Pterostilbene Inhibits the Melanogenesis Activity in UVB-Irradiated B164A5 Cells

Pterostilbene is a potent antioxidant and anti-inflammatory agent. However, its chemopreventive effects via anti-tyrosinase activity and inhibitory effects on melanin content have not been reported previously. Hence, this study aimed to investigate the anti-melanogenic activity of pterostilbene on UVB-irradiated B164A5 mouse melanoma cells. The effects of pterostilbene and resveratrol on cell viability were determined by MTT assay, whereas melanin content and tyrosinase assay were employed to assess melanogenesis activity. Western blot analysis was performed to determine the tyrosinase expression. Based on the MTT assay, the IC50 value of pterostilbene on UVB-irradiated B164A5 cells was 34.0 ± 3.43 μM, in comparison to resveratrol (>100 μM). Next, 5 and 10 μM pterostilbene showed a significant dose-dependent inhibition ( P < .01) of tyrosinase activity in UVB-irradiated B164A5 cells at 37.14 ± 2.71% and 58.36 ± 6.8%, respectively. The findings from the tyrosinase assay also confirmed the downregulation of tyrosinase expression in UVB-irradiated B164A5 cells as measured by Western blot analysis. Finally, 10 μM pterostilbene showed a significantly decreased melanin content ( P < .01) in UVB-irradiated B164A5 cells, at 27.34 ± .98 μg/mL. In conclusion, pterostilbene showed anti-melanogenic activity that was 10 times more potent than resveratrol in the UVB-irradiated B164A5 cell.

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Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

Reproduction ◽

10.1530/rep.0.1250495 ◽

2003 ◽

pp. 495-507 ◽

Cited By ~ 6

Author(s):

SA Joshi ◽

S Shaikh ◽

S Ranpura ◽

VV Khole

Keyword(s):

Western Blot ◽

Postnatal Development ◽

Blot Analysis ◽

Western Blot Analysis ◽

Polyclonal Antiserum ◽

Cognate Gene ◽

Specific Proteins ◽

Androgen Regulation ◽

Dose Dependent

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.

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FGF21 Ameliorates Hepatic Fibrosis by Multiple Mechanisms

10.21203/rs.3.rs-418625/v1 ◽

2021 ◽

Author(s):

Fanrui Meng ◽

Mir Hassan Khoso ◽

Kai Kang ◽

Qi He ◽

Yukai Cao ◽

...

Keyword(s):

Protein Expression ◽

Western Blot ◽

Hepatic Fibrosis ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cell Model ◽

Dependent Manner ◽

Rt Pcr ◽

Mrna And Protein Expression ◽

Dose Dependent

Abstract Previous study reports that FGF21 could ameliorate hepatic fibrosis, but its mechanisms have not been fully investigated. In this study, three models were used to investigate the mechanism by which FGF21 alleviates liver fibrosis. CCL4 and DMN were respectively used to induce hepatic fibrosis animal models. Our results demonstrated that liver index and liver function were deteriorated in both models. HE and Masson’s staining showed that the damaged tissue architectonics were observed in the mice of both models. Treatment with FGF21 significantly ameliorated these changes. ELISA analysis showed that the serum levels of IL-1β, IL-6 and TNF-α were significantly elevated in both models. However, administration of FGF21 significantly reduced these inflammatory cytokines. RT-PCR and Western blot analysis showed that mRNA and protein expression of collagenI, α-SMA and TGF-β were significantly decreased by treatment with FGF21. PDGF-BB stimulant was used to establish the experimental cell model in HSCs. RT-PCR and Western blot analysis demonstrated that the expression of collagenI and α-SMA were significantly upregulated by this stimulant in model group. Interestingly, our results showed that mRNA and protein expression of leptin were also significantly induced in PDGF-BB treated HSCs. Administration of FGF21 could significantly reduce leptin expression in a dose dependent manner and these effects were reversed in siRNA (against β-klotho) transfected HSCs. Furthermore, the leptin signaling pathways related protein p-ERK/t-ERK, p-STAT3/STAT3 and TGF-β were significantly downregulated by FGF21 treatment in a dose dependent manner. The expression of SOCS3 and Nrf-2 were enhanced by treatment with FGF21. The underlying mechanism may be that FGF21 regulates leptin-STAT3 axis via Nrf-2 and SOCS3 pathway in activated HSCs.

