Qiangzhi decoction protects mice from influenza A pneumonia through inhibition of inflammatory cytokine storm / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the preventive effects of Qiangzhi Decoction (, QZD) on influenza A pneumonia through inhibition of inflammatory cytokine storm in vivo and in vitro.</p><p><b>METHODS</b>One hundred ICR mice were randomly divided into the virus control, the Tamiflu control and the QZD high-, medium-, and low-dose groups. Mice were infected intranasally with influenza virus (H1N1) at 10 median lethal dose (LD50). QZD and Tamiflu were administered intragastrically twice daily from day 0 to day 7 after infection. The virus control group was treated with distilled water alone under the same condition. The number of surviving mice was recorded daily for 14 days after viral infection. The histological damage and viral replication and the expression of inflammatory cytokines were monitored. Additionally, the suppression capacity on the secretion of regulated on activation normal T cells expressed and secreted (RANTES) and tumor necrosis factor-α (TNF-α) in epithelial and macrophage cell-lines were evaluated.</p><p><b>RESULTS</b>Compared with the virus control group, the survival rate of the QZD groups significantly improved in a dose-dependent manner (P<0.05), the viral titers in lung tissue was inhibited (P<0.05), and the production of inflammatory cytokines interferon-γ (IFN-γ), interleukin-6 (IL-6), TNF-α, and intercellular adhesion molecule-1 (ICAM-1) were suppressed (P<0.05). Meanwhile, the secretion of RANTETS and TNF-α by epithelial and macrophage cell-lines was inhibited with the treatment of QZD respectively in vitro (p<0.05) CONCLUSIONS: The preventive effects of QZD on influenza virus infection might be due to its unique cytokine inhibition mechanism. QZD may have significant therapeutic potential in combination with antiviral drugs.</p>

Real-time elastography for quantitative assessment of liver fibrosis in a rat model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of real-time elastography for quantitative evaluation of liver fibrosis in a rat model.</p><p><b>METHODS</b>A total of 70 male Wistar rats were included in the group for dimethylnitrosamine (DMN)-induced liver injury, and 10 saline-injected rats were used as normal control. Hepatic injury was induced by a single intraperitoneal injection of DMN at a dose of 50 mg/kg of body weight. Nine or ten rats in the group with DNM injected and one or two rats in the normal control group were randomly selected and sacrificed at each of the following post-injection time: day 5, 7, 10, 14, 21, 24, and 28. And their livers were taken for pathology analysis. All the rats underwent real-time elastography before sacrificed in order to acquire area ratio of low-strain region (% AREA) and liver fibrosis index (LF index) which were compared with the stage of liver fibrosis and grade of necroinflammatory pathologically. By the different data, Spearman correlation analysis, rank-sum test or receiver operating characteristic curve was used.</p><p><b>RESULTS</b>Among 58 successfully modeled rats, there were nine, 13, 14 and 12 rats of S1, S2, S3 and S4 liver fibrosis on pathology, respectively, which were with or without mild necroinflammatory. The other 10 rats were found to be S0 with severe necroinflammatory. Values of LF index and % AREA both increased with liver fibrosis stage (P less than 0.05). There was certain correlation between LF index and liver fibrosis stage (r=0.643, P=0.000), so was % AREA and liver fibrosis stage (r=0.662, P=0.000). As for LF index, Areas under the receiver operating characteristic curve (Az) was 0.943, 0.890, 0.743 and 0.821 for the diagnosis of hepatic fibrosis S1 or higher, S2 or higher, S3 or higher and S4, respectively; as for % AREA, they were 0.948, 0.883, 0.772 and 0.842, respectively. However, we found a significant difference for LF index or % AREA between S0 with and without severe inflammatory activity rats (P=0.005 and P=0.017).</p><p><b>CONCLUSION</b>Real-time elastography is available for quantitative assessment of liver fibrosis in rats induced by DMN, but severe inflammatory activity can affect its accuracy.</p>

