• Molecules and Cells
• /
• v.38 no.4
• /
• pp.327-335
• /
• 2015

Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic ${\alpha},{\beta}$-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

• Molecules and Cells
• /
• v.39 no.4
• /
• pp.352-357
• /
• 2016

Vertebrate neurogenesis requires inhibition of endogenous bone morphogenetic protein (BMP) signals in the ectoderm. Blocking of BMPs in animal cap explants causes the formation of anterior neural tissues as a default fate. To identify genes involved in the anterior neural specification, we analyzed gene expression profiles using a Xenopus Affymetrix Gene Chip after BMP-4 inhibition in animal cap explants. We found that the xCyp26c gene, encoding a retinoic acid (RA) degradation enzyme, was upregulated following inhibition of BMP signaling in early neuroectodermal cells. Whole-mount in situ hybridization analysis showed that xCyp26c expression started in the anterior region during the early neurula stage. Overexpression of xCyp26c weakly induced neural genes in animal cap explants. xCyp26c abolished the expression of all trans-/cis-RA-induced posterior genes, but not basic FGF-induced posterior genes. Depletion of xCyp26c by morpholino-oligonucleotides suppressed the normal formation of the axis and head, indicating that xCyp26c plays a critical role in the specification of anterior neural tissue in whole embryos. In animal cap explants, however, xCyp26c morpholinos did not alter anterior-to-posterior neural tissue formation. Together, these results suggest that xCyp26c plays a specific role in anterior-posterior (A-P) neural patterning of Xenopus embryos.

• Molecules and Cells
• /
• v.21 no.1
• /
• pp.42-51
• /
• 2006

We investigated the mechanism of contraction induced by S1P in esophageal smooth muscle cells. Western blot analysis demonstrated that $S1P_1$, $S1P_2$, $S1P_3$, and $S1P_5$ receptors existed in the cat esophagus. Only penetration of EDG-5 ($S1P_2$) antibody into permeabilized cells inhibited S1P-induced contraction. Pertussis toxin (PTX) also inhibited contraction, suggesting that it was mediated by $S1P_2$ receptors coupled to a PTXsensitive $G_i$ protein. Specific antibodies to $G_{i2}$, $G_q$ and $G_{\beta}$ inhibited contraction, implying that the S1P-induced contraction depends on PTX-insensitive $G_q$ and $G_{\beta}$ dimers as well as the PTX-sensitive $G_{i2}$. Contraction was not affected by the phospholipase $A_2$ inhibitor DEDA, or the PLD inhibitor ${\rho}$-chloromercuribenzoate, but it was abolished by the PLC inhibitor U73122. Incubation of permeabilized cells with $PLC{\beta}3$ antibody also inhibited contraction. Contraction involved the activation of a PKC pathway since it was affected by GF109203X and chelerythrine. Since $PKC{\varepsilon}$ antibody inhibited contraction, $PKC{\varepsilon}$ may be required. Preincubation of the muscle cells with the MEK inhibitor PD98059 blocked S1P-induced contraction, but the p38 MAP kinase inhibitor SB202190 did not. In addition, co-treatment of cells with GF 109203X and PD98059 did not have a synergistic effect, suggesting that these two kinases are involved in the same signaling pathway. Our data suggest that S1P-induced contraction in esophageal smooth muscle cells is mediated by $S1P_2$ receptors coupled to PTX-sensitive $G_{i2}$ proteins, and PTX-insensitive $G_q$ and $G_{\beta}$ proteins, and that the resulting activation of the $PLC{\beta}3$ and $PKC{\varepsilon}$ pathway leads to activation of a p44/p42 MAPK pathway.

• Molecules and Cells
• /
• v.43 no.1
• /
• pp.66-75
• /
• 2020

Saturated fatty acids contribute to β-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca²⁺ depletion followed by notable store-operated Ca²⁺ entry. Subsequent elevation of cytosolic Ca²⁺ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca²⁺ uniporter (MCU) is the major route for Ca²⁺ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca²⁺ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal β-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and reduced depolarization-triggered Ca²⁺ influx likely due to the inactivation of voltage-gated Ca²⁺ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca²⁺]_i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca²⁺]_i elevation and defective [Ca²⁺]_i transients. Extracellular Ca²⁺ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca²⁺ uptake via MCU upregulation is a mechanism by which pancreatic β-cells are able to alleviate cytosolic Ca²⁺ overload and its detrimental consequences.

