Safety and efficacy of postoperative adjuvant therapy with atezolizumab and bevacizumab after radical resection of hepatocellular carcinoma.

BACKGROUND: The effects of postoperative adjuvant therapy for high-risk recurrent hepatocellular carcinoma (HCC) in immunotherapy are still under investigation. This study evaluated the preventive effects and safety of postoperative adjuvant therapy, including atezolizumab, and bevacizumab, against the early recurrence of HCC with high-risk factors. METHODS: The complete data of HCC patients who underwent radical hepatectomy with or without postoperative adjuvant therapy after two-year follow-up were analyzed retrospectively. The patients were divided into high-risk or low-risk groups based on HCC pathological characteristics. High-risk recurrence patients were divided into postoperative adjuvant treatment and control groups. Due to the difference in approaches in postoperative adjuvant therapies, they were divided into transarterial chemoembolization (TACE), atezolizumab, and bevacizumab (T + A), and combination (TACE+T + A) groups. The two-year recurrence-free survival rate (RFS), overall survival rate (OS), and associated factors were analyzed. RESULTS: The RFS in the high-risk group was significantly lower than that in the low-risk group (P = 0.0029), and the two-year RFS in the postoperative adjuvant treatment group was significantly higher than that in the control group (P = 0.040). No severe complications were observed in those who received atezolizumab and bevacizumab or other therapy. CONCLUSION: Postoperative adjuvant therapy was related to two-year RFS. TACE, T + A, and the combination of these two approaches were comparable in reducing the early recurrence of HCC without severe complications.

Effects of l-arginine on endometrial estrogen receptor α/ß and progesterone receptor expression in nutrient-restricted sheep.

This study aimed to determine the effects of l-arginine (L-Arg) supplementation on steroid hormone receptors in non-pregnant ovine endometrium. All experimental ewes were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on estrogen receptor α/ß (ERα/ß) and progesterone receptor (PGR) expression in the ovine endometrium were assessed. Our results showed that levels of ERß and PGR expression were significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on ERß and PGR expression (p < 0.05). Also, expression of endometrial ERα was substantially increased (p < 0.05) by L-Arg supplementation. Furthermore, ERα/ß and PGR were mainly detected in the endometrial luminal epithelium and glandular epithelium. Therefore, we isolated and identified endometrial epithelial cells (EECs) from sheep. Different concentrations of L-Arg were added to investigate the effects on ERα/ß and PGR in EECs. The expression levels of endothelial nitric oxide synthase, ERß, and PGR were significantly increased in response to low-concentration (200 µmol) L-Arg supplementation, which subsequently decreased with a high concentration (800 µmol) (p < 0.05). Otherwise, ERα expression was remarkably increased at both L-Arg concentrations in EECs (p < 0.05). Overall, the results indicated that L-Arg performed crucial roles in the regulation of ovine steroid hormone receptor expression in the endometrium. The results of this study provide a theoretical basis and technical means for the normal function of endometrium in response to low nutrient levels.

Effects of l-arginine on endometrial microvessel density in nutrient-restricted Hu sheep.

Nutrient deficiency in ruminants can lead to estrus cycle disorders, a decreased pregnancy rate, and reduce birth weight. l-arginine (L-Arg), an important amino acid, can improve uterine homeostasis in pregnant sheep and prevent intrauterine growth restriction (IUGR). However, most studies of L-Arg have been conducted on pregnant sheep and few have reported the effects of L-Arg on microvessel density (MVD) in the non-pregnant ovine endometrium. The processes of normal uterine cyclical development and implantation are dependent on a balanced of endometrial MVD. In this study, female Hu sheep were randomly assigned to either a control group (n = 6), a nutrient-restricted group (n = 6), or an L-Arg supplemented nutrient-restricted group (n = 6). The effects of L-Arg on MVD in ovine endometrium were then studied. Our results showed that ovine endometrial MVD was significantly increased by nutrient restriction, but L-Arg counteracted the effect of nutrient restriction on MVD (P < 0.05). Levels of angiogenic growth factors (including VEGFA, VEGFR2, and FGF2) had significant increases (P < 0.05) in endometrium of nutrient restriction on sheep, but that L-Arg supplementation substantially decreased (P < 0.05) their expressions in nutrient restriction sheep. Furthermore, oxidative stress caused by nutrient restriction was also alleviated by L-Arg supplementation in the ovine endometrium. Overall, the results suggested that L-Arg has crucial roles in maintaining the balance of endometrial MVD and angiogenic growth factors, and increasing anti-oxidation capability in the endometrium of nutrient-restricted sheep. This study will provide a theoretical basis and technical means for the normal development of endometrial microvessels in low nutrition level.

