Antitumor Effects and Related Mechanisms of Penicitrinine A, a Novel Alkaloid with a Unique Spiro Skeleton from the Marine Fungus Penicillium citrinum.

Penicitrinine A, a novel alkaloid with a unique spiro skeleton, was isolated from a marine-derived fungus Penicillium citrinum. In this study, the isolation, structure and biosynthetic pathway elucidation of the new compound were described. This new compound showed anti-proliferative activity on multiple tumor types. Among them, the human malignant melanoma cell A-375 was confirmed to be the most sensitive. Morphologic evaluation, apoptosis rate analysis, Western blot and real-time quantitative PCR (RT-qPCR) results showed penicitrinine A could significantly induce A-375 cell apoptosis by decreasing the expression of Bcl-2 and increasing the expression of Bax. Moreover, we investigated the anti-metastatic effects of penicitrinine A in A-375 cells by wound healing assay, trans-well assay, Western blot and RT-qPCR. The results showed penicitrinine A significantly suppressed metastatic activity of A-375 cells by regulating the expression of MMP-9 and its specific inhibitor TIMP-1. These findings suggested that penicitrinine A might serve as a potential antitumor agent, which could inhibit the proliferation and metastasis of tumor cells.

A multicenter, randomized, observation-controlled clinical trial to evaluate the efficacy and safety of thymalfasin adjuvant therapy in patients with HBV-related HCC after curative resection - first announcement of the protocol.

Hepatocellular carcinoma (HCC), the third leading cause of cancer-related death worldwide, is a disease of immune microenvironment. Chronic Hepatitis B virus (HBV) infection, also an immune-related disease, is the major etiological factor for HCC especially in Asia. As an immune regulator, which has pleiotropic activities on T cells, nature killer cells and dendritic cells and so on, the efficacy of thymalfasin on HCC patients has been proven by several pilot studies as an adjuvant therapy. Combination of thymalfasin significantly improved survival and prolonged the time to tumor recurrence in patients who received transcatheter arterial chemoembolization after tumor resection. An improvement in patients' immunity has also been demonstrated. However, there is no large-scale randomized controlled study so far in resectable HCC patients. To confirm the role of thymalfasin adjuvant therapy in patients with HBV-related HCC after curative resection, a large-scale multicenter randomized controlled trial has been planned in China to investigate the effect of thymalfasin (1.6 mg twice a week for 12 months) on 2-year recurrence-free survival rate and tumor immune microenvironment. Here is the first announcement of the study protocol (ClinialTrials.gov Identifier: NCT02281266).

Laparoscopic segmental colectomy for colonic lymphangiomas: a definitive, minimally invasive surgical option.

Colonic lymphangioma is an unusual benign malformation. We herein describe two cases. A 36-year-old woman was admitted with one year of intermittent abdominal pain; colonoscopy, abdominopelvic computed tomography and endoscopic ultrasonography (EUS) revealed enlarged cystic masses at the ascending colon. In another 40-year-old man, colonoscopy and EUS revealed an asymptomatic lobulated cystic mass with four small sessile polyps at the sigmoid colon. Both patients underwent laparoscopic segmental colectomy. Both masses were histologically confirmed as cystic lymphangiomas, and the patients were discharged without complications. The management of colonic lymphangioma depends on the individual situation; close surveillance or endoscopic therapy may be appropriate for asymptomatic lesions smaller than 2.5 cm in diameter. Surgical intervention can be considered for larger lesions or in patients who develop complication risks. Laparoscopic segmental colon resection may be recommended to excise relatively large submucosal lesions because it is a definitive, minimally invasive intervention with a fast postoperative recovery.

Effects of CO2 pneumoperitoneum on the expression of thymidine kinase 1 and Ki67 in colorectal carcinoma cells.

