Glypican-3-specific cytotoxic T lymphocytes induced by human leucocyte antigen-A*0201-restricted peptide effectively kill hepatocellular carcinoma cells in vitro

OBJECTIVE@#To investigate potential human leucocyte antigen (HLA)-A2-restricted epitope peptides of glypican-3 (GPC3) and determine the cytotoxicity of peptide-specific cytotoxic T lymphocytes (CTLs) against hepatocellular carcinoma (HCC) cells.@*METHODS@#The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3 SMMC 7721 and HepG2 cells was detected using IFN-γ based enzyme-linked immunospot and lactate dehydrogenase release assays in vitro.@*RESULTS@#A total of six peptides were identified for bindings to HAL-A2 and the GPC3 522-530 and GPC3 229-237 peptides with HLA-A*0201 molecules displayed high binding affinity and stability. The CTLs induced by the GPC3 522-530 or positive control GPC3 144-152 peptide responded to the peptide by producing IFN-γ, which were abrogated by treatment with anti-HLA-A2 antibody. The GPC3 522-530-specific CTLs responded to and killed SMMC 7721 and HepG2 cells, instead of GPC3-silenced SMMC 7721 or HepG2 cells. GPC3 522-530-specific CTLs response to HCC cells was blocked by anti-HLA-A2 antibody.@*CONCLUSIONS@#The GPC3 522-530 peptide contains antigen-determinant and its specific CTLs can effectively kill HCC in a HLA-A2-restricted and peptide-dependent manner. Our findings suggest that this peptide may be valuable for development of therapeutic vaccine.

Glypican-3-specific cytotoxic T lymphocytes induced by human leucocyte antigen-A*0201-restricted peptide effectively kill hepatocellular carcinoma cells in vitro

Objective To investigate potential human leucocyte antigen (HLA)-A2-restricted epitope peptides of glypican-3 (GPC3) and determine the cytotoxicity of peptide-specific cytotoxic T lymphocytes (CTLs) against hepatocellular carcinoma (HCC) cells. Methods The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3

C-reactive protein/albumin ratio as a novel inflammation-based prognostic index for predicting outcomes of patients with colorectal cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the association of C-reactive protein/albumin ratio (CAR) with the prognosis of patients with colorectal cancer and compare the prognostic value of CAR with other inflammation-based prognostic scoring systems.</p><p><b>METHODS</b>We retrospectively evaluated 163 newly diagnosed colorectal cancer patients in Nanfang Hospital between January, 2007 and December, 2014. All recommended cutoff values of the clinicopathological factors were defined using receiver- operating characteristic (ROC) curve analyses. We evaluated the prognostic value of CAR in comparison with Glasgow Prognostic Score (GPS) and neutrophil lymphocyte ratio (NLR) with the area under the ROC curve. Univariate and multivariate analyses using the Cox proportional hazards model were performed to identify the factors closely associated with overall survival of the patients. Kaplan-Meier analysis was used to compare overall survival curves between patients with a high CAR and those with a low CAR.</p><p><b>RESULTS</b>The recommended cutoff value of CAR was 0.132. Kaplan-Meier analysis and log rank test demonstrated a significant difference in the overall survival between patients with a low CAR (<0.132) and those with a high CAR (≥0.132) (2157.0∓395.3 vs 1661.0∓136.4 days, P<0.001). The area under the ROC curve of CAR, NLR and GPS was 0.656, 0.550 and 0.642, respectively, indicating a better prognostic value of CAR. Univariate analyses showed that age, C-reactive protein, albumin, CAR, NLR, GPS, platelet, TMN stage, Dukes stage and chemotherapy regimens were associated with the overall survival of the patients (P<0.05). Multivariate analyses showed that TMN stage [HR=1.689 (95%CI: 1.146-2.488), P=0.008] and Dukes stage [HR=2.447 (95%CI: 1.349-4.441), P=0.003] were associated with the overall survival of the patients.</p><p><b>CONCLUSIONS</b>Similar to the previously reported inflammation-based prognostic systems (GPS and NLR), CAR is useful for predicting the survival of patients with colorectal cancer and can be complementary to the two prognostic scoring systems.</p>

