丹参酮对人肺癌细胞株的增殖抑制作用及其分子机理

目的 观察丹参酮ⅡA对人肺腺癌细胞 (SPC A 1)的增殖抑制作用及其分子机理。方法 体外培养的SPC A 1细胞经无毒剂量 ( 0 .5 μg/ml)的丹参酮ⅡA处理 5天后 ,观察细胞形态学、增殖动力学、细胞周期分布及肿瘤相关基因表达改变 ,并以全反式维甲酸及顺铂作为对照。结果 SPC A 1细胞经丹参酮ⅡA处理后 ,细胞生长明显减慢 ,集落形成率显著降低。FCM细胞周期分析 :S期细胞显著减少 ,G0 /G1 期细胞则明显增加。丹参酮ⅡA可使细胞p5 3、p2 1表达显著升高而CDKN2 明显降低。结论 丹参酮ⅡA对SPC A 1细胞增殖具有显著抑制作用 ,其分子机理可能是丹参酮ⅡA能显著上调p5 3、p2 1表达和下调CDKN2 的表达而抑制DNA的合成。

探讨亚甲蓝的光动力作用

目的:探讨亚甲蓝的光动力作用及其在CPD5的PDT中是否表现出抗氧化特性。方法:采用细胞排染试验(1)对比观察了亚甲蓝和CPD5对胰癌细胞的光动力效果。(2)观察了亚甲蓝对CPD5光动力作用的影响。结果:(1)亚甲蓝的PDT效果不如CPD5。(2)CPD5浓度在(0.625～5)μg/ml范围内时,由于有亚甲蓝存在,CPD5的治疗作用下降;CPD5浓度在(0.078125～0.3125)μg/ml范围内时,亚甲蓝表现出光敏化作用。结论:(1)亚甲蓝的光动力效果不理想。(2)体外光动力过程中,未发现亚甲蓝的抗氧化性质。

Aberrant Methylation in CpG Islands of pl5 and pl6 Tumor Suppressor Genes in Pancreatic Cancer Tissue

Objective： To detect the aberrant methylation patterns in the CpG islands of p16 and p15 tumor suppressor genes, and to analyze its correlation with pancreatic carcinogenesis and with clinicopathological characteristics of patients with pancreatic cancer （PC）. Methods： The methylation-specific polymerase chain reaction （MSP） method was used to monitor methylation patterns in the CpG islands of p15 and p16 genes from 29 cases of PC and 3 cases of chronic pancreatitis （CP） paraffin-embedded tissue, as well as 2 cases of normal liver tissues and 12 cases of normal blood samples. Results： p15 and p16 genes were detected to show unmethylation patterns and no amplification using methylation-specific primers in control group. The aberrant methylation rates of p16 in carcinoma tissue and adjacent noncarcinoma tissue were 37.9% （11 of 29 cases） and 34.5% （10 of 29 cases） respectively. Of the 11 aberrant methylated samples, 5 showed complete methylation and 6 hemimethylation. The methylation rates of p15 gene in carcinoma tissue and adjacent noncarcinoma tissue were 27.5% （8/29） and 24.4% （7/29） respectively. Of the 8 aberrant methylated samples, 3 showed complete methylation and 5 hemimethylation. In 6 PC samples, aberrant methylation in CpG islands of both p15 and p16 genes existed simultaneously. The aberrant methylation patterns in CpG islands of p15 and p16 genes had no close correlation with the clinicopathological characteristics （age, sex, smoking, volume of primary tumor, differentiation, clinical stage and histological classification） of the patients with PC （P〉0.05）. Conclusion： The aberrant methylation in CpG islands of p15 and p16 genes could be regarded as an early molecular event in PC and had no close correlation with the clinicopathological characteristics of the patients with PC.

Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity

AIM： To investigate the inhibitory effect of small interfering RNA （siRNA） on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity. METHODS： A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with LipofectamineTM 2000. The down regulation of survivin expression was detected by semi-quantitive RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry. RESULTS： The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semlquantitive RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation. CONCLUSION： The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce apoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene taerapy of pancreatic cancer.