Over-expression of caveolin-1 inhibits proliferation and invasion of pancreatic carcinoma cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of caveolin-1 on the biologic behavior of pancreatic carcinoma cell line panc1 cells in vitro.</p><p><b>METHODS</b>Eukaryotic expression vectors containing human caveolin-1 gene was stably transfected into panc1 cells with Lipofectamine2000. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western plotting. The cell growth activity was examined by MTT assay. Anchorage-independent growth was detected by colony formation assay in soft agar. Flow cytometry was used to analyze the cell cycle and apoptosis. Cell invasion assay was used for evaluating cell invasion capacity. The relative phosphorylation level of EGFR, c-Raf, Mek, Erk, p38 and SAPK/JNK were detected by Western blotting.</p><p><b>RESULTS</b>Three transfected cell clones overexpressing caveolin-1 were obtained. Comparing with the panc1 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of flow cytometry showed that over-expression of caveolin-1 resulted in the cell cycle arrest at G(0)/G(1) phase and increased the apoptotic cell fraction. Cell invasion assay showed that overexpression of caveolin-1 significantly inhibited the panc1 cell invasion. Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR, c-Raf, Mek and Erk while did not affect the activity of p38 and SAPK/JNK.</p><p><b>CONCLUSION</b>Over-expression of caveolin-1 inhibits the growth and invasion of pancreatic carcinoma cells in vitro. These phenotypes may be correlated with the inhibition of EGFR-c-Raf-Mek-Erk signaling pathway.</p>

Clonality and Ki-67 protein expression in gastric carcinoma and precancerous lesions / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clonality of gastric carcinoma and precancerous lesions and its relationship with Ki-67 protein expression.</p><p><b>METHODS</b>Formalin-fixed paraffin embedded tissues were collected from 174 cases of gastric endoscopic biopsies and surgical removed specimens. The lesional tissues were isolated by Laser Capture Microdissection. Methylation sensitive restriction enzyme (HpaII) digestion and polymerase chain reaction (PCR) were used to detect the clonality at the polymorphic human androgen receptor gene locus on the X chromosome. PCR products were analyzed by capillary electrophoresis using applied Biosystems 3730 DNA Analyzer. In addition, a two-step immunohistochemical staining EnVision method was used to detect the expression of Ki-67 protein.</p><p><b>RESULTS</b>The frequency of detection of monoclonality and expression rate of Ki-67 were found increased in a stepwise fashion from gastrointestinal metaplasia, low grade intraepithelial neoplasia, high grade intraepithelial neoplasia to intestinal carcinoma (15.63%, 5/32; 22.22%, 10/45; 69.44%, 25/36 and 100.0%, 20/20; respectively). The presence of clonal proliferation was correlated with Ki-67 expression in low grade intraepithelial neoplasia (P < 0.01).</p><p><b>CONCLUSIONS</b>The presence of clonal proliferation and increased Ki-67 are increasingly detected in the lesions along the multi-step gastric carcinogenesis model. Clonal status is associated with the expression rate of Ki-67 to a certain extent, suggesting a combined application of both markers may be useful in assessing early stages of gastric carcinoma.</p>

Chronic hepatitis B virus infection and the methylation status of p16INK4A promoter / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the relationship among HBV-associated histopathological indexes, x gene mutations and the methylation status of p16INK4A promoter in liver with chronic hepatitis B virus infection, in order to illustrate their role in p16INK4A hypermethylation and HCC progression.</p><p><b>METHODS</b>Twenty-three cases of surgically resected HBV-associated hepatocellular carcinoma and twenty-five fine needle aspiration biopsy cases of chronic hepatitis B were chosen for this study. The methylation status of the p16INK4A promoter in tumors, their corresponding peritumoral samples and chronic hepatitis B cases was determined by methylation-specific polymerase chain reaction (MSP). EnVision two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by real-time fluorescence quantitative PCR. Polymerase chain reaction and the direct sequencing method was used for mutation analysis of HBV x gene.</p><p><b>RESULTS</b>In peritumoral samples (P = 0. 025) and chronic hepatitis B cases (P = 0.029), the expression of HBx protein in methylated groups was all significantly higher than that in unmethylated groups of p16INK4A gene. But in tumors, there was no such significant difference. Other HBV antigens including HBsAg and HBcAg, tissue HBV DNA levels and point mutations of HBV x gene did not show a relationship with the methylation status of p16INK4A gene.</p><p><b>CONCLUSION</b>The data suggest that p16INK4A hypermethylation correlated closely with higher HBx expression in precancerous lesions. HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hepermethylation of p16INK4A promoter.</p>

Effects of caveolin-1 on biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line GU / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro.</p><p><b>METHODS</b>Eukaryotic expression vector of human caveolin-1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTT assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy.</p><p><b>RESULTS</b>The expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erkl/2.</p><p><b>CONCLUSION</b>Overexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.</p>

