• Biotechnology and Bioprocess Engineering:BBE
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• 제6권5호
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• pp.332-336
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• 2001

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q$\_$Tpo/)when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant-enhancement in q$\_$Tpo/ at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhanced q$\_$Tpo/. When the enhancement of the q$\_$Tpo/ of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (γ$^2$=0.80, p<0.001, ANOVA), which suggested that an enhancement of q$\_$Tpo/ after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhanced q$\_$Tpo/ can be done upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.

• 대한한방내과학회지
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• 제28권1호
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• pp.12-24
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• 2007

Objective : To investigate the effect of Samilshinkihwan(SISKW) on white rats with deteriorated immunity caused by methotrexate. Methods : The test articles were dosed once a day for 14 days by gastric gavage at a dosage 1000, 500 and 250mg/kg/10ml of SISKW from 2 days after last MTX-dosing, and changes in body weight, spleen weight, total blood leukocyte numbers, total lymphocyte numbers, B and T lymphocyte ratio, CD3+CD4+, CD3+/CD8+ and CD4+/CD8+ T lymphocyte ratio in the blood and spleen were measured. In addition, the serum interleukin (IL)-2 levels and the productivity of IL-2 of splenic cells were also demonstrated in this study. Results : The changes on body weight increased significantly in the 1000mg/kgof SISKW group. The changes on the spleen weight, the total blood leukocytenumbers, the total lymphocyte numbers in the blood and spleen, the ratio of T-cell in the blood and spleen and the ratio of CD3+CD4+ T-cell in spleen increased significantly in all SISKW groups as compared with the control group. The ratio of CD4+/CD8+ T-cells in blood increased significantly. The serum IL-2 levels and productivity of IL-2 of splenic cells increased significantly in 1000 and 500mg/kg SISKW groups as compared with the control group. Conclusions : Samilshinkihwanhas an effect of increasing immune responses, especially on cellular immune responses, in white rats with deteriorated immunity caused by methotrexate.

• 대한한방소아과학회지
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• 제22권1호
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• pp.123-147
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• 2008

Objectives This study is to investigate how dose Ikhwang-San can be effective on SD rats which deteriorated immunity caused by methotrexate. Methods The test sample were dosed once a day for 14 days by gastric gavage at the beginning of dosage 1000, 500 and 250㎎/㎏/10㎖ from 2 days after last MTX-dosing, and the changes of the body and spleen weight, total number of blood leukocytes, total number of lymphocytes, the percentage of B-cell, T-cell, CD3+CD4+ T-cell, CD3+CD8+ T-cell and CD4+/CD8+ T-cell ratios in the blood and spleen were observed. Results The changes of the body and spleen weight, the total number of blood leukocytes, the total number of lymphocyte in the blood and spleen were significantly increased in IHS Extracts groups comparing with the control group. The percentage of B-cell, T-cell, CD3+CD4+ T-cell in the blood and spleen were significantly increase in IHS groups and comparing with the control group. The ratio of CD4+/CD8+ T-cell in blood and spleen was significantly increased in IHS Extracts groups comparing with the control group. Conclusions According to those results, Ikhwang-San has good immunostimulating effect on SD rats which had deteriorated immunity caused by methotrexate.

• 대한한방소아과학회지
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• 제21권1호
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• pp.227-252
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• 2007

Objectives : In order to investigate the effect of Bobitang on SD rats with deteriorated immunity caused by methotrexate. Methods : The test sample were dosed once a day for 14 days by gastric gavage at a dosage 1,000, 500 and $250mg/kg/10m{\ell}$ from 2 days after last MTX-dosing, and the changes on body weight and gains, spleen weight and total blood leukocyte numbers, total lymphocyte numbers, the percentage of B-cell, T-cell, CD3+CD4+ T-cell, CD3+CD8+ T-cell and CD4+/CD8+ T-cell ratios in the blood and spleen were observed. Results : The changes on body weight gains, the spleen weight, the total blood leukocyte numbers, the total lymphocyte numbers in the blood and spleen, the ratio of T-cell in the blood and spleen, the ratio of CD3+CD4+ T-cell in the blood and spleen were increased significantly in BBT Extracts groups as compared with control group. The ratio of B-cell in the blood and spleen was not increased significantly in BBT Extracts groups as compared with control group. The percentage of CD3+CD8+ T-cell in the blood and spleen was decreased significantly in BBT Extracts groups as compared with control group. The ratio of CD4+/CD8+ T-cell in blood and spleen was increased significantly in BBT Extracts group as compared with control group. Conclusions : According to the above results, Bobi-Tang has an effect of increasing immune responses on SD rats with deteriorated immunity caused by methotrexate.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.121-129
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• 2002

