Persistence of side population cells with high drug efflux capacity in pancreatic cancer

AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell line PANC-1 and its proportion change when cultured with Gemcitabine, was detected by Hoechst 33342 staining and FACS analysis. The expression of ABCB1 and ABCG2 was detected by real- time PCR in either SP cells or non-SP cells. RESULTS: SP cells do exist in PANC-1, with a median of 3.3% and a range of 2.1-8.7%. After cultured with Gemcitabine for 3 d, the proportion of SP cells increased significantly (3.8% ± 1.9%, 10.7% ± 3.7%, t = 4.616, P = 0.001 < 0.05). ABCB1 and ABCG2 expressed at higher concentrations in SP as compared with non-SP cells (ABCB1: 1.15 ± 0.72, 5.82 ± 1.16, t = 10.839, P = 0.000 < 0.05; ABCG2: 1.16 ± 0.75, 5.48 ± 0.94, t = 11.305, P = 0.000 < 0.05), which may contribute to the efflux of fluorescent staining and drug resistance. CONCLUSION: SP cells with inherently high resistance to chemotherapeutic agents do exist in pancreatic cancers, which may be candidate cancer stem cells contributing to the relapse of the tumor.

Antibody-platinum(IV)prodrugs conjugates for targeted treatment of cutaneous squamous cell carcinoma

Antibody-drug conjugates(ADCs)are a new type of targeting antibodies that conjugate with highly toxic anticancer drugs via chemical linkers to exert high specificity and efficient killing of tumor cells,thereby attracting considerable attention in precise oncology therapy.Cetuximab(Cet)is a typical antibody that offers the benefits of good targeting and safety for individuals with advanced and inoperable cutaneous squamous cell carcinoma(cSCC);however,its anti-tumor activity is limited to a single use.Cisplatin(CisPt)shows good curative effects;however,its adverse effects and non-tumor-targeting ability are major drawbacks.In this study,we designed and developed a new ADC based on a new cytotoxic platinum(IV)prodrug(C8Pt(IV))and Cet.The so-called antibody-platinum(IV)prodrugs conjugates,named Cet-C8Pt(IV),showed excellent tumor targeting in cSCC.Specifically,it accurately delivered C8Pt(IV)into tumor cells to exert the combined anti-tumor effect of Cet and CisPt.Herein,metabolomic analysis showed that Cet-C8Pt(IV)promoted cellular apoptosis and increased DNA damage in cSCC cells by affecting the vitamin B6 metabolic pathway in tumor cells,thereby further enhancing the tumor-killing ability and providing a new strategy for clinical cancer treatment using antibody-platinum(IV)prodrugs conjugates.

Effects of Taxotere on invasive potential and multidrug resistance phenotype in pancreatic carcinoma cell line SUIT-2

INTRODUCTIONDevelopment of drug-resistance to chemotherapyand subsequent metastasis of tumor are primarilyresponsible for treatment failure and the death fromcancer. There have been many previous studies onthe relationship between expression of multidrugresistance (MDR) phenotype P-glycoprotein (P-gp)and the malignant properties of tumors, but theresults are often conflicting[1-8]. The difference intumor types or MDR phenotype induced by specificagents might account for this discrepancy. Taxotere(TXT), a member of the family of taxanes, hasantitumor activity through its effect of promotingthe polymerization of tubulin[9,10].

Stem cell technology for antitumor drug loading and delivery in oncology

The main aim of antineoplastic treatment is to maximize patient benefit by augmenting the drug accumulation within affected organs and tissues,thus incrementing drug effects and,at the same time,reducing the damage of non-involved tissues to cytotoxic agents.Mesenchymal stromal cells(MSC)represent a group of undifferentiated multipotent cells presenting wide self-renewal features and the capacity to differentiate into an assortment of mesenchymal family cells.During the last year,they have been proposed as natural carriers for the selective release of antitumor drugs to malignant cll,s thus optimizing cytotoxic action on cancer cll,while significantly reducing adverse side efect on healthy cells.MSC chemotherapeutic drug loading and delivery is an encouraging new area of cell therapy for several tumors,especially for those with unsatisfactory prognosis and limited treatment options available.Although some experim ental models have been sucesfuly developed,phase I dinical studies are needed to confirm this potential application of cell therapy,in particular in the case of primary and secondary lung cancers.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1