MicroRNA-200b suppresses cell invasion and metastasis by inhibiting the epithelial-mesenchymal transition in cervical carcinoma.

The expression of microRNA (miR)-200b is suppressed in numerous tumor types, leading to epithelial-mesenchymal transition, which enables solid tissue epithelial cancers to invade and metastasize. The present study assessed the role of miR-200b in cervical cancer with the aim of clarifying the underlying pathophysiological mechanisms and to identify potential strategies for its prevention and treatment of cervical cancer. Reverse­transcription quantitative PCR revealed that miR­200b was downregulated in invasive cervical carcinoma tissues compared with that in normal adjacent tissues. A Transwell migration assay indicated that transfection of cervical cancer cells with miR­200b mimics significantly inhibited their migratory potential, while migration was enhanced in cells transfected with miR­200b inhibitor. Furthermore, western blot analysis indicated a negative correlation between miR­200b and mesenchymal marker vimentin as well as matrix metalloproteinase­9, which has a key role in tumor invasion and metastasis. In addition, a positive correlation between miR­200b and the epithelial marker E­cadherin was revealed by western blot and immunofluorescence. The results of the present study suggested that miR­200b suppressed the migratory potential of cervical carcinoma cells and therefore their ability to metastasize by inhibiting the epithelial-mesenchymal transition, which may be utilized for the treatment of cervical cancer.

MicroRNA-200b inhibits epithelial-mesenchymal transition and migration of cervical cancer cells by directly targeting RhoE.

Previous studies have identified microRNA-200b (miR-200b) as a powerful regulator of epithelial-mesenchymal transition (EMT) via the control of gene expression. EMT is a critical event that is associated with the initiation of malignant tumor metastasis. A lack of E-cadherin expression and overexpression of vimentin are hallmarks of EMT. It is well­known that RhoE, which is associated with regulation of the actin cytoskeleton and migration via alterations in cell motility, regulates the expression of E-cadherin, matrix metalloproteinase-9 (MMP-9) and vimentin. However, it remains to be elucidated whether miR­200b may alter the molecular behavior of RhoE. The present study aimed to determine whether miR­200b was able to regulate the EMT of cervical cancer, in order to control metastasis. In addition, the correlation between miR­200b and RhoE, E­cadherin and vimentin expression was investigated. Notably, miR­200b was shown to inhibit the function of RhoE and suppress the EMT of cervical cancer. Furthermore, HeLa cells were transfected with miR­200b mimics or inhibitors, and the protein expression levels of E­cadherin, MMP­9, vimentin and RhoE were subsequently detected. A Transwell assay was also conducted, in order to observe the metastatic ability of the HeLa cells. In addition, a luciferase reporter assay was performed using luciferase reporter vectors containing the full length 3'­untranslated region (UTR) of RhoE; miR­200b was able to significantly suppress relative luciferase activity by targeting the 3'­UTR of RhoE. These results suggested that miR­200b may markedly inhibit metastatic potential by regulating cell EMT and inhibiting RhoE; therefore, miR-200b may be considered an effective target for the treatment of patients with highly metastatic cervical cancer.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer.

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Organoid cultures derived from patients with advanced prostate cancer.

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. Whole-exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

Posttranslational control of Cdc25 degradation terminates Drosophila's early cell-cycle program.

In most metazoans, early embryonic development is characterized by rapid mitotic divisions that are controlled by maternal mRNAs and proteins that accumulate during oogenesis. These rapid divisions pause at the midblastula transition (MBT), coinciding with a dramatic increase in gene transcription and the degradation of a subset of maternal mRNAs. In Drosophila, the cell-cycle pause is controlled by inhibitory phosphorylation of Cdk1, which in turn is driven by downregulation of the activating Cdc25 phosphatases. Here, we show that the two Drosophila Cdc25 homologs, String and Twine, differ in their dynamics and that, contrary to current models, their downregulations are not controlled by mRNA degradation but through different posttranslational mechanisms. The degradation rate of String protein gradually increases during the late syncytial cycles in a manner dependent on the nuclear-to-cytoplasmic ratio and on the DNA replication checkpoints. Twine, on the other hand, is targeted for degradation at the onset of the MBT through a switch-like mechanism controlled, like String, by the nuclear-to-cytoplasmic ratio, but not requiring the DNA replication checkpoints. We demonstrate that posttranslational control of Twine degradation ensures that the proper number of mitoses precede the MBT.

