Chemotherapeutic Drug Sensitivity Research Articles

Role of the forkhead transcription factor FOXO-FOXM1 axis in cancer and drug resistance

The forkhead transcription factors FOXO and FOXM1 have pivotal roles in tumorigenesis and in mediating chemotherapy sensitivity and resistance. Recent research shows that the forkhead transcription factor FOXM1 is a direct transcriptional target repressed by the forkhead protein FOXO3a, a vital downstream effector of the PI3K-AKT-FOXO signaling pathway. Intriguingly, FOXM1 and FOXO3a also compete for binding to the same gene targets, which have a role in chemotherapeutic drug action and sensitivity. An understanding of the role and regulation of the FOXO-FOXM1 axis will impact directly on our knowledge of chemotherapeutic drug action and resistance in patients, and provide new insights into the design of novel therapeutic strategy and reliable biomarkers for prediction of drug sensitivity.

Effects of Astragalus polysaccharides on P-glycoprotein efflux pump function and protein expression in H22 hepatoma cells in vitro

BackgroundAstragalus polysaccharides (APS) are active constituents of Astragalus membranaceus. They have been widely studied, especially with respect to their immunopotentiating properties, their ability to counteract the side effects of chemotherapeutic drugs, and their anticancer properties. However, the mechanism by which APS inhibit cancer and the issue of whether that mechanism involves the reversal of multidrug resistance (MDR) is not completely clear. The present paper describes an investigation of the effects of APS on P-glycoprotein function and expression in H22 hepatoma cell lines resistant to Adriamycin (H22/ADM).MethodsH22/ADM cell lines were treated with different concentrations of APS and/or the most common chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine. Chemotherapeutic drug sensitivity, P-glycoprotein function and expression, and MDR1 mRNA expression were detected using MTT assay, flow cytometry, Western blotting, and quantitative RT-PCR.ResultsWhen used alone, APS had no anti-tumor activity in H22/ADM cells in vitro. However, it can increase the cytotoxicity of certain chemotherapy drugs, such as Cyclophosphamid, Adriamycin, 5-Fluorouracil, Cisplatin, Etoposide, and Vincristine, in H22/ADM cells. It acts in a dose-dependent manner. Compared to a blank control group, APS increased intracellular Rhodamine-123 retention and decreased P-glycoprotein efflux function in a dose-dependent manner. These factors were assessed 24 h, 48 h, and 72 h after administration. APS down regulated P-glycoprotein and MDR1 mRNA expression in a concentration-dependent manner within a final range of 0.8–500 mg/L and in a time-dependent manner from 24–72 h.ConclusionAPS can enhance the chemosensitivity of H22/ADM cells. This may involve the downregulation of MDR1 mRNA expression, inhibition of P-GP efflux pump function, or both, which would decrease the expression of the MDR1 protein.

Sa1787 Regulation of Chemotherapy Resistance Through Epithelial-Mesenchymal Transition by Zinc Finger E-Box-Binding Proteins

Introduction: Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) and cancer stem cell functions. We have demonstrated recently that ZEBs are upregulated in esophageal squamous cell carcinoma (ESCC) cells with mesenchymal characteristics and increased malignant potential such as anchorage independent growth, invasiveness and tumorigenicity upon xenograft transplantation (Cancer Res. 2011;71:683647). However, the functional roles of ZEBs in chemotherapeutic resistance remain to be elucidated. Methods: Primary tumor tissues from patients who underwent surgery with or without neoadjuvant chemotherapy were analyzed. ESCC cell lines and In Vitro transformed human esophageal cells EPC2T were treated with either 5-fluorouracil or cisplatin, and subjected to WST-1 colorimetric cell proliferation assays, phase contrast imaging, flow cytometry, quantitative RT-PCR andWestern blotting analyses. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2 in a regulatable manner (Tet-On system). Results: 70% of primary tumor tissues from patients who received neoadjuvant chemotherapy (n=10) contained ZEB1 positive ESCC cells, reminiscent of EMT. By contrast, ZEB1 positive ESCC cells were found in 33% of patients without neoadjuvant chemotherapy (n=51). EMT was present in a subset of cultured ESCC cells. The percentage of mesenchymal cells correlated with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and ZEBs. Moreover, flow cytometry revealed that the mesenchymal ESCC cells express a high level of CD44, a cancer stem cell marker, implicated in invasion, metastasis and chemoresistance. Interestingly, chemotherapeutic drug sensitivity was reduced as a function of the frequency of mesenchymal subset of the cells when four different cell lines were compared. In two cell lines (TE1 and TE5) with moderate levels of mesenchymal cells, the cells surviving 5-fluorouracil treatment were predominantly mesenchymal. In HCE7 cells displaying fully mesenchymal characteristics, knockdown of either ZEB1 or ZEB2 increased 5-fluorouracil sensitivity. Moreover, TGF-β induced EMT in EPC2T cells, resulted in a significantly increased cell viability rate upon cisplatin treatment and that was antagonized by knockdown of either ZEB1 or ZEB2. Conclusions: These data demonstrate that ZEB1 and ZEB2 confer chemotherapy resistance through EMT-mediated enrichment of a unique subset of ESCC cells defined by a CD44 upregulation. Neoadjuvant therapy may induce ZEB1 positive cells, thus having broader implications upon EMT and cancer therapy.

