Management of granulomatous lobular mastitis: an international multidisciplinary consensus (2021 edition).

Granulomatous lobular mastitis (GLM) is a rare and chronic benign inflammatory disease of the breast. Difficulties exist in the management of GLM for many front-line surgeons and medical specialists who care for patients with inflammatory disorders of the breast. This consensus is summarized to establish evidence-based recommendations for the management of GLM. Literature was reviewed using PubMed from January 1, 1971 to July 31, 2020. Sixty-six international experienced multidisciplinary experts from 11 countries or regions were invited to review the evidence. Levels of evidence were determined using the American College of Physicians grading system, and recommendations were discussed until consensus. Experts discussed and concluded 30 recommendations on historical definitions, etiology and predisposing factors, diagnosis criteria, treatment, clinical stages, relapse and recurrence of GLM. GLM was recommended as a widely accepted definition. In addition, this consensus introduced a new clinical stages and management algorithm for GLM to provide individual treatment strategies. In conclusion, diagnosis of GLM depends on a combination of history, clinical manifestations, imaging examinations, laboratory examinations and pathology. The approach to treatment of GLM should be applied according to the different clinical stage of GLM. This evidence-based consensus would be valuable to assist front-line surgeons and medical specialists in the optimal management of GLM.

Dexmedetomidine reduces enteric glial cell injury induced by intestinal ischaemia-reperfusion injury through mitochondrial localization of TERT.

This study was performed to uncover the effects of dexmedetomidine on oxidative stress injury induced by mitochondrial localization of telomerase reverse transcriptase (TERT) in enteric glial cells (EGCs) following intestinal ischaemia-reperfusion injury (IRI) in rat models. Following establishment of intestinal IRI models by superior mesenteric artery occlusion in Wistar rats, the expression and distribution patterns of TERT were detected. The IRI rats were subsequently treated with low or high doses of dexmedetomidine, followed by detection of ROS, MDA and GSH levels. Calcein cobalt and rhodamine 123 staining were also carried out to detect mitochondrial permeability transition pore (MPTP) and the mitochondrial membrane potential (MMP), respectively. Moreover, oxidative injury of mtDNA was determined, in addition to analyses of EGC viability and apoptosis. Intestinal tissues and mitochondria of EGCs were badly damaged in the intestinal IRI group. In addition, there was a reduction in mitochondrial localization of TERT, oxidative stress, whilst apoptosis of EGCs was increased and proliferation was decreased. On the other hand, administration of dexmedetomidine was associated with promotion of mitochondrial localization of TERT, whilst oxidative stress, MPTP and mtDNA in EGCs, and EGC apoptosis were all inhibited, and the MMP and EGC viability were both increased. A positive correlation was observed between different doses of dexmedetomidine and protective effects. Collectively, our findings highlighted the antioxidative effects of dexmedetomidine on EGCs following intestinal IRI, as dexmedetomidine alleviated mitochondrial damage by enhancing the mitochondrial localization of TERT.

Dexmedetomidine inhibits mitochondria damage and apoptosis of enteric glial cells in experimental intestinal ischemia/reperfusion injury via SIRT3-dependent PINK1/HDAC3/p53 pathway.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury commonly occurs during perioperative periods, resulting in high morbidity and mortality on a global scale. Dexmedetomidine (Dex) is a selective α2-agonist that is frequently applied during perioperative periods for its analgesia effect; however, its ability to provide protection against intestinal I/R injury and underlying molecular mechanisms remain unclear. METHODS: To fill this gap, the protection of Dex against I/R injury was examined in a rat model of intestinal I/R injury and in an inflammation cell model, which was induced by tumor necrosis factor-alpha (TNF-α) plus interferon-gamma (IFN-γ) stimulation. RESULTS: Our data demonstrated that Dex had protective effects against intestinal I/R injury in rats. Dex was also found to promote mitophagy and inhibit apoptosis of enteric glial cells (EGCs) in the inflammation cell model. PINK1 downregulated p53 expression by promoting the phosphorylation of HDAC3. Further studies revealed that Dex provided protection against experimentally induced intestinal I/R injury in rats, while enhancing mitophagy, and suppressing apoptosis of EGCs through SIRT3-mediated PINK1/HDAC3/p53 pathway in the inflammation cell model. CONCLUSION: Hence, these findings provide evidence supporting the protective effect of Dex against intestinal I/R injury and its underlying mechanism involving the SIRT3/PINK1/HDAC3/p53 axis.

