Concentrations Of Adriamycin Research Articles

RELATIVE ELECTRO PERMEABILITY AND ELECTRIC PULSE EFFECTIVENESS IN HUMAN BREAST ADEN CARCINOMA: AN IN VITRO STUDY

Objective(s) This study was carried out in order to evaluate the effects of electrochemotherapy, electrical pulses and chemotherapeutic drugs on the killing of cancerous cells and their probable synergistic effects. Materials and Methods Electrochemotherapy treatments conducted on MCF-7 cell line derived from human breast adenocarcinoma tumor using four chemotherapeutic drugs including bleomycin, cisplatin, adriamycin and cyclophosphamide and six electrical doses. Cell survival assayed using MTT method, 72 hrs after the treatment; also the killing effects of each drug and electric dose determined. Finally, “Relative Pulse Effectiveness” and “Relative Electropermeability Effectiveness” calculated. Results All electrical doses decreased cell survival, significantly for bleomycin and cisplatin, however, they were only, significant in high concentration of cyclophosphamide and adriamycin. For the applied drugs, “Relative Electropermeability Effectiveness” was more than one (1.00), except for adriamycin. Conclusion It seems that for the diffusion of molecules into cells, application of high duration electric pulses is more efficient for high molecular weight drugs while for low molecular weight drugs, strong pulses are more effective. In intermediate molecular weight, there is no difference between increasing the pulse strength and/or duration to achieve additional electropermeability. Electropermeability effect of different electric doses and electrochemotherapy efficiency can be evaluated by “REE” and “RPE”, respectively.

Reversal effect of MDR1 and MDR3 gene silencing by shRNA on resistance of breast carcinoma cells to adriamycin

Objective To investigate the effect of MDR1 and MDR3 gene silence by shRNA of human breast carcinoma cell line MCF-7/Adr,and explore the role of MDR1 and MDR3 in adriamycin-resistance of breast carcinoma cells. Methods shRNA plasmid vector specifically targeting MDR1 and MDR3 gene was transfected into cells. The control group was transfected with empty vector. The concentration of adriamycin was detected by the flow cytometry (FCM). Cell apoptosis was analysed by FITC-Annexin-V/PI double staining. Cell viability and the IC50 of adriamycin on MCF-7/Adr cells were determined by MTT method. MDR1 and MDR3 mRNA were assessed by RT-PCR. P-gp expression was detectedby immunochemistry. Results After treatment with ABCB1 and ABCB4 shRNA plasmid vector, the apoptosis of MCF-7/Adr cells was (30.21±1.65)%and (22.07±2.17)% respectively. Compared with untransfecedgroup and empty vector transfection group the difference was significant(P＜0.01). MDR1 and MDR3 shRNAcould increase cellular adriamycin accumulation of MCF-7/Adr cells. MCF-7/Adr cells viability and the IC50were significantly decreased after transfection. Compared with untransfeced group and empty vector transfectiongroup, the mRNA level of MDR1 and MDR3 in MCF-7/Adr cells were decreased by (89.5±0.8)%and(85.1±1.2)%, the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. Immunochemistry proved that the expression of p-gp was significantly inhibited. Compared with untransfeced group and empty vector transfection group the difference was significant (P＜0.05). Conclusion The shRNA can effectively and specifically silence the expression of MDR1 and MDR3 gene, reverse the adriamycin-resistance mediated by P-gp in MCF-7/Adr cells. The reversal effect of adriamycin-resistance by shRNA of MDR1 is more effective than that of MDR3. Key words: Drug resistance, neoplasm; Epirubicin; Apoptosis; shRNA

Reversal of P-Glycoprotein-Dependent Resistance to Adriamycin by 5-bromotetrandrine in K562/A02 Cell Line.