Abstract 1785: MET/VEGFR dual inhibition and prominent safety profile of TAS-115 are favorable for the combination with chemotherapeutic drugs

Abstract MET is known as a proto-oncogene, which involves in growth, migration, metastasis and angiogenesis in tumor progression. Recently, it is reported that MET also functions as chemo and EGFR-TKI resistant factor in cancer cells. Particularly, HGF/MET signal seemed to reduce the potency of conventional chemotherapeutic drugs in clinical. Meanwhile, VEGF/VEGFR signal plays an important role in angiogenesis in tumor growth, and is essential for the survival signal of endothelial cells(ECs) to protect from the damage by chemotherapeutic drugs. Some VEGFR inhibitors have already been approved for the treatment of several carcinomas with monotherapy. Many clinical combination trials with VEGFR inhibitor and chemotherapeutic agents have been tried, but its therapeutic benefit are restricted. It is highly desire for improving therapeutic benefit by combination therapy with VEGFR inhibitor and standard chemotherapy. TAS-115, a novel oral MET/VEGFR dual inhibitor, demonstrated not only potent efficacy but also prominent safety profiles achieved by improvement of PK profile and cellular selectivity. Potent MET/VEGFR dual inhibition provides significant anti-tumor effect from lower dose for TAS-115. On the one hand, the improvement of PK profile and cell selectivity led to better tolerability. As a result, therapeutic window (MTD/ED50) of TAS-115 became wider than that of the pre-existing VEGFR or MET/VEGFR inhibitors. These profiles give TAS-115 the long-term dosing at full effective dose without severe toxicity. Moreover, in vitro study, HGF completely suppressed the induction of apoptosis by 5-FU or Paclitaxel in cancer cells expressed MET. Meanwhile, VEGF abolished the cytotoxic effect by these chemotherapeutic drugs against ECs. It was suggested that harmonization of both HGF and VEGF in tumor greatly contributed to the resistance against conventional chemotherapeutic drugs. The MET/VEGFR dual inhibition by TAS-115 improves the sensitivity of chemotherapeutic drugs for both cancer cells and ECs via suppression of function as chemo-resistance factor by MET and VEGFR. In fact, the combination therapies with TAS-115 and conventional chemotherapeutic drugs caused tumor regression or potent growth inhibition without sever body weight loss in several xenograft models. Notably, TAS-115 markedly enhanced anti-tumor effect of Paclitaxel, and brought tumor shrinkage of approximately 80% in the combination. More important feature is that TAS-115 has disturbed rapid tumor re-growth following Paclitaxel treatment and the higher safety profiles of TAS-115 permits combination therapy with chemotherapeutic drugs in chronic treatment. It is expected TAS-115 demonstrates prominent therapeutic benefit in combination with chemotherapy based on the synergistic action of the potent MET/VEGFR dual inhibition and higher safety profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1785. doi:1538-7445.AM2012-1785