Objective Retinoblastoma(RB)is a prevalent type of eye cancer in youngsters.Prospero homeobox 1(Prox1)is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic,hepatocyte,pancreatic,heart,lens,retinal,and cancer cells.The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance,as well as to explore the underlying Notch1 mechanism.Methods Human RB cell lines(SO-RB50 and Y79)and a primary human retinal microvascular endothelial cell line(ACBRI-181)were used in this study.The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction(RT-qPCR)and Western blotting.Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay.Drug-resistant cell lines(SO-RB50/vincristine)were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance.We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1.Finally,a xenograft model was constructed to assess the effect of Prox1 on RB in vivo.Results Prox1 was significantly downregulated in RB cells.Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine.Notch1 was involved in Prox1’s regulatory effects.Notch1 was identified as a target gene of Prox1,which was found to be upregulated in RB cells and repressed by increased Prox1 expression.When pcDNA-Notch1 was transfected,the effect of Prox1 overexpression on RB was removed.Furthermore,by downregulating Notch1,Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo.Conclusion These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1,implying that Prox1 could be a potential therapeutic target for RB.

Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-x_L mRNA in Extended Drug Resistance Leukemia Cell Line HL- 60/E6

To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.

Quantitative characterization of cell physiological state based on dynamical cell mechanics for drug efficacy indication

Cell mechanics is essential to cell development and function,and its dynamics evolution reflects the physiological state of cells.Here,we investigate the dynamical mechanical properties of single cells under various drug conditions,and present two mathematical approaches to quantitatively characterizing the cell physiological state.It is demonstrated that the cellular mechanical properties upon the drug action increase over time and tend to saturate,and can be mathematically characterized by a linear timeinvariant dynamical model.It is shown that the transition matrices of dynamical cell systems significantly improve the classification accuracies of the cells under different drug actions.Furthermore,it is revealed that there exists a positive linear correlation between the cytoskeleton density and the cellular mechanical properties,and the physiological state of a cell in terms of its cytoskeleton density can be predicted from its mechanical properties by a linear regression model.This study builds a relationship between the cellular mechanical properties and the cellular physiological state,adding information for evaluating drug efficacy.

Efficacy of Bee Products(Anzer Honey,Pollen and Propolis)in Detection and Healing of Damage Induced by Antidiabetic Drug Vildagliptin/Metformin Hydrochloride in Healthy Human Pancreatic Cells:Cytotoxic,Genotoxic and Biochemical Studies

Background and Objective Although drugs are powerful therapeutic agents,they have a range of side effects.These side effects are sometimes cellular and not clinically noticeable.Vildagliptin/metformin hydrochloride is one of the most widely used oral antidiabetic drugs with two active ingredients.In this study,we investigated its harmful effects on the metabolic activation system in healthy human pancreatic cells“hTERT-HPNE”,and we aimed to improve these harmful effects by natural products.To benefit from the healing effect,we used the unique natural products produced by the bees of the Anzer Plateau in the Eastern Black Sea Region of Turkey.Methods Cytotoxic and genotoxic effects of the drug were investigated by different tests,such as MTT,flow cytometry-apoptosis and comet assays.Anzer honey,pollen and propolis were analyzed by gas chromatography/mass spectrometry(G/C-MS).A total of 19 compounds were detected,constituting 99.9%of the samples.Results The decrease in cell viability at all drug concentrations was statistically significant compared to the negative control(P<0.05).A statistically significant decrease was detected in the apoptosis caused by vildagliptin/metformin hydrochloride with the supplementation of Anzer honey,pollen and propolis in hTERT-HPNE cells(P<0.05).Conclusion This study can contribute to other studies testing the healing properties of natural products against the side effects of oral antidiabetics in human cells.In particular,Anzer honey,pollen and propolis can be used as additional foods to maintain cell viability and improve heal damage and can be evaluated against side effects in other drug studies.

