• Molecules and Cells
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• v.44 no.1
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• pp.50-62
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• 2021

Among all cancer types, lung cancer ranks highest worldwide in terms of both incidence and mortality. The crosstalk between lung cancer cells and their tumor microenvironment (TME) has begun to emerge as the "Achilles heel" of the disease and thus constitutes an attractive target for anticancer therapy. We previously revealed that crosstalk between lung cancer cells and endothelial cells (ECs) induces chemoresistance in multicellular tumor spheroids (MCTSs). In this study, we demonstrated that factors secreted in response to crosstalk between ECs and lung cancer cells play pivotal roles in the development of chemoresistance in lung cancer spheroids. We subsequently determined that the expression of hypoxia up-regulated protein 1 (HYOU1) in lung cancer spheroids was increased by factors secreted in response to crosstalk between ECs and lung cancer cells. Direct interaction between lung cancer cells and ECs also caused an elevation in the expression of HYOU1 in MCTSs. Inhibition of HYOU1 expression not only suppressed stemness and malignancy, but also facilitated apoptosis and chemosensitivity in lung cancer MCTSs. Inhibition of HYOU1 expression also significantly increased the expression of interferon signaling components in lung cancer cells. Moreover, the activation of the PI3K/AKT/mTOR pathway was involved in the HYOU1-induced aggression of lung cancer cells. Taken together, our results identify HYOU1, which is induced in response to crosstalk between ECs and lung cancer cells within the TME, as a potential therapeutic target for combating the aggressive behavior of cancer cells.

• Journal of Life Science
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• v.22 no.9
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• pp.1145-1151
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• 2012

Aspirin and its deacetylated form, sodium salicylate (NaSal), have been shown to exert chemopreventive activities against many human cancers including those of the colon, lung, and breast. Previously, we showed that combined treatment of NaSal and genistein synergistically induced apoptosis in A549 lung cancer cells, indicating that these two natural chemicals could be used in combination for cancer therapy. In this study, we examined effects of NaSal/genistein combined treatment on other cancer cells and in three-dimensional multicellular tumor spheroid (MTS) and in an in vitro solid tumor model. We found that the combined treatment induces apoptosis in the HCT116 cells and the A549 cells, but not in the MCF-7 cells. Interestingly, the MCF-7 cells responded to the NaSal/genistein combined treatment by undergoing cell death when they were cultivated as MTS. The combined treatment induced apoptosis at an earlier stage in the MCF-7 MTS culture. However, when the MCF-7 MTS was cultivated for a longer period, it induced necrosis rather than apoptosis. We further found that the apoptotic pattern observed in MCF-7 MTS was incomplete: the chromatins were condensed and fragmented, but the nuclear membrane was still intact. Taken together, these results demonstrate that the NaSal/genistein combined treatment induces incomplete apoptosis and necrosis in the MCF-7 MTS culture system.

• Radiation Oncology Journal
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• v.7 no.2
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• pp.149-156
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• 1989

Multicellular tumor spheroids of HeLa cells have been grown in a static culture system. Samples of spheroids were exposed for 2 h to graded concentration of cis-platinum and its analogue, carboplatin, and then response assayed by survival of clonogenic cells. The purpose of present experiment is to clarify the effectiveness of these platinum compounds and to evaluate intrinsic radiosensitivity of cells using spheroids of HeLa cells as an experimental in vitro model. Variations of the drug sensitivity of monolayers as well as spheroids were also evaluated in cell-survival curves. In cis-platinum concentration-survival curve, there was a large shoulder extending as far as $Cq=3.4{\mu}M$, after which there was exponential decrease in survival curve having a Co Value of $1.2{\mu}M$ in spheroids. While the Co for the spheroids was essentially no significant change, but Cq value was larger than that of monolayers. This suggest that the effect of cis-platinum is greater En the monolayer with actively proliferaing cells than hypoxic one. In the carboplatin concentration-survival curves, the Co value of spheroids was $15.0{\mu}M$ and the ratio with the Co from monolayer cell $(32.5{\mu}M)$ was 0.40, thus indicating that the spheroids had a greater sensitivity to carboplatin than monolayers. Therefore, the effect of carboplatin is mainly on the deeper layers of spheroids acting as hypoxic cell sensitizer. The enhanced effect was obtained for monolayer cells using combined X-ray and carboplatin treatment 2 hours before irradiation. The result shown in isobologram analysis for the level of surviving fraction at 0.01 indicated that the effect of two agents was trusty supra-additive. From this experimental data, carboplatin has excited much recent interest as one of the most promising, since it is almost without nephrotoxicity and causes less gastrointestinal toxicity than cis-platinum. Interaction between carboplatin and radiation might play an important role for more effective local tumor control.

• Journal of Pharmaceutical Investigation
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• v.34 no.5
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• pp.393-399
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• 2004

For the efficient determination of activity against solid tumors, an in vitro tumor model that resembles the condition of in vivo solid tumors, is required. The purpose of this study was to establish a rapid culture method and viability assay for an in vitro 3-dimensional tumor model, multicellular spheroid (MCS). Among 12 human cancer cell lines, a few cell lines including DLD-1 (human colorectal carcinoma cells) formed fully compact MCS which was adequate for in vitro viability assay. DLD-1 MCS showed steady growth reaching $700\;{\mu}m$ diameter after 11 day culture. DLD-1 cells grown as MCS showed significant increase in $G_0/G_1$ phase compared to the monolayer cells (73.9% vs 45.7%), but necrotic regions or apoptotic cells were not observed. The cells cultured as MCS showed resistance to 5-FU (10.3 fold higher $IC_{50}$) compared to monolayers, however, tirapazamine (a hypotoxin) showed similar activity in both culture systems. In summary, MCS may be a valid in vitro model for activity screening of anticancer agents against human solid tumors and also exploitable for studying molecular markers of drug resistance in human solid tumors.