Inhibited effects of exogenous caveolin-1 on the growth of laryngeal squamous cell carcinoma cell line Hep-2, experiments in vitro and in vivo / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the effect of exogenous caveolin-1 on the growth of laryngeal squamous cell carcinoma and its mechanisms.</p><p><b>METHODS</b>Eukaryotic expression vectors containing human caveolin-1 gene were transfected into Hep-2 cell line, the positive clones with high expression of caveolin-1 were identified by fluorescence quantitative real time reverse transcriptase-polymerase chain reaction and Western Blot. Cell proliferation viability was tested by methyl thiazolyl tetrazolium assay, the protein expression of epidermal growth factor receptor (EGFR), P-EGFR, extracellular signal-regulated kinase 1, 2 (Erk1, 2), P-Erkl, 2 and caveolin-1 were detected by Western Blot. The combination of caveolin-1 and EGFR were studied by immunoprecipitation and Western Blot. The in vivo antitumor activity of caveolin-1 was tested in Hep-2 xenograft tumor models in athymic nude mice, and the protein expressions of P-EGFR, P-Erk1, 2 and caveolin-1 were examined by immunohistochemistry.</p><p><b>RESULTS</b>Three of caveolin-1 stably transfected Hep-2 cell clones were established. MTT assay showed that the proliferation of caveolin-1 overexpression Hep-2 cell clones decreased significantly comparing with the control. Immunoprecipitation and western Blot showed that caveolin-1 and EGFR were combined in Hep-2 cell line. Comparing with the parental cell line and cells transfected with control vector, there were the lower phosphorylation of EGFR and Erk1, 2 in the caveolin-1 overexpression Hep-2 cell clones. In the xenograft tumor models in nude mice, caveolin-1 overexpression Hep-2 cell clones showed the slower growth, smaller tumor size and the lower phosphorylation of EGFR and Erk1, 2.</p><p><b>CONCLUSIONS</b>Overexpression of caveolin-1 inhibits growth of Hep-2 cell line in vitro and in vivo, arresting EGFR-MAPK signal pathway may involve in its mechanism.</p>

Application of detecting gene rearrangement in diagnosing and typing of primary non-Hodgkin's lymphoma in nasal cavity / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To evaluate the significance of immunoglobulin heavy chain (IgH) gene rearrangement for B-cell lymphoma and T-cell receptor (TCR) gene rearrangement for T-cell lymphoma and NK/T-cell lymphoma in diagnosing and typing of primary non-Hodgkin's lymphoma (NHL) in nasal cavity.</p><p><b>METHODS</b>Semi-nested polymerase chain reaction ( PCR) with two pairs of primers was used to detect monoclonal IgH gene rearrangement in paraffin-embedded tissues from 11 patients with B-cell lymphoma, and one-stepped PCR with two pairs of primers was used to detect T-cell receptor gene rearrangement from 23 patients with NK/T-cell lymphoma and 20 patients with T-cell lymphoma. Ten patients with nasal polyp were detected with all the primers by PCR respectively.</p><p><b>RESULTS</b>Among the 54 patients with an evaluable PCR results, 10 of 11 (90. 9% ) B-cell lymphomas were positive for monoclonal IgH gene rearrangement, 17 of 20 (85. 0% ) T-cell lymphomas and 10 of 23 (43. 5% ) NK/T-cell lymphomas were positive for monoclonal TCR gene rearrangement. Ten patients with nasal polyp were negative for all detection.</p><p><b>CONCLUSIONS</b>Detecting gene rearrangement was an efficient method in auxiliary diagnosing and typing of primary NHL in nasal cavity; Using semi-nested PCR or one-stepped PCR with two pairs of primers can enhance the positive rate of gene rearrangement detection.</p>

Clonality study of palmar fibromatosis / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clonality of palmar fibromatosis by molecular genetic analysis of X chromosome inactivation pattern at a polymorphic site of human androgen receptor gene (HUMARA).</p><p><b>METHODS</b>Twelve female cases of palmar fibromatosis were enrolled into this study. Hematoxylin and eosin-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected by laser capture microdissection technology in order to obtain the proliferative spindle cells. Tumor cells isolated from rectal adenocarcinoma in a female patient were used as positive control. The genomic DNAs were extracted with phenol and chloroform, digested with methylation-sensitive restriction endonuclease HpaII, and amplified by polymerase chain reaction (PCR) using primers targeted to a highly polymorphic short tandem repeat of HUMARA. The amplimers were separated on vertical 8% non-denaturing polyacrylamide gels and the patterns were visualized with ethidium bromide stain.</p><p><b>RESULTS</b>The methodology for clonality analysis was validated in the positive control using rectal adenocarcinoma cells. Among the 12 cases studied, PCR amplification failed in 3 samples and 1 sample showed homozygosity which was not suitable for further analysis. Eight samples were successfully amplified and showed a random X chromosome inactivation pattern, suggesting polyclonality in these lesions.</p><p><b>CONCLUSIONS</b>Palmar fibromatosis is a polyclonal condition and should be considered as a form of non-neoplasmic fibroblastic proliferation.</p>

