Cytotoxic effect of a polymer�Ccopper(II) complex containing 2,2��-bipyridyl ligand on human lung cancer cells

The cytotoxic activity of the polymer–copper(II) complex, [Cu(bpy)₂(BPEI)]Cl₂·4H₂O (bpy = 2,2′-bipyridine, BPEI = branched polyethyleneimine) on NCI-H460 human lung cancer cells was determined adopting MTT assay and specific staining techniques. Many of the cultured NCI-H460 cancer cells treated with this complex suffered loss of viability and death mostly through apoptosis and/or necrosis as evidenced by the changes in the nuclear morphology and cytoplasmic features.     

Consistent interactions between tumor cell IL-6 and macrophage TNF-α enhance the growth of human prostate cancer cells in the bone of nude mouse

To test the hypothesis that tumor-associated macrophages (TAMs) enhance the growth and metastasis of human prostate cancer in the bone, we evaluated the effects of decreasing interleukin-6 (IL-6) production by tumor cells and TAMs in a mouse model of bone metastasis. Human PC-3MM2 cells that produce IL-6 were transfected with lentivirus containing IL-6 small hairpin RNA (shRNA) or nonspecific RNA and injected into the tibias of nude mice treated intraperitoneally every 5days for 5weeks with phosphate-buffered saline (PBS), liposomes containing PBS, or liposomes containing clodronate (to decrease the number of macrophages). Transfection of PC-3MM2 cells with IL-6 shRNA significantly decreased cellular expression of IL-6 and the number of TAMs and osteoclasts in bone tumors, which correlated with significant decreases in tumor size, bone lysis, and incidence of lymph node metastasis. Treatment of mice with clodronate liposomes significantly decreased the number of TAMs and osteoclasts in the bone tumors, the expression of IL-6 in the PC3-MM2 cells, and the production of tumor necrosis factor (TNF)-α by TAMs. These findings correlated with a significant decrease in tumor size, bone lysis, and lymph node metastasis. Knocking down IL-6 in tumor cells and decreasing TAMs was associated with the lowest incidences of bone tumors and lymph node metastasis. These results suggest that TAMs enhance the growth of prostate cancer cells in the bone.     

Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway

Aim:

To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.

Methods:

Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.

Results:

Plumbagin (2.5–40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC₅₀ value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5–20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.

Conclusion:

Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.     

Resveratrol analog, 3,5,2′,4′-tetramethoxy-trans-stilbene, potentiates the inhibition of cell growth and induces apoptosis in human cancer cells

Resveratrol, a trihydroxystilbene found in grapes and several plants, has been shown to be active in inhibiting multistage carcinogenic process. Using resveratrol as the prototype, we synthesized several analogs and evaluated their growth inhibitory effect using cultured human cancer cells. In the present report we show that one of the resveratrol analogs, 3, 5,2',4'-tetramethoxy-trans-stilbene, potentiated the inhibition of cancer cell growth. Prompted by the strong growth inhibitory activity of the compound (IC50; 0.8 microg/ml) compared to resveratrol (IC50; 18.7 microg/ml) in cultured human colon cancer cells (Col2), we performed an action mechanism study using the compound. The compound induced the accumulation of cellular DNA contents in the sub-G0 phase DNA contents of the cell cycle by in a time-dependent manner. The morphological changes were also consistent with an apoptotic process. This result indicated that the compound induced apoptosis of cancer cells, and may be a candidate for use in the development of potential cancer chemotherapeutic or cancer chemopreventive agents.     

PPAR-γ ligand promotes the growth of APC-mutated HT-29 human colon cancer cells in vitro and in vivo

