• Journal of Ginseng Research
• /
• v.46 no.3
• /
• pp.418-425
• /
• 2022

Background: Sorafenib is effective in treating hepatoma, but most patients develop resistance to it. STAT3 signaling has been implicated in sorafenib resistance. Artesunate (ART) and 20(R)-ginsenoside Rg3 (Rg3) have anti-hepatoma effects and can inhibit STAT3 signaling in cancer cells. This study aimed to evaluate the effects of Rg3 in combination with ART (Rg3-plus-ART) in overcoming sorafenib resistance, and to examine the involvement of STAT3 signaling in these effects. Methods: Sorafenib-resistant HepG2 cells (HepG2-SR) were used to evaluate the in vitro anti-hepatoma effects of Rg3-plus-ART. A HepG2-SR hepatoma-bearing BALB/c-nu/nu mouse model was used to assess the in vivo anti-hepatoma effects of Rg3-plus-ART. CCK-8 assays and Annexin V-FITC/PI double staining were used to examine cell proliferation and apoptosis, respectively. Immunoblotting was employed to examine protein levels. ROS generation was examined by measuring DCF-DA fluorescence. Results: Rg3-plus-ART synergistically reduced viability of, and evoked apoptosis in HepG2-SR cells, and suppressed HepG2-SR tumor growth in mice. Mechanistic studies revealed that Rg3-plus-ART inhibited activation/phosphorylation of Src and STAT3 in HepG2-SR cultures and tumors. The combination also decreased the STAT3 nuclear level and induced ROS production in HepG2-SR cultures. Furthermore, overactivation of STAT3 or removal of ROS diminished the anti-proliferative effects of Rg3-plus-ART, and removal of ROS diminished Rg3-plus-ART's inhibitory effects on STAT3 activation in HepG2-SR cells. Conclusions: Rg3-plus-ART overcomes sorafenib resistance in experimental models, and inhibition of Src/STAT3 signaling and modulation of ROS/STAT3 signaling contribute to the underlying mechanisms. This study provides a pharmacological basis for developing Rg3-plus-ART into a novel modality for treating sorafenib-resistant hepatoma.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.20 no.4
• /
• pp.844-852
• /
• 2006

This study was to evaluate the effect of Yeongyupaedog-san (YGPDS) on mouse Thl and Th2 cells' differentiation and ovalbumin (OVA)-induced allergic inflammation. The proliferation of mouse CD4 T cells and the secretion of Th1/Th2 cytokines under the influence of YGPDS extract were measured as well as the amount of ${\beta}-hexosaminidase$ in RBL-2H3 cells and the levels of $TNF-{\alpha}$ and 1L-6 secretion in Raw264.7 cells. BALB/c mice were orally administered with YGPDS extract and simultaneously inoculated with OVA to induce allergic reaction and measure the level of total IgE, OVA-specific IgE and the production of IFN- g, IL-4, IL-5 by the spleen cells. When mouse CD4 T cell were stimulated with anti-CD3 and anti-CD28 for 48 hours in various concentrations of YGPDS extract, it increased proliferation of CD4 cells by 11% in $100\;{\mu}g/^{ml}$ concentration but it showed an inhibition by 37% at $200\;{\mu}g/^{ml}$ CD4 T cells under Th1/Th2 polarizing conditions for 3 days with YGPDS resulted in mild decrease of IFN- g in Thl cells and significant decrease of IL-4 in Th2 cells at $500\;{\mu}g/^{ml}\;and\;100\;{\mu}g/^{ml}$ by 18% and 21%, respectively. YGPDS extract had a dose-dependent inhibitory effect on antigen-induced release of ${\beta}-hexosaminidase$ in RBL-2H3 cells. Treatment of YGPDS extract on LPS stimulated Raw 264.7 cells showed dose-dependent decrease in TNF-n production. Oral administration of YGPDS extract on OVA-induced allergic mice showed an inhibitory effect on the levels of total serum IgE and OVA-specific IgE by 25% and 34% , respectively. Culture of spleen cells with OVA resulted in significant increase of IFN- g by 44% and significant decrease of IL-4 and IL-5 by 56%, and 24%, respectively. The results show that YGPDS does not strongly induce mouse T cells to transform into Thl or Th2 but it has an anti-allergic effect in vitro, and that it also corrects the unbalance between the reactions of Th cells in allergic diseases.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.20 no.6
• /
• pp.1467-1476
• /
• 2006