• Experimental and Molecular Medicine
• /
• v.50 no.11
• /
• pp.6.1-6.12
• /
• 2018

Morphine tolerance remains a challenge in the management of chronic pain in the clinic. As shown in our previous study, the dopamine D2 receptor (D2DR) expressed in spinal cord neurons might be involved in morphine tolerance, but the underlying mechanisms remain to be elucidated. In the present study, selective spinal D2DR blockade attenuated morphine tolerance in mice by inhibiting phosphatidylinositol 3 kinase (PI3K)/serine-threonine kinase (Akt)-mitogen activated protein kinase (MAPK) signaling in a ${\mu}$ opioid receptor (MOR)-dependent manner. Levo-corydalmine (l-CDL), which exhibited micromolar affinity for D2DR in D2/CHO-K1 cell lines in this report and effectively alleviated bone cancer pain in our previous study, attenuated morphine tolerance in rats with chronic bone cancer pain at nonanalgesic doses. Furthermore, the intrathecal administration of l-CDL obviously attenuated morphine tolerance, and the effect was reversed by a D2DR agonist in mice. Spinal D2DR inhibition and l-CDL also inhibited tolerance induced by the MOR agonist DAMGO. l-CDL and a D2DR small interfering RNA (siRNA) decreased the increase in levels of phosphorylated Akt and MAPK in the spinal cord; these changes were abolished by a PI3K inhibitor. In addition, the activated Akt and MAPK proteins in mice exhibiting morphine tolerance were inhibited by a MOR antagonist. Intrathecal administration of a PI3K inhibitor also attenuated DAMGO-induced tolerance. Based on these results, l-CDL antagonized spinal D2DR to attenuate morphine tolerance by inhibiting PI3K/Akt-dependent MAPK phosphorylation through MOR. These findings provide insights into a more versatile treatment for morphine tolerance.

• Biomolecules & Therapeutics
• /
• v.29 no.5
• /
• pp.551-561
• /
• 2021

Thyroid cancer is the most common endocrine malignancy. Patients with well-differentiated thyroid cancers, such as papillary and follicular cancers, have a favorable prognosis. However, poorly differentiated thyroid cancers, such as medullary, squamous and anaplastic advanced thyroid cancers, are very aggressive and insensitive to radioiodine treatment. Thus, novel therapies that attenuate metastasis are urgently needed. We found that both PDGFC and PDGFRA are predominantly expressed in thyroid cancers and that the survival rate is significantly lower in patients with high PDGFRA expression. This finding indicates the important role of PDGF/PDGFR signaling in thyroid cancer development. Next, we established a SW579 squamous thyroid cancer cell line with 95.6% PDGFRA gene insertion and deletions (indels) through CRISPR/Cas9. Protein and invasion analysis showed a dramatic loss in EMT marker expression and metastatic ability. Furthermore, xenograft tumors derived from PDGFRA geneedited SW579 cells exhibited a minor decrease in tumor growth. However, distant lung metastasis was completely abolished upon PDGFRA gene editing, implying that PDGFRA could be an effective target to inhibit distant metastasis in advanced thyroid cancers. To translate this finding to the clinic, we used the most relevant multikinase inhibitor, imatinib, to inhibit PDGFRA signaling. The results showed that imatinib significantly suppressed cell growth, induced cell cycle arrest and cell death in SW579 cells. Our developed noninvasive apoptosis detection sensor (NIADS) indicated that imatinib induced cell apoptosis through caspase-3 activation. In conclusion, we believe that developing a specific and selective targeted therapy for PDGFRA would effectively suppress PDGFRA-mediated cancer aggressiveness in advanced thyroid cancers.

