PPARγ-dependent pathway in the growth-inhibitory effects of K562 cells by carotenoids in combination with rosiglitazone

Background

Carotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression.

Methods

The effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting.

Results

Rosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone.

Conclusions

PPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells.

General significance

Carotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.     

Direct and protective effects of single or combined addition of vincristine and ε-viniferin on human HepG2 cellular oxidative stress markers in vitro

The objective of this study is to examine the direct effects of low doses and high doses of ε-viniferin, a substance known to be an antioxidant, and vincristine sulphate, a chemotherapeutic agent, alone and in combination [ε-viniferin + vincristine] on HepG2 cell strain, as well as evaluate oxidative stress after incubation periods of 3, 6, and 24 h. Direct effect was determined right after the incubation period; however, for protective effect, antioxidant protection response was determined after the treatment for 1 h with 500 μM H₂O₂, which is an oxidative stressor. For this purpose, superoxide dismutase was determined for enzyme activity, and lipid hydroperoxide (LPO) and reduced glutathione concentrations were studied as indicators of oxidative stress. Results show that low [3.63 µM vincristine + 3.75 µM ε-viniferin] and high [11.25 µM vincristine + 15.8 µM ε-viniferin] doses of combination groups showed similar direct antioxidant effect on LPO levels as protective when compared to the H₂O₂ control group (p < 0.05). Superoxide dismutase enzyme showed a direct antioxidant effect in low and high dose combination groups. In addition, when the incubation period was increased to 24 h, a protective effect was observed in both dose groups (p < 0.05). Reduced glutathione activities showed a direct effect in the low dose combination group, and a protective effect in both the low and high doses in the 24 h. These results show that combined usage of drugs in HepG2 cell strain possesses a protective effect against exogenically produced oxidative stress conditions.     

Behavioural measures reflect pain-mitigating effects of meloxicam in combination with Tri-Solfen® in mulesed Merino lambs

Flystrike costs the Australian industry $173 to 280 M per annum and 70% to 80% of Merino lambs are currently mulesed to reduce the risk of flystrike. To alleviate welfare concerns there has been widespread adoption of analgesics to mitigate the pain associated with mulesing. The objective of this experiment was to determine the effectiveness of Tri-Solfen® and meloxicam (Metacam® 20) at reducing pain-related behavioural responses to mulesing in Merino lambs. One hundred and forty Merino lambs were allocated to one of seven treatment groups: (1) non-mulesed (Control); (2) mulesed with no pain relief; (3) subcutaneous (s.c.) meloxicam administered 15 min before mulesing; (4) Tri-Solfen® administered at time of mulesing; (5) Tri-Solfen® and saline injection (s.c.) 15 min before mulesing; (6) Tri-Solfen® and meloxicam (s.c.) 15 min before mulesing; and (7) meloxicam (s.c.) at time the of mulesing. Behavioural responses such as standing, walking and lying were measured every 15 min for 6 h on the day of marking and for up to 2 h for 4 days thereafter. Standing (hunched v. normal) and walking (stiff v. normal) behaviours were then categorised into pain- and normal-related behaviours while lying remained in its own category. Mulesed lambs with no pain relief displayed significantly more pain-related behaviours than Control lambs during the 6 h post-mulesing (1.22 v. 0.22 out of a total score of 3; RSD=1.15). Lambs that received a combination of pain relief displayed significantly less pain-related behaviour than mulesed lambs with no pain relief on the day of mulesing (0.85 v. 1.22 out of a total score of 3; RSD=1.15). Administration of meloxicam or Tri-Solfen® on their own had minimal if any significant effect on pain-related behaviours on the day of mulesing. The results of this experiment support the use of pain-related behaviours to measure the efficacy of analgesics and the use of multimodal analgesia during mulesing of lambs.     

Antagonic effects of oestradiol in interaction with IGF-1 on proliferation of lactotroph cells in vitro

The effects of IGF-1, 17 β oestradiol and its functional interaction on lactotrophs cell proliferation were evaluated. In addition we investigated the involvement of PKC α, ɛ and phosphorilated ERK, in the mitogenic process. Primary cell cultures of adenohypophysis from female Wistar rats were studied in serum free conditions. The proliferation of lactotrophs was determined by double immunostaining for BrdU and PRL. The incubation with IGF-1 5, 30 or 100 ng/ml during 48 or 72 h increased lactotrophs proliferation two–threefold depending on IGF-1 concentration. Co-incubation of IGF-1 (30 ng/ml) with genistein (25 μM) or BIM (0.5 or 2 μM), lowered of tyrosine kinase receptor or of PKC respectively, inhibited the induced IGF-1 lactotrophs proliferation. 17 β oestradiol (1, 10 or 100 nM) had not mitogenic effect, whereas in the presence of serum PRL cells proliferation was stimulated. Co-incubation with 1 nM oestradiol and IGF-1 significantly decreased the lactotroph BrdU-labelling achieved with IGF-1. PKC α, ɛ and ERK1/2 levels measured by western blot augmented in the presence of IGF-1 and were inhibited with the addition of genistein, supporting a participation of these enzymes in the proliferate process. Co-incubation of IGF-1 with 1 nM oestradiol decreased both PKC isoforms and activated ERK1/2 levels, suggesting that oestradiol would exert its antiproliferative effect by acting on the signalling pathway of IGF-1. The results revealed antagonic effects of oestradiol on lactotroph proliferation depending on its concentration and the presence of IGF-1.     