This study was to evaluate the effect of Bulhwangeumjeonggi-san (BS) on mouse Th1 and Th2 cells' differentiation and ovalbumin (OVA)-induced allergic inflammation. The proliferation of mouse CD4 T cells and the secretion of Th1/Th2 cytokines under the influence of BS extract were measured as well as the amount ${\beta}$-hexosaminidase in RBL-wH3 cells and the levels of TNF-${\alpha}$ and IL-6 secretion in Raw264.7 cells. BALB/c mice were orally administered with BS extract and simultaneously inoculated with OVA to induce allergic reaction and measure the level of total lgE, OVA-specific lgE and the production of IFN- g, IL-4, IL-5 by the spleen cells. When mouse CD4- T cell were stimulated with anti-CD3 and anti-CD28 for 48 hours in various concentrations of BS extract, it increased proliferation of CD4 cells by 14% in 50 ${\mu}g/m{\ell}$ concentration but it showed an inhibition in higher concentrations. CD4 T cells under Th1/Th2 polarizing conditions for 3 days with BS resulted in mild decrease of IFN- g in Th1 cells and mild increase of IL-4 in Th2 cell at 50 ${\mu}g/m{\ell}$ but the level of IL-4 decreased by 18% at 100 ${\mu}g/m{\ell}$. BS extract had a dose-dependent inhibitory effect on antigen-induced release of ${\beta}$-hexosaminidase in RBL-2H3 cells. Treatment of BS extract on LPS stimulated Raw 264.7 cells showed dose-dependent decrease in TNF-${\alpha}$ production. Oral administration of BS extract on OVA-induced allergic mice showed an inhibitory effect on the levels of total serum lgE and OVA-specific lgE by 50% and 55%, respectively. Culture of spleen cells with OVA resulted in significant increase of IFN- g by 25% and significant decrease of IL-4 and IL-5 by 53%, and 38%, respectively. The results show that BS does not strongly induce mouse T cells to transform into Th1 or Th2 but it has an anti-allergic effect in vitro, and that it also corrects the unbalance between the reactions of Th cells in allergic diseases.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.28 no.4
• /
• pp.709-727
• /
• 2001

The pattern of programmed cell death(PCD) has been examined during the early developmental period of development in mouse embryos, from embryonic day 4.5(E4.5) to E11.5 Embryos from Balb/c breedings were harvested at various embryonic stages between E4.5 and El1.5. Cell death was analysed by in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) staining in tissue sections and whole embryos. At the blastocyst stage(E4.5), a very few apoptotic cells were found in the inner cell mass of the blastocyst. In the early egg cylinder stage(35.0-5.5), a few apoptotic cells were detected in the embryonic ectoderm, the embryonic endoerm and the proamniotic cavity. In the advanced egg cylinder stage(E5.5-6.5), TUNEL-posifive cells were observed in the extra-embryonic ectoderm and extra-embryonic endoderm as well as in the embryonic ectoderm, embryonic visceral endoderm and proamniotic cavity. In the streak stage(E6.75-7.75), many TUNEL-positive cells were found in the ectoplacental cone. In contrast, only very few apoptotic cells were found in the chorion and extra-embryonic endoderm in extra-embryonic regions. In intra-embryonic region, a few apoptotic cells were randomly found in the embryonic ectoderm, mesoderm and visceral endoderm. At the early somitogenesis stage(E8.0-8.5), most apoptotic cells were observed in the most cranial portion of neural fold (neural ectoderm and adjacent ectoderm). At the mid somitogenesis stage(39.0-9.5), the otic placode first showed TUNEL-positive at this stage. Small number of TUNEL-positive cells were also first seen around optic placode and branchial arches. Three streams of TUNEL-positive cells were clearly seen in the cranial region at 59.5-9.75. At E10.5, apoptotic cells were localized in the developing eye, the junctional portion of medial nasal, lateral nasal and maxillary processes, the lateral portion of branchial arches, the junction of bilateral mandibular processes, and apical ectodermal ridges of limb buds. At E11.5, apoptotic cells were noticeably decreased in most area, except the developing limbs and several somites in the tail region. In this study, the global temporospatial pattern of PCD throughout early development of mouse embryos was discussed. It may provide the basis for further studies on its role in the morphogenesis of the embryo.

