Holotransferrin Enhances Selective Anticancer Activity of Artemisinin against Human Hepatocellular Carcinoma Cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance （ p 〈 0. 05, p 〈 0.01 ）. The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis

AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism.METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice.Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected.RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%)and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75± 1.12%,P＜0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807)was significantly lower than that in control group (22.330 ± 5.696, P＜ 0.01).CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.

Inhibitory effects of genistein on metastasis of human hepatocellular carcinoma

AIM:To investigate the inhibitory effects of genistein on metastasis of MHCC97-H hepatocellular carcinoma cells and to explore the underlying mechanism.METHODS:MHCC97-H hepatocellular carcinoma cells were exposed to genistein.A cell attachment assay was carried out in a microculture well pre-coated with fibronectin.The invasive activity of tumor cells was assayed in a transwell cell culture chamber,and cell cycle and apoptosis were evaluated by a functional assay.In addition,the expression and phosphorylation of FAK were detected by Western blotting.In situ xenograft transplantation of hepatocellular carcinoma was performed in 12 nude mice and lung metastasis of hepatocellular carcinoma was observed.RESULTS:Genistein signifi cantly inhibited the growth of MHCC97-H cells in vitro.Adhesion and invasiveness of MHCC97-H cells were inhibited in a concentration-dependent fashion,and the inhibitory effect of genistein was more potent in the 10 μg/mL and 20 μg/ mL genistein-treated groups.Genistein caused G0/G1 cell cycle arrest,an S phase decrease,and increased apoptosis.The expression and phosphorylation of FAK in MHCC-97H cells were signifi cantly decreased.In situ xenograft transplantation of hepatocellular carcinoma was also significantly suppressed by genistein.The number of pulmonary micrometastatic foci in the genistein group was signifi cantly lower compared with the control group(12.3±1.8 vs 16.6±2.6,P<0.05).CONCLUSION:Genistein appears to be a promising agent in the inhibition of metastasis of hepatocellular carcinoma.

Over-expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma

AIM: To describe the significant over-expression of fibroblast growth factor receptor 3 (FGFR3), which is a signal transduction and cell proliferation related gene in hepatocellular carcinoma (HCC).METHODS: Following DNA microarray, Northern blot and quantitative real-time PCR were employed to confirm FGFR3 expression difference in HCC tissues and surrounding non-neoplastic liver tissue. FGFR3 expression levels were further determined by immunohistochemical study in 43 cases of HCC.RESULTS: Northern blot results showed the significant over-expression of FGFR3 in HCC tissues, which was consistent with that from DNA microarray. Quantitative real-time PCR demonstrated that the mean ratio of FGFR3 mRNA to glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA in HCC tissue was 0.250, whereas the ratio in non-neoplastic liver tissue was 0.014. Statistical analyses of 43 cases of HCC revealed that HCC scored higher than the matched non-neoplastic liver tissues.Examination of clinicopathological features revealed a strong correlation of over-expression of FGFR3 with poor tumor differentiation and high nuclear grade.CONCLUSION: Over-expression of FGFR3 may play an important role in liver carcinogenesis. FGFR3 may be an ideal candidate as a molecular marker in the diagnosis of HCC and a potential therapeutic target.

Enhanced radioimmunotherapeutic efficacy of a monoclonal antibody cocktail against SMMC-7721 human hepatocellular carcinoma

The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal atibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. boioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I labeled Hepama-1 and 131Llabeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 Mab alone The MAb cocktail could target a greater number of hepstoma cells and increase the magnitude of hepatoma cen uptde of radioamibodies. The in vjtro results explain the enhanced effect of the MAb cocktail in in vjvo model system.

Antitumor activity of an hTERT promoter-regulated tumor-selective oncolytic adenovirus in human hepatocellular carcinoma

AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, and in vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice. RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015. Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells. CONCLUSION: hTERT promoter-regulated replicativeadenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.