Effect of glypican-3 on the proliferation of human hepatoma cell line MHCC97-L in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct glypican-3 (GPC3)-green fluorescent protein eukaryotic expression vector pEGFP-c3-GPC3, and analyze the effect of GPC3 on the proliferation of human hepatoma cell line MHCC-97L.</p><p><b>METHODS</b>The eukaryotic expression vector pEGFP-c3-GPC3 was constructed with recombinant DNA technique and transfected into MHCC-97L cells via Lipofectamine 2000. The cells stably expressing GPC3 were screened by flow cytometry and G418. The mRNA expression of GPC3 was detected by RT-QPCR method, and the protein expression by Western blotting and fluorescence microscope. The effect of GPC3 gene on the growth of the cells was examined by MTT assay.</p><p><b>RESULTS</b>Restriction endonuclease analysis and DNA sequencing verified correct construction of the recombinant plasmid. The green fluorescence was detected in the transfected MHCC-97L cells under fluorescence microscope. RT-QPCR and Western blotting both confirmed successful expression of GPC3 in MHCC-97L cells. The growth curve showed a significant acceleration of the proliferation of the transfected MHCC97-Lsol;GPC3 cells as compared with MHCC97-L and MHCC97-L/C3 cells (P<0.001).</p><p><b>CONCLUSION</b>We have successfully constructed the eukaryotic expression vector pEGFR-c3-GPC3, which allows stable GPC3 expression in MHCC97-L/GPC3 cells. The upregulation of GPC3 expression can stimulate the growth of hepatoma cell line MHCC97-L in vitro.</p>

Gefitinib enhances the radiosensitivity of nasopharyngeal carcinoma cell line CNE2 in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the radiosensitizing effect of gefitinib on nasopharyngeal carcinoma cell line CNE2 in vitro.</p><p><b>METHODS</b>Nasopharyngeal carcinoma cell line CNE2 was cultured in RP2MI 1640. MTT assay was performed to evaluate the cell proliferation changes in response to gefitinib treatment and the radiosensitizing effect of gefitinib. The cell survival curves and sensitive enhancement ratio (SERs) were obtained with a clonogenic assay. Flow cytometry analysis was applied to detect the cell cycle changes and cell apoptosis.</p><p><b>RESULTS</b>MTT assay showed that cells exposed to gefitinib and radiation had a significantly lower survival ratio compared to the cells with radiation exposure only (0.582∓0.012 vs 0.398∓0.016, P=0.002), with a SER of 1.535∓0.134. The S phase cell percentage was significantly decreased and G(2)-M phase cells increased in gefitinib plus radiation group (P=0.000), suggesting a synergistic effect of gefitinib and radiation.</p><p><b>CONCLUSION</b>Gefitinib can enhance the radiosensitivity of nasopharyngeal carcinoma CNE2 cells in vitro possibly by inhibiting cell proliferation, inducing cell apoptosis, and causing changes in the cell cycle distribution.</p>

Effects of silencing H-ras gene by RNA interference on cetuximab-sensitivity of cetuximab-resistant human nasopharyngeal carcinoma cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the changes of the sensitivity of cetuximab-resistant human nasopharyngeal carcinoma (hNPC) cells 5-8F/Erbitux to cetuximab by silencing H-ras gene with RNA interference (RNAi).</p><p><b>METHODS</b>The 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect the protein levels of H-ras and K-ras. Real-time PCR was employed to detect the expression of H-ras and K-ras. H-ras-shRNA plasmids (shRNA vector carrying the H-ras gene) were constructed and transferred into 5-8F/Erbitux cells. The gene and protein expression levels of H-ras and the changes of the sensitivity of 5-8F/Erbitux cells to cetuximab after transfection were measured, respectively.</p><p><b>RESULTS</b>After treatment with cetuximab for 3 and 5 days, the resistance index (RI) of the 5-8F/Erbitux cells was 1.2 and 1.1, and the protein levels of H-ras and K-ras in 5-8F/Erbitux cells were 0.798 +/- 0.019 and 0.190 +/- 0.011, respectively, significantly higher than that in the 5-8F cells (P<0.001). The gene expressions of H-ras and K-ras in 5-8F/Erbitux cells were 1.260 +/- 0.114 and 0.850 +/- 0.006, respectively. Compared with 5-8F cells, the former was higher (P = 0.016) and the latter was lower (P = 0.000). After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of H-ras gene and protein, and the cell apoptosis and inhibition rates increased significantly (P<0.001).</p><p><b>CONCLUSIONS</b>H-ras siRNA can reverse cetuximab-resistance of 5-8F/Erbitux cells through down-regulation of H-ras gene expression, indicating that the generation of cetuximab-resistance in 5-8F/Erbitux cells is associated with amplification and overexpression of the H-ras gene.</p>

