• Herbal Formula Science
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• v.27 no.1
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• pp.53-63
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• 2019

Objectives : In this study, we investigated the combination effects of Cinnamomi cortex Ethanol Extract (CcEE) and hyperthermia in the human AGS gastric cancer cell line. Methods : AGS cells were treated with the indicated concentrations of CcEE (0, 50 or $60{\mu}g/mL$) for 1h prior to hyperthermia. And then incubated for a further 30 min at the indicated temperatures (37, 42 or $43^{\circ}C$) in a humidified incubator containing 5% $CO_2$ or a thermostatically controlled water bath for hyperthermia. The cell viability was measured by MTT assay, Morphology assay and Trypan blue assay. To investigate the possible molecular signaling pathways, the activation of mitogen-activated protein kinase (MAPK) proteins (ERK, p38 and JNK) and expression of various anti-apoptotic proteins such as Caspase-3, Caspase-9, p53, Cyclin D1 and MMP-2 were assessed by Western blot analysis. In addition, Annexin V and 7-amino-actinomycin D (7-AAD) staining was performed to examine the apoptotic mechanism. Results : Combination of CcEE with hyperthermia effectively suppressed the cell viability and changed cellmorphology compared with CcEE or hyperthermia treatment alone. Combined treatment also abated the expression of Caspase-3, Caspase-9, Cyclin D1 and MMP-2. Whereas, the expression level of p53 was up-regulated by co-treatment. Moreover, combination treatment enhanced phosphorylation of ERK, p38 and JNK. In addition, this combination increased anti-cancer effect by inducing cell death through the apoptosis. Conclusions : Taken together, all these findings suggest that the combination treatment with CcEE and hyperthermia may have therapeutic potential as a promising approach to patients with stomach cancer.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.775-783
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• 2021

Sea cucumber, Holothuria scabra, is a well-known traditional Asian medicine that has been used for suppressing inflammation, promoting wound healing, and improving immunity. Moreover, previous studies demonstrated that the extract from H. scabra contains many bioactive compounds with potent inhibitory effect on tumor cell survival and progression. However, the effect of the methanolic extract from the body wall of H. scabra (BWMT) on human prostate cancer cells has not yet been investigated. In this study, we aimed to investigate the effects and underlying mechanism of BWMT on prostate cancer cell viability and metastasis. BWMT was obtained by maceration with methanol. The effect of BWMT on cell viability was assessed by MTT and colony formation assays. The intracellular ROS accumulation was evaluated using a DCFH-DA fluorescence probe. Hoechst 33342 staining and Annexin V-FITC/PI staining were used to examine the apoptotic-inducing effect of the extract. A transwell migration assay was performed to determine the anti-metastasis effect. BWMT significantly reduced cell viability and triggered cellular apoptosis by accumulating intracellular ROS resulting in the upregulation of JNK and p38 signaling pathways. In addition, BWMT also inhibited the invasion of PC3 cells by downregulating MMP-2/-9 expression via the ERK pathway. Consequently, our study provides BWMT from H. scabra as a putative therapeutic agent that could be applicable against prostate cancer progression.

• Journal of Microbiology and Biotechnology
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• v.30 no.6
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• pp.893-902
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• 2020

Propolis is a resinous substance that is collected by Apis mellifera from plant sources and is used in traditional medicine. To study the phytochemical constituents and apoptotic potential of Jordanian propolis extract against different cancer cell lines, propolis was extracted using methanol, hexane, and ethyl acetate and was fractionated using chromatographic methods. Cytotoxicity was assessed using MTT and LDH assays. The apoptotic potential was investigated using florescence microscopy, multicaspase assay, Annexin-V and dead cell assay, and cell cycle assay. The phytochemical constituents were analyzed using GC-MS. The methanol extract of propolis exhibited cytotoxic potential against all cell lines tested. The IC₅₀ values of the methanol extract were 47.4, 77.8, 91.2, and 145.0 ㎍/ml for HepG2, LoVo, MDAMB231, and MCF7 cell lines, respectively. The IC₅₀ values of the F1 fraction were 31.6 (MDAMB231), 38.9 (HepG2), 36.7 (LoVo) and 75.5 (MCF7) ㎍/ml. On further purification using thin-layer chromatography, the IC₅₀ values of the F1-3 fraction were found to be 84.31(HepG2), 79.2 (MCF7), 70.4 (LoVo), and 68.9 (MDAMB231) ㎍/ml, respectively. The anticancer potential of the F1 fraction was confirmed through the induction of apoptosis and cell cycle arrest at the G0/G1 phase. The GC-MS analysis of the F1 fraction revealed the presence of 3-methyl-4-isopropylphenol (29.44%) as a major constituent. These findings indicate the potential of propolis extract as a cancer therapy. However, further investigation is required to assess the acute and subacute toxicity of the most active fraction.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.2
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• pp.581-590
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• 2016

