Human Esophageal Squamous Cell Cancer Research Articles

Upregulation of sex-determining region Y-box 9 (SOX9) promotes cell proliferation and tumorigenicity in esophageal squamous cell carcinoma.

Sex-determining region Y-box 9 (SOX9), a vital transcription factor, play important roles in numerous biological and pathological processes. However, the clinical significance and biological role of SOX9 expression has not been characterized in human esophageal squamous cell cancer (ESCC). Herein, we found that SOX9 was markedly upregulated, at both mRNA and protein level, in ESCC cell lines and ESCC tissues and that SOX9 expression was significantly correlated with tumor clinical stage, T classification, N classification, M classification, pathological differentiation, and shorter overall survival. The proliferation and tumorigenicity of ESCC cells were dramatically induced by SOX9 overexpression but were inhibited by SOX9 knockdown both in vitro and in vivo. Moreover, we demonstrated that upregulation of SOX9 increased the expression of phosphorylated Akt, the cyclin-dependent kinase (CDK) regulator cyclin D1, phosphorylated forkhead box O (FOXO)1, and phosphorylated FOXO3, but SOX9 downregulation decreased their expression, whereas the levels of the CDK inhibitors p21Cip1 and p27Kip1 were attenuated in SOX9-transduced cells. Taken together, our results suggest that SOX9 plays an important role in promoting the proliferation and tumorigenesis of ESCC and may represent a novel prognostic marker for the disease.

Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.

Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late-phase severe adverse effects independently of the TP53 status in vitro. Our findings indicated the feasibility of the combination of Ad-FIR with DNA damaging agents for future esophageal cancer treatment.

Abstract 4032: Functional analysis of exosomes derived from human esophageal squamous cell carcinoma

Abstract Exosomes are small membrane vesicles (30-100 nm) released by many types of cells including cancer cells, and are thought to play an important role in cell-to-cell communications. Exosomes contain lipids, proteins, and nucleic acids such as mRNAs or microRNAs, they are tought to have relationship with tumor growth or metastasis. Our laboratory was previously reported the level of microRNA-1246, expressed mainly in tumor tissues of esophageal squamous cell carcinoma (ESCC), was significantly high in exosomes extracted from the serum of ESCC patients. However, the exosome dynamics and transportation between cancer cells and the other cells are still unclear. In this study, we assessed the cell-to-cell communication via exosomes with fluorescent imaging and analyzed the effect of cancer cell derived exosome to tumor progression. In order to image and analyze the movement of cancer cell-derived exosomes, green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes, was expressed in human esophageal squamous cancer cell line TE2. Exosomes were extracted from supernatant with ultracentrifuge method. Extracted GFP-tagged exosomes were added to the other cells to obtain the images of transportation. The effects of exosomes to tumor progression were also assessed with cell proliferation assay, invasion oassay, and migration assay. Our data indicates that cancer cell derived exosomes affect tumor progression. Citation Format: Yasunori Matsumoto, Masayuki Kano, Yasunori Akutsu, Takanori Nishimori, Naoyuki Hanari, Isamu Hoshino, Kentaro Murakami, Takeshi Toyozumi, Hiroshi Suito, Masahiko Takahashi, Nobufumi Sekino, Yoshihiro Nabeya, Hisahiro Matsubara. Functional analysis of exosomes derived from human esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4032. doi:10.1158/1538-7445.AM2015-4032

Silencing stathmin-modulating efficiency of chemotherapy for esophageal squamous cell cancer with paclitaxel.