Hepatic cancer stem cells and drug resistance: Relevance in targeted therapies for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of most common malignancies in the world. Systemic treatments for HCC, particularly for advanced stages, are limited by the drug resistance phenomenon which ultimately leads to therapy failure. Recent studies have indicated an association between drug resistance and the existence of the cancer stem cells (CSCs) as tumor initiating cells. The CSCs are resistant to conventional chemotherapies and might be related to the mechanisms of the ATP Binding Cassette (ABC) transporters and alterations in the CSCs signaling pathways. Therefore, to contribute to the development of new HCC treatments, further information on the characterization of CSCs, the modulation of the ABC transporters expression and function and the signaling pathway involved in the self renewal, initiation and maintenance of the cancer are required. The combination of transporters modulators/inhibitors with molecular targeted therapies may be a potent strategy to block the tumoral progression. This review summarizes the association of CSCs, drug resistance, ABC transporters activities and changes in signaling pathways as a guide for future molecular therapy for HCC.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells

AIM:To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P【0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P【0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P【0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P【0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P【0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.

Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells

AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.

Salvianolic acid B modulates the expression of drug-metabolizing enzymes in HepG2 cells

BACKGROUND: Enzymes involved in drug and xenobiotic metabolism have been considered to exist in two groups: phase I and phase II enzymes. Cytochrome P450 isoenzymes (CYPs) are the most important phase I enzymes in the metabolism of xenobiotics. The products of phase I metabolism are then acted upon by phase II enzymes, including glutathione S-transferases (GSTs). Herbs that inhibit CYPs such as CYP3A4 or that induce GSTs may have the potential to protect against chemical carcinogenesis since the mutagenic effects of carcinogens are often mediated through an excess of CYP-generated reactive intermediates. This study was designed to investigate the effects of salvianolic acid B (Sal B), a pure compound extracted from Radix Salviae Miltiorrhizae, a Chinese herb, on cell proliferation and CYP1A2 and CYP3A4 mRNA expression in the presence or absence of rifampicin, a potent inducer of CYPs and GST protein expression in HepG2 cells. METHODS: HepG2 cells were incubated with different concentrations of Sal B. Cell proliferation was determined by SYTOX-Green nucleic acid staining. CYP3A4 and CYP1A2 mRNA expression was assayed by real-time PCR. GST protein expression was analyzed by Western blotting. RESULTS: Low concentrations of Sal B (0-20 μmol/L) had no significant effects on cell proliferation, while higher concentrations (100-250 μmol/L) significantly inhibited proliferation in a concentration-dependent manner. Ten μmol/L Sal B, but not 1 μmol/L, down-regulated CYP3A4 and CYP1A2 mRNA expression after 24 hours of incubation, whereas both 1 and 10 μmol/L Sal B down-regulated CYP3A4mRNA expression after 96 hours of incubation; moreover, 1 and 10 μmol/L Sal B inhibited CYP3A4 mRNA expression induced by rifampicin. Both 1 μmol/L and 10 μmol/L Sal B increased GST expression. CONCLUSION: Sal B inhibits CYP3A4 and CYP1A2 mRNA expression and induces GST expression in HepG2 cells.

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells

AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.

Induced pluripotent stem cells for therapy personalization in pediatric patients:Focus on drug-induced adverse events

