Unexpected discovery of intrahepatic cholangiocarcinoma during follow-up in two cases of liver cysts: case report.

Background: The liver cyst is commonly treated by hepatobiliary surgery. Generally, most patients show no apparent symptoms and often get diagnosed accidentally during the imaging examinations. In addition, most patients with liver cysts follow a benign course, with fewer severe complications and rare occurrences of malignant changes. Therefore, based on disease characteristics and healthcare costs, long-term regular follow-up of liver cysts are rarely performed clinically. Case Description: Here, we reported two previously treated or observed cases for liver cysts, where intrahepatic neoplastic lesions were found unexpectedly at the liver cyst during follow-up. These two patients' clinical manifestations and laboratory examinations lacked specificity with unclear pre-operative diagnosis, whereas the post-operative pathology confirmed cholangiocarcinoma. One of the patients was a 64-year-old female with right upper abdominal distension. She underwent cyst fenestration for a liver cyst 3 years ago. In the latest admission, imaging examination revealed a tumor in the left inner lobe of the liver. The tumor was located in the exact fenestration location, and the pathological diagnosis of cholangiocarcinoma was made after surgical resection. The patient received Lenvatinib post-operatively and had no recurrence during the follow-up. Another patient, a 68-year-old woman, was asymptomatic, but the liver margin was palpable under the ribs on her physical examination. She had a previous diagnosis of liver cysts and was on regular yearly follow-up. In the last follow-up, a tumor was found close to a cyst. It was diagnosed as intrahepatic cystadenocarcinoma before surgery; however, the pathological features after surgical resection were more consistent with the cholangiocarcinoma. The patient had lung metastases 2 months after the surgery, but her condition improved after receiving targeted therapy and immunotherapy. Moreover, she is alive to this day. Conclusions: We reported 2 cases of intrahepatic cholangiocarcinoma discovered accidentally during the follow-up of hepatic cysts. The location of the malignant tumor coincided with the location of the cyst, making the clinical differential diagnosis problematic. Therefore, it is necessary to be vigilant about the possibility of combined malignant tumors for the follow-up of complex cysts, as early detection and treatment may help improve the prognosis of these patients. After surgery, multimodal therapy, including chemotherapy, immunotherapy, and targeted therapy, is helpful.

Effect of CLIC1 gene silencing on proliferation, migration, invasion and apoptosis of human gallbladder cancer cells.

This study aimed to explore the effects of CLIC1 gene silencing on proliferation, migration, invasion and apoptosis of human gallbladder cancer (GBC). GBC and normal gallbladder tissues were extracted for the detection of mRNA and protein expressions of CLIC1. GBC-SD and NOZ cells in the logarithmic growth phase were selected to conduct the experiment. Three different siRNA recombined expression vectors were established using CLIC1 as a target at different sites. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were, respectively, used to detect the CLIC1 mRNA and protein expressions. MTT assay was performed to detect the cell proliferation. Flow cytometry was applied to measure the cell apoptosis and cell cycle distribution. The variations of cell migration and invasion were evaluated using Transwell assay. GBC tissues showed higher CLIC1 mRNA and protein expressions than normal gallbladder tissues. The CLIC1 mRNA and protein expressions in the CLIC1 siRNA group were significantly lower than those in the NC and blank groups. Compared with the NC and blank groups, the CLIC1 siRNA group showed a significant decrease in cell proliferation but an obvious increase in apoptosis rate in GBC cells. Besides, in the CLIC1 siRNA group, cell percentage in G0/G1 and G2/M phase was gradually increased but decreased in S phases. The migration and invasion abilities in GBC cells were significantly lower than those in the NC and blank groups. Our study demonstrates that CLIC1 gene silencing could promote apoptosis and inhibit proliferation migration and invasion of GBC cells.

CUL4B promotes metastasis and proliferation in pancreatic cancer cells by inducing epithelial-mesenchymal transition via the Wnt/ß-catenin signaling pathway.

