OBJECTIVE: To investigate the influence of Ku80 inhibition on the chemotherapeutic sensitivity of the T-acute lymphoblastic leukemia(T-ALL) cell line Jurkat, and to explore the potential mechanism. METHODS: The transcription and expression level of Ku80 in 6 hematological malignant cell lines were detected by RT-qPCR and Western blot, respectively. The expression of Ku80 in Jurkat cells was detected by Western blot after transfection with the recombinant shKu80 lentiviral vector. The proliferation capacity of Jurkat cells was explored by CCK-8 after Ku80 inhibition. The colony formation ability, apoptosis, and γH2AX(a protein marker of DNA damage) expression in Jurkat cells were investigated after Ku80 silencing and co-treated with etoposide(VP16) for 4 hours through soft agar assay, flow cytometry and Western blot, respectively. RESULTS: The mRNA level and protein expression of Ku80 were both highest in Jurkat among 6 hematological malignant cell lines. Ku80 expression was successfully down regulated in Jurkat cells after relative plasmid transfected. The proliferative ability of cells was significantly decreased after Ku80 inhibition(P < 0.05). The colony formation capacity of Jurkat cells was obviously reduced and the cells apoptosis and γH2AX expression were increased after Ku80 inhibition, with or without VP16 incubation. CONCLUSION Targeted silencing of Ku80 could enhance the sensitivity of VP16 in Jurkat cells, which might be associated with the elevated level of DNA damage accumulation.
Humans ; Ku Autoantigen/metabolism* ; Jurkat Cells ; Apoptosis/drug effects* ; Cell Proliferation ; Etoposide/pharmacology* ; DNA Damage ; Cell Line, Tumor ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma

Extracellular matrix (ECM) has been implicated in tumor progress and chemosensitivity. Ovarian cancer brings a great threat to the health of women with a significant feature of high mortality and poor prognosis. However, the potential significance of matrix stiffness in the pattern of long non-coding RNAs (lncRNAs) expression and ovarian cancer drug sensitivity is still largely unkown. Here, based on RNA-seq data of ovarian cancer cell cultured on substrates with different stiffness, we found that a great amount of lncRNAs were upregulated in stiff group, whereas SNHG8 was significantly downregulated, which was further verified in ovarian cancer cells cultured on polydimethylsiloxane (PDMS) hydrogel. Knockdown of SNHG8 led to an impaired efficiency of homologous repair, and decreased cellular sensitivity to both etoposide and cisplatin. Meanwhile, the results of the GEPIA analysis indicated that the expression of SNHG8 was significantly decreased in ovarian cancer tissues, which was negatively correlated with the overall survival of patients with ovarian cancer. In conclusion, matrix stiffening related lncRNA SNHG8 is closely related to chemosensitivity and prognosis of ovarian cancer, which might be a novel molecular marker for chemotherapy drug instruction and prognosis prediction.
Female ; Humans ; Cisplatin/pharmacology* ; Elasticity/physiology* ; Etoposide ; Extracellular Matrix/physiology* ; Ovarian Neoplasms/metabolism* ; RNA, Long Noncoding/metabolism*

BACKGROUND: Small cell lung cancer (SCLC) is characterized by poor differentiation, high malignancy and rapid growth fast, short double time, early and extensive metastatic malignancy. In clinical, chemotherapy is the main treatment method, while resistance to multiple chemotherapy drugs in six to nine months has been a major clinical challenge in SCLC treatment. Therefore, It has important clinical value to building SCLC aninimal model which is similar to patients with SCLC. Animal model of xenotransplantation (PDX) from the patients with small cell lung cancer can well retain the characteristics of primary tumor and is an ideal preclinical animal model. The study is aimed to establish SCLC PDX animal model and induce the chemoresistance model to help to study the mechanism of chemoresistance and individual treatment. METHODS: Fresh surgical excision or puncture specimens from SCLC patients were transplanted into B-NSGTM mice subcutaneous tissues with severe immunodeficiency in one hour after operation the B-NSGTM mice subcutaneous in 1 hour, and inject chemotherapy drugs intraperitoneally after its tumor growed to 400 mm³ with EP which is cisplatin 8 mg/kg eight days and etoposide 5 mg/kg every two days until 8 cycles. Measure the tumor volum and mice weights regularly, then re-engrafted the largest tumor and continue chemotherapy. RESULTS: Nine cases were conducted for B-NSG mice modeling. Three of nine cases could be engrafted to new B-NSG mice at least two generation. The SCLC PDX animal models have been established successfully. After adopting chemotherapy drugs, the chemoresistance PDX models have been established. High homogeneity was found between xenograft tumor and patient's tumor in histopathology, immunohistochemical phenotype (Syn, CD56, Ki67). CONCLUSIONS The SCLC PDX animal model and the chemoresistance PDX animal model have been successfully constructed, the success rate is 33%, which provides a platform for the clinical research, seeking for biological markers and choosing individual treatment methods of SCLC.
Animals ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cisplatin ; administration & dosage ; Disease Models, Animal ; Drug Resistance, Neoplasm ; Etoposide ; administration & dosage ; Female ; Humans ; Interleukin Receptor Common gamma Subunit ; deficiency ; genetics ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Small Cell Lung Carcinoma ; drug therapy ; metabolism ; pathology ; Transplantation, Heterologous ; methods ; Xenograft Model Antitumor Assays

