells and to further analyze the immunoregulatory effects of 1, 25-(OH)2D3 on Der p treated P815 mast cells. Methods Different concentrations of Der p and 1, 25-(OH)2D3 were used alone or in combination to stimulate P815 mast cells. The supernatants of the stimulated cell culture were analyzed by enzyme-linked immunosorbent assay (ELISA) for the detection of IL-4. The stimulated cells were collected and analyzed by real-time PCR and Western blot assays for the detection of TLR4 at mRNA and protein levels, respectively. Results (1) TLR4 expression was detected in P815 cells. The expression of TLR4 was enhanced in P815 cells treated with various concentrations of Der p. A significant dose-dependent up-regulation of TLR4 was observed in P815 cells after incubation with Der p for 36 h. (2) Der p promoted the release of IL-4 in P815 cells (P 0.05). (4) 10-8 mol/L of 1, 25(OH)2D3 promoted the Der p-induced expression of TLR4 in P815 cells (P<0.01). However, 1, 25(OH)2D3 inhibited the release of IL-4 in a dose-dependent manner (P<0.05 or P<0.01). Conclusion (1) Der p could promote the inflammation and allergic reaction through up-regulating TLR4 and IL-4 in mast cells. (2) The possible mechanism for the inhibitory of 1, 25(OH)2D3 on Der p-induced immune responses was due to the suppression of Th2-type immune responses through inhibiting the synthesis and secretion of IL-4 in mast cells. Key words: Mast cells; P815 cells; Dermatophagoides pteronyssinus; TLR4; 1, 25(OH)2D3

Limonoids and Triterpenoids from Dysoxylum mollissimum var. glaberrimum.

Seven new limonoids, dysomollides A-G (1-7), and two new cycloapotirucallane-type triterpenoids, dysomollins A and B (8 and 9), together with three known compounds, dysoxylumin A (10) and toonapubesins A (11) and B (12), were isolated from the twigs of Dysoxylum mollissimum var. glaberrimum. The structures of 1-9 were elucidated on the basis of spectroscopic methods. Compound 10 showed inhibitory activity against A549 cells with an IC50 value of 2.1 μM, and compound 11 exhibited activity against P388 cells with an IC50 value of 6.7 μM.

Chojalactones A-C, cytotoxic butanolides isolated from Streptomyces sp. cultivated with mycolic acid containing bacterium.

The soil-derived bacterium, Streptomyces sp. CJ-5, was cocultured with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. The combined culture method significantly enhanced the production of the secondary metabolites in Streptomyces sp. CJ-5, leading to the isolation of three novel butanolide chojalactones A-C (1-3), with unusual γ-butyrolactone scaffolds. The complete structures, including the absolute configurations of 1-3, were determined based on spectroscopic data and total syntheses. In methylthiazole tetrazolium (MTT) assays, 1 and 2 showed moderate cytotoxicity against P388 cells.

Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Piceatanol: Anti-Cancer Compound From Gewang Seed Extract

Evaluation of antioxidant, antibacterial, antifungal and cytotoxic effects of Clausena suffruticosa ethanolic root extractMd. Atiar Rahman, Joti Sankhar Chakma, Saiful Islam, Nazim Uddin Ahmed

Fruit Extract from A Sechium edule Hybrid Induce Apoptosis in Leukaemic Cell Lines but not in Normal Cells

The antiproliferative potential of a crude extract from the chayote hybrid H-837-07-GISeM® and its potential for apoptosis induction were assessed in leukaemic cell lines and normal mouse bone marrow mononuclear cells (BM-MNCs). The extract strongly inhibited the proliferation of the P388, J774, and WEHI-3 cell lines (with an IC50 below 1.3 μg·mL−1), reduced cell viability, and induced apoptotic body production, phosphatidylserine translocation, and DNA fragmentation. However, the extract had no effect on BM-MNCs. We postulate that these properties make the extract a good candidate for an anti-tumour agent for clinical use.

Matrix metalloproteinase-9 is required for vasculogenic mimicry by clear cell renal carcinoma cells

