Modulation of Immunosuppression by Oligonucleotide-Based Molecules and Small Molecules Targeting Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that exert suppressive function on the immune response. MDSCs expand in tumor-bearing hosts or in the tumor microenvironment and suppress T cell responses via various mechanisms, whereas a reduction in their activities has been observed in autoimmune diseases or infections. It has been reported that the symptoms of various diseases, including malignant tumors, can be alleviated by targeting MDSCs. Moreover, MDSCs can contribute to patient resistance to therapy using immune checkpoint inhibitors. In line with these therapeutic approaches, diverse oligonucleotide-based molecules and small molecules have been evaluated for their therapeutic efficacy in several disease models via the modulation of MDSC activity. In the current review, MDSC-targeting oligonucleotides and small molecules are briefly summarized, and we highlight the immunomodulatory effects on MDSCs in a variety of disease models and the application of MDSC-targeting molecules for immuno-oncologic therapy.

Role of STAT3 as a Negative Regulator in Mac2- Binding Protein Expression / 대한진단검사의학회지

BACKGROUND: Mac-2 binding protein (Mac-2BP) is a secreted glycoprotein from the culture fluid of several human cancer cells, especially breast, lung, and gastric cells. Mac-2BP plays a role in immune response and cell adhesion activity in patients with various cancer and infectious diseases. In this study, we attempted to identify the regulators of Mac-2BP expression at the transcriptional level. METHODS: To determine the effect of epidermal growth factor (EGF) to Mac-2BP expression in gastric cancers, we constructed the different lengths of Mac-2BP promoter plasmids and measured the promoter activity and Mac-2BP expression. In addition to investigating the role of signal transducer and activator of transcription3 (STAT3) or human telomerase reverse transcriptase (hTERT) as a regulator of Mac-2BP, we transfected the small interfering RNA (siRNA) specific for STAT3 or hTERT, and Mac-2BP level was observed by a quantitative ELISA. RESULTS: EGF treatment could suppress the Mac-2BP transcription in HEK293 or gastric cancer cell lines (SNU-638 or AGS). In 5'-deleted promoter experiment, pGL3-Mac Pro-2377 transfected cells showed a decreased luciferase activity compared to pGL3-Mac Pro-2277. We also identified that (-2,366/-2,356) on Mac-2BP promoter is a putative STAT3 binding site and suppression of STAT3 with STAT3 specific siRNA increased the Mac-2BP level, suggesting the role of STAT3 as a negative regulator, in contrast to hTERT, which is known as a positive regulator. CONCLUSIONS: EGF signal is critical for the Mac-2BP expression, and more importantly, STAT3 could work as a negative regulator, while hTERT as a positive regulator in Mac-2BP transcription.

Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors / 대한진단검사의학회지

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.

Modulation of Telomerase Activity and Human Telomerase Reverse Transcriptase Expression by Caspases and Bcl-2 Family Proteins in Cisplatin-Induced Cell Death / 대한진단검사의학회지

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.

C-Terminal Serine Rich Region of IKK-beta Interacts with Bfl-1, a Homologue of Bcl-2 / 대한진단검사의학회지

BACKGROUND: Bcl-2 family proteins play a central role in regulating apoptosis. In human, over 20 members of this family have been identified to date. Bfl-1, a member of the Bcl-2 family, has been known to retard apoptosis in various cell lines. However, the function of Bfl-1 remains unclear. METHODS: In order to investigate the Bfl-1 function, we employed yeast two-hybrid system to identify the proteins which are capable of interacting with Bfl-1. The interaction of inhibitor kappaB kinase-beta (IKK-beta) and Bfl-1 was confirmed using glutathione S-transferase pull down assays. To determine which regions of IKK-beta were required for interaction with Bfl-1, we constructed 12 deletion mutants of IKK-beta and 5 deletion mutants of Bfl-1. RESULTS: Bfl-1 interacted with the C-terminal region of IKK-beta which is a subunit of IKK complex, and IKK-beta activity is very important in the NF-kappaB related pathway. In addition, the amino acids 673-745 of IKK-beta were important for Bfl-1 interactions, and amino acids 1-484 of Bfl-1, including Bcl-2 homology domains (BH1, BH2, BH3, BH4), were crucial for IKK-beta interactions. CONCLUSIONS: IKK beta C-terminus contains many serine residues as binding partner of Bfl-1. Our results suggested that Bfl-1 is involved in the NF-kappaB activation through interaction of IKK-beta and Bfl-1. Further studies need to be performed to understand functions of the IKK-beta and Bfl-1 associated with the regulation of the NF-kappaB activation pathway.