Lapatinib plus capecitabine in treating HER2-positive advanced breast cancer: efficacy, safety, and biomarker results from Chinese patients / 癌症

Overexpression of human epidermal growth factor receptor-2 (HER2) in metastatic breast cancer (MBC) is associated with poor prognosis. This single-arm open-label trial (EGF109491; NCT00508274) was designed to confirm the efficacy and safety of lapatinib in combination with capecitabine in 52 heavily pretreated Chinese patients with HER2-positive MBC. The primary endpoint was clinical benefit rate (CBR). Secondary endpoints included progression-free survival (PFS), time to response (TTR), duration of response (DoR), central nervous system (CNS) as first site of relapse, and safety. The results showed that there were 23 patients with partial responses and 7 patients with stable disease, resulting in a CBR of 57.7%. The median PFS was 6.34 months (95% confidence interval, 4.93-9.82 months). The median TTR and DoR were 4.07 months (range, 0.03-14.78 months) and 6.93 months (range, 1.45-9.72 months), respectively. Thirteen (25.0%) patients had new lesions as disease progression. Among them, 2 (3.8%) patients had CNS disease reported as the first relapse. The most common toxicities were palmar-plantar erythrodysesthesia (59.6%), diarrhea (48.1%), rash (48.1%), hyperbilirubinemia (34.6%), and fatigue (30.8%). Exploratory analyses of oncogenic mutations of PIK3CA suggested that of 38 patients providing a tumor sample, baseline PIK3CA mutation status was not associated with CBR (P = 0.639) or PFS (P = 0.989). These data confirm that the lapatinib plus capecitabine combination is an effective and well-tolerated treatment option for Chinese women with heavily pretreated MBC, irrespective of PIK3CA status.

Efficacy and safety of bevacizumab plus chemotherapy in Chinese patients with metastatic colorectal cancer: a randomized phase III ARTIST trial / 癌症

The efficacy and safety of bevacizumab with modified irinotecan, leucovorin bolus, and 5-fluorouracil intravenous infusion (mIFL) in the first-line treatment of metastatic colorectal cancer (mCRC) has not been well evaluated in randomized clinical trials in Chinese patients. We conducted a phrase III trial in which patients with previously untreated mCRC were randomized 2:1 to the mIFL [irinotecan (125 mg/m(2)), leucovorin (20 mg/m(2)) bolus, and 5-fluorouracil intravenous infusion (500 mg/m(2)) weekly for four weeks every six weeks] plus bevacizumab (5 mg/kg every two weeks) group and the mIFL group, respectively. Co-primary objectives were progression-free survival (PFS) and 6-month PFS rate. In total, 214 patients were enrolled. Our results showed that addition of bevacizumab to mIFL significantly improved median PFS (4.2 months in the mIFL group vs. 8.3 months in the bevacizumab plus mIFL group, P < 0.001), 6-month PFS rate (25.0% vs. 62.6%, P < 0.001), median overall survival (13.4 months vs. 18.7 months, P = 0.014), and response rate (17% vs. 35%, P = 0.013). Grades 3 and 4 adverse events included diarrhea (21% in the mIFL group and 26% in the bevacizumab plus mIFL group) and neutropenia (19% in the mIFL group and 33% in the bevacizumab plus mIFL group). No wound-healing complications or congestive heart failure occurred. Our results suggested that bevacizumab plus mIFL is effective and well tolerated as first-line treatment for Chinese patients with mCRC. Clinical benefit and safety profiles were consistent with those observed in pivotal phase III trials with mainly Caucasian patients.

