Samlee Mankhetkorn

Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols.

BackgroundMultidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFκB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFκB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFκB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents.ResultsAlthough the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFκB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism.ConclusionsThis demonstrates that different classes of natural NFκB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis.

Quercetin, Siamois 1 and siamois 2 induce apoptosis in human breast cancer MDA-MB-435 cells xenograft in vivo

We sought to investigate the apoptosis-inducing activities of quercetin, Siamois 1, and Siamois 2 against invasive estrogen-receptor negative MDA-MB 435 cells xenografted in athymic nude mice. This study clearly demonstrated that these compounds exhibited apoptosis-inducing activities in cell culture system. Quercetin (20 μg/mL), Siamois 1 (100 μg/mL), and Siamois 2 (200 μg/mL) can induce apoptotic cell death by 40± 5%, 44 ± 14 %, and 31 ± 13 %, respectively. Two-fold of IC50 of these compounds were clearly found to induce apoptosis in breast tumor tissue which can be determined by 99mTc-annexin V scintigraphy and histological staining. This is the first report that the apoptosis-inducing effects of quercetin, Siamois 1, and Siamois 2 on the MDA-MB 435 cell in vitro were effectively extrapolated to the in vivo situation. These compounds might be considered as a simple dietary supplement and with further clinical investigation for their use as a nutrition-based intervention in breast cancer treatment.

PEG-OCL micelles for quercetin solubilization and inhibition of cancer cell growth

In this study, quercetin (QCT), a flavonoid with high anticancer potential, was loaded into polymeric micelles of PEG-OCL (poly(ethylene glycol)-b-oligo(ε-caprolactone)) with naphthyl or benzyl end groups in order to increase its aqueous solubility. The cytostatic activity of the QCT-loaded micelles toward different human cancer cell lines and normal cells was investigated. The results showed that the solubility of QCT entrapped in mPEG750-b-OCL micelles was substantially increased up to 1 mg/ml, which is approximately 110 times higher than that of its solubility in water (9 μg/ml). The average particle size of QCT-loaded micelles ranged from 14 to 19 nm. The QCT loading capacity of the polymeric micelles with naphthyl groups was higher than that with benzyl groups (10% and 6%, respectively). QCT-loaded, benzyl- and naphthyl-modified micelles effectively inhibited the growth of both sensitive and resistance cancer cells (human erythromyelogenous leukemia cells (K562) and small lung carcinoma cells (GLC4)). However, the benzyl-modified micelles have a good cytocompatibility (in the concentration range investigated (up to 100 μg/ml), they are well tolerated by living cells), whereas their naphthyl counterparts showed some cytotoxicity at higher concentrations (60-100 μg/ml). Flow cytometry demonstrated that the mechanism underlying the growth inhibitory effect of QCT in its free form was inducing cell cycle arrest at the G2/M phase. Benzyl-modified micelles loaded with QCT also exhibited this cycle arresting the effect of cancer cells. In conclusion, this paper shows the enhancement of solubility and cell cycle arrest of QCT loaded into micelles composed of mPEG750-b-OCL modified with benzyl end groups. These micelles are therefore considered to be an attractive vehicle for the (targeted) delivery of QCT to tumors.

Spectrophotometric Characterization of Behavior and the Predominant Species of Flavonoids in Physiological Buffer: Determination of Solubility, Lipophilicity and Anticancer Efficacy

The objectives of this study were to investigate the behavior of flavonoids in an aqueous physiological buffer and to determine the structural and functional group substitution which is responsible for their anticancer action. The de- protonated anionic form of 7 flavonoids can easily be determined using spectrophotometry, and owing to its charged state, is highly soluble in aqueous physiological buffer and is not prone to aggregation. The protonated form of these 7 flavon- oids is much less soluble and tends to aggregate following precipitation. For all flavonoids studied except catechin and 5,5� -dihydroxy-6,7,3� ,4� -tetramethoxyflavone, it was possible to determine the rates of deprotonation; pKa value of eri- odictyol, apigenin, kaempferol, quercetin, WP 279, and WP 283 was equal to 7.00, 8.72, 7.86, 8.30, 7.70 and 9.90, respec- tively. The methoxyl group substitutions in place of hydrogen atoms and/or hydroxyl groups at various positions of car- bon atoms in ring A, B and C particularly WP 283 resulted in an increase in the solubility, lipophilicity, and specifically its anticancer efficacy (by 60-fold). The neutral forms of flavonoids are predominantly active molecules and the active sites responsible for anticancer activity are found in ring A and C, especially C4=O, C5-OH and C2=C3.