BACKGROUND: To investigate thymidine kinase 1(TK-1) and Ki67 expression levels of human colorectal carcinoma cells line SW480 after exposure to a simulated laparoscopic carbon dioxide (CO2) pneumoperitoneum environment at different pressures and lengths of exposure time. METHODS: The effects of the simulated laparoscopic CO2 pneumoperitoneum environment at different CO2 pressures (6, 9, 12, and 15 mmHg) and exposure times (2 and 4 h) on TK-1 and Ki67 of SW480 cells were assessed by flow cytometry and reverse transcription (RT-PCR). Cells cultured in a standard environment were used as the control group (at 37 °C, 5% CO2). RESULTS: In this study, TK-1 and Ki67 in SW480 cells tended to decrease with the increase of CO2 pressure and exposure time. Significantly lower expression levels were observed at 0 and 24 h of culture after exposure to both at 12 and 15 mmHg, as compared with the control group at 6 and 9 mmHg (p < 0.05). The expression of TK-1 and Ki67 levels increased up to a plateau of the control group after 48 and 72 h (p > 0.05). With the CO2 pneumoperitoneum exposure time prolonging, the expression of TK-1 and Ki67 levels in 12 or 15 mmHg was lower than in 2 h (p < 0.05). In the same exposure time, the transcription level of TK-1 and Ki67 decreased significantly in 12 and 15 mmHg CO2 pneumoperitoneum groups (p < 0.05) and returned to the basal level of control group after being cultivated for 48 h (p > 0.05). In the same pressure, the difference of TK-1 mRNA between the groups of 2 and 4 h was also significant. CONCLUSION: The expression levels of TK-1 and Ki67 were suppressed temporarily after the continuous CO2 insufflation in higher pressure (at 12 and 15 mmHg). The higher the pressure of CO2 insufflation, the more the inhibiting effects of TK-1 and Ki67 will be. The longer the time of CO2 insufflation, the more significantly their expression decreased.

The marine fungal metabolite, dicitrinone B, induces A375 cell apoptosis through the ROS-related caspase pathway.

Dicitrinone B, a rare carbon-bridged citrinin dimer, was isolated from the marine-derived fungus, Penicillium citrinum. It was reported to have antitumor effects on tumor cells previously; however, the details of the mechanism remain unclear. In this study, we found that dicitrinone B inhibited the proliferation of multiple tumor types. Among them, the human malignant melanoma cell, A375, was confirmed to be the most sensitive. Morphologic evaluation, cell cycle arrest and apoptosis rate analysis results showed that dicitrinone B significantly induced A375 cell apoptosis. Subsequent observation of reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) reduction revealed that the apoptosis induced by dicitrinone B may be triggered by over-producing ROS. Further studies indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. Caspase-9, caspase-8 and caspase-3 were activated during the process, leading to PARP cleavage. The pan-caspase inhibitor, Z-VAD-FMK, could reverse dicitrinone B-induced apoptosis, suggesting that it is a caspase-dependent pathway. Our data for the first time showed that dicitrinone B inhibits the proliferation of tumor cells by inducing cell apoptosis. Moreover, compared with the first-line chemotherapy drug, 5-fluorouracil (5-Fu), dicitrinone B showed much more potent anticancer efficacy, suggesting that it might serve as a potential antitumor agent.

Effect of CO2 pneumoperitoneum on the expression of the chemokine receptors CXCR4 and CCR7 in colorectal carcinoma cells in vitro.

