Po Li Wei

Overexpression and Activation of the α9-Nicotinic Receptor During Tumorigenesis in Human Breast Epithelial Cells

BACKGROUNDnLarge epidemiological cohort studies in the United States have indicated that active and passive smoking are associated with increased breast cancer risk. However, there was no direct evidence of an effect of tobacco carcinogens on the cellular molecules involved in breast tumorigenesis.nnnMETHODSnReverse transcription-polymerase chain reaction was used to determine the expression of all of the nicotinic acetylcholine receptor (nAChR) subunits in 50 human breast cancer samples and to determine the expression of the alpha9-nAChR subunit in 276 surgical and laser capture microdissected breast tumor vs normal tissue pairs. Stable MDA-MB-231 breast cancer cell lines were established in which expression of the alpha9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the alpha9-nAChR subunit could be conditionally overexpressed by removal of doxycycline from the culture fluid. Cell proliferation and soft agar assays and tumor growth in nude mice were used as measures of cell transformation. All statistical tests were two-sided.nnnRESULTSnIn 186 (67.3%) of the 276 paired samples, alpha9-nAChR mRNA was expressed at (mean 7.84-fold) higher levels in breast cancers than in surrounding normal tissue. Stable expression of alpha9-nAChR short interfering RNA in MDA-MB-231 cells attenuated nicotine-stimulated proliferation and growth in soft agar and reduced tumor volume when the cells were introduced as xenografts in SCID mice (n = 5 mice per group; mean tumor volume at 6 weeks treatment in mice injected with Si alpha9 cells = 995.6 mm(3), in mice injected with parental cells = 2993.2 mm(3), difference = 1997.6 mm(3), 95% confidence interval [CI] = 1705 to 2290.2 mm(3), P = .009). Long-term treatment of MCF-10A normal breast epithelial cells with either nicotine or its active metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, triggered precancerous transformation as defined by soft agar assay. Inducible overexpression of alpha9-nAChR in MCF-10A cell xenografts in nude mice substantially increased tumor growth (n = 5 mice per group; DOX+, mean tumor volume without nicotine vs with nicotine = 266.2 vs 501.6 mm(3), difference = 235.4 mm(3), 95% CI = 112.7 to 358 mm(3), P = .009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm(3), difference = 277.4 mm(3), 95% CI = 98.1 to 456.7 mm(3), P = .016; mean tumor volume in the presence of nicotine, DOX+ vs DOX- = 501.6 vs 898.6 mm(3), difference = 397 mm(3), 95% CI = 241.3 to 552.6 mm(3), P = .009).nnnCONCLUSIONnThe alpha9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.

Tea polyphenol (−)‐epigallocatechin‐3‐gallate inhibits nicotine‐ and estrogen‐induced α9‐nicotinic acetylcholine receptor upregulation in human breast cancer cells

SCOPEnThe aim of this research was to explore whether the tea-polyphenol (-)-epigallocatechin-3-gallate (EGCG) could be used as a potential agent for blocking smoking (nicotine, Nic)- or hormone (estradiol, E2)-induced breast cancer cell proliferation through inhibition of a common signaling pathway.nnnMETHODS AND RESULTSnTo explore whether Nic (>0.1 μM, 24 h) and E2 (>1 nM, 24 h) significantly increased α9-nicotinic acetylcholine (α9-nicotinic acetylcholine receptor (nAChR)) mRNA and protein expression levels, real-time PCR and immunoblotting analysis experiments were performed in human breast cancer (MCF-7) cells. Luciferase promoter activity experiment was performed to test the α9-nAChR promoter activity affected by Nic, E2 or EGCG. The results indicate that treatment with EGCG (1 μM) profoundly decreases Nic- and E2-induced MCF-7 proliferation by down regulating α9-nAChR expression. The α9-nAChR promoter activity is significantly induced by 24-h treatment with Nic (10 μM) or E2 (10 nM) (>1.8 and ∼2.3-fold, respectively) in MCF-7 cells. Pretreatment with EGCG eliminated the Nic- and E2-induced α9-nAChR promoter-dependent luciferase activity. We further demonstrate that combined treatment with EGCG profoundly inhibits [3H]-Nic/ α9-nAChR binding activity in breast cancer cells.nnnCONCLUSIONSnWe found that the EGCG could be used as an agent for blocking smoking (Nic)- or hormone (E2)-induced breast cancer cell proliferation by inhibiting of α9-nAChR signaling pathway. This study reveals the novel antitumor mechanisms of EGCG, and these results may have significant applications for chemopreventive purposes in human breast cancer.