Cytostatic effect of xanthone-loaded mPEG-b-p(HPMAm-Lac2) micelles towards doxorubicin sensitive and resistant cancer cells.

Xanthone exhibits several medicinal activities and especially it inhibits the growth of cancer cells. However, the use of xanthone is limited because of its low aqueous solubility and systemic toxicity. In the present study xanthone was loaded into poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-dilactate] mPEG-b-p(HPMAm-Lac(2)) micelles in order to overcome these drawbacks. It was shown that xanthone could be loaded in these micelles up to 2 mg/mL with ~100% entrapment efficiency and ~20% loading capacity. The average particle diameter of the xanthone loaded mPEG-b-p(HPMAm-Lac(2)) micelles as determined by dynamic light scattering ranged from 84 to 112 nm. In vitro assays showed that xanthone in its free form as well as loaded in polymeric micelles had a high cytotoxicity towards both doxorubicin sensitive and, importantly, resistant cancer cells. On the other hand empty mPEG-b-p(HPMAm-Lac(2)) micelles did not show any cytotoxicity towards normal cells (PBMCs). Interestingly, the cytostatic effect of xanthone towards normal cells was masked when loaded in the micelles. The mechanism of cell growth inhibition by xanthone-loaded polymeric micelles was mediated through induction of apoptosis, as evidenced from a subdiploid peak of propidium iodide stained cells using flow cytometric analysis. From the results of this study it can be concluded that xanthone has potent anticancer activity not only on sensitive but also on doxorubicin resistant cancer cell lines. mPEG-b-p(HPMAm-Lac(2)) micelles are therefore attractive delivery systems of xanthone for the treatment of cancer.

Antioxidant activity and cytotoxicity of Cyrtosperma johnstonii extracts on drug sensitive and resistant leukemia and small cell lung carcinoma cells

Context: The number of patients with cancer is increasing. New therapeutic agents to overcome drug-resistant tumors are urgently needed. Cyrtosperma johnstonii N.E. Br. (Araceae) is used for treatment of cancer in Thai traditional medicine. This study aimed to evaluate antioxidant activity and cytotoxicity of C. johnstonii extracts on human cancer cells. Materials and methods: Dried powder of C. johnstonii rhizomes was extracted with several solvents. The 0.1 mg/ml extract solution was tested for antioxidant activity by 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Color formation from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to determine cell viability. Standardization of the extract was performed by high-performance liquid chromatography (HPLC) with photodiode array detector at 254 and 360 nm. Cell cycle arrest was evaluated by flow cytometry after 5 min, 12 h and 24 h treated with 20 µg/ml of the acetone extract. Results: The acetone extract exhibited the highest phenolic content and antioxidant activity (TEAC and EC values = 19.2 ± 0.14 and 19.2 ± 0.31 mM/mg, respectively). The IC50 values for leukemia ranged from 11 ± 1 to 29 ± 3 µg/ml and from 5 ± 2 to 6 ± 0 µg/ml for small cell lung carcinoma cells. Cell cycle arrest occurred at the G2/M phase followed by apoptosis. HPLC analysis revealed that rutin is the major constituents of the extract. Discussion and conclusion: The acetone extract of C. johnstoni is a promising source of natural antioxidants and anticancer. The extract inhibits cancer cells effectively with less effect on normal cells.

Modeling of C-SNARF-1 pH Fluorescence Properties: Towards Calibration Free Optical Fiber pH Sensing for in Vivo Applications

Organic functions of the human body are related to biological constants. Variations of these constants, among them pH, induce pathological troubles. The general goal of our work is to fabricate a fluorescent pH sensor at the end of an optical fiber for in vivo pH measurements. One difficulty using fluorescence indicators is the need to perform an accurate calibration. In this communication, we present methods used to simplify and potentially avoid calibration procedures of fluorescence indicators. The first method concerns the simplification of calibration procedures making them independent of the indicator’s concentration, path length and equipment used. The second method concerns modelling the fluorescence emission of the molecules as a function of pH only. This model is used to fit the exact shape of C-SNARF-1 fluorescence spectra obtained at any pH. Subsequently, the pH of a solution can be computed with an accuracy of 0.1 pH unit without the calibration procedure employed up to now. These methods constitute the first steps toward calibration free pH measurements. They can be applied to any fluorescent indicator exhibiting a dual emission peak. As a conclusion, this is the first time that fluorescence properties of C-SNARF-1 are fully mathematically