In vitro effects of an in silico-modelled 17β-estradiol derivative in combination with dichloroacetic acid on MCF-7 and MCF-12A cells

Objectives: To investigate anti‐proliferative properties of a novel in silico‐modelled 17β‐oestradiol derivative (C9), in combination with dichloroacetic acid (DCA), on MCF‐7 and MCF‐12A cells. Materials and methods: xCELLigence system was employed to determine optimal seeding number for cells, and crystal violet assay was used to assess cell number and to determine IC₅₀ value (24 h) for combination treatment. Light and fluorescent microscopy techniques were used to morphologically detect types of cell death. Flow cytometry was used to analyse cell cycle and apoptosis. Results: Optimal seeding number for 96‐well plates was determined to be 5000–10 000 cells/well for both MCF‐7 and MCF‐12A cells. IC₅₀ for MCF‐7 cells of the combination treatment after 24 h was 130 nm of C9 in conjunction with 7.5 mm of DCA (P < 0.05). In contrast, the same concentration inhibited cell population growth by only 29.3% for MCF‐12As after 24‐h treatment (P < 0.05). Morphological studies revealed lower cell density of both types of combination‐treated cells. Flow cytometric analyses demonstrated increase in sub‐G₁ phase in combination‐treated MCF‐7 cells. Conclusions: These results demonstrate that the novel 17β‐oestradiol derivative C9, in combination with DCA is a potent anti‐proliferation treatment, with properties of selectivity towards tumourigenic cells. Thus, this warrants further studies as a potential combination chemotherapeutic agent for further cancer cell lines.     

Apoptotic process of porcine intestinal M cells

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer’s patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18⁺/PCNA⁺ cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18⁺/alkaline phosphatase⁺ cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex. This investigation was supported by a Grant-in-Aid for Scientific Research (16658107) from the Ministry of Education, Culture, Sports, Science and Technology, by two grants (Prion Project and Secure and Healthy Livestock Farming Project) from the Ministry of Agriculture, Forestry and Fisheries, and by a grant from the Naito Foundation.     

6 Iodo-δ-lactone: A derivative of arachidonic acid with antitumor effects in HT-29 colon cancer cells

BackgroundIL-δ (5-hydroxy-6 iodo-8,11,14-eicosatrienoic delta lactone) an iodinated arachidonic acid (AA) derivative, is one of the iodolipids biosynthesized by the thyroid. Although IL-δ regulates several thyroid parameters such as cell proliferation and goiter growth it was found that this iodolipid inhibits the growth of other non thyroid cell lines.ObjectivesTo study the effect of IL-δ on cell proliferation and apoptosis in the colon cancer cell line HT-29.ResultsTreatment with IL-δ reduced cell viability in a concentration-dependent manner: 1 μM 20%, 5 μM 25%, 10 μM 31%, 50 μM 47% and caused a significant decrease of PCNA expression (25%). IL-δ had pro-apoptotic effects, evidenced by morphological features of programmed cell death such as pyknosis, karyorrhexis, cell shrinkage and cell blebbing observed by fluorescence microscopy, and an increase in caspase-3 activity and in Bax/Bcl-2 ratio (2.5 after 3 h of treatment). Furthermore, IL-δ increased ROS production (30%) and lipid peroxidation levels (19%), suggesting that apoptosis could be a result of increased oxidative stress. A maximum increase in c-fos and c-jun protein expression in response to IL-δ was observed 1 h after initiation of the treatment. IL-δ also induced a tumour growth delay of 70% compared to the control group in NIH nude mice implanted with HT-29 cells.ConclusionOur study shows that IL-δ inhibits cell growth and induces apoptosis in the colon cancer cell line, HT-29 and opens the possibility that IL-δ could be a potential useful chemotherapy agent.     

Protective effects of Peroxiredoxin 6 overexpression on amyloid β-induced apoptosis in PC12 cells

Abstract

Oxidative stress triggered by amyloid beta (Aβ) accumulation contributes substantially to the pathogenesis of Alzheimer's disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aβ_25–35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aβ_25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aβ_25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.     