KDM4B promotes DNA damage response via STAT3 signaling and is a target of CREB in colorectal cancer cells.

Resistance to radiotherapy is a major limitation for the successful treatment of colorectal cancer (CRC). Recently, accumulating evidence supports a critical role of epigenetic regulation in tumor cell survival upon irradiation. Lysine Demethylase 4B (KDM4B) is a histone demethylase involved in the oncogenesis of multiple human cancers but the underlying mechanisms have not been fully elucidated. Here we show that KDM4B is overexpressed in human colorectal cancer (CRC) tumors and cell lines. In CRC cells, KDM4B silencing induces spontaneous double-strand breaks (DSBs) formation and potently sensitizes tumor cells to irradiation. A putative mechanism involved suppression of Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway, which is essential for efficient repair of damaged DNA. Overexpression of STAT3 in KMD4B knockdown cells largely attenuates DNA damage triggered by KDM4B silencing and increases cell survival upon irradiation. Moreover, we find evidence that transcription factor CAMP Responsive Element Binding Protein (CREB) is a key regulator of KMD4B expression by directly binding to a conserved region in KMD4B promoter. Together, our findings illustrate the significance of CREB-KDM4B-STAT3 signaling cascade in DNA damage response, and highlight that KDM4B may potentially be a novel oncotarget for CRC radiotherapy.

Effect of recurrent severe hypoglycemia on cognitive performance in adult patients with diabetes: A meta-analysis.

The purpose of this study was to investigate the existence and extent of cognitive impairment in adult diabetes mellitus (DM) patients with episodes of recurrent severe hypoglycemia, by using meta-analysis to synthesize data across studies. PubMed, EMBASE and Cochrane library search engines were used to identify studies on cognitive performance in DM patients with recurrent severe hypoglycemia. Random-effects meta-analysis was performed on seven eligible studies using an inverse-variance method. Effect sizes, which are the standardized differences between the experimental group and the control group, were calculated. Of the 853 studies, 7 studies met the inclusion criteria. Compared with control subjects, the adult DM patients with episodes of recurrent severe hypoglycemia demonstrated a significantly lowered performance on memory in both types of DM patients, and poor performance of processing speed in type 2 DM patients. There was no significant difference between adult DM patients with and those without severe hypoglycemia in other cognitive domains such as general intelligence, executive function, processing speed and psychomotor efficiency. Our results seem to confirm the hypothesis that cognitive dysfunction is characterized by worse memory and processing speed in adult DM patients with a history of recurrent severe hypoglycemia, whereas general intelligence, executive function, and psychomotor efficiency are spared.

Expression of TREM-1 mRNA in acute pancreatitis.

AIM: To explore the expression of triggering receptor expressed on myeloid cells (TREM-1) mRNA in acute pancreatitis (AP). METHODS: Using the reverse transcription polymerase chain reaction (RT-PCR), we examined the expression of TREM-1 mRNA in 10 cases of mild acute pancreatitis (MAP), 8 cases of severe acute pancreatitis (SAP), and 10 cases of healthy control subjects. And we also examined the expression of TREM-1 mRNA in 14 cases of AP (including 10 MAP and 4 SAP) before treatment, after successful therapy and clinically cured. RESULTS: The expression of TREM-1 mRNA in the groups of MAP, SAP patients and healthy control subjects was 0.771+/-0.274, 1.092+/-0.331 and 0.459+/-0.175, respectively; there was a significant difference among the three groups (P<0.05). And there was also a significant difference between the AP patients (0.914+/-0.341) and healthy control subjects (0.459+/-0.175) (P<0.05). Moreover, in the 14 cases of AP, before treatment, after successful therapy and clinically cured, the expression of TREM-1 mRNA was 0.905+/-0.226, 0.739+/-0.169 and 0.633+/-0.140, respectively, and there was a significant difference among the three stages (P<0.05). CONCLUSION: The expression of TREM-1 mRNA in the patients with AP increases obviously, and correlates with the degree of AP. Furthermore, the expression of TREM-1 mRNA is distinctly different at the different stages of AP. It indicates TREM-1 may play an important role in the occurrence and development of AP.

Mechanisms of inhibition of growth of human pancreatic carcinoma implanted in nude mice by somatostatin receptor subtype 2.