Total serum DNA and DNA integrity: diagnostic value in patients with hepatitis B virus-related hepatocellular carcinoma.

AIMS: This study aimed to test the diagnostic utility of the total serum cell-free DNA (cfDNA) and DNA integrity index for detection of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: We initially evaluated the sodium iodide (NaI) method, Triton/Heat/Phenol (THP) protocol and QIAamp Kit for cfDNA extraction. Then cfDNA was isolated from the sera of 80 patients with HBV-related HCC, 80 patients with chronic HBV infection and 50 healthy subjects, and quantified by real-time quantitative polymerase chain reaction (qPCR) amplification of beta-actin genomic DNA fragments using two sets of primers of 100 and 400 bp. DNA integrity was calculated as the ratio of 400 bp to 100 bp ß-actin fragments. RESULTS: The THP approach was not only superior to the other two methods in terms of DNA quantity, but also was simpler, more rapid, and less costly. Serum DNA integrity in HCC patients was significantly higher than that in HBV patients or healthy controls. As for total cfDNA levels, although a significant difference was found between HCC patients and healthy individuals, no significant difference was found between HBV patients with and without HCC. DNA integrity was associated with tumour size, TNM stage, lymph node and distant metastasis. DNA integrity had a higher sensitivity and specificity in discriminating HCC from HBV patients than total DNA. CONCLUSIONS: The THP method is preferred for extraction of cfDNA. DNA integrity is a promising molecular biomarker for detecting HCC in patients with chronic HBV infection; it reflects the progression and metastatic potential of the tumour.

Total serum DNA and DNA integrity: diagnostic value in patients with hepatitis B virus-related hepatocellular carcinoma.

AIMS: This study aimed to test the diagnostic utility of the total serum cell-free DNA (cfDNA) and DNA integrity index for detection of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: We initially evaluated the sodium iodide (NaI) method, Triton/Heat/Phenol (THP) protocol and QIAamp Kit for cfDNA extraction. Then cfDNA was isolated from the sera of 80 patients with HBV-related HCC, 80 patients with chronic HBV infection and 50 healthy subjects, and quantified by realtime quantitative polymerase chain reaction (qPCR) amplification of beta-actin genomic DNA fragments using two sets of primers of 100 and 400 bp. DNA integrity was calculated as the ratio of 400 bp to 100 bp ß-actin fragments. RESULTS: The THP approach was not only superior to the other two methods in terms of DNA quantity, but also was simpler, more rapid, and less costly. Serum DNA integrity in HCC patients was significantly higher than that in HBV patients or healthy controls. As for total cfDNA levels, although a significant difference was found between HCC patients and healthy individuals, no significant difference was found between HBV patients with and without HCC. DNA integrity was associated with tumour size, TNM stage, lymph node and distant metastasis. DNA integrity had a higher sensitivity and specificity in discriminating HCC from HBV patients than total DNA. CONCLUSIONS: The THP method is preferred for extraction of cfDNA. DNA integrity is a promising molecular biomarker for detecting HCC in patients with chronic HBV infection; it reflects the progression and metastatic potential of the tumour.

[Adult-to-adult living-related donor liver transplantation: report of 2 cases].

OBJECTIVE: To investigate the feasibility and safety of adult-to-adult living-related donor liver transplantation using a right lobe graft. METHODS: The clinical data of 2 cases of living-related donor liver transplantation performed between July, 2010 and November, 2010 were analyzed. RESULTS: Liver transplantation was performed using a right lobe graft including the middle hepatic vein in one case and a right lobe graft without the middle hepatic vein in the other. The ratio of graft volume to standard liver volume was 46.2% and 47.3% in the two cases, with GR/WR of 0.83 and 0.80, and donor residue liver of 42.1% and 39.5%, respectively. The donor operation lasted for 6.5 h and 5 h in the two cases with blood loss of about 200-250 ml without blood transfusion. The donors recovered uneventfully without any surgical complications, whose liver function was normal 7 days after the operation, and were discharged 14 days and 16 days after the surgery, respectively. The recipient operation lasted for 8 h and 7 h with blood loss of about 800-1000 ml. The right hepatic vein, hepatic artery, portal vein and bile duct reconstruction were performed by end-to-end anastomoses in the 2 recipients. Bile duct anastomosis stricture occurred in the first recipient 2 months after transplantation and was treated with percutaneous transhepatic cholangiography and drainage. The second recipient recovered smoothly without any complications. The recipients have so far survived 9 months and 5 months, respectively. CONCLUSION: Adult-to-adult living-related donor liver transplantation is a safe and effective option for treatment of end-stage liver diseases in the context of cadaveric liver graft shortage.

