Esophageal squamous cell carcinoma (ESCC) shows high frequency and mortality in Asian regions, including China. Previous analysis of genomic DNA of ESCC using comparative genomic hybridization indicated that amplification of the chromosome 5p regions is a common event in ESCC cell lines and patient cases of Hong Kong Chinese origin, and the results suggested that the genes located in the chromosome 5p regions may play crucial roles in the molecular pathogenesis of ESCC. Our previous studies on ESCC confirmed the tumorigenic and overexpression properties of a novel gene JS-1 located in chromosome 5p15.2 upstream to δ-catenin. In the present study, another novel gene JK-1 which is located at 5p15.1 downstream to δ-catenin was characterized for its roles in the pathogenesis of ESCC. Thirteen ESCC cell lines and 30 surgical specimens of esophageal tumors were studied for the overexpression of JK-1 using multiplex RT-PCR analysis. The transforming capacity of overexpression of JK-1 was also investigated by transfecting NIH 3T3 and HEK 293 cells with the expression vector cloned with JK-1, followed by the soft agar and foci formation assays. JK-1 was overexpressed in 9/13 (69%) of the ESCC cell lines and 9/30 (30%) of the ESCC patient cases. Both NIH 3T3 and HEK 293 cells acquired the properties of anchorage-dependent and-independent growth when JK-1 was overexpressed. Most significantly, subcutaneous sarcomas were formed in all (3/3) the athymic nude mice after NIH 3T3 cells overexpressing JK-1 were injected subcutaneously. Our results thus indicated that JK-1 is commonly overexpressed in ESCC and has a prominent capacity to transform normal cells. Our overall results thus provide the first evidence that the overexpression of JK-1 and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC.

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to ‰ catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC. The present study also forms the ground work for further identification of novel mechanisms of molecular carcinogenesis in ESCC and other cancers.

Previous studies have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) exhibited apoptotic properties in various solid and non-solid tumors in vitro. However, the inhibitory actions of GSE on oncogenic expression and telomerase activity in esophageal squamous cell carcinoma (ESCC) have not been studied before. In the present study, the anti-cancer effects of GSE were demonstrated in three ESCC cell lines (HKESC-1, HKESC-2 and SLMT-1) by MTS and anchorage-independent clongenicity assays, expression studies on oncogenes at 11q13 (CCND1, INT2, FGF4 and EMS1) and real-time quantitative telomeric repeat amplification protocol assay to show the inhibitory effect of GSE on telomerase in ESCC. The means of MTS 50 of GSE for the ESCC cell lines and non-tumor NIH 3T3 cells were 21 and 163 μg/ml respectively. The anchorage-independent clongenicity assay showed that SLMT-1 cells lost their colony-forming potential which was dose-dependent to GSE. Moreover, GSE demonstrated dosedependent suppression on the expression of INT2, EMS1 and FGF4, and inhibition of telomerase activity in the ESCC cell lines. Our overall results thus provide the first evidence that the anti-cancer effects of GSE on ESCC involve the suppression of oncogenic expression and inhibition of telomerase activity. Our findings also offer a new opportunity for the future development of GSE as a novel anti-cancer agent for ESCC and possibly for other cancers.

No abstract

Abstract.Chinese practitioners have employed the use of traditional Chinese medicine as an anti-cancer agent since the ancient period. Different combinations have been formulated for various purposes. Some have been claimed for postchemotherapy use but their direct actions on cancer cells may not be significantly reported. In the present study, we have tested the possible anti-leukemia potential of a combination regimen including crocodile egg extract, wild radix ginseng and natural Ganoderma lucidum (CGG extract) on acute myelogenous leukemia (AML) in vitro. A water soluble CGG extract was prepared and its antiproliferative activity was tested on the KG1a AML cell line and two freshly prepared bone marrow aspirate samples isolated from patients with de novo AML during presentation by a MTS/PMS assay. Furthermore, the possible activity of the CGG extract on the regeneration potential of KG1a cells was also investigated using a semi-solid methyl-cellulose colony formation assay. Lastly, the acute toxicity of CGG extract was further examined by a single high-dose oral feeding to rats. We found that the CGG extract could possess significant antiproliferative activity on AML cells. A strong colony formation inhibition was further demonstrated on KG1a cells. After feeding the rats with an excessive dose of CGG extract, we observed no development of acute toxicity. We concluded that the CGG extract has growth inhibitory potential on KG1a cells and AML bone marrow samples in vitro. An in vivo toxicity test revealed that no acute toxicity was observed after feeding the rats a high dosage of the CGG extract. Further animal model tests are necessary to investigate the possible chronic toxicity of the CGG extract.

Abstract. Cantharidin isolated from Mylabris caraganae and other insects is used traditionally as an anti-cancer drug especially on hepatoma and leukaemia. Previously, we demonstrated that the novel synthetic cantharidin analogue CAN 032 possessed apoptotic activity on two human hepatoma cell lines Hep3B hepatocellular carcinoma and SKHep-1 liver adenocarcinoma. However, its underlying mechanistic action on cancer cells remained unclear. Herein, we furthered our work by making use of KG1a acute myelogenous leukaemia (AML) and K562 chronic myelogenous leukaemia (CML) as experimental models. As anticipated, both leukaemia cell lines were sensitive to the cytotoxic action of CAN 032. The activity of CAN 032 was both dose-and time-course-dependent. CAN 032 readily inhibited the colony formation potential of both leukaemia cell lines. KG1a AML treated with CAN032 decreased G1 phase cell population, mitochondrial membrane potential collapse, caspase 3 activation and hence DNA fragmentation. Pre-incubation of leukaemia cells with the general caspase inhibitor Z-VAD-FMK could partially reversed the apoptotic action of CAN 032. This result suggested that the caspasedependent pathway is necessary for the apoptotic action of CAN 032. CAN 032 provides a new direction for novel drug discovery in experimental cancer therapy.

Cantharidin isolated from Mylabris caraganae and other insects has been used as an anti-cancer drug in China for many years. However, its toxicity on the renal system and suppression effect on bone marrow limits its usage clinically. A synthetic analogue of cantharidin (CAN 037) has been shown to have cytotoxic effect on the SK-Hep 1 hepatoma cell line but its underlying working principle remains undefined. Here we further report the action of CAN 037 on an acute myelogenous leukaemia (AML) cell line, KG1a. [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium] (MTS) assay was used to demonstrate the cytotoxicity of CAN 037 on KG1a cells. Morphological changes of CAN 037-treated leukaemia cells were recorded under an inverted microscope. Possible activation of caspase 3, 8 and 9 from KG1a cells was also investigated. KG1a AML cells were sensitive to CAN 037. Morphological changes including cell shrinkage and loss of colony formation ability were observed. Caspase 3, 8 and 9 activity was elevated, whereas pre-incubating the KG1a cells with the generic caspase inhibitor z-VAD-fmk could only partially reverse the CAN 037-induced cell death. In addition to the SK-Hep-1 hepatoma cell line, CAN 037 is also effective in inducing the death of KG1a AML cells in vitro. Apoptosis is involved in the action of CAN 037 including the activation of the caspase family. Caspase-dependent cell death pathway may be necessary but not essential in CAN 037-induced apoptosis of KG1a cells. Further consideration of the structural activity relationship of CAN 037 may provide opportunities to improve its therapeutic value.