Overexpression of NDRG1: relationship with proliferative activity and invasiveness of breast cancer cell line and breast cancer metastasis / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between NDRG1 and metastasis of breast cancer and the effects of NDRG1 overexpression on the proliferation and invasion of breast cancer cells.</p><p><b>METHODS</b>NDRG1 was detected at its protein level by immunohistochemistry (IHC) and its messenger RNA (mRNA) was detected by real-time reverse transcriptase-polymerase chain reaction (real time RT-PCR) in clinical breast cancer specimens. Liposome was used to transiently transfer NDRG1 into MDA-MB-231, a highly invasive human breast cancer cell line. The proliferation of MDA-MB-231 was measured by Bromodeoxy Uridine (BrdU) incorporation assay and the transfection effect on cell cycle distribution was determined by fluorescence assisted cell sorting (FACS). The invasive ability of the transfected cells was investigated by reconstituted matrigel invasion and polycarbonate filters migration experiments.</p><p><b>RESULTS</b>NDRG1 expressions at protein and mRNA levels in tumors of patients with lymph node metastases were significantly lower as compared with those with localized breast cancers (P < 0.01). The amount of NDRG1 mRNA in MCF7, a relatively non-invasive breast cancer cell line, was 10.8 times higher than that in MDA-MB-231 cells (P < 0.01). The BrdU incorporation rate declined significantly (P < 0.05) in NDRG1 overexpressing MDA-MB-231 cells. An increase of the cell population at G(0)/G(1) phase was observed 48 hours post-transfection along with a decrease of cell population at S phase. Overexpression of NDRG1 significantly retarded the invasiveness of MDA-MB-231 cells in matrigel-coated invasion chambers (P < 0.05), when compared to cells transfected with control vectors. However, the migration abilities of cells with or without the transfection were virtually identical.</p><p><b>CONCLUSIONS</b>NDRG1 expression reversely correlates with breast cancer metastasis and progression, and may serve as a prognostic biomarker for predicting early metastasis. The inhibition of proliferation and invasion demonstrated by our MDA-MB-231 transfection experiments implies that NDRG1 is a tumor metastasis suppressor gene and may be a new candidate for gene therapy against human breast cancer.</p>

Expression and significance of hTERT mRNA in breast carcinoma and its relation to p53 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>This study was designed to investigate the significance of hTERT mRNA in breast carcinogenesis and to explore the diagnostic efficacy, and to study the effect of tumor suppressor gene p53 on the expression of hTERT mRNA.</p><p><b>METHODS</b>The expression of hTERT mRNA was examined by in situ hybridization in 12 cases of normal breast tissue nearby cancer, 7 of simple ductal hyperplasia, 20 of atypical hyperplasia, 18 of ductal carcinoma in situ and 25 with invasive ductal carcinoma. The expression of p53 protein were examined by immunohistochemistry in 43 carcinomas.</p><p><b>RESULTS</b>hTERT was not detected in normal breast tissue nearby cancer and simple ductal hyperplasia. The positive rate of hTERT mRNA in atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 25.0%, 83.3% and 88.0%, respectively. The prevalence and intensity of hTERT mRNA expression were much greater in carcinoma than those in simple or atypical hyperplasia and normal breast tissue nearby cancer (P < 0.05). The expression of hTERT was not correlated with tumor size and lymph node metastasis (P > 0.05). The positive correlation between hTERT mRNA and p53 was found in breast carcinoma (r = 0.5540, P < 0.01).</p><p><b>CONCLUSION</b>hTERT mRNA expression is closely related to the malignant transformation of breast tissue. Semi-quantitative detection of hTERT mRNA expression in situ is helpful in differentiated diagnosis of carcinoma in situ and atypical hyperplasia. Inactivation of p53 may play a role in the transcriptive activation of hTERT gene in breast carcinoma.</p>