Establishment of a Primary Pleomorphic Xanthoastrocytoma Cell Line

Anaplastic pleomorphic xanthoastrocytoma is an aggressively growing, malignant, and eventually fatal tumor of the central nervous system. Testing chemotherapeutic drug sensitivity under in vitro conditions would be a useful strategy to determine sensitive or resistant drugs for fatal brain cancers. To establish primary cell cultures of excised tumor tissue from pleomorphic xanthoastrocytoma-bearing patients and to test their sensitivity against various anticancer chemotherapy drugs. Prepared suspensions of the excised tumor tissue from a patient who had a recurrent grade 3 pleomorphic xanthoastrocytoma was cultured in culture dishes until cells began to grow. Immunofluorescent and immunohistochemical visualizations were performed using confocal and light microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in comparison with ³H-thymidine incorporation assay was used to test cellular toxicity of several anticancer drugs. We established vigorously growing primary cells of the tumor. Drug sensitivity testing was conducted successfully. Primary cell cultures of surgically removed tumor tissues may be useful in studies of cancer biology and chemotherapeutic drug sensitivity for recurrent malignant brain tumors, particularly for anaplastic pleomorphic xanthoastrocytoma.

Abstract 5194: Notch regulates squamous differentiation, cell plasticity and tumor heterogeneity in esophageal carcinoma

Abstract Introduction: The Notch receptor family regulates cell fates and may act as a tumor suppressor in the squamous epithelia. While squamous differentiation is maintained in esophageal squamous cell carcinoma (ESCC) and its precursor lesions, the roles of Notch in esophageal tumor biology remain elusive. Methods: Transformed human esophageal cells expressing EGFR, p53R175H and cyclin D1 (EPC2-T) and ESCC cell lines were stably transduced with ICN1, an active form of NOTCH1 (N1) in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or gamma-secretase inhibitors (GSI). 8xCSL-luciferase reporter was transiently transfected to assess Notch activity. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2. Squamous epithelium was reconstituted in organotypic (3D) culture, a form of tissue engineering. In addition, cell growth was assessed in soft agar and immunodeficient mice. Quantitative RT-PCR, Western blotting, immunohistochemistry (IHC) and flow cytometry were done to determine gene expression. Primary ESCC tissues (n=20) were analyzed by IHC. Results: In primary ESCC, N1 was localized to well-differentiated tumor cell nests expressing involucrin (IVL), a squamous differentiation marker, while downregulated in poorly-differentiated cells lacking IVL within a single tumor tissue, implying N1 in tumor heterogeneity. In cell lines, ICN1 activated Notch-dependent transcription and squamous differentiation in the absence of DNMAML1. In 3D culture, DNMAML1 and GSI not only blocked squamous differentiation but induced massive invasion and epithelial-to-mesenchymal transition (EMT) with Wnt activation as suggested by loss of E-cadherin and nuclear localization of β-catenin. In agreement, DNMAML1 upregulated LEF1, Zinc finger E-box binding proteins ZEB1 and ZEB2, transcription factors all essential in EMT and maintenance of cancer stem cells, facilitating transforming growth factor (TGF)-β1-induced EMT in EPC2-T cells. Moreover, DNMAML1 enriched a unique subset of CD44-bright and CD24-dim cells and augmented colony formation, tumorigenicity as well as chemotherapeutic drug resistance against Cisplatin. Importantly, Cre-mediated removal of chromosomally integrated floxed DNMAML1 reactivated Notch to allow terminal differentiation with IVL induction. Moreover, ZEB knockdown greatly impaired TGF-β-mediated EMT while restoring chemotherapeutic drug sensitivity. Conclusions: These data indicate that Notch activation promotes squamous differentiation whereas Notch inhibition enriches poorly differentiated tumor cells with cancer stem cell potential with drug resistance, which involves ZEB and Wnt activation, thus providing a novel mechanistic insight into how Notch signaling may contribute to tumor heterogeneity in ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2011-5194

