Emergence of clade 2.3.4.4b novel reassortant H5N1 high pathogenicity avian influenza virus in South Korea during late 2021.

High pathogenicity H5N1 avian influenza viruses pose a threat to both animal and human health worldwide. In late 2020, outbreaks of H5 high pathogenicity avian influenza viruses belonging to clade 2.3.4.4b emerged in Europe, following on from outbreaks in East Asia in earlier years. However, very recent studies show that clade 2.3.4.4b H5N1, rather than 2.3.4.4b H5N8, has become predominant in wild birds and has infected poultry in several countries. In this study, we describe isolation of a novel H5N1 virus from a captured mandarin duck in South Korea, and another H5N1 virus from a quail farm. We performed genetic analysis of these two viruses to identify their origin and to determine their relationship with the clade 2.3.4.4b H5N1 viruses currently circulating in Europe. Based on our results, it is presumed that the novel H5N1 virus isolated in Korea originated from an unknown reassortant between clade 2.3.4.4b H5N8 viruses circulating from 2020 and other Eurasian viruses, with additional reassortment of genes and point mutations that discriminate them from the recently reported H5N1 virus in Europe.

Lithium chloride promotes neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy / 中国病理生理杂志

AIM:To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process .METHODS:MSCs were isolated and cultured in vitro.The cells were divided into LiCl group and control group .MSCs were treated withβ-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons .The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associ-ated protein 1 light chain 3 ( LC3) were measured by immunofluorescence method and Western blot .An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells.The protein expression of NSE and MAP-2 were determined by Western blot .RESULTS: After induction, the ex-pression of NSE and MAP-2 were increased .The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05).After induction, the number of LC3-positive dots and the expression of LC3-Ⅱin LiCl group were greater than those in control group (P<0.05).The expres-sion of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated -cells, and on the contra-ry, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA.CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy .

[Bacteria community in different aged Coptis chinensis planting soil revealed by PCR-DGGE analysis].

In order to reveal the cause of disease occurred in the process of Coptis chinensis growth, this paper studied the bacterial species diversity index of different aged rhizospheric and non-rhizospheric soil planting normal or sick C. chinensis by using PCR-DGGE technique. The representative DGGE bands were chosen to be cloned, and sequenced, the phylogeny were constructed. The results showed that the bacterial communities were very different between the normal and diseased soil samples of C. chinensis, and the diversity index (H) of diseased soil samples were higher than that of normal soil samples. Sequencing analysis of representative cloned DGGE bands showed that the unculturable bacteria were the dominant groups, and bacteria belonged to genus Bacillus, Acidovorax, Acinetobacter, uncultured Kluyvera, and uncultured Comamonas were also existing, but the reported plant pathogenic bacteria were not found in the C. chinensis planting soil. The density and brightness of clone band d in diseased soil samples was higher than that in normal soil sample, and sequencing analysis showed that it belonged to genus Acidovorax. Obviously, during the process of C. chinensis growth, the rhizospheric bacteria population changed, and the quantity of bacteria belong Acidovorax increased, which probably resulted in the disease occurred during C. chinensis growth.

Ki-67 index as a prognostic factor of subsequent lapatinib-based therapy in HER2-positive metastatic breast cancer with resistance to trastuzumab.

Prognostic factor analysis has been conducted to determine whether the parameters of clinical data and biomarkers would predict differential progression-free survival (PFS) or overall survival (OS) from lapatinib-based therapy in patients with primary or acquired resistance to trastuzumab. Treatment with lapatinib plus capecitabine for HER2-positive metastatic breast cancer (MBC) with primary or acquired resistance to trastuzumab was analyzed retrospectively. Tumor biomarkers, which came from the biopsies before the starting of lapatinib therapy, were evaluated by immunohistochemistry (IHC). Prognostic factors related to PFS or OS of the lapatinib therapy were assessed by univariate and multivariate analysis. Ki-67 index and liver metastases were the significant prognostic factors for predicting PFS of subsequent lapatinib therapy in the univariate analysis and the multivariate analysis. The risk for disease progression in patients who had a Ki-67 index<40% was 59% less than that in patients had Ki-67 ≥ 40 (HR = 0.41, 95% CI, 0.23-0.74, P = 0.003). TTP of prior trastuzumab therapy, liver metastases, and the number of metastatic sites were three independent prognostic factors of subsequent lapatinib therapy. Ki-67 index was the significant prognostic factors for predicting PFS of the subsequent second line targeted therapy in patients with trastuzumab resistance.