Endoscopic ultrasound-guided fine-needle aspiration cytology diagnosis of solid pseudopapillary tumor of the pancreas: A case report and literature review

We describe the clinical, imaging and cytopathological features of solid pseudopapillary tumor of the pancreas (SPTP) diagnosed by endoscopic ultrasound- guided (EUS-guided) fine-needle aspiration (FNA). A 17-year-old woman was admitted to our hospital with complaints of an unexplained episodic abdominal pain for 2 mo and a short history of hypertension in the endocrinology clinic. Clinical laboratory examinations revealed polycystic ovary syndrome, splenomegaly and low serum amylase and carcinoembryonic antigen (CEA) levels. Computed tomography (CT) analysis revealed a mass of the pancreatic tail with solid and cystic consistency. EUS confirmed the mass, both in body and tail of the pancreas, with distinct borders, which caused dilation of the peripheral part of the pancreatic duct (major diameter 3.7 mm). The patient underwent EUS-FNA. EUS-FNA cytology specimens consisted of single cells and aggregates of uniform malignant cells, forming microadenoid structures, branching, papillary clusters with delicate fibrovascular cores and nuclear overlapping. Naked capillaries were also seen. The nuclei of malignant cells were round or oval, eccentric with fine granular chromatin, small nucleoli and nuclear grooves in some of them. The malignant cells were periodic acid Schiff (PAS)-Alcian blue (+) and immunocytochemically they were vimentin (+), CA 19.9 (+), synaptophysin (+), chromogranin (-), neuro-specific enolase (-), a1- antitrypsin and a1-antichymotrypsin focal positive. Cytologic findings were strongly suggestive of SPTP. Biopsy confirmed the above cytologic diagnosis. EUS- guided FNA diagnosis of SPTP is accurate. EUS findings,cytomorphologic features and immunostains of cell block help distinguish SPTP from pancreatic endocrine tumors, acinar cell carcinoma and papillary mucinous carcinoma.

Persistence of side population cells with high drug efflux capacity in pancreatic cancer

AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell line PANC-1 and its proportion change when cultured with Gemcitabine, was detected by Hoechst 33342 staining and FACS analysis. The expression of ABCB1 and ABCG2 was detected by real- time PCR in either SP cells or non-SP cells. RESULTS: SP cells do exist in PANC-1, with a median of 3.3% and a range of 2.1-8.7%. After cultured with Gemcitabine for 3 d, the proportion of SP cells increased significantly (3.8% ± 1.9%, 10.7% ± 3.7%, t = 4.616, P = 0.001 < 0.05). ABCB1 and ABCG2 expressed at higher concentrations in SP as compared with non-SP cells (ABCB1: 1.15 ± 0.72, 5.82 ± 1.16, t = 10.839, P = 0.000 < 0.05; ABCG2: 1.16 ± 0.75, 5.48 ± 0.94, t = 11.305, P = 0.000 < 0.05), which may contribute to the efflux of fluorescent staining and drug resistance. CONCLUSION: SP cells with inherently high resistance to chemotherapeutic agents do exist in pancreatic cancers, which may be candidate cancer stem cells contributing to the relapse of the tumor.

Apoptosis of human pancreatic cancer cells induced by Triptolide

AIM: To investigate apoptosis in human pancreatic cancer cells induced by Triptolide (TL), and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax. METHODS: Human pancreatic cancer cell line SW1990 was cultured in DMEM media for this study. MTT assay was used to determine the cell growth inhibitory rate in vitro. Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment. RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax. RESULTS: TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner. TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h, the apoptotic rates of human pancreatic cancer cells increased significantly. RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not. CONCLUSION: TL is able to induce the apoptosis in human pancreatic cancer cells. This apoptosis may be mediated by up-regulating the expression of apoptosis- associated caspase-3 and bax gene.

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3

AIM: To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) in the proliferation and infiltration of pancreatic cancer cell line SW1990. METHODS: Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro . Supernatant fluid of cultured PSCs and SW1990 cells was collected. Expression of GAL-3 in SW1990 cells and PSCs was detected by ELISA, RT-PCR and Western blotting. Proliferation of cultured PSCs and SW1990 cells was measured by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Infiltration of SW1990 cells was detected by a cell infiltration kit. RESULTS: SW1990 cells expressed GAL-3 and this was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells stimulated proliferation of PSCs via GAL-3. GAL-3 antibody inhibited SW1990 cell proliferation, while the supernatant fluid of PSCs stimulated proliferation of SW1990 cells through interaction with GAL-3 protein. The supernatant fluid of PSCs enhanced the invasiveness of SW1990 cells through interaction with GAL-3. CONCLUSION: GAL-3 and PSCs were involved in the proliferation and infiltration process of pancreatic cancer cells.

Reversal of the phenotype by K-ras^(val12) silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1

AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism. METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-rasval12 and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-Avasval12. After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus Adh1-K-rasval12 was obtained. Stable suppression of K-rasval12 was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry. RESULTS: We obtained adenovirus AdHl-K-rasval12 carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-rasval12, the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-rasval12-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdHl-p53, respectively) (P<0.05). CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-rasval12 infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.