Adverse drug reactions(ADRs)are major clinical problems,particularly in special populations such as pediatric patients.Indeed,ADRs may be caused by a plethora of different drugs leading,in some cases,to hospitalization,disability or even death.In addition,pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs,leading,in some cases,to more severe consequences.To improve the comprehension,and thus the prevention,of ADRs,the set-up of sensitive and personalized assays is urgently needed.Important progress is represented by the possibility of setting up groundbreaking patient-specific assays.This goal has been powerfully achieved using induced pluripotent stem cells(iPSCs).Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body,this model may be accurate in predicting drug toxicity,especially when this toxicity is related to individual genetic differences.This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs,with particular attention to drugs used in the pediatric field.We especially focused on the intestinal,hepatic,pancreatic,renal,cardiac,and neuronal levels,also discussing progress in organoids creation.The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine,liver,pancreas,kidney,heart,and brain.Based on the existing knowledge,these models are powerful and promising tools in multiple clinical applications including toxicity screening,disease modeling,personalized and regenerative medicine.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells(HDDPCs) and HDDPC-derived iPS cells

The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a Piggy Bac（PB）-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing td Tomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Effects of glucocorticoids on the growth and chemosensitivity of carcinoma cells are heterogeneous and require high concentration of functional glucocorticoid receptors

AIM： To determine how glucocorticoids （GCs） may affect the growth and chemosensitivity of common carcinoma cells. METHODS： The effect of dexamethasone （DEX） on growth and chemosensitivity was assessed in 14 carcinoma cell lines. The function of GC receptors （GR） was assessed by MMTV reporter assay. Overexpression of GR was done by in vitro transfection and expression of a GR-expressing vector. Immunohistochemical stain of tissues and ceils were done by PA1-511A, an anti-GR monodonal antibody. RESULTS： DEX inhibited cell growth of four （MCF-7, MCF- 7/MXR1, MCF-7/TPT300, and HeLa）, increased cisplatin cytoxicity of one （SiHa）, and decreased dsplatin cytotoxicity of two （H460 and Hep3B） cell lines. The GR content of the seven cell lines affected by DEX was significantly higher than those of the seven cell lines unaffected by DEX （5.2±2.5×10^4 sites/cell vs1.3±1.4×10^4 sites/cell, P= 0.005）. Only two DEX-unresponsive cell lines {NPC-TW01 and NPC- TW04） oontained high GR amounts in the range （1.9-8.1×10^4 sites/cell） of the seven DEX-responsive cell lines. The GR function of NPC-TW01 and NPC-TW04, however, was foundto be impaired. The importance of high cellular amount of GR in mediating DEX susceptibility of the cells was further exemplified by GR dose-dependent drug resistance to cisplatin of AGS, a cell line with low GR content and was unaffected by DEX before transfection of GR-expressing vector. Immunohistochemical studies of human cancer tissues showed that 5 of the 45 （11.1%） breast cancer and 43 of the 85 （50.6%） non-small cell lung cancer had high GR contents at the ranges of the GC-responsive carcinoma cell lines. CONCLUSION： The growth and chemosensitivity of human carcinomas with high GR contents may be affected by GC. However, in light of the heterogeneous and even contradictive effects of GC on these cells, routine examination of GR contents of human carcinoma tissues may not be clinically useful until other markers that help predict the ultimate effect of GC on individual patients are identified.

Multifactorial nature of hepatocellular carcinoma drug resistance: Could plant polyphenols be helpful?

Primary hepatocellular carcinoma （HCC） is a quite frequent tumor which results in high mortality and most often exhibits a poor response to present drug therapies. Clearly, a thorough understanding of the biological bases of this malignancy might suggest new strategies for its treatment. Here we examine the evidences that both ＂pharmacological＂ mechanisms （e.g. drug transporter or detoxification enzyme over-expression） and alterations in other critical factors, including the IAPs （Inhibitory of Apoptosis Proteins）, involved in enhancement of cell survival and proliferation may determine the therapeutic resistance of HCC; we also underline the possible role in the process of the activation of transcription factors, like NF-κB, capable of contemporaneously up-regulating the mechanisms discussed. On this basis, we finally comment on the possible use of natural multi-targeted antitumoral agents like plant polyphenols to achieve sensitization to treatments in HCC.

