Jung-Hoon Yoon

Doxorubicin-loaded solid lipid nanoparticles to overcome multidrug resistance in cancer therapy

UNLABELLED In the present study we developed doxorubicin-loaded solid lipid nanoparticles (SLN-Dox) using biocompatible compounds, assessed the in vitro hemolytic effect, and examined their in vivo effects on drug retention and apoptosis intensity in P-glycoprotein-overexpressing MCF-7/ADR cells, a representative Dox-resistant breast cancer cell line. Our SLNs did not show hemolytic activity in human erythrocytes. In comparison with Dox, SLN-Dox efficiently enhanced apoptotic cell death through the higher accumulation of Dox in MCF-7/ADR cells. Therefore, SLN-Dox have potential to serve as a useful therapeutic approach to overcome the chemoresistance of adriamycin-resistant breast cancer. FROM THE CLINICAL EDITOR Doxorubicin loaded solid lipid nanoparticles (SLN-Dox) were studied in a cell line representative of doxorubicin resistant breast cancer. The nanoparticles did not show hemolytic activity; furthermore, they efficiently enhanced apoptotic cell death through higher accumulation of doxorubicin in cancer cells. This approach may be viable in overcoming the chemoresistance of adriamycin resistant breast cancer.

Epigallocatechin-3-gallate suppresses the expression of HSP70 and HSP90 and exhibits anti-tumor activity in vitro and in vivo

BackgroundEpigallocatechin-3-gallate (EGCG), one of the major catechins in green tea, is a potential chemopreventive agent for various cancers. The aim of this study was to examine the effect of EGCG on the expression of heat shock proteins (HSPs) and tumor suppression.MethodsCell colony formation was evaluated by a soft agar assay. Transcriptional activity of HSP70 and HSP90 was determined by luciferase reporter assay. An EGCG-HSPs complex was prepared using EGCG attached to the cyanogen bromide (CNBr)-activated Sepharose 4B. In vivo effect of EGCG on tumor growth was examined in a xenograft model.ResultsTreatment with EGCG decreased cell proliferation and colony formation of MCF-7 human breast cancer cells. EGCG specifically inhibited the expression of HSP70 and HSP90 by inhibiting the promoter activity of HSP70 and HSP90. Pretreatment with EGCG increased the stress sensitivity of MCF-7 cells upon heat shock (44°C for 1 h) or oxidative stress (H2O2, 500 μM for 24 h). Moreover, treatment with EGCG (10 mg/kg) in a xenograft model resulted in delayed tumor incidence and reduced tumor size, as well as the inhibition of HSP70 and HSP90 expression.ConclusionsOverall, these findings demonstrate that HSP70 and HSP90 are potent molecular targets of EGCG and suggest EGCG as a drug candidate for the treatment of human cancer.

Full-field optical coherence microscopy for identifying live cancer cells by quantitative measurement of refractive index distribution.

The feasibility of identifying cancer cells by measuring the refractive index (RI) distribution across a single live cell with ultrahigh resolution full-field optical coherence microscopy (FF-OCM) is presented. The FF-OCM is utilized to quantify integral RI distributions of unmodified cells without any cell treatments and used as a biophysical indicator for diagnosing cell malignancy. Firstly, the physical thickness distribution of the cell adherent to a culture dish is measured by taking a series of 0.6 µm resolved en-face tomograms. Subsequently, from the en-face image of the bottom surface of the cell or the top surface of the dish, the phase gain image of the cell is extracted. Then, from these two measurements the axially averaged RI map of the cell is extracted. The implemented FF-OCM system had a 0.8 µm axial resolution and the phase measurement sensitivity of the system was around 124 mrad. With the system, RI maps of several living cell lines of normal and cancer cells were constructed and quantitatively analyzed. The experiments showed that cancer cells had higher RI than normal ones. This approach using the FF-OCM has significant potential for cancer diagnosis and dynamic cell analysis as in situ label-free biophysical assay.

Polo-like Kinase 1 Phosphorylates Heat Shock Transcription Factor 1 and Mediates Its Nuclear Translocation during Heat Stress

Heat shock transcription factor 1 (HSF1) is activated by pathophysiologic stresses and activation leads to an increased cellular level of heat shock proteins (Hsp(s)). Although the activation of HSF1 occurs via multiple stress-induced processes such as hyperphosphorylation, the exact cellular mechanism of HSF1 activation is still unclear. Here we show polo-like kinase 1 (PLK1) and HSF1 interact in vivo using the tandem affinity purification system. Although the interaction between HSF1 and PLK1 is increased by thermal stress, overexpression of PLK1 did not affect HSF1 trimerization or DNA binding activity. This interaction results in the phosphorylation of HSF1 on serine 419 by PLK1. Interestingly, mutation of serine 419 to alanine inhibited heat-stress induced HSF1 nuclear translocation. Our results suggest that the phosphorylation of HSF1 by PLK1 is an essential step for HSF1 nuclear translocation by heat stress.