This study determines whether cullin 4B (CUL4B) promotes pancreatic cancer (PC) metastasis by inducing epithelial-mesenchymal transition (EMT) via the Wnt/ß-catenin signaling pathway. A total of 64 PC patients were enrolled in this study. Human PC cell lines were distributed into blank, negative control, shCUL4B, PLOC, PLOC-CUL4B, and PLOC-CUL4B + siRNA-ß-catenin groups. The expressions of CUL4B, Wnt/ß-catenin signaling pathway-related proteins, and EMT-related proteins were determined using RT-qPCR and Western blotting. The positive expressions of CUL4B and ß-catenin protein in tissues were detected by immunohistochemistry. MTT assay and flow cytometry was performed for cell proliferation and cell cycle, scratch test, and transwell assay for cell migration and invasion ability. CUL4B and ß-catenin were expressed at a higher level in PC tissues than in paracancerous tissues though paracancerous tissues had higher expressions of CUL4B and ß-catenin than normal tissues. The PLOC-CUL4B group showed increased CUL4B, Wnt, ß-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; decreased E-cadherin expression; accelerated cell proliferation; increased S-phase cell percentages; increased cell migration ability; more liver metastases; and enlarged tumor than the PLOC and PLOC-CUL4B + siRNA-ß-catenin groups. The shCUL4B group showed decreased CUL4B, Wnt, ß-catenin, LEF-1, c-Jun, Cyclin D1, N-cadherin, Vimentin, Snail, and ZEB1 expression; increased E-cadherin expression; decelerated cell proliferation; decreased S-phase cell percentages; reduced cell migration ability; less liver metastases; and decreased tumor weight than the blank and negative control groups. We demonstrate that CUL4B promotes PC metastasis by inducing EMT via the Wnt/ß-catenin signaling pathway. Therefore, CUL4B might be the clinical target for treating PC.

Octyl Ester of Ginsenoside Rh2 Induces Apoptosis and G1 Cell Cycle Arrest in Human HepG2 Cells by Activating the Extrinsic Apoptotic Pathway and Modulating the Akt/p38 MAPK Signaling Pathway.

Ginsenoside Rh2 is a potential active metabolite of ginseng that has antitumor activity against a variety of tumor cells. Previously, we reported that Rh2-O, an octyl ester derivative of ginsenoside Rh2, had a higher anticancer activity than Rh2 through activating the intrinsic apoptotic pathway. In this study, we found that the extrinsic apoptotic pathway was also involved in Rh2-O-induced HepG2 cells apoptosis as evidenced by the up-regulation of Fas, FasL, TNFR1, and TNF-α as well as the cleavage of caspase 8. Moreover, flow cytometric analysis demonstrated that Rh2-O induced G1 cell cycle arrest in HepG2 cells. Rh2-O-induced G1 phase arrest was accompanied by the down-regulation of cyclin D3 and cyclin E and cyclin-dependent kinases (CDK) 4 and 6 and the up-regulation of p21WAF1/CIP1 and p27KIP1. In addition, Rh2-O down-regulated the phosphorylation of Akt, and its inhibitor LY294002 promoted Rh2-O-induced G1 phase arrest. Rh2-O treatment also activated p38 MAPK, JNK, and ERK expression. Inhibitors of p38 MAPK (SB203580), but not those of JNK (SP600125) or ERK (PB98095), promoted Rh2-O-induced G1 phase arrest in HepG2 cells. These results indicated that the disruption of Akt and p38 MAPK cascades played a pivotal role in Rh2-O-induced G1 phase arrest.

Suppression of angiogenesis and tumor growth in vitro and in vivo using an anti-angiopoietin-2 single-chain antibody.

Hepatocellular carcinomas (HCCs) are tumors with a highly developed vascular architecture. HCC cells require access to blood vessels for growth and metastasis; therefore, the inhibition of angiogenesis represents a potential therapeutic target for HCC that may reduce the mortality and morbidity from HCC. Various attempts to develop an anti-angiogenic therapy have been made in past decades; however, modest results have been achieved in clinical trials and the challenge of HCC treatment remains. Single-chain antibodies (scFv) are characterized by low molecular weight, low immunogenicity, high penetration and a short half-life, and are easy to produce on a large scale by genetic engineering. Accordingly, an scFv against a specific angiogenic regulator, such as angiopoietin (Ang), may be a promising anti-angiogenic therapy for HCC. Our previous study indicated that an imbalanced expression of angiopoietin-2 (Ang-2) vs. angiopoietin-1 (Ang-1) in HCCs contributes to initiation of neovascularization and promotes the angiogenesis and progression of HCCs. Therefore, we suggest that specific Ang-2-targeting interventions may be valuable in the treatment of HCC via remodeling the neovascular network and changing the tumor microenvironment. In this study, a prokaryotic expression vector of Ang-2 was constructed and purified human Ang-2 protein was isolated. An scFv against human Ang-2 (scFv-Ang2) was identified and purified via phage display technology, and the effects of scFv-Ang2 in vitro and in vivo on HCC in nude mice were evaluated. The results show that scFv-Ang2 inhibits vascular endothelial growth factor (VEGF) and Ang-2 induces the proliferation, migration and tubule formation of human umbilical vein endothelial cells (HUVECs) in vitro. In the in vivo assay, statistical indices, including tumor weight and volume, metastases to lungs, CD31 expression and the microvessel density (MVD) count in the scFv-Ang2-treated group of mice were significantly lower than those in the control group (P<0.05). In conclusion, the successfully generated scFv-Ang2 showed significant inhibitory effects on the angiogenesis and tumor growth of human HCC in vitro and in vivo.

