人膀胱癌细胞原代培养及体外药物敏感性实验研究

目的研究不同个体表浅性膀胱癌组织对抗癌药物及其联合应用的敏感性。方法采用肿瘤细胞原代培养技术和MTT比色法测定了59例膀胱癌组织对灌注抗癌药物及其联合应用的抑制率和敏感性。结果14例失败,45例获得成功,总体可评价率为76.3%,不同个体对不同抗癌药物的抑制率存在明显差异;除MTX对膀胱癌G1和G3级平均抑制率差异有统计学意义外,其它抗癌药物对不同个体膀胱癌细胞的平均抑制率与膀胱癌的病理分级、分期均无关;抗癌药物对初发膀胱癌的敏感率和平均抑制率均高于复发膀胱癌;2种不同作用机制的抗癌药物联合应用对不同个体膀胱癌细胞的敏感率和平均抑制率均高于单一药物,二者敏感率和平均抑制率差异有统计学意义。结论应根据药敏结果,选择相对敏感的抗癌药物,并将作用于不同机制、细胞不同周期的抗癌药物联合,制订个体化膀胱灌注治疗方案,以提高疗效,最大限度减少副反应,减少肿瘤复发率。

蛋白磷酸酶Ⅰ(PPP1R3)在直肠癌细胞体外侵袭转移的生物学活性研究

目的探讨研究蛋白磷酸酶I(protein phosphatase1 regulatory subunit 3,PPP1R3)在直肠癌SW480细胞中的黏附、运动、迁移和侵袭能力的生物学活性。方法用PPP1R3、EGFR抗体、VEGFR抗体干预SW480细胞后,分别对其行细胞运动实验、黏附实验、迁移及侵袭实验,分析PPP1R3对SW480细胞运动、黏附、迁移和侵袭能力的影响及其作用机制。结果随着PPP1R3浓度的增加,直肠癌SW480细胞的黏附、运动、迁移和侵袭能力明显减退(P<0.05)。而且穿过人工基底膜的能力明显减低(P<0.05)。结论 PPP1R3能够抑制直肠癌SW480体外的侵袭、转移作用,可为PPP1R3基因表达缺失的直肠癌术后患者提供放化疗、生物靶向治疗依据。

磁场对肿瘤细胞抑制作用的试验与分析

目的:用磁场进行小鼠荷瘤和人体的离体癌细胞抑制试验。方法:用装备有NdFeB永磁材料的仪器产生非均匀磁场(磁通密度为0.20T,梯度为0.07T/cm)对荷瘤小鼠和人体的离体癌细胞每天照射0.5小时,12天后解剖检查试验结果。结果:磁场对荷瘤小鼠的癌细胞抑止率达61%(P<0.002),胸腺平均比化疗组重8mg(P<0.01),癌组织的纤维包膜是对照组的2～3倍。谷丙转氨酶(SGPT)低于化疗组的平均值(P<0.001),肝、心肌细胞无损伤;白细胞(WBC)高于化疗组的平均值(P<0.02),免疫功能提高;血红蛋白(Hb)低于化疗组的平均值(P<0.05),局部缺氧,癌细胞呈片状坏死,部分癌组织呈孤岛状。磁疗组无明显毒副作用反应。重复性实验结果相近。小鼠和人体离体癌细胞实验显著性检验无显著性意义。结论:磁场在一定的梯度范围是可以有效的抑制小鼠肿瘤的生长。

Transfection of the Human Sodium/Iodide Symporter(NIS) Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.