OBJECTIVES: Several studies reported that somatostatin receptor subtypes, especially subtype 2 (SSTR2), exerted their cytostatic and/or cytotoxic effects on various types of tumors. The aim of this study was to investigate the antitumor effect of SSTR2 gene transfer to the pancreatic cancer cell line PC-3 and the mechanisms involved in this effect. METHODS: The full-length human SSTR2 cDNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection; positive clones were screened by G418, and stable expression of SSTR2 was detected by the immunohistochemical SABC method and RT-PCR. Athymic mice were separately xenografted with SSTR2-expressing cells (experimental group), vector control, and mock control cells. TUNEL assay was used to determine the apoptotic index (AI) in the tumors of these groups. The immunohistochemical SP method was used to determine expression of apoptosis-regulating genes Bcl-2 and Bax and re-expression of SSTR2 and to assess intratumoral microvessel density (MVD). Moreover, tumor volume and weight were compared among these 3 groups. RESULTS: Restoration of SSTR2 was observed in the experimental group both in vitro and in vivo. The AI was significantly higher in the experimental group (3.39 +/- 0.84%) compared with that in the vector control (0.69 +/- 0.08%) and mock control (0.68 +/- 0.09%) (P < 0.05). MVD was significantly lower in the experimental group (6.30 +/- 1.71) than that in the vector control (12.64 +/- 1.69) and mock control (13.50 +/- 1.86) (P < 0.05). Furthermore, a significant decrease in Bcl-2 and increase in Bax protein expression were detected in the experimental group compared with the vector control and mock control (P < 0.05). A significant negative correlation of protein expression between Bcl-2/Bax ratio and SSTR2 was observed in these tumors (P < 0.05). Tumor volume and weight were significantly decreased in the experimental group compared with the vector control and mock control (P < 0.05) groups. However, no significant differences were observed between the vector control and mock control (P > 0.05). CONCLUSION: Re-expression of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, induces apoptosis, which may be mediated via down-regulation of Bcl-2 and up-regulation of Bax (alteration of Bcl-2/Bax ratio) and inhibits tumor angiogenesis in pancreatic carcinoma, resulting in inhibition of tumor growth.

Effect of octreotide on human pancreatic cancer cells after transfected with somatostatin receptor type 2 gene.

AIM: To observe the effect of octreotide on apoptosis rate of human pancreatic cancer cells PC-3 after transfected with somatostatin receptor type 2 (SST2) gene. METHODS: SST2 plasmid was transfected into PC-3 cells by liposome. Result of transfection was detected by immunocytochemical staining and Western blotting. Apoptosis rates of PC-3 cells under different dosages of octreotide were measured by MTT assay and flow cytometry (FCM). RESULTS: Apoptosis rate caused by octreotide of transfected PC-3 cells was 7.56+/-1.06% at the dosage of 0.20 microg/mL, 9.25+/-1.73% at the dosage of 0.40 microg/mL and 14.18+/-2.71% at the dosage of 0.80 microg/mL. Apoptosis rate caused by octreotide of non-transfected PC-3 cells was 5.76+/-0.75% at the dosage of 0.20 microg/mL, 6.69+/-0.80% at the dosage of 0.40 microg/mL and 7.26+/-1.28% at the dosage of 0.80 microg/mL. Transfected PC-3 cells growth inhibition rate caused by octreotide was 9.36+/-1.34% at the dosage of 0.20 microg/mL, 12.03+/-1.44% at the dosage of 0.40 microg/mL and 20.23+/-4.21% at the dosage of 0.80 microg/mL. Non-transfected PC-3 cells growth inhibition rate caused by octreotide was 6.44+/-0.66% at the dosage of 0.20 microg/mL, 7.65+/-0.88% at the dosage of 0.40 microg/mL and 9.29+/-1.32% at the dosage of 0.80 microg/mL. We found that octreotide caused higher apoptosis rate and inhibition rate in transfected groups than in non-transfected groups (P<0.05) at the tested dosages (0.20, 0.40 and 0.80 microg/mL). CONCLUSION: Deficiency of SST2 was probably the major reason why octreotide had little effect on PC-3 cells. Transfecting SST2 gene could strengthen the ability of octreotide of killing PC-3 cells. It provided an experimental evidence for using both octreotide and transfection with SST2 gene on clinical treatment of pancreatic cancer.