Berberine hydrochloride: anticancer activity and nanoparticulate delivery system

BackgroundBerberine hydrochloride is a conventional component in Chinese medicine, and is characterized by a diversity of pharmacological effects. However, due to its hydrophobic properties, along with poor stability and bioavailability, the application of berberine hydrochloride was hampered for a long time. In recent years, the pharmaceutical preparation of berberine hydrochloride has improved to achieve good prospects for clinical application, especially for novel nanoparticulate delivery systems. Moreover, anticancer activity and novel mechanisms have been explored, the chance of regulating glucose and lipid metabolism in cancer cells showing more potential than ever. Therefore, it is expected that appropriate pharmaceutical procedures could be applied to the enormous potential for anticancer efficacy, to give some new insights into anticancer drug preparation in Chinese medicine.Methods and resultsWe accessed conventional databases, such as PubMed, Scope, and Web of Science, using “berberine hydrochloride”, “anti-cancer mechanism”, and “nanoparticulate delivery system” as search words, then summarized the progress in research, illustrating the need to explore reprogramming of cancer cell metabolism using nanoparticulate drug delivery systems.ConclusionWith increasing research on regulation of cancer cell metabolism by berberine hydrochloride and troubleshooting of issues concerning nanoparticulate delivery preparation, berberine hydrochloride is likely to become a natural component of the nanoparticulate delivery systems used for cancer therapy. Meanwhile, the known mechanisms of berberine hydrochloride, such as decreased multidrug resistance and enhanced sensitivity of chemotherapeutic drugs, along with improvement in patient quality of life, could also provide new insights into cancer cell metabolism and nanoparticulate delivery preparation.

Overexpression of Smac/DIABLO in Hep-2 Cell Line: Possible Role in Potentiating the Sensitivity of Chemotherapeutic Drugs

The major obstacles for tumor chemotherapy are drug resistance and/or adverse effects on the host. In the present study we investigated the role of the second mitochondria-derived activator of caspase (Smac/DIABLO) in the action of cisplatin (DDP), 5-fluorouracil (5-FU), and the combination of both in Hep-2 cells. Hep-2 laryngeal carcinoma cells exposed to DDP, 5-FU and the combination of both were investigated. Cell viability was determined by MTT assay. Apoptosis was measured by Ho.33342 and PI double staining and flow cytometry. The expression of Smac/DIABLO at the mRNA and protein level was assayed by RT-PCR and Western blotting. DDP, 5-FU and the combination of both drugs reduced the cell survival rates in a concentration- and time-dependent manner. The drug combination not only exerted a stronger inhibitory effect, but also at a lower concentration compared with the single drugs. Apoptosis was concomitant in a caspase-dependent manner. The expression of Smac/DIABLO increased significantly at both mRNA and protein levels after cell exposure to the combination compared with single drugs. Smac/DIABLO plays a pivotal role in attaining a synergistic effect in Hep-2 cells in response to this combined strategy.

Resistance-related proteinin rectal cancer tissue expression and significanee of combi-nation chemotherapy withLigustrazine

：Objective To investigateresistance-related protein in tumor tissue expression and significance of combinationchemotherapy with Liguslrazine.Methods In this paper, drug-resistant cancer patientsresected tumor tissue was isolated preparation of cell suspension. Study of P-170, MRP andLRP by using flow cytometry and Western-Blotting to elucidate the mechanism of drugresistance, and use traditional Chinese medicine Ligustrazine reversal of multidrugresistance experiments.Results LOVO/5-FU resistance in the mechanism of drug-resistantmainly by increasing P-170, MRP protein expression. Chinese medicine Ligustrazine can beadopted to reduce P-170, MRP protein expression to sensitize drug-resistant rectal cancercells.Conclusions Ligustrazine can increase the resistance of cancer cells tochemotherapeutic drug sensitivity, then the Ligustrazine can effectively increase the ORRin patients with short-term effect, worthy to be popularized.

Mitochondrial DNA depletion promotes impaired oxidative status and adaptive resistance to apoptosis in T47D breast cancer cells