Microsatellite Alterations on Chromosome 8 of Hepatocellular Carcinoma

Objective: To study the features of microsatellite alterations on chromosome 8 and their asso- ciation with clinicopathological characteristics of hepatocellular carcinoma (HCC). Methods: Ten highly- polymorphic microsatellite markers on chromosome 8 were selected to be detected for loss of heterozygosity (LOH), microsatellite instability (MSI) and allelic imbalance (AI) in 56 HCC using MegaBACE 500 auto- matic DNA analysis system. Results: LOH was found in 37 of 56 HCC (66.1%) on at least 1 locus, and the top three loci were D8S261(53.5%), D8S1721(52.5%) and D8S1771(52.5%). LOH frequency on D8S277 was signi?cantly higher in cases positive for serum HBsAg than in those negative for HBsAg (P <0.01). Similarly, LOH on D8S261, D8S298 and D8S1733 occurred more frequently in patients with negative HB- sAg than those with positive HBsAg (P <0.01). LOH on D8S298 and D8S1771 was more frequent in those tumors larger than 3 cm in size (P <0.05 or P <0.01). LOH frequencies of D8S1721 were signi?cantly higher in the patients with absent or not intact tumor capsule than in those with intact tumor capsule (P <0.05). LOH on D8S298 and D8S1771 was more frequently detected in tumors with intrahepatic metastasis than in those without intrahepatic metastasis (P <0.01). MSI was found in 12.5% (7/56) cases, and AI was found in 19.6% (11/56), no correlation was found between MSI and AI and clinicopathological character- istics of HCC. Conclusion: Frequent microsatellite alterations on chromosome 8 existed in HCC. LOH, which represents tumor suppressor gene pathway, plays a more important role in hepatocarcinogenesis; MSI representing mismatch repair gene pathway ranks next. LOH at a particula locus is associated with the clinicopathological parameters of human HCC.

Characterization of Two Human Lung Adenocarcinoma Cell Lines by Reciprocal Chromosome Painting

Lung cancer is a leading cause of cancer death worldwide. Some lung cancer patients correlate with a gas of radon besides smoking. To search for common chromosomal aberrations in lung cancer cell lines established from patients induced by different factors, a combined approach of chromosome sorting, forward and reverse chromosome painting was used to characterize karyotypes of two lung adenocarcinoma cell lines： A549 and GLC-82 with the latter line derived from a patient who has suffered long-term exposure to environmental radon gas pollution. The chromosome painting results revealed that complex chromosomal rearrangements occurred in these two lung adenocarcinoma cell lines. Thirteen and twenty-four abnormal chromosomes were identified An A549 and GLC-82 cell lines, respectively. Almost half of abnormal chromosomes in these two cell lines were formed by non-reciprocal translocations, the others were derived from deletions and duplication/or amplification in some chromosomal regions. Furthermore, two apparently common breakpoints, HSA8q24 and 12q14 were found in these two lung cancer cell lines.

Stable knockdown of heparanase expression in gastric cancer cells in vitro

AIM:To develop short hairpin RNA(shRNA)against heparanase,and to determine its effects on heparanase expression and the malignant characteristics of gastric cancer cells. METHODS:Heparanase-specific shRNA was constructed and transferred into cultured the gastric cancer cell line SGC-7901.Stable subclonal cells were screened by G418 selection.Heparanase expression was measured by reverse transcriptase-polymerase chain reaction(RT-PCR),real-time quantitative PCR and Western blotting.Cell proliferation was detected by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)colorimetry and colony formation assay. The in vitro invasiveness and metastasis of cancer cells were measured by cell adhesion assay,wound healingassay and matrigel invasion assay.The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells. RESULTS:Stable transfection of heparanase-specific shRNA,but not of scrambled shRNA and mock vector,resulted in reduced mRNA and protein levels of heparanase.The shRNA-mediated knockdown of heparanase did not affect the cellular proliferation of SGC-7901 cells.However,the in vitro invasiveness and metastasis of cancer cells were decreased after knockdown of heparanase.Moreover,transfection of heparanase-specific shRNA decreased the in vitro angiogenesis capabilities of SGC-7901 cells. CONCLUSION:Stable knockdown of heparanase can efficiently decrease the invasiveness,metastasis and angiogenesis of human gastric cancer cells.In contrast,stable knockdown of heparanase does not affect the cell proliferation.

Nuclear β-catenin expression as a prognostic factor in advanced colorectal carcinoma

AIM： To investigate the changing pattern of β-catenin expression and its prognostic value in advanced colorectal cancer （CRC）. METHODS： Archival tumor samples were analyzed for β-catenin using immunohistochemistry （IHC） in 95 patients with advanced CRC. RESULTS： Membranous β-catenin expression was found in the normal colorectal epithelium. Almost 100% of CRC cases showed membranous and cytoplasmic expression, and 55 （58%） cases showed nuclear expression. In univariate （Kaplan-Meier） survival analysis, only the nuclear index （NI） was a significant predictor of disease free survival （DFS） （P = 0.023; n = 35）, with a NI above the median associated with longer DFS （34.2 too） than those with a NI below the median （15.5 too） （P = 0.045, ANOVA）. The other indices were not significant predictors of DFS, and none of the three tested indices （for membranous, cytoplasmic, or nuclear expression） predicted diseasespecific survival （DSS）. However, when dichotomized as positive or negative nuclear expression, the former was a significant predictor of more favorable DFS （P = 0.041） and DSS （P = 0.046）. CONCLUSION： Nuclear β-catenin expression provides additional information in predicting patient outcome in advanced CRC.

