Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings

Purpose

To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms.

Method

CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo.

Results

Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl.

Conclusion

Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML.     

Does Iterative Reconstruction Lower CT Radiation Dose: Evaluation of 15,000 Examinations

Purpose

Evaluation of 15,000 computed tomography (CT) examinations to investigate if iterative reconstruction (IR) reduces sustainably radiation exposure.

Method and Materials

Information from 15,000 CT examinations was collected, including all aspects of the exams such as scan parameter, patient information, and reconstruction instructions. The examinations were acquired between January 2010 and December 2012, while after 15 months a first generation IR algorithm was installed. To collect the necessary information from PACS, RIS, MPPS and structured reports a Dose Monitoring System was developed. To harvest all possible information an optical character recognition system was integrated, for example to collect information from the screenshot CT-dose report. The tool transfers all data to a database for further processing such as the calculation of effective dose and organ doses. To evaluate if IR provides a sustainable dose reduction, the effective dose values were statistically analyzed with respect to protocol type, diagnostic indication, and patient population.

Results

IR has the potential to reduce radiation dose significantly. Before clinical introduction of IR the average effective dose was 10.1±7.8mSv and with IR 8.9±7.1mSv (p*=0.01). Especially in CTA, with the possibility to use kV reduction protocols, such as in aortic CTAs (before IR: average14.2±7.8mSv; median11.4mSv /with IR:average9.9±7.4mSv; median7.4mSv), or pulmonary CTAs (before IR: average9.7±6.2mSV; median7.7mSv /with IR: average6.4±4.7mSv; median4.8mSv) the dose reduction effect is significant(p*=0.01). On the contrary for unenhanced low-dose scans of the cranial (for example sinuses) the reduction is not significant (before IR:average6.6±5.8mSv; median3.9mSv/with IR:average6.0±3.1mSV; median3.2mSv).

Conclusion

The dose aspect remains a priority in CT research. Iterative reconstruction algorithms reduce sustainably and significantly radiation dose in the clinical routine. Our results illustrate that not only in studies with a limited number of patients but also in the clinical routine, IRs provide long-term dose saving.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

The Effects of Cadmium-Zinc Interactions on Biochemical Responses in Tobacco Seedlings and Adult Plants

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.     

Discovery and SAR analysis of 5-chloro-4-((substituted phenyl)amino)pyrimidine bearing histone deacetylase inhibitors

Histone deacetylases (HDACs) are validated targets for the development of anticancer drugs in epigenetics. In the discovery of novel HDAC inhibitors with anticancer potency, the 5-chloro-4-((substituted phenyl)amino)pyrimidine fragment is assembled as a cap group into the structure of HDAC inhibitors. The SAR revealed that presence of small groups (such as methoxy substitution) is beneficial for the HDAC inhibitory activity. In the enzyme inhibitory selectivity test, compound L20 exhibited class I selectivity with IC₅₀ values of 0.684 µM (selectivity index of >1462), 2.548 µM (selectivity index of >392), and 0.217 µM (selectivity index of >4608) against HDAC1, HDAC2 and HDAC3 compared with potency against HDAC6 (IC₅₀ value of >1000 µM), respectively. In the antiproliferative assay, compound L20 showed both hematological and solid cancer inhibitory activities. In the flow cytometry, L20 promoted G0/G1 phase cell cycle arrest and apoptosis of K562 cells.     

Ionizing Radiation Potentiates Dihydroartemisinin-Induced Apoptosis of A549 Cells via a Caspase-8-Dependent Pathway

