Sang-Hyun Song

Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanism in gastric cancer cells

DLC-1 (deleted in liver cancer) gene is frequently deleted in hepatocellular carcinoma. However, little is known about the genetic status and the expression of this gene in gastric cancer. In this study, Northern and Southern analysis showed that seven of nine human gastric cancer cell lines did not express DLC-1 mRNA, but contained the DLC-1 gene. To identify the mechanism of the loss of DLC-1 mRNA expression in these cell lines, we investigated the methylation status of DLC-1 gene by using methylation-specific PCR (MSP) and Southern blot, and found that five of seven DLC-1 nonexpressing gastric cancer cell lines were methylated in the DLC-1 CpG island. Treatment with 5-aza-2′-deoxycytidine (5-Aza-dC) induced DLC-1 mRNA expression in the gastric cancer cell lines that have the methylated alleles. Studies using SNU-601 cell line with methylated DLC-1 alleles revealed that nearly all CpG sites within DLC-1 CpG island were methylated, and that the in vitro methylation of the DLC-1 promoter region is enough to repress DLC-1 mRNA expression, regardless of the presence of transcription factors capable of inducing this gene. In all, 29 of 97 (30%) primary gastric cancers were also shown to be methylated, demonstrating that methylation of the DLC-1 CpG island is not uncommon in gastric cancer. In addition, we demonstrated that DLC-1 mRNA expression was induced, and an increase in the level of acetylated H3 and H4 was detected by the treatment with trichostatin A (TSA) in two DLC-1 nonexpressing cell lines that have the unmethylated alleles. Taken together, the results of our study suggest that the transcriptional silencing of DLC-1, by epigenetic mechanism, may be involved in gastric carcinogenesis.

AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity.

AKAP12/Gravin, one of the A-kinase anchoring proteins (AKAPs), functions as a kinase scaffold protein and as a dynamic regulator of the β2-adrenergic receptor complex. However, the biological role of AKAP12 in cancer development is not well understood. The AKAP12 gene encodes two major isoforms of 305 and 287 kDa (designated AKAP12A and AKAP12B, respectively, in this report). We found that these two isoforms are independently expressed and that they are probably under the control of two different promoters. Moreover, both isoforms were absent from the majority of human gastric cancer cells. The results from methylation-specific PCR (MSP) and bisulfite sequencing revealed that the 5′ CpG islands of both AKAP12A and AKAP12B are frequently hypermethylated in gastric cancer cells. Treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor efficiently restored the expression of AKAP12 isoforms, confirming that DNA methylation is directly involved in the transcriptional silencing of AKAP12 in gastric cancer cells. Hypermethylation of AKAP12A CpG island was also detected in 56% (10 of 18) of primary gastric tumors. The restoration of AKAP12A in AKAP12-nonexpressing cells reduced colony formation and induced apoptotic cell death. In conclusion, our results suggest that AKAP12A may function as an important negative regulator of the survival pathway in human gastric cancer.

Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using NF-κB as a possible target

Purpose Recently, many studies have shown that celecoxib induces apoptosis in various cancer cells by different mechanisms depending on the cell type. This study examined the apoptotic effect of celecoxib in cervical cancer cells and its mechanism.Methods Cell viability was measured by MTT assay and apoptosis was examined by DNA fragmentation and flow cytometry. Western blotting and immunoprecipitation were used to explore various mechanisms of celecoxib-induced apoptosis. The activation of NF-κB was confirmed by EMSA.Results Celecoxib induced apoptosis independent of COX-2 activity. This event accompanied the activation of caspase-8 and -9 with Bid cleavage and the loss of mitochondrial membrane potential. The protective effect of caspase-8 and -9 inhibitors on celecoxib-induced apoptosis suggests the importance of caspase-8 and -9 activation in this apoptotic pathway. Fas/FADD-mediated apoptotic pathway was detected only in C33A cells, demonstrated by the immunoprecipitation of Fas-FADD in celecoxib-treated cells and the protective effect of FADD dominant negative mutant. Finally, NF-κB appeared to be involved in celecoxib-induced apoptosis, as revealed by increased NF-kB DNA binding activity in a time-dependent manner and attenuation of its proapoptotic effect by N-tosyl-L-phenylalanyl-chloromethyl ketone, an NF-kB blocker.Conclusions These data show that caspase-8 and -9 are involved in the apoptotic effect of celecoxib in cervical cancer cells. This requires the FADD-dependent pathway in a cell type-specific manner. In addition, NF-κB may play a key role in celecoxib-induced apoptosis.