Inhibitory effect of ZD6474 combined with adriamycin on MCF-7 human breast cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the killing effect of ZD6474 combined with adriamycin (ADM) on MCF-7 human breast cancer cells.</p><p><b>METHODS</b>The inhibitory effects of ZD6474 and ADM alone and in combination on the proliferation of MCF-7 cells were assessed by MTT assay. The cell cycle and cell apoptosis were detected by flow cytometry.</p><p><b>RESULTS</b>ZD6474 and ADM both significantly inhibited the proliferation of MCF-7 cells, showing a synergistic effect of their reactions in combined use (P<0.05). ZD6474 or ADM alone caused cell cycle arrest at G0/G1 and S phases, respectively. Combined use of the two drugs resulted in significant reduction of the M-phase cell percentage and cell cycle arrest at G0/G1 and S phases. The coadministration of the drugs significantly increased the apoptosis rate of the cells as compared with ZD6474 or ADM treatment alone (P<0.05).</p><p><b>CONCLUSIONS</b>ZD6474 and ADM show a synergistic effect in inhibiting the proliferation and inducing apoptosis of MCF-7 cells.</p>

Short-term therapeutic effect and safety of endostar combined with XELIRI regimen in the treatment of advanced colorectal cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the short-term efficacy and toxicity of endostar in combination with XELIRI as the second-line treatment for advanced colorectal cancer.</p><p><b>METHODS</b>Twenty-one patients with advanced colorectal cancer were treated with intravenous infusion of endostar (15 mg/day for 14 consecutive days) and irinotecan (250 mg/m(2), single dose on the first day), and oral administration of capecitabine (1.0 mg/m(2), twice daily for 14 days), and the treatment cycle was repeated every 21 days. The efficacy and toxicity of the treatments were evaluated according to RECIST and NCI-CTCAE3.0 standard, respectively.</p><p><b>RESULTS</b>The overall response rate was 9.5% in these patients, with a median time to progression (mTTP) of 3.9 months. The main adverse effects associated with the treatment included leucopenia, nausea/vomiting and peripheral neuritis.</p><p><b>CONCLUSION</b>Endostar combined with XELIRI is effective and safe as the second-line treatment for advanced colorectal cancer, and further clinical investigation is warranted.</p>

Effect of anti-EGFR monoclonal antibody on chemosensitivity of human colon cancer cells and the mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of anti-EGFR monoclonal antibody on the chemosensitivity of human colon cancer cells and explore the possible molecular mechanism.</p><p><b>METHODS</b>The inhibitory effect of irinotecan (CPT-11), oxaliplatin (L-OHP) and fluorouracil (5-Fu), used alone or in combination with anti-EGFR monoclonal antibody, on the proliferation of LoVo cells in vitro was assessed by MTT assay. The expressions of PI3K and Akt protein in the treated cells were examined by Western blotting, and their mRNA expressions were detected by RT-PCR.</p><p><b>RESULTS</b>Both h-R3 and C-225 treatments significantly increased the chemosensitivity of LoVo cells to irinotecan and oxaliplatin. 5-Fu and h-R3 coadministered showed a synergistic effect on the cells, but 5-Fu and C-225 had an antagonistic action. Treatment with C-225 or h-R3 resulted in lowered expressions of PI3K and Akt in LoVo cells.</p><p><b>CONCLUSION</b>Anti-EGFR monoclonal antibody can increase the chemosensitivity of human colon cancer cells to most chemotherapeutic drugs, and such effect might be attributed to the blocking of PI3K/Akt signaling pathway by these antibodies.</p>