Summary PPAR-γ has been known to induce suppression, differentiation and reversal of malignant changes in colon cancer in vitro. However, there are several reports that PPAR-γ ligands enhance colon polyp development in APC^min mice in vivo. These contradictory results have not yet been thoroughly explained. To explain the contradictory results, we analyzed the effects of different concentrations of the PPAR-γ agonist, 15-deoxy-D12, 14-prostaglandin (15-d Δ PGJ2) and pioglitazone, on APC gene-mutated colon cancer cell lines (HT-29). We measured cell growth and suppression by cell count and MTT assay and analyzed the expression of β-catenin and c-Myc protein by Western blot. In addition, we inoculated HT-29 cells into APC^min mice to compare tumor size. High concentrations (10–100 μM/L 15-d Δ PGJ2 and pioglitazone) of PPAR-γ ligand suppressed growth, while low concentrations (0.01–1 μM/L 15-d Δ PGJ2 and pioglitazone) of PPAR-γ ligand promoted growth. In particular, the effects of 0.1 μM/L 15-d Δ PGJ2 and pioglitazone on cell growth were statistically significant (P = 0.003, P = 0.001, respectively). Tumor growth was associated with an increase in β-catenin and c-Myc expression. The growth of xenograft tumors was greater in PPAR-γ ligand-treated mice than in control mice (control vs day 14: P = 0.024, control vs day 28: P = 0.007). The expression of β-catenin and c-Myc protein were also elevated in PPAR-γ-treated mouse tissues. PPAR-γ ligand can promote the growth of APC-mutated HT-29 colon cancer cells in vitro and in vivo. In addition, the tumor promoting effect seems to be associated with an increase in β-catenin and c-Myc expression. We think that well-controlled clinical trials should be conducted to confirm our results and to verify clinical applications.     

Two new 8-O-4′-type lignans from the stem of Schima superba and their cell growth inhibitory activities against human cancer cell lines

Two new lignans (1 and 2) were isolated from the EtOH extracts of the stem of Schima superba, and elucidated as (7R,8S)-1-(3,4-dimethoxyphenyl)-2-O-(2-methoxy-4-omegahydroxypropylphenyl)propane-1,3-diol (1) and threo-1-(4-hydroxy-3-methoxyphenyl)-1-methoxy-2-{4-[1-formyl-(E)-vinyl]-2-methoxyphenoxy}-3-propanol (2) by spectral analysis. Compounds 1 and 2 showed cell growth inhibitory activity against HeLa, CNE, HepG-2, and HEp-2 cell lines. Compound 1 exhibited significant cytotoxicities with IC₅₀ values of less than 4 μg/ml against all the tested cell lines.     

Characterization of ligand binding to the σ(1) receptor in a human tumor cell line (RPMI 8226) and establishment of a competitive receptor binding assay

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 μM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.     

Antiproliferative effects of novel urea derivatives against human prostate and lung cancer cells; and their inhibition of β-glucuronidase activity

Twenty-one novel urea derivatives were synthesized and their structures characterized by mass, NMR, IR, and UV spectroscopy. These compounds were evaluated for their antiproliferative profile against human PC-3 (prostate) and NCI-H460 (lung) cancer cell lines. Among them, compound 21 N-(3-nitrophenyl)-N′-(1-phenylethyl)urea was found to be active against both PC-3 (IC₅₀ ± SEM: 20.13 ± 0.91 μM) and NCI-H460 (GI₅₀: 22 ± 2.6 μM) cell lines; hence has the potential to be further studied as anticancer agent. These compounds were also investigated for their ability to inhibit urease, β-glucuronidase, and phosphodiesterase enzymes. N-(2,6-Dimethylphenyl)-N′-(4′-nitrophenyl)urea (1) demonstrated 90 % inhibition of β-glucuronidase enzyme (IC₅₀ ± SEM: 3.38 ± 0.043 μM).     

Possible contribution of β-glycosidases and caspases in the cytotoxicity of novel glycoconjugates in colon cancer cells

