• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7473-7478
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• 2014

Cholangiocarcinoma (CCA) is a fatal cancer with poor prognosis and less than 10% of CCA patients can be offered surgical cure. Conventional chemotherapy results in unfavorable outcomes. At present, plant-derived compounds are gaining interest as potential cancer therapeutics, particularly for treatment-refractory cancers. In this study, antitumor activity of tiliacorinine, the major alkaloid isolated from a tropical plant, on CCA was first demonstrated. Antiproliferative effects of tiliacorinine on human CCA cell lines were investigated using SRB assays. Acridine orange/ethidium bromide staining, flow cytometric analysis and DNA laddering assays were used for apoptotic determination. Apoptosis-related proteins were verified by Western blotting and antitumor activity of tiliacorinine in vivo was demonstrated in CCA xenografted mice. Tiliacorinine significantly inhibited proliferation of human CCA cell lines with $IC_{50}$ $4.5-7{\mu}M$ by inducing apoptosis through caspase activation, upregulation of BAX, and downregulation of $Bcl_{xL}$ and XIAP. Tiliacorinine considerably reduced tumor growth in CCA xenografted mice. These results demonstrated antitumor effects of tiliacorinine on human CCA in vitro and in vivo. Tiliacorinine may be an effective agent for CCA treatment.

• Archives of Pharmacal Research
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• 제23권2호
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• pp.121-127
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• 2000

Cannabigerol (1, CBG), methyl 4-[(2E)-3,7-dimethyl-2,6-octad ienyl)oxy]-3-methoxybenzoate (2, DTM), 5-fluorouracil (3, FU) as a reference, and cannabidiol (4, CBD) were tested for their growth inhibitory effects against KB(ATCC NO, OCL 17) cell lines using two different assays, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and the sulforhod-amine B protein (SRB) assay. These compounds showed inhibitory activity in vitro in the micromolar range against KB cell lines. In general, the antitumor activities of these compounds (1, 2, 3 and 4) were dose-dependent over the micromolar concentration range of 1 to 100 M. The comparison of $IC_{50}$ values of these compounds in tumor cell lines showed that their susceptibility to these compounds decreases in the following order: DTM > CBD > 5-FU > CBG by MTT assay and DTM = CBD > 5-FU > CBG by SRB assay. CBG 1, DTM 2, 5-FU 3, and CBD 4 were tested for their cytotoxic effects on NIH 3T3 fibroblasts using two different assays, the MTT assay and SRB assay. These compounds exhibited potent cytotoxic activities in vitro in the micromolar range against NIH 3T3 fibroblasts. In general, the cytotoxic acivities of these compounds (1, 2, 3 and 4) were dose-dependent over the micromolar concentraion range of 1 to 100 M. The comparison of $CD_{50}$ values of these compounds in NIH 3T3 fibroblasts shows that their susceptibility to these compounds in decreases the following order(:) CBD > 5-FU > DTM > CBG by MTT assay, CBD > 5-FU > CBG > DTM by SRB assay. These results suggest that DTM 2 has the most growth-inhibitory activity against KB cell lines.

• Mass Spectrometry Letters
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• 제3권1호
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• pp.21-24
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• 2012

$5{\alpha}$-Dihydrotestosterone (DHT) is the primary active metabolite of testosterone, catalyzed by $5{\alpha}$-reductase ($5{\alpha}R$) in the skin, prostate, and liver. In this study, the $5{\alpha}R$ activity in rat liver S9 fraction in the presence of a NADPH-generating system was evaluated and compared by gas chromatography-mass spectrometry (GC-MS)-based in vitro assays. Testosterone and a $5{\alpha}R$ inhibitor, finasteride, were added to the S9 fractions and incubated at $37^{\circ}C$ for 1 h. Both testosterone and DHT were quantitatively measured and compared with two different GC-MS-based steroid profiling techniques. DHT was not detected by conventional GC-MS analysis in the absence of finasteride when the concentration of testosterone in the S9 fraction was less than $0.2{\mu}M$, whereas the isotope-dilution GC-MS (GC-IDMS) system was able to evaluate the $5{\alpha}R$ activity. Because the S9 fraction contains more reactive enzymes and is easier to collect from tissues compared with a microsomal solution, the combination of the S9 fraction and GC-IDMS technique may be a promising assay for evaluating the $5{\alpha}R$ activity in large-scale clinical studies.

