Vincent W.S. Liu

Long-term follow-up of chronic hepatitis B patients treated with interferon alfa

BACKGROUND There is very little data on the long-term outcome of patients with chronic hepatitis B after interferon therapy. A 6-year follow-up of two interferon trials in chronic hepatitis B patients is reported. METHODS One hundred twenty-eight Chinese adults with chronic hepatitis B who received interferon therapy were followed for 19-79 months (median 41 months). Twenty-nine patients lost hepatitis B e antigen and two also lost hepatitis B surface antigen within 1 year of treatment. RESULTS Seven (24%) responders reactivated. Twenty-eight (28%) nonresponders had sustained clearance of hepatitis B e antigen during follow-up. Delayed clearance of hepatitis B e antigen occurred more frequently in nonresponders who had elevated pretreatment serum transaminase levels. (P = 0.002). Serum hepatitis B virus DNA became undetectable by polymerase chain reaction assay in both responders who lost hepatitis B surface antigen but in only 8 (17%) patients who lost hepatitis B e antigen only. Delayed clearance of hepatitis B surface antigen was not seen in any of the 48 patients who had sustained clearance of hepatitis B e antigen. CONCLUSIONS Contrary to reports from Western countries, complete elimination of markers of hepatitis B virus infection was uncommon in Chinese patients with chronic hepatitis B who underwent interferon therapy despite similar duration of follow-up.

Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells.

Tumor suppressor effect of follistatin-like 1 in ovarian and endometrial carcinogenesis—a differential expression and functional analysis

Endometrial and ovarian cancers are the most common and the most lethal gynecologic malignancies worldwide, respectively. By performing differential expression analysis using annealing control primer-based reverse transcription (RT)-polymerase chain reaction (PCR) on pooled complementary DNA (cDNA) from 45 endometrial and 36 ovarian cancers and their non-tumor samples, reduced expression of the follistatin-like 1 (FSTL1) was identified. Downregulation of FSTL1 was further confirmed on individual samples and cell lines by quantitative real-time RT-PCR and western blotting. For in vitro functional study, full-length cDNA of FSTL1 was cloned and transiently transfected into the ovarian cancer cell line Ovca420 and endometrial cancer cell line AN3CA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell count demonstrated significantly slower proliferation rate. By terminal uridine deoxynucleotidyl transferase dUTP nick end labeling and flow cytometric analysis, higher apoptotic activity and a remarkable increase in sub-G(1) cell population were observed in transfected cells, suggesting that FSTL1 induced apoptosis in cancer cells. Subsequent messenger RNA and protein expression analysis on downstream apoptotic molecules revealed upregulation and/or activation of FAS, FASLG, TRADD, Caspase-3, Caspase-7 and PARP by FSTL1 transfection, suggesting that FSTL1-induced apoptosis may be initiated mainly by FAS/FASLG death receptor-ligand binding. Cell migration and invasion assays demonstrated a remarkably lower cell migration and invasion capability in FSTL1-transfected cells in relation to downregulation of matrix metallopeptidase-2. Our findings suggested that a tumor suppressor role of FSTL1 may be important in ovarian and endometrial carcinogenesis.

High frequency of mitochondrial genome instability in human endometrial carcinomas

To investigate the occurrence of somatic mitochondrial DNA (mtDNA) mutations in human primary endometrial carcinomas, we sequenced the D-loop region, the 12S and 16S rRNA genes of mtDNA of cancer tissues and their matched normal controls. About 56% (28 out of 50) of cases carry one or more somatic changes in mtDNA including deletion, point mutation and mitochondrial microsatellite instability (mtMSI), namely the change in length of short base-repetitive sequences of mtDNA. In particular, mtMSI was frequently detected in 89% (25 out of 28) of all the cases carrying somatic changes followed by point mutations (25%; seven out of 28) and deletion (3.5%; one out of 28). The CCCCCTCCCC sequences located in the Hypervariable Regions I and II of the D-loop and 12S rRNA gene are instability hot spot regions in endometrial carcinomas. It is suggested that errors in replication may account for the high frequency of mtMSI in human endometrial carcinomas. The relatively high prevalence of mtMSI may be a potential new tool for detection of endometrial cancer.