Epigenetic-based therapies in cancer: progress to date.

Epigenetic gene silencing is a hallmark of cancer cells. Two important types of epigenetic changes are DNA methylation and histone modification. These modifications are catalysed by DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), resulting in chromatin structure changes and gene inactivation. Interestingly, inhibition of these enzymes is known to induce differentiation or apoptosis of cancer cells. Therefore, DNMTs and HDACs have become attractive therapeutic targets. In recent years, many different DNMT and HDAC inhibitors have been developed, and multiple molecular mechanisms through which these agents exert anti-cancer effects have been identified. While a large number of clinical trials are ongoing, hypomethylating agents and HDAC inhibitors seem to be promising for treating several types of cancer. Moreover, developing effective strategies of combining epigenetic therapy with conventional chemotherapy will be one of the major challenges in the future.We briefly review current advances in epigenetic therapies with a focus on recently reported clinical trials.

The effect of nitric oxide on cyclooxygenase-2 (COX-2) overexpression in head and neck cancer cell lines.

The overexpression of cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) has been previously reported in head and neck squamous cell carcinoma (HNSCC), as well as in many cancers. We hypothesized that endogenous nitric oxide (NO) might increase the expression of COX‐2 in cancer cells. Therefore, we investigated the cross‐talk between NO and the prostaglandin (PG) pathways in HNSCC cell lines. We found that COX‐2 and iNOS expressions were elevated simultaneously. On adding the NO donor, SNAP, the PGE2 level was increased 2–20 times due to increased COX‐2 expression. This increase of COX‐2 expression by SNAP or PMA (potent inducer of both iNOS and COX‐2) was blocked to various degrees by NO scavengers and NOS inhibitors (L‐NAME and 1400W). Also, the expression of COX‐2 in resting cells was inhibited by NOS inhibitors. Moreover, COX‐2 expression, induced by SNAP, was inhibited by ODQ, a soluble guanylate cyclase (sGC) inhibitor. The effect of dibutyryl‐cGMP on COX‐2 expression was similar to that of SNAP. These results imply that endogenous or exogenous NO activates sGC and that the resulting increase of cGMP induces a signaling that upregulates the expression of COX‐2 in HNSCC cell lines. We also observed that NO increased COX‐2 expression in different cancer cell lines, including cervic and gastric cancer cell lines. These findings further support the notion that NO can be associated with carcinogenesis through the upregulation of COX‐2, and that NOS inhibitor may be also useful for cancer prevention.

RAD51C-Deficient Cancer Cells Are Highly Sensitive to the PARP Inhibitor Olaparib

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

Inhibition of histone deacetylase 10 induces thioredoxin-interacting protein and causes accumulation of reactive oxygen species in SNU-620 human gastric cancer cells

Histone deacetylase (HDAC)10, a novel class IIb histone deacetylase, is the most similar to HDAC6, since both contain a unique second catalytic domain. Unlike HDAC6, which is located in the cytoplasm, HDAC10 resides in both the nucleus and cytoplasm. The transcriptional targets of HDAC10 that are associated with HDAC10 gene regulation have not been identified. In the present study, we found that knockdown of HDAC10 significantly increased the mRNA expression levels of thioredoxin-interacting protein (TXNIP) in SNU-620 human gastric cancer cells; whereas inhibition of HDAC1, HDAC2, and HDAC6 did not affect TXNIP expression. TXNIP is the endogenous inhibitor of thioredoxin (TRX), which acts as a cellular antioxidant. Real-time PCR and immunoblot analysis confirmed that inhibition of HDAC10 induced TXNIP expression. Compared to class I only HDAC inhibitors, inhibitors targeting both class I and II upregulated TXNIP, indicating that TXNIP is regulated by class II HDACs such as HDAC10. We further verified that inhibition of HDAC10 induced release of cytochrome c and activated apoptotic signaling molecules through accumulation of reactive oxygen species (ROS). Taken together, our results demonstrate that HDAC10 is involved in transcriptional downregulation of TXNIP, leading to altered ROS signaling in human gastric cancer cells. How TXNIP is preferentially regulated by HDAC10 needs further investigation.