Glycoconjugates represent a recent trend in cancer chemotherapy that adopts the concept of selective prodrug/drug targeting of tumor cells by selectively binding to specific transmembrane glucose transporters. Following preferential uptake of sugar conjugates into cancer cells, they are presumably subject to enzymatic cleavage by specific β-glycosidases to liberate the free active cytotoxic aglycones that act selectively on cancer cells and spare other noncancerous ones. In this sense, the cytotoxicity of an array of newly synthesized glycoconjugates, including curcumin β-glucoside, perillyl alcohol β-glucoside, perillyl alcohol β-galactoside, diethylstilbesterol β-glucoside and diethylstilbesterol β-galactoside have been investigated over 24–96 h in a panel of human colon cancer cells namely, Caco-2, HT29 and T84 cells. The role of β-glycosidases and caspases in the bioactivation and cytotoxicity of these compounds has been addressed in the current study. All the glycoconjugates have proven cytotoxic efficacy in a time-dependent manner. Curcumin β-glucoside was the most potent amongst all glycoconjugates tested. The sensitivity rank order of tumor cells towards all β-glucosides was Caco-2 > HT29 > T84. This sensitivity ranking was well correlated with β-glucosidase activity assessed in these cell lines. Unlike perillyl alcohol galactoside, the cytotoxicity rank order for diethylstilbesterol β-galactoside was not coping with the β-galactosidase activity detected. Apoptosis was assessed by fluorometric assay of caspase-3 and caspase-9 activities. Initiation and activation of apoptosis were increased in all colon cancer cells following exposure to most of the glycoconjugates, and this was well correlated with the cytotoxicity rank order of these prodrugs. Enzymatic cleavage of glycoconjugates was accomplished using a host of hydrolytic enzymes and cleavage kinetics was determined using HPLC. The glycoconjugates were only cleaved by β-glucosidases and β-galactosidases, but not by pancreatic lipase or hepatic esterase. Taken together, one could conclude that β-glucosidases and β-galactosidases are crucial for the bioactivation and cytotoxicity of these glycoconjugates. Also, initiation and activation of apoptosis in tumor cells may contribute, at least partly, for the cytotoxicity of these sugar conjugates.     

A role for L-��-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Background and purpose:

Increased circulating levels of L-α-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

Experimental approach:

Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex® invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves.

Key results:

GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [³⁵S]GTPγS to cell membranes (pEC₅₀ 6.47 ± 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces.

Conclusions and implications:

LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Phase II trial of 4′-deoxydoxorubicin (esorubicin) in hormone resistant prostate cancer

Summary Fifteen patients with hormone resistant advanced prostate cancer were treated with anthracycline analog 4-deoxydoxorubicin (Esorubicin). No patient had objective evidence of tumor regression. Six patients (40%) were classified using the National Prostatic Cancer Project criteria as having stable disease after two courses of therapy. Treatment was associated with significant hematologic toxicity with 50% of patients experiencing grade III or IV neutropenia. Clinical cardiac toxicity was not observed. Further trials of 4 -deoxydoxorubicin do not appear to be warranted in advanced prostate cancer.     

Berbamine, a novel nuclear factor κB inhibitor, inhibits growth and induces apoptosis in human myeloma cells

Aim:

We sought to investigate the effect of berbamine on the growth of human multiple myeloma cell line KM3 and elucidate the mechanism of its action.

Methods:

MTT assay was used to determine the inhibitory effect of berbamine alone or combined with chemotherapeutic drugs. Flow cytometry was performed to characterize cell cycle profile in response to berbamine treatment. Western blot was used to measure the protein levels of p65, IκB Kinase α (IKKα), TNFAIP3 (A20), IκBα, p-IκBα, cyclinD1, Bcl-2, BAX, Bcl-x_L, Bid, and survivin.

Results:

Berbamine inhibits the proliferation of KM3 cells in a dose- and time-dependent manner. Combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO) resulted in enhanced inhibition of cell growth. Flow cytometric analysis revealed that KM3 cells were arrested at G₁ phase and apoptotic cells increased from 0.54% to 51.83% for 36 h. Morphological changes of cells undergoing apoptosis were observed under light microscope. Berbamine treatment led to increased expression of A20, down-regulation of IKKα, p-IκBα, and followed by inhibition of p65 nuclear localization. As a result, NF-κB downstream targets such as cyclinD1, Bcl-x_L, Bid and survivin were down-regulated.

Conclusion:

Berbamine inhibits the growth of KM3 cells by inducing G₁ arrest as well as apoptosis. Berbamine blocks NF-κB signaling pathway through up-regulating A20, down-regulating IKKα, p-IκBα, and then inhibiting p65 nuclear translocation, and resulting in decreased expression of the downstream targets of NF-κB. Our results suggest that berbamine is a novel inhibitor of NF-κB activity with remarkable anti-myeloma efficacy.     