Antitumor Activity of Novel Indirubin Derivatives in Rat Tumor Model

Purpose: The novel indirubin derivatives 5′-nitro-indirubinoxime, 5′-fluoro-indirubinoxime, and 5′-trimethylacetamino-indirubinoxime were designed and tested for antitumor activity both in vitro and in vivo using rat tumor model. Experimental Design: Three-week-old male Sprague-Dawley rats were inoculated s.c. on the left flank with 107 RK3E-ras rat kidney epithelial cells harboring k-ras gene. Alternatively, 5 × 106 RK3E-ras cells were injected into the oral mucosa. Indirubin derivative treatment began on the 3rd or 6th day after oral or s.c. cell injection, respectively. Indirubin derivatives were directly injected into the tumor every other day for a total of five times. Animals were monitored daily and tumor volume was measured by caliper. Results: Indirubin derivatives showed potent antiproliferative activity on various human cancer cells and oncogenic RK3E-ras rat kidney cells, with IC50 ranging from 1 to 12 μmol/L. Treatment with indirubin derivatives induced the activation of caspase-7 followed by apoptosis in RK3E-ras cells. Indirubin derivatives showed strong antitumor activity in rat solid and oral tumor models. Direct injection of indirubin derivatives every other day for 10 days induced significant inhibition of tumor growth in Sprague-Dawley rats bearing RK3E-ras-induced tumors. Histologically, treatment with indirubin derivatives caused significant inhibition of tumor formation with increased apoptosis and decreased tumor cell proliferation. Conclusions: Our data showed that novel indirubin derivatives 5′-nitro-indirubinoxime, 5′-fluoro-indirubinoxime, and 5′-trimethylacetamino-indirubinoxime effectively arrested the tumor growth by inhibiting cell proliferation and inducing apoptosis. These findings provide the potential value of indirubin derivatives as novel candidates for antitumor agents.

Amurensin G, a Potent Natural SIRT1 Inhibitor, Rescues Doxorubicin Responsiveness via Down-Regulation of Multidrug Resistance 1

The transition from a chemotherapy-responsive cancer to a chemotherapy-resistant one is accompanied by increased expression of multidrug resistance 1 (MDR1, p-glycoprotein), which plays an important role in the efflux from the target cell of many anticancer agents. We recently showed that a Forkhead box-containing protein of the O subfamily 1 (FoxO1) is a key regulator of MDR1 gene transcription. Because nuclear localization of FoxO1 is regulated by silent information regulator two ortholog 1 (SIRT1) deacetylase, we wondered whether SIRT1 dominates MDR1 gene expression in breast cancer cells. Overexpression of SIRT1 enhanced both FoxO reporter activity and nuclear levels of FoxO1. Protein expression of MDR1 and gene transcriptional activity were also up-regulated by SIRT1 overexpression. In addition, SIRT1 inhibition reduced both nuclear FoxO1 levels and MDR1 expression in doxorubicin-resistant breast cancer cells (MCF-7/ADR) cells. A potent SIRT1 inhibitor, amurensin G (from Vitis amurensis), was identified by screening plant extracts and bioassay-guided fractionation. The compound suppressed FoxO1 activity and MDR1 expression in MCF-7/ADR cells. Moreover, pretreatment of MCF-7/ADR cells with 1 μg/ml amurensin G for 24 h increased cellular uptake of doxorubicin and restored the responsiveness of MCF-7/ADR cells to doxorubicin. In xenograft studies, injection of 10 mg/kg i.p. amurensin G substantially restored the ability of doxorubicin to inhibit MCF-7/ADR-induced tumor growth. These results suggest that SIRT1 is a potential therapeutic target of MDR1-mediated chemoresistance and that it may be possible to develop amurensin G as a useful agent for chemoresistance reversal.

Role of autophagy in chemoresistance: Regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA–PKcs and PARP-1

Capsaicin treatment was previously reported to reduce the sensitivity of breast cancer cells, but not normal MCF10A cells, to apoptosis. The present study shows that autophagy is involved in cellular resistance to genotoxic stress, through DNA repair. Capsaicin treatment of MCF-7 cells induced S-phase arrest and autophagy through the AMPKα-mTOR signaling pathway and the accumulation of p53 in the nucleus and cytosol, including a change in mitochondrial membrane potential. Capsaicin treatment also activated δ-H2AX, ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and poly(ADP-ribose) polymerase (PARP)-1. Genetic or pharmacological disruption of autophagy attenuated capsaicin-induced phospho-ATM and phospho-DNA-PKcs and enhanced apoptotic cell death. ATM inhibitors, including Ku55933 and caffeine, and the genetic or pharmacological inhibition of p53 prevented capsaicin-induced DNA-PKcs phosphorylation and stimulated PARP-1 cleavage, but had no effect on microtubule-associated protein light chain 3 (LC3)-II levels. Ly294002, a DNA-PKcs inhibitor, boosted the capsaicin-induced cleavage of PARP-1. In M059K cells, but not M059J cells, capsaicin induced ATM and DNA-PKcs phosphorylation, p53 accumulation, and the stimulation of LC3II production, all of which were attenuated by knockdown of the autophagy-related gene atg5. Ku55933 attenuated capsaicin-induced phospho-DNA-PKcs, but not LC3II, in M059K cells. In human breast tumors, but not in normal tissues, AMPKα, ATM, DNA-PKcs, and PARP-1 were activated and LC3II was induced. The induction of autophagy by genotoxic stress likely contributes to the sustained survival of breast cancer cells through DNA repair regulated by ATM-mediated activation of DNA-PKcs and PARP-1.