PTEN mutation, methylation and expression in breast cancer patients.

The tumor suppressor gene, PTEN, has previously been demonstrated to be involved in breast tumorigenesis and tumor progression. The aim of the present study was to investigate the expression and significance of PTEN in breast carcinomas, to detect the mutation frequency of PTEN in sporadic breast carcinoma tissues and to determine the association between PTEN promoter methylation and gene expression. Immunohistochemical methods were used to analyze the expression of the PTEN gene in 146 cases of breast carcinoma and 10 cases of normal breast tissue closely adjacent to the carcinoma. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used to analyze conformation polymorphisms in 45 breast carcinoma and 10 normal breast tissues. Point mutations of abnormal single stranded conformation were detected by DNA sequencing. The methylation of the PTEN promoter was analyzed by methylation-specific PCR. Expression of PTEN was detected in 57.5% (84/146) of patients with breast carcinoma. By contrast, PTEN expression was detected in 100% of normal samples. Expression of PTEN was found to negatively correlate with the tumor size, the pathological stage and the expression of the estrogen receptor (ER) and the progesterone receptor (PR) in breast cancer. The 2-year disease-free survival of patients with a high expression of PTEN was higher compared with those with low PTEN expression (P<0.05). Missense mutations in exon 2 of PTEN were identified in 1/45 breast cancer cases. PTEN promoter methylation was detected in 31.1% (14/45) of breast carcinomas, of which 64.3% (9/14) were associated with a loss of PTEN expression. The tumor suppressor gene, PTEN, was abnormally expressed in the breast carcinomas. The number of PTEN mutations were low (1/45) in the sporadic breast cancer cases analyzed in the present study and PTEN promoter methylation may have been the main mechanism leading to the decreased expression of PTEN. These results indicate that PTEN is important for the tumorigenesis, development and prognosis of breast cancer.

[Analysis of the factors affecting pathologic complete response to neoadjuvant chemotherapy in breast cancer patients].

OBJECTIVE: To analyze the factors affecting pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) in breast cancer patients. METHODS: A retrospective cohort study was carried out to analyze the clinical data of 141 breast cancer patients treated with neoadjuvant chemotherapy. The factors affecting pCR and the changes of tumor receptor status before and after treatment were analyzed. RESULTS: Among all the 141 patients, 21 patients (14.9%) achieved pCR. The rate of pCR achieved by regimens of anthracycline combined with taxane was higher (16.8%, 19/113) than that by anthracycline-containing regimens (7.1%, 1/14). The dose intensity of anthracycline had a significant correlation with pCR rate (P < 0.05). The pCR rate in the relative dose intensity of taxane ≥ 0.85 arm was higher than that of < 0.85 arm (P = 0.02). Eighty patients (56.7%) had completed more than 4 cycles of chemotherapy and the median time to achieve pCR was 6 (3 to 10) cycles. The pCR rate had a significant difference between patients < 6 and ≥ 6 cycles (7.1% vs. 22.5%,P = 0.01). Multivariate analysis showed that tumor size measured by palpation ≤ 5 cm and ≥ 6 chemotherapy cycles were significantly related with pCR rate (P < 0.05). In all the 21 pCR patients, the pre-treatment ER(-), PR(-), HER-2(-) statuses were in 14, 14 and 17 patients, respectively. The status of ER, PR, HER-2 of most patients (74.2%, 69.7% and 87.7%, respectively) was not changed after treatment. Among the patients with changes in receptor status, ER changed from negative to positive was in the majority (37.1%, 13/35 vs. 12.9%, 4/31, P < 0.05), and the percentage of changes in PR and HER-2 status had no significant differences. CONCLUSIONS: The regimens of anthracycline combined with taxane can achieve a higher pCR rate. The lymph node and receptor status before therapy have no significant correlation with pCR. Patients who have primary tumor size ≤ 5 cm, ≥ 6 chemotherapy cycles and enough dose intensity are easier to achieve pCR. The receptor status before and after therapy should be determined, and according to any positive results, physicians can chose HER-2 targeted therapy and/or endocrine therapy after surgery to benefit the patients.

[Clinical observation of sunitinib treatment for refractory advanced breast cancer ulcer].