• Advances in Traditional Medicine
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• 제8권3호
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• pp.266-278
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• 2008

Erythrina stricta, a deciduous tree widely used traditionally in indigenous system of medicine for various ailments such as rheumatism, fever, leprosy, epilepsy etc. The leaves of Erythrina stricta was extracted with ethanol (70%) and used for the evaluation of various in vitro antioxidant assays which includes H - donor activity, nitric oxide scavenging, superoxide anion scavenging, reducing ability, hydroxyl radical, hydrogen peroxide scavenging, total phenolic content, total flavonoid content, total antioxidant activity by thiocyanate and phosphomolybdenum method, metal chelating, $\beta$-carotene bleaching, total peroxy radical assays. The pro-oxidant activity was measured using bleomycin-dependent DNA damage. Ex vivo models like lipid peroxidation and erythrocyte haemolysis were also used to study the antioxidant property of the extract. The various antioxidant activities were compared with suitable standard antioxidants such as ascorbic acid, butylated hydroxyl toluene, $\alpha$-tocopherol, curcumin, quercetin and Trolox. The generation of free radicals viz. $O_2^{{\cdot}-}$, $OH^{\cdot}$, $H_2O_2$, $NO^{\cdot}$ and peroxyl radicals were effectively scavenged by the ethanolic extract of Erythrina stricta. In all the methods, the extract offered strong antioxidant activity in a concentration dependent manner. The total phenolic content, flavonoid content and total antioxidant activity in Erythrina stricta were determined as microgram (g) pyrocatechol, quercetin and $\alpha$-tocopherol equivalent/mg respectively. The extract did not exhibit any prooxidant activity when compared with ascorbic acid. The results obtained in the present study clearly indicates that Erythrina stricta scavenges free radicals and reduces lipid peroxidation, ameliorating the damage imposed by oxidative stress in different disease conditions and serve as a potential source of natural antioxidant.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제50권4호
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• pp.206-215
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• 2024

Objectives: The current in vitro study aimed to assess the effects of ascorbic acid augmented albumin platelet-rich fibrin (AA Alb-PRF) on the wound healing activity of human gingival fibroblasts (HGFs) purported to be a regenerative biomaterial in surgical procedures. Materials and Methods: All assays were performed on three HGF groups, group I: complete media; group II: Alb-PRF, and group III: AA Alb-PRF. Alb-PRF was prepared following the protocol by Fujioka-Kobayashi et al. (2021). For preparation of AA Alb-PRF, 2,500 ㎍ AA was added to the blood pre-centrifugation. All groups were subjected to 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to estimate cell viability and proliferation, scratch assay for migration (0, 4, 12, and 24 hours) and transwell migration assay for chemotactic migration assessment (24 hours). Outcome variables were optical density (OD) for MTT assay, percentage of wound closure in scratch assay, and number of migrated cells in transwell migration assay. One-way ANOVA for MTT and transwell migration assays and two-way ANOVA for scratch assay with Bonferroni correction were performed with significance set at P<0.05. Results: Cell viability and proliferation (OD: 0.684±0.003 and proliferation: 28%) and wound closure (49.92%±1.62% at 4 hours and 61.39%±0.88% at 12 hours) were significantly higher in group III, while group II demonstrated the maximum number of HGFs migrating across the transwell membrane (9.25±2.49) with P<0.05. Conclusion: HGFs demonstrated a significant increase in viability and proliferation along with rapid wound closure in the presence AA Alb-PRF compared to Alb-PRF alone, indicating additional beneficial effects of AA. Thus, AA Alb-PRF potentiates the wound healing activity of HGFs and could be employed in oral, maxillofacial, and periodontal surgeries as a regenerative biomaterial.

• 동아시아식생활학회지
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• 제15권3호
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• pp.315-322
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• 2005

In this study we report on the estrogen activity of silkworm pupa and herb extracts in vitro. The estrogenic activity of these resources was investigated by competition binding assays with estrogen receptor $\alpha(ER{\alpha})\;or\;ER{\beta}$, and viability of MCF-7 cells, a human breast cancer cell line. Saturation ligand-binding analysis of $ER{\alpha}\;and\;ER{\beta}$ revealed that all plant extracts competed with estrogen ligand for binding to both ER subtypes with a similar preference and degree and competed stronger with ligand for binding to $ER{\beta}\;than\;to\;ER{\alpha}$. The highest $ER{\alpha}-binding$ sample was silkworm pupa aqueous extract The highest $ER{\beta}-binding$ sample was silkworm pupa oil. These samples were further tested for bioactivity based on their ability to regulate cell growth rate in ER(+) breast cancer cell line, MCF-7 cells. Our studies showed that silkworm pupa, soritae, sesame, yam, pueraria, malt, ginseng, Polygonum multiflorum, and Curcuma longa significantly stimulated the growth of MCF-7 cells (P<0.05). In summary, these results suggested that silkworm pupa and herbs might be useful as potential phytoestrogens.