Objective: The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro.Methods: Drug sensitivity was determined using the MTT assay. Adriamycin (ADM) accumulation, the protein levels of P-glycoprotein (P-gp) and the apoptotic changes were analyzed by fluorospectrophotometry, respectively. The mRNA levels of P-gp was determined by RT-PCR.Results: BrTet at 0.25, 0.5 and reversed ADM resistance in MDR K562/A02 cells dose-dependently and its potency was greater than that of Tet at the same concentrations. The IC50 of ADM for K562 and K562/A02 cells were 55.122 mg/l and 1.1373 mg/l, respectively. Treating K562/A02 cells with BrTet(1uM)and TTD(1uM)both for 48 hours partially restored the sensitivity of K562/A02 cells to ADM (IC50 were 4.7729 mg/l and 13.584 mg/l respectively) but had not effect on K562 cells. The fold reversal (FR) were 11.55 and 4.06 respectively. K562/A02 cells showed apoptotic characteristics after treated with Brtet and Tet both for 48 hours compared with control group(apoptosis rate was 61.1%, 11.1% and 9.9%,respectively); Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of ADM in K562/A02 cells in a dose-dependent manner. The fluorescence intensity of intracellelar ADM in K562/A02 cells treated with ADM(1mg/L)was 33% of that in K562 cells. BrTet and Tet elevated the intracellular ADM concentration in K562/A02 cells up to 52% and 69%,respectively. BrTet also inhibited the overexpression of P-gp in K562/A02 cells. The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5 and 97.97.The P-gp expression was down after treated with BrTet and TTD (65.05 and 54.86). The mdr1 mRNA was also down regulated.Conclusions: BrTet showed significant MDR reversal activity in vitro. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs. BrTet may be a promising MDR modulator for eventual assessment in the clinic.

MDR-reversing effect of Extractum trametes robiniophila murr on human hepatocellular carcinoma HepG2/ADM in vitro

目的 探讨槐耳清膏体外逆转人肝癌细胞多药耐药性的作用及可能的机制.方法 MTT法检测槐耳清膏的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性;流式细胞仪检测细胞内阿霉素浓度;RT-RCR检测MDR1基因的表达.结果 槐耳清膏在0.01g/L以下剂量时对HepG2和HepG2/ADM耐药细胞株的抑制率均＜10%,半数抑制率(IC50)分别为0.917 g/L和1.66 g/L,无细胞毒剂量即0.008 g/L的槐耳清膏能部分逆转HepG2/ADM细胞的耐药性,与之合用使耐药肝癌细胞对阿霉素、顺铂、丝裂霉素、氟尿嘧啶的相对逆转效率分别为79%、73.5%、97.4%和86.7%,HepG2/ADM细胞内阿霉素浓度明显增加,MDR1表达下降了33.7%.结论 槐耳清膏具有体外逆转人肝癌细胞多药耐药性的作用,可能与下调MDR1表达、增加细胞内化疗药物积累有关。

The roles of four multi-drug resistance proteins in hepatocellular carcinoma multidrug resistance

The roles of multi-drug resistance protein 1 (MDR1), multi-drug resistance related protein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) in the multi-drug resistance (MDR) of hepatocellular carcinoma (HCC) were studied. By exposing HepG2 cell line to progressively increased concentrations of adriamycin (ADM), HepG2 multi-drug resistant subline (HepG2/ADM) was induced. The MDR index of HepG2/ADM was detected by using MTT. The expressions of the four MDR proteins in the three cell lines (L02, HepG2, HepG2/ADM) were investigated at mRNA and protein levels by real-time RT-PCR and Western blot respectively. Our results showed that when the ADM concentration was under 100 microg/L, HepG2 could easily be induced to be drug-resistant. The IC(50) of the HepG2/ADM to ADM was 282 times that of HepG2. The expression of MDR1 and BCRP mRNA in HepG2/ADM cells were 400 and 9 times that of HepG2 cells respectively while there was no difference in the mRNA expressions of MRP1 and LRP. There was no difference between HepG2 and L02 cells in the mRNA expressions of the four genes. At the protein level, the expressions of MDR1, BCRP and LRP but MRP1 in HepG2/ADM were significantly higher than those of HepG2 and L02. Between HepG2 and L02, there was no difference in the expressions of four genes at the protein level. HepG2/ADM is a good model for the study of MDR. The four genes are probably the normally expressed gene in liver. The expressions of MDR1 and BCRP could be up-regulated by anti-cancer agents in vitro. The MDR of HCC was mainly due to the up-regulation of MDR1 and BCRP but MRP1 and LRP. These findings suggest they may serve as targets for the reversal of MDR of HCC.