• The Korean Journal of Physiology
• /
• v.23 no.2
• /
• pp.237-252
• /
• 1989

Effect of a $Na^+$ gradient on $Ca^{2+}$ uptake was studied in isolated sarcolemmal vesicles of cat ileal longitudinal muscle. $Ca^{2+}$ uptake was markedly stimulated in the presence of an outwardly directed $Na^+$ gradient. External $Na^+$, monensin and A23187 abolished the $Na^+-dependent$ $Ca^{2+}$ uptake. Monovalent cations such as $K^+$, $Li^+$, $Rb^+$, $Cs^+$ and choline could not substitute for $Na^+$ in enhancement of $Ca^{2+}$ uptake. Divalent cations such as $Ba^{2+}$, $Sr^{2+}$, $Mn^{2+}$ and $Cd^{2+}$ but not $Mg^{2+}$ inhibited the $Na^+-dependent$ $Ca^{2+}$ uptake. Increase in external pH in the range of 6.0 to 8.0 stimulated the $Na^+-dependent$ $Ca^{2+}$ uptake. Amiloride inhibited the $Na^+-dependent$ $Ca^{2+}$ uptake at concentrations above 0.5 mM, whereas diltiazem or vanadate did not. The apparent Km of the $Na^+-dependent$ $Ca^{2+}$ uptake for $Ca^{2+}$ was 18.2 ${\mu}M$ and apparent Vmax was 689.7 pmole/mg protein/5 sec. Kinetic analysis of the $Na^+-dependent$ $Ca^{2+}$ uptake showed a noncompetitive interaction between internal $Na^+$ and external $Ca^{2+}$. The dependence of $Ca^{2+}$ uptake on internal $Na^+$ showed sigmoidal kinetics and Hill coefficient for internal $Na^+$ was 2.52. Inside positive membrane potential generated by imposing an inwardly directed $K^+$ gradient and valinomycin significantly stimulated the $Na^+-dependent$ $Ca^{2+}$ uptake. These results indicate that a $Na^+-Ca^{2+}$ exchange system exists in the sarcolemmal membranes isolated from cat ileal longitudinal muscle and it might operate as an electrogenic process.

• Microbiology and Biotechnology Letters
• /
• v.29 no.3
• /
• pp.181-185
• /
• 2001

The naturally occurring chemical indole-3-carbinol (13C), found in vegetables of the Brassica genus, is a promising anticancer agent that was shown previ- ously to induce a Gl cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. The anticancer activity of 13C and the possible mechanisms of its action were explored in a human hepatocellular carcinoma cell line, HepG2. Treatment of HepG2 cells with 13C suppressed the growth of the cells. The growth sup- pression caused by 13C ($IC_{50}$/: 444$\mu$M) was found to be partially due to its ability to stop the cell cycle in HepG2 cells. Western blot analysis for the Gl phase artiest demonstrated that the expression-levels of cyclin-dependent kinase (Cdk4, Cdk6) and cyclic D were reduced strongly after treatment of Hep72 cells with 13C (4007M) for 24- 72 hrs. Furthermore, I3C selectively abolished the expression of Cdk6 in a dose- and time-dependent manner, and accordingly, inhibited the phosphorylation of retinoblastoma. Interestingly, after the HepG2 cells reached their max- imal growth arrest, the level of the p21, a well-known Cdk inhibitor, increased significantly. Therefore, it could be considered that the Gl arrest of HepG2 cells treated with 13C was due to the indirect inhibition of Cdk4/6 activities by p21 Western blot analysis for G2/M phase arrest of demonstrated the levels of Cdc2 and cyclin Bl werer reduced dramatically after the treatment of HepG2 cells with 13C ($40\mu$M) for 24-72 hrs. flow cytometry of propidium iodide-stained HepG2 cells revealed that 13C induces a Gl (53%,72hr incubation) and G2 (25%,24hr incubation) cell cycle arrest. Thus, our observations have uncovered a previously undefined antiproliferative pathway for r3C that implicates Cdk4/6 and Cdc2 as a target for cell cycle control in human HepG2 cells. However, the 13C-medi- ated cell cycle arrest and repression of Cdk4/6 production did not affect the apoptotic induction of HepG2 cell.

