G. G. Chen

Oestrogen mediates the growth of human thyroid carcinoma cells via an oestrogen receptor – ERK pathway

Abstract.  Objectives: Although thyroid cancer occurs much more frequently in females, the role of sex hormones in thyroid carcinogenesis is unknown. In this study, it has been investigated how 17β‐oestradiol (E2) influenced proliferation and growth of thyroid cancer cells. Materials and Methods: Cell proliferation and its related molecules were examined in thyroid papillary carcinoma cells (KAT5), follicular thyroid carcinoma cells (FRO) and anaplastic carcinoma cells (ARO). Levels of oestrogen receptor (ER) α and β were regulated by their agonists (PPT and DPN), antagonists and siRNA. Results: E2 promoted cell proliferation. Such an effect was positively related to ERα but negatively to ERβ; PPT enhanced cell proliferation while DPN inhibited it. PPT increased Bcl‐2 expression while DPN decreased it. DPN also elevated Bax expression. PPT elevated the level of phosphorylated extracellular signal‐regulated kinase 1/2 (pERK1/2), suggesting a positive role of ERK1/2 in E2‐induced cell proliferation. Knockdown of ERα significantly attenuated E2‐mediated Bcl‐2 and pERK1/2 expression. In contrast, knockdown of ERβ markedly enhanced them. Conclusions: Oestrogen stimulates proliferation of thyroid cancer cells, associated with increase in Bcl‐2 and decrease in Bax levels in an ERK1/2‐related pathway. Imbalance between ERα and ERβ may contribute to thyroid carcinogenesis.

Induction of thyroid papillary carcinoma cell proliferation by estrogen is associated with an altered expression of BCL-XL

PURPOSEOne of the features of thyroid carcinoma is its predilection for women of reproductive age relative to men. An increased risk has also been documented in women who have used estrogens for gynecologic reasons. The aim of this study was to explore the mechanism by which sex hormones contribute to the development of thyroid carcinoma, which is not well understood at present. MATERIALS AND METHODSIn this study, we investigated the effects of estradiol and testosterone on cell proliferation in a human thyroid papillary carcinoma cell line (KAT5) by MTT assay. We also studied the expression of estrogen receptors and the levels of anti-apoptotic Bcl-xL protein, pro-apoptotic Bax protein, and messenger RNA in the cells by Western blot and reverse transcriptase polymerase chain reaction analysis. RESULTSThe results showed that estradiol promotes cell proliferation when compared with cells treated with testosterone and untreated cells, and that the growth-promoting effect of estradiol was attenuated by tamoxifen. The expression of Bcl-xL was markedly increased in a dose-dependent manner, resulting in an elevated ratio of Bcl-xL to Bax. DISCUSSIONWe conclude that estradiol promotes KAT5 cell proliferation and that the underlying mechanism may be associated with up-regulation of Bcl-xL expression. The data provide insight into the molecular mechanism underlying the epidemiologic data that shows a two- to threefold increased prevalence of thyroid carcinoma in women relative to men. From the therapeutic point of view, the finding that estradiol enhances anti-apoptotic signaling pathways may be significant in the search for novel prevention and treatment strategies of thyroid carcinomas.

Differential gene expression of hepatocellular carcinoma using cDNA microarray analysis.

A cDNA microarray technique, which allows simultaneous analysis of differential expression of mRNA of over 4000 known human genes, was utilized to study the gene expression in 10 pairs of HCC and nontumorous tissues from ethnic Chinese patients in Hong Kong. A total of 211 genes were found to be highly expressed and 147 genes were downregulated in more than 1 out of 10 of the HCC pairs. The results were significant by two-tailed Wilcoxon test (P < or = 0.05 with 95% confidence) on the intensity of each DNA spot of the 10 HCC pairs. Six genes were highly expressed and 10 genes were downregulated in more than 30% of HCC pairs. Results are consistent with other published reports using traditional differential display, subtractive hybridization, or immunohistochemical staining methods. We also detected that beta-actin and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), which have been commonly used as an internal standard control in mRNA expression studies, were highly expressed in HCC when compared with nontumorous tissue. It is concluded that cDNA microarray analysis is an effective method in the detection of differential gene expression in HCC.