OBJECTIVE: To observe the preliminary efficacies and adverse events of sunitinib in the treatment of metastatic breast cancer ulcer. METHODS: From December 2008 to May 2010, patients with advanced breast cancer ulcer took a single sunitinib. The dosage was adjusted on the basis of adverse events. And clinical response was evaluated. RESULTS: Nine patients with advanced breast cancer ulcer finished the treatment. The objective response and the clinical benefit time to progression of sunitinib were 3 and 7 patients with metastatic breast cancer ulcer, and the median time to progression (TTP) was 2.0 months. The most common adverse events included fatigue, hand-foot syndrome, neutropenia, thrombocytopenia and hypertension. CONCLUSION: Single-agent sunitinib treatment of refractory advanced breast cancer ulcer has marked efficacies. However, neutropenia, thrombocytopenia and hypertension are the major dose-limited toxicities.

[Efficacy and prognostic analysis of retreatment with trastuzumab-based therapy after multi-line targeted therapy resistance in HER2-positive metastatic breast cancer].

OBJECTIVE: To evaluate the predictive factors for efficacy and prognosis of retreatment trastuzumab in the patients with HER2 positive metastatic breast cancer (MBC) developing successive resistance to multi-line targeting therapies. METHODS: The data of 29 patients with HER2 positive MBC were collected from July 2008 to July 2010 at our department. All patients were treated with trastuzumab, lapatinib and retreated with trastuzumab sequentially. Twenty-one patients progressed during the initial trastuzumab therapy. All patients were treated with lapatinib to disease progression and retreated with trastuzumab to disease progression or death subsequently. A Log-rank test was used for univariate analysis and a Cox regression model was employed for multivariate analysis. RESULTS: The efficacy showed no significant difference between the patients with progression or those without progression during the initial trastuzumab therapy. The time-to-progression (TTP) of prior lapatinib therapy was an influencing factor of median progression-free survival (PFS) (P < 0.0001) and the duration from discontinuation of lapatinib to trastuzumab retreatment an influencing factor of median overall survival (OS) (P = 0.008) of trastuzumab retreatment in our univariate analysis. The median PFS of trastuzumab retreatment for patients with TTP of lapatinib therapy > 12 weeks (hazard ratio (HR) = 0.02, P = 0.003) or whose duration of double trastuzumab treatment ≤ 1 year (HR = 0.26, P = 0.03) was significantly prolonged in multivariate analysis. Meanwhile, the death risk of patients whose duration from discontinuation of lapatinib to trastuzumab retreatment ≤ 4 weeks decreased 89% as compared with trastuzumab retreatment (HR = 0.11, P = 0.004). CONCLUSION: TTP of prior lapatinib therapy and the duration of double trastuzumab treatment are two predictive factors of PFS of trastuzumab retreatment. And the duration from discontinuation of lapatinib to trastuzumab retreatment is an important independent prognostic factor for trastuzumab retreatment. The patients with HER2 positive MBC should be treated continually with anti-HER2 targeted therapy.

ß-methasone-containing biodegradable poly(lactide-co-glycolide) acid microspheres for intraarticular injection: effect of formulation parameters on characteristics and in vitro release.

A sustained drug release system based on the injectable poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with ß-methasone was prepared for localized treatment of rheumatic arthritis. The microscopy and structure of microspheres were characterized by scanning electron microscope (SEM) and Fourier transform infrared (FTIR). The effects of various formulation parameters on the properties of microspheres and in vitro release pattern of ß-methasone were also investigated. The results demonstrated that increase in drug/polymer ratio led to increased particle size as well as drug release rate. Increase in PLGA concentration led to increased particle size, but decreased burst release. The drug encapsulation efficiency increased sharply by increasing polyvinyl alcohol (PVA) concentration in the aqueous phase from 1.5 to 2.0%. ß-methasone release rate decreased considerately with decreasing OP (organic phase)/AP (aqueous phase) volume ratio. Stirring rate had significantly influence on the particle size and encapsulation efficiency. Independent of formulation parameters, ß-methasone was slowly released from the PLGA microspheres over 11 days. The drug release profile of high drug loaded microspheres agree with Higuchi equation with a release mechanism of diffusion and erosion, that of middle drug loaded microspheres best agreed with Hixcon-Crowell equation and controlled by diffusion and erosion as well. The low drug loaded microspheres well fitted to logarithm normal distribution equation with mechanism of purely Fickian diffusion.

[Chinese HER2 positive early breast cancer trastuzumab adjuvant therapy: preliminary outcomes].