The mutation and reduction of mitochondrial DNA (mtDNA) have been extensively detected in human cancers. The effects of mitochondrial dysfunction are particularly important in breast cancer, because estrogen-mediated metabolites generate large quantities of local reactive oxygen species in the breast, which directly bind to mtDNA and facilitate neoplastic transformation. To further elucidate the molecular roles of mtDNA in breast cancer, we determined the oxidative status of a breast tumor cell line lacking mtDNA (T47D ρ⁰) and analyzed its susceptibility after exposure to various anticancer drugs as well as different proapoptotic signals. Our data showed that T47D ρ⁰ cells generated significantly increased levels of lactate with concomitantly reduced oxygen consumption and ATP production compared with the wild-type (WT). The amount of reactive oxygen species generation in ρ cells was lowered to approximately 12% that of parental cells, as evidenced by the oxidation of redox-sensitive probes. Although mtDNA depletion did not affect the expression of superoxide dismutase or its activity, the activities of antioxidant enzymes, catalase and glutathione peroxidase, were significantly higher in ρ⁰ cells compared with WT cells. In addition, mtDNA-depleted cells displayed a decreased sensitivity and accumulation of chemotherapeutic drugs (doxorubicin, vincristine, and paclitaxel), potentially because of the upregulated expression of multidrug resistance 1 (MDR1) gene and its product P-glycoprotein. When compared with their WT counterparts, T47D ρ⁰ cells were also more resistant to apoptosis induced by varying concentrations of staurosporine and anti-Fas antibody. Altogether, our results indicate the importance of intact mtDNA for maintaining the proper intracellular oxidative status. These data provide evidence for a possible role of mtDNA content reduction in acquiring an apoptosis-resistant phenotype during breast tumor progression and might contribute to effective therapeutic strategies for this common malignancy.

Fractionated irradiation induced radio-resistant esophageal cancer EC109 cells seem to be more sensitive to chemotherapeutic drugs

BackgroundChemo-radiotherapy, a combination of chemotherapy and radiotherapy, is the most frequent treatment for patients with esophageal cancer. In the process of radiotherapy, the radiosensitive cancer will become a radio-resistant one.MethodsIn order to detect the chemotherapeutic drug sensitivity in radio-resistant cancer cells and improve the therapy efficiency, we firstly established a radio-resistant esophageal cancer cell model (referred to as EC109/R) from the human esophageal squamous cell carcinoma cell line EC109 through fractionated irradiation using X-rays. The radio-sensitivity of EC109/R cells was measured by clonogenic assay. To detect the drug sensitivity for EC109/R compared to its parent cells, we employed MTT method to screen the effectiveness of five different drugs commonly used in clinical therapy. The ratio of apoptosis was examined by flow cytometry.ResultsEC109/R cells were more sensitive to 5-fluorouracil, doxorubicin, paclitaxel and etoposide, but tolerant to cisplatin compared to its original cells.ConclusionOur study implies that fractionated irradiation induced radio-resistant esophageal cancer cell is more sensitive to certain kind of chemotherapeutic drugs. It provides evidence for choosing the sequence of radiotherapy and chemotherapy in esophageal cancer.

Anti-tumor action and clinical application of proteasome inhibitor

Ubiquitin-proteasome pathway mediates the degradation of cell protein, and cell cycle, gene translation and expression, antigen presentation and inflammatory development. Proteasome inhibitor can inhibit growth and proliferation of tumor cell, induce apoptosis and reverse multipledrug resistance of tumor cell, increase the sensitivity of other chemotherapeutic drugs and radiotherapy, and is a novel class of potent anti-tumor agents.

Effect of silencing cyclin D1 gene on chemotherapeutic drug sensitivity in K562 cells in vitro

Objective Cyclin D1 gene plays a significant role in regulating cell cycle progression.It is reported that over-expression of cyclin D1 gene is intimately associated with origination,development and prognosis of tumor and is associated with tumor cells resistance to chemotherapy drug.Suppression of cyclin D1 protein expression leads to cellular chemosensitization.This study was to determine whether this effect also existed in chronic leukemia cell line K562 by inhibiting the expression of cyclin D1 protein through RNA interference.Methods Plasmid vectors expressing small hairpin RNA (shRNA) targeting at cyclin D1 gene were constructed and transfected into K562 cells by chitosan.Cyclin D1 protein was examined using Western Blotting analysis.The cell cycle and apoptosis were determined by flow cytometry.Cellular chemosensitization was evaluated by MTY assay. Results Expression of cyclin D1 protein was markedly down-regulated after transfection with pshRNA-419 and pshRNA-575 at 48 h.Down-regulation of cyclin D1 protein could affect the redistribution of cell cycle,induce apoptosis of K562 cells,decrease 50％inhibitoryconcentration (IC50) of adriamycin and enhance cellular chemosensitization.But there had no above biological effects observed after transfection with blank vector and control vector of m-pshRNA-790 at 48 h.Conclusion K562 cells could be chemosensitized by the down-regulation of cyclin D1 expression through RNA interference. Key words: Cyclin D1; RNA interference; K562 cells; Microbial sensitivity tests