[Comparison of subtotal colectomy with antiperistaltic cecoproctostomy and total colectomy with ileoproctostomy in treating slow transit constipation].

OBJECTIVE: To compare clinical outcome and quality of life of subtotal colectomy with antiperistaltic cecoproctostomy and total colectomy with ileorectal anastomosis (TAC-IRA) in patients with severe slow transit constipation (STC). METHODS: Of the 56 patients enrolled in this study from January 1999 to June 2008, 32 cases underwent subtotal colectomy with antiperistaltic cecoproctostomy, and 20 patients underwent TAC-IRA. The patients' clinical characteristics, operative data, postoperative outcome, functional result and gastrointestinal quality of life index (GIQLI) survey were compared between the two groups. RESULTS: All patients were followed up for 1-7 years (median, 4 years). The basic clinical characteristics between the two groups was comparable. During the follow-up period, the number of daily bowel movements in the subtotal colectomy group was significantly fewer than that in TAC-IRA group (2.5+/-0.8 vs. 3.4+/-0.8; P=0.000). The Wexner continence score was significantly lower in subtotal colectomy group (4.4+/-1.6 vs. 5.8+/-1.9; P=0.011), and the GIQLI score in subtotal colectomy group was significantly higher than that in the TAC-IRA group (120.7+/-7.5 vs. 111.1+/-12.0; P=0.005). CONCLUSION: Subtotal colectomy with antiperistaltic cecoproctostomy appeared to be the superior treatment than the TAC-IRA for selected patients with slow transit constipation for improved functional outcomes and quality of life.

[Efficacy comparison of subtotal colectomy with antiperistaltic cecoproctostomy and total colectomy with ileorectal anastomosis for patients with slow transit constipation].

OBJECTIVE: To compare the efficacy between subtotal colectomy with antiperistaltic cecoproctostomy and total colectomy with ileorectal anastomosis (TAC-IRA) for patients with severe refractory slow transit constipation(STC). METHODS: During 1999 to 2002, TAC-IRA was the preferred procedure for 20 STC patients in our department. From 2003 to 2005, 17 STC patients underwent subtotal colectomy plus antiperistaltic cecoproctostomy. Clinical data of the two groups were collected and compared retrospectively. RESULTS: There were no significant differences in basic preoperative clinical data between the two groups. During the follow-up period, the time of daily defecation in the antiperistaltic cecoproctostomy group was less than that of TAC-IRA group (2.4+/-0.9 vs 3.4+/-0.8, P=0.0014), meanwhile the Wexner continence score was significantly lower in the antiperistaltic cecoproctostomy group (4.3+/-1.8 vs 5.8+/-1.9, P=0.0223). Barium enema after subtotal colectomy showed that residual ascending colon and cecum presented a sign of "reservoir". CONCLUSION: Subtotal colectomy with antiperistaltic cecoproctostomy is a better method for appropriately selected patients with STC than TAC-IRA.

[Long-term results of subtotal colectomy with antiperistaltic cecoproctostomy in chronic slow-transit constipation].

OBJECTIVE: To assess the long-term results after subtotal colectomy with antiperistaltic cecoproctostomy in idiopathic chronic slow-transit constipation. METHODS: Between January 2003 and February 2004, 14 patients with chronic slow-transit constipation and 2 patients with mixed constipation underwent subtotal colectomy with antiperistaltic cecoproctostomy. The following information was collected during follow-up (mean 3 years): number of bowel movement, stool consistency, complications, quality of life and degree of satisfaction. RESULTS: There was no mortality or major postoperative complications. One month after the operation, bowel frequency was a mean of 4 daily, with a semi-liquid stool consistency. After 3 years, bowel frequency was a mean of 2 daily, with a semi-solid stool consistency. Although no patient used antidiarrheal medicine, laxatives continued to be used by one case with mixed chronic constipation. All patients reported a good or improved quality of life and satisfied with the results. Two patients developed adhesive ileus post operation. There was no diarrhea or incontinence occurred during the follow-up. CONCLUSIONS: Subtotal colectomy with end-to-end antiperistaltic cecoproctostomy for appropriately selected patients with slow-transit constipation results in consistent relief of constipation and satisfactory outcome.