Paclitaxel (PTX) is broadly considered the drug of choice for treating human esophageal squamous cell cancer (ESCC). However, PTX resistance often ultimately leads to treatment failure. stathmin, or Op18, is a ubiquitously expressed 19-kDa cytosolic phosphoprotein that can integrate various cellular regulatory signals. stathmin overexpression could lead to resistance to chemotherapeutic agents. In this study we investigated the effect of stathmin gene silencing, using small interfering RNA (stathmin siRNA), on the efficacy of PTX in ESCC. Transfection of stathmin siRNA could significantly inhibit stathmin mRNA and protein levels in ESCC cell lines EC9706 and Eca-109. The silencing of stathmin combined with PTX significantly inhibited the proliferation of EC9706 and Eca-109 cells, with a significantly higher proportion of cells at G2/M phase and this antiproliferative effect was accompanied by an increase in apoptosis rates and morphology changes of EC9706 and Eca-109. Thus, combined chemotherapeutic agent PTX and stathmin siRNA could potentially enhance the therapeutic outcomes of PTX in treating ESCC.

Abstract 1676: The HSP90 inhibitor 17-AAG improves chemoresistance of cisplatin-resistant esophageal squamous cell carcinoma cell lines

Abstract [Background] Although cisplatin is a platinum drug that is widely used to treat esophageal squamous cell carcinoma, acquired chemoresistance develops during the course of treatment and is often the reason for treatment failure. Overcoming chemoresistance is essential for improving the outcomes of patients. Heat shock protein 90 (HSP90) is known to be overexpressed in various cancer cells, and its inhibition by small molecules, either alone or in combination, has shown promise in the treatment of solid malignancies. In the present study, we evaluated the synergistic effect of combining cisplatin with the HSP90 inhibitor, 17-N-allylamino-17-demethoxy geldanamycin (17-AAG), on two cisplatin-resistant human esophageal squamous cancer cell lines, KYSE30 and KYSE150. [Results] The results obtained demonstrated the synergistic inhibitory effects of cisplatin and 17-AAG on the growth of KYSE30 and KYSE150 cells. Cell growth and cell number were more effectively reduced by the combined treatment with cisplatin and 17-AAG than by the treatment with either cisplatin or 17-AAG alone. Western blotting revealed that the combined action of cisplatin and 17-AAG cleaved poly (ADP-ribose) polymerase (PARP) and caspase 3. Time lapse imaging revealed that the activations of caspase 3/7 preceded the cell death induced by the combined treatment of cisplatin and 17-AAG. These demonstrated that the reductions in both cell growth and cell number were mediated by apoptosis. Time-course experiments showed that reductions in X-linked inhibitor of apoptosis protein (XIAP) and phosphorylated Akt were concomitant with apoptosis. [Discussion] The results of the present study demonstrated that 17-AAG synergized with cisplatin and induced apoptosis in cisplatin-resistant esophageal squamous cell carcinoma cell lines, and also that modulation of the Akt/XIAP pathway may underlie this synergistic effect. [Conclusion] Combination therapy with cisplatin and a HSP90 inhibitor may represent a promising strategy to overcome cisplatin resistance in esophageal squamous cell carcinoma. Citation Format: Takashi Ui, Kazue Morishima, Shin Saito, Yuji Sakuma, Hirofumi Fujii, Yoshinori Hosoya, Yoshikazu Yasuda, Toshiro Niki. The HSP90 inhibitor 17-AAG improves chemoresistance of cisplatin-resistant esophageal squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1676. doi:10.1158/1538-7445.AM2014-1676

Inhibitory effects of maslinic acid upon human esophagus, stomach and pancreatic cancer cells

Apoptotic, anti-invasive and anti-migratory effects of maslinic acid (MA) at 4, 8, or 16 µM in human esophageal squamous cancer cell line, OE33; gastric cancer cell line, SGC-7901; and pancreatic cancer cell line, Panc-1, were examined. MA treatments inhibited OE33 and SGC-7901 cells growth at 21–66% and 32–75%, respectively; but lowered Panc-1 viability at 13–27% only. MA treatments increased cleaved caspase-3 and Bax expression, and raised caspase-3 and caspase-8 activities in OE33 and SGC-7901 cells. MA treatments also increased DNA fragmentation and decreased reactive oxygen species production in these two cell lines. MA treatments declined invasion and migration in OE33 and SGC-7901 cells, and lowered vascular endothelial growth factor and transforming growth factor-beta1 levels in these cells. MA suppressed hypoxia-inducible factor-1alpha, matrix metalloproteinase (MMP)-2 and MMP-9 expression in OE33 and SGC-7901 cells. These findings indicated that this triterpene was a potent agent against esophagus and stomach cancers.