Preliminary study of combination therapy with interferon-α and zinc in chronic hepatitis C patients with genotype 1b

We have evaluated the efficacy of interferon-α (IFN-α) plus zinc therapy in hepatitis C patients with genotype 1b, poor responders for IFN alone. Ten patients were injected with 10 MU of IFN-α every day for 4 wk, followed by three times a week for 20 wk (control group). Nine patients took 300 mg of zinc sulfate a day orally during IFN-α therapy (zinc sulfate group), and 15 patients took IFN-α and 150 mg of polaprezinc (polaprezinc group). On the d 8 of IFN therapy, circadian zinc levels in serum elevated significantly in the polaprezinc group compared to the zinc sulfate group or control group. Serum ALT levels normalized in 73.3% of the polaprezinc group, 55.6% of the zinc sulfate group, and 40.0% of the control group at 6 mo after the end of IFN therapy. Sustained eradication for the hepatitis C virus RNA judged at the end of the 6-mo follow-up period was higher in the polaprezinc group than in the zinc sulfate group (53.3% vs 11.1%, p<0.05) or the control group (20.0%). No clinical side effects of zinc were observed at the dose used. The data suggest that polaprezinc is expected to increase the therapeutic response of IFN-α for chronic hepatitis C with genotype 1b.     

”In vivo cryotechnique” in combination with replica immunoelectron microscopy for caveolin in smooth muscle cells

A novel ”in vivo cryotechnique” with replica immunoelectron microscopy was developed for detecting caveolin localization on replica membranes prepared directly from living smooth muscle cells. After quick-freezing mouse duodenal walls by our ”in vivo cryotechnique”, the specimens were prepared for freeze-fracture and deep-etch replica membranes. Then they were treated with 5% SDS and 0.5% collagenase to keep some antigens on the replica membranes. The immunogold method could be used to clarify the localization of the caveolin antigen in relation to three-dimensional ultrastructures of living smooth muscle cells. Our new cryotechnique can provide native organization of functional molecules in living cells. Accepted: 7 October 1999     

Dimerization of aurein 1.2: effects in structure, antimicrobial activity and aggregation of Cândida albicans cells

Antimicrobial peptides (AMPs) are a promising solution to face the antibiotic-resistant problem because they display little or no resistance effects. Dimeric analogues of select AMPs have shown pharmacotechnical advantages, making these molecules promising candidates for the development of novel antibiotic agents. Here, we evaluate the effects of dimerization on the structure and biological activity of the AMP aurein 1.2 (AU). AU and the C- and N-terminal dimers, (AU)₂K and E(AU)₂, respectively, were synthesized by solid-phase peptide synthesis. Circular dichroism spectra indicated that E(AU)₂ has a “coiled coil” structure in water while (AU)₂K has an α-helix structure. In contrast, AU displayed typical spectra for disordered structures. In LPC micelles, all peptides acquired a high amount of α-helix structure. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, while this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action of AU. Notably, the antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. The ability to aggregate yeast cells makes dimeric versions of AU attractive candidates to inhibit the adhesion of C. albicans to biological targets and medical devices, preventing disease caused by this fungus.     

Paradoxal pharmacologic effects observed withβ-blocker agents on cardiac cells in culture

Summary The direct effects of propranolol and metoprolol were studied on cultured neonatal rat ventricular myocytes by recording cellular contraction with a video signal analyzer of cells movements. The experiments were performed on 3-d-old cultures forming a synchronously beating monolayer. The spontaneous beating frequency of preparations depended on cultures and varied from 100 to 330 beats/min with a peak for the interval 140 to 149 beats/min. Propranolol and metoprolol were tested at 1, 5, 10μM and 10, 50, 100μM, respectively. At these doses the twoβ-blockers induced chronotropic and inotropic effects in the same range. The two agents induced rapid and short-lasting negative inotropic effects, which were dose-dependent and delayed negative chronotropic effects even with the lowest doses. In some preparations paradoxal effects were evidenced: an increase of the amplitude or frequency of the contractions was observed. The results obtained with 5μM propranolol or 50μM metoprolol could be separated in two groups depending on the basal beating rate (〈 or 〉 to 150 beats/min). In cultures with a rapid frequency, the drugs had a marked negative chronotropic effect and, surprisingly, a positive inotropic effect was observed. These findings confirm the interdependence of the two parameters frequency and amplitude of contraction on this model, and evidence the interest of taking into account the control parameters before any interpretation.     