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Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.2759.2759 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2759-2759 ◽

Cited By ~ 1

Author(s):

Varun V Prabhu ◽

Amriti Lulla ◽

Christina L Kline ◽

Peter J Van den Heuvel ◽

Mala K. Talekar ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Single Agent ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent Decrease ◽

Dose Dependent

Abstract ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM). In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM. We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results. In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells. In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. Disclosures Prabhu: Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

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A Novel Method for Screening Anti-Angiogenic Compounds: The Chorionicallantoic Membrane (CAM)/Feather Bud (FB) Assay.

Blood ◽

10.1182/blood.v112.11.1896.1896 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1896-1896

Author(s):

Haiming Chen ◽

Cathy S Wang ◽

Mingjie Li ◽

Eric Sanchez ◽

Jennifer Li ◽

...

Keyword(s):

Zoledronic Acid ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Rt Pcr ◽

New Agents ◽

Angiogenic Activity ◽

Marked Inhibition ◽

Dose Dependent Fashion ◽

Dose Dependent

Abstract Enhanced angiogenesis is a hallmark of solid tumors and hematological malignancies, and anti-angiogenic therapeutic approaches have recently shown significant benefit in the clinic. As a result, many anti-angiogenic agents are currently in early development. Very few methods have been used to evaluate the anti-angiogenic activity of these agents using an ex vivo assay. Unfortunately, currently available methods are both time consuming and costly. We have developed a novel approach to test the anti-angiogenic activity of new agents in a rapid, accurate and inexpensive way. This model consists of using a combined chorioallantoic membrane (CAM) and feather bud (FB) assay. The CAM already has a well developed vascular network and provides an ideal microenvironment and the FB serves as an active biological testing tool for evaluating angiogenesis. FB is a component of epithelial and mesenchymal cells. The method consists of using fertilized chick eggs incubated horizontally at 37.5°C in a humidified incubator and windowed by day 8. Another set of E8 chicken embryonic skins are collected under a dissecting microscope to isolate FB. The FB is treated with drugs or control reagents and implanted onto the CAM. The eggs are sealed with an adhesive tape and incubated for an additional 2–4 days. The endothelial cells of CAM proliferate and migrate into the FB after two days. After 4 days of culture, both blood vessel formation and FB development are determined by microscopy. New blood vessels in FB are analyzed by H&E and immunohistochemical (IHC) staining and expression of endothelial genes and proteins using RT-PCR and Western blot analysis, respectively. First, we establish that the compound being tested should only affect endothelial proliferation or migration and not kill the epithelial and mesenchymal tissues. We have used this new method to investigate several compounds. First, we evaluated the anti-angiogenic agent fumagillin (1μM) and minocycline (100nM). Although neither drug had any cytotoxic effects on the epithelial and mesenchymal tissues when cultured alone, marked inhibition of FB development occurred on the CAM in a dose-dependent fashion with both drugs as determined by microscopy and IHC. In addition, Western blot analysis showed marked inhibition of Tie-2 protein expression in a dose-dependent fashion in the presence of these drugs. Zoledronic acid, a potent bisphosphonate which has recently been shown to harbor anti-angiogenic activity, was found to markedly inhibit FB development in the presence of this drug at a concentration of 10 μM whereas less effect was observed at 2 μM. This drug did not have any direct effect on epithelial and mesenchymal cells when these tissues were cultured alone. We then examined gene and protein expression of the FB cells on CAM that were treated with zoledronic acid. Both FLK-1 and Tie-2 transcript and protein levels were significantly reduced in a dose-dependent fashion following treatment with zoledronic acid as assessed by RT-PCR and Western blot analysis. We are currently testing the potential anti-angiogenic effects of many other novel drugs using this new model. Overall, the present findings demonstrate that the CAM/ FB angiogenesis model is likely to be a reliable, fast, sensitive, and economical system to screen the anti-angiogenic effects of new agents.

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Semaphorin 3A Inhibits Inflammation in Chondrocytes under Excessive Mechanical Stress

Mediators of Inflammation ◽

10.1155/2018/5703651 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 13

Author(s):

Chikako Sumi ◽

Naoto Hirose ◽

Makoto Yanoshita ◽

Mami Takano ◽

Sayuri Nishiyama ◽

...