Increased expression levels of S100A4 associated with hypoxia-induced invasion and metastasis in esophageal squamous cell cancer

Here, we explored the expression of S100A4 in esophageal squamous cell cancer (ESCC) tissues and investigated its role in hypoxia-induced invasion and metastasis in ESCC cell lines EC-1 and EC-9706. Immunohistochemistry analysis demonstrated that S100A4 was overexpressed in human ESCC tissues especially in ESCC tissues with deep invasion and lymph node metastasis. Hypoxia-induced S100A4 overexpression was observed in EC-1 and EC-9706 cells, in which it was associated with invasion and metastasis. Furthermore, we used EC-1 and EC-9706 cells again to upregulate or knockdown the expression S100A4 to investigate the mechanism role of S100A4 in hypoxia-induced invasion and metastasis in ESCC cells. And the results showed that S100A4 played an important role in promoting the invasion and metastasis of EC-1 and EC-9706 cells under hypoxia. Therefore, S100A4 overexpression might be an important mechanism by which hypoxia induced invasion and metastasis, and S100A4 could also be a potential target for the treatment of ESCC.

TRAP1 Shows Clinical Significance and Promotes Cellular Migration and Invasion through STAT3/MMP2 Pathway in Human Esophageal Squamous Cell Cancer

Tumor necrosis factor receptor-associated protein 1 (TRAP1), an important member of mitochondrial heat shock protein 90 family, is involved in multiple biological processes in several types of tumors. However, its pathological role in esophageal squamous cell cancer (ESCC) remains unknown. Herein, we demonstrated the clinical value of TRAP1, and its role in apoptosis and motility in ESCC. The clinical potential of TRAP1 was investigated through immunohistochemical analysis in 328 ESCC samples, which revealed that strong TRAP1 expression was associated with increased risk of lymph node metastasis, while high TRAP1 expression correlated with poor prognosis. Expression of TRAP1 was found to be an independent prognostic factor for patients with ESCC. Additionally, the upregulation of TRAP1 antagonized cisplatin-induced apoptosis while its downregulation sensitized cells to cisplatin-induced apoptosis. As revealed by the transwell assay, TRAP1 overexpression promoted cellular migration and invasion as compared to the control groups. In contrast, silencing of endogenous TRAP1 expression attenuated the ability of migration and invasion. Finally, the molecular mechanism investigated in the present study demonstrated that TRAP1-mediated migration and invasion occurred through STAT3/MMP2 signaling pathway. In conclusion, TRAP1 may be considered as a molecular predictive marker for prognosis and a novel molecular candidate for therapeutic target in ESCC.

Akt-mediated transforming growth factor-β1-induced epithelial-mesenchymal transition in cultured human esophageal squamous cancer cells.

Epithelial-mesenchymal transition (EMT) has a crucial role during embryonic development and has also come under intense scrutiny as a mechanism through which esophageal squamous cell cancer (ESCC) progresses to become metastatic. Transforming growth factor beta (TGF-β)-mediated EMT has been observed in a variety of cell types and has been identified as the main inducer of EMT in many types of cancer. Akt activity is involved in TGF-β-mediated EMT; however, its precise relationship and role in EMT in ESCC has not been well explained to date. Our data demonstrated that in human ESCC tissues Akt and its activated form, phosphorylated-Akt (p-Akt), were overexpressed; in addition, Akt and p-Akt were negatively correlated with epithelial cadherin (E-cadherin). In EC-9706 cells, exogenous TGF-β1 could induce EMT and at the same time could increase the EC-9706 cell invasive and metastatic ability. Moreover, Akt knockdown by small-interfering RNA could attenuate the EMT induced by TGF-β1 by increasing the epithelial marker E-cadherin and decreasing the mesenchymal marker Vimentin. Silencing Akt expression could decrease the migration ability of EC-9706 cells efficiently. In short, Akt is likely to have a more important role in the EMT induced by TGF-β1 in EC-9706 and may contribute to the invasive and metastatic ability of EC-9706. Akt may be an effective therapeutic in advanced and metastatic ESCC.