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4249-4254
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• 2013

Background: Parafibromin is a protein encoded by the HRPT2 (hyperparathyroidism 2) oncosuppressor gene and its down-regulated expression is involved in pathogenesis of parathyroid, breast, gastric and colorectal carcinomas. This study aimed to clarify the effects of parafibromin expression on the phenotypes and relevant mechanisms of DLD-1 colon carcinoma cells. Methods: DLD-1 cells transfected with a parafibromin-expressing plasmid were subjected to examination of phenotype, including proliferation, differentiation, apoptosis, migration and invasion. Phenotype-related proteins were measured by Western blot. Parafibromin and ki-67 expression was detected by immunohistochemistry on tissue microarrays. Results: The transfectants showed higher proliferation by CCK-8, better differentiation by electron microscopy and ALP activity and more apoptotic resistance to cisplatin by DNA fragmentation than controls. There was no difference in early apoptosis by annexin V, capase-3 activity, migration and invasion between DLD-1 cells and their transfectants. Ectopic parafibromin expression resulted in down-regulated expression of smad4, MEKK, GRP94, GRP78, $GSK3{\beta}$-ser9, and Caspase-9. However, no difference was detectable in caspase-12 and -8 expression. A positive relationship was noted between parafibromin and ki-67 expression in colorectal carcinoma. Conclusions: Parafibromin overexpression could promote cell proliferation, apoptotic resistance, and differentiation of DLD-1 cells.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.12
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• pp.5139-5145
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• 2016

There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to $100{\mu}g/mL$), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of $10{\mu}g/mL$ and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with $100{\mu}g/mL$ of fraction E or F for 96 hr increased the fraction of differentiated cells up to $14.8{\pm}3.56%$ and $16.5{\pm}2.08%$ respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells ($56.8{\pm}3.7%$ and $67.4{\pm}4.2%$, $p{\leq}0.01$). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells.

• Journal of Ginseng Research
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• v.43 no.1
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• pp.95-104
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• 2019

Background: Oxidized low-density lipoprotein (ox-LDL) causes vascular endothelial cell inflammatory response and apoptosis and plays an important role in the development and progression of atherosclerosis. Ginsenoside compound K (CK), a metabolite produced by the hydrolysis of ginsenoside Rb1, possesses strong anti-inflammatory effects. However, whether or not CK protects ox-LDL-damaged endothelial cells and the potential mechanisms have not been elucidated. Methods: In our study, cell viability was tested using a 3-(4, 5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay. Expression levels of interleukin-6, monocyte chemoattractant protein-1, tumor necrosis factor-${\alpha}$, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 were determined by enzyme-linked immunosorbent assay and Western blotting. Mitochondrial membrane potential (${\Delta}{\Psi}m$) was detected using JC-1. The cell apoptotic percentage was measured by the Annexin V/ propidium iodide (PI) assay, lactate dehydrogenase, and caspase-3 expression. Apoptosis-related proteins, nuclear factor $(NF)-{\kappa}B$, and mitogen-activated protein kinases (MAPK) signaling pathways protein expression were quantified by Western blotting. Results: Our results demonstrated that CK could ameliorate ox-LDL-induced human umbilical vein endothelial cells (HUVECs) inflammation and apoptosis, $NF-{\kappa}B$ nuclear translocation, and the phosphorylation of p38 and c-Jun N-terminal kinase (JNK). Moreover, anisomycin, an activator of p38 and JNK, significantly abolished the anti-apoptotic effects of CK. Conclusion: These results demonstrate that CK prevents ox-LDL-induced HUVECs inflammation and apoptosis through inhibiting the $NF-{\kappa}B$, p38, and JNK MAPK signaling pathways. Thus, CK is a candidate drug for atherosclerosis treatment.