Tobacco-specific carcinogen enhances colon cancer cell migration through α7-nicotinic acetylcholine receptor

Objective:To study the mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-enhanced migration of colon cancer cells. Background Data:Long-term cigarette smoking increases the risk of colorectal cancer mortality. Tobacco-specific carcinogen, NNK, was reported to increase DNA synthesis of colon cancer cells. Since metastasis is the major cause of cancer death, the influence of NNK on the migration of colon cancer cells remains to be determined. Methods:Receptor for NNK in colon cancer cells was identified by polymerase chain reaction (PCR) and real-time PCR. The influence of NNK on migration of colon cancer cells was evaluated by transwell and wound-healing assay. Receptor-mediated migration was studied by both inhibitor and small interfering RNA. Results:α7 nicotinic acetylcholine receptor, α7-nAChR, was identified in 2 colon cancer cell lines, HT29 and DLD-1. NNK enhanced HT29 cell migration in both transwell and wound-healing assays. NNK also enhanced DLD-1 cell migration in dose dependent manner. We used inhibitor and siRNA to demonstrate that α7-nAChR mediated NNK-enhanced colon cancer cell migration and downregulation of E-cadherin were involved in NNK-enhanced migration of colon cancer cells. Furthermore, Snail and ZEB1, 2 major transcription repressors of E-cadherin in colon cancers, were induced by NNK treatment. Conclusions:Tobacco specific carcinogen, NNK, enhanced colon cancer metastasis through α7-nAChR and E-cadherin—one of the hallmarks of epithelial mesenchymal transition—and its transcription repressors. Therefore, smoking should be avoided in the patients with colorectal cancer.

Nicotine Enhances Colon Cancer Cell Migration by Induction of Fibronectin

BackgroundLong-term cigarette smoking increases the risk of colorectal cancer mortality. Tobacco’s addictive toxin, nicotine, was reported to increase DNA synthesis of colon cancer cells. Because metastasis is the major cause of cancer death, the influence of nicotine on the migration of colon cancer cells remains to be determined.MethodsThe influence of nicotine on the migration of colon cancer cells was evaluated using transwell assay. Nicotine receptor-mediated migration was studied by using both inhibitors and small interfering RNA (siRNA). The role of COX-2 signal was studied using pharmacological inhibitors. The expression of epithelial mesenchymal transition (EMT) marker and COX-2 signal was evaluated using real-time polymerase chain reaction (PCR).ResultsNicotine enhanced DLD-1 and SW480 cell migration in a dose-dependent manner. We used inhibitors and siRNA to demonstrate that α7-nAChR mediates nicotine-enhanced colon cancer cell migration and upregulates fibronectin expression, which is involved in nicotine-enhanced migration. Furthermore, COX-2 signal was induced by nicotine treatment and is involved in nicotine-enhanced fibronectin expression.ConclusionsNicotine, tobacco’s additive toxin, enhances colon cancer metastasis through α7-nAChR and fibronectin—a mesenchymal marker for epithelial mesenchymal transition. Furthermore, COX-2 signal was involved in the induction of fibronectin. Therefore, smoking may play role in the progression of colon cancer.

Crosstalk between nicotine and estrogen-induced estrogen receptor activation induces α9-nicotinic acetylcholine receptor expression in human breast cancer cells