OBJECTIVE: To evaluate the efficacy and safety of 1-year adjuvant trastuzumab (herceptin) versus 1-year non-trastuzumab observation in Chinese patients with HER2-positive early breast cancer during a median follow-up of 1 year. METHODS: The HERA trial was an international, multicenter, randomized, open-label, phase III trial comparing treatment with trastuzumab for 1 and 2 years with observation after standard adjuvant chemotherapy, radiotherapy or both in patients with HER2-positive node-positive or high-risk node-negative early breast cancer. The primary endpoint was disease-free survival. Secondary end points included recurrence-free survival, distant disease-free survival, overall survival and cardiac safety. The first planned interim analysis comparing the efficacy and safety of treatment with trastuzumab for 1 year versus observation were completed in April 2005. Only the outcomes of recruited Chinese patients were reported. RESULTS: A total of 122 Chinese patients from 8 participating centers were included for planned interim analysis. And they were divided into trastuzumab (n = 68) and observation (n = 54) groups. Three and eight disease-free survival events were observed in the trastuzumab and observation groups respectively. Two-year disease-free survival rates were 92.9% and 81.4% respectively (P = 0.0489); 2-year recurrence-free survival and distant disease-free survivals were 98.1% vs 81.4% (P = 0.0064) and 98.1% vs 83.3% (P = 0.0117) respectively. Trastuzumab was generally well-tolerated with a decent safety profile. Severe cardiotoxicity was not observed. CONCLUSION: One-year treatment with adjuvant trastuzumab improves disease-free survival, recurrence-free survival and distant disease-free survival in Chinese patients with HER2-positive early breast cancer.

[A clinical study of reasonable doses of docetaxel salvage therapy for patients with metastatic breast cancer].

OBJECTIVE: To evaluate the correlation of clinical effects and reasonable doses of docetaxel salvage therapy for patients with metastatic breast cancer. METHODS: We reviewed retrospectively the clinical records of patients with metastatic breast cancer treated with docetaxel and statistically analyzed the correlation between clinical effects and reasonable doses of docetaxel. RESULTS: The objective response rate and clinical benefit rate of docetaxol in patients with metastatic breast cancer were 27.0% and 35.0%, respectively, and the median progression free survival (PFS) was 5.0 (3.8 - 6.3) months. In the analysis at a single dose level, the clinical benefit rate and PFS of the ≥ 90.0 mg/m(2) docetaxel group were superior to that of the < 90.0 mg/m(2) group (P = 0.008, P = 0.045). Multi-dose level group stratified analysis showed that the docetaxel < 75.0 mg/m(2) group was better than the 75.0 - 84.9 mg/m(2) PFS group (P = 0.018), and the ≥ 95.0 mg/m(2) group was better than the 75.0 - 84.9 mg/m(2) group (P = 0.048). In patients who received >third line treatment or previously received paclitaxel adjuvant therapy, the PFS of the ≥ 94.9 mg/m(2) docetaxel group was 6.0 months, better than the 3.0 months of the 75.0 ∼ 84.9 mg/m(2) group (P = 0.031; P = 0.021). CONCLUSION: There is a clear correlation between clinical effects and reasonable doses of docetaxel salvage therapy in patients with metastatic breast cancer.

Evaluation of treatment response for breast cancer: are we entering the era of "biological complete remission"?

Breast cancer is one of the most common malignancies in women. The post-operative recurrence and metastasis are the leading causes of breast cancer-related mortality. In this study, we tried to explore the role of circulating tumor cell (CTC) detection combination PET/CT technology evaluating the prognosis and treatment response of patients with breast cancer; meanwhile, we attempted to assess the concept of "biological complete remission" (bCR) in this regard. A 56-year-old patient with breast cancer (T(2)N(1)M(1), stage IV left breast cancer, with metastasis to axillary lymph nodes and lungs) received 6 cycles of salvage treatment with albumin-bound paclitaxel plus capecitabine and trastuzumab. Then, she underwent CTC detection and PET/CT for efficacy evaluation. CTC detection combination PET/CT is useful for the evaluation of the biological efficacy of therapies for breast cancer. The bCR of the patient appeared earlier than the conventional clinical imaging complete remission and promised the histological (pathological) complete remission. The integrated application of the concepts including bCR, imageological CR, and histological CR can achieve the early and accurate assessment of biological therapeutic reponse and prognosis of breast cancer.

[Analysis of chemotherapeutic efficacies in metastatic triple-negative breast cancer].