BACKGROUND: The ability of pneumoperitoneum in laparoscopic surgery to promote proliferation and metastasis of colorectal cancer has become a focus of research in the field of minimally invasive surgery. The aim of this research was to investigate the effect of CO2 pneumoperitoneum under different pressures and exposed times on the expression of chemokine receptors in colorectal carcinoma cells. METHODS: We constructed an in vitro pneumoperitoneum model. SW480 colon carcinoma cells were exposed to CO2 pneumoperitoneum under different pressures (6, 9, 12, and 15 mmHg) for 1, 2, and 4 hours. These cells were then cultivated under the same conditions as normal SW480 colon carcinoma cells without CO2 pneumoperitoneum (control group), treated at 37°C, and 5% CO2. The expression of the chemokine receptors CXC receptor 4 (CXCR4) and chemokine C receptor 7 (CCR7) was detected by immunocytochemistry and reverse transcriptase polymerase chain reaction after being cultivated for 0, 24, 48, and 72 hours. RESULTS: Immunocytochemistry showed that CXCR4 expression in SW480 cells was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups for the same exposure times compared with controls (P < 0.05). CCR7 expression in SW480 cells was significantly decreased in the 12 and 15 mmHg CO2 pneumoperitoneum-treated groups compared with controls (P < 0.05). CXCR4 and CCR7 expression increased up to the level of the control group after 24 and 48 hours (P > 0.05). If the CO2 pneumoperitoneum pressure increased, CXCR4 and CCR7 expression decreased at all exposure times. If the CO2 pneumoperitoneum exposure time prolonged, there were no significant differences in CXCR4 and CCR7 expression under the same pressure. Under all exposure times, CXCR4 and CCR7 mRNA expression was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups (P < 0.05) compared with controls, and it increased up to the level of controls after being cultivated for 48 hours (P > 0.05). If the CO2 pneumoperitoneum pressure increased (with all exposure times) and exposure time prolonged (under the same pressure), there were no significant differences in CXCR4 and CCR7 expression. CONCLUSIONS: CXCR4 and CCR7 expression is temporarily affected after continuous CO2 pneumoperitoneum treatment. The high pressure of CO2 pneumoperitoneum plays an important role in suppressing the expression of these chemokine receptors. Different lengths of time of exposure to a CO2 pneumoperitoneum-like environment do not change CXCR4 and CCR7 expression.

[Intracorporeal Roux-en-Y reconstruction after laparoscopic distal gastrectomy: a report of 20 cases].

OBJECTIVE: To investigate the safety and feasibility of intracorporeal Roux-en-Y reconstruction after laparoscopic distal gastrectomy. METHODS: Clinical data of 20 patients undergoing laparoscopic distal gastrectomy and intracorporeal Roux-en-Y reconstruction in our hospital from August 2012 to March 2013 were analyzed retrospectively. RESULTS: Totally laparoscopic distal gastrectomy was successfully performed in all the patients. The mean operation time was (190.8±53.6) min, the mean operative blood loss was(122.4±57.7) ml, and mean number of harvested lymph node was 31.2±5.7. Tumor-free proximal margin was confirmed by pathological examination in all the patients. The mean time to first flatus and hospital stay were (2.6±1.6) d and (8.1±2.0) d. One case developed pulmonary infection postoperatively, but no anastomosis related complication was observed. CONCLUSION: Intracorporeal Roux-en-Y reconstruction after laparoscopic distal gastrectomy is safe and feasible.

[Effect of PC cell-derived growth factor RNA interference on biological behavior of esophageal squamous carcinoma cells].

OBJECTIVE: To investigate the effect of PC cell-derived growth factor (PCDGF) RNA interference on esophageal squamous carcinoma cells Eca-109 in vitro. METHODS: The PCDGF-shRNA expression vector was transfected into the Eca-109 cells by liposome. After transfection, the mRNA and protein expressions of PCDGF were detected by RT-PCR and Western-blot respectively. Cell Counting Kit-8 (CCK-8) assay and Boyden chamber method were performed to measure the cell proliferation and invasion ability respectively. RESULTS: The expression levels of PCDGF mRNA and protein were both decreased in Eca-109 cells transfected with PCDGF-shRNA expression vector (transfection group). Twenty-four, 48 and 72 h after transfection, the cells proliferation in the transfection group was inhibited, and the inhibition rate was 20.4%, 21.1% and 20.9% respectively. The cell proliferation activity in the transfection group was significantly lower than that in the non-transfection group, liposome group and negative vector group (all P<0.05). The number of cell migration in the non-transfection group,negative vector group, liposome group and transfection group was 118.8±12.0, 100.8±9.0, 114.3±4.7, and 53.5±16.3 respectively. The differences were statistically significant between the transfection group and the other 3 groups (all P<0.05). CONCLUSIONS: PCDGF RNA interference can inhibit the proliferation and invasion abilities of esophageal squamous carcinoma cells in vitro. PCDGF gene may be the new target of gene therapy.

Effects of γ-synuclein on the tumorigenicity and metastasis of colon cancer SW1116 cells in vitro and in vivo.