BackgroundVasculogenic mimicry (VM), a new pattern of tumor microcirculation system, has been proved to be important for tumor growth and progression and may be one of the causes of antiangiogenesis resistance. Matrix metalloproteinase-9 (MMP9) was shown to correlate with VM formation in some other cancers. However, the relationship between VM formation and MMP9 in renal cell carcinoma (RCC) has not been determined. MethodsThe VM formation and MMP9 expressions were analyzed by CD34/periodic acid–Schiff dual staining and immunohistochemistry in 119 RCC specimens. We used a well-established 3-dimention culture model to compare VM formation in 786-O, 769-P, and HK-2 cell lines in vitro. MMP9 expressions on either messenger RNA or protein levels were compared among the cell lines by quantitative polymerase chain reaction or Western blot. To determine further the relationship between MMP9 and VM in RCC, 786-O and 769-P were treated with specific MMP9 inhibitor or small interfering RNA. VM formation, cell migration, and invasion were subsequently assessed by 3-dimention culture, wound-healing, and transwell assays. ResultsImmunohistochemistry demonstrated both VM formation and MMP9 overexpression were positively associated with clinical staging, pathological grade, and metastasis (P<0.01). VM formation was closely correlated with MMP9 overexpression in RCC (r = 0.602, P<0.01). Lower MMP9 expression level was observed in normal kidney cell line HK-2, which was unable to form VM on Matrigel, whereas higher expression of MMP9 was found in VM-forming cancer cell lines 786-O and 769-P. Inhibition of MMP9 not only disrupted VM formation in 786-O and 769-P but also reduced cell migration and invasion. ConclusionsThese results indicate an intimate relationship between MMP9 overexpression and VM formation in RCC. Treatments targeting VM formation by inhibiting the activity of MMP9 could be beneficial in RCC therapy.

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.

シアル酸結合レクチン(SBL)のマウス白血病細胞に対するアポトーシス誘導機構

シアル酸結合レクチン(SBL)のマウス白血病細胞に対するアポトーシス誘導機構

A New Flavonoid Derivative as Cytotoxic Compound Isolated from Ethyl Acetate Extract of Macaranga gigantifolia Merr. Leaves

A new prenylated flavonoid compound 5,7,3’,4’-tetrahydroxy-3,6-diprenylflavone (1) has been isolated from methanol extract of the leaves of Macaranga gigantifolia Merr. Isolation was performed using chromatography methods and their structures were elucidated based on spectroscopic data. cytotoxic activity against murine leukemia P-388 cell line conducted using MTT method. Compound 1 showed strong cytotoxic activity with IC50 value 6.19μg/mL

Multitarget Effects of Quercetin in Leukemia

This study proposes to investigate quercetin antitumor efficacy in vitro and in vivo, using the P39 cell line as a model. The experimental design comprised leukemic cells or xenografts of P39 cells, treated in vitro or in vivo, respectively, with quercetin; apoptosis, cell-cycle and autophagy activation were then evaluated. Quercetin caused pronounced apoptosis in P39 leukemia cells, followed by Bcl-2, Bcl-xL, Mcl-1 downregulation, Bax upregulation, and mitochondrial translocation, triggering cytochrome c release and caspases activation. Quercetin also induced the expression of FasL protein. Furthermore, our results demonstrated an antioxidant activity of quercetin. Quercetin treatment resulted in an increased cell arrest in G1 phase of the cell cycle, with pronounced decrease in CDK2, CDK6, cyclin D, cyclin E, and cyclin A proteins, decreased Rb phosphorylation and increased p21 and p27 expression. Quercetin induced autophagosome formation in the P39 cell line. Autophagy inhibition induced by quercetin with chloroquine triggered apoptosis but did not alter quercetin modulation in the G1 phase. P39 cell treatment with a combination of quercetin and selective inhibitors of ERK1/2 and/or JNK (PD184352 or SP600125, respectively), significantly decreased cells in G1 phase, this treatment, however, did not change the apoptotic cell number. Furthermore, in vivo administration of quercetin significantly reduced tumor volume in P39 xenografts and confirmed in vitro results regarding apoptosis, autophagy, and cell-cycle arrest. The antitumor activity of quercetin both in vitro and in vivo revealed in this study, point to quercetin as an attractive antitumor agent for hematologic malignancies.

Abstract 4388: Targeted VHL activation by RNAa inhibits renal cell carcinoma

Abstract Von Hippel Lindau (VHL) is a tumor suppressor protein classically known to target the α subunit of hypoxia inducible factor (HIF) for proteasomal degradation. Emerging evidence has underscored a novel role for VHL in both cell cycle regulation and extracelluar matrix assembly. Recent studies have reported that chemically synthesized small duplex RNAs complementary to target promoter sequence, also known as small activating RNA (saRNA), can specifically induce gene expression in cancer cells, which might be associated with epigenetic changes and exhibited long-lasting gene activation for nearly 2 weeks. In the present study, we designed 5 candidate dsRNAs (dsVHL-58, dsVHL-93, dsVHL-395, dsVHL-515 and dsVHL-675) targeted for the VHL promoter at sites ranging from -675 to -58 relative to the transcription start site. Compared with controls, dsVHL-675 induced VHL expression by approximately 10-fold in renal cell carcinoma 769-P (VHL methylated) cells. Transfection of a dsVHL-675 into 769-P cells significantly inhibited cell proliferation in dose dependent manner. In addition, single transfection of dsVHL-675 induced VHL gene expression for 14 days. Functional analysis of dsVHL-675 transfected cells revealed that dsVHL-675 induced apoptosis by activating caspase 3 in dose dependent manner. Knockdown experiments confirmed that induction of apoptosis by dsVHL-675 required Ago2 and TNRC6A. This study suggests that targeted activation of VHL by dsVHL-675 may be explored as a novel therapy for the treatment of renal cell carcinoma. Citation Format: Jong Soon Kang, Moo Rim Kang, Jieun Yun, Soo Jin Oh, Ki Hwan Park, Chang Woo Lee. Targeted VHL activation by RNAa inhibits renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4388. doi:10.1158/1538-7445.AM2014-4388