CdS Quantum Dots as Fluorescence Probes for Detection of Adriamycin Hydrochloride

Water-soluble CdS quantum dots(CdS-QDs) capped with thioglycolic acid were easily prepared, and a detection method of adriamycin was presented based on the fluorescence quenching of CdS-QDs. It was found that a complex could be formed between cetyltrimethyl ammonium bromide (CTAB) and CdS-QDs by using electrostatic interaction in Britton-Robinson (BR) buffer at pH = 7.00, and the strong fluorescence emission of the complex was observed at 500 nm when the complex was excited at 378 nm. The presence of adriamycin, however, could strongly quench the fluorescence through hydrophobic interaction. The overall quenching percentage as a function of adriamycin concentration matches the Stern-Volmer equation very well. These properties make CdS-QDs a potential fluorescence probe for the detection of adriamycin. The detection limit(3σ) of adriamycin is approximately 10−9 mol/L.

Self-assembled nanoparticles based on linoleic-acid modified carboxymethyl-chitosan as carrier of adriamycin (ADR)

Abstract Linoleic-acid (LA) is covalently conjugated to carboxymethyl-chitosan via a 1-ethyl-3-(3-dimethylaminopropyyl)-carbodiimide-mediated (EDC-mediated) reaction to generate self-aggregated chitosan nanoparticles by sonication. The average diameter of the particles is 417.8 nm, with a size range from 222.8 nm to 1028 nm. Adriamycin (ADR) can be physically entrapped into the self-aggregates. The drug loading experiments indicate that the loading capacity and efficiency increase with increasing concentration of ADR. ADR is slowly released from chitosan self-aggregates for about 3 days. Furthermore, the effects of drug controlled release become more obvious on increasing the pH value.

Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK-AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and the combination of MK-AS and ADM has a much better in vitro and in vivo synergism.

Reversal of adriamycin resistance in human mammary cancer cells by small interfering RNA of MDR1 and MDR3 genes

The purpose of this paper is to investigate the reversal effect of small interfering RNA (siRNA) targeting MDR1 and MDR3 genes on the resistance of MCF-7/ADR cells to adriamycin. siRNA plasmid vector targeting MDR1 and MDR3 genes was transfected into MCF-7/ADR cells, and then was stained with Annexin-V FITC (fluorescein isothiocyanate conjugated) to detect the early stage cell apoptosis by flow cytometry (FCM). 50% inhibition concentration (IC50) of adriamycin for MCF-7/ADR cells was determined by MTT method. MDR1 and MDR3 mRNA was assessed by RT-PCR. Treatment of MCF-7/ADR cells with the two kinds of siRNAs resulted in a reversal of adriamycin resistance of MDR to different extents. 1) The apoptosis efficiency of MDR1 and MDR3 siRNA vector after transfection was (18.21+/-1.65) % and (9.07+/-2.16) % respectively (P<0.05), and there was significant differences in the apoptosis efficiency between p Suppressor Neo vector and the MDR1 siRNA or MDR3 siRNA vector (P<0.01); 2) The reversal effect of MDR1 siRNA is higher than that of MDR3 siRNA (P<0.05); 3) The expression of MDRI and MDR3 mRNA can be restrained by p Suppressor Neo MDR1 and MDR3 siRNA respectively, and the reduction in the mRNA level was in a time-dependent manner (P<0.01). MDR1 and MDR3 gene silencing can enhance intracellular adriamycin accumulation in MCF-7/ADR cells, improve sensitivity of MCF-7/ADR cells to adriamycin, and induce cell apoptosis. The reversal effect of adriamycin resistance by siRNA of MDR1 was more effective than that of MDR3.