OBJECTIVE: To investigate the effects of chemotherapeutic agents widely used in clinical practice on major histocompatibility complex class I-related chain A and B (MICA/B) expression in breast cancer cells, and to explore the molecular mechanisms involved. METHODS: We examined MICA/B mRNA and surface protein expressions in breast cancer cells treated with chemotherapeutic agents by real-time RT-PCR and flow cytometry respectively. The blocking effects of ataxia telangiectasia mutated and Rad3-related kinase (ATM/ATR) inhibitor caffeine and nuclear factor κB (NF-κB) inhibitor pynolidine dithiocarbamate (PDTC) on etoposide-upregulated MICA/B mRNA and surface protein expressions were investigated. Electrophoretic mobility shift assay (EMSA) was taken to investigate whether etoposide enhanced the binding of NF-κB to MICA/B gene promoter. RESULTS: Three topoisomerase inhibitors etoposide, camptothecin and doxorubicine upregulated MICA and MICB mRNA expressions in breast cancer cell MCF-7. Comparing to no-drug-treated cells, MICA mRNA levels increased to (1.68±0.17), (2.54±0.25) and (3.42±0.15) fold, and levels of MICB mRNA increased to (1.82±0.24), (1.56±0.05) and (5.84±0.57) fold respectively in cancer cells treated by etoposide at the concentrations of 5, 20 and 100 μmol/L (P<0.05). MICA and MICB mRNA levels also increased significantly when MCF-7 cells were incubated with camptothecin or doxorubicine at the specific concentrations (P<0.05). MICB mRNA expression also increased slightly in another breast cancer cell SK-BR-3 treated by topoisomerase II inhibitors etoposide and camptothecin (P<0.05). Furthermore, etoposide and camptothecin upregulated MICA/B surface protein expression in MCF-7 cells (P<0.05), and the upregulation was found in both living and apoptotic cells. Our study showed that etoposide induced-MICA/B expression in MCF-7 was inhibited by caffeine at different concentrations. When cancer cells were treated by caffeine with 1, 5 and 10 mmol/L, MICA mRNA levels decreased from (3.75±0.25) to (0.89±0.05), (0.81±0.02) and (0.48±0.04) fold respectively (P<0.001), and MICB mRNA levels decreased from (6.85±0.35) to (1.36±0.13), (0.76±0.06) and (0.56±0.03) fold (P<0.05), while MICA/B protein levels decreased from (3.42±0.05) to (1.32±0.03), (1.21±0.06) and (1.14±0.03) fold (P<0.001), indicating that etoposide-induced MICA/B expression was inhibited by ATM/ATR inhibitor. Similarly, NF-κB inhibitor PDTC also inhibited MICA/B mRNA and protein expressions induced by etoposide significantly when MCF-7 cells were incubated with PDTC at the concentrations of 10, 50 and 100 μmol/L (P<0.05), indicating that NF-κB was also involved in this process. EMSA showed that the binding of NF-κB to MICA/B promoter enhanced in MCF-7 cells after etoposide treatment. CONCLUSION Topoisomerase inhibitor increased MICA/B mRNA and protein expressions in breast cancer cells, indicating that chemotherapeutic agents might increase the recognizing and killing ability of immunocytes to breast cancer cells. ATM/ATR and NF-κB pathways might be involved in it.
Antineoplastic Agents/pharmacology* ; Ataxia Telangiectasia Mutated Proteins/physiology* ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Doxorubicin ; Etoposide/pharmacology* ; Histocompatibility Antigens Class I ; Humans ; I-kappa B Proteins ; NF-kappa B/physiology* ; RNA, Messenger ; Topoisomerase Inhibitors ; Up-Regulation

OBJECTIVETo investigate the changes of ID1 expression in tumor cells treated with etoposide, cisplatin and ultraviolet (UV) irradiation, and explore the effect of ID1 on chemotherapeutic drug- and UV-induced apoptosis.