Aberrant Activation of ERK/FOXM1 Signaling Cascade Triggers the Cell Migration/Invasion in Ovarian Cancer Cells

Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor essential for cell cycle progression. Numerous studies have documented that FOXM1 has multiple functions in tumorigenesis and its elevated levels are frequently associated with cancer progression. Here, we characterized the role of ERK/FOXM1 signaling in mediating the metastatic potential of ovarian cancer cells. Immunohistochemical (IHC), immunoblotting and semi-quantitative RT-PCR analyses found that both phospho-ERK and FOXM1 were frequently upregulated in ovarian cancers. Intriguingly, the overexpressed phospho-ERK (p<0.001) and FOXM1 (p<0.001) were significantly correlated to high-grade ovarian tumors with aggressive behavior such as metastasized lymph node (5 out of 6). Moreover, the expressions of phospho-ERK and FOXM1 had significantly positive correlation (p<0.001). Functionally, ectopic expression of FOXM1B remarkably enhanced cell migration/invasion, while FOXM1C not only increased cell proliferation but also promoted cell migration/invasion. Conversely, inhibition of FOXM1 expression by either thiostrepton or U0126 could significantly impair FOXM1 mediated oncogenic capacities. However, the down-regulation of FOXM1 by either thiostrepton or U0126 required the presence of p53 in ovarian cancer cells. Collectively, our data suggest that over-expression of FOXM1 might stem from the constitutively active ERK which confers the metastatic capabilities to ovarian cancer cells. The impairment of metastatic potential of cancer cells by FOXM1 inhibitors underscores its therapeutic value in advanced ovarian tumors.

Primary peritoneal malignant mixed Müllerian tumors

Primary peritoneal malignant mixed Müllerian tumors (MMMTs) are rarely reported in the literature.

Melatonin as a negative mitogenic hormonal regulator of human prostate epithelial cell growth: potential mechanisms and clinical significance.

Abstract:  Circannual variation in the human serum levels of prostate‐specific antigen, a growth marker of the prostate gland, has been reported recently. The present study was conducted to investigate the role of the photoperiodic hormone melatonin (MLT) and its membrane receptors in the modulation of human prostate growth. Expression of MT1 and MT2 receptors was detected in benign human prostatic epithelial tissues and RWPE‐1 cells. MLT and 2‐iodomelatonin inhibited RWPE‐1 cell proliferation and up‐regulated p27Kip1 gene and protein expression in the cells. The effects of MLT were blocked by the nonselective MT1/MT2 receptor antagonist luzindole, but were not affected by the selective MT2 receptor antagonist 4‐phenyl‐2‐propionamidotetraline. Of note, the antiproliferative action of MLT on benign prostate epithelial RWPE‐1 cells was effected via increased p27Kip1 gene transcription through MT1 receptor‐mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel, a signaling process which has previously been demonstrated in 22Rv1 prostate cancer cells. Taken together, the demonstration of the MT1/PKA+PKC/p27Kip1 antiproliferative pathway in benign and malignant prostate epithelial cell lines indicated the potential importance of this MLT receptor‐mediated signaling mechanism in growth regulation of the human prostate gland in health and disease. Collectively, our data support the hypothesis that MLT may function as a negative mitogenic hormonal regulator of human prostate epithelial cell growth.

Melatonin MT1 receptor‐induced transcriptional up‐regulation of p27Kip1 in prostate cancer antiproliferation is mediated via inhibition of constitutively active nuclear factor kappa B (NF‐κB): potential implications on prostate cancer chemoprevention and therapy