The primary aim of this study was to elucidate the role of the estrogen receptor (ER), a transcription factor involved in the nicotine- and 17β-estradiol (E2)-mediated up-regulation of α9-nAChR gene expression. A real-time polymerase chain reaction (PCR) assay was used to quantify the α9-nAChR mRNA expression levels of surgically isolated (nxa0=xa0339) and laser-capture microdissected tissues (ER+ versus ER−, nxa0=xa06 per group). Chromatin immunoprecipitation (ChIP) and luciferase-promoter activity assays were used to investigate the ER-mediated transcriptional regulation of α9-nAChR gene expression. We observed that breast tumors with higher α9-nAChR mRNA expression levels (i.e., a mean fold ratio in the tumor/normal-paired samples of greater than tenfold) were associated with the lowest 5-year disease-specific survival rate (50%, dead/alivexa0=xa04/4, totalxa0=xa08 patients, Pxa0=xa00.006), in contrast to breast tumors with low levels (i.e., a mean fold ratio of less than onefold) of α9-nAChR expression (88%, dead/alivexa0=xa03/22, totalxa0=xa025 patients). Furthermore, higher α9-nAChR mRNA expression levels were preferentially detected in ER+ tumor tissues in comparison to ER− tumor tissues (ER+ versus ER− patients: nxa0=xa0160 vs. 72; mean fold ratios of α9-nAChR expressionxa0=xa011xa0±xa03 vs. 6.7xa0±xa02.3 fold, respectively). In vitro promoter-binding assays demonstrated that the ER is a major transcription factor that mediates nicotine- and E2-induced up-regulation of α9-nAChR gene expression in MCF-7 cells. In conclusion, our data indicate that the ER plays a central role in mediating α9-nAChR gene up-regulation in response to either nicotine or E2 stimulation.

Nicotine Promotes Cell Migration Through Alpha7 Nicotinic Acetylcholine Receptor in Gastric Cancer Cells

BackgroundThe objective was to study the mechanism of nicotine-enhanced migration of gastric cancer cells. Long-term cigarette smoking increases the risk of gastric cancer mortality. Tobacco-specific mitogen, nicotine, was reported to correlate with cancer progression on gastric cancer. Since metastasis is the major cause of cancer death, the influence of nicotine on the migration of gastric cancer cells remains to be determined.Materials and MethodsThe influence of nicotine on migration of gastric cancer cells was evaluated by transwell assay and wound-healing migration assay. Receptor-mediated migration was studied by both inhibitor and small interfering RNA.ResultsAlpha7 nicotinic acetylcholine receptor, alpha7-nAChR, was identified in gastric cancer cell lines, AGS cells. Nicotine enhanced AGS cell migration in transwell assay and wound-healing migration assay in a dose-dependent manner. We used inhibitor and siRNA to demonstrate that alpha7-nAChR mediated nicotine-enhanced gastric cancer cell migration through downregulation E-cadherin and upregulation ZEB-1 and snail.ConclusionsTobacco-specific mitogen, nicotine, enhanced gastric cancer metastasis through alpha7-nAChR and suppression of E-cadherin level—one of the hallmarks of epithelial to mesenchymal transition. Therefore, patients with gastric cancer should avoid smoking.

Oncological and functional outcomes of intersphincteric resection for low rectal cancer

BACKGROUNDnThe intersphincteric resection technique has been used to extend the opportunity for sphincter preservation in patients with very low rectal cancer. The aim of this study is to assess the long-term oncological and functional outcomes of intersphincteric resection.nnnMETHODSnPatients with extraperitoneal rectal cancer were treated and retrospectively chart reviewed. The oncological and functional outcomes were evaluated. Comparisons of the overall disease-free survival and recurrence were analyzed for the different surgical procedures.nnnRESULTSnFrom July 2002 to August 2009, 162 patients with extraperitoneal rectal cancer were retrospectively chart reviewed. One-hundred one patients (62.3%) underwent low anterior resection, 26 patients (16%) received radical proctectomy and intersphincteric resection with coloanal anastomosis, and 23 (14.2%) had abdominoperineal resection. The sphincter preservation rate was 80%. In the intersphincteric resection group, overall survival rates at 3 and 5 y were 83% and 83%, and disease-free survival at 3 and 5 y were 82% and 76%, respectively. The mean stool frequency was 4.7 per 24 h. There were 38.1% of patients suffering from stool fragmentation, and 23.8% had nocturnal defecation. About one-third of the patients required antidiarrheal medications. Overall, 90.8% of patients were satisfied with the functional results of surgery.nnnCONCLUSIONSnOur data show intersphincteric resection for low rectal cancer is feasible and safe. Preoperative radiotherapy may negatively affect symptom-specific quality of life.