Liver targeting and the delayed drug release of the nanoparticles of adriamycin polybutylcyanoacrylate in mice

Liver targeting drug delivery systems can improve the curative effects and relieve the cytotoxicity of the chemotherapy drugs in the treatment of liver diseases. Nanoparticles carrying therapeutic drugs are currently under hot investigation with great clinical significance. This study was aimed to investigate the different tissue distribution of the adriamycin polybutylcyanoacrylate nanoparticle (ADM-PBCA-NP) in the mice body after an injection via lateral tail vein, and to study the liver targeting effects of ADM-PBCA-NP in different diameters on normal mice liver. One hundred and eighty Kunming mice were randomly divided into 6 groups with 30 mice in each group (5 treatment groups of ADM-PBCA-NP in the different diameter ranges, non-conjugated free adriamycin injection was employed as the control group). A single dose of either conjugated or free adriamycin equaled 2 mg/kg of body weight was delivered via the tail vein. Five mice in each trail were sacrificed at 5, 15, 30 minutes, 1, 5 and 12 hours postinjection, respectively. The adriamycin concentrations in the respectively collected liver, kidney, spleen, heart, lung and plasma were demonstrated using a high performance liquid chromatography with fluorescence detector. Compared with the control group, adriamycin was hardly detected in the heart muscle of the treatment groups (P < 0.05). The nanoparticle-conjugated adriamycin was cleaned up quickly from the kidney tissue. The adriamycin concentrations of the mice liver and spleen in the experimental groups were significantly higher than that in the control group, except for the group with the nanoparticles diameters of (22.3 +/- 6.2) nm (P < 0.05). The ADM-PBCA-NP in (101.0 +/- 20.3) nm diameter had the highest liver distribution, and the second highest adriamycin distribution in liver was the group of (143.0 +/- 23.5) nm diameter (P < 0.05). Moreover, adriamycin was released slowly in the liver during the detection period in the experimental groups. ADM-PBCA-NP in (22.3 +/- 6.2) nm diameter was not distributed in the tissue of the liver, kidney, heart, spleen, and lung. ADM-PBCA-NP in 100 - 150 nm diameter range has the best liver targeting with a characteristic of slow medicine release. It also decreases the medicine distribution in the heart, kidney and lung. In the treatment of liver cancer, the polybutylcyanoacrylate nanoparticles system has a good liver targeting ability, which increases the anticancer activity and markedly decreases the toxicity of adriamycin.

Adriamycin-induced oxidative stress, activation of MAP kinases and apoptosis in isolated cardiomyocytes

Background: Adriamycin (ADR) induced heart failure is associated with an increase in oxidative stress and apoptosis. Changes due to ADR in mitogen-activated protein kinases (ERK1/2 and p38 MAPKs), pro- and anti-apoptotic proteins (Bax and Bcl-xl) and apoptosis were examined in isolated adult rat cardiomyocytes. Methods: Isolated adult rat cardiomyocytes were exposed to different concentrations of ADR (0.1, 1 and 10 μmol/L) and analyzed at different post-treatment durations. Antioxidant, trolox (20 μmol/L) was used to determine involvement of oxidative stress on these changes. Results: Total ERK1/2 and p38 did not show any significant change. However, phosphorylated ERK1/2 showed a rapid increase (497%) after 5 min of ADR treatment, peaking (610%) at 10 min followed by a decline to submaximal levels at 30 min (280%) and 60 min (247%). Phosphorylated p38 showed no changes until after 30 min, peaking (284%) at 1 h, followed by a small decline at 2 h. These changes were found to be dose-dependent (0.1, 1 and 10 μmol/L of ADR). Adriamycin induced apoptosis was confirmed by Hoechst staining. The ratio of Bax/Bcl-xl increased in a dose-dependent manner. At 10 μmol/L, this ratio increased to a maximum at 1 h, remained steady at the level for up to 12 h, followed by a decline below the base line at 24 h. Antioxidant, trolox modulated the ADR-induced increase in phosphorylation of ERK1/2 and p38 MAPKs as well as in the ratio of Bax/Bcl-xl. Conclusion: It is suggested that ADR activates MAP kinases, followed by an activation of pro-apoptotic protein Bax which results in cardiomyocyte apoptosis and these effects appear to be mediated by ADR-induced oxidative stress.

Adriamycin thermotherapy through the hepatic artery using VX2 carcinoma in rabbit liver as a model

OBJECTIVE It has been reported that heating can enhance sensitivity of rabbit VX2 cells to adriamycin and increase the intracellular concentration of adriamycin. This study was designed to evaluate the anti-tumor effect of interventional hyperthermia and interventional thermochemotherapy on VX2 carcinoma in rabbit liver.