METHODSIn the present study, upon onset of apoptosis induced by various kinds of inducers such as etoposide, cisplatin and UV irradiation, the expression level of ID1 was detected by Western blot and real-time PCR. We also analyzed the half-life of ID1 protein and stability of ID1 mRNA respectively by cycloheximide inhibition test and RT-PCR. Annexin-V assay was carried out to evaluate the contribution of ID1 protein to chemotherapeutic drug- and UV-induced apoptosis.

RESULTSID1 expression presented a profound down-regulation in the HCT116 cells treated with etoposide, cisplatin and UV irradiation(P<0.05 for all). The apoptosis in the UV irradiation group, cisplatin group, etoposide group was (58.70±1.55)%, (35.80±0.92)% and (21.00±0.72)%, respectively, significantly higher than that of the control group(1.10±0.07)%, (1.20±0.13)% and (3.50±0.23)% (P<0.05 for all). Upon etoposide treatment, ID1 expression level was decreased via induction of mRNA instability, but not the protein degradation changes. Additionally, ectopic expression of ID1 in the HCT116 cells alleviated etoposide-, cisplatin- and UV-induced apoptosis. The results of flow eytometry revealed that the percentage of apoptotic cells in the ID1 group under the treatment of etoposide, cisplatin and UV irradiation was (23.80±0.82)%, (17.80±1.34)% and (13.40±0.53)%, respectively, significantly lower than that in the empty vector group (41.10±1.61)%, (30.40±2.67)% and (22.50±3.47)% (P<0.05 for all).

CONCLUSIONSThese observations indicate that the treatment with etoposide reduces the amount of ID1 by induction of mRNA instability, and exogenously introduced ID1 protects cells against etoposide-, cisplatin- and UV irradiation-induced apoptosis. Inhibition of the ID1 bioactivity may become a new strategy in cancer treatment.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Cisplatin ; pharmacology ; Down-Regulation ; Etoposide ; pharmacology ; HCT116 Cells ; drug effects ; metabolism ; radiation effects ; Half-Life ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Ultraviolet Rays

OBJECTIVETo investigate the role of RLIP76 in regulating multi-drug resistance in small cell lung cancer (SCLC), and to analyze the relationship between its expression and prognosis.

METHODSThe expressions of RLIP76 protein and gene were detected by Western blotting and real-time PCR (RT-PCR) in both the chemosensitive SCLC H69 cell line and chemoresistant H69AR cell line, respectively. siRNA was transfected into the H69AR cells to inhibit RLIP76 expression, and eGFP-RLIP76 was transfected into the H69 cells to enhance RLIP76 expression. The drug-sensitivity of cells to chemotherapeutic drugs (ADM, DDP, VP-16) were detected by CCK8 assay. The expression of RLIP76 in the SCLC tissues was detected by immunohistochemistry. The relationship of RLIP76 expression with clinicopathological features and prognosis of the patients was analyzed.

RESULTSThe expression of RLIP76 in H69AR cells was 13.675 ± 0.983, significantly higher than 1.074 ± 0.107 in the H69 cells (P < 0.01). The drug-sensitivities of H69AR cells to chemotherapeutic drugs were significantly increased when the expression of RLIP76 was down-regulated (P< 0.001). The sensitivities of H69 cells to chemotherapeutic drugs ADM, DDP and VP-16 were significantly decreased after transfection with eGFP-RLIP76 up-regulating the RLIP76 expression (P = 0.003). The positive expression rates were 61.3% and 9.4% in the SCLC tumor tissues and para-cancerous tissues, respectively (P < 0.01). The expression of RLIP76 was significantly correlated with clinical stage, chemosensitivity and overall survival of the SCLC patients (P < 0.05).