Recent evidence suggests the involvement of γ-synuclein in tumorigenesis and tumor progression. The present study was designed to further clarify the effects of γ-synuclein on the biological features of colon cancer cells in vitro and in vivo. We constructed the eukaryotic expression vector and siRNA vector and selected stable transfectants to respectively upregulate and downregulate γ-synuclein expression in SW1116 cells. we found that silencing of γ-synuclein significantly attenuated SW1116 cell growth and colony formation in vitro (P<0.05), and overexpression of γ-synuclein moderately enhanced cell growth and colony formation, but not significantly when compared with the parental SW1116 cells and empty vector-transfected cells (P>0.05). Meanwhile, overexpression of γ-synuclein significantly facilitated SW1116 cell migration, invasion and adhesion to human liver sinusoidal endothelial cells (HLSECs) in vitro (P<0.05), and the effects were less attenuated by γ-synuclein knockdown (P>0.05). Furthermore, γ-synuclein promoted these malignant phenotypes in a γ-synuclein expression quantity-dependent manner not only in vitro but also in the in vivo expression. stable cells were injected subcutaneously into the right flank, and injected intrasplenically in nude mice. γ-synuclein knockdown suppressed the tumorigenicity of SW1116 cells in mice, which presented significantly smaller tumor masses on day 6 over a 30-day period, compared with empty vector cells (P<0.05). Meanwhile, overexpression of γ-synuclein led to a profound augmentation of liver metastasis in nude mice, not only in macroscopic appearance but also in the size and weight of livers (P<0.05). These results provide strong evidence that suggests γ-synuclein plays a positive role in the progression of colorectal cancer.

Omentoplasty in preventing anastomotic leakage of oesophagogastrostomy following radical oesophagectomy with three-field lymphadenectomy.

OBJECTIVES: Anastomotic leakage is a major cause of mortality in oesophageal surgery. Whether omentoplasty after oesophagogastrostomy could reduce anastomotic leakage is still controversial. The aim of this study is to investigate the function of omentoplasty to reinforce cervical oesophagogastrostomy after radical oesophagectomy with three-field lymphadenectomy. METHODS: A total of 184 patients who underwent radical oesophagectomy with three-field lymphadenectomy took part in this prospective study. Patients were randomized to receive either the omentoplasty or non-omentoplasty. In the omentoplasty group, the omentum was wrapped around the oesophagogastric anastomosis after oesophagogastrostomy. Age, gender, location of carcinoma, stage, body mass index, diabetes, coronary artery disease, peripheral vascular disease and performance of omentoplasty were recorded. The anastomotic leakage and stricture and recurrence site were followed up for three years after the operation. RESULTS: The two groups were comparable in terms of age, gender, location of carcinoma, stage, body mass index, diabetes, coronary artery disease and peripheral vascular disease (P > 0.05). In contrast to the non-omentoplasty group with a postoperative anastomotic leakage rate of 9.8%, the omentoplasty subjects demonstrated a significantly lower rate of 3.3% (P < 0.05). No lethal leakage was found in the omentoplasty group, while two non-omentoplasty patients developed incurable empyema and mediastinitis due to leakage and ultimately died. The rate of incidence of anastomotic stricture in the omentoplasty and non-omentoplasty groups were 4.3% and 2.2% respectively. Of the five cases of death during the hospital stay, two were found in the omentoplasty group and three in non-omentoplasty. There was no significant difference of lethal leakage, stricture and death rate between the two groups (P > 0.05). The hospital stay was significantly longer for non-omentoplasty patients, compared with that for the omentoplasty subjects (P < 0.05). Tumour recurrence in lymphatic- or haematogenous metastasis was similar in both groups (P > 0.05). CONCLUSION: Omentoplasty may prevent anastomotic leakage of oesophagogastrostomy following radical oesophagectomy with three-field lymphadenectomy.

[Advantage of perisplenic hilar lymph node dissection by laparoscopy-assisted total gastrectomy (D2) over conventional open total gastrectomy for advanced gastric cancer].