Effect of hydroxyapatite nanoparticles on the growth and p53/c-Myc protein expression of implanted hepatic VX2 tumor in rabbits by intravenous injection.

AIM: To evaluate the effect of hydroxyapatite nano-particles (Nano HAP) by intravenous injection on the inhibition of implanted hepatic VX(2) tumor growth in rabbits and cell p53/c-Myc protein expression. METHODS: 60 hepatic VX(2) tumor-bearing rabbits was randomly divided into five groups. Nano HAP collosol 20 mg/kg, 40 mg/kg, 5-FU solutions 20 mg/mL, mixed liquor of 5-FU solution 20 mg/mL and Nano HAP collosol 20 mg/kg were infused by vein, normal saline conducted as the control. The general state, weight, liver function and gross tumor volume were detected dynamically. The expression of p53 and c-Myc gene protein in tumor tissue was detected by immunohistochemistry methods. RESULTS: The growth of implanted hepatic VX(2) tumors was significantly inhibited in all therapy groups, 3 wk after the injection, the tumor control rates in Nano HAP collosol groups were 25.5% and 32.5% respectively, and the gross tumor volumes were obviously less than that of control group. (24.81 +/- 5.17 and 22.73 +/- 4.23 vs 33.32 +/- 5.26, P<0.05). The tumor control rate of 5-FU group was 43.7% (18.74 +/- 4.40 vs 33.32 +/- 5.26, P<0.05), but the general state of the animals after injection aggravated; and the adverse reaction in the drug combination group obviously decreased. Due to the effect of Nano HAP, the positive expression of tumor associated the mutated p53 and c-Myc in tumor tissue was decreased obviously compared with the control group. CONCLUSION: Nano HAP has evident inhibitory action on rabbit implanted hepatic VX(2) tumor in vivo, which may be the result of decreasing the expression of the mutated p53 and c-myc, and drug combination can obviously decrease the adverse reaction of 5-FU.

Growth inhibition of high-intensity focused ultrasound on hepatic cancer in vivo.

AIM: To investigate the damaging effect of high-intensity focused ultrasound (HIFU) on cancer cells and the inhibitory effect on tumor growth. METHODS: Murine H22 hepatic cancer cells were treated with HIFU at the same intensity for different lengths of time and at different intensities for the same length of time in vitro, the dead cancer cells were determined by trypan blue staining. Two groups of cancer cells treated with HIFU at the lowest and highest intensity were inoculated into mice. Tumor masses were removed and weighed after 2 wk, tumor growth in each group was confirmed pathologically. RESULTS: The death rate of cancer cells treated with HIFU at 1 000 W/cm2 for 0.5, 1, 2, 4, 8, and 12 s was 3.11+/-1.21%, 13.37+/-2.56%, 38.84+/-3.68%, 47.22+/-5.76%, 87.55+/-7.32%, and 94.33+/-8.11%, respectively. A positive relationship between the death rates of cancer cells and the length of HIFU treatment time was found (r = 0.96, P<0.01). The death rate of cancer cells treated with HIFU at the intensity of 100, 200, 400, 600, 800, and 1 000 W/cm2 for 8 s was 26.31+/-3.26%, 31.00+/-3.87%, 41.97+/-5.86%, 72.23+/-8.12%, 94.90+/-8.67%, and 99.30+/-9.18%, respectively. A positive relationship between the death rates of cancer cells and the intensities of HIFU treatment was confirmed (r = 0.98, P<0.01). The cancer cells treated with HIFU at 1 000 W/cm2 for 8 s were inoculated into mice ex vivo. The tumor inhibitory rate was 90.35% compared to the control (P<0.01). In the experimental group inoculated with the cancer cells treated with HIFU at 1 000 W/cm2 for 0.5 s, the tumor inhibitory rate was 22.9% (P<0.01). By pathological examination, tumor growth was confirmed in 8 out of 14 mice (57.14%, 8/14) inoculated with the cancer cells treated with HIFU at 1 000 W/cm2 for 8 s, which was significantly lower than that in the control (100%, 15/15, P<0.05). CONCLUSION: HIFU is effective on killing or damage of H22 hepatic cancer cells in vitro and on inhibiting tumor growth in mice ex vivo.