MK615 inhibits pancreatic cancer cell growth by dual inhibition of Aurora A and B kinases

AIM:To investigate the anti-neoplastic effect of MK615, an anti-neoplastic compound isolated from Japanese apricot, against human pancreatic cancer cells in vitro. METHODS: Three human pancreatic cancer cell lines PANC-1, PK-1, and PK45H were cultured with MK615 at concentrations of 600, 300, 150, and 0 μg/mL. Growth inhibition was evaluated by cell proliferation assay, and killing activity was determined by lactate dehydrogenase (LDH) assay. Expression of Aurora A and B kinases was detected by real-time polymerase chain reaction (PCR) and Western blotting. Cell cycle stages were evaluated by flow cytometry. RESULTS: The growth inhibitory rates of MK615 at 150, 300, and 600 μg/mL were 2.3% ± 0.9%, 8.9% ± 3.2% and 67.1% ± 8.1% on PANC1 cells, 1.3% ± 0.3%, 8.7% ± 4.1% and 45.7 ± 7.6% on PK1 cells, and 1.2 ± 0.8%, 9.1% ± 2.1% and 52.1% ± 5.5% on PK45H cells, respectively (P <0.05). The percentage cytotoxicities of MK615 at 0, 150, 300, and 600 μg/mL were 19.6% ± 1.3%, 26.7% ± 1.8%, 25.5% ± 0.9% and 26.4% ± 0.9% in PANC1 cells, 19.7% ± 1.3%, 24.7% ± 0.8%, 25.9% ± 0.9% and 29.9% ± 1.1% in PK1 cells, and 28.0% ± 0.9%, 31.2% ± 0.9%, 30.4% ± 1.1% and 35.3 ± 1.0% in PK45H cells, respectively (P < 0.05). Real-time PCR and Western blotting showed that MK615 dually inhibited the expression of Aurora A and B kinases. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. CONCLUSION: MK615 exerts an anti-neoplastic effect on human pancreatic cancer cells in vitro by dual inhibition of Aurora A and B kinases.

Effect of blocking IGF-I receptor on growth of human hepatocellular carcinoma cells

AIM： To study the expression level and localization of insulin-like growth factor -Ⅰ receptor （IGF-IR） in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody （αIR3） on the growth of HepG2 cells. METHODS： The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry. The influences of αIR3 on proliferation and apoptosis were examined by the 3- （4, 5-dimethylthiazol-2-yl）-2, 5- diphenyltetrazolium bromide （MTT） assay and electron microscopy, respectively. Flow cytometry （FCM） was applied for the analysis of cell cycle and apoptosis was observed under electron microscope. RESULTS： IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1 μg/mL αIR3 for 48 h in vitro, the cell growth index （GI） of HepG2 cells was significantly higher than that of control （103.41% ys 100%, P 〈 0.01）. However, the αIR3 for 24 h at final concentration of 4.0 μg/mL made the GI of HepG2 cells lower than that of control （93.37% vs 100%, P 〈 0.01）. Compared with control, treated with αIR3 for 48 h at final concentrations ranging from 2.0 μg/mL to 4.0 μg/mL markedly reduced the GIs of HepG2 cells （97.63%, 97.16%, 95.13%, 92.53% vs 100%, P 〈 0.05 or P 〈 0.01）, treated with αIR3 for 72 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL decreased the GIs of HepG2 cells obviously （95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P 〈 0.01）, and treated with αIR3 for 96 h at final concentrations ranging from 0.5 μg/mL to 4.0 μg/mL made GIs of HepG2 cells lower significantly （88.86%, 83.97%, 79.81%, 77.24%, 70.51% vs 100%, P 〈 0.05or P 〈 0.01）. Moreover, treated with αIR3 from 24 h to 96 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also, αIR3 treatment for 72 h at final concentration from 0.5 μg/mL to 2.0 μg/mL increased the proportion of G0/G1 phase cells（61.73%, 67.1%, 83.7%,76.87% vs 44.47%, P 〈 0.01） and significantly decreased that of S phase cells（28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P 〈 0.01）, in contrast to the proportion of G2/M phase cells. The apoptotic rates of HepG2 cells were increased more than that of control （7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P 〈 0.01）. CONCLUSION： The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGF- IR. The blockage of IGF-IR with αIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.