OBJECTIVE: To explore the chemotherapeutic efficacies and prognostic factors of metastatic triple-negative breast cancer. METHODS: The clinicopathologic data of 151 patients with metastatic triple-negative breast cancer were collected from September 1994 to November 2011 and their clinicopathologic characteristics, recurrence and survival were analyzed. RESULTS: Platinum plus taxol or vinorelbine was significantly higher than others for these patients (42.1% vs 23.1%, P = 0.022). The median overall survivals of those on first-line chemotherapy with partial remission, stable disease and progressive disease were 29.6, 24.7 and 13.1 months respectively. The differences were statistically significant (P = 0.045). Two or three-line chemotherapy showed no obvious statistical relationship with total overall survival. Simple factor analysis showed that the number of metastasis, visceral metastases and the efficacies of first-line chemotherapy were correlated with overall survival (all P < 0.05). Multivariate Cox regression showed that disease-free survival and the efficacies of first-line chemotherapy were the independent prognostic factors of metastatic triple-negative breast cancer. CONCLUSION: The combined chemotherapy of platinum may achieve better efficacies in the treatment of metastatic triple-negative breast cancer. And the efficacies of first-line chemotherapy are closely correlated with survival.

Gene expression profiling for breast cancer prognosis in Chinese populations.

To investigate a quantitative reverse transcription polymerase chain reaction (QRT-PCR) assay different from 21-gene assay which can be used to prognosticate the risk of recurrence in patients with estrogen receptor (ER) positive, lymph node (LN) negative breast cancer. To accurately determine the relationship between the Recurrence Score (RS) derived from our assay and the risk of distant recurrence in Chinese patients with LN negative and positive breast cancer through the analysis of paraffin tissues. We obtained archival paraffin-embedded tissues from patients with invasive breast cancer and varying axillary lymph node involvement. QRT-PCR reaction was performed by using the method of SYBR Green I dye with primers. Expression of the 21-genes was converted to RS by a prespecified algorithm. We then assessed the probability of the test to accurately predict distant recurrence-free survival in this retrospective cohort. Ninety-three patients were eligible based on gene expression profiles. In our population, most breast cancer patients were premenopausal (82.6%), at early stage (93.6%) and ER positive (91.4%). Median follow-up was 65.9 months. The 5-year recurrence-free survival rate for the group was 58.8%. The concordance between the reverse transcription-PCR and immunohistochemical (IHC) measurement for ER, progesterone receptor (PgR), and HER-2 determinations was high and comparable. High RS was predictive of an elevated risk of relapse (p < 0.001). In subgroups of patients, RS had significantly predictive performance both in node-negative (p = 0.009) and node-positive patients (p = 0.038). Multivariable analysis showed that nodal status, adjuvant hormonal therapy and RS were significantly related to prognosis. RS category is a better predictor than the other risk assessment criteria or clinicopatholic features, with which we can determine more accurately the risks for recurrence of various patients. We have established an easy and economical QRT-PCR assay and validated in concordance with IHC measurements for ER, PgR, and HER-2. RS was associated with distant recurrence among Chinese patients with hormone receptor (HR) positive breast cancer. This study may promote the use of RS estimated from the expression of the 21-gene set for prognostication and routine clinical diagnostic application in Chinese populations.

[Efficacy and safety of regimens of capecitabine-based chemotherapy in the treatment of advanced breast cancer].

OBJECTIVE: To evaluate retrospectively the efficacy and toxicity of capecitabine-based chemotherapy in the treatment of advanced breast cancer. METHODS: Three hundred and seventy-six patients with advanced breast cancer were treated with capecitabine-based chemotherapy regimens in our department from Sep 2002 to Sep 2009. They were divided into 3 groups. The group 1 was treated with capecitabine 1000 mg/m(2) orally twice daily on d1-d14, repeated every 3 weeks. The group 2 was treated with capecitabine as group 1, and combined with docetaxel 60 - 75 mg/m(2) intravenous infusion on d1, repeated every 3 weeks. The group 3 was treated with capecitabine as group 1, and combined with vinorelbine 25 mg/m(2) intravenous infusion on d1 and d8, repeated every 3 weeks. The median treatment period of treatment was 3 cycles. RESULTS: Among the 376 patients, 218 patients were evaluable for response. In the group 1 the objective response rate (ORR) was 12.8% and the clinical benefit rate (CBR) was 21.6%. The CBR but not ORR of first line therapy with capecitabine was 35.2%, significantly higher than that of more than first line therapy (17.1%, P < 0.01). The ORRs for group 2 and group 3 were 53.8% and 36.4%, respectively. In the group 2 there was no significant difference in the ORR between the first line therapy and more than first line therapy. In the group 3 the ORR of first line therapy of NX regimen was 36.4%, significantly higher than that of more than first line therapy (16.7%, P < 0.01). CONCLUSIONS: The capecitabine-based chemotherapy is effective and tolerable, and can be used not only in first line but also more than first line therapy. The single agent maintenance chemotherapy after response to combined chemotherapy can prolonge the duration of treatment for patients with metastatic breast cancer.