Delivery of Superparamagnetic Polymeric Micelles Loaded with Quercetin to Hepatocellular Carcinoma Cells

The aim of this study is to develop co-encapsulation of quercetin (QCT) and superparamagnetic iron oxide nanoparticles (SPIONs) into methoxy-poly(ethylene glycol)-b-oligo(ɛ-caprolactone), mPEG750-b-OCL-Bz micelles (QCT-SPION-loaded micelles) for inhibition of hepatitis B virus-transfected hepatocellular carcinoma (HepG2.2.15) cell growth. QCT-SPION-loaded micelles were prepared using film hydration method. They were spherical in shape with an average size of 22-55 nm. The best QCT-SPION-loaded micelles showed entrapment efficiency and loading capacity of QCT at 70% and 3.5%, respectively, and of SPIONs at 15% and 0.8%, respectively. Transverse (T2) relaxivity of SPIONs was 137 mM-1s-1. SPION clusters present inside the core of QCT-SPION-loaded micelles increased T2 relaxivity value (246 mM-1s-1) indicating the good magnetic resonance imaging sensitivity of QCT-SPION-loaded micelles in comparison with SPIONs. QCT-SPION-loaded micelles could be taken up by HepG2.2.15 cells and showed higher cytotoxicity than QCT. Furthermore, these cells were arrested by QCT-SPION-loaded micelles at the G0/G1 phase of cell cycle. QCT-SPION-loaded micelles accumulated in the vicinity of Neodymium Iron Boron (NdFeB) magnetic disc, resulting in the potent inhibition of cancer cell growth at the strong magnetic field strength. In conclusion, mPEG750-b-OCL-Bz micelles are a promising multi-functional vehicle for co-delivery of QCT and SPIONs for disease monitoring and therapies of hepatocellular carcinoma.

3D modeling of keloid scars in vitro by cell and tissue engineering

Keloids are pathologic scars defined as dermal fibrotic tumors resulting from a disturbance of skin wound healing process. Treatments against keloids are multiple, sometimes empirical and none of them really provides an effective tool for physicians. The lack of effective treatments is correlated with the poor understanding of keloid pathogenesis. To fill this gap, researchers need strong models mimicking keloids as closely as possible. The objective of this study was to establish in vitro a new reconstructed keloid model (RKM), by combining fibroblasts extracted from the three major area of a keloid (center, periphery, non-lesional) in a three-dimensional biomaterial. To this aim, fibroblasts of three keloid locations were extracted and characterized, and then integrated in a hydrated collagen gel matrix during a three-step procedure. The heterogeneity of fibroblasts was assessed according to their proliferative and remodeling capacities. RKMs were further visualized and characterized by both light and scanning electron microscopy. This reconstructed keloid model should be very useful for investigating keloid fibroblasts function in conditions mimicking in vivo situation. Moreover, RKM should also be a suitable model for either drug study and discovery or innovative approaches using medical devices both during cancer and cancer-like disease investigation.

Visualization of Inflammation at Early Stage of Lung Cancer in Xenografted Temporally Immunosuppression Rats by Ferrioxamine Magnetic Resonance Imaging

Physiological responses such as chronic inflammation and angiogenesis could be used as biomarkers for early detection of cancer with noninvasive imaging modalities. The present study reports the application of magnetic resonance imaging instrument to image the binding of ferrioxamine with hemin that allows visualizing the chronic inflammation foci of lung tissue of immunocompromised rats xenografted using small cell lung carcinoma. A low concentration of ferrioxamine (0.05 ± 0.02 μM·kg−1 of rat weight) deposited on tissue outside the vasculature was found to diffuse across the capillary walls to the interstitial space and inflammation foci, which provided a clear enhancement of T1-weighted gradient-echo sequence images. Ferrioxamine imaging allowed the determination of inflammatory sites and their localization in 3D fat-suppressed maximum intensity projections. The smallest dimension of foci that can be clearly determined is about 0.1 mm3. In concomitant to the in vivo imaging, analysis of histological tissue section showed the development of inflammatory sites. This study provides evidence that medical imaging instrument such as MRI scanner allows researchers to correlate images taken with MRI with those using high-resolution microscopy. Moreover, ferrioxamine is a useful molecular probe for determining chronic inflammation particularly at the very early stages of cancer.