A novel PPAR alpha／gamma dual agonist inhibits cell growth and induces apoptosis in human glioblastoma T98G cells

AIM: To examine the effect of a novel peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist TZD 18 on cell proliferation and apoptosis in human glioblastoma T98G cells and its possible mechanism. METHODS: RTPCR, MTT, TUNEL, Flow cytometry, and Western blot analysis were employed. RESULTS: TZD18 inhibited the growth of T98G cells in a concentration-dependent manner, which was associated with a GI to S cell cycle arrest. Besides, significant apoptosis was induced after treatment with a non-toxic dose of TZD 18. During the process, the expression of Bcl-2 protein was down-regulated, while that of Bax and p27^kip proteins was up-regulated, and the activity of caspase-3 was elevated. However, this effect appeared to be PPARα and PPARγ/independent since their antagonists could not reverse this effect. CONCLUSIONS: TZD18, a novel PPARα/γ dual agonist, inhibited cell growth and induce apoptosis in human glioblastoma T98G cells in vitro, indicating a therapeutic potential for TZD18 in the treatment of glioblastoma.     

Interaction of vascular endothelial cells with leukocytes, platelets and cancer cells in inflammation, thrombosis and cancer growth and metastasis

Adhesion and migration of mammalian cells are of crucial importance in a number of biological events, such as fertilization, embryogenesis, pattern, tissue and organ formation, and in a variety of physiological and pathological processes, including lymphocyte trafficking, leukocyte recruitment, hemostasis, wound healing, tumor angiogenesis and cancer metastasis. All these     

Tretinoin or retinol enhancement of lymphokine-activated killer cell proliferation and cytotoxicity against human bladder cancer cells in vitro

目的：研究维Ａ酸（Ｔｒｅ）或维生素Ａ（Ｒｅｔ）对膀胱肿瘤患者淋巴因子激活的杀伤细胞（ＬＡＫ细胞）的增殖和对膀胱肿瘤细胞的细胞毒作用．方法：分别用细胞计数和ＭＴＴ法测定ＬＡＫ细胞的增殖和细胞毒作用．结果：Ｔｒｅ或Ｒｅｔ１０－１００ｎｍｏｌ·Ｌ－１加强由白细胞介素２（ＩＬ２）诱导的ＬＡＫ细胞的增殖和对膀胱肿瘤细胞的杀伤作用．结论：Ｔｒｅ或Ｒｅｔ增强膀胱肿瘤患者ＬＡＫ细胞增殖及对膀胱肿瘤的细胞毒作用．     

Glycogen synthase kinase 3β inhibitors induce apoptosis in ovarian cancer cells and inhibit in-vivo tumor growth

Ovarian cancer is the most lethal gynecological malignancy among US women. Paclitaxel/carboplatin is the current drug therapy used to treat ovarian cancer, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3β (GSK3β), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3β inhibitors (GSK3βi) were synthesized, selected, and tested in vitro using SKOV3 and OVCA432 serous ovarian cancer cell lines. From a panel of 10 inhibitors, GSK3βi 9ING41 was found to be the most effective in vitro. 9ING41 induced apoptosis as indicated by 4',6-diamidino-2-phenylindole-positive nuclear condensation, poly (ADP-ribose) polymerase cleavage, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The mechanism for apoptosis was through caspase-3 cleavage. GSK3βi upregulated phosphorylation of the inhibitory serine residue of GSK3β in OVCA432 and SKOV3 cell lines and also inhibited phosphorylation of the downstream target glycogen synthase. An in-vivo xenograft study using SKOV3 cells demonstrated that tumor progression was hindered by 9ING41 in vivo. The maximum tolerated dose for 9ING41 was greater than 500 mg/kg in rats. Pharmacokinetic analysis showed 9ING41 to have a bioavailability of 4.5% and to be well distributed in tissues. Therefore, GSK3β inhibitors alone or in combination with existing drugs may hinder the growth of serous ovarian cancers.