Preparation and characterization of self-assembled nanoparticles based on glycol chitosan bearing adriamycin

An anthracycline drug, adriamycin, was chemically conjugated onto the backbone of glycol chitosan via an acid-labile cis-aconityl linkage. The physicochemical characteristics of the glycol chitosan–adriamycin (GC–ADR) conjugates were investigated by dynamic light scattering, atomic force microscopy, and fluorescence spectroscopy. The GC–ADR conjugates were capable of forming nano-sized self-aggregates in an aqueous medium, when the adriamycin content in the conjugate was in the range of 2.0–5.0 wt.%. The self-aggregates were spherical in shape, and had mean diameters of 238–304 nm, depending on the adriamycin content. The critical aggregation concentrations of the conjugates, estimated by the fluorescence quenching method, were as low as 1.0–2.5×10−2 mg/ml. The size of self-aggregates was not affected by the polymer concentration in the range from 50 to 2,000 μg/ml, and was maintained up to 8 days in phosphate-buffered saline (pH 7.4), indicating high colloidal stability. The release of adriamycin from self-aggregates was significantly dependent on the pH of the medium due to the cis-aconityl linkage; e.g., the amount of adriamycin released for 4 days was 7.3±0.3% at pH 7, whereas it was 29.3±1.9% at pH 4. The cell viability results demonstrated that free adriamycin shows more potent cytotoxicity than the conjugates, primarily attributed to the sustained release of adriamycin from self-aggregates. In conclusion, the self-aggregates, formed by GC–ADR conjugates, might be useful for the site-specific delivery of adriamycin in a sustained manner.

Interstitial Hyperthermia With Intra-arterial Injection of Adriamycin for Malignant Glioma

A new method for treating malignant glioma by concurrent intra-arterial injection of adriamycin during thermotherapy was performed in seven patients with malignant glioma, four males and three females, with five cases of glioblastoma and two of anaplastic oligodendroglioma. Adriamycin was intra-arterially injected at a dose of 20 mg via the common carotid artery during thermotherapy. The heating procedure was repeated three times combined with chemotherapy in one therapy course, and a total of nine therapy courses were performed in the seven patients. All patients tolerated the protocol well. Based on post-therapy computed tomography, five of the therapy courses achieved partial response, one course resulted in disease progression, and the remaining three courses showed no change. The median time to progression was 3.4 months and the overall median length of survival following stereotactic biopsy was 13.2 months. Facial flushing was observed during eight therapy courses, and extensive alopecia in six therapy courses. Intracystic concentrations of adriamycin were determined in three patients, and marked increases were observed. Intra-arterial injection chemotherapy during hyperthermia is a promising therapeutic method for treatment of malignant glioma with few adverse effects.

Development and characterization of multidrug resistant human hepatocarcinoma cell line in nude mice

To establish a multidrug resistant (MDR) cell sub-line from the human hepatocarcinoma cell line (HepG2) in nude mice. HepG2 cell cultures were incubated with increasing concentrations of adriamycin (ADM) to develop an ADM-resistant cell subline (HepG2/ADM) with cross-resistance to other chemotherapeutic agents. Twenty male athymic BALB/c-nu/nu mice were randomized into HepG2/nude and HepG2/ADM/nude groups (10 in each group). A cell suspension (either HepG2 or HepG2/ADM) was injected subcutaneously into mice in each group. Tumor growth was recorded, and animals were sacrificed 4-5 wk after cell implantation. Tumors were prepared for histology, and viable tumor was dispersed into a single-cell suspension. The IC50 values for a number of chemotherapeutic agents were determined by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (MTT) assay. Rhodamine-123 retention/efflux and the level of resistance-associated proteins were determined by flow cytometry. The mRNA expression of mdr1, mrp and lrp genes was detected using reverse transcriptase polymerase chain reaction (RT-PCR) in HepG2/nude and HepG2/ADM/nude groups. The appearances of HepG2/nude cells were slightly different from those of HepG2/ADM/nude cells. Similar tumor growth curves were determined in both groups. A cross-resistance to ADM, vincristine, cisplatin and 5-fluorouracil was seen in HepG2/ADM/nude group. The levels of P-glycoprotein and multidrug resistance-associated proteins were significantly increased. The mRNA expression levels of mdr1, mrp and lrp were higher in HepG2/ADM/nude cells. ADM-resistant HepG2 subline in nude mice has a cross resistance to chemotherapeutic drugs. It may be used as an in vivo model to investigate the mechanisms of MDR, and explore the targeted approaches to overcoming MDR.