Expression levels and significance of hypoxia inducible factor-1 alpha and vascular endothelial growth factor in human colorectal adenocarcinoma.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a transcription factor, is overexpressed in common human cancers and their metastases. This study aimed at determining the expression levels of HIF-1alpha and vascular endothelial growth factor (VEGF) in SW480 cells and in colorectal adenocarcinoma tissue and ascertaining whether HIF-1alpha and VEGF play important roles in tumor angiogenesis. METHODS: HIF-1alpha mRNA expression was analyzed using in situ hybridization and RT-PCR. HIF-1alpha and VEGF protein were detected in SW480 cells and colorectal adenomas and adenocarcinomas by immunohistochemistry using streptavidin/peroxidase (SP). Western blot was used to detect HIF-1alpha protein extracted from SW480 cells. Microvessel density (MVD) in colorectal carcinomas was determined by anti-CD34 immunostaining in colorectal carcinomas. RESULTS: Optical density values representing HIF-1alpha mRNA expression levels were found to be significantly higher in SW480 cells in hypoxic conditions than in cells under normoxic conditions (P < 0.05) or in hypoxic conditions but treated with genistein (P < 0.05). The levels of HIF-1alpha and VEGF protein expression in SW480 cells were significantly higher in the hypoxia group than in the normoxia group (P < 0.01, P < 0.05, respectively) and hypoxia/genistein group (P < 0.01, P < 0.05, respectively). The positive expression rates of HIF-1alpha mRNA changed dramatically when comparing colorectal adenomas with adenocarcinomas of different Dukes' stages (P < 0.05). HIF-1alpha mRNA was also expressed at higher levels in adenocarcinomas than that in adenomas (P < 0.01). HIF-1alpha protein expression correlated significantly with VEGF protein expression and MVD. CONCLUSIONS: Hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinomas. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinomas.

[A study of the inhibition effect of HIFU and its mechanism of action on the proliferation of human breast cancer cells].

OBJECTIVE: To investigate the killing effect of high intensity focused ultrasound (HIFU) on human tumor cells and study the mechanism of its action. METHODS: Human breast cancer cells MCF-7 were treated with different intensity of HIFU in vitro and incubated. The killing effect and proliferation inhibition effect of HIFU on tumor cells were investigated with the methods of trypan blue exclusion assay, MTT assay and colony formation assay. And the cell cycle, apoptotic cells and the expressions of P53, BCL-2, FAS and HSP70 were measured with flow cytometry. RESULTS: After HIFU treatment, the death rates of cancer cell increased, the proliferation of cell waned (P < 0.05), the number of colony formation decreased (P < 0.001), the cell number increased in G0/G1 phase (P < 0.05) and decreased in S phase, the apoptotic indices increased (P < 0.01), the expression of HSP70 was enhanced, and no significant changes of P53, BCL-2, FAS expressions were found (P > 0.05). CONCLUSION: HIFU produces significant effects such as killing, proliferation inhibition and colony formation inhibition on human breast cancer cells; its mechanism of action may be associated with the inhibition of DNA synthesis. In addition, HIFU treatment can induce apoptotic cell death, which may not be associated with the mediation and regulation of P53, BCL-2 and FAS genes.

[Relationship of hypoxia-inducible factor 1 alpha (HIF-1alpha) gene expression with vascular endothelial growth factor (VEGF) and microvessel density (MVD) in human colorectal adenoma and adenocarcinoma].

BACKGROUND & OBJECTIVE: Hypoxia-inducible factor-1 alpha (HIF-1alpha), a transcriptional factor response to hypoxia plays an important role in tumor angiogenesis. This study was designed to examine the expression of HIF-1alpha gene and its relationship with vascular endothelial growth factor (VEGF) protein and microvessel density (MVD). METHODS: HIF-1alpha gene expression was analyzed using in situ hybridization, and VEGF expression levels were determined by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinomas. Microvessel density (MVD) was determined by anti-CD34 immunostaining. RESULTS: Positive expression of HIF-1alpha mRNA were found in 67.8% (42/62) of the colorectal adenocarcinomas and 44.4% (8/18) of the adenomas. The mean percentage of HIF-1alpha mRNA positive cells increased gradually with the development from Dukes'stage A to stage C+D (P< 0.05). The frequencies of HIF-1alpha positive cells in pathologic stage of the specimens were as follows: adenoma 8%, Dukes A 14%, Dukes B 23%, Dukes C+D 35%. The positive expression rate of VEGF protein in colorectal adenocarcinoma group was significantly higher than that in colorectal adenoma group (59.7% vs 33.3%,P< 0.05). During colorectal tumor progression, the expression of HIF-1alphamRNA was positively correlated with the VEGF protein expression and MVD (r(s) = 0.768 P< 0.01 and r(s) = 0.683 P< 0.05, respectively). CONCLUSION: These results suggest that tumor angiogenesis induced by HIF-1alpha mRNA and VEGF protein might play an important role in tumorigenesis of colorectal adenoma and progression of colorectal cancer.