[Mechanisms of inhibition of growth of human pancreatic carcinoma implanted in nude mice by somatostatin receptor subtype 2].

OBJECTIVE: To investigate the antitumor effect of somatostatin receptor subtype 2 (SSTR2) gene transfection into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect. METHODS: The full-length human SSTR2 cDNA was introduced into the pancreatic cancer cell line PC-3 by lipofect amine-mediated transfection. Positive clones were screened by G418 and stable expression of SSTR2 was detected by immunocytochemistry SABC method and RT-PCR. Fifteen athymic mice were randomly divided into 3 groups of 5 mice to be xenografted with SSTR2-expressing cells (experimental group), empty vector control group, and control group cells respectively. The weight of mice and the size of tumor were measured every week. Eight weeks later the mice were killed and the tumors were taken out. TUNEL assay was used to determine the apoptotic index (AI) in these tumors. Immunohistochemistry SP method was used to determine the expressions of apoptosis regulating genes, Bcl-2 and Bax, and intratumoral. microvessel density (MVD). Moreover, the tumor volume and weight were compared among these three groups. RESULTS: The AI was significantly higher in the experimental group (3.39% +/- 0.84%) compared with the empty vector control group (0.69% +/- 0.08%) and control group (0.68% +/- 0.09%) (both P < 0.05). The significant decrease in Bcl-2, and increase in Bax protein expressions were detected in the experimental group compared with the empty vector control group and control group (both P < 0.05). MVD was significantly lower in the experimental group (6.3 +/- 1.7) than those in the empty vector control group (12.6 +/- 1.7) and control group (13.5 +/- 1.9) (P < 0.05). Moreover, the tumor volume and weight were significantly lower in the experimental group as compared with the empty vector control group and control group (P < 0.05). However, no significant differences in all the indicators were observed between the empty vector control group and the control group (both P > 0.05). CONCLUSIONS: Re-expression of SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, can induce apoptosis which may be mediated via down-regulation of Bcl-2 and up-regulation of Bax, and inhibit tumor angiogenesis in pancreatic carcinoma, resulting in inhibition of tumor growth.

Anti-angiogenic effects of somatostatin receptor subtype 2 on human pancreatic cancer xenografts.

Somatostatin receptor subtypes, especially subtype 2 (SSTR2), exert their antitumor (cytostatic and/or cytotoxic) and anti-angiogenic effects. Here we aimed to investigate the anti-angiogenic effect of SSTR2 gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect. The full-length human SSTR2 complementary DNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection, and stable expression of SSTR2 was detected by immunohistochemistry and RT-PCR. Athymic mice were separately xenografted with SSTR2-expressing cells (experimental group), vector control and mock control cells. Intratumoral microvessel density (MVD) was assessed by immunohistochemistry. Immunohistochemistry and RT-PCR were used to determine the expression of angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase (MMP)-2 in xenograft tumors. MVD was significantly lower in the experimental group (5.16 +/- 1.34) than that in the vector control (16.52 +/- 2.25) and mock control (15.32 +/- 2.53) (P < 0.05). The immunohistochemical assay showed a significant decrease in the expression of VEGF, bFGF and MMP-2 protein in the experimental group compared with the vector control and mock control, considering both the integral optical density and area of staining (P < 0.05). RT-PCR showed a significant reduction of VEGF, bFGF and MMP-2 mRNA expression in the experimental group compared with the vector control and mock control (P < 0.05). Thus, introduction of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, exerts its anti-angiogenic effects by down-regulating the expression of the factors, which are involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a promising strategy of gene therapy for pancreatic cancer.

Antiangiogenic effect of somatostatin receptor subtype 2 on pancreatic cancer cell line: Inhibition of vascular endothelial growth factor and matrix metalloproteinase-2 expression in vitro.