Nicotine as a mitogenic stimulus for pancreatic acinar cell proliferation

Cell proliferation is an important process in life for growth of normal and cancer cells. The signal transduction pathways activated during this process are strictly regulated. This editorial focuses on the role of nicotine, a mitogen, in the induction of signaling pathways resulting in proliferation of pancreatic tumor cells and compares these events with those in normal acinar cells isolated from the rat pancreas. The data shows striking similarities between these two cellular systems. In addition, the editorial reviews very recent literature of the contribution of MAPK signaling in cell lines associated with human diseases. A prospective cellular model of nicotine induced activation of MAPK cascade is presented.

PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG

AIM:To investigate the silencing effects of pAdshRNA-pleiotrophin(PTN) on PTN in pancreatic cancer cells,and to observe the inhibition of pAd-shRNA-PTN on neurite outgrowth from dorsal root ganglion(DRG) neurons in vitro.METHODS:PAd-shRNA-PTN was used to infect pancreatic cancer BxPC-3 cells;assays were conducted for knockdown of the PTN gene on the 0th,1st,3rd,5th,7th and 9th d after infection using immunocytochemistry,real-time quantitative polymerase chain reaction(PCR),and Western blotting analysis.The morphologic changes of cultured DRG neurons were observed by mono-culture of DRG neurons and co-culture with BXPC-3 cells in vitro.RESULTS:The real-time quantitative PCR showed that the inhibition rates of PTN mRNA expression in the BxPC-3 cells were 20%,80%,50% and 25% on the 1st,3rd,5th and 7th d after infection.Immunocytochemistry and Western blotting analysis also revealed the same tendency.In contrast to the control,the DRG neurons co-cultured with the infected BxPC-3 cells shrunk;the number and length of neurites were significantly decreased.CONCLUSION:Efficient and specific knockdown of PTN in pancreatic cancer cells and the reduction in PTN expression resulted in the inhibition of neurite outgrowth from DRG neurons.

Suppression of matrix metalloproteinase-2 via RNA interference inhibits pancreatic carcinoma cell invasiveness and adhesion

AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.

Fascin promotes the motility and invasiveness of pancreatic cancer cells

AIM:To explore the role of actin-bundling protein, fascin during the progression of pancreatic cancer. METHODS:The plasmid expressing human fascin-1 was stably transfected into the pancreatic cancer cell line MIA PaCa-2. The proliferation, cell cycle, motility, scattering, invasiveness and organization of the actin filament system in fascin-transfected MIA PaCa-2 cells and control non-transfected cells were determined. RESULTS:Heterogeneous overexpression of fascin markedly enhanced the motility, scattering, and inva-siveness of MIA PaCa-2 cells. However, overexpression of fascin had minimal effect on MIA PaCa-2 cell pro-liferation and cell cycle. In addition, cell morphology and organization of the actin filament system were distinctly altered in fascin overexpressed cells. When transplanted into BALB/c-nu mice, fascin-transfected pancreatic cancer cells developed solid tumors at a slightly slower rate, but these tumors displayed more aggressive behavior in comparison with control tumors. CONCLUSION: Fascin promotes pancreatic cancer cell migration, invasion and scattering, thus contributes to the aggressive behavior of pancreatic cancer cells.

KAI1 is a potential target for anti-metastasis in pancreatic cancer cells

AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t test.RESULTS: By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively (P < 0.05).CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of pancreatic cancer after direct injection into pancreatic adenocarcinoma. These results show that the suppressed invasion and motor function of pancreatic cancer cells may be a key reason why the KAI1 gene controls pancreatic cancer cell metastasis.

ERK signaling pathway may induce gemcitabine chemoresistance in pancreatic cancer cell line SW1990 by regulating the expression of mdr-1 and RRM1 gene