MicroRNA-133b downregulation and inhibition of cell proliferation, migration and invasion by targeting matrix metallopeptidase-9 in renal cell carcinoma

microRNA (miRNA)-133b has been revealed to be downregulated in head and neck/oral, bladder, human non-small cell lung, colorectal and esophageal squamous cell cancer. The present study examined the expression of miR-133b in renal cell carcinoma (RCC) cell lines and the effects of miRNA-133b on RCC cell proliferation, migration and invasion. Quantitative polymerase chain reaction was used to detect the expression of miR-133b in RCC cell lines. Following transfection of miR-133b, the expression of miR-133b was examined and a cell viability assay, cell migration assay, cell invasion assay, western blot analysis and luciferase assay were conducted in RCC cell lines. The present study revealed that miRNA‑133b was downregulated and inhibited cell proliferation, migration and invasion in 786-O and A498 cells. In addition, to the best of our knowledge, the present study provided the first evidence that miRNA-133b may directly target matrix metallopeptidase 9 (MMP-9) in RCC. The present study also provided evidence that miRNA-133b suppresses cell proliferation, migration and invasion by targeting MMP-9 in RCC cell lines. These results suggested that miRNA-133b may be used for the development of novel molecular markers and therapeutic approaches to inhibit the metastasis of RCC.

Overexpression of RUNX3 Inhibits Malignant Behaviour of Eca109 Cells in Vitro and Vivo

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.

Frequent amplification of ORAOV1 gene in esophageal squamous cell cancer promotes an aggressive phenotype via proline metabolism and ROS production.

Chromosomal band 11q13 seems to be one of the most frequently amplified lesions in human cancer, including esophageal squamous cell cancer (ESCC). The oral cancer overexpressed 1 (ORAOV1) gene has been identified within this region, but its detailed biological function in human ESCC remains largely unclear. In our clinical samples of stage III ESCC, ORAOV1 amplification was observed in 49 of 94 cases (53%). ORAOV1 amplification was significantly associated with a poorly differentiated histology and tumors located in the upper or middle esophagus. Patients with ORAOV1 amplification tended to have a shorter survival period, although the difference was not significant. To investigate the function of ORAOV1, we created ORAOV1--overexpressed ESCC cell lines that exhibited increased cellular proliferation and colony formation, compared with in vitro controls. In vivo, ORAOV1-overexpressed cells exhibited a significantly increased tumorigenicity and a significantly larger tumor volume and poorer differentiation than controls. The peptide mass fingerprinting technique demonstrated that ORAOV1 bound to pyrroline-5-carboxylate reductase (PYCR), which is associated with proline metabolism and reactive oxygen species (ROS) production. Then, ORAOV1-overexpressed cell lines were resistant to stress treatment, which was cancelled by PYCR-knockdown. In addition, the ORAOV1-overexpressed cell line had a higher intracellular proline concentration and a lower ROS level. Our findings indicate that the ORAOV1 gene is frequently amplified in ESCC, enhances tumorigenicity and tumor growth, and is associated with a poorly differentiated tumor histology via proline metabolism and ROS production. ORAOV1 could be a novel target for the treatment of ESCC.

The HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) synergizes with cisplatin and induces apoptosis in cisplatin-resistant esophageal squamous cell carcinoma cell lines via the Akt/XIAP pathway

Although cisplatin (CDDP) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC), acquired chemoresistance remains a major problem. Combination therapy may represent one strategy to overcome this resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition by small molecules, either alone or in combination, has shown promise in the treatment of solid malignancies. In the present study, we evaluated the synergistic effects of combining CDDP with the HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) on two CDDP-resistant human esophageal squamous cancer cell lines, KYSE30 and KYSE150. The results obtained demonstrated the synergistic inhibitory effects of CDDP and 17-AAG on the growth of KYSE30 and KYSE150 cells. Cell growth and cell number were more effectively reduced by the combined treatment with CDDP and 17-AAG than by the treatment with either CDDP or 17-AAG alone. Western blotting revealed that the combined action of CDDP and 17-AAG cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3, which demonstrated that the reduction in both cell growth and cell number was mediated by apoptosis. Time-course experiments showed that reduction in X-linked inhibitor of apoptosis protein (XIAP) and phosphorylated Akt were concomitant with apoptosis. The results of the present study demonstrate that 17-AAG synergizes with CDDP and induces apoptosis in CDDP-resistant ESCC cell lines, and also that modulation of the Akt/XIAP pathway may underlie this synergistic effect. Combination therapy with CDDP and an HSP90 inhibitor may represent a promising strategy to overcome CDDP resistance in ESCC.

Combination treatment with paclitaxel and doxorubicin inhibits growth of human esophageal squamous cancer cells by inactivation of Akt

Despite the fact that paclitaxel and doxorubicin are widely used as chemotherapy agents against several types of cancer, their combined effects on esophageal squamous cell carcinoma (ESCC) have never been fully elucidated. The present study was designed to investigate the biological effects of paclitaxel and doxorubicin in ESCC cells. Combination treatment with paclitaxel and doxorubicin significantly inhibited the proliferation of TE-12 cells in a dose-and time-dependent manner compared to treatment with paclitaxel or doxorubicin alone. FACS analysis showed that the percentage of cells in the G2/M phase was significantly increased at 12 h after treatment with the combination. Increased p-cdc2, p-Wee1 and p53 protein levels were observed, while Akt activation was suppressed by combination treatment with paclitaxel and doxorubicin. In addition, treatment with paclitaxel plus doxorubicin significantly increased apoptosis as indicated by increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-7 and -9 levels. These results suggest that combination treatment with paclitaxel and doxorubicin induced G2/M cell cycle arrest and apoptosis in human ESCC cells by suppressing Akt activity. These findings highlight the potent apoptotic effect of combination therapy with paclitaxel and doxorubicin in ESCC cells and the potential clinical benefits of these two drugs in esophageal cancer.

O3–053 - ORAOV1 is Amplified in Esophageal Squamous Cell Cancer and Related to Tumor Growth and Poorly Differentiated Tumor

Background: Chromosomal band 11q13 seems to be one of the most frequently amplified lesions in human cancer including esophageal squamous cell cancer (ESCC). Oral cancer overexpressed 1 (ORAOV1) was identified within this region, which is presumably a candidate treatment target in oral squamous cell cancer. Although recent study has demonstrated that yeast orthologue of ORAOV1 is related to reactive oxygen species (ROS), its detail biological function in human ESCC remains unclear. Methods: To investigate the function, we made ORAOV1 overexpressed ESCC cell lines using stable viral transfection technique. In order to search for the binding protein with ORAOV1, peptide mass fingerprinting and co-immunoprecipitation technique were used. In addition, ORAOV1 amplification was evaluated using the copy number assay in 94 clinical samples of stage III ESCC and the association with clinical characteristics was analyzed. Results: ORAOV1 overexpressed ESCC cell lines showed increased cellular proliferation and colony formation compared with control cells in vitro. In vivo, ORAOV1 overexpressed cells exhibited a significantly larger tumor volume and poorlier differentiated tumor than control cells. Peptide mass fingerprinting and co-immunoprecipitation technique demonstrated that ORAOV1 bound to pyrroline-5-carboxylate reductase, which is associated with proline metabolism and ROS production. Then, ORAOV1 overexpressed cells produced lower ROS level than control cells and were resistant to stress treatment. In clinical samples, ORAOV1 amplification of more than 4 copies was observed in 49 cases (53%). ORAOV1 amplification was significantly associated with poorly differentiated and upper or middle located tumor (P = 0.033 and 0.012, respectively). Patients with ORAOV1 amplification tended to have shorter (but not significant) survival than non-amplification patients (median overall survival, 21.6 vs 33.7 months, P = 0.16). Conclusion: ORAOV1 is frequently amplified in ESCC, enhances tumor growth, and is associated with poorly differentiated tumor via proline metabolism and ROS production. Our findings indicate that ORAOV1 can become a novel treatment target in ESCC.