• Journal of Animal Science and Technology
• /
• v.48 no.4
• /
• pp.493-502
• /
• 2006

The slaughter weight (wt) is a most important economic factor in swine roduction. The present study was undertaken to determine if it is feasible to increase the slaughter wt of finishing pigs to 130kg without significantly affecting the growth efficiency and carcass quality. One hundred and sixty gilts and 160 barrows born from lean-type Yorkshire×Landrace (YL)-dam and Duroc (D)-sire lines, which are most widely used for terminal breeding for pork production in Gyeongsangnam-do, were randomly allocated into 16 pens under a 2 (sex)×2 (slaughter wt; 110 vs. 130kg) factorial arrangement of treatments. The animals were fed ad libitum a diet containing 3,200kcal DE/kg and 15.5% crude protein. After slaughter at the predetermined wt, yields of trimmed primal cuts and physicochemical characteristics of the longissimus dorsi muscle (LD) were measured or analyzed. Average daily gain was greater (P<0.01) in barrows than in gilts (0.86 vs. 0.79kg), but it was not different between the 110- and 130-kg slaughter wt groups (P>0.05). Backfat thickness was greater in the 130- vs. 110-kg group in both sexes (gilts: 21.6 vs. 17.6 mm, barrows: 25.1 vs. 20.8 mm). The yield percentage of belly was greater in barrows than in gilts (21.2 vs. 20.5%) and also in the 130-kg vs. 110-kg group (21.4 vs. 20.2%). Major physicochemical characteristics of LD including the color, percentage of 48-h drip loss, 24-h pH and percentages of crude protein and fat were not influenced by slaughter wt. Marginal profit of the 130-kg vs. 110-kg market pig was approximately 󰠏20,000 won/head, which resulted primarily from the heavy carcass weight of the former exceeding the upper limit of the A- or B-grade. However, if it had not been for the carcass weight limit of the current grading system, a 130-kg market pig would have had a potential marginal profit greater than 20,000 won. Results suggest that slaughter wt of lean-line finishing pigs can be increased to 130kg without significantly compromising the growth efficiency and carcass quality and accordingly, the upper weight limit of the ‘good-grade’ carcass needs to be increased or abolished to accommodate the larger market pigs.

• The Korean Journal of Nuclear Medicine
• /
• v.35 no.2
• /
• pp.83-88
• /
• 2001

Purpose: The purpose of this study was to ascertain whether radiation adaptive response could be induced by high dose I-131 therapy in patients with differentiated thyroid cancer. Materials and Methods: Lymphocytes from 21 patients (7 males, 14 females, mean age $55{\pm}12$ years) were collected before and after administration of 5,550 MBq (150 mCi) I-131. They were exposed to a challenge dose of 1 Gy gamma rays using a Cs-137 cell irradiator. The number of ring-form (R) and dicentric (D) chromosomes was counted under the light microscope, and used to calculate the frequency of chromosomal aberration. Ydr, which was defined as the sum of R and D divided by the total number of counted lymphocytes. Results: Ydr in patients before I-131 therapy ($0.09{\pm}0.01$) was not different from that of controls ($0.08{\pm}0.01$). Ydr was significantly increased to $0.13{\pm}0.02$ (p<0.0001) after I-131 therapy. Increase of Ydr after the challenge irradiation of 1 Gy was significantly lower in patients after I-131 therapy than before I-131 therapy ($0.17{\pm}0.03\;vs\;0.21{\pm}0.02$, p<0.0001). Cycloheximide (CHM), an inhibitor of protein synthesis, abolished this effect. Ydr after CHM ($0.20{\pm}0.01$) was significantly higher than Ydr after I-131 therapy ($0.17{\pm}0.03$, p<0.0001), but was not different from Ydr before I-131 therapy ($0.21{\pm}0.02$).Conclusion: High dose I-131 therapy induces an adaptive response in peripheral lymphocytes of patients with well-differentiated thyroid cancer, which is associated with protein synthesis.