Objective： To investigate the relationship between extracellular signal-regulated kinase （ERK） pathway, multidrug resistance gene （mdr-1）, ribonucleotide recluctase M1 （RRM1） gene and their roles in gemcitabine （GEM） chemoresistance in pancreatic cancer cell line SW1990. Methods： The GEM-resistance cell model was constructed by a stepwise method. Immunohistochemistry was used to measure the expression of ERK protein （ERK1/2） in the established cell strains in a semiquantitative way. The mRNA expression of mdr-1 and RRM1 were detected by RT-PCR. MTT assay was performed to determine the IC50 value. Results： The established GEM-resistant cell strains were able to gain stable growth and passage ability in the medium contained different concentration levels of GEM （0, 30, 60, 100, 150 and 200 nmol/L）. The expression of ERK protein, mdr-1 and RRM1 gene were elevated accompanied by the increase of GEM concentration. There was a highly positive correlation between mdr-1, RRM1 expression and GEM-resistanca level （r = 0.960, P = 0.002 and r = 0.966, P = 0.002）. The expression of ERK protein also correlated with the mdr-1 and RRM1 level （r = -0.943, P = 0.005 and r = -0.883, P = 0.02）. At the GEM-resistance level of 200 nmol/L, the grey scale value of ERK1/2 was 164.22 ±13.17, mdr-1/β-actin and RRM1/β-actin were 1.41 ±0.04 and 1.45 ± 0.18, respectively; after treated with ERK pathway inhibitor U0126, these values synchronously decreased to 186.85 ± 13.14, 0.2 3± 0.02 and 0.21 ± 0.03, respectively; inversely, the ERK1/2 grey scale value was 106.55 ± 16.45, mdr-l/β-actin and RRMl/β-actin were 1.50± 0.07 and 1.52 ± 0.12, respectively, which presented a tendency of synchronously increase after treated with ERK pathway activator EGF. The IC50 values in GEM-resistant cells of 0 nmol/L and 200 nmol/L levels were 4.104 and 10.20, respectively. After treated with U0126, these values decreased to 3.26 and 4.50, respectively; while treated with EGF, the IC50 values increased to 8.89 and 17.17, respectively. Conclusion： The ERK pathway may induce the GEM-chemoresistance in pancreatic cell line SW1990 through the participation in the regulation of the mdr-1 and RRM1 gene expression.

Apoptosis of pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid in combination with horseradish peroxidase

AIM： To explore the mechanisms underlying the apoptosis of human pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid （IAA） in combination with horseradish peroxidase （HRP）. METHODS： BXPC-3 cells derived from human pancreatic cancer were exposed to 40 or 80 μmol/L IAA and 1.2 μg/mL HRP at different times. Then, Mn- assay was used to detect the cell proliferation. Flow cytometry was performed to analyze cell cycle. Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling assay was used to detect apoptosis. 2,7-Dichlorofluorescin diacetate uptake was measured by confocal microscopy to determine free radicals. Level of malondialdehyde （MDA） and activity of superoxide dismutase （SOD） were measured by biochemical methods. RESULTS： IAA/HRP initiated growth inhibition of BXPC-3 cells in a dose- and time-dependent manner. Flow cytometry revealed that the cells treated for 48 h were arrested at G1/G0. After exposure to 80 μmol/L IAA plus 1.2 μg/mL HRP for 72 h, the apoptosis rate increased to 72.5‰, which was nine times that of control. Content of MDA and activity of SOD increased respectively after treatment compared to control. Meanwhile, IAA/HRP stimulated the formation of free radicals. CONCLUSION： The combination of IAA and HRP can inhibit the growth of human pancreatic cancer BXPC-3 cells in vitro by inducing apoptosis.

Effect of gemcitabine on the expression of apoptosis-related genes in human pancreatic cancer cells

AIM： TO investigate the expression of genes involved in the gemcitabine-induced cytotoxicity in human pancreatic cancer cells. METHODS： A human pancreatic cancer cell line, PANC-1, was cultured. 1 × 10^4 PANG-1 cells were plated in 96-well microtiter plates. After being incubated for 24 h, gemcitabine was added to the medium at concentrations ranging 2.5 -1 000 mg/L. The AlamarBlue dye method was used for cell growth analysis. DNA fragmentation was quantitatively assayed using a DNA fragmentation enzyme-linked immunosorbent assay （ELISA） kit. PAP and TP53INP1 mRNA expression was determined using the reverse transcription-polymerase chain reaction with semi-quantitative analysis. The expression of GSK-3β and phospho-GSK-3β proteins was examined with Western blot analysis. RESULTS： The IC50 for the drug after a 48-h exposure to gemcitabine was 16 mg/L. The growth of PANC-1 cells was inhibited by gemcitabine in a concentration-dependent manner （P〈 0.0001） and the cell growth was also inhibited throughout the time course （P〈0.0001）. The DNA fragmentation rate in the gemcitabine-treated group at 48 h was 44.7 %, whereas it was 25.3 % in the untreated group. The PAP mRNA expression was decreased after being treal^l with gemcitabine, whereas the TP53INP1 mRNA was increased by the gemcitabine treatment. Western blot analysis showed that phospho- GSK-3βZ^ser9 was induced by the gemcitabine treatment. CONCLUSION： Gemcitabine suppresses PANC-1 cell proliferation and induces apoptosis. Apoptosis is considered to be associated with the inhibition of PAPand GSK-3β, and the activation of TP53INP1 and pospho- GSK-3β^ser9 .