Efficacy of 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a in photodynamic therapy of human esophageal squamous cancer cells

The present study investigated the effects of 2-(1-hexyloxyethyl)-2-devinylpyro pheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) on in vitro cell survival and in vivo tumor growth derived from human esophageal squamous cancer cells (Eca109). A cell counting kit 8 (CCK8) assay was used to assess the phototoxicity of HPPH-mediated PDT in cultured Eca109 cells. The inhibition of tumor growth was determined by the changes in the relative tumor volume (RTV) and tumor weight. The results revealed that HPPH, in the range of 0.005–1 μg/ml, exhibited no cytotoxicity in the Eca109 cells without light exposure and that the in vitro efficiency of HPPH-mediated PDT was higher compared with that of Photofrin®-mediated PDT. The in vivo results indicated that graded doses of HPPH-mediated PDT significantly inhibited the xenograft tumor growth derived from the Eca109 cells in a dose-dependent manner. The inhibition efficacy of 0.6 and 1.0 mg/kg HPPH-mediated PDT was similar to that of 10 mg/kg Photofrin-mediated PDT. Furthermore, HPPH possessed a lower toxicity than Photofrin at the dose that achieved the same efficacy in mice bearing Eca109 subcutaneous tumors. The histopathological findings indicated that the tumor tissues in the photosensitizer (PS)-treated mice demonstrated varying degrees of necrosis. HPPH and Photofrin exhibited vascular cytotoxicity on the treated tumors. In conclusion, the present study demonstrated that the phototoxicity of HPPH-mediated PDT is higher than that of Photofrin-mediated PDT of the same dose. HPPH possessed lower toxicity than Photofrin at the dose that achieved the same efficacy. Therefore, HPPH may be a promising agent for treating human esophageal squamous cell cancer (ESCC).

The glioma-associated oncogene homolog 1 promotes epithelial–mesenchymal transition in human esophageal squamous cell cancer by inhibiting E-cadherin via Snail

The glioma-associated oncogene homolog 1 (GLI1) family of zinc finger transcription factors is the nuclear mediator of the Hedgehog pathway that regulates genes essential for various stages of tumor development and progression. However, the role and mechanism by which high expression of GLI1 contributes to the invasion and metastasis of human esophageal squamous cell cancer (ESCC) has not been fully elucidated. In the present study, we demonstrated that GLI1 was over-expressed in human ESCC tissues, especially in ESCC tissues with deep invasion and lymph-node metastasis. Moreover, GLI1 was also over-expressed in ESCC cell lines and correlated with the aggressiveness of ESCC cell lines. In addition, GLI signaling pathway agonist purmorphamine could increase the invasion and metastasis ability of ESCC cells in vitro. There is increasing evidence for the contribution of epithelial-mesenchymal transition (EMT) to ESCC invasion and metastasis, therefore we investigated GLI1's role in EMT. Our results showed that high expression of GLI1 dampened expression of E-cadherin and enhanced the expression of Vimentin, and it also improved the expression of Snail, indicative of its role in EMT occurrence. Mechanistic studies showed that down-expression of Snail reversed GLI1 activation-regulated expression of EMT markers, suggesting the role of Snail in GLI1-mediated EMT. Taken together, our results had revealed that GLI1 could participate in the invasion and metastasis of ESCC through EMT. These studies indicated that in ESCC, GLI1 could be a useful target for cancer prevention and therapy.