AIM: To investigate the anti-angiogenic effect of somatostatin receptor subtype 2 (SSTR2) gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect. METHODS: The full length human SSTR2 cDNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection. Positive clones were screened by G418 and stable expression of SSTR2 was detected by immunohistochemistry SABC methods and RT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect vascular endothelial growth factor (VEGF) levels in the cell culture supernatants of SSTR2-expressing cells, vector control and mock control cells. Furthermore, the expressions of VEGF and matrix metalloproteinase-2 (MMP-2) were detected by immunohistochemistry SABC methods and RT-PCR in these cells. RESULTS: VEGF levels in the cell culture supernatants were significantly reduced in the SSTR2-expressing cells (first week, 172.63+/-21.2 ng/L and after two months, 198.85+/-26.44 ng/L) compared with the vector control (first week, 790.39+/-86.52 ng/L and after two months, 795.69+/-72.35 ng/L) and mock control (first week, 786.42+/-90.62 ng/L and after two months, 805.32+/-84.36 ng/L) (P<0.05). The immunohistochemical assay showed a significant reduction of the integral optical density of VEGF and MMP-2 in the SSTR2-expressing cells (42.25+/-8.6 and 70.5+/-6.25, respectively) compared with the vector control (85.75+/-12.9 and 110.52+/-13.5, respectively) and mock control (82.6+/-9.28 and 113.56+/-9.62, respectively) (P<0.05). Conversely, the average gray value of VEGF and MMP-2 was significantly increased in the SSTR2-expressing cells (121.56+/-8.43 and 134.46+/-19.95, respectively) compared with the vector control (55.72+/-5.6 and 62.26+/-12.68, respectively) and mock control cells (58.48+/-6.2 and 65.49+/-9.16, respectively) (P<0.05). Moreover, the expressions of VEGF mRNA and MMP-2 mRNA were significantly reduced in the SSTR2-expressing cells (0.1384+/-0.017 and 0.2343+/-0.070, respectively) compared with the vector control (1.024+/-0.117 and 0.806+/-0.119, respectively) and mock control (1.085+/-0.105 and 0.714+/-0.079, respectively) (P<0.05). CONCLUSION: The expression of reintroduced human SSTR2 gene exerts its antiangiogenic effects by down-regulating the expressions of the factors involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a new strategy of gene therapy for pancreatic cancer.

Survivin expression induced by doxorubicin in cholangiocarcinoma.

AIM: To study the role of survivin expression induced by chemotherapy agent (doxorubicin) in the development and anti-chemotherapy of cholangiocarcinoma. METHODS: Expression of survivin was detected by SP immunohistochemical technique in 33 cases of cholangiocarcinoma, 28 cases of adjacent noncancerous bile duct, and 5 cases of benign bile duct lesions. Low concentration of doxorubicin (0.05 mg/l) was added in cultured cholangiocarcinoma cell line (QBC939). The expression of survivin was detected by RT-PCR and Western blot at 24 h and 48 h after adding doxorubicin. RESULTS: Survivin was expressed in 24 of 33 cholangiocarcinoma cases (72.7%). In contrast, no expression of survivin in adjacent noncancerous and benign bile duct lesions was observed (P<0.01). No correlation was found between survivin expression and clinical features. Doxorubicin could markedly (P<0.001) up-regulate survivin mRNA and protein expression of QBC939 cells. CONCLUSION: Overexpression of survivin in cholangiocarcinomas may play an important role in the development of cholangiocarcinoma, its relationship with prognosis of cholangiocarcinoma deserves further investigation. Higher expression of survivin is induced by doxorubicin in QBC939. Survivin expression may resist apoptosis induced by chemotherapy agents.

Alteration of somatostatin receptor subtype 2 gene expression in pancreatic tumor angiogenesis.

AIM: To explore the difference of somatostatin receptor subtype 2 (SST2R) gene expression in pancreatic cancerous tissue and its adjacent tissue, and the relationship between the change of SST2R gene expression and pancreatic tumor angiogenesis related genes. METHODS: The expressions of SST2R, DPC4, p53 and ras genes in cancer tissues of 40 patients with primary pancreatic cancer, and the expression of SST2R gene in its adjacent tissue were determined by immunohistochemiscal LSAB method and EnVision(TM) method. Chi-square test was used to analyze the difference in expression of SST2R in pancreatic cancer tissue and its adjacent tissue, and the correlation of SST2R gene expression with the expression of p53, ras and DPC4 genes. RESULTS: Of the tissue specimens from 40 patients with primary pancreatic cancer, 35 (87.5%) cancer tissues showed a negative expression of SST2R gene, whereas 34 (85%) a positive expression of SST2R gene in its adjacent tissues. Five (12.5%) cancer tissues and its adjacent tissues simultaneously expressed SST2R. The expression of SST2R gene was markedly higher in pancreatic tissues adjacent to cancer than in pancreatic cancer tissues (P<0.05). The expression rates of p53, ras and DPC4 genes were 50%, 60% and 72.5%, respectively. There was a significant negative correlation of SST2R with p53 and ras genes (chi(1)(2)=9.33, chi(2)(2)=15.43, P<0.01), but no significant correlation with DPC4 gene (chi(2)=2.08, P>0.05). CONCLUSION: There was a significant difference of SST2R gene expression in pancreatic cancer tissues and its adjacent tissues, which might be one cause for the different therapeutic effects of somatostatin and its analogs on pancreatic cancer patients. There were abnormal expressions of SST2R, DPC4, p53 and ras genes in pancreatic carcinogenesis, and moreover, the loss or decrease of SST2R gene expression was significantly negatively correlated with the overexpression of tumor angiogenesis correlated p53 and ras genes, suggesting that SST2R gene together with p53 and ras genes may participate in pancreatic cancerous angiogenesis.