Abstract:  Our laboratory has recently demonstrated a melatonin MT1 receptor‐mediated antiproliferative signaling mechanism in androgen receptor (AR)‐positive prostate epithelial cells which involves up‐regulation of p27Kip1 through dual activation of Gαs/protein kinase A (PKA) and Gαq/protein kinase C (PKC) in parallel, and down‐regulation of activated AR signaling via PKC stimulation. The aim of the present investigation was to identify the transcription factor that mediates melatonin’s up‐regulatory effect on p27Kip1 in LNCaP and 22Rv1 prostate cancer cells. Deletion mapping and reporter assays of the p27Kip1 promoter revealed that the putative melatonin‐responsive transcription factor binds to a 116 base‐pair region of the promoter sequence, which contains a potential nuclear factor kappa B (NF‐κB) binding site. When the NF‐κB binding site was abolished by site‐directed mutagenesis, the stimulatory effect of melatonin on p27Kip1 promoter activity was mitigated. Notably, melatonin inhibited the DNA binding of activated NF‐κB via MT1 receptor‐induced PKA and PKC stimulation. Furthermore, melatonin’s up‐regulatory effect on p27Kip1 transcription and consequent cell antiproliferation were abrogated by NF‐κB activator but mimicked by NF‐κB inhibitor. The results indicate that inhibition of constitutively active NF‐κB via melatonin MT1 receptor‐induced dual activation of (Gαs) PKA and (Gαq) PKC can de‐repress the p27Kip1 promoter leading to transcriptional up‐regulation of p27Kip1. MT1 receptor‐mediated inhibition of activated NF‐κB signaling provides a novel mechanism supporting the use of melatonin in prostate cancer chemoprevention and therapy.

Frequent occurrence of mitochondrial microsatellite instability in the D-loop region of human cancers

Abstract: We analyzed the occurrence of mitochondrial microsatellite instability (mtMSI) in 262 pairs of female cancer tissues with the matched normal controls. mtMSI was detected in only 4 of 12 microsatellites found in the mitochondrial genome (3 in the D‐loop and 1 in the 12S rRNA gene). Interestingly, 95.6% (87/91) of mtMSI was detected in the D‐loop, namely, at nucleotide positions 303‐315, 514‐523, and 16184‐16193. This demonstrates that the D‐loop is a hotspot for mtMSI. Different incidences of mtMSI at these three microsatellites were found in the four cancer types (including cervical, endometrial, ovarian, and breast). Together with those mtMSI reported in other studies, the differential occurrence of mtMSI at each of the markers in the D‐loop region was observed, indicating that the extent of mtMSI varies from one cancer to another. Although the mechanisms of generation and functional impact of mtMSI are still not clear, the high incidence of mtMSI in the D‐loop and its broad distribution in human cancers render it a potential marker for cancer detection.

Differential Functions of Growth Factor Receptor–Bound Protein 7 (GRB7) and Its Variant GRB7v in Ovarian Carcinogenesis

Purpose: Aberrant overexpression of growth factor receptor–bound protein 7 (GRB7) and its variant GRB7v has been found in numerous human cancers. The goal of this study was to characterize the functions of GRB7 and GRB7v in the ovarian carcinogenesis and to investigate the differential roles of GRB7 and GRB7v in the modulation of signaling pathways. Experimental Design: Quantitative reverse transcription–PCR, Western blot, and immunohistochemical analyses were used to evaluate the levels of GRB7 and GRB7v. The cellular localization, functions, and signaling pathways regulated by GRB7 and GRB7v were investigated by enforced expression of GRB7 and GRB7v. Results: Quantitative reverse transcription–PCR and Western blot analyses showed that GRB7 and GRB7v were frequently upregulated in ovarian cancer samples. The overexpressed GRB7 (P = 0.009) and GRB7v (P = 0.017) were significantly correlated with high-grade ovarian cancer. Immunohistochemical analysis on ovarian cancer tissue array confirmed that the upregulated GRB7 was significantly correlated with high-grade ovarian cancer (P = 0.001). Confocal microscopy analysis showed that GRB7 and GRB7v predominately localized in cytoplasm of ovarian cancer cells, consistent with their roles as signaling adaptors. Enforced expression of GRB7 promoted cell proliferation, migration, and invasion, whereas GRB7v only increased cell proliferation and anchorage-independent growth ability. With the treatment of specific kinase inhibitors, we showed that both GRB7 and GRB7v promoted cell proliferation through activating extracellular signal-regulated kinase signaling, whereas GRB7 enhanced cell migration/invasion by activating c-Jun NH2 terminal kinase signaling. Conclusions: Our studies implicate that the overexpressed GRB7 and GRB7v are associated with high-grade tumors and exert distinct tumorigenic functions through regulating different signaling pathways in ovarian cancer cells. Clin Cancer Res; 16(9); 2529–39. ©2010 AACR.