Elevated Maspin Expression Is Associated with Better Overall Survival in Esophageal Squamous Cell Carcinoma (ESCC)

Tumor suppressor maspin is a differentially regulated gene in the progression of many types of cancer. While the biological function of maspin in blocking tumor invasion and metastasis is consistent with the loss of maspin expression at the late stage of tumor progression, the differential expression and the biological significance of maspin in early stage of tumor progression appear to be complex and remain to be elucidated. In the current study, we examined the expression of maspin in 84 esophageal squamous cell carcinoma (ESCC) cases (stages I–III) and 55 non-tumor adjacent esophageal tissue specimens by immunohistochemical (IHC) staining. The correlation of maspin with clinicopathological parameters was analyzed. Compared to normal esophageal squamous tissue where 80% (47/55) of the cases expressed maspin at a low to moderate level, all ESCC specimens (100% (84/84)) were positive for maspin expression at a moderate to high level. ESCC with low or moderate maspin expression had significantly shorter postoperative survival rates compared to those that had high maspin expression (p<0.001). Since the correlation of maspin with ESCC histology and the correlation of maspin with ESCC prognosis seem to be at odds, we further investigated the biological function of maspin in ESCC using the established ESCC cell lines. The expression of maspin in five human esophageal squamous cancer cell lines (T12, E450, KYSE150, EC109, and KYSE510) was examined by the Western blot. ESCC cell line KYSE510 that did not express maspin and was stably transfected by maspin cDNA or an empty vector. The resulting transfected cells were characterized in vitro. Maspin expression significantly inhibited cell proliferation, motility and matrigel invasion. Taken together, our data suggest that the transient up-regulation of maspin in the early development of ESCC may be a defense mechanism against further transition towards more malignant phenotypes, ultimately slowing down ESCC tumor progression.

Restoring KLF5 in Esophageal Squamous Cell Cancer Cells Activates the JNK Pathway Leading to Apoptosis and Reduced Cell Survival

Esophageal cancer is the eighth most common cancer in the world and has an extremely dismal prognosis, with a 5-year survival of less than 20%. Current treatment options are limited, and thus identifying new molecular targets and pathways is critical to derive novel therapies. Worldwide, more than 90% of esophageal cancers are esophageal squamous cell cancer (ESCC). Previously, we identified that Krüppel-like factor 5 (KLF5), a key transcriptional regulator normally expressed in esophageal squamous epithelial cells, is lost in human ESCC. To examine the effects of restoring KLF5 in ESCC, we transduced the human ESCC cell lines TE7 and TE15, both of which lack KLF5 expression, with retrovirus to express KLF5 upon doxycycline induction. When KLF5 was induced, ESCC cells demonstrated increased apoptosis and decreased viability, with up-regulation of the proapoptotic factor BAX. Interestingly, c-Jun N-terminal kinase (JNK) signaling, an important upstream mediator of proapoptotic pathways including BAX, was also activated following KLF5 induction. KLF5 activation of JNK signaling was mediated by KLF5 transactivation of two key upstream regulators of the JNK pathway, ASK1 and MKK4, and inhibition of JNK blocked apoptosis and normalized cell survival following KLF5 induction. Thus, restoring KLF5 in ESCC cells promotes apoptosis and decreases cell survival in a JNK-dependent manner, providing a potential therapeutic target for human ESCC.

Effects of metformin on cell proliferation and apoptosis in human esophageal squamous cancer cell line Eca109

Effects of metformin on cell proliferation and apoptosis in human esophageal squamous cancer cell line Eca109