CONCLUSIONSOur results suggest that RLIP76 is involved in the regulation of small cell lung cancer multidrug resistance. RLIP76 may serve as a potential target gene to evaluate the chemosensitivity and clinical prognostic for small cell lung cancer.

ATP-Binding Cassette Transporters ; metabolism ; physiology ; Antineoplastic Agents ; pharmacology ; Cisplatin ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Etoposide ; pharmacology ; GTPase-Activating Proteins ; metabolism ; physiology ; Humans ; Lung Neoplasms ; drug therapy ; metabolism ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; drug therapy ; metabolism ; Transfection ; Up-Regulation

OBJECTIVETo investigate the role of SALL4 in regulating multi-drug resistance in small cell lung cancer (SCLC), and to evaluate its clinical significance.

METHODSThe expression of SALL4 protein and gene was detected by Western blot and real-time PCR (RT-PCR) in both H69 and H69AR cell lines, respectively. SALL4 expression in H69AR was blocked by the siRNA, and then the drug-sensitivities of H69AR cell lines to chemotherapeutic drugs such as cisplatin, doxorubicin, and etoposide were evaluated by cell counting kit assay. SALL4 expression was also examined by immunohistochemistry, and correlated with patients' clinicopathological features and prognosis.

RESULTSThe expression of SALL4 was significantly increased in H69AR cells than in the H69 cells (P < 0.01). Down-regulation of SALL4 increased the drug-sensitivities of H69AR cells to chemotherapeutic drugs (P = 0.02). The expression of SALL4 was significantly increased in SCLC than in para-carcinoma tissues (P < 0.01). SALL4 expression correlated with clinical stage, chemosensitivity and overall survival (P < 0.05), but not with patients' age and gender.

CONCLUSIONSALL4 is involved in the regulation of multidrug resistance in SCLC; SALL4 may be a potential target gene to evaluate the chemosensitivity and clinical prognosis for SCLC.

Antineoplastic Agents ; pharmacology ; Cisplatin ; pharmacology ; Down-Regulation ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Etoposide ; pharmacology ; Humans ; Lung Neoplasms ; drug therapy ; metabolism ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; drug therapy ; metabolism ; Transcription Factors ; genetics ; physiology

OBJECTIVETo investigate the role of homeobox gene A5 (HOXA5) in multidrug resistance of human small cell lung cancer (SCLC) cells and the possibility of using HOXA5 as the therapeutic targets for SCLC treatment.

METHODSWe examined HOXA5 mRNA and protein expressions in chemosensitive human SCLC cells (H69) and the multidrug-resistant SCLC cells (H69AR) using quantitative real-time PCR and immunoblotting. HOXA5 expression was then enhanced or suppressed by transfection of the cells with HOXA5 expression plasmids or small interference RNA (siRNA), and the chemosensitivity of transfected cells to cisplatin (DDP) and etoposide (VP-16) was evaluated using cell counting kit-8 (CCK8) assay.

RESULTSH69 cells showed a 8.99-fold higher expression of HOXA5 than H69AR cells. HOXA5 knockdown caused obvious reductions in the chemosensitivity of H69 cells to DDP and VP-16 with increased cells in G0/G1 phase; conversely, HOXA5 enhancement resulted in an increased sensitivity of H69AR cells to DDP and VP-16.

CONCLUSIONHOXA5 may play an important role in multidrug resistance of SCLC and can be a potential therapeutic target in clinical treatment of SCLC.

Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cisplatin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Etoposide ; pharmacology ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Immunoblotting ; Lung Neoplasms ; metabolism ; pathology ; Plasmids ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; metabolism ; pathology ; Transfection