OBJECTIVE: To compare the number of harvested perisplenic hilar lymph nodes by laparoscopy-assisted total gastrectomy (LATG) and conventional open total gastrectomy (OTG) for advanced upper and middle gastric cancer. METHODS: Three hundred twelve patients with advanced gastric cancer treated in a single institution between Sept 2008 and Jan 2011 were included in this study. They were divided into two groups: the LATG group and OTG (D2) group. All the surgical operations were performed by one surgeon or under his supervision. The lymph node clearance outcomes of the patients treated by those two surgical procedures were analyzed. RESULTS: The harvested lymph node numbers of the two groups were (29.57 ± 9.62) and (29.38 ± 11.22) respectively, statistically with no significant difference (P = 0.875). The numbers of lymph node dissected around the splenic area in the LATG group and OTG group (Section 10, 11 group) were (2.01 ± 1.34) and (1.33 ± 1.11), respectively, indicating a significant difference (P = 0.000). The numbers of lymph nodes dissected around the celiac region (Section 7, 8, 9, 11p and 12a(2) group) were (7.90 ± 3.41) and (7.22 ± 2.65), respectively, with a non-significant difference (P = 0.050). There were also no significant differences while comparing with the numbers of lymph nodes dissected in the cardiac area (group 1, 2), pyloric region (5, 6 group) and the greater and lesser omentum area (group 3 and 4) between the two groups (P = 0.605, P = 0.248, P = 0.262). CONCLUSION: Short-term results of this study indicate that laparoscopy-assisted total gastrectomy (D2) is better than conventional open surgery in perisplenic hilar lymph node dissection.

[Application of serum proteomic mass spectrum analysis in breast cancer].

OBJECTIVE: To analyze the characteristics of serum proteins mass spectra in healthy controls, benign breast tumors, and CA15-3 negative or CA15-3 positive breast cancer patients by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). METHODS: Tissue samples of 113 cases of breast cancer (93 case of CA15-3 negative, 20 case of CA15-3 positive), 103 cases of benign breast tumor and 92 cases of healthy controls were examined and analyzed by SELDI and protein chip (CM10) techniques. Biomarker Pattern Software (BPS) was used to detect the protein peaks significantly different between them and establish a diagnostic pattern which was further evaluated by a blind test. RESULTS: Twelve significantly different protein peaks were found in serum samples between breast cancer patients and healthy controls. Eleven significantly different peaks were found between benign breast tumor patients and healthy controls. By combined analysis of those three different protein mass spectra, the peak 15 952 was found to be significantly different between breast cancer group and healthy controls, and the peak 7985 was significantly different among breast cancer group, benign breast tumor group and health controls. The blind test with the differential proteins for the serum samples of 93 cases of CA15-3 negative breast cancer and 36 cases of benign breast tumors showed that the sensitivity was 80.6% and specificity was 91.7%. The blind test in 20 cases of CA15-3 positive breast cancer and 36 cases of benign breast tumors showed that the sensitivity was 75.0% and specificity was 91.7%. Four significantly different protein peaks were found between the benign breast tumor patients and CA15-3 negative breast cancer patients. No significantly different protein were found between CA15-3 negative and CA15-3 positive patients. CONCLUSION: Significantly different protein peaks can be screened out in breast cancer, benign breast tumor patients and healthy controls by SELDI-TOF-MS analysis.

[A prospective randomized control trial of the approach for laparoscopic right hemi-colectomy:medial-to-lateral versus lateral-to-medial].

OBJECTIVE: To compare the medial-to-lateral approach with the lateral-to-medial approach in laparoscopic right hemi-colectomy for right colon cancer. METHODS: A prospective randomized controlled trial was performed in the Fujian provincial tumor hospital between January 2007 and July 2009. Forty-eight cases with right colon cancer were randomly divided into two groups:medial-to-lateral laparoscopic right hemi-colectomy group(group M) and lateral-to-medial laparoscopic right hemi-colectomy group(group L). Primary outcome(operative time) and secondary outcomes (estimated blood loss, intra-operative complication, post-operative complication, number of lymph node retrieval, hospital stay) were compared between two groups. RESULTS: Operative time was(122.5+/-25.8) min in group M and (162.9+/-30.9) min in Group L (P=0.01). Estimated blood loss was(55.8+/-36.2) ml in group M and (104.6+/-58.2) ml in group L(P=0.01). There were no significant differences between the two groups in intra-operative complications(4.2% vs 8.3%, P=1.00), post-operative complications (8.3% vs 16.7%, P=0.66), number of lymph node retrieval (17.4+/-3.2 vs 17.8+/-3.4, P=0.67), and hospital stay[(7.8+/-2.2) d vs (8.0+/-3.6) d, P=0.81]. CONCLUSION: The medial-to-lateral approach reduces operative time and blood loss in laparoscopic right hemi-colectomy as compared with the lateral-to-medial approach.