• Parasites, Hosts and Diseases
• /
• v.24 no.2
• /
• pp.145-158
• /
• 1986

This study was undertaken to purify cystic fluid (CF) antigen of Taenia solium metacestodes by affinity chromatogaphy using specific monoclonal antibody(McAb) and to characterize the antigenicity of the purified antigen. The hybridoma cell lines, prepared by fusion between mouse plasmacytoma and spleen cells from BALB/c mice immunized with CF, secreted antibodies reacting to various helminthic antigens. Majority of cell lines reacted to CF only but some also reacted to parenchymal antigen of T. solium metacestodes, adult T. saginata, sparganum, hydatid cystic fluid, Paragonimus westermani and Clonorchis sinensis, either in combination with CF, other antigens or independently. Cloned cells derived from monoclonal lines also produced antibodies reacting either to CF only or to other helminthes in combination or independently. These results indicated that CF of T. solium metacestodes contained proteins which possessed antigenic determinants not only specific to CF but also cross reactive with the afore-mentioned helminthes. CF of T. solium metacestodes was purified by affinity chromatography using the McAb which reacted to CF and parenchymal antigens. The affinity-purified antigen (A-Ag) and unbound pool CF (U-Ag) were separated. A-Ag showed 2 protein bands by disc-PAGE whereas CF exhibited 6 bands and U-Ag consisted of all bands CF had. The diagnostic significance of A-Ag was evaluated by ELISA in human neurocysticercosis and other helminthic and neurologic diseases. By A-Ag, the levels of the specific IgG antibody, as shown by absorbance in sera and CSF, were lower than those of CF and U-Ag. Accordingly, the sensitivity was about 70% of CF and U-Ag. However, the nonspecific positive reactions to CF and U-Ag, observed in sparganosis, T. saginata infection and paragonimiasis did not occur when A-Ag was used. These results indicated that the affinity-purified A-Ag had the higher specificity but the lower sensitivity as a diagnostic antigen in cysticercosis, probably because it only detected a single or limited numbers of monospecific antibodies among the diverse polyclonal antibodies produced in the patients with neurocysticercosis.

• Korean Journal of Veterinary Research
• /
• v.35 no.3
• /
• pp.543-552
• /
• 1995

This study was undertaken to investigate the eftects of Cd administered ad libitum for 6 weeks on the cellular immune responses of Balb/c mice. The results were summarized as follows; 1. The mice fed 25, 50 and 100ppm Cd drank as much as control, but the mice fed 200ppm Cd drank significantly less water after Cd exposure than did control. Increasing rates of body weight of Cd-fed mice for 6 weeks were as this, control group 27.0%, Cd administered groups(25, 50, 100 and 200ppm) 28.54%, 28.31%, 20.49% and 18.04%, respectively. 2. Absolute spleen to body weight(mg/g) of control, 25, 50, 100 and 200ppm Cd administered groups were $4.34{\pm}0.23$, $4.20{\pm}0.54$, $4.80{\pm}0.87$, $4.25{\pm}0.32$ and $4.40{\pm}0.32$, respectively. Splenic cellularity(${\times}10^7$) of control was $24.29{\pm}5.98$ but increased to $27.72{\pm}5.48$, $32.96{\pm}8.44$, $28.32{\pm}8.76$ and $29.64{\pm}4.08$ in 25, 50, 100 and 200ppm Cd-fed groups, respectively. 3. Total $CD_4{^+}$ cells(${\times}10^7$) of control, 25, 50, 100 and 200ppm Cd-fed groups were $9.15{\pm}2.24$, $10.40{\pm}2.04$, $12.04{\pm}3.08$, $10.20{\pm}3.16$ and $10.80{\pm}1.48$, respectively and total $CD_8{^+}$ cells(${\times}10^7$) of these groups were $2.32{\pm}0.56$, $2.54{\pm}0.27$, $3.12{\pm}0.80$, $2.25{\pm}0.70$ and $2.24{\pm}0.28$, in order. On the other hand, $CD_4{^+}/CD_8{^+}$ ratios in total cells were increased significantly except for 50ppm Cd-fed group($3.88{\pm}0.01$). And that of control was $3.97{\pm}0.02$, but those of 25, 100 and 200ppm were $4.35{\pm}0.01$, $4.54{\pm}0.03$ and $4.81{\pm}0.03$. 4. Phagocytosis rates of peritoneal macrophages were increased significantly in 25 and 50ppm Cd groups($36.34{\pm}9.45$ and $37.15{\pm}9.22$, respectively), but 100 and 200ppm groups showed similar rates($18.20{\pm}3.04$ and $19.48{\pm}3.22$ respectively) to that of control($21.43{\pm}3.62$). 5. In mitogen-induced splenocyte proliferation, various concentraions of $CdCl_2(10^{-4}-10^{-7}M)$ were added to mitogen-stimulated culture in vitro. Splenocyte proliferation induced by LPS was decreased dose dependently, but proliferation by Con-A was increased slightly in concentrations of $10^{-7}-10^{-6}M$. 6. Significant cytotoxicity of splenocytes with $CdCl_2$ were shown at $10^{-4}M$ treated group, especially at 24 hrs. From these results, it could be concluded that Cd might modulate the immune responses by modifying a distribution of T cell subpopulations.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.1
• /
• pp.265-271
• /
• 2014