Farnesoid X receptor expression is reduced in human hepatocellular carcinoma

Farnesoid X receptor (FXR, NR1H4) is a member of nuclear hormone receptor superfamily. Previously studies showed that FXR-/- mice spontaneously developed liver tumors when they aged, however, the relevance of which to human hepatocellular carcinoma (HCC) is unclear. The aim of this study is to observe whether FXR expression is also downregulated in HCC and discuss the mechanism of the reduced FXR expression in HCC. Expression of FXR and small heterodimer partner (SHP) was measured by real-time PCR and immunohistochemical technique. Effect of pro-inflammatory cytokines on expression of FXR and its promoter activity were determined in primary hepatocytes or HepG2 and Huh7 cell lines. Our results showed that expression of FXR and its target gene SHP in human HCC was strongly downregulated compared to the normal liver tissues. In addition, pro-inflammatory cytokines were able to decrease FXR expression by inhibiting the FXR promoter activity. In conclusion this work demonstrates FXR expression is strongly downregulated in human HCC, which may be caused by decreased FXR promoter activity, suggesting a potential role of FXR in human HCC development.

The study of anti-hepatitis drug dimethyl dicarboxylate biphenyl on invasion of human hepatocellular carcinoma MHCC97-H cells and its active mechanisms

Objective: To assess the anti-invasive effect of DDB and its possible active mechanism in human hepatocellular carcinoma MHCC97-H with high metastasis potential. Methods: MTT assay was used to evaluate the cytotoxicity of DDB to MHCC97-H cells and the anti-adhesion of DDB on MHCC97-H cells to laminin (LN) and fibronectin (FN). The anti-invasive effect of DDB was detected by the transwell chamber experiment. VEGF, nm23-H1 and uPAR mRNA transcriptions were determined by RT-PCR assay. The secretion and expression of α-fetal protein (AFP) were analyzed by ELISA and flow cytometry, respectively. Results: DDB at non-cytotoxic concentrations (10, 50 and 100 μmol/L) obviously inhibited the adhe- sion of MHCC97-H on LN and FN. In the transwell chamber experiment, the inhibition rates of the invasion of DDB 50 and 100 μmol/L on MHCC97-H cells were 25.8% and 32.3%, respectively. By RT-PCR assay, DDB 50 and 100 μmol/L decreased VEGF, nm23-H1 and uPAR mRNA expressions in MHCC97-H cells. The ELISA assay showed that 50, 100 and 200 μmol/L DDB decreased the AFP secretion of MHCC97-H cells, the inhibitory rates were 16.5%, 17.5% and 48.5%, respectively. DDB also decreased the expression of AFP in MHCC97-H cells by flow cytometry assay. Conclusion: DDB, an anti-hepatitis drug, at non-cytotoxic concentrations showed significant anti-invasion effect in human hepatocellular carcinoma MHCC97-H cells, and the inhibition of VEGF, nm23-H1 and uPAR expression should contribute to the anti-invasion property of DDB.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Preliminary Study of Oxidative Stress in Human Hepatocellular Carcinoma and Adjacent Normal Liver Tissues

OBJECTIVE The antioxidative system in human hepatocellular carcinoma was investigated. METHODS The activities of cytosolic catalase (CAT), superoxide dismu-tase, glutathione peroxidase (GSH-Px), glutathione S-tranferase and levels of reduced glutathione, total protein thiols and malondialdehyde were assayed in 10 cases of hepatocellular carcinoma and adjacent normal liver. RESULTS Hepatoma tissues showed higher activities of CAT, GSH -Px and lower content of total antioxidative capacity compared to adjacent normal liver tissue (P<0.05). CONCLUSION These findings suggest that the antioxidative defense-related enzymes and antioxidants are largely regulated in hepatoma cells. However, the mechanism which is not clear requires further investigation.

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential

Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient like metastatic model of human HCC in nude mice (LCI-D20)and a Iow metastatic model of human HCC in nude mice LCI-D35 ) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding.Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-Iincreased gradually following tumor progression in LCID20 model, and correlated with tumor size and AFP level,Phasic expression of tissue intercellular adhesion molecule-I in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including antiangiogenesis, antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.

Cloning of differentially expressed genes in human hepatocellular carcinoma and nontumor liver

INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.