Effect of all-trans retinoic acid on drug sensitivity and expression of survivin in LoVo cells

All-trans retinoic acid (ATRA) can influence the tumor cell proliferation cycle, and some chemotherapeutic drugs are cycle specific. In this study, we hypothesize that ATRA can enhance chemotherapeutic drug sensitivity by affecting the cell cycle of tumor cells. The cell cycle of LoVo cells was evaluated using flow cytometry (FCM). Cell viability was analyzed using the MTT assay. The morphologic changes in the treated LoVo cells were measured with acridine orange (AO)/ethidium bromide (EB) staining. Expression of survivin in LoVo cells was analyzed by immunofluorescence assay. After LoVo cells were treated with ATRA, the G0/G1 ratio of the tumor cells increased and the cell ratio of S- and G2/M-phase decreased. Viability of the cells decreased significantly after combined treatment with ATRA and 5-fluorouracil (5-FU) or mitomycin c (MMC) and was evaluated by fluorescence microscopy. Expression level of survivin in the tumor cells decreased after ATRA combination treatment. ATRA enhances drug sensitivity of the LoVo cell line to cell cycle-specific agents and inhibits the expression of survivin in LoVo cells. The combination of ATRA and 5-FU or MMC promoted cell apoptosis, and the mechanism involved in apoptosis may be related to inhibition of survivin gene expression.

The transglutaminase 2 gene ( TGM2 ), a potential molecular marker for chemotherapeutic drug sensitivity, is epigenetically silenced in breast cancer

Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme capable of catalyzing protein cross-links. TG2-dependent cross-links are important in extracellular matrix integrity and it has been proposed that this TG2 activity establishes a barrier to tumor spread. Furthermore, TG2 controls sensitivity to the chemotherapeutic drug doxorubicin. Both doxorubicin sensitivity and TG2 expression are highly variable in cultured human breast cancer cell lines and inspection of the human gene (termed TGM2) determined that a canonical CpG island exists within its 5' flank. These features, when combined with its potential tumor suppressor activity, make TG2 an attractive candidate for epigenetic silencing. Consistent with this, we observed that culturing breast tumor cells with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-azadC) resulted in a robust increase in TG2 expression. Analysis of DNA harvested from cultured lines and primary breast tumor samples indicated that TGM2 often displays aberrant hypermethylation and that there is a statistically significant correlation between gene methylation and reduced expression. Finally, we observed that doxorubicin-resistant MCF-7/ADR cells do not show TGM2 silencing but that doxorubicin-sensitive MCF-7 cells do and that culturing MCF-7 cells on 5-azadC and subsequently restoring TG2 expression reduced sensitivity to doxorubicin. This work indicates that the TGM2 gene is a target for epigenetic silencing in breast cancer and suggests that this aberrant molecular event is a potential marker for chemotherapeutic drug sensitivity.

SiRNA‐MEDIATED BCL‐2 AND BCL‐XL GENE SILENCING SENSITIZES HUMAN HEPATOBLASTOMA CELLS TO CHEMOTHERAPEUTIC DRUGS

1. The aim of the present study was to investigate the changes in chemotherapeutic drug sensitivity of HepG2 cells transfected with Bcl-2 and Bcl-xl siRNA expression vectors. 2. Bcl-2 and Bcl-xl siRNA and negative siRNA expression vectors were constructed and stably transfected into HepG2 cells. Reverse transcriptase-polymerase chain reaction was used to detect the target gene expression, and the Bcl-2, Bcl-xl, Bax and caspase-3 protein levels were measured using western blots and immunofluorescence. The sensitivity of the cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and flow cytometry. 3. The Bcl-2 and Bcl-xl gene expression and corresponding protein levels in Bcl-2 siRNA, Bcl-xl siRNA and Bcl-2/Bcl-xl siRNA transfected cells were reduced compared with negative siRNA transfected or untreated cells. The Bax protein level remained unaltered but the caspase-3 level was enhanced when Bcl-2 and Bcl-xl protein levels were reduced. The MTT results demonstrated that Bcl-2 and Bcl-xl transfected cells exhibited increased sensitivity to 5-FU or HCPT. Flow cytometry demonstrated that the sub G1 cell population increased in Bcl-2/Bcl-xl siRNA co-transfected and Bcl-xl siRNA and Bcl-2 siRNA transfected cells when compared with negative siRNA or untreated cells. The latter trend was strengthened further in the presence of 5-FU or HCPT. 4. Thus, Bcl-2 and Bcl-xl siRNA-mediated gene silencing, in combination with chemotherapy, may be a potential therapeutic strategy against human hepatoblastoma.