Interleukin-7 (IL-7) is a potent anti-apoptotic cytokine that enhances immune effector cell functions and is essential for lymphocyte survival. While it known to induce differentiation and proliferation in some haematological malignancies, including certain types of leukaemias and lymphomas, little is known about its role in solid tumours, including breast cancer. In the current study, we investigated whether IL-7 could enhance the in vivo antitumor activity of tumor-reactive $CD8^+$ T cells with induction of IFN-${\gamma}$ in a murine breast cancer model. Human IL-7 cDNA was constructed into the eukaryotic expression plasmid pcDNA3.1, and then the recombinational pcDNA3.1-IL-7 was intratumorally injected in the TM40D BALB/C mouse graft model. Serum and intracellular IFN-${\gamma}$ levels were measured by ELISA and flow cytometry, respectively. $CD8^+$ T cell-mediated cytotoxicity was analyzed using the MTT method. Our results showed that IL-7 administration significantly inhibited tumor growth from day 15 after direct intratumoral injection of pcDNA3.1-IL-7. The anti-tumor effect correlated with a marked increase in the level of IFN-${\gamma}$ and breast cancer cells-specific CTL cytotoxicity. In vitro cytotoxicity assays showed that IL-7-treatment could augment cytolytic activity of $CD8^+$ T cells from tumor bearing mice, while anti-IFN-${\gamma}$ blocked the function of $CD8^+$ T cells, suggesting that IFN-${\gamma}$ mediated the cytolytic activity of $CD8^+$ T cells. Furthermore, in vivo neutralization of $CD8^+$ T lymphocytes by CD8 antibodies reversed the antitumor benefit of IL-7. Thus, we demonstrated that IL-7 exerts anti-tumor activity mainly through activating $CD8^+$ T cells and stimulating them to secrete IFN-${\gamma}$ in a murine breast tumor model. Based on these results, our study points to a potential novel way to treat breast cancer and may have important implications for clinical immunotherapy.

• Current Research on Agriculture and Life Sciences
• /
• v.7
• /
• pp.83-97
• /
• 1989

This study was carried out to obtain basic information necessary for aggregation and in-vitro culture of mouse embryos by treating phytohemagglutinin-p (PHA-P). The 4-, 8-cell and morula embryos were obtained from female mice of albino BALE/C, CBA and C57BL strains, those were injected 5 i.u pregenant mare serum gonadotrophin and 5 i.u human chorionic gonadotrophin to superovulation. The zona pellucidia was removed by placing the embryos in Acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P. The pairs of zona free embryos were subjected to aggregation by glassneedle in BMOC-3 containing 5 ug/ml PHA-P. The aggregation embryos were cultured in Brinster's mouse ova culture-3(BMOC-3) medium under the gas phase of 5% $CO_2$ in air $37^{\circ}C$ for 13 to 50 hours. The results obtained in this study are summarised as follows : 1. When 4-, 8-cell and morula embryos were zona-freed in acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P, and cultured in vitro to blastocysts, the 4- and 8-cell embryos showed slightly less development rates than the morula one did, and solution of 5 ug/ml PHA-P brought some higher development rate than negative control. 2. As 2, 5 or 10 ug/ml PHA-P was added to the solution to aggregate 4-, 8-cell or morula embryos, 2 ug/ml solution represented slightly lower aggregation rate than the higher levels solutions, and 4- and 8-cell embryos showed higher rates than morula one did (P<.05). 3. In respect to the development rates of aggregated embryos to morula no significant difference was found among PHA-P levels and between 4-and 8-cell embryos. With respect to those of aggregated embryos to blastocysts the different levels of PHA-P showed similar results, however, the 4- and 8-cell embryos represented higher rates than the morula one did (P<.05). 4. The mean time necessary for development of aggregated 4-, 8-cell and morula embryos to blastocysts were 38.5-40, 26-27 and 19-20hrs. Respectively in solution for aggregation. 5. The aggregation rates of embryos were 34-94%, when treated protease or/and PHA-P. Supplementation of 5 ug/ml PHA-P to the solution for aggregation showed a trend demonstrating higher aggregation rate compared to negative control, although no significance was found. However, 4- and 8-cell embryos represented significantly higher aggregation rates than the morula one did (P<.05). 6. The development rates of 4- and 8-cell embryos to morula were 52.7-84.7 and 73.8-87.2%, respectively, showing no significant difference between two cell stages. However, the aggregation rates of embryos treated with solution containing PHA-P were higher than negative control (P<.05). 7. The development rates of 4- and 8-cell and morula embryos to blastocysts were 41.7-77.7 78.7-83.0 and 0-19.2%, respectively. The rates of 4-cell embryos treated with PHA-P were significant higher than the negative control (P<.05). The 8-cell and morula embryos also showed more rates when treated PHA-P.