Mast cells and human hepatocellular carcinoma

AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patient characteristics.METHODS: Tissue sections of 22 primary HCCs were histochemically stained with toluidine blue, in order to be able to quantify the MCs in and around the neoplasm using a computer-assisted image analysis system. HCC was staged and graded by two independent pathologists. To identify the sinusoidal capillarisation of each specimen 3μm thick sections were histochemically stained with sirius red, and semi-quantitatively evaluated by two independent observers. The data were statistically analysed using Spearman′s correlation and Student′s t-test when appropriate.RESULTS: MCs density did not correlate with the age or sex of the patients, the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, or the stage or grade of the HCC. No significant differences were found between the MCs density of the patients with and without hepatitis C virus infection, but they were significantly higher in the specimens showing marked sinusoidal capillarisation.CONCLUSION: The lack of any significant correlation between MCs density and the stage or grade of the neoplastic lesions suggests that there is no causal relationship between MCs recruitment and HCC. However, as capillarisation proceeds concurrently with arterial blood supply during hepatocarcinogenesis, MCs may be considered of primary importance in the transition from sinusoidal to capillary-type endothelial cells and the HCC growth.

A novel TRPC6-dependent mechanism of TGF-β-induced migration and invasion of human hepatocellular carcinoma cells

Mechanisms on cancer cell migration and invasion have been major topics of cancer research and anti-cancer therapy development. Among the multiple cell signaling pathways involved in cell migration, those elicited by transforming growth factorβ(TGF-β) have attracted tremendous attention. The TGF-βpolypeptide cytokines include four isoforms:TGF-β1, TGF-β2, TGF-β3, and TGF-β4, which are secreted mainly from cells of white blood cell lineage, such as macrophages, T cells and platelets. However, TGF-βcan also be synthesized and released from fibroblasts and

Transarterial chemoembolization combined with recombinant human adenovirus type 5 H101 prolongs overall survival of patients with intermediate to advanced hepatocellular carcinoma: a prognostic nomogram study

Background: Patients with intermediate to advanced hepatocellular carcinoma(HCC) are most commonly treated with transarterial chemoembolization(TACE). Previous studies showed that TACE combined with recombinant human adenovirus type 5(H101) may provide a clinical survival benefit. In the present study, we aimed to determine the survival benefit of TACE with or without H101 for patients with intermediate to advanced HCC and to develop an e ective nomogram for predicting individual survival outcomes of these patients.Methods: We retrospectively collected data from 590 patients with intermediate to advanced HCC who were treated at Sun Yat?sen University Cancer Center between January 2007 and July 2015. After propensity score matching, 238 patients who received TACE with H101(TACE with H101 group) and 238 patients who received TACE without H101(TACE group) were analyzed. Overall survival(OS) was evaluated using the Kaplan–Meier method; the nomogram was developed based on Cox regression analysis. Discrimination and calibration were measured using the concordance index(c?index) and calibration plots.Results: Clinical and radiologic features were similar between the two groups. OS rates were significantly lower in the TACE group than in the TACE with H101 group(1?year OS rate, 53.8% vs. 61.3%; 2?year OS rate, 33.4% vs. 44.2%; 3?year OS rate, 22.4% vs. 40.5%; all P < 0.05). Multivariate Cox regression analysis for the entire cohort showed that alpha?fetoprotein level, alkaline phosphatase level, tumor size, metastasis, vascular invasion, and TACE with or without H101 were independent factors for OS, all of which were included in the nomogram. Calibration curves showed good agreement between nomogram?predicted survival and observed survival. The c?index of the nomogram for predict?ing OS was 0.716(95% confidence interval 0.686–0.746).Conclusions: TACE plus H101 extends the survival of patients with intermediate to advanced HCC. Our proposed nomogram provides individual survival prediction and stratification for patients with intermediate to advanced HCC who receive TACE with or without H101.

EXPRESSION OF CELLULAR ONCOGENES IN HUMAN PRIMARY HEPATOCELLULAR CARCINOMA

With DNA-mRNA hybridization in situ technique, the expression of five oncogenes, c-N-ras, c-Ki-ras. c-Ha-ras, c-myc and c-fos, was observed in two cases of human hepatocellular carcinoma. The expression of c-N-ras & c-fos was greatly enhanced in tumor tissues of the two cases, and about 25% -50% of the tumor cells showed positive expression. The other three oncogenes namely c-Ki-ras, c-Ha-ras & c-myc, were not detected in these two carcinomas or in the non-cancerous liver tissues adjacent to the carcinomas. It is surmised that c-N-ras and c-fos may play coordinative role in maintaining the malignant phenotype of human primary hepatocellular carcinoma.