Suppression of esophageal cancer cell growth using curcumin,(-)-epigallocatechin-3-gallate and lovastatin

AIM:To determine the effects of curcumin,(-)-epigallocatechin-3-gallate (EGCG),lovastatin,and their combinations on inhibition of esophageal cancer.METHODS:Esophageal cancer TE-8 and SKGT-4 cell lines were subjected to cell viability methyl thiazolyl tetrazolium and tumor cell invasion assays in vitro and tumor formation and growth in nude mouse xenografts with or without curcumin,EGCG and lovastatin treatment.Gene expression was detected using immunohistochemistry and Western blotting in tumor cell lines,tumor xenografts and human esophageal cancer tissues,respectively.RESULTS:These drugs individually or in combinations significantly reduced the viability and invasion capacity of esophageal cancer cells in vitro.Molecularly,these three agents reduced the expression of phosphorylated extracellular-signal-regulated kinases (Erk1/2),c-Jun and cyclooxygenase-2 (COX-2),but activated caspase 3 in esophageal cancer cells.The nude mouse xenograft assay showed that EGCG and the combinations of curcumin,EGCG and lovastatin suppressed esophageal cancer cell growth and reduced the expression of Ki67,phosphorylated Erk1/2 and COX-2.The expression of phosphorylated Erk1/2 and COX-2 in esophageal cancer tissue specimens was also analyzed using immunohistochemistry.The data demonstrated that 77 of 156 (49.4%) tumors expressed phosphorylated Erk1/2 and that 121 of 156 (77.6%) esophageal cancers expressed COX-2 protein.In particular,phosphorylated Erk1/2 was expressed in 23 of 50 (46%) cases of esophageal squamous cell carcinoma (SCC) and in 54 of 106 (50.9%) cases of adenocarcinoma,while COX-2 was expressed in 39 of 50 (78%) esophageal SCC and in 82 of 106 (77.4%) esophageal adenocarcinoma.CONCLUSION:The combinations of curcumin,EGCG and lovastatin were able to suppress esophageal cancer cell growth in vitro and in nude mouse xenografts,these drugs also inhibited phosphorylated Erk1/2,c-Jun and COX-2 expression.

Expression of 6 MicroRNAs in Prostate Cancer and Its Significance

OBJECTIVE Numerous microRNAs (miRNAs) are deregulatedin human cancers. The experimental evidence supports thatmiRNAs plays a role in the initiation and progression of humanmalignancies.The present study was undertaken to evaluatethe differential expression of 6 miRNAs as biomarker for earlydetection of prostate cancer, and then to determine whether theexpression profiling of these miRNAs could predict the prognosisof prostate cancer.METHODS The expression profilings of these 6 miRNAs wereinvestigated using the method of locked nucleic acid (LNA)-modified oligonucleotide in situ hybridization (ISH). And thetechnology of tissue microarray (TMA) was employed using theformalin-fixed, paraffin-embedd (FFPE) specimens taken from52 patients with prostate carcinoma (PCa) and 38 patients withbenign prostatic hyperplasia (BPH).RESULTS The rates of positive expression for 6 miRNAs (miR-15b, miR-16, let-7g, miR- 96,miR-182 and miR-183) were 26.92%,15.38%, 15.38%, 67.31%, 61.54% and 71.15% in the specimens ofprostate cancer, and 57.89%, 76.32%, 68.42%, 44.74%, 31.58%,47.37% in the tissues of benign prostatic hyperplasia, respectively.The expressions of all 6 miRNAs between the prostate cancer andbenign prostatic hyperplasia tissues were significantly different(P < 0.05). The positive rate of these 6 miRNAs was significantlyrelated to the Gleason Grading of prostate cancer (P < 0.01). Therewas no significant correlation between the expression of thesemiRNAs and age and the concentration of serum PSA of thepatient (P >0.05). We also found that the expression of miR-15b,miR-96 and miR-182 correlated with clinical stages of tumor (P <0.05). The expression of miR-96 correlated with lobus prostatae oftumor invasion (P < 0.01), but the expressions of the remaining fivemiRNAs were not correlated with that (P >0.05). In addition, theexpression of miR-15b was negatively related to that of miR-96,miR-182 and miR-183, respectively (P < 0.01, r < 0.00).There wasa positive correlation among the expressions of miR-96, miR-182and miR-183 in prostate cancer (P < 0.01, r >0.00). The expressionof miR-16 was positively related to that of miR-let-7g (P < 0.01, r >0.00).CONCLUSION The results suggest that miRNA expressionprofiling could have relevance to the biological and clinicalbehavior of prostate cancer,and they might be importantbiomarkers for early detection and prognostic assessment ofprostate cancer.