This report is designed to explore the molecular mechanism by which dihydroartemisinin (DHA) and ionizing radiation (IR) induce apoptosis in human lung adenocarcinoma A549 cells. DHA treatment induced a concentration- and time-dependent reactive oxygen species (ROS)-mediated cell death with typical apoptotic characteristics such as breakdown of mitochondrial membrane potential (Δψ_m), caspases activation, DNA fragmentation and phosphatidylserine (PS) externalization. Inhibition of caspase-8 or -9 significantly blocked DHA-induced decrease of cell viability and activation of caspase-3, suggesting the dominant roles of caspase-8 and -9 in DHA-induced apoptosis. Silencing of proapoptotic protein Bax but not Bak significantly inhibited DHA-induced apoptosis in which Bax but not Bak was activated. In contrast to DHA treatment, low-dose (2 or 4 Gy) IR induced a long-playing generation of ROS. Interestingly, IR treatment for 24 h induced G2/M cell cycle arrest that disappeared at 36 h after treatment. More importantly, IR synergistically potentiated DHA-induced generation of ROS, activation of caspase-8 and -3, irreparable G₂/M arrest and apoptosis, but did not enhance DHA-induced loss of Δψ_m and activation of caspase-9. Taken together, our results strongly demonstrate the remarkable synergistic efficacy of combination treatment with DHA and low-dose IR for A549 cells in which IR potentiates DHA-induced apoptosis largely by enhancing the caspase-8-mediated extrinsic pathway.     

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus

Background

Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin.

Results

The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5x10⁵ cfu/ml to 10³ cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 μg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against β-lactamase in concentrations manner.

Conclusions

So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits β-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.     

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H2O2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.     

Benefits and Risks of the Hormetic Effects of Dietary Isothiocyanates on Cancer Prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1–5 µM) to promote cell proliferation to 120–143% of the controls in a number of human cell lines, whilst at high levels (10–40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10–20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint.     

Mometasone and desloratadine additive effect on eosinophil survival and cytokine secretion from epithelial cells

Background

Although antihistamines and topical corticosteroids are used in combination to treat allergic rhinitis, their additive effect has not been yet demonstrated. The aim was investigate the antiinflammatory additive effect of mometasone and desloratadine on cytokine and sICAM-1 secretion by epithelial cells, and on eosinophil survival stimulated by human epithelial cells secretions from nasal mucosa and polyps.

Methods

Epithelial cells obtained from nasal mucosa or polyps were stimulated with 10% fetal bovine serum in presence of mometasone (10^-11M-10^-5M) with/without desloratadine (10^-5M). Cytokine and sICAM-1 concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated during 4 days with epithelial cell secretions with (10^-11M-10^-5M) and/or desloratadine (10^-5M) and survival assessed by Trypan blue. Results are expressed as percentage (mean ± SEM) compared to control.

Results

Fetal bovine serum stimulated IL-6, IL-8, GM-CSF and sICAM-1 secretion. In mucosa and polyp epithelial cells, mometasone inhibited this induced secretion while desloratadine inhibited IL-6 and IL-8. The combination of 10^-5M desloratadine and 10^-9M mometasone reduced IL-6 secretion (48 ± 11%, p < 0.05) greater extent than mometasone alone (68 ± 10%) compared to control (100%). Epithelial cell secretions induced eosinophil survival from day 1 to 4, this effect being inhibited by mometasone. At day 4, the combination of mometasone (10^-11M) and desloratadine (10^-5M) provoked an increased inhibition of eosinophil survival induced by cell secretions (27 ± 5%, p < 0.01) than mometasone (44 ± 7%) or desloratadine (46 ± 7%) alone.

Conclusions

These results suggest that the combination of desloratadine and mometasone furoate have a greater antinflammatory effect in an in vitro model of eosinophil inflammation than those drugs administered alone.     

Dyhidro-β-agarofurans natural and synthetic as acetylcholinesterase and COX inhibitors: interaction with the peripheral anionic site (AChE-PAS), and anti-inflammatory potentials

In order to find molecules of natural origin with potential biological activities, we isolate and synthesise compounds with agarofuran skeletons (epoxyeudesmanes). From the seeds of Maytenus disticha and Maytenus magellanica we obtained six dihydro-β-agarofurans, and by means of the Robinson annulation reaction we synthesised five compounds with the same skeleton. The structures were established on the basis of NMR, IR, and MS. The evaluated compounds showed inhibitory activity on the acetylcholinesterase enzyme and on the COX enzymes. Compound 4 emerged as the most potent in the acetylcholinesterase inhibition assay with IC₅₀ 17.0 ± 0.016 µM on acetylcholinesterase (AChE). The compounds evaluated were shown to be selective for AChE. The molecular docking, and the propidium displacement assay suggested that the compounds do not bind to the active site of the enzyme AChE, but rather bind to the peripheral anionic site (PAS) of the enzyme, on the other hand, the natural compound 8, showed the best inhibitory activity on the COX-2 enzyme with an IC₅₀ value of 0.04 ± 0.007 µM. The pharmacokinetic profile calculated in silico using the SWISSADME platform shows that these molecules could be considered as potential drugs for the treatment of neurodegenerative diseases such as AD.     