The co-expression of USP22 and BMI-1 may promote cancer progression and predict therapy failure in gastric carcinoma.

Recent experimental evidence support the model in which the simultaneous induction of BMI-1 and USP22 is critical during cancer progression. Whether this model may affect gastric cancer (GC) progression is worthy of additional study. In this study, we examined the significance of the USP22 and BMI-1 expression in GC (n = 219), non-cancerous mucosa (n = 37), and lymph node metastasis (n = 37). The protein expression level of USP22 and BMI-1 were concomitantly up-regulated from non-cancerous mucosa to primary carcinoma and from carcinomas to lymph node metastasis (P < 0.001). A statistical correlation was observed between USP22 and BMI-1 expression in GC tissues (n = 219, r = 0.634, P < 0.001) and in lymph node metastasis (n = 37, r = 0.689, P < 0.001). The incidence of positive expression was 57.08% for USP22, 49.32% for BMI-1, and 45.21% for USP22/BMI-1 in 219 GC tissues, respectively. Co-positive of USP22/BMI-1 was significantly correlated with gross features (x(2) = 14.256, P < 0.001), differentiation (x(2) = 5.872, P = 0.015), pT classification (x(2) = 18.486, P < 0.001), pN classification (x(2) = 9.604, P = 0.002), pM classification (x(2) = 32.766, P < 0.001), and AJCC stage (x(2) = 58.278, P < 0.001). Notably, high USP22/BMI-1 expression was significantly associated with shorter disease-specific survival (P < 0.001). By Cox regression analysis, co-positive of USP22/BMI-1 was found to be an independent prognostic factor (P = 0.002). Our results indicated the simultaneous activation of USP22 and BMI-1 may associate with GC progression and therapy failure.

[Clinical study of blood type A donor liver transplantation in type O recipients].

OBJECTIVE: To study the clinical effect and feasibility of blood type A donor liver transplantation in blood type O recipients. METHODS: The clinical data were analyzed in 6 blood type O patients receiving transplantation of the liver grafts from blood type A donors. The clinical effect and outcomes of the transplantations were evaluated to assess the feasibility of ABO incompatible liver transplantation between type A donors and type O recipients. RESULTS: The operations and the postoperative recovery were smooth in all the 6 recipients. Only one patient died 5 months postoperatively due to liver tumor metastasis, and the other 5 patients survived with the longest survival reaching 14 months. Acute graft rejection occurred in one patient 1 week after the operation on account of abnormally elevated serum bilirubin level, which was successfully managed with immediate methylprednisolone therapy. No such complications as acute graft rejection, bile duct stenosis or bile leakage was found in the other patients. CONCLUSION: Blood type A donor liver transplantation in type O recipient is feasible in emergency or other special conditions.

[The effect of As2O3 on induction of apoptosis and inhibition of telomerase activity in colon cancer LS-174T cells].

OBJECTIVE: To study the impact of arsenic trioxide (As2O3) on human colorectal carcinoma LS-174T cells and their activity of telomerase. METHODS: LS-174T cells and xenograft model of nude mice were treated with As2O3. The inhibitory effect of As2O3 on survival of LS-174T cells was determined by MTT assay. Apoptosis was determined by electron microscopy and fluorescence microscopy. Cell cycle was assessed by flow cytometry. Telomerase activity in LS-174T cells was determined by PCR-ELISA kit. RESULTS: With the increasing concentration of As2O3, the ratio of living cells to dead cells decreased significantly, and the IC50 value was 5.23 micromol/L. Apoptosis curve appeared after 24 h and cells turned to apoptosis in a time-dependent manner. As2O3 inhibited the telomerase activity in cell extraction, obviously in a concentration-dependent and time-dependent manner. Inhibitiory effect of As2O3 on xenograft model of nude mice was observed by tumor volume and weight measurement, showing a significant difference between As2O3 and control groups (P < 0.05). CONCLUSION: Both the experiments in vitro and in vivo showed an inhibitory effect of As2O3 on colonrectal cancer S-174T cell growth, probably by induction of apoptosis and inhibition of telomerase activity.