Testican-1-mediated epithelial–mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer

Human epidermal growth factor receptor 2 (HER2)-directed treatment using trastuzumab has shown clinical benefit in HER2-positive gastric cancer. Clinical trials using lapatinib in HER2-positive gastric cancer are also currently underway. As with other molecularly targeted agents, the emergence of acquired resistance to HER2-directed treatment is an imminent therapeutic problem for HER2-positive gastric cancer. In order to investigate the mechanisms of acquired resistance to HER2-directed treatment in gastric cancer, we generated lapatinib-resistant gastric cancer cell lines (SNU216 LR) in vitro by chronic exposure of a HER2-positive gastric cancer cell line (SNU216) to lapatinib. The resultant SNU216 LR cells were also resistant to gefitinib, cetuximab, trastuzumab, afatinib and dacomitinib. Interestingly, SNU216 LR cells displayed an epithelial–mesenchymal transition (EMT) phenotype and maintained the activation of MET, HER3, Stat3, Akt and mitogen-activated protein kinase signaling in the presence of lapatinib. Using gene expression arrays, we identified the upregulation of a variety of EMT-related genes and extracellular matrix molecules, such as Testican-1, in SNU216 LR cells. We showed that the inhibition of Testican-1 by small interfering RNA decreased Testican-1-induced, MET-dependent, downstream signaling, and restored sensitivity to lapatinib in these cells. Furthermore, treatment with XAV939 selectively inhibited β-catenin-mediated transcription and Testican-1-induced EMT signaling, leading to G1 arrest. Taken together, these data support the potential role of EMT in acquired resistance to HER2-directed treatment in HER2-positive gastric cancer, and provide insights into strategies for preventing and/or overcoming this resistance in patients.

Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances anti-tumor effects of the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in triple-negative breast cancer cells.

IntroductionOlaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, has been found to have therapeutic potential for treating cancers associated with impaired DNA repair capabilities, particularly those with deficiencies in the homologous recombination repair (HRR) pathway. Histone deacetylases (HDACs) are important for enabling functional HRR of DNA by regulating the expression of HRR-related genes and promoting the accurate assembly of HRR-directed sub-nuclear foci. Thus, HDAC inhibitors have recently emerged as a therapeutic agent for treating cancer by inhibiting DNA repair. Based on this, HDAC inhibition could be predicted to enhance the anti-tumor effect of PARP inhibitors in cancer cells by blocking the HRR pathway.MethodsWe determined whether suberoylanilide hydroxamic acid (SAHA), a HDAC inhibitor, could enhance the anti-tumor effects of olaparib on breast cancer cell lines using a cytotoxic assay, cell cycle analysis, and Western blotting. We evaluated how exposure to SAHA affects the expression of HRR-associated genes. The accumulation of DNA double strand breaks (DSBs) induced by combination treatment was assessed. Induction of autophagy was monitored by imaging green fluorescent protein-tagged microtubule-associated protein 1A/1B-light chain 3 (LC3) expression following co-treatment with olaparib and SAHA. These in vitro data were validated in vivo using a human breast cancer xenograft model.ResultsTriple-negative breast cancer cell (TNBC) lines showed heterogeneous responses to the PARP and HDAC inhibitors. Co-administration of olaparib and SAHA synergistically inhibited the growth of TNBC cells that expressed functional Phosphatase and tensin homolog (PTEN). This effect was associated with down-regulation of the proliferative signaling pathway, increased apoptotic and autophagic cell death, and accumulation of DNA damage. The combined anti-tumor effect of olaparib and SAHA was also observed in a xenograft model. These data suggest that PTEN expression in TNBC cells can sensitize the cell response to simultaneous inhibition of PARP and HDAC both in vitro and in vivo.ConclusionOur findings suggest that expression of functional PTEN may serve as a biomarker for selecting TNBC patients that would favorably respond to a combination of olaparib with SAHA. This provides a strong rationale for treating TNBC patients with PTEN expression with a combination therapy consisting of olaparib and SAHA.

RAD001 shows activity against gastric cancer cells and overcomes 5-FU resistance by downregulating thymidylate synthase

We evaluated RAD001, an inhibitor of the mammalian target of rapamycin (mTOR) in human gastric cancer cell lines and determined the molecular mechanisms. RAD001 has marked growth inhibitory activity against the SNU-1 and SNU-216 cells. It inhibited phosphorylation of mTOR and S6K, and induced G1 cell cycle arrest. Synergistic growth-inhibitory effects in combination with 5-fluorouracil (5-FU) was identified. Furthermore, RAD001 conferred sensitivity to 5-FU-resistant cell lines by downregulating thymidylate synthase (TS). In conclusion, RAD001 showed growth inhibitory activity against gastric cancer cells and acted synergistically with cytotoxic agents such as 5-FU by downregulating TS.