• Tuberculosis and Respiratory Diseases
• /
• v.57 no.5
• /
• pp.425-433
• /
• 2004

Background : Induction of oral tolerance (OT) has been known to prevent allergic inflammation in acute asthma model within 4 weeks. However it is remained whether induction of OT may effectively prevent allergic inflammation in chronic asthma model over 4 weeks. We observed the effect of induction of OT on allergic inflammation and airway remodeling in chronic asthma model up to 8 weeks. Methods : 5-week-old female BALB/c mice divided into 4 groups-control group, asthma group, low dose OT group, and high dose OT group. To induce oral tolerance mice were fed ovalbumin (OVA) before sensitization with OVA and aluminum hydroxide-1 mg for 6 consecutive days in the low dose OT group and 25 mg once in the high dose OT group. Mice in the asthma group were fed phosphate buffered saline instead of OVA. After sensitization followed by repeated challenge with aerosolized 1% OVA during 6 weeks, enhanced pause (Penh), inflammatory cells, IL-13, and IFN-${\gamma}$ levels in bronchoalveolar lavage (BAL) fluids as well as OVA-specific IgE, IgG1, and IgG2a levels in serum were measured. In addition the degree of goblet cell hyperplasia and peribronchial fibrosis were observed from lung tissues by PAS and Masson's trichrome stain. Results : Both OT groups showed a significant decrease in Penh, inflammatory cells, IL-13, and IFN-${\gamma}$ levels in BAL fluids as well as OVA-specific IgE, IgG1, and IgG2a levels in serum compared with the asthma group (P<0.05). In addition, the degree of goblet cell hyperplasia and peribronchial fibrosis were significantly attenuated in both OT groups compared with the asthma group (P<0.01). Conclusion : These results suggest that induction of OT may effectively prevent allergic inflammation as well as airway remodeling even in chronic asthma model up to 8 weeks.

• The Korean Journal of Nuclear Medicine
• /
• v.37 no.6
• /
• pp.393-401
• /
• 2003

Objective: Pullulan derivatives (PD) can be used to make self-assembled hydrogel nanoparticles which are responsive to ionic strength. The aim of this study is to evaluate the potential of PD as a retaining carrier of radioisotope inside tumors. Materials and Methods: Four types of PD were evaluated which included pullulan acetate (PA), succinylated PA (SPA), PA-DTPA and SPA-DTPA conjugates. They were radiolabeled with Tc-99m. Labelling efficiencies were determined at 30 min, 1, 2, 4 and 12 hours after radiolabeling. CT-25 colon cancer cells were subcutaneously injected into Balb/c mice. After 2 weeks of subcutaneous injection, Tc-99m-labelled PD (Tc-99m-PD) were injected into the tumors. Whole body images of mice were obtained at 30 min, 1, 2, and 12 hr after intratumoral injection. All twenty mice were grouped into four groups by largest diameter; control A (largest diameter = 5 mm, n = 5), control B (largest diameter = 10 mm, n = 5), pullulan A (largest diameter = 5 mm, n = 5), pllulan B (largest diameter = 10 mm, n = 5). Dynamic images were obtained for 1 hour after intratumoral injection. Static images were obtained at 1 hr, 2 hr, 3 hr and 4 hr after intratumoral injection with Tc-99m pertechnetate and Tc-99m-PA. Target-to-background ratios and retention rates were calculated. Results: Labeling efficiencies of PA, SPA, PA-DTPA and SPA-DTPA were $94.5{\pm}5.9%,\;97.8{\pm}3.5%\;94.2{\pm}3.8%,\;and\;92.5{\pm}6.2%$, respectively (p>0.05). Percent retention rates (%RR) of PA and PA-DTPA were significantly higher than those of control, however, those of SP-DTPA and SPA became similar to control at 4 and 12 hr, respectively. %RR of pullulan A and pullulan B at 1, 4 and 8 hr is significantly higher than that of control (p < 0.05). However, %RR between pullulan A and pullulan B were similar. Conclusion: The lonic strength dependent PD-nanoparticles are retained in the tumor. No difference of %RR according to tumor size was noted. Therapeutic application of PD labelled with beta- or alpha- emitting radionuclides can be expected.