[Exploration on the increased sensitivity to docetaxel and reversing mechanism of drug resistance of antisense survivin RNA in gastric cancer cell line SGC7901 cells].

OBJECTIVE: To explore the effects of survivin antisense RNA on apoptosis and chemosensitivity to docetaxel in gastric cancer line SGC7901 cells, and its relation to mdr-1. METHODS: survivin antisense eukaryotic vector anti-pcDNA3-svv was transfected into SGC7901 cells by electorporation and positive clone was screened out. survivin protein and mdr-1 mRNA were determined by Western blot and RT-PCR. Apoptosis-inducing effect was examined by electron microscopy. Sensitivity to docetaxel was examined by MTT. Expression of mdr-1 and survivin mRNA were detected in the SGC7901 cells after drug-resisitance induction. RESULTS: The expression of survivin protein of SGC7901 cells after transfection reduced significantly than that of non-transfected cells. MDR indexes of transfection group and non-transfection group were 0.196 +/- 0.013 and 3.126 +/- 0.019, respectively. The IC50 of transfection group to docetaxel was (16.7 +/- 1.98) microg/L and non-transfection group was (55.7 +/- 1. 89) microg/L, with a statistically significant difference. Expression of survivin mRNA in drug-resistant cells decreased along with the decreasing of mdr-1. CONCLUSION: Antisense surivivin RNA can induce apoptosis in gastric cancer cells and increase sensitivity to docetaxel. The reversing mechanism of drug resistance is related with decreasing of mdr-1.

Expression and correlation of CD44v6, vascular endothelial growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-9 in Krukenberg tumor.

AIM: To explore the expression and correlation of CD44v6, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and matrix metalloproteinase (MMP)-9 in Krukenberg and primary epithelial ovarian carcinoma. METHODS: The expressions of CD44v6, VEGF, MMP-2 and MMP-9 were detected by immunohistochemical method in 20 cases of normal ovarian tissues, 38 cases of Krukenberg tumor and 45 cases of primary epithelial ovarian carcinoma. RESULTS: The expression of CD44v6 (primary epithelial ovarian carcinoma tissue vs normal ovarian tissue: chi(2) = 4.516, P = 0.034; Krukenberg tumor tissue vs normal ovarian tissue: chi(2) = 19.537, P = 0.001) and VEGF (primary epithelial ovarian carcinoma tissue vs normal ovarian tissue: P = 0.026; Krukenberg tumor tissue vs normal ovarian tissue: chi(2) = 22.895, P = 0.001) was significantly higher in primary epithelial ovarian carcinoma tissue and Krukenberg tumor tissue than in normal ovarian tissue. The positive expression rate of MMP-2 and MMP-9 was 0% in the normal ovarian tissue. The positive expression rate of CD44v6 (chi(2) = 10.398, P = 0.001), VEGF (chi(2) = 13.149, P = 0.001), MMP-2 (chi(2) = 33.668, P = 0.001) and MMP-9 (chi(2) = 38.839, P = 0.001) was remarkably higher in Krukenberg tumor than in primary epithelial ovarian carcinoma. The correlation of CD44v6, VEGF, MMP-2, and MMP-9 was observed in primary epithelial ovarian carcinoma and Krukenberg tumor. CONCLUSION: CD44v6, VEGF, MMP-2, and MMP-9 are involved in ovarian carcinoma, gastric cancer and Krukenberg tumor. Detection of CD44v6, VEGF, MMP-2 and MMP-9 may contribute to the diagnosis of ovarian carcinoma, gastric cancer, and Krukenberg tumor.

[Clinicopathologic significance of CD105 mRNA expression in human breast carcinoma].

OBJECTIVE: To study endoglin (CD105) gene expression in breast cancer and its clinicopathologic significance. METHODS: In 40 patients with breast cancers, CD105 mRNA was detected at center and periphery of tumor and at nearby normal tissue by RT-PCR. RESULTS: The difference in CD105 mRNA expressions between cancer and normal breast tissue was significant (t = 12.08, P < 0.05), and the expression was significantly higher at the tumor periphery than at the tumor center (t = 7.52, P < 0.05). CD105 over-expression was related to lymph node metastases (t = 2.71, P < 0.05), but not to age, tumor size, pathologic grade or pathologic type (P > 0.05). CONCLUSION: CD105 over-expression may play a crucial role in the progression of breast cancer and lymph node metastasis.