Treatment of hepatoma with liposome-encapsulated adriamycin administered into hepatic artery of rats

AIM： To observe the therapeutic effects of liposomeencapsulated adriamycin （LADM） on hepatoma in comparison with adriamycin solution （FADM） and adriarnycin plus blank liposome （ADM ＋ BL） administered into the hepatic artery of rats. METHODS： LADM was prepared by pH gradient-driven method. Normal saline, FADM （2 mg/kg）, ADM＋BL （2 mg/kg）, and LADM （2 mg/kg） were injected via the hepatic artery in rats bearing liver W256 carcinosarcoma, which were divided into four groups randomly. The therapeutic effects were evaluated in terms of survival time, tumor enlargement ratio, and tumor necrosis degree. The difference was determined with ANOVA and Dunnett test and log rank test. RESULTS： Compared to FADM or ADM ＋ BL, LADM produced a more significant tumor inhibition （tumor volume ratio： 1.243±0.523 vs 1.883±0.708, 1.847±0.661, P 〈 0.01）, and more extensive tumor necrosis. The increased life span was prolonged significantly in rats receiving LADM compared with FADM or ADM＋BL （231.48 vs 74.66, 94.70） （P 〈 0.05）. CONCLUSION： The anticancer efficacies of adriamycin on hepatoma can be strongly improved by liposomal encapsulation through hepatic arterial administration.

RNAi knockdown of PIK3CA preferentially inhibits invasion of mutant PIK3CA cells

AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms. METHODS: The mutation of PIK3CA in exons 9 and 20 of gastric cancer cell lines HGC-27, SGC-7901, BGC-823, MGC-803 and MKN-45 was screened by polymerase chain reaction (PCR) followed by sequencing. BGC-823 cells harboring no mutations in either of the exons, and HGC-27 cells containing PIK3CA mutations were employed in the current study. siRNA targeting PIK3CA was chemically synthesized and was transfect- ed into these two cell lines in vitro . mRNA and protein expression of PIK3CA were detected by real-time PCR and Western blotting, respectively. We also measured phosphorylation of a serine/threonine protein kinase (Akt) using Western blotting. The proliferation, migration and invasion of these cells were examined separately by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT), wound healing and Transwell chambers assay. RESULTS: The siRNA directed against PIK3CA effectively led to inhibition of both endogenous mRNA and protein expression of PIK3CA, and thus significantly down-regulated phosphorylation of Akt (P < 0.05). Furthermore, simultaneous silencing of PIK3CA resulted in an obvious reduction in tumor cell proliferation activity, migration and invasion potential (P < 0.01). Intriguing, mutant HGC-27 cells exhibited stronger invasion ability than that shown by wild-type BGC-823 cells. Knockdown of PIK3CA in mutant HGC-27 cells contributed to a reduction in cell invasion to a greater extent than in non-mutant BGC-823 cells. CONCLUSION: siRNA mediated targeting of PIK3CA may specifically knockdown the expression of PIK3CA in gastric cancer cells, providing a potential implication for therapy of gastric cancer.

The promoting molecular mechanism of alphafetoprotein on the growth of human hepatoma Bel7402 cell line

AIM: The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402. METHODS: Cell proliferation enhanced by AFP was detected by MTT assay, 3H-thymidine incorporation and S-stage percentage of cell cycle analysis. With radioactive labeled 125I-AFP for receptor binding assay; cAMP accumulation, protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium (Ca2+i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N- ras, p 53, and p21( ras ) in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively. RESULTS: It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay, (3)H-thymidine incorporation and S-phase percentage up to 2-fold. Two subtypes of AFP receptors were identified in the cells with Kds of 1.3 x 10(-9)mol.L(-1) and 9.9 x10(-8)mol. (-1)L respectively. Pretreatment of cells with AFP resulted in a significant increase (625%) in cAMP accumulation. The activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61.2% at treatment time point 2, 6, 12 and 24 hours. The level of intracellular calcium were elevated after the treatment of alpha-fetoprotein and achieved to 204% at 4 min. The results also showed that AFP(20mg.L(-1)) could upregulate the expression of N- ras oncogenes and p 53 and p21( ras ) in Bel 7402 cells. In the later case,the alteration were 81.1%(12h) and 97.3%(12h) respectively compared with control. CONCLUSION: These results demonstrate that AFP is a potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells. Its growth-regulatory effects are mediated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.