Hyperthermia in Cancer Therapy

Many malignant cell lines exhibit a therapeutic response to supernormal temperatures. Selective destruction of tumor cells has been observed following moderate hyperthermia (42° to 43° C) in vivo, and tumor eradication by heat has been achieved without normal tissue morbidity. Thermal cell killing appears to be independent of oxygen tension, and the sensitivity of S-phase cells to thermal damage is complementary to that for cellular radiation response. Hyperthermia is therefore a promising adjunct to radiotherapy. At the Claire Zellerbach Saroni Tumor Institute, Mount Zion Hospital and Medical Center, San Francisco, the differential thermal sensitivity of malignant cells is being studied to achieve improved tumor control in patients refractory to more conventional treatments. Preliminary results of a two-year clinical trial indicated increased local objective responses when hyperthermia and radiation were used in combination.     

The role of Rho-associated kinase inhibitor,Y-27632 on primary culture of ovine spermatogonial stem cells

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old’s lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days’ culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.     

HZ08 Reverse P-Glycoprotein Mediated Multidrug Resistance In Vitro and In Vivo

BackgroundMultidrug efflux transporter P-glycoprotein (P-gp) is highly expressed on membrane of tumor cells and is implicated in resistance to tumor chemotherapy. HZ08 is synthesized and studied in order to find a novel P-gp inhibitor.MethodsMDCK-MDR1 monolayer transport, calcein-AM P-gp inhibition and P-gp ATPase assays were used to confirm the P-gp inhibition capability of HZ08. Furthermore, KB-WT and KB-VCR cells were used to evaluate the P-gp inhibitory activity of HZ08 both in vitro and in vivo.ResultsResults showed that HZ08 was more potent than verapamil in MDCK-MDR1 monolayer transportation model. Meanwhile, P-gp ATPase assay and calcein-AM P-gp inhibition assay confirmed that HZ08 inhibited P-gp ATPase with a calcein-AM IC50 of 2.44±0.31μM. In addition, significantly greater in vitro multidrug resistance reversing effects were observed when vincristine or paclitaxel was used in combination with 10μM HZ08 compared with 10μM verapamil. Moreover, HZ08 could significantly enhance the sensitivity of vincristine with a similar effect like verapamil in both KB-WT and KB-VCR tumor xenograft models.ConclusionsThe novel structure HZ08 could be a potent P-gp inhibitor.     

Juxtaposition of the changes in intracellular calcium and force during staircase potentiation at 30 and 37°C

Ca²⁺ entry during the action potential stimulates muscle contraction. During repetitive low frequency stimulation, skeletal muscle undergoes staircase potentiation (SP), a progressive increase in the peak twitch force induced by each successive stimulus. Multiple mechanisms, including myosin regulatory light chain phosphorylation, likely contribute to SP, a temperature-dependent process. Here, we used the Ca²⁺-sensitive fluorescence indicators acetoxymethyl (AM)-furaptra and AM-fura-2 to examine the intracellular Ca²⁺ transient (ICT) and the baseline Ca²⁺ level at the onset of each ICT during SP at 30 and 37°C in mouse lumbrical muscle. The stimulation protocol, 8 Hz for 8 s, resulted in a 27 ± 3% increase in twitch force at 37°C and a 7 ± 2% decrease in twitch force at 30°C (P < 0.05). Regardless of temperature, the peak rate of force production (+df/dt) was higher in all twitches relative to the first twitch (P < 0.05). Consistent with the differential effects of stimulation on twitch force at the two temperatures, raw ICT amplitude decreased during repetitive stimulation at 30°C (P < 0.05) but not at 37°C. Cytosolic Ca²⁺ accumulated during SP such that baseline Ca²⁺ at the onset of ICTs occurring late in the train was higher (P < 0.05) than that of those occurring early in the train. ICT duration increased progressively at both temperatures. This effect was not entirely proportional to the changes in twitch duration, as twitch duration characteristically decreased before increasing late in the protocol. This is the first study identifying a changing ICT as an important, and temperature-sensitive, modulator of muscle force during repetitive stimulation. Moreover, we extend previous observations by demonstrating that contraction-induced increases in baseline Ca²⁺ coincide with greater +df/dt but not necessarily with higher twitch force.     

Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation

DNA damage-induced S phase (S) checkpoint includes inhibition of both replicon initiation and chain elongation. The precise mechanism for controlling the two processes remains unclear. In this study, we showed that Hus1-deficient mouse cells had an impaired S checkpoint after exposure to DNA strand break-inducing agents such as camptothecin (CPT) (≥1.0 µM), or ionizing radiation (IR) (≥15 Gy). The Hus1-dependent S checkpoint contributes to cell resistance to CPT. This impaired S checkpoint induced by CPT or IR in Hus1-deficient cells reflected mainly the chain elongation step of DNA replication and was correlated with the reduction of dissociation of PCNA from DNA replication foci. Although Hus1 is required for Rad9 phosphorylation following exposure of cells to CPT or IR, Hus1-deficient cells showed normal activation of ATR/CHK1 and ATM kinases at doses where the checkpoint defects were manifested, suggesting that Hus1 is not a component of the sensor system for activating these pathways in S checkpoint induced by CPT or IR.     

Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-κB and Src Protein Kinase Signaling Pathways

Objective

Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.

Methods

Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.

Results

The individual IC₅₀ of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC₅₀ values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.

Conclusions

Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.     

Significant effects of biologic therapy on lipid profiles and insulin resistance in patients with rheumatoid arthritis

IntroductionThe goal of this study was to investigate (1) the associations of rheumatoid arthritis (RA)-related inflammation or rheumatoid factor/anti-cyclic citrullinated peptide (anti-CCP) positivity with lipid profiles and insulin resistance (IR), (2) the effects of biologic therapy on lipid profiles and IR, and (3) potential predictors for the presence of subclinical atherosclerosis.MethodsSerum levels of lipid profiles were determined by enzymatic methods in 32 adalimumab-treated patients, 16 etanercept-treated patients, 24 tocilizumab-treated patients, and 20 biologic-naïve patients. Atherogenic index, which corresponds to the ratio of total cholesterol to high-density lipoprotein cholesterol (HDL-C), was calculated. IR was measured by homeostasis model assessment. Pro-inflammatory cytokine levels were examined by enzyme-linked immunosorbent assay. Common carotid artery intima-media thickness was determined by using sonography.ResultsThere was an inverse correlation between disease activity (disease activity score for 28 joints, or DAS28) and low-density lipoprotein cholesterol (LDL-C) levels (r = −0.226, P <0.05) and a positive correlation between DAS28 and IR (r = 0.361, P <0.005). Anti-CCP-positive patients had significantly higher DAS28 and IR compared with anti-CCP-negative patients. There was also a positive correlation between IR and levels of interleukin-6 or tumor necrosis factor-alpha (TNF-α). HDL-C levels significantly increased in patients receiving 6-month anti-TNF-α therapy, and levels of total cholesterol, LDL-C, and triglyceride increased in tocilizumab-treated patients. IR significantly decreased in patients under biologic therapy but was unchanged in biologic-naïve patients. Age, IR, and DAS28 were significant predictors of severe subclinical atherosclerosis (odds ratios of 1.08, 2.77, and 2.52, respectively).ConclusionsSignificant associations of RA-related inflammation with lipid profiles and IR indicate the involvement of RA in atherosclerosis pathogenesis. Biologic therapies were associated with IR reduction without change in atherogenic index, but their beneficial effects on atherosclerosis reduction need to be verified in the future.     

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.