Keyword(s):

Gene Expression ◽

Western Blot ◽

Mechanical Stress ◽

Inflammatory Cytokines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Dependent Manner ◽

Semaphorin 3A ◽

Neuropilin 1 ◽

Dose Dependent

Background. Excessive mechanical stress causes inflammation and destruction of cartilage and is considered one of the cause of osteoarthritis (OA). Expression of semaphorin 3A (Sema3A), which is an axon guidance molecule, has been confirmed in chondrocytes. However, there are few reports about Sema3A in chondrocytes, and the effects of Sema3A on inflammation in the cartilage are poorly understood. The aim of this study was to examine the role of Sema3A in inflammation caused by high magnitude cyclic tensile strain (CTS). Methods. Expression of Sema3A and its receptors neuropilin-1 (NRP-1) and plexin-A1 (PLXA1) in ATDC5 cells was examined by Western blot analysis. ATDC5 cells were subjected to CTS of 0.5 Hz, 10% elongation with added Sema3A for 3 h. Gene expression of IL-1β, TNF-ɑ, COX-2, MMP-3, and MMP-13 was examined by qPCR analysis. Furthermore, the phosphorylation of AKT, ERK, and NF-κB was detected by Western blot analysis. Results. Added Sema3A inhibited the gene expression of inflammatory cytokines upregulated by CTS in a dose-dependent manner. Addition of Sema3A suppressed the activation of AKT, ERK, and NF-κB in a dose-dependent manner. Conclusions. Sema3A reduces the gene expression of inflammatory cytokines by downregulating the activation of AKT, ERK, and NF-κB pathways in ATDC5 cells under CTS.

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Identification of a 71 kDa protein as a putative non-genomic membrane progesterone receptor in boar spermatozoa

Journal of Endocrinology ◽

10.1677/joe.1.05607 ◽

2005 ◽

Vol 184 (2) ◽

pp. 417-425 ◽

Cited By ~ 12

Author(s):

S Jang ◽

L S H Yi

Keyword(s):

Progesterone Receptor ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Membrane Fraction ◽

Cytosolic Fraction ◽

Dependent Manner ◽

Boar Spermatozoa ◽

Dose Dependent ◽

Ligand Blot

A putative non-genomic progesterone receptor was identified by Western blot analysis from the membrane fraction but not the cytosolic fraction of boar spermatozoa using monoclonal antibody (mAb) C-262. When the membrane and the cytosolic fractions of boar liver, kidney, uterus and spermatozoa were analyzed with mAb C-262, protein bands with molecular masses of 86 and 120 kDa were detected from the cytosolic fraction of the uterus, whereas a 71 kDa protein was detected from the membrane fraction of spermatozoa. Apparently, while the 86 and 120 kDa proteins from the uterus correspond to the genomic progesterone receptor isoforms A and B in boar, the 71 kDa protein of the sperm membrane fraction seems to be a novel membrane-associated progesterone receptor. Ligand blot assay of the membrane and the cytosolic fractions of boar spermatozoa performed with peroxidase-conjugated progesterone revealed that only the 71 kDa membrane protein binds specifically to progesterone, reinforcing the results obtained from the Western blot analysis. Also ligand blot assays performed in the presence of mAb C-262 demonstrated that mAb C-262 inhibited progesterone binding to the 71 kDa protein in a dose-dependent manner. Ligand blot assays performed in the presence of free progesterone, RU486 or estrogen revealed that binding of peroxidase-conjugated progesterone to the 71 kDa protein was inhibited by free progesterone and RU486 in a dose-dependent manner but not by estrogen, which further confirms that progesterone binds to the 71 kDa protein specifically. Furthermore, the progesterone-induced acrosome reaction was inhibited by mAb C-262 in a dose-dependent manner. These results strongly imply that spermatozoa possess a progesterone receptor in a membrane-bound form and can be influenced by progesterone via non-genomic progesterone receptor.