Ultrasound-mediated microbubble destruction enhances LMP-1 gene transfection into dendritic cells in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the transfection efficiency and the optimal conditions of delivering latent membrane protein-1 (LMP-1) gene to dendritic cells (DCs) by ultrasound exposure combined with contrast agent.</p><p><b>METHODS</b>Human DCs were cultured in vivo and transfected with the recombinant plasmid pEGFP-C3-LMP1 under varying conditions including ultrasound intensities, exposure time and microbubble contrast agent concentration. The transfection efficiency was assessed by fluorescent microscopy and flow cytometry, and the cell viability by trypan blue exclusion test.</p><p><b>RESULTS</b>An exposure time of 60 s at MI 1.0 with a microbubble contrast agent concentration of 20% resulted in the optimal effect of delivering the recombinant plasmid pEGFP-C3-LMP1 into the DCs, with a transfection efficiency of (14.37∓2.12)%. Over 90% of the transfected cells were viable after the transfection.</p><p><b>CONCLUSION</b>Microbubble contrast agent combined with ultrasound exposure can enhance the delivery of recombinant plasmid pEGFP-C3-LMP1 into the DCs.</p>

In vivo and in vitro stability of ¹³¹I-Herceptin and its form of existence in the blood of rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the in vivo and in vitro stability of (131)I-Herceptin and its form of existence in the blood.</p><p><b>METHODS</b>Herceptin was labelled with iodine-131 using the Iodogen method. (131)I-Herceptin was stored at 4 degrees celsius for 3, 24, 48, 72 and 96 h, and the radiochemical purity (RCP) was measured by high performance liquid chromatography (HPLC). Five rabbits received injections of (131)I-Herceptin and at 1, 3, 6, 24, 48, 72, 96 and 120 h after the injection, blood samples were taken to measure the RCP of (131)I-Herceptin in the serum, and the radio count of the serum and blood cells was calculated.</p><p><b>RESULTS</b>The baseline RCP of (131)I-Herceptin was (94.9±2.7)%. The RCP was stable after placement at 4 degrees celsius for not over 72 h (F=15.985, P<0.001), but was significantly lowered to (82.6±2.8)% after preservation for over 72 h (t=9.971, P<0.001). Within the time of 1.0 to 96 h after injection in rabbits, (131)I-Herceptin existed mainly in the serum with a radio count of 81%-87%; 24 h after the injection, the RCP of (131)I-Herceptin in the serum was significantly lowered to (75.4±3.9)% (t=6.564, P<0.001).</p><p><b>CONCLUSION</b>Storage at 4 degrees celsius for no more than 72 h does not obviously affect the activity of (131)I-Herceptin in terms of RCP. After injection in rabbits, (131)I-Herceptin exists mainly in the serum and its radiochemical purity remains stable within 24 h, after which obvious degradation occurs.</p>

Correlation between ras gene and the resistance of nasopharyngeal carcinoma to cetuximab / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the correlation of ras gene to the drug resistance of nasopharyngeal carcinoma to cetuximab.</p><p><b>METHODS</b>Cultured 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. MTT assay was used to determine the IC50 (half inhibitory concentration) of cetuximab and the drug resistance index (RI). Western blotting was employed to detect the protein levels of H-ras and K-ras. Real-time PCR was used to detect the expression of H-ras and K-ras. Gene sequencing was performed to identify potential mutations in H-ras and K-ras genes.</p><p><b>RESULTS</b>We successfully induced cetuximab-resistant 5-8F/Erbitux hNPC cells by stepwise exposure to increasing doses of cetuximab. After treatment with cetuximab for 3 and 5 days, the RI of 5-8F/Erbitux cells was 1.2 and 1.1, respectively. The 5-8F/Erbitux cells had increased levels of H-ras and K-ras protein expressions (P<0.001) and enhanced gene expressions of H-ras (P=0.016) and ras-p21 (P=0.113) with decreased K-ras gene expression (P=0.000). Sequence analysis identified no mutations in the H-ras and K-ras genes in codons 12, 13, 59, and 61.</p><p><b>CONCLUSION</b>Gene amplification and overexpression of H-ras is the major mechanism that causes the drug resistance of 5-8F/Erbitux cells to cetuximab.</p>