Protective effect of ε-viniferin on β-amyloid peptide aggregation investigated by electrospray ionization mass spectrometry

Abnormal β-amyloid peptide accumulation and aggregation is considered to be responsible for the formation and cerebral deposition of senile plaques in the brains of patients with Alzheimer's disease (AD). Inhibition of the formation of β-amyloid (Aβ) fibrils would be an attractive therapeutic target for the treatment of AD. Resveratrol and its derivatives exhibit a broad range of pharmacological properties such as protection against cardiovascular diseases and cancers, as well as promoting antiaging effects. We reported previously that ε-viniferin glucoside (VG), a resveratrol-derived dimer, strongly inhibits Aβ (25-35) fibril formation in vitro. In this study, we investigated the effects of VG on the aggregation of the full-length peptides (Aβ (1-40) and Aβ (1-42)) and on the β-amyloid-induced toxicity in PC12 cells. VG inhibited Aβ cytotoxicity and the non-covalent complex between VG and Aβ was observed by electrospray ionization mass spectrometry.     

High level expression of functional human IgMs in human PER.C6® cells

Natural IgM antibodies play an important role in the body’s defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6® cells – a human cell line - to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6® cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.     

High level expression of functional human IgMs in human PER.C6? cells

Natural IgM antibodies play an important role in the body''s defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6® cells—a human cell line—to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6® cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.Key words: PER.C6®, IgM, expression, hexamer, pentamer, J chain     

Cell cycle-specific changes in β-adrenergic receptor concentrations in C6 glioma cells

The concentration of β-adrenergic receptors in synchronized C6 cells is lowest during mitosis and G₁ (1000–2000 receptors/cell). A rapid increase in β-receptor concentration occurs in early S phase to 9000 receptors/cell, followed by a decrease during G₂ to the low concentration found at mitosis. In non-synchronized C6 cell populations, mitotic cells have one-half of the average β-receptor concentration of the population as a whole, confirming the observations made with synchronized cells.     

Phase I/II study of adoptive transfer of γδ T cells in combination with zoledronic acid and IL-2 to patients with advanced renal cell carcinoma

Human Vγ2 Vδ2-bearing T cells have recently received much attention in cancer immunotherapy. In this study, we conducted a phase I/II clinical trial of the adoptive transfer of γδ T cells to patients with advanced renal cell carcinoma. Eleven patients who had undergone nephrectomy and had lung metastasis were enrolled. Peripheral blood γδ T cells obtained from the patients were stimulated ex vivo with 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), a synthetic pyrophosphomonoester antigen, and transferred in combination with zoledronic acid (Zol) and teceleukin (recombinant human interleukin-2). Expanded γδ T cells exhibited potent cytotoxic activity against tumor cells in vitro, and the proportion of peripheral blood γδ T cells among CD3⁺ cells typically peaked three to 5 days after transfer. Tumor doubling time was prolonged in all 11 patients, and the best overall responses were 1 CR, 5 SD, and 5 PD, as defined based on Response Evaluation Criteria in Solid Tumors (RECIST). Although ten patients developed adverse reactions of grade ≥3, they were likely to have been the result of the concomitant infusion of Zol and IL-2, and most symptoms swiftly reverted to normal during the course of treatment. In conclusion, this clinical trial demonstrated that our regimen for the adoptive transfer of γδ T cells in combination with Zol and IL-2 was well tolerated and that objective clinical responses could be achieved in some patients with advanced renal cell carcinoma.     

Anti-melanogenic effects of δ-tocotrienol are associated with tyrosinase-related proteins and MAPK signaling pathway in B16 melanoma cells

Tocotrienols are known to possess potent antioxidant, anticancer, and cholesterol lowering activities. Being able to rapidly penetrate the skin, these vitamin E isoforms have been explored for potential treatment against melanoma. This study aimed to elucidate the mechanism involved in the anti-melanogenic effects of δ-tocotrienol (δT3) in B16 melanoma cells. Results showed that at 20 μM of δT3 significantly inhibited melanin formation and ROS generation. Treatment with δT3 also effectively suppressed the expression of melanogenesis-related proteins, including MC1R, MITF, TYRP-1, and TYRP-2. More importantly, we observed that the mitogen-activated protein kinase (MAPK) pathway was involved in mediating δT3's inhibitory effect against melanin production. Specifically, δT3 treatment markedly induced the activation of extracellular signal-regulated kinases (ERK). The use of ERK activation inhibitor (PD98059) abrogated the δT3-mediated downregulation expression melanogenesis-related proteins and restored melanin production. Furthermore, siRNA targeting ERK effectively blocked the δT3-induced repression of tyrosinase and TYRP-1 expression. These results suggest that δT3's inhibitory effect against melanogenesis is mediated by the activation of ERK signaling, thereby resulting in downstream repression of melanogenesis-related proteins and the subsequent melanin production. These data provide insight to δT3's effect and the targeting of ERK signaling for treatment against melanogenesis.