The effect of HER2/neu overexpression on p53 gene expression, cell proliferation and sensitivity to gamma-irradiation via the PI3K/Akt pathway in breast cancer cell MCF7 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of HER2/neu overexpression on the wild p53 gene expression, cell proliferation and sensitivity to gamma-irradiation via phosphatidylinositol 3-kinase (PI3K) pathway in human breast cancer cell MCF7.</p><p><b>METHODS</b>Lipofectin-mediated gene transfection method was used to transfer HER2/neu into MCF7 cells. Expression of HER2/neu, p53, Akt and p-Akt protein after PI3K pathway inhibitor LY294002 treatment was determined by Western blot. Cell proliferation and cell surviving fraction after gamma-irradiation treatment were assayed by MTT.</p><p><b>RESULTS</b>Eighteen of HER2/neu stably transfected MCF7 cell clones were established, one of them was HER2/neu overexpressing. HER2/neu overexpressing MCF7 cells showed higher p-Akt expression and lower p53 expression than those of parental MCF7 cells, which could be abrogated by LY294002. HER2/neu overexpressing MCF7 cells had higher proliferation rate and lower sensitivity to gamma-irradiation than those of parental MCF7 cells, which could be opposed by LY294002.</p><p><b>CONCLUSION</b>Overexpression of HER2/neu induces reduced expression of wild-type p53 protein, relatively high cell proliferation and low sensitivity to gamma-irradiation in breast cancer cell MCF7 by activating PI3K/Akt pathway, which may contribute to therapeutic resistance in some breast cancer patients with wild-type p53 gene status.</p>

Correlation study of expression levels of prostate-specific membrane antigen and prostate-specific antigen with Gleason score of prostate carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the correlation of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) expression with Gleason score of prostate carcinoma.</p><p><b>METHODS</b>Monoclonal antibodies against epitopes of PSMA extracellular domain were prepared, with which the expression of PSMA of prostate carcinoma (PC) was determined by immunohistochemical staining. Correlation of its expression with Gleason score of PC was statistically analyzed, and compared with that of PSA.</p><p><b>RESULTS</b>Eight hybridoma cell lines secreting monoclonal antibodies specific for PSMA were prepared. PSMA expression level was positively correlated with Gleason score. In poorly differentiated prostate carcinoma, the expression intensity of PSMA was higher than that of medium-and well-differentiated prostate carcinoma (P < 0.01). However, there was no correlation between level of PSA expression and Gleason score (P > 0.05).</p><p><b>CONCLUSION</b>PSMA expression level may be used as a useful surrogate marker in Gleason grading of prostate carcinoma. It may be a more suitable target than PSA in antibody mediated immunotherapy against poorly differentiated prostate carcinoma which is usually not sensitive to hormonal therapy.</p>

Overexpression of HER2/neu downregulates wild p53 protein expression via PI3K and Ras/Raf/MEK/ERK pathways in human breast cancer cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of HER2/neu overexpression on wild p53 protein expression and to delineate the related signal pathways.</p><p><b>METHODS</b>Lipofectin method was used to transfer HER2/neu into human breast tumor cell line MCF7. Overexpression of HER2/neu was then determined by Western blot. Western blot was also used to detect the quantity of p53, Akt, p-Akt, p-Raf, p-MEK, p-ERK protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect p53 mRNA expression. PI3K pathway inhibitor LY294002 and MEK inhibitor U0126 were used to block the two pathways. The subsequent effect on p53 protein expression was then determined.</p><p><b>RESULTS</b>HER2/neu-overexpressed MCF7 clone (MCF7-neu3) was successfully established, in which the amount of HER2/neu protein was 13 times more than that in parental MCF7 cells. The amount of p53 protein in MCF7-neu3 was 40% of that in parental MCF7 cells (P < 0.01), while there was no difference on p53 mRNA level. There were 2.5, 2.0, 1.6 and 1.6 fold increase in the amount of p-Akt, p-Raf, p-MEK, p-ERK protein respectively in MCF7-neu3 to that in parental MCF7 cells (P < 0.01). When treated with LY294002 or U0126 for 24 hours, the amount of wild p53 protein in MCF7-neu3 cells was 1.7 or 1.5 times higher than those in DMSO treated cells. There were 4.7 or 5.3 times increase in the p53 protein when MCF7-neu3 cells were treated with LY294002 or U0126 for 48 hours (P < 0.01). Similar results were not seen in MDA-MB-453 cells which contained mutant p53.</p><p><b>CONCLUSIONS</b>HER2/neu overexpression can activate PI3K and Ras/Raf/MEK/ERK pathways, resulting in reduction of wild p53 protein expression. This may be the molecular mechanism responsible for the poor prognosis and therapeutic non-responsiveness in HER2/neu-overexpressed breast cancer patients.</p>