Value of carcinoembryonec antigen in biochemotherapy response monitoring in patients with Her-2-positive advanced breast cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the value of serum carcinoembryonec antigen (CEA) in monitoring the response to biochemotherapy by Herceptin plus taxol (TAX) in patients with Her-2-positive advanced breast cancer.</p><p><b>METHODS</b>The changes in serum CEA level were investigated retrospectively after two cycles of biochemotherapy in 83 patients with Her-2-positive advanced breast cancer. The correlations between the changes and radiological objective response were analyzed.</p><p><b>RESULTS</b>After two cycles of biochemotherapy, the clinical benefit rate (CBR) was 81.9%. In the 60 patients with lowered CEA level, the CBR was 85.0% (51/60), with a non-response rate of 15.0% (9/60); in contrast, the CBR was only 34.8% in 23 patients with elevated CEA, with a non-response rate of 65.2%, showing significant difference between the two groups (P<0.05).</p><p><b>CONCLUSION</b>Serum CEA level can be used to monitor the therapeutic effect of biochemotherapy in patients with Her-2-positive advanced breast cancer.</p>

Expression of PAK4 in breast cancer and benign breast pathological changes / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the role of p21-activated kinase-4 (PAK4) in the occurrence, progression and metastasis of breast cancer.</p><p><b>METHOD</b>PAK4 expression was detected in 35 cases of normal breast, 22 breast cystic hyperplasia, 28 breast adenofibroma, 37 breast cancer (including 7 non-invasive cancer, 9 early invasive cancer and 21 invasive cancer) and 13 metastatic breast cancer tissues using immunohistochemistry for a comparison of PAK4 expression and distribution.</p><p><b>RESULTS</b>PAK4 was expressed mainly in the cytoplasm of the cancer cells, occasionally in the cell nuclei, and virtually not expressed in the matrix surrounding the breast cells. PAK4 positivity rates increased in the order of normal breast tissues, benign changes (including breast cystic hyperplasea and breast adenoma), breast cancer and metastatic cancer tissues; in the cancer tissues, the positivity rates increased in the order of non-invasive breast tumor, early invasive tumor and invasive tumor tissues.</p><p><b>CONCLUSION</b>PAK4 is closely correlated to the progression and metastasis of breast cancer and may become a new diagnostic and therapeutic target of breast cancer.</p>

Inhibitory effect of SHP-1 gene transfer on the proliferation of breast cancer cell line MDA-MB-231 / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe SHP-1 protein expression in breast cancer cell line MDA-MB-231 before and after SHP-1 gene transfer and its effect on the proliferation of MDA-MB-231 cells.</p><p><b>METHODS</b>The eukaryotic expression vector pEGFP-C3-SHP-1 was constructed and transfected into breast cancer cell line MDA-MB-231 via Lipofectamine 2000, and the positive clones were selected using G418. SHP-1 expression in MDA-MB-231 cells was detected with immunocytochemistry and Western blotting, and the cell growth curve was observed using MTT assay.</p><p><b>RESULTS</b>SHP-1 was highly expressed in transfected MDA-MB-231 cells, whose proliferation was significantly inhibited (P<0.05).</p><p><b>CONCLUSION</b>SHP-1 gene transfer into MDA-MB-231 cells results in inhibition of the cell proliferation.</p>