Using recombinant Chinese hamster ovary (CHO) cells, strategies for developing high producers for the recombinant human Transforming Growth $Factor-{\beta}1$ ($TGF-{\beta}1$) protein are proposed and their physiological characteristics in cell cultures were investigated. $TGF-{\beta}1$ is a pleiotrophic polypeptide involved in various biological activities, including cell growth, differentiation, and deposition of extracellular matrix proteins. The CHO cells included human $TGF-{\beta}1$ cDNA in conjunction with a dihydrofolate reductase (DHFR) gene, which was cotransfected into the cells to amplify the transfected $TGF-{\beta}1$ cDNA. As a first-round screening of the transfected cells, a relatively high $TGF-{\beta}1$-producing cell line was selected, and then, it acquired a resistance to increasing concentrations of methotrexate (MTX) up to $60{\mu}M$,resulting in a significant improvement in its $TGF-{\beta}1$ biosynthetic ability. After applying a monoclonal selection strategy to the MTX-resistant cells, more productive cells were screened, including the APP-3, App-5, and App-8 cell lines. These high producers were compared with two other cell lines (AP-l cell line without amplification of transfected $TGF-{\beta}1$ cDNA and nontransfectant of $TGF-{\beta}1$ cDNA) in terms of cell growth, $TGF-{\beta}1$ productivity, sugar uptake, and byproduct formation, in the presence or absence of MTX in the culture medium. Consequently, both monoclonal selection as well as an investigation of the physiological characteristics were found to be needed for the efficient screening of higher $TGF-{\beta}1$ producers, even after the transfection and amplification of the transfected gene.

• Asian Pacific Journal of Cancer Prevention
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• 제15권19호
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• pp.8377-8382
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• 2014

Background: Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy worldwide. Cancer development and progression require inactivation of tumor suppressor genes and activation of proto-oncogenes. The well recognized mechanism of action demonstrated for chemotherapeutic agents is induction of apoptosis via reactivation of p53. In this context, we evaluate the efficacy of IV and oral routes of our novel PH and temperature sensitive doxorubicin-methotrexate-loaded nanoparticles (DOX-MTX NP) in affecting p53 profile in an OSCC rat model. Methods: In this study, 120 male rats were divided into 8 groups of 15 animals each. The new formulated DOX-MTX NP and free doxorubicin were IV and orally given to rats with 4-nitroquinoline-1-oxide induced OSCC. Results: Results showed that both DOX and DOX-MTX-NP caused significant increase in mRNA levels of P53 compared to the untreated group (p<0.000). With both DOX and DOX-MTX NP, the IV mode was more effective than the oral (gavage) route (p<0.000). Surprisingly, in oral mode, p53 mRNA was not affected in DOX treated groups (p>0.05), Nonetheless, both IV and oral administration of MTX-DOX NP showed superior activity (~3 fold) over free DOX in reactivation of p53 in OSCC (p<0.000). The effectiveness of oral route in group treated with nanodrug accounts for the enhanced bioavailability of nanoparticulated DOX-MTX compared to free DOX. Moreover, in treated groups, tumor stage was markedly related to the amount of p53 mRNA (p<0.05). Conclusion: Both oral and IV application of our novel nanodrug possesses superior activity over free DOX-in up-regulation of p53 in a OSCC model and this increase in p53 level associated with less aggressive tumors in our study. Although, impressive results obtained with IV form of nanodrug (-21 fold increase in p53 mRNA level) but both forms of nanodrug are effective in OSCC, with less toxicity normal cells.

• The Korean Journal of Physiology and Pharmacology
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• 제16권1호
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• pp.11-16
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• 2012

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; $25{\mu}g/ml$) and methotrexate (MTX; $50{\mu}g/ml$), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptase-polymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.141-149
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• 2006

Tamoxifen (TAM), a non-steroidal anti estrogen anticancer drug and chemopreventive agent for breast cancer, have caused cholestasis in liver. The potent hepatocarcinogenicity of this drug has been reported. Methotrexate (MTX) is dihydrofolate reductase inhibitor which interfaces with the synthesis for urine nucleotide and dTMP. And it may cause atrophy, necrosis and steatosis in liver. These two anticancer drug have well-known hepatotoxicity. So, in this study we compare the gene expression pattern of antitumor agent TAM and MTX, using the cDNA microarray. We have used 4.8 K cDNA microarray to identify hepatotoxicity-related genes in 5-week-old male Sprague-Dawley (SD) rats. Confirm the pattern of gene expression, we have used Real time PCR for targeted gene. In the case of MTX, Protease related gene (Ctse, Ctsk) and Protein kinase (Pctk 1) have shown specific expression pattern. And in the case of TAM, apoptosis related gene (Pdcd 8) and signal transduction related gene (kdr) have significantly up regulated during treatment time. Gene related with growth factor, lipid synthesis, chemokins were significantly changed. From the result of this study, the information about influence of TAM and MTX to hepatoxicity will provide.

