Cytotoxicity of Goniothalamin on the Human Hepatocellular Carcinoma HEPG2 Cell Line

Goniothalamin is a biologically active styrylpyrone derivative isolated from various Goniothalamus sp., belonging to the Annonacae family. This plant extract has been reported to be cytotoxic towards several tumor cell lines such as pancreas carcinoma (PANC-1), gastric carcinoma (HGC-27) and breast carcinoma (MCF-7). The purpose of this study was to examine and characterize the in vitro cytotoxicity effect of goniothalamin on the human hepatocellular carcinoma HepG2 cells and normal liver Chang cells and also to study the morphological and biochemical changes of goniothalamin-treated HepG2 and Chang cells. Goniothalamin (2.3 -150 μM; 24, 48 and 72 hours) treatment to HepG2 and Chang cells resulted in a dose and time dependent inhibition of cell growth as assessed by MTT and LDH assays. The data suggest that goniothalamin selectively inhibits HepG2 cells (IC50 of MTT= 4.6(±0.23) μM; IC50 of LDH= 5.20(±0.01) μM for 72 hours) with less inhibition of growth in Chang cells (IC50 of MTT= 35.0(±0.09) μM; IC50 of LDH= 32.5(± 0.04) μM for 72 hours. The cytotoxic activity of goniothalamin on HepG2 cells was confirmed by Trypan blue dye exclusion assay. Goniothalamin reduced the number of viable cells (non-stained) associated with an increase on the number of non-viable cells (stained) and the Viability Indexes were 52 ± 1.73% for HepG2 cells and 62 ± 4.36% for Chang cells at IC50 after 72 hours. Cells were exposed to goniothalamin at lowest concentration (2.3 μM), IC50 (of MTT results), and highest concentration (150 μM) for 24, 48, or 72 hours and then examined for effects on cell cycle (using the flow cytometry) or proliferation (using the BrdU ELISA assay). The cytotoxic activity of goniothalamin was related to the inhibition of DNA synthesis, as revealed by the reduction of BrdU incorporation. At 72 hours with the lowest goniothalamin concentration of 2.3 μM, the normal liver Chang cells retained 97.6% of control proliferation while the liver cancer HepG2 cells were reduced to 19.8% of control proliferation. Goniothalamin caused the accumulation of hypodiploid apoptotic cells in cell cycle analysis by flow cytometry. Goniothalamin arrested HepG2 and Chang cells in the G2/M phase with different degrees. Light microscopy examination of HepG2 and Chang cells exposed to different concentrations of goniothalamin up to 72 h demonstrated changes in cellular morphology; i.e. cell rounding followed by a loss of adherence with subsequent cell shrinkage and blebbing. In addition, the apoptotic cells were more abundant in goniothalamin-treated HepG2 cells (84 ± 4.58%) for 72 hours than in untreated cell (4 ± 2.65%) upon measurement by TUNEL staining. In view of the toxicity of goniothalamin, the kind of cell death, namely apoptosis or necrosis, was assessed. Therefore, staining with fluorescence labeled annexin V in combination with propidium iodide was performed on HepG2 and Chang cells exposed to goniothalamin. The laser scanning cytometry of propidium iodide and annexin V-