Study of Cytotoxic Effects of K562/Adm Cells by Low Frequency Ultrasound Enchance Adriamycin.

Objective The aim of this study is to investigate and analysis the effect of ultrasound potentiate the cytotoxicity of adriamycin and reverse drug resistance on leukemia drug resistance K562/Adm cell line in vitro, to find out the mechanism of the reverse effect of ultrasound exposure.Methods Human leukemia adriamycin resistant strain K562/Adm as target cells, were treated by adriamycin singly in group Adm, by ultrasound exposure singly in group US and by adriamycin prior to ultrasound exposure in group Adm+US. The dosages that didn't attribute to cell killing immediately were detected. Trypan blue dye exclusion test and MTT assay were used to determine the sensitivity of K562/Adm. Wright's staining and transmission electron microscope were used to detect the apoptosis and structure change of K562/Adm cells. Flow cytometry was used to analysis intracellular drug concentration and electron microscopic scanning was used to observe the membrane change. Immunocytochemistrial method was used to evaluate the expressions of P-gp.Results At 0.17W•cm−2 and lower acoustic intensity, ultrasound didn't result in K562/Adm acute cells destruction; and 0.5 W•cm−2 ultrasonic intention could make cell killed rapidly after K562/Adm cells were irritated by ultrasound exposure singly. Significant differences were obtained between ultrasound treated and untreated cells in the presence of various concentrations of adriamycin. If the same concentration of cytotoxic agents were used, more cells were killed if sonication was applied. ultrasound for 30s at 20kHz, 0.17W•cm−2 intensity almost could not damage K562/Adm cells but dramatically decrease adriamycin concentration which induce cell achieve IC50;There were some morphological alterations in cells irradiated by ultrasound, Nearly all the treated cells by ultrasound exhibited small holes with diameter about 1~2 μm in the K562/Adm cell surface; the intracellular adriamycin accumulation in group Adm+US were prompted compared with Adm group and controlled group. Many apoptotic phenomena were observed in Adm+US group, show many vesicle and the form of apoptotic body, but there were no change in US group compared with controlled group. And the expression of P-gp protein had no significant difference between before and after ultrasound eradiated.ConclusionsHigher ultrasound intensity could make K562/Adm cells killed rapidly, and lower ultrasonic level could potentiate the cytotoxicity of adriamycin to K562/Adm cells and reverse drug resistance on K562/Adm cells.Ultrasonic cavitation and sonoporation are the main mechanisms of the synergism between adriamycin and low-level sonication; the ultrasonically induced increase in intracellular drug accumulation.The expression of P-gp had no change in US group compare with controlled group.

Selective Killing of Adriamycin-Resistant (G2 Checkpoint-Deficient and MRP1-Expressing) Cancer Cells by Docetaxel

Chemotherapy of cancer is limited by toxicity to normal cells. Drug resistance further limits the therapy. Here, we investigated selective killing of drug-resistant cancer cells by antagonistic drug combinations, which can spare (because of drug antagonism) normal cells. We used paired cell lines that are resistant to Adriamycin due to either expression of MRP1 or lack of G2 checkpoints. The goal was to selectively kill Adriamycin-resistant cancer cells with Docetaxel (Taxotere), while protecting parental (Adriamycin-sensitive) cells, using cytostatic concentrations of Adriamycin. Taxotere kills cells in mitosis. Therefore, by arresting parental cells in G2, 20 to 40 ng/mL of Adriamycin prevented cell death caused by Taxotere. Also, Adriamycin prevented the effects of Taxotere in normal human lymphocytes. In contrast, Taxotere selectively killed MRP1-expressing leukemia cells, which did not undergo G2 arrest in the presence of Adriamycin. Also, in the presence of Adriamycin, HCT116-p21-/- cancer cells with a defective G2 checkpoint entered mitosis and were selectively killed by Taxotere. Finally, 20 ng/mL of Adriamycin protected normal FDC-P1 hematopoietic cells from Taxotere. Whereas parental cells were protected by Adriamycin, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD90598 potentiated the cytotoxic effect of Taxotere selectively in Raf-1-transformed FDC-P1 leukemia cells. We propose a therapeutic strategy to prevent normal cells from entering mitosis while increasing apoptosis selectively in mitotic cancer cells.