Glucose-Regulated Protein 78 (GRP78) Mediated the Efficacy to Curcumin Treatment on Hepatocellular Carcinoma

BackgroundGlucose-regulated protein 78 (GRP78) plays an important role in the therapeutic treatment and progression of cancer. However, little is known about the effect of GRP78 expression to curcumin in hepatocellular carcinoma (HCC).Materials and MethodsIn this study, we generated GRP78 knockdown cells (GRP78KD) by a short interfering RNA (siRNA) technique. The antiproliferation effects of curcumin were determined by MTT assay, TUNEL assay, and cell cycle determination.ResultsWe found that GRP78KD cells were more resistant to curcumin treatment compared with the parental cells in MTT assay. The apoptosis cell population was increased in scrambled-siRNA cells treated with curcumin compared with GRP78KD cells in cell cycle distribution and TUNEL assays. Finally, we found that knocking down GRP78 causes resistance to curcumin treatment through the suppression of caspase-3 and caspase-8 expression levels.ConclusionsWe conclude that the expression level of GRP78 may contribute to the therapeutic effect of curcumin on HCC cells.

MicroRNA-200a/b influenced the therapeutic effects of curcumin in hepatocellular carcinoma (HCC) cells

MicroRNAs (miRNAs) play an essential role in regulating gene expression in normal and malignant cells. Expression of the microRNA-200 (miR-200) family has been correlated with malignancy in cancers. However, whether miR-200a/b plays a role in curcumin-mediated treatment of hepatocellular carcinoma (HCC) is unknown. We performed miRNA array analyses in two different HCC cell lines (HepG2 and HepJ5). The expression patterns of miR-200 family members were assessed with real-time PCR. We overexpressed miR-200 family members using a lentiviral system and selected stably transduced clones with antibiotics. The anticancer effects of curcumin on J5-200a, J5-200b, and J5-control cells were assessed by MTT assay, flow cytometry cell cycle analysis, and TUNEL assay. We found that HepG2 cells, which were more resistant to curcumin treatment than HepJ5 cells, expressed higher levels of miR-200a/b. The MTT assay revealed that the overexpression of miR-200a/b in HepJ5 cells conferred enhanced resistance to curcumin treatment compared with the control cells. By cell cycle analysis and TUNEL assay, we found that apoptosis was increased dramatically in J5-control cells compared with J5-200a and J5-200b cells after curcumin treatment. Finally, we evaluated the levels of Bcl-2, Bax, and Bad, and found a decrease of Bcl-2 levels and increase of Bad levels in the J5-control cells treated with curcumin. The expression levels of miR-200a/b might determine the therapeutic efficacy of curcumin on HCC cells.

Intersphincteric resection for very low rectal cancer: clinical outcomes of open versus laparoscopic approach and multidimensional analysis of the learning curve for laparoscopic surgery

BACKGROUNDnLaparoscopic rectal cancer surgery is regarded as more complex because of its technical difficulties in pelvic exposure, dissection, and sphincter preservation. This study therefore aimed to investigate the feasibility of laparoscopic resection for low rectal cancer using intersphincteric resection (ISR) and to assess its short-term oncological outcomes. Further, we intended to analyze the learning curve for laparoscopic surgery and identify the factors influencing the learning curve.nnnMETHODSnPatients with low rectal cancer who received open or laparoscopic ISR were retrospectively chart reviewed. The surgical and oncological outcomes were evaluated. Comparisons of operating time, estimated blood loss, surgical outcomes, and histopathologic status were analyzed. Also, operating time was used as a technical indicator for learning curve analysis.nnnRESULTSnThe mean estimated blood loss was 265 mL (range, 100-800 mL) in the open group and 104 mL (range, 30-250 mL) in the laparoscopic group. There was a significant difference between these two groups (Pxa0<xa00.001). Operative experience analysis showed that the mean operating time was 402.1 min (range, 210-570 min) in the first stage and 331.4 min (range, 210-450 min) in the second stage, and on pathologic examination the mean number of lymph nodes harvested was 11.1 (range, 5-21) in the first stage and 18.3 (range, 11-31) in the second stage, with statistical differences between these two stages (Pxa0=xa00.034 and Pxa0=xa00.004, respectively). Multifactorial analysis showed that operating time was associated with surgeons experience (<18 or ≥18 cases) (odds ratioxa0=xa02.918, 95% CI 1.078-7.902). Protective stoma creation was also associated with surgeons experience (odds ratioxa0=xa03.999, 95% CI 1.153-13.86).nnnCONCLUSIONSnOur data show that laparoscopic ISR for low rectal cancer is feasible and safe. Surgeons experience improved operating time and postoperative complications.