Role of PTEN promoter methylation in tamoxifen-resistant breast cancer cells

Tamoxifen (TAM) resistance is a serious clinical problem in the treatment of breast cancer. Here, we found that S-adenosylmethionine (SAM) and DNA methyltransferase1 (DNMT1) expression are up-regulated in TAM-resistant breast cancer (TAMR-MCF-7) cells. We further focused on whether increased SAM with DNMT1 overexpression in TAMR-MCF-7 cells lead to aberrant methylation of the PTEN gene promoter and its therapeutic potential. Methylation-specific PCR analyses revealed that two sites within the PTEN promoters were methylated in TAMR-MCF-7 cells, which resulted in down-regulation of PTEN expression and increase in Akt phosphorylation. Both the loss of PTEN expression and the increased Akt phosphorylation in TAMR-MCF-7 cells were completely reversed by 5-aza-2′-deoxycytidine (5-Aza), a DNMT inhibitor. 5-Aza inhibited the basal cell proliferation rate of TAMR-MCF-7 cells and intraperitoneal injection of 5-Aza significantly suppressed TAMR-MCF-7 tumor growth in a xenograft study. Immunohistochemistry showed that PTEN expression in TAM-resistant human breast cancer tissues was lower than in TAM-responsive cases. These results suggest that methylation of the PTEN promoter related to both SAM increase and DNMT1 activation contributes to persistent Akt activation and are potential therapeutic targets for reversing TAM resistance in breast cancer.

Apicidin, a histone deaceylase inhibitor, induces both apoptosis and autophagy in human oral squamous carcinoma cells.

Apicidin acts as a potent histone deacetylases (HDAC) inhibitor and the precise mechanism for its anti-tumor activity in human oral squamous cell carcinoma (OSCC) cells has not been examined. The aim of this study was to evaluate the anti-tumor efficacy of apicidin through apoptosis and autophagy in OSCC cells. Cells were treated with apicidin and cell death was quantified. Cell cycle and apoptosis were measured using flow cytometry assay, immunoblot. Autophagy was characterized by the increase of LC3B-II and the formation of acidic vesicular organelles (AVOs). Apicidin significantly inhibited the proliferation of OSCC cells in a dose-dependent manner. Apicidin markedly up-regulated p21(WAF1) led to G2/M phase arrest. Apicidin significantly increased the number of apoptotic cells compared to untreated control. Apicidin induced not only apoptosis but also autophagy in OSCC cells. Apicidin dramatically increased the levels of LC3 type II expression, ATG5 protein expression and the accumulation of AVOs. Inhibition of autophagy enhanced apicidin-mediated cytotoxicity through an increase in apoptosis. These results suggest that apicidin exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence of apicidin-induced autophagy and autophagy inhibition enhances apicidin-mediated apoptosis in OSCC cells.

Novel Role of Pin1 Induction in Type II Collagen-Mediated Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints and subsequent destruction of cartilage and bone. Inflammatory mediators such as PGs and proinflammatory cytokines contribute to RA progress. Pin1, a peptidyl prolyl isomerase, plays important pathophysiological roles in several diseases, including cancer and neurodegeneration. We found that both Pin1 and cyclooxygenase-2 (COX-2) were highly expressed in ankle tissues of type II collagen-induced RA mice. HTB-94 cells overexpressing Pin1 and primary cultured human chondrocytes showed increased basal expression of proinflammatory proteins (COX-2, inducible NO synthase, TNF-α, and IL-1β). Site-directed mutagenesis revealed that Pin1-mediated transcriptional activation of COX-2 was coordinately regulated by NF-κB, CREB, and C/EBP. Gel shift, reporter gene, and Western blot analyses confirmed that NF-κB, CREB, and C/EBP were consistently activated in chondrocytes overexpressing Pin1. Treatment of RA mice with juglone, a chemical inhibitor of Pin1, significantly reduced RA progress and COX-2 expression in the ankle tissues. Moreover, juglone dose dependently decreased the basal COX-2 expression in primary cultured chondrocytes from RA patients. These results demonstrate that Pin1 induction during RA progress stimulates proinflammatory protein expression by activating NF-κB, CREB, and C/EBP, and suggest that Pin1 is a potential therapeutic target of RA.