Non-mitogenic form of acidic fibroblast growth factor protects against graft-versus-host disease without accelerating leukemia.

Acid fibroblast growth factor (aFGF) has been shown to prevent epithelial damage under various conditions, suggesting its potential to inhibit GVHD. However, because aFGF receptors are expressed on tumor cells, it may possibly offset the graft-vs.-tumor (GVT) effects of allogeneic bone marrow transplantation (allo-BMT). Here, we addressed these questions in a B6→B6D2F1 allo-BMT model. Although aFGF administration attenuated GVHD in non-leukemic recipients, aFGF treatment markedly accelerated death in mice that received recipient-type tumor (P815) cells along with allo- or syngeneic-BMT. Similar protection against GVHD was achieved by administration of a non-mitogenic form of aFGF (naFGF). Importantly, GVT effects were fully preserved in naFGF-treated recipients. Furthermore, aFGF, but not naFGF, significantly enhanced P815 cell proliferation both in vitro and in vivo. Our data indicate that the tumor-promoting, but not GVHD-protecting, effect of aFGF largely depends on its mitogenic activity, and suggest that naFGF may provide a safer approach to inhibiting GVHD in patients with malignancies.

Panobinostat synergizes with bortezomib to induce endoplasmic reticulum stress and ubiquitinated protein accumulation in renal cancer cells.

BackgroundInducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. Inhibition of histone deacetylase (HDAC) 6 by the HDAC inhibitor panobinostat hinders the refolding of unfolded proteins by increasing the acetylation of heat shock protein 90. We investigated whether combining panobinostat with the proteasome inhibitor bortezomib would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby causing ubiquitinated proteins to accumulate and induce ER stress.MethodsCaki-1, ACHN, and 769-P cells were treated with panobinostat and/or bortezomib. Cell viability, clonogenicity, and induction of apoptosis were evaluated. The in vivo efficacy of the combination was evaluated using a murine subcutaneous xenograft model. The combination-induced ER stress and ubiquitinated protein accumulation were assessed.ResultsThe combination of panobinostat and bortezomib induced apoptosis and inhibited renal cancer growth synergistically (combination indexes <1). It also suppressed colony formation significantly (p <0.05). In a murine subcutaneous tumor model, a 10-day treatment was well tolerated and inhibited tumor growth significantly (p <0.05). Enhanced acetylation of the HDAC6 substrate alpha-tubulin was consistent with the suppression of HDAC6 activity by panobinostat, and the combination was shown to induce ER stress and ubiquitinated protein accumulation synergistically.ConclusionsPanobinostat inhibits renal cancer growth by synergizing with bortezomib to induce ER stress and ubiquitinated protein accumulation. The current study provides a basis for testing the combination in patients with advanced renal cancer.

Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice.

Acquired mutations in KIT are driver mutations in systemic mastocytosis (SM). Here, we tested the role of SHP2/PTPN11 phosphatase in oncogenic KIT signaling using an aggressive SM mouse model. Stable knock-down (KD) of SHP2 led to impaired growth, colony formation, and increased rates of apoptosis in P815 cells. This correlated with defects in signaling to ERK/Bim, Btk, Lyn, and Stat5 pathways in P815-KD cells compared to non-targeting (NT). Retro-orbital injections of P815 NT cells in syngeneic DBA/2 mice resulted in rapid development of aggressive SM within 13-16 days characterized by splenomegaly, extramedullary hematopoiesis, and multifocal liver tumors. In contrast, mice injected with P815 SHP2 KD cells showed less disease burden, including normal spleen weight and cellularity, and significant reductions in mastocytoma cells in spleen, bone marrow, peripheral blood and liver compared to NT controls. Treatment of human mast cell leukemia HMC-1 cells or P815 cells with SHP2 inhibitor II-B08, resulted in reduced colony formation and cell viability. Combining II-B08 with multi-kinase inhibitor Dasatinib showed enhanced efficacy than either inhibitor alone in blocking cell growth pathways and cell viability. Taken together, these results identify SHP2 as a key effector of oncogenic KIT and a therapeutic target in aggressive SM.