Engineered T Cell Therapies from a Drug Development Viewpoint

Cancer is one of the leading causes of death worldwide. Recent advances in cellular therapy have demonstrated that this platform has the potential to give patients with certain cancers a second chance at life. Unlike chemical compounds and proteins, cells are living, self-replicating drugs that can be engineered to possess exquisite specificity. For example, T cells can be genetically modified to express chimeric antigen receptors (CARs), endowing them with the capacity to recognize and kill tumor cells and form a memory pool that is ready to strike back against persisting malignant cells. Anti-CD19 chimeric antigen receptor T cells (CART19s) have demonstrated a remarkable degree of clinical efficacy for certain malignancies. The process of developing CART19 essentially follows the conventional “one gene, one drug, one disease” paradigm derived from Paul Ehrlich’s “magic bullet” concept. With major players within the pharmaceutical industry joining forces to commercialize this new category of “living drugs,” it is useful to use CART19 as an example to examine the similarities and differences in its development, compared with that of a conventional drug. In this way, we can assimilate existing knowledge and identify the most effective approach for advancing similar strategies. This article reviews the use of biomarker-based assays to guide the optimization of CAR constructs, preclinical studies, and the evaluation of clinical efficacy;adverse effects (AEs);and CART19 cellular kinetics. Advanced technologies and computational tools that enable the discovery of optimal targets, novel CAR binding domains, and biomarkers predicting clinical response and AEs are also discussed. We believe that the success of CART19 will lead to the development of other engineered T cell therapies in the same manner that the discovery of arsphenamine initiated the era of synthetic pharmaceuticals.

Lethal Effect of Benzene Nitrogen Mustard Glucoside Derivate on K562 Cells

A new synthesized benzene nitrogen mustard was converted into glycosyl donor-trichloroacetimidate that was glycosylated with p-nitrophenol（glycosyl donors） to form β-lactosyl p-nitrobenzene under the protection of acetyl in a stereoselective manner, was prepared and evaluated for its cytotoxicity towards cultured K562 cell line. Methylthiazoy tetrazolium（MTT） assay, transmission electron microscopy（TEM）, flow cytometry（FCM） and immunohistochemistry were utilized to explore the mechanisms of how the compound arrests the growth of HCT-T cells. This new synthesed benzene nitrogen mustard glucoside derivate（BNMGD） presented a lower toxicity to normal cells, but is significantly more toxic to K562 cells compared with nitrogen mustard, meanwhile it can induce the apoptosis of K562 cells. These results indicate that the new synthesized BNMGD can inhibit the growth of K562 cells and induce the apoptosis, and its cytotoxicity towards cultured K562 cell line is much more effective than that of nitrogen mustard.

THE EFFECT OF HYPO－OSMOLAR SOLUTIONS WITH HIBITANE ON SHED CANCER CELLS IN PERITONEAL CAVITY OF PATIENTS WITH GASTRIC CANCER

For 100 patients with gastric cancer, intraoperative intraperitoneal perfusion(IOIPP)using double-distilled water(D.D.W)with hibitane 37℃ for 5 minutes was performed.The anticancer effects were assessed cytologically in pre-IOIPP and post-IOPP specimens of the abdominal effusion and/or lavaged peritoneal fluid.The radical gastrectomy was performed in 65 of 93 cases with advanced gastric cancer, shed cancer cells in peritoneal cavity were detected in 16 of 65 cases with pre-IOIPP(24.62%)and to 3 of post-IOIPP cases(4.62%).There was significant difference between the two groups(P<0.01) . Shed cancer cells were inactivated in vivo estimated by the trypan-blue staining technique in 7 cases with radical surgery.But the effect of the IOIPP on shed cancer calls was not significant in cases with peritoneal metastasis. Also there were shed cancer cells in peritoneal cavity of patients with SS cancer histologically before the IOIPP(15. 56%)and iatrogenic diffusion of cancer cells during operation(7/24)in thisarticle.The above results indicated that the IOIPP with hypo-osmolar solution containing hibitane, 37℃ for 5 min was indispensable and useful for the killing of peritoneal shed cancer cells in order to prevent postoperative peritoneal recurrence in patients with radical surgery of advanced gastric cancer regardless of conditions of serosal invasion.