[Effects of 5-Aza-2'-deoxycitydine and trichostatin A on expression and apoptosis of ALDH1a2 gene in human bladder cancer cell lines].

OBJECTIVE: To study the effect on promoter de-methylation, expression of ALDH1a2 gene and cell apoptosis by treated with 5-Aza-dC and TSA in five human bladder cancer cell lines. METHODS: Human bladder cancer cell lines RT-4, 253J, 5637, BIU-87 and T24 were cultured and treated with 5-Aza-dC and(or) TSA. The expression of the ALDH1a2 gene was detected by RT-PCR and Western blot. The methylation status of gene promoter was determined by MSP, and the cell cycle profile was established by flow cytometry. RESULTS: ALDH1a2 was silenced in five human bladder cancer cell lines. Re-expression of ALDH1a2 was detected after treated with 5-Aza-dC alone or TSA in combination. ALDH1a2 transcript was marked in each cell lines combined with 5-Aza-dC and TSA treatment which showed a synergistic effect on expression of ALDH1a2 transcript. Early apoptotic was the main mode of apoptosis and death of human bladder cancer cell lines induced by 5-Aza-dC and TSA. The percentage of early apoptotic cells was 1.4% in control group and 2.8% in TSA group, however, 20.2% in 5-Aza-dC group and 33.8% in 5-Aza-dC + TSA group, respectively. The groups of TSA, 5-Aza-dC and 5-Aza-dC + TSA were significantly different from control group (P < 0.05). CONCLUSIONS: Aberrant methylation of ALDH1a2 gene is the main cause for gene transcriptional inactivation. Re-expression of ALDH1a2 gene and cell apoptosis are detected after either treatment with 5-Aza-dC alone or in combination with TSA.

[Analysis of lymph node metastases in early esophageal carcinoma and treatment regimens].

OBJECTIVE: To investigate the incidence of lymph node metastasis (LNM) in early esophageal carcinoma and the techniques of dissection. METHODS: Standard three-field dissection was performed in patients with small superficial esophageal carcinoma detected by endoscopy from 1993 - 2007. The lymph node metastases in different regions were identified by histopathology. The survival rate of the cases was analyzed. RESULTS: A total of 149 patients with early esophageal carcinoma were identified by postoperative pathological examination. The overall lymph node metastasis (LNM) rate was 22.8%, and the degree of LNM was 2.4% in all fields. Most lymph node metastases from upper thoracic esophageal carcinoma were found in cervical and the right upper mediastinal nodes. The LNM from middle thoracic esophageal carcinoma were approximately equal in the cervical, mediastinal, and abdominal lymph nodes, and abdominal lymph node metastasis predominated in lower thoracic esophageal carcinoma. The metastatic rate of LNM adjacent to the right recurrent laryngeaal nerve was the highest (44.1%). Significant differences were shown among the rates of LNM in relation to different macroscopic pattern, depth of invasion and differentiation of tumor (P < 0.01), but not to the longitudinal length of tumor (P > 0.05). The overall 5-year survival rate was 77.9%. It was 87.0% in patients without LNM, and 47.1% in those with LNM. CONCLUSION: Lymph node metastasis in early esophageal carcinoma is in a high frequency. Patients with tumor invasion into the mucosa or lamina propria but without lymph node metastasis may undergo a local operation such as endoscopic mucosectomy and have a good prognosis. Patients with tumor invasion into the muscularis mucosae or submucosa should be treated with radical surgery with three-field lymphadenectomy, especially, to dissect the lymph nodes adjacent to the recurrent laryngeal nerve.