Celecoxib inhibits proliferation and induces apoptosis via prostaglandin E2 pathway in human cholangiocarcinoma cell lines.

AIM: To evaluate the roles and mechanisms of celecoxib in inducing proliferation inhibition and apoptosis of human cholangiocarcinoma cell lines. METHODS: Cyclooxygenase-2-overexpressing human cholangiocarcinoma cell line QBC939 and cyclooxygenase-2-deficient human cholangiocarcinoma cell line SK-CHA-1 were used in the present study. The anti-proliferative effect was measured by methabenzthiazuron (MTT) assay; apoptosis was determined by transferase-mediated dUTP nick end labeling (TUNEL) detection and transmission electron microscopy (TEM). Cell cycle was analyzed by flow cytometry (FCM). The PGE(2) levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). RESULTS: Celecoxib suppressed the production of PGE(2) and inhibited the growth of QBC939 cells. Celecoxib at 10, 20, and 40 micromol/L inhibited PGE(2) production by 26 %, 58 %, and 74 % in QBC939 cells. The PGE(2) level was much lower constitutively in SK-CHA-1 cells (18.6+/-3.2) compared with that in QBC939 (121.9+/-5.6) cells (P<0.01) and celecoxib had no significant influence on PGE(2) level in the SK-CHA-1 cells. The PGE(2) concentration in SK-CHA-1 cells also reduced but not significantly after treatment with celecoxib. The PGE(2) concentration in SK-CHA-1 cells was (16.5+/-2.9) ng/well, (14.8+/-3.4) ng/well, (13.2+/-2.0) ng/well and (12.6+/-3.1) ng/well respectively, when pre-treated with 1 micromol/L, 10 micromol/L, 20 micromol/L and 40 micromol/L of celecoxib for 48 h (P>0.05, vs control). The anti-proliferation effect of celecoxib (20 micromol/L) on QBC939 cells was time-dependent, it was noticeable on day 2 (OD490=0.23+/-0.04) and became obvious on day 3 (OD490=0.31+/-0.07) to day 4 (OD490= 0.25+/-0.06), and the OD490 in the control group (day 1) was 0.12+/-0.03 (P<0.01, vs control). The anti-proliferation effect of celecoxib could be abolished by the addition of 200 pg/mL PGE(2). The proliferation of SK-CHA-1 cells was inhibited slightly by celecoxib, the cell density OD490 in the presence of celecoxib and in control group was 0.31+/-0.04 and 0.42+/-0.03 respectively on day 2 (P>0.05), 0.58+/-0.07 and 0.67+/-0.09 respectively on day 3 (P>0.05), and 0.71+/-0.08 and 0.78+/-0.06 respectively on day 4 (P>0.05). Celecoxib induced proliferation inhibition and apoptosis by G(1)-S cell cycle arrest: the percentage of QBC939 cells in G(0)-G(1) phase after treatment with 40 micromol/L (74.66+/-6.21) and 20 micromol/L (68.63+/-4.36) celecoxib increased significantly compared with control cells (54.41+/-5.12, P<0.01). The percentage of SK-CHA-1 cells in G(0)-G(1) phase after treatment with various concentrations of celecoxib didn't change significantly compared with control cells. The TUNEL index was much higher in QBC939 cells treated with 20 micromol/L celecoxib for 2 d (0.063+/-0.018) and for 4 d (0.102+/-0.037) compared with control cells (0.017+/-0.004, P<0.01). CONCLUSION: The current in vitro study indicates that inhibition of proliferation and induction of apoptosis in human cholangiocarcinoma cells by cyclooxygenase-2 specific inhibitor celecoxib may involve in COX-dependent mechanisms and PGE(2) pathway. Celecoxib as a chemopreventive and chemotherapeutic agent might be effective primarily on COX-2-expressing cholangiocarcinoma.