• International Journal of Advanced Culture Technology
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• 제6권4호
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• pp.109-115
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• 2018

Garlic is a medicinal plant used throughout the world for its anti-inflammatory, antioxidant, and antiplatelet activities. Chitosan is a natural polysaccharide obtained from chitin, and derivatives of chitosan have been shown to inhibit platelet aggregation and adhesion. We hypothesized that fermented preparations of these products may possess stronger antiplatelet effects than the non-fermented forms owing to the increased bioavailability of the bioactive compounds produced during fermentation. Therefore, we compared these compounds via in vitro and ex vivo platelet aggregation assays by using standard light transmission aggregometry and ex vivo granule secretions from rat platelets. We found that fermented preparations exerted more potent and significant inhibition of platelet aggregation both in vitro and ex vivo. Likewise, ATP release from dense granules of platelets was also significantly inhibited in fermented preparation-treated rat platelets compared to that in non-fermented preparation-treated ones. We concluded that fermented preparations exerted more potent effects on platelet function both in vitro and ex vivo, possibly as a result of the increased bioavailability of active compounds produced during fermentation. We therefore suggest that fermented products may be potent therapeutics against platelet-related CVDs and can be used as antiplatelet and antithrombotic agents.

• Asian-Australasian Journal of Animal Sciences
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• 제16권9호
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• pp.1261-1267
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• 2003

We have investigated whether HspBP1, a Hsp70 binding protein, could have effect on the assembly of the bovine progesterone receptor (bPR) with a chaperone complex consisting of bovine Hsp90 (bHsp90), bovine Hsp70 (bHsp70), Hop, Ydj-1, and p23. The bPR, isolated in its native conformation, loses its function to interact with progesterone hormone in the absence of this protein complex. However, in the presence of bHsp90, bHsp70, Hop, p23 and Ydj-1, its function could be restored in vitro. Our findings here indicate that the inclusion of HspBP1 to five-protein system prevented the proper assembly of progesterone receptor-chaperone complex and induce the loss of bPR ability to interact with hormone. Immunoprecipitation assays of bPR with HspBP1 show that the presence of HspBP1 did not have any effect on the assembly of Ydj-1 and bHsp70 with the progesterone receptor. However, further assembly of Hsp90, Hop and p23 was completely prevented and the function of the bPR was lost. In vitro competition and protein folding assays indicated that the binding of HspBP1 to bHsp70 prevented the ternary complex formation of bHsp70, bHsp90, and Hop. These results indicate that HspBP1 is a negative regulator of the assembly of Hsp90, Hop and Hsp70, and thus, prevent the proper maturation of unliganded bPR with chaperones assembly system.

• Asian Pacific Journal of Cancer Prevention
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• 제15권7호
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• pp.3211-3217
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• 2014

Background: The current study investigated the effects of Piper longumine on radio-sensitization of human breast cancer MDA-MB-231 cells and underlying mechanisms. Materials and Methods: Human breast cancer MDA-MB-231 cells were cultured in vitro and those in logarithmic growth phase were selected for experiments divided into four groups: control, X-ray exposed, Piper longumine, and Piper longumine combined with X-rays. Conogenic assays were performed to determine the radio-sensitizing effects. Cell survival curves were fitted by single-hit multi-target model and then the survival fraction (SF), average lethal dose ($D_0$), quasi-threshold dose ($D_q$) and sensitive enhancement ratio (SER) were calculated. Cell apoptosis was analyzed by flow cytometry (FCM). Western blot assays were employed for expression of apoptosis-related proteins (Bc1-2 and Bax) after treatment with Piper longumine and/or X-ray radiation. The intracellular reactive oxygen species (ROS) level was detected by FCM with a DCFH-DA probe. Results: The cloning formation capacity was decreased in the group of piperlongumine plus radiation, which displayed the values of SF2, D0, Dq significantly lower than those of radiation alone group and the sensitive enhancement ratio (SER) of D0 was1.22 and 1.29, respectively. The cell apoptosis rate was increased by the combination treatment of Piper longumine and radiation. Piper longumine increased the radiation-induced intracellular levels of ROS. Compared with the control group and individual group, the combination group demonstrated significantly decreased expression of Bcl-2 with increased Bax. Conclusions: Piper longumine at a non-cytotoxic concentration can enhance the radio-sensitivity of MDA-MB-231cells, which may be related to its regulation of apoptosis-related protein expression and the increase of intracellular ROS level, thus increasing radiation-induced apoptosis.

• Molecules and Cells
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• 제39권2호
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• pp.149-155
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• 2016

In the heart, excitation-contraction (E-C) coupling is mediated by $Ca^{2+}$ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the $Ca^{2+}$ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich $Ca^{2+}$ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202-231). Second, in vitro binding assays were conducted to examine the $Ca^{2+}$ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped $Ca^{2+}$ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such $Ca^{2+}$ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of $Ca^{2+}$ into SR at intermediate $Ca^{2+}$ concentrations.