IL-12 Production and Subsequent Natural Killer Cell Activation by Necrotic Tumor Cell-loaded Dendritic Cells in Therapeutic Vaccinations

BACKGROUND: Immunization of dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. In this study, we examined whether the uptake of necrotic tumor cells could modulate DC phenotypes and whether the immunization of necrotic tumor cell-loaded DCs could elicit efficient tumor specific immune responses followed by a regression of established tumor burdens. METHODS: We prepared necrotic tumor cell-pulsed DCs for the therapeutic vaccination and investigated their phenotypic characteristics, the immune responses induced by these DCs, and therapeutic vaccine efficacy against colon carcinoma in vivo. Several parameters including phagocytosis of tumor cells, surface antigen expression, chemokine receptor expression, IL-12 production, and NK as well as CTL activation were assessed to characterize the immune response. RESULTS: DCs derived from mouse bone marrow efficiently phagocytosed necrotic tumor cells and after the uptake, they produced remarkably increased levels of IL-12. A decreased CCR1 and increased CCR7 expression on DCs was also observed after the tumor uptake, suggesting that antigen uptake could induce DC maturation. Furthermore, co-culturing of DCs with NK cells in vitro enhanced IL-12 production in DCs and IFN-gamma production in NK cells, which was significantly dependent on IL-12 production and cell-to-cell contact. Immunization of necrotic tumor cell-loaded DCs induced cytotoxic T lymphocytes as well as NK activation, and protected mice against subsequent tumor challenge. In addition, intratumoral or contra-lateral immunization of these DCs not only inhibited the growth of established tumors, but also eradicated tumors in more than 60% of tumor-bearing mice. CONCLUSION: Our data indicate that production of IL-12, chemokine receptor expression and NK as well as CTL activation may serve as major parameters in assessing the effect of tumor cell-pulsed DC vaccine. Therefore, DCs loaded with necrotic tumor cells offer a rational strategy to treat tumors and eventually lead to prolonged survival.

The Effect of Induced Heat Shock Protein 33 in Human Corneal Epithelial Cell

PURPOSE: The purposes of study was to assess the expression patterns of heat shock protein 33 (HSP33) after gene transfection and to evaluate the protective effects of heat shock protein 33 from apoptosis and oxidative stress in transfected human corneal epithelial cell. METHODS: The cultured human corneal epithelial cells were divided control and experimental group. Experimental group was transfected with HSP 33. After transfection, the experimental group was treated with etoposide (induced apoptosis) and hydrogen peroxide (induced oxidative stress). The expression patterns of HSP 33 was0 examined by western blot. The viability (cell protection rate of heat shock protein) and protective effect against apoptosis after etoposide and hydrogen peroxide treatment were measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Expression of Heat shock protein 33 was increased in the transfection group compared with non-transfection group. The increased cell number (viability=anti-apoptotic effect of heat shock protein) of transfection group with induced HSP 33 etoposide and hydrogen peroxide treatment show that Hsp 33 appeared to have protective effect in Human corneal epithelial cell from apoptosis and oxidative stress. CONCLUSIONS: These data suggest that experimentally induced heat shock protein 33 could protect etoposide-generated apoptosis and hydreogen peroxide generated oxidative stress in human corneal epithelial cell.

Detection of Hepatitis B Virus X Antigen and Anti-X Antibody in Sera of HBV Infected Patients by ELISA using Recombinant X Proteins Expressed in E. coli and Anti-HBx Monoclonal Antibodies / 대한임상병리학회지

BACKGROUND: The X gene is the smallest coding region of the hepatitis B virus (HBV) genome. Several studies reported that X gene-encoded protein may be related to viral replication, and possibly used as a new marker indicative of HBV infection. However, its practical application as a diagnostic reagent remains limited. In this study, we developed anti-X monoclonal antibodies using recombinant hepatitis B virus X (HBx) proteins and investigated the humoral immune responses against HBx in sera of HBV-infected patients by enzyme-linked immunosorbent assay (ELISA). METHODS: Sera of 47 HBV-associated patients and 12 normal controls were studied. Using recombinant HBx expressed in Escherichia coli, seven clones of monoclonal anti-HBx antibodies were developed. The binding site and activity of each monoclonal antibody were determined by ELISA and Western blot analysis, and antibodies that gave the best signals in both assays were selected for the detection of HBx antigen. An ELISA to detect anti-X was also constructed by using recombinant HBx proteins. RESULTS: Clinical samples from patients with liver cirrhosis and hepatocellular carcinoma (HCC) were more than 60% positive for anti-HBx antibody. The positive rate of X antigen in patients with liver cirrhosis and HCC was 27% and 33%, respectively. None of acute hepatitis patients and chronic asymptomatic carriers were positive for HBx antigen or anti-X antibody. The present ELISA system detected circulating HBx with a dynamic range from 5 to 1000 ng per milliliter and the specificity of the assay was also acceptable. The analysis of binding site and activity of monoclonal antibodies performed by ELISA were in agreement with Western blotting results. CONCLUSIONS: ELISA using recombinant HBx and monoclonal antibodies showed good sensitivity and corresponded well with immunoblotting results. For the clinical application of this assay, however, further study is needed on the relationship between HBx and the progression of the disease.