The sequence-dependent cytotoxic effect of trastuzumab in combination with 5-Fluorouracil or cisplatin on gastric cancer cell lines.

The effect of trastuzumab on patients with HER-2/neu (HER2)-positive gastric cancer has been confirmed in a phase III clinical trial (ToGA study). However, the optimized sequence and synergic mechanism of trastuzumab and chemotherapy are not clear. Our study investigated the effects and mechanisms of trastuzumab in combination with 5-Fluorouracil (5-Fu) or cisplatin (DDP) on gastric cancer cell lines. Flow cytometry was used to determine HER2 expression and cell cycle. MTT assay was performed to evaluate cytotoxicity. Western blotting and RT-PCR were used to analyze signaling transduction and mRNA expression. Sequential 5-Fu followed by trastuzumab and trastuzumab plus DDP followed by trastuzumab produced the best inhibitory effects. Inhibition of HER2-PI3K-AKT signal transduction, downregulation of nucleotide excision repair cross-complementation 1 (ERCC1), and interference with cell cycle distribution may elucidate the synergism between trastuzumab and chemotherapy. These results provide some evidence for designing a rational regime when trastuzumab is being considered to be used in patients with gastric cancer.

[Expression of Gemcitabine-resistance-related gene and polymorphism of ribonucleotide reductase M1 gene promoter in Gemcitabine-resistant A549/Gem and NCI-H460/Gem cell lines].

OBJECTIVE: To assay the expression of cytidine deaminase (CDA), ribonucleotide reductase subunit 1 (RRM1), phosphatase and tensin homologue deleted from chromosome 10 (PTEN), excision repair cross-complementation group 1 (ERCC1), deoxycytidine kinase (dCK) and RRM1(-)37A/C polymorphism, which have been shown relevant to gemcitabine resistance in two human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem, so as to make clear how do they vary during the course of acquiring resistance to gemcitabine. METHODS: The human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem were established in our Department by repeated clinical serum peak concentration and gradually increasing doses. Real-time fluorescent quantitative PCR was used to examine the expression of CDA, RRM1, PTEN, ERCC1, dCK and RRM1(-)37A/C polymorphism in those cell lines at different time points during their induction process. RESULTS: The resistance indexes of A549/Gem and NCI-H460/Gem cells reached 163.228 and 181.684, and then remained stable at 115.297 and 129.783, respectively. The expression of CDA, RRM1, PTEN and ERCC1 varied along with the changing gemcitabine resistance indexes, but expression of dCK did not change apparently. The wild type promoter was able to amplify the genomic DNA in different induction stages of A549/Gem and NCI-H460/Gem cells, but allelotype did not, indicating that the gene type of A549/Gem, NCI-H460/Gem and their parental cells remaining still wild type. CONCLUSION: Compared with their parental cells, the expressions of CDA, RRM1, PTEN and ERCC1 in human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem rise, the expression of dCK changes inapparently, therefore, their gene type are remaining wild type.

[Combination with SN-38 on human colon cancer LoVo cells].

OBJECTIVE: To observe the anti-proliferation effect of bevacizumab and SN-38 (active metabolite of irinotecan), and investigate the possible mechanisms of these two agents. METHODS: Human colon cancer LoVo cells were cultured under hypoxic conditions. Inhibition of cell proliferation was evaluated by MTT assay. The drug modulation on HIF-1alpha, VEGF, ERK and AKT were assessed by the following assays. The mRNA expression of HIF-1alpha and VEGF were measured by RT-PCR. The protein expression of HIF-1alpha, ERK and AKT were evaluated by Western blot analysis, and VEGF by ELISA assay. RESULTS: Among different combination schedules, Bevacizumab given after SN-38 show most synergistic anti-proliferation effect. Under hypoxic conditions, the expression of HIF-1alpha and VEGF increased as time accumulated, Bevacizumab combined with SN-38 almost completely inhibited the expression of HIF-1alpha and VEGF. Moreover, the MAP kinase pathway was involved in the drug modulation of HIF-1alpha and VEGF. CONCLUSION: These findings suggest the anti-proliferation effect of bevacizumab and SN-38 was schedule-dependent, and the synergistic effect of Bevacizumab and SN-38 was related to drug modulation of the HIF-1alpha and MAP kinase pathway.