The gastrin gene promoter is regulated by p73 isoforms in tumor cells

p73, a new p53 family member, is a transcription factor that is increasingly recognized in cancer research as an important player in tumorigenesis as well as in chemotherapeutic drug sensitivity. Despite the substantial structural and functional similarities to p53, accumulating evidence suggests that p53 and p73 may differently regulate their transcriptional targets. In this study, we have investigated the role of p73 in regulation of the gastrin gene promoter. Gastrin is a peptide hormone and an important factor in determining the progression of a number of human malignancies. Our results show that p73 can bind to the gastrin promoter. This leads to transcriptional upregulation of gastrin mRNA. We also found that the levels of gastrin and p73 transcripts correlate in primary gastric tumors. Taken together, our results demonstrate a novel mechanism for regulation of gastrin gene transcription and support a concept that p53 and p73 may have different biological roles in tumors.

Antagonism of HSV-tk Transfection and Ganciclovir Treatment on Chemotherapeutic Drug Sensitivity

Our study focused on the influence of herpes simplex virus thymidine kinase (HSV-tk) expression and ganciclovir (GCV) treatment on the sensitivity of C6 glioma cells to frequently used chemotherapeutic drugs, i.e. adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), and methotrexate (MTX). Transfection with HSV-tk revealed an increased sensitivity to GCV and CDDP and a decreased sensitivity to ADR and MTX. No significant differences were found in sensitivity to 5-FU. Combined treatment in a HSV-tk negative cell line revealed an additive effect when GCV was combined with ADR, whereas an antagonistic effect was found when GCV was combined with CDDP, 5-FU, or MTX. Comparable results were obtained in an HSV-tk positive cell line, apart from CDDP, which showed an additive effect. In conclusion, both HSV-tk transfection and subsequent GCV treatment can influence the sensitivity of tumor cells to various chemotherapeutic drugs in an antagonistic manner. Therefore, combining HSV-tk/GCV gene therapy with chemotherapy might not always be beneficial.

Mitotic Checkpoint: A Therapeutic Target in the Treatment of Human Cancer

The most common feature of human cancer cells is aneuploidy and a defective mitotic checkpoint is thought to be responsible. Since a group of anticancer drugs target the mitotic checkpoint, this review will discuss the association of mitotic checkpoint with chemotherapeutic drug sensitivity and suggest a novel therapeutic tool to achieve chemosensitization in human cancer cells. Keywords: eukaryotic cells, dna replication, chromosome, cell cycle checkpoints, kinetochores

P73 isoforms can induce T-cell factor-dependent transcription in gastrointestinal cells.

A new p53 family member, p73, and its isoform DeltaNp73 are increasingly recognized in cancer research as important players in tumorigenesis, as well as in chemotherapeutic drug sensitivity. Despite substantial structural similarities to p53, accumulating evidence suggests that p53 and p73 may play different roles in human tumorigenesis. In this study, we have investigated the role of p73 and DeltaNp73 in upper gastrointestinal tumorigenesis. Our results indicate that p73 and DeltaNp73 are frequently overexpressed in >60% of primary adenocarcinomas of the stomach and esophagus. We have demonstrated that this overexpression can lead to the suppression of p73 transcriptional and apoptotic activity in gastrointestinal cells. Moreover, it induces beta-catenin up-regulation and T-cell factor/lymphocyte enhancement factor-dependent transcription. Wild-type p53, but not mutant p53, can inhibit this effect. Our results demonstrate a novel mechanism for activation of beta-catenin in gastrointestinal tumors and support the concept that overexpression of p73 isoforms can play an important role in tumorigenesis.