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Cancer cells are selectively eliminated through UPR driven apoptosis during treatment with Aquilegia nivalis extracts

10.21203/rs.3.rs-234542/v1 ◽

2021 ◽

Author(s):

Nazia Hilal ◽

Ozaira Qadri ◽

Irshad A Nawchoo ◽

Seema Akbar ◽

Khalid Majid Fazili

Keyword(s):

Cell Cycle ◽

Western Blot ◽

Dna Fragmentation ◽

Cell Lines ◽

Cancer Cell ◽

Blot Analysis ◽

Western Blot Analysis ◽

Methanolic Extract ◽

Dapi Staining ◽

Dose Dependent

Abstract BackgroundAquilegia nivalis Flax Jackson, also called Aquilegia vulgaris sub sp. nivalis (Bak.) Brühl or columbine, locally known as “Zoe-neel”, is a wild edible plant traditionally used as an anti-inflammatory medicine by the local nomadic tribes inhabiting the Himalayas of Jammu and Kashmir. The plant has been used as herbal medicine since middle ages in treating ailments that include chronic rhinitis and various infectious diseases. The extracts from the plant possess antioxidant properties and have been reported to be hepatoprotective in rats. Our preliminary studies, however, pointed to hitherto unexplored anti-apoptotic potential of the plant which lead us to carry the in-depth study using breast cancer cell lines to validate its anti-cancerous properties and explore the affected pathways.MethodsMTT assay was used to draw the dose response curve and evaluate the effect of increasing concentrations of the extract on cell lines to determine the appropriate dosage to be used for further experimentation. DNA fragmentation analysis was followed through gel electrophoresis and DAPI staining was pursued by phase contrast microscopy to study apoptosis. Quantitative PCR was used to study the expression of UPR signaling and RIDD markers at the level of mRNA. Western blot analysis was used in studying the expression of the various markers of the signaling pathways. The cell cycle analysis was carried out using flow cytometry.ResultsMTT assay revealed that the methanolic extract of the plant (ANME) was selectively cytotoxic to various cancer cell lines as revealed by lower IC50 values relative to normal cell lines. The results of cell cycle analysis were similar as ANME caused Sub G1 arrest of the cell cycle. DNA fragmentation analysis, DAPI staining and western blot analysis for PARP and caspases revealed that the extract selectively induced apoptosis in cancerous cell lines. UPR markers p-Ire1α and Xbp1 splicing were consistently alleviated in a dose dependent manner, the rate of phosphorylation of eIF2a and ATF4 also decreased with increasing concentration of ANME. The RT PCR results of the RIDD marker, Blos1S1 revealed a similar dose dependent association. The methanolic extract was especially chosen for it could be easily internalized by the cells and any resultant potential bioactive compounds could gain access to the cells because of their hydrophobic nature.ConclusionOur results suggest that ANME causes deactivation of UPR signaling pathway facilitating apoptosis selectively in cancerous cells, paving the way forward for a novel approach in cancer therapeutics.

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Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽

10.1530/reprod/119.1.15 ◽

2000 ◽

pp. 15-23 ◽

Cited By ~ 15

Author(s):

K Jewgenow ◽

M Rohleder ◽

I Wegner

Keyword(s):

In Vitro Fertilization ◽

Zona Pellucida ◽

Antigenic Determinants ◽

Vitro Fertilization ◽

Contraceptive Vaccine ◽

Sperm Binding ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

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Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

Journal of Neurosurgery ◽

10.3171/2010.7.gks10972 ◽

2010 ◽

Vol 113 (Special_Supplement) ◽

pp. 228-235 ◽

Cited By ~ 4

Author(s):

Qiang Jia ◽

Yanhe Li ◽

Desheng Xu ◽

Zhenjiang Li ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Western Blot ◽

Gamma Knife ◽

Blot Analysis ◽

Western Blot Analysis ◽

Glioma Cells ◽

C6 Glioma ◽

C6 Glioma Cells ◽

C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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Antioxidant effects and in vitro cytotoxicity on human cancer cell lines of flavonoid-rich Flamboyant (Delonix regia (Bojer) Raf.) flower extract

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666201029154746 ◽

2020 ◽

Vol 21 ◽

Author(s):

Putthiporn Khongkaew ◽

Phanphen Wattanaarsakit ◽

Konstantinos I. Papadopoulos ◽

Watcharaphong Chaemsawang

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Flower Extract ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Murine Leukemia Cell ◽

Dose Dependent

Background: Cancer is a noncommunicable disease with increasing incidence and mortality rates both worldwide and in Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Introduction: Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. Methods and Results: In the present report, using High Performance Liquid Chromatography (HPLC), we demonstrate in the DRE, the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30 % w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01 % w/w respectively while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 µg/ml and 53.65±7.24 µg/ml respectively. Discussion: Our cancer cell line studies using the MTT assay demonstrated DREs potent and dose dependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Conclusion: Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract’s lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

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