Off-Isocenter Winston-Lutz Test for Stereotactic Radiosurgery/Stereotactic Body Radiotherapy

The single-isocenter technique in linear accelerator-based stereotactic radiosurgery/stereotactic body radiotherapy (SRS/SBRT) has been broadly used to treat multiple lesions. However, quantitative study to verify that the mechanical field center coincides with the radiation field center when both are off from the isocenter has never been performed. We developed an innovative method to measure this accuracy, called the off-isocenter Winston-Lutz test, and here we provided a practical clinical guideline to implement this technique. We used ImagePro V.6 to analyze images of a Winston-Lutz phantom obtained using a Varian 21EX linear accelerator with an electronic portal imaging device, set up as for single-isocenter SRS/SBRT for multiple lesions. We investigated asymmetry field centers that were 3 cm and 5 cm away from the isocenter, as well as performing the standard Winston-Lutz test. We used a special beam configuration to acquire images while avoiding collision, and we investigated both jaw and multileaf collimation. For the jaw collimator setting, at 3 cm off-isocenter, the mechanical field deviated from the radiation field by about 2.5 mm;at 5 cm, the deviation was above 3 mm, up to 4.27 mm. For the multileaf collimator setting, at 3 cm off- isocenter, the deviation was below 1 mm;at 5 cm, the deviation was above 1 mm, up to 1.72 mm, which was 72% higher than the tolerance threshold. These results indicated that the further the asymmetry field center is from the machine isocenter, the larger the deviation of the mechanical field from the radiation field, and the distance between the center of the asymmetry field and the isocenter should not exceed 3 cm in our clinic. We recommend that every clinic that uses linear accelerator, multileaf collimator-based SRS/SBRT perform the off-isocenter Winston-Lutz test in addition to the standard Winston-Lutz test and use their own deviation data to create planning guideline.

Anti-rheumatic Drug Iguratimod(T-614) Alleviates Cancer-induced Bone Destruction via Down-regulating Interleukin-6 Production in a Nuclear Factor-κB-dependent Manner

Cytokines are believed to be involved in a “vicious circle” of progressive interactions in bone metastasis. Iguratimod is a novel anti-rheumatic drug which is reported to have the capability of anti-cytokines. In this study, a rat model was constructed to investigate the effect of iguratimod on bone metastasis and it was found that iguratimod alleviated cancer-induced bone destruction. To further explore whether an anti-tumor activity of iguratimod contributes to the effect of bone resorption suppression, two human breast cancer cell lines MDA-MB-231 and MCF-7 were studied. The effect of iguratimod on tumor proliferation was detected by CCK-8 assay and flow cytometry. The effects of iguratimod on migration and invasion of cancer cells were determined by wound-healing and Transwell assays. Results showed that high dose（30 μg/m L） iguratimod slightly suppressed the proliferation of cancer cells but failed to inhibit their migration and invasion capacity. Interestingly, iguratimod decreased the transcription level of IL-6 in MDA-MB-231 cells in a concentration-dependent manner. Moreover, iguratimod partially impaired NF-κB signaling by suppressing the phosphorylation of NF-κB p65 subunit. Our findings indicated that iguratimod may alleviate bone destruction by partially decreasing the expression of IL-6 in an NF-κB-dependent manner, while it has little effect on the tumor proliferation and invasion.

Amiloride and guggulsterone suppression of esophageal cancer cell growth in vitro and in nude mouse xenografts

Esophageal adenocarcinoma is increasing in the US and Western countries and frequent gastresophageal reflux or gastresophageal reflux disease carrying gastric acid and bile acid could contribute to esophageal adenocarcinogenesis. This study was designed to detect the expression of gastric acid-inducing gene Na＋/H＋ exchanger-1 （NHE-1） ex vivo and then to explore targeting of NHE-1 expression or activity to control esophageal cancer cell viability in vitro and in nude mouse xenografts. The data showed that NHE-1 was highly expressed in esophageal adenocarcinoma tissues （66 of 101 cases [65.3%｜, but not in normal esophageal squamous cell epithelium （1 of 26 cases [3.8~0]）. Knockdown of NHE-1 expression using NHE-1 shRNA or inhibition of NHE-1 activity using the NHE-1 inhibitor amiloride suppressed viability and induced apoptosis in esophageal cancer cells. Molecularly, amiloride inhibited expression of cyclooxygenase-2 and matrix metallopeptidase-9 but not NHE-1 mRNA in esophageal cancer cells. A combination of amiloride and guggulsterone （a natural bile acid receptor inhibitor） showed more than additive effects in suppressing esophageal cancer cell growth in vitro and in nude mouse xenografts. This study suggests that inhibition of NHE-1 expression or activity or combination of amiloride and guggulsterone could be useful in control of esophageal adenocarcinoma.

Lost expression of thyroid hormone receptor-β1 mRNA in esophageal cancer

Thyroid hormone receptors （TR）, ligand-mediated transcription factors, regulate cell growth, differentiation, and apoptosis. In humans, two different genes encode TR-α and TR-β and they are often co-expressed in various tissues at different levels. To explore the role of TR in esophageal cancer, we analyzed expression of TR-β1 mRNA （most abundantly expressed in the majority of normal cells） in normal and malignant esophageal tissue specimens using in situ hybridization. The TR-β1 mRNA was detected in 92.3% （96 of 104） of normal esophageal mucosa, whereas TR-β1 mRNA was only detected in 55.8% （58 of 104 cases） of esophageal squamous cell carcinoma specimens （P〈 0.00001）. Expression of TR-β1 mRNA was associated with well-differentiated cancers （P〈 0.001）. Furthermore, we determined whether the loss of heterozygosity （LOH） in TR-β1 gene locus would be responsible for the lost TR-β1 expression. Analysis of 73 esophageal tissue specimens generated 39 informative cases, 17 of which showed LOH （43.6%） but only 9 of these 17 cases were correlated with lost TR-β1 expression. This study demonstrated that expression of TR-β1 mRNA was lost in esophageal cancer tissues, which may be due to multiple mechanisms.