• Journal of Gynecologic Oncology
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• 제29권6호
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• pp.89.1-89.8
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• 2018

Objective: Highly effective chemotherapy for patients with low-risk gestational trophoblastic neoplasia (GTN) is associated with almost a 100% cure rate. However, 20%-30% of patients treated with chemotherapy need to change their regimens due to severe adverse events (SAEs) or drug resistance. We examined the treatment outcomes of second-line chemotherapy for patients with low-risk GTN. Methods: Between 1980 and 2015, 281 patients with low-risk GTN were treated. Of these 281 patients, 178 patients were primarily treated with 5-day intramuscular methotrexate (MTX; n=114) or 5-day drip infusion etoposide (ETP; n=64). We examined the remission rates, the drug change rates, and the outcomes of second-line chemotherapy. Results: The primary remission rates and drug resistant rates of 5-day ETP were significantly higher (p<0.001) and significantly lower (p=0.002) than those of 5-day MTX, respectively. Forty-seven patients (26.4%) required a change in their chemotherapy regimen due to the SAEs (n=16) and drug resistance (n=31), respectively. Of these 47 patients failed the first-line regimen, 39 patients (39/47, 82.9%) were re-treated with single-agent chemotherapy, and 35 patients (35/39, 89.7%) achieved remission. Four patients failed second-line, single-agent chemotherapy and eight patients (17.0%) who failed first-line regimens were treated with combined or multi-agent chemotherapy and achieved remission. Conclusions: Patients with low-risk GTN were usually treated with single-agent chemotherapy, while 20%-30% patients had to change their chemotherapy regimen due to SAEs or drug resistance. The second-line regimens of single-agent chemotherapy were effective; however, there were several patients who needed multiple agents and combined chemotherapy to achieve remission.

• 대한한의학회지
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• 제38권1호
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• pp.34-45
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• 2017

Objectives: This study aimed to investigate the effect of banhasasim-tang intravenous herbal acupuncture (BST-IVHA) on emesis induced by chemotherapy in rats. Methods: This study used methotrexate(MTX)-induced Rat-Pica model. The rats were randomly allocated into seven groups; normal group, two saline groups, four Banhasasim-tang(BST) groups (groups treated with BST-IVHA). All the experimental animals except those in the normal group were injected with MTX. Those in the pre-treatment groups were treated with saline injection (saline group) or BST-IVHA (BST group) before MTX injection. Those in the post-treatment groups were treated with saline injection or BST-IVHA after MTX injection. Two different dosages of BST-IVHA solution (low dose; BST-1 group, high dose; BST-2 group) were used. The changes in body weight, food intake, and kaolin consumption at 24h, 48h, and 60h were monitored and analyzed. Results: 1. No significant change was found in body weight. 2. The food intake at 48h was increased significantly in the BST-1 pre-treatment group($19.89{\pm}0.01g$) compared to the pre-saline group($18.68{\pm}0.26g$). 3. The kaolin consumption was significantly decreased in the BST-1 pre-treatment group at 24h($0.24{\pm}0.02g$) and 60h($0.36{\pm}0.14g$), in the BST-2 pre-treatment group at 48h($0.02{\pm}0.01g$) and 60h($0.80{\pm}0.31g$) compared to the pre-saline group($24h:0.81{\pm}0.37g$, $48h:0.76{\pm}0.43g$, $60h:1.56{\pm}0.03g$). The kaolin consumption was also significantly decreased in the in the BST-1 post-treatment group at 24h($0.05{\pm}0.02g$), 48h($0.64{\pm}0.06g$) and 60h($0.14{\pm}0.05g$), in the BST-2 post-treatment group at 48h($0.01{\pm}0.01g$) and 60h($0.01{\pm}0.01g$) compared to the post-saline group($24h:0.51{\pm}0.4g$, $48h:3.58{\pm}0.33g$, $60h:2.5{\pm}0.2g$). Conclusions: BST-IVHA showed an anti-emetic effect in MTX-induced rat-pica model. This result suggests that BST-IVHA could be an effective treatment for chemotherapy-induced emesis.