[Inducement of FLICE inhibitory protein antisense oligonucleotides on apoptosis of human gastric cancer cell line BGC823].

BACKGROUND & OBJECTIVE: Cellular FLICE inhibitory protein (cFLIP) plays an important role in cell apoptosis, researches of antisense oligonucleotides (ASODN) of cFLIP gene may provide a new method or protocol for treatment of human gastric cancer. This study was to explore effect of cFLIP ASODN on apoptosis of human gastric cancer cell line BGC823. METHODS: Human cervical cancer cell line HeLa was used as control, cFLIP ASODN was introduced into BGC823 cells and HeLa cells, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detect cFLIP(L/S) (cellular FLIP(Short) and cellular FLIP(long)) mRNA and protein. The 5'FAM-conjugated ASODN was created complementary to a sequence that included the start site of FLIP open reading frame. After introducing, MTT was used to detect cell inhibition rate,TUNEL and flow cytometry (FCM) were used to detect cell apoptosis, and Western blot was used to detect protein level of cFLIP. RESULTS: The encoding mRNA and protein of cFLIP(L) and cFLIP(S) can be detected in both HeLa and BGC823 cells. MTT revealed that cFLIP ASODN significantly inhibited proliferation of BGC823 cells (P< 0.05) in a concentration-dependent manner. TUNEL staining detected positive FLIP expression, specific apoptotic peak can be detected before G1 peak by FCM, and Western blot revealed that protein level of cFLIP(L) and cFLIP(S) decreased significantly (P< 0.05). CONCLUSION: The cFLIP(L/S) mRNA and encoded proteins expressed in both HeLa and BGC823 cells. ASODN may down-regulate cFLIP(L/S) protein level, and initiate apoptosis of BGC823 cells.

Peptide nucleic acids arrest the growth of gastric cancer cells SGC7901.

BACKGROUND: Peptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells. METHODS: Heteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay. RESULTS: PNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99.4% after 7 days; the rate of cloning inhibition was 98.2% after 8 days; whereas for oligonucleotide groups, at the same concentration, the percentages were 50.1% and 67.5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P < 0.05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P < 0.05). At the same time, the impact on cell morphology was observed. CONCLUSIONS: The results showed that PNAs are potent antisense reagents. The telomerase-associated therapies are very promising for the treatment of malignant tumours.

Expression of cyclooxygenase-2 and clinicopathologic features in human gastric adenocarcinoma.

AIM: To study the expression of cyclooxygenase-2 (COX-2) gene in gastric cancer and the relationship between COX-2 expression and clinicopathologic features of gastric cancer. METHODS: With reference to the expression of beta-actin gene, COX-2 mRNA level was examined in cancerous tissues and adjacent noncancerous mucosa from 33 patients by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Quantitation of relative band Adj volume counts was performed using molecular Analyst for windows software. The COX-2 index was determined from the band Adj volume counts ratio of COX-2 to constitutively expressed actin. RESULTS: The COX-2 index in gastric carcinoma was significantly higher than that in normal mucosa (0.5966+/-0.2659 vs 0.2979+/-0.171, u=5.4309, P<0.01). Significantly higher expression of COX-2 mRNA was also observed in patients with lymph node involvement than that in those without (0.6775+/-0.2486 vs 0.4105+/-0.2182, t=2.9341, P<0.01). Furthermore, the staging in the UICC TNM classification significantly correlated with COX-2 overexpression (F=3.656, P<0.05), the COX-2 index in stage III and IV was significantly higher than those in stage I and II (q=3.2728 and q=3.4906, P<0.05). The COX-2 index showed no correlation with patient's age, sex, blood group, tumor location, gross typing, depth of invasion, differentiation, and the greatest tumor dimension (P>0.05). CONCLUSION: Expression of COX-2 mRNA in gastric carcinoma was significantly higher, which may enhance lymphatic metastasis in patients with gastric carcinoma. The staging in the UICC TNM classification was significantly correlated with COX-2 over-expression. COX-2 may contribute to progression of tumor in human gastric adenocarcinoma.