• Journal of Life Science
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• v.29 no.1
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• pp.9-17
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• 2019

As tumors develop, they encounter microenvironmental stress, such as hypoxia and glucose depletion, due to poor vascular function, thereby leading to necrosis, which is observed in solid tumors. Necrotic cells are known to release cellular cytoplasmic contents, such as high mobility group box 1 (HMGB1), into the extracellular space. The release of HMGB1, a proinflammatory and tumor-promoting cytokine, plays an important role in promoting inflammation and metabolism during tumor development. Recently, HMGB1 was shown to induce the epithelial-mesenchymal transition (EMT) and metastasis. However, the underlying mechanism of the HMGB1-induced EMT, invasion, and metastasis is unclear. In this study, we showed that noninvasive breast cancer cells MCF-7 formed tightly packed, rounded spheroids and that the cells in the inner regions of a multicellular tumor spheroid (MTS), an in vitro model of a solid tumor, led to necrosis due to an insufficient supply of O2 and glucose. In addition, after 7 d of MTS culture, the EMT was induced via the transcription factor Snail. We also showed that HMGB1 receptors, including RAGE, TLR2, and TLR4, were induced by MTS culture. RAGE, TLR2, and TLR4 shRNA inhibited MTS growth, supporting the idea that RAGE/TLR2/TLR4 play critical roles in MTS growth. They also prevented MTS culture-induced Snail expression, pointing to RAGE/TLR2/TLR4-dependent Snail expression. RAGE, TLR2, and TLR4 shRNA suppressed the MTS-induced EMT. In human cancer tissues, high levels of RAGE, TLR2, and TLR4 were detected. These findings demonstrated that the HMGB-RAGE/TLR2/TLR4-Snail axis played a crucial role in the growth of the MTS and MTS culture-induced EMT.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.129-130
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• 1994

The science of toxicology is the understanding of the mechanisms by which exogenous agents produce deleterious effects in biological systems. The actions of chemicals such as drugs are ultimately exerted at the cellular and gene levels. Over the past decade. several in vitro alternative methods such as cultured cells for assessing the toxicity of various xenobiotics have been proposed to reduce the use of animals. In this workshop three advanced methods will be presented. These methods are novel important models for toxicologic studies. Dr. Tabuchis group has establishcd two immortalized gastric surface mucosa cell lines from the pminary cultore of gastric fundic mucosal cells of adult transgenic mice harboring a temperature sensitive simian virus 40 large T-anugen gene. As the immortalized cell lines of various tissues possess unique characteristics to maintain their normal functions for several months, these cell lines are extremely useful for not only toxicity testing but also pharmacological screening in new drug development. Professor Funatsu have studied the formation of spherical multicelluar aggregates of adult rat hepatocytes(spheroid) having tissue like structure. The sphcroid shown thre is a prototype module of an artificial liver support system. Thus, the urea synthesis activity of the artificial liver was maintained at least to days in 100％ rat blood plasma. Dr. Takezawa and his coworkers have developed a novel culture system of multicellular spheroids considered 〃organoids〃 by utilizing a thermo-responsive polymer as a substratum of anchorage dependent cells. His final goal is to reconstitute the organoids of various normal organs, e.g., liver, skin etc. and also abnormal deseased organs such as tumor.

• Journal of Life Science
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• v.21 no.7
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• pp.953-960
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• 2011

Necrosis is characterized by the cell membrane rupture and release of the cellular contents, including high-mobility group box 1 protein (HMGB1), into the extracellular microenvironment. HMGB1 acts as a transcriptional regulator in nuclei, but exerts a pro-inflammatory and tumor-promoting cytokine activity when released into the extracellular space. Its overexpression is associated with tumor progression and chemoresistance. Thus, HMGB1 acts as a clinically important molecule in tumor biology. In this study, we examined whether HMGB1 affects cell death induced by anti-cancer drugs. Here we show that HMGB1 prevented cisplatin (alkylating agent)-induced apoptosis and switched the cell fate to necrosis in MCF-7, MDA-MB231, and MDA-MB361 cells. Similar apoptosis-to-necrosis switch effects of HMGB1 were observed in cells treated with 4-HC, another alkylating agent. In contrast, HMGB1 did not exert any significant effects on docetaxel (DOC)-induced apoptosis in MCF-7 cells. We also show that cisplatin-induced apoptosis was switched to necrosis in MCF-7 multicellular tumor spheroids (MTS) that were cultured for 8 days and had necrotic cores, but DOC-induced apoptosis was prevented without the apoptosis-to-necrosis switch. Finally, the levels of RAGE, a receptor of HMGB1, were increased with extended culture of MTS. These findings demonstrate that HMGB1 switches alkylating agent-induced apoptosis to necrosis, suggesting that the strategy to prevent necrosis occurring as an undesirable action of alkylating agent-based chemotherapy should be delineated to improve the efficacy of chemotherapy for cancer.