The Mitotic Spindle Checkpoint Is a Critical Determinant for Topoisomerase-based Chemotherapy

A novel strategy in cancer therapy is the induction of mitotic cell death by the pharmacological abrogation of cell cycle checkpoints. UCN-01 is such a compound that overrides the G2 cell cycle arrest induced by DNA damage and forces cells into a deleterious mitosis. The molecular pathways leading to mitotic cell death are largely unknown although recent evidence indicates that mitotic cell death represents a special case of apoptosis. Here, we demonstrate that the mitotic spindle checkpoint is activated upon chemotherapeutic treatment with topoisomerase II poisons and UCN-01. Cells that are forced to enter mitosis in the presence of topoisomerase inhibition arrest transiently in a prometaphase like state. By using a novel pharmacological inhibitor of the spindle checkpoint and spindle checkpoint-deficient cells we show that the spindle checkpoint function is required for the mitotic arrest and, most importantly, for efficient induction of mitotic cell death. Thus, our results demonstrate that the mitotic spindle checkpoint is an important determinant for the outcome of a chemotherapy based on the induction of mitotic cell death. Its frequent inactivation in human cancer might contribute to the observed resistance of tumor cells to these chemotherapeutic drugs.

Combined effects of Cantide and chemotherapeutic drugs on inhibition of tumor cells’ growthin vitroandin vivo

To investigate the combination effect of hTERT antisense oligonucleotide "Cantide" and three chemotherapeutic drugs (cisplatin, 5-fluorouracil (5-FU) and adriamycin (ADM)) on inhibiting the proliferation of HepG2, BGC and A549 cell lines in vitro, and to investigate the efficacy of Cantide used in combination with cisplatin (DDP) in vivo. Cantide was transfected into these tumor cells by Lipofectin, and cell growth activity was calculated by microcytotoxicity assay. In vivo study, cells of HepG2 were implanted in Balb/c nude mice for 4 d. Then Cantide, DDP and Cantide+DDP were given intraperitoneally for 24 d respectively. The body weights of the tumor-bearing animals and their tumor mass were measured later to assess the effect of combination therapy in the nude mice. To evaluate the interaction of Cantide and these chemotherapeutic drugs, SAS software and Jin Zhengjun method were used. Combination treatments with 0.1 micromol/L Cantide reduced the IC50 of DDP, 5-FU and ADM from 1.07, 4.15 and 0.29 microg/mL to 0.25, 1.52 and 0.12 microg/mL respectively. The inhibition ability of DDP, 5-FU and ADM respectively in combination with Cantide in these tumor cells was higher than that of these drugs alone (P<0.0001). And synergism (Q > or = 1.15) was observed at the lower concentration of DDP (< or = 1 microg/mL), 5-FU (< or = 10 microg/mL) and ADM (< or = 0.1 microg/mL) with combination of Cantide. In vivo, combination treatment with Cantide and DDP produced the greater growth inhibition of human liver carcinoma cells HepG2 in nude mice (0.65+/-0.19 g tumor) compared with that when only one of these drugs was used (Cantide group: 1.05+/-0.16 g tumor, P = 0.0009<0.001; DDP group: 1.13+/-0.09 g tumor, P = 0.0001<0.001). These findings indicate that Cantide may enhance therapeutic effectiveness of chemotherapeutic drugs over a wide range of tumor cells in vitro, and the combination use of Cantide and DDP can produce much higher inhibition rates, as compared with when either of these drugs was used only in vivo.

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402/R

To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2,000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhIFN-gamma, monoclonal anti-CD3 antibody, rhIL-1alpha as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5 x 10(10) of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.