Bile from a patient with anomalous pancreaticobiliary ductal union promotes the proliferation of human cholangiocarcinoma cells via COX-2 pathway.

AIM: To explore the effects of COX-2 gene in the proliferative activity induced by bile from anomalous pancreaticobiliary ductal union (APBDU) on human cholangiocacinoma cell line. METHODS: Bile sample from APBDU and normal bile sample were used for this study. The proliferative effect of bile was measured by methabenzthiazuron (MTT) assay; COX-2 mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Cell cycle was analyzed by flow cytometry (FCM), and the PGE(2) levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). RESULTS: Bile from APBDU can significantly promote the proliferation of human cholangiocarcinoma QBC939 cells compared with normal bile (P=0.005) and up-regulated remarkably their COX-2 mRNA expression (P=0.004). The proliferative activity of APBDU bile can be abolished by addition of cyclooxygenase-2 specific inhibitor celecoxib. CONCLUSION: Bile from APBDU can promote the proliferation of human cholangiocarcinoma QBC939 cells via COX-2 pathway.

Proliferative activity of bile from congenital choledochal cyst patients.

AIM: To explore the potential carcinogenicity of bile from congenital choledochal cyst (CCC) patients and the mechanism of the carcinogenesis in congenital choledochal cyst patients. METHODS: 20 bile samples from congenital choledochal cyst patients and 10 normal control bile samples were used for this study. The proliferative effect of bile was measured by using Methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry (FCM), and the PGE(2) levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: CCC bile could significantly promote the proliferation of human cholangiocarcinoma QBC939 cells compared with normal bile (P=0.001) and negative control group (P=0.002), and the proliferative effect of CCC bile could be abolished by addition of cyclooxygenase-2 specific inhibitor celecoxib (20 microM). The QBC939 cells proliferative index was increased significantly after treated with 1 % bile from CCC patient (P=0.008) for 24 h, the percentage of S phase (29.48+/-3.27)% was increased remarkably (P<0.001) compared with normal bile (11.72+/-2.70) %, and the percentage of G0/G1 phase (54.19+/-9.46) % was decreased remarkably (P=0.042) compared with normal bile (69.16+/-10.88) %, however, bile from CCC patient had no significant influence on apoptosis of QBC939 cells (P=0.719). CONCLUSION: Bile from congenital choledochal cyst patients can promote the proliferation of human cholangiocarcinoma QBC939 cells via COX-2 and PGE(2) pathway.

Expression of cyclooxygenase-1 and -2 in extra-hepatic cholangiocarcinoma.

OBJECTIVE: To investigate the expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in extra-hepatic cholangiocarcinoma and the relationship between their expression and clinicopathological parameters. METHODS: COX-1 and COX-2 were detected in 56 extra-hepatic cholangiocarcinomas, including 31 matched tissues originating from non-tumorous bile ductal tissue adjacent to tumours and 6 normal bile ductal tissues, by immunohistochemistry strept avidin-biotin complex using isozyme selective antibodies. RESULTS: There was no difference in expression of COX-1 between carcinomas (96%, 54/56) and noncancerous specimens (94%, 29/31, P>0.05) or normal bile ductal tissues (100%, 6/6, P>0.05). The positive rate of COX-2 expression in extra-hepatic cholangiocarcinomas (86%, 48/56) was significantly higher than their matched tissues (39%, 12/31, P<0.01) and normal bile ductal tissues (0%, 0/6, P<0.01). Overexpression of COX-2 in extra-hepatic cholangiocarcinoma was related to the metastasis of lymph nodes, distant organs or tissues (P<0.05) as well as the degree of tumour differentiation (P<0.05). CONCLUSIONS: The overexpression of COX-2 plays a crucial role in the carcinogenesis and development of extra-hepatic cholangiocarcinoma, indicating that COX-2 may serve as a target for chemoprevention of extra-hepatic cholangiocarcinoma.