Relationship between the insulin-like growth factor 1 receptor signaling pathway and the resistance of nasopharyngeal carcinoma to cetuximab / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To establish a cetuximab-resistant human nasopharyngeal carcinoma 5-8F/Erbitux cell line and preliminarily study the relationship between the insulin-like growth factor 1 receptor (IGF-1R) signaling pathway and the resistance of nasopharyngeal carcinoma to cetuximab.</p><p><b>METHODS</b>A nasopharyngeal cancer cell line, 5-8F, with high epidermal growth factor receptor (EGFR) expression and cetuximab sensitivity, was selected as study object. The cetuximab-resistant 5-8F/Erbitux cell line was induced by stepwise selection after exposure to increasing doses of cetuximab. The IC(50) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the resistance index (RI) was calculated. The growth curves of 5-8F and 5-8F/Erbitux cells were plotted and the doubling times were calculated by cell counting assay. The cell cycle was detected by flow cytometry. Cross-resistance profiles of 5-8F/Erbitux cells to 5-Fu, Taxol and DDP were tested by MTT assay. Expression levels of P-gP, IGF-1R and P-IGF-1R of 5-8F and 5-8F/Erbitux cells were determined by Western blot analysis and MDR1 gene by real-time fluorescent quantitative PCR.</p><p><b>RESULTS</b>A cetuximab-resistant human nasopharyngeal carcinoma cell line 5-8F/Erbitux was successfully established and their resistance index (RI) were 1.2 and 1.1, respectively, at 3 d and 5 d of the cetuximab treatment. The doubling times of 5-8F and 5-8F/Erbitux cells were 26.63 h and 142.30 h, respectively. Flow cytometry demonstrated that 5-8F/Erbitux cells showed an increased population at G(0)/G(1) phase (P < 0.001) and reduced population at S phase (P < 0.001), compared with 5-8F cells. The 5-8F/Erbitux cells showed cross-resistance to 5-Fu (RI = 3.95, P < 0.01) and some resistance to Taxol as well as enhanced sensitivity to DDP (P > 0.05 for all). The 5-8F/Erbitux cells also had increased levels of P-gP, IGF-1R and P-IGF-1R compared with 5-8F cells (P < 0.001 for all). Expression of MDR1 gene was not detected in 5-8F cells and only very weak expression in 5-8F/Erbitux cells.</p><p><b>CONCLUSION</b>Cetuximab-resistant 5-8F/Erbitux cells have no common multidrug resistance like that induced by traditional chemotherapeutic drugs. The excessive activation of the IGF-1R signaling pathway is probably one of the mechanisms that caused resistance of 5-8F/Erbitux cells to cetuximab.</p>

Effects of Poly I:C in inducing growth inhibition and apoptosis of human hepatocellular carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect and mechanism of Poly I:C in inducing growth inhibition and apoptosis of human hepatocellular carcinoma SMMC-7721 cells.</p><p><b>METHODS</b>SMMC-7721 cells were treated with different doses of Poly I:C for 24, 48, and 72 h, and the cell growth inhibition rate was analyzed with CCK-8 assay. The cell cycle and the apoptosis were analyzed using flow cytometry with Annexin-V and PI staining, and quantitative RT-PCR analysis were used to detect the expression of TLR3, TRIF, and IFN-beta mRNA in cells.</p><p><b>RESULTS</b>In the cells exposed to Poly I:C at low, moderate, and high doses, the inhibitory rates was the highest in high-dose Poly I:C group, and at a given Poly I:C dose, prolonged exposure resulted in significantly increased cell growth inhibition rate (P<0.05). Flow cytometry showed that Poly I:C induced cell apoptosis in a time- and dose-dependent manner and significantly increased the percentage of G1-phase cells as compared with that in the control group. The mRNA level of TLR3, TRIF, and IFN-beta were also increased following Poly I:C treatment in comparison with the control group.</p><p><b>CONCLUSION</b>Poly I:C can induce significant growth inhibition and apoptosis of SMMC-7721 cells in a dose- and time-dependent manner possibly by causing cell cycle arrest and TLR3 signaling pathway activation that leads to IFN-beta production and cell apoptosis.</p>

Retrovirus-mediated SHP-1 gene expression in human breast cancer MDA-MB-231 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a retrovirus-mediated expression system carrying human SHP-1 gene to transfer SHP-1 gene in human breast cancer MDA-MB-231 cells.</p><p><b>METHODS</b>The full-length SHP-1 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line MCF-7 over-expressing SHP-1 protein. The gene fragment was inserted into the vector pLNCX2 to construct the recombinant retroviral plasmid, which was transfected into the packaging cell PT67 via Lipofectamine2000. A cell line stably producing the virus was selected with G418. MDA-MB-231 cells was infected with the virus, and the expression of SHP-1 gene in the positive cell clone was detected with Western blotting.</p><p><b>RESULTS</b>A 1.8 kb cDNA fragment of SHP-1 gene was obtained from MCF-7 cells and successfully inserted into the pLNCX2. A stable cell clone PT67/SHP-1 and virus supernatant were obtained. Expression of SHP-1 protein was detected in the cells infected with the virus.</p><p><b>CONCLUSION</b>The recombinant retroviral vector carrying SHP-1 gene has been successfully constructed and MDA- MB-231/SHP-1 cell line expressing SHP-1 has been obtained to allow further functional study of SHP-1 in breast cancer.</p>