Primate-specific microRNA-637 inhibits tumorigenesis in hepatocellular carcinoma by disrupting signal transducer and activator of transcription 3 signaling†

MiR‐637 (microRNA‐637) is a primate‐specific miRNA belonging to the small noncoding RNA family, which represses gene regulation at the post‐transcriptional expression level. Although it was discovered approximately 5 years ago, its biomedical significance and regulatory mechanism remain obscure. Our preliminary data showed that miR‐637 was significantly suppressed in four HCC cell lines and, also, in most of the hepatocellular carcinoma (HCC) specimens, thereby suggesting that miR‐637 would be a tumor suppressor in HCC. Simultaneously, the enforced overexpression of miR‐637 dramatically inhibited cell growth and induced the apoptosis of HCC cells. The transcription factor, signal transducer and activator of transcription 3 (Stat3), is constitutively activated in multiple tumors, and aberrant Stat3 activation is linked to the promotion of growth and desensitization of apoptosis. Our study showed that Stat3 tyrosine 705 phosphorylation and several Stat3‐regulated antiapoptotic genes were down‐regulated in miR‐637 mimics‐transfected and Lv‐miR637‐infected HCC cells. In addition, miR‐637 overexpression negatively regulated Stat3 phosphorylation by suppressing autocrine leukemia inhibitory factor (LIF) expression and exogenous LIF‐triggered Stat3 activation and rescued cell growth in these cells. A nude mice model also demonstrated the above‐described results, which were obtained from the cell model. Furthermore, we found that LIF was highly expressed in a large proportion of HCC specimens, and its expression was inversely associated with miR‐637 expression. Conclusion: Our data indicate that miR‐637 acted as a tumor suppressor in HCC, and the suppressive effect was mediated, at least in part, by the disruption of Stat3 activation. (HEPATOLOGY 2011)

Increased expression of nucleophosmin/B23 in hepatocellular carcinoma and correlation with clinicopathological parameters

Nucleophosmin (NPM, B23, numatrin, NO38) is an abundant nucleolar phosphoprotein involved in multiple cellular functions. Previous evidence indicates that high-level expression of NPM causes uncontrolled cell growth and suggests that NPM may have oncogenic potential. In this study, we examined NPM expression in 103 paired cases of hepatocellular carcinoma (HCC), 12 cases of hepatic focal nodular hyperplasia, 17 cases of liver tissue adjacent to a hepatic haemangioma, and series of array tissues from normal human organs and malignancies using a monoclonal antibody against NPM and reverse transcription–PCR techniques, Western blot analysis, immunohistochemistry, and immunocytofluorescence. Our data indicated that NPM expression was significantly higher in HCC than in the non-malignant hepatocytes (P<0.001). Nucleophosmin was weakly expressed in hepatocytes from a 5-month-old embryo and in stationary hepatocytes of healthy adults. Moreover, enhanced expression of NPM in HCC correlated with the level of PCNA (R2=0.5639) and with the clinical prognostic parameters such as serum alpha fetal protein level, tumour pathological grading, and liver cirrhosis (P<0.05). Our results suggest that NPM may play an important role in the progression of tumorigenesis and that NPM may serve as a potential marker for HCC.

The contributions of oestrogen receptor isoforms to the development of papillary and anaplastic thyroid carcinomas.

Oestrogen (E2) is known to promote the proliferation of thyroid papillary carcinoma cells (KAT5). However, the molecular mechanism responsible is not well understood. In the study reported herein, the localization of ER alpha (ERα) and beta (ERβ) in KAT5 and anaplastic carcinoma cells (FRO) was studied by immunofluorescence staining and by immunoblotting the proteins in subcellular fractions. Cell proliferation and apoptosis were also determined together with the expression of relevant proteins. The pattern of the subcellular localization of ERα and ERβ differed between papillary and anaplastic cancer. Upon E2 treatment, the level of ERα increased in the nuclei of papillary cancer cells but ERβ remained unchanged. The level of mitochondrial ERβ surpassed that of ERα in anaplastic cancer cells. The different locations of ERα and ERβ in KAT5 and FRO agreed with the finding that E2 promoted the proliferation of KAT5 but inhibited or did not affect that of FRO cells, and with the proposed functions of these two receptors. E2 inhibited the level of Bax in the mitochondria of papillary cancer, followed by a decrease of cytochrome c and/or apoptosis‐inducing factor (AIF) release from the mitochondria into the cytosol. However, in anaplastic cancer, E2 promoted the expression of Bax in the mitochondria and the release of cytochrome c and/or AIF from mitochondria into the cytosol. Our results may explain the differences in epidemiology and responses to anti‐tumour therapy between papillary and anaplastic cancer in terms of the subcellular localization of ER isoforms. In conclusion, the findings provide evidence to support the observation that E2 is an important factor in the development of thyroid cancer. The subcellular localization of ERα and ERβ may account for the different pathogenesis of thyroid papillary and anaplastic cancers. Copyright

Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein

As a mitochondrial membrane death ligand, Bid oligomerises Bak to release cytochrome C and its deficiency renders hepatocytes resistant to apoptosis induced by Fas. The Bid level in hepatocellular carcinoma (HCC) is unknown. In this report, we examined the expression of Bid protein and mRNA in HCC cancerous tissues and their corresponding non-cancerous ones. The effect of the hepatitis B x protein (HBx) on the expression of Bid was also evaluated by transfecting hepatoma cells with the HBx gene. The results showed that the expression of Bid was significantly lower in cancerous tissues than that in their corresponding non-cancerous tissues. Immunohistochemical study revealed that Bid molecule was mainly localised in hepato-cytoplasm. Some nuclei were also positive for Bid antigen though to a lesser degree. In vitro experiments demonstrated that the expression of Bid in cells transfected with HBx was significantly lower than that in the cells without HBx transfection. This finding suggests that HBx may play a causative role in the reduction of Bid expression in HCC. This in vitro result is, to some degree, supported by clinical data that all the HCC examined are positive for hepatitis B virus (HBV). We conclude from this data that the expression of Bid in HCC is significantly decreased and the reduction of Bid may result from a mechanism associated with HBx, a major hepatocarcinogenic product from HBV. The imbalance of increased anti-apoptosis and decreased pro-apoptosis seen in HCC is a critical mechanism leading to the uncontrolled growth of tumour cells. Therefore, this study suggests that a deficiency in the expression of Bid may contribute to the development of such an imbalance in HCC.

Calcium-mediated activation of PI3K and p53 leads to apoptosis in thyroid carcinoma cells.

Abstract.The molecular mechanism responsible for cadmium-induced cell death in thyroid cancer cells (FRO) is unknown. We demonstrated that apoptosis of FRO cells induced by cadmium was concentration and time dependent. Cadmium caused the rapid elevation of intracellular calcium and induced phosphorylation of Akt, p53, JNK, ERK and p38. Inhibition of PI3K/Akt attenuated the cadmium-induced apoptosis, but the inhibition of JNK inhibitor, ERK or p38 aggravated it, indicating that activation of PI3K/Akt was a pro-apoptosis signal in response to cadmium treatment, whereas the activation of stress-activated protein kinase JNK, ERK and p38 functioned as survival signals to counteract the cadmium-induced apoptosis. Buffering of the calcium response attenuated mitochondrial impairment, recovered the cadmium-activated Akt, p53, JNK, ERK and p38, and subsequently blocked the apoptosis. These results suggested that apoptosis induced by cadmium in FRO cells was initiated by the rapid elevation of intracellular calcium, followed by calcium-mediated activation of PI3K/Akt and mitochondrial impairment.

Bid Stands at the Crossroad of Stress-Response Pathways

Bid, a BH3-only Bcl-2 family member, is proven to be a pivotal molecule for the regulation of tumorigenesis by its multiple functions in promoting apoptosis, survival and proliferation. Growing evidence supports that Bid has double roles with respect to stress-response. In most cases it functions in a truncated form, but the cleavage of Bid may not be an absolute requirement for Bid to be pro-apoptotic. Full-length Bid can also translocate to and activate the mitochondria without cleavage. Bid has emerged as a central player linking death signals through surface death receptors to the core apoptotic mitochondrial pathway. Bid is also involved in DNA damage response, and the phosphorylated Bid may negatively regulate its pro-apoptotic function independent of the BH3 domain. This review surveys recent developments in understanding the molecular mechanisms of Bid activation and its roles in regulating the cross-talk of cell cycle arrest and apoptosis.

Hepatitis B virus x protein induces autophagy via activating death-associated protein kinase

Hepatitis B virus x protein (HBX), a product of hepatitis B virus (HBV), is a multifunctional protein that regulates viral replication and various cellular functions. Recently, HBX has been shown to induce autophagy; however, the responsible mechanism is not fully known. In this study, we established stable HBX‐expressing epithelial Chang cells as the platform to study how HBX induced autophagy. The results showed that the overexpression of HBX resulted in starvation‐induced autophagy. HBX‐induced autophagy was related to its ability to dephosphorylate/activate death‐associated protein kinase (DAPK). The block of DAPK by its siRNA significantly counteracted HBX‐mediated autophagy, confirming the positive role of DAPK in this process. HBX also induced Beclin 1, which functions at the downstream of the DAPK‐mediated autophagy pathway. Although HBX could activate JNK, a kinase known to participate in autophagy in certain conditions, the change in JNK failed to influence HBX‐induced autophagy. In conclusion, HBX induces autophagy via activating DAPK in a pathway related to Beclin 1, but not JNK. This new finding should help us to understand the role of autophagy in HBX‐mediated pathogenesis and thus may provide targets for intervening HBX‐related disorders.