Cloning and characterization of a novel gene (C17orf25) from the deletion region on chromosome 17p13.3 in hepatocelular carcinoma

Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open rea(ling frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17or25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma.

Impact of obesity on the surgical outcome following repeat hepatic resection in Japanese patients with recurrent hepatocellular carcinoma

AIM: To evaluate the impact of obesity on the posto- perative outcome after hepatic resection in patients with hepatocellular carcinoma (HCC). METHODS: Data from 328 consecutive patients with primary HCC and 60 patients with recurrent HCC were studied. We compared the surgical outcomes between the non-obese group (body mass index: BMI < 25 kg/m2) and the obese group (BMI ≥ 25 kg/m2). RESULTS: Following curative hepatectomy in patients with primary HCC, the incidence of postoperative complications and the long-term prognosis in the non- obese group (n = 240) were comparable to those in the obese group (n = 88). Among patients with recurrent HCC, the incidence of postoperative complications after repeat hepatectomy was not significantly different between the non-obese group (n = 44) and the obese group (n = 16). However, patients in the obese group showed a significantly poorer long-term prognosis than those in the non-obese group (P < 0.05, five-yearsurvival rate; 51.9% and 92.0%, respectively). CONCLUSION: Obesity alone may not have an adverse effect on the surgical outcomes of patients with primary HCC. However, greater caution seems to be required when planning a repeat hepatectomy for obese patients with recurrent HCC.

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

AIM： To construct an expression plasmid encoding human wild-type midkine （MK） and enhanced green fluorescence protein （EGFP） fusion protein （MK-EGFP）, and to analyze the subcellular localization of MK in different cardnoma cell lines. METHODS： Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS： Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION： This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

DIFFERENCE IN BIOLOGICAL CHARACTERISTICS AND SENSITIVITY TO CHEMOTHERAPY AND RADIOTHERAPY BETWEEN INTRAHEPATIC AND EXTRAHEPATIC CHOLANGIOCARCINOMA CELLS IN VITRO

Objective To investigate and compare the biological characteristics and sensitivity to chemotherapy and radiotherapy of intrahepatic and extrahepatic cholangiocarcinoma cells in vitro.Methods The intrahepatic and extrahepatic cholangiocarcinoma cell lines were established,and cells with steady passage were chosen to study the biological characteristics including morphology,growth dynamics,chromosome,and levels of cancer antigen(CA)125,CA19-9,alpha-fetoprotein(AFP),and carcino-embryonic antigen(CEA).Meanwhile,MTT assay was used to determine the sensitivity of both kinds of cells to 6 chemotherapeutic drugs,including cisplatin,paclitaxel,harringtonine,5-fluorouracil,vincristine,and aclacimomycin,and the inhibitory rate of cells under the irradiation of 10 Gy ray was also measured.Results The intrahepatic cholangiocarcinoma cells were mostly fusiform in shape,and extrahepatic cholangiocarcinoma cells were mostly round or polygon in shape.Their doubling time was 26.3 hours and 23.1 hours,respectively.Their average number of chromosomes was 59(range,38-84)and 67(range,49-103),respectively.The chromosome karyotypes of most intrahepatic cholangiocarcinoma cells were hyperdiploid and hypotriploid,while hypertriploid was predominant in extrahepatic cholangiocarcinoma cells.The level of CA 125 in supernatant of extrahepatic cholangiocarcinoma cells increased obviously,while levels of other determined tumor markers in both kinds of cells were all within normal range.The intrahepatic cholangiocarcinoma cells were low sensitive to cisplatin and paclitaxel,but not sensitive to the other 4 chemotherapeutic drugs.The extrahepatic cholangiocarcinoma cells were high sensitive to cisplatin,but not sensitive to the other 5 drugs.Both kinds of cells had poor sensitivity to radiotherapy.Conclusions Intrahepatic and extrahepatic cholangiocarcinoma cells show differences in shape,doubling time,chromosome karyotype,tumor marker level,and chemosensitivity,whereas they both have poor radiosensitivity.Though they are similar in histopathology,they have different growth characteristics and have discrepancy in treatment and prognosis.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Effect of Coriolus Versicolor Polysaccharide-B on the Biological Characteristics of Human Esophageal Carcinoma Cell Line Eca109