• Biomolecules & Therapeutics
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• v.26 no.3
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• pp.328-334
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• 2018

Because of the unsatisfactory treatment options for breast cancer (BC), there is a need to develop novel therapeutic approaches for this malignancy. One such strategy is chemotherapy using non-toxic dietary substances and botanical products. Studies have shown that Panduratin A (PA) possesses many health benefits, including anti-inflammatory, anti-bacterial, anti-oxidant and anticancer activities. In the present study, we provide evidence that PA treatment of MCF-7 BC cells resulted in a time- and dose-dependent inhibition of cell growth with an $IC_{50}$ of $15{\mu}M$ and no to little effect on normal human MCF-10A breast cells. To define the mechanism of these anti-proliferative effects of PA, we determined its effect critical molecular events known to regulate the cell cycle and apoptotic machinery. Immunofluorescence and flow cytometric analysis of Annexin V-FITC staining provided evidence for the induction of apoptosis. PA treatment of BC cells resulted in increased activity/expression of mitochondrial cytochrome C, caspases 7, 8 and 9 with a significant increase in the Bax:Bcl-2 ratio, suggesting the involvement of a mitochondrial-dependent apoptotic pathway. Furthermore, cell cycle analysis using flow cytometry showed that PA treatment of cells resulted in G0/G1 arrest in a dose-dependent manner. Immunoblot analysis data revealed that, in MCF-7 cell lines, PA treatment resulted in the dose-dependent (i) induction of $p21^{WAF1/Cip1}$ and p27Kip1, (ii) downregulation of Cyclin dependent kinase (CDK) 4 and (iii) decrease in cyclin D1. These findings suggest that PA may be an effective therapeutic agent against BC.

• Archives of Pharmacal Research
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• v.27 no.3
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• pp.305-313
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• 2004

The effect of diazoxide, a $K^{+}$channel opener, on apoptotic cell death was investigated in HepG2 human hepatoblastoma cells. Diazoxide induced apoptosis in a dose-dependent manner and this was evaluated by flow cytometric assays of annexin-V binding and hypodiploid nuclei stained with propidium iodide. Diazoxide did not alter intracellular $K^{+}$concentration, and various inhibitors of $K^{+}$channels had no influence on the diazoxide-induced apoptosis; this implies that $K^{+}$channels activated by diazoxide may be absent in the HepG2 cells. However, diazoxide induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration, and this was completely inhibited by the extracellular $Ca^{2+}$ chelation with EGTA, but not by blockers of intracellular $Ca^{2+}$ release (dantrolene and TMB-8). This result indicated that the diazoxide-induced increase of intracellular $Ca^{2+}$ might be due to the activation of a Ca2+ influx pathway. Diazoxide-induced $Ca^{2+}$ influx was not significantly inhibited by either voltage-operative $Ca^{2+}$ channel blockers (nifedipinen or verapamil), or by inhibitors of $Na^{+}$, $Ca^{2+}$-exchanger (bepridil and benzamil), but it was inhibited by flufenamic acid (FA), a $Ca^{2+}$-permeable nonselective cation channel blocker. A quantitative analysis of apoptosis by flow cytometry revealed that a treatment with either FA or BAPTA, an intracellular $Ca^{2+}$ chelator, significantly inhibited the diazoxide-induced apoptosis. Taken together, these results suggest that the observed diazoxide-induced apoptosis in the HepG2 cells may result from a $Ca^{2+}$ influx through the activation of $Ca^{2+}$-permeable non-selective cation channels. These results are very significant, and they lead us to further suggest that diazoxide may be valuable for the therapeutic intervention of human hepatomas.

• Journal of Life Science
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• v.29 no.12
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• pp.1305-1313
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• 2019

Licochalcone (LC), isolated from the roots of Glycyrrhiza inflata has multiple pharmacological effects including anti-inflammatory and anti-tumor activities. To date, Licochalcone C (LCC) has induced apoptosis and inhibited cell proliferation in oral and bladder cancer cells, but lung cancer has not yet been studied. In addition, no study reported LCC-induced autophagy in cancer until now. The present study was designed to investigate the effect of LCC on gefitinib-sensitive and -resistant lung cancer cells and elucidate the mechanism of its action. The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay data showed that LCC significantly inhibited cell viability in non-small cell lung cancer (NSCLC) HCC827 (gefitinib-sensitive) and HCC827GR (gefitinib-resistant) cell lines. Interestingly, Annexin V/7-aminoactinomycin D double staining and cell cycle analysis showed an apoptosis rate within about 20% at the highest concentration of LCC. LCC induced G2/M arrest by reducing the expression of the cell cycle G2/M related proteins cyclin B1 and cdc2 in NSCLC cell lines. Treatment of LCC also induced autophagy by increasing the expression of the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) and the protein autophagy-related gene 5 involved in the autophagy process. In addition, LCC increased the production of reactive oxygen species (ROS), and the cell viability was partially restored by treatment with the ROS inhibitor N-acetyl-L-cysteine. In western blotting analysis, the expression of cdc2 was increased and LC3 was decreased by the simultaneous treatment of NAC and LCC. These results indicate that LCC may contribute to anti-tumor effects by inducing ROS-dependent G2/M arrest and autophagy in NSCLC. In conclusion, LCC treatment may be useful as a potential therapeutic agent against NSCLC.