More than 95% of the thyroid carcinomas are well differentiated types showing favorable prognosis. However, only a few therapeutic options are available to treat the patients with undifferentiated thyroid carcinomas, especially with refractory thyroid carcinomas that are not amenable to surgery or radioiodine ablation. We investigated the anticancer effects of 20 chemotherapy and hormonal therapy drugs on 8 thyroid carcinoma cell lines. In vitro chemosensitivity was tested using the adenosine-triphosphate-based chemotherapy response assay (ATP-CRA). The tumor inhibition rate (TIR; or cell death rate) or half maximal inhibitory concentration (IC50) was analyzed to interpret the results. Of the 12 chemotherapy drugs, etoposide (178.9 index value in follicular carcinoma cell line) and vincristine (211.7 in Hurthle cell carcinoma cell line) were the most active drugs showing the highest chemosensitivity, and of the 8 additional drugs, trichostatin A (0.03 microg/mL IC50 in follicular carcinoma cell line) showed favorable outcome having the anticancer effect. In our study, the result of etoposide and vincristine show evidence as active anticancer drugs in thyroid carcinoma cell lines and trichostatin A seems be the next promising drug. These drugs may become an innovative therapy for refractory thyroid carcinomas in near future.
Adenosine Triphosphate/chemistry/pharmacology/therapeutic use ; Antineoplastic Agents/chemistry/*pharmacology/therapeutic use ; Apoptosis/drug effects ; Cell Line, Tumor ; Etoposide/chemistry/pharmacology/therapeutic use ; Humans ; Hydroxamic Acids/chemistry/pharmacology/therapeutic use ; Thyroid Neoplasms/drug therapy ; Vincristine/chemistry/pharmacology/therapeutic use

OBJECTIVETo investigate the changes of drug sensitivity of spindle poison-induced polyploid tumor cells to chemotherapeutic agents and its possible mechanism.

METHODSNocodazole in a dose of 100 ng/ml was used to induce polyploidization in a breast cancer cell line MDA-MB-231 cells. The polyploid cells (T-MDA-MB-231) were sorted by flow cytometry. The morphological changes and proliferation of T-MDA-MB-231 cells were compared with that of MDA-MB-231 cells. The cell growth inhibition was assessed by MTT assay. The cells were treated with paclitaxel, docetaxel, vincristine, epirubicin, 5-Fu, VP16 and oxaliplatin, respectively. Those cells were labeled with annexin V-FITC/PI and analyzed by flow cytometry. Bcl-2 was knocked down in T-MDA-MB-231 cells using SiRNA and their growth inhibition was evaluated by MTT assay to evaluate the reversing effect of Bcl-2-silencing on drug resistance.

RESULTSThe polyploid T-MDA-MB-231 cells grew in vitro continuously and maintained constant DNA content. They had a larger cell size, and grew more slowly than MDA-MB-231 cells. The IC(50(s)) of T-MDA-MB-231 cells were significantly higher than that of the MDA-MB-231 cells: paclitaxel: (6.37 ± 0.07) vs. (2.05 ± 0.83) µmol/L; docetaxel: (32.98 ± 1.48) vs. (11.95 ± 0.98) µmol/L; vincristine: (35.28 ± 1.66) vs. (14.58 ± 0.94) µmol/L; oxaliplatin: (19.07 ± 0.45) vs. (9.75 ± 1.05) µmol/L; 5-Fu: (85.49 ± 3.21) vs. (31.35 ± 1.51) µmol/L; and epirubicin: (0.53 ± 0.06) vs. (0.15 ± 0.01) µmol/L, (all P < 0.05). The IC(50(s)) of VP16 in T-MDA-MB-231 cells was (2.85 ± 0.50)µmol/L, significantly lower than the (12.20 ± 1.55) µmol/L in MDA-MB-231 cells (P < 0.05), and that of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (19.59 ± 0.48) µmol/L, significantly higher than the (12.20 ± 1.55) µmol/L in the MDA-MB-231 cells (P < 0.05). The IC(50(s)) of docetaxel of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (21.52 ± 0.68) µmol/L, significantly decreased and lower than that before Bcl-2 silencing (32.98 ± 1.48) µmol/L.

CONCLUSIONSOur results indicate that polyploid tumor cells induced by spindle poison Nocodazole are more resistant to most of chemotherapeutic drugs. Downregulation of Bcl-2 increases the sensitivity of polyploid cells to docetaxel. The high expression of Bcl-2 may be one of the drug resistance mechanisms of polyploid tumor cells. The polyploid tumor cells are relatively sensitive to VP16, suggesting that VP16 might be an effective candidate drug for treatment of chemoresistant polyploid tumors.

Antineoplastic Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Resistance, Neoplasm ; Epirubicin ; pharmacology ; Etoposide ; pharmacology ; Female ; Fluorouracil ; pharmacology ; Gene Knockdown Techniques ; Humans ; Inhibitory Concentration 50 ; Nocodazole ; pharmacology ; Organoplatinum Compounds ; pharmacology ; Paclitaxel ; pharmacology ; Polyploidy ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Taxoids ; pharmacology ; Vincristine ; pharmacology