Objective To investigate the effect of Coriolus versicolor polysaccharide-B （CVPs-B） on the biological characteristics of human esophageal carcinoma cell line Ecal09 in vitro. Methods The cells of experimental group （EG） were cultured in DMEM with 10% FCS and 150μg/mL CVPs-B, the cells of control group （CG） were cultured in DMEM with 10% FCS without CVPs-B. MTT reduction assay was performed to detect the effect of CVPs-B on the proliferation of Ecal09 cells after the compound was administrated in varying concentrations. The living conditions of the Ecal09 cells were determined using trypan blue exclusion. Then, cell growth curves were drawn. Flow cytometry was performed to detect the effect of CVPs-B on the apoptosis and cell cycle of Ecal09. Results In comparison with the CG, a marked decrease in the proliferation of Eca09 cells was observed in the EG, after incubation with CVPs-B. The survival rate of Eca09 cells decreased as the time of CVPs-B incubation prolonged. Comparing the cell cycles and apoptotic rates between the two groups, the proportions of cells in the G0/G1, S, and G2/M phases in the EG were found to be （68.4±3.7）%, （13.9±2.1）%, and （17.7±1.4）%, respectively, after 24 h incubation with CVPs-B. The cells had an apoptotic rate of （9.7±0.7）%. On the other hand, the proportions of the G0/G1, S, and G2/M cells of the CG were found to be （53.9±3.6）%, （26.6±2.8）%, and （19.5±2.3）%, respectively, with an apoptotic rate of （5.7±1.4）%. In comparison with the CG cells, significant cell growth in the G0/G1 phase was observed in the EG （P〈0.05）. Furthermore, a significant decrease in the number of cells in the S phase was observed （P〈0.05） in the EG. Conclusions CVPs-B can inhibit proliferation and enhance apoptosis of Ecal09 cells and may be useful in the treatment of esophageal carcinoma.

In Vitro Lethal Effect of Photodynamic Therapy on Human Pancreatic Cancer Cells and Its Major Influencing Factors

OBJECTIVE To investigate the in vitro lethal effect of photo- dynamic therapy （PDT） using the photosensitizer hematoporphyrin on the human pancreatic cancer cell line Panc-1, the major influencing factors and the mechanisms of treatment. METHODS Three factors--the time needed for photosensitizer and cell incubation, the photosensitizer concentration （PhoC） and the exposure dose （ExpD）--were examined with different levels of these factors. Optical density （OD） was used as a measure of CCK-8 in the experiment, and was converted to the rate of cell survival. The separate effect of each factor on the photodynamic action was studied, and the interactions were investigated. The effects of different incubation times and PhoC levels on the fluorescence intensity （FI） of the intracellular photosensitizer were determined, and the mechanisms of these factors leading to the therapeutic effects of PDT discussed. RESULTS An increase in the photosensitizer and cell incubation time, an increase of PhoC, and enhancement of the ExpD, produced a corresponding decrease in the rate of Panc-1 cell survival after PDT （P 〈 0.05）. PDT achieved its maximum lethal effects 16 h after starting the incubation, with a PhoC of 10 mg/L and an ExpD of 20 J/cm2; at these levels a synergistic interaction between PhoC and the ExpD occurred, decreasing the cell survival rate （P 〈 0.05）. Neither simple administration of photosensitizer without ExpD （0 J/cm2） or illumination in the absence of PhoC （0 mg/L） affected the rate of cell survival （P 〉 0.05）. With an increase of PhoC and lengthening of the incubation time, the FI of the intracellular photosensitizer accordingly increased （P 〈 0.05）, and attained its maximum value at a PhoC of 10 mg/L and 36 h after the incubation. With an increase of PhoC, the FI of the photosensitizer, hematoporphyrin, in the solution increased progressively at first and then decreased （fluorescence quenching）. CONCLUSION PDT with the photosensitizer hematoporphyrin has clear lethal effects on the human pancreatic cancer cell line Panc-1, but the presence of a photosensitizer and laser irradiation by themselves do not have independent lethal effects. The three influencing factors--the time for photosensitizer and cell incuba- tion, PhoC and ExpD--correlate positively with the PDT response, within certain limits. Beyond these limits, the PDT response does not significantly increase. The main mechanism of the PDT response lies in the effect of these factors on the level of the intracellular photosensitizer and the fluorescence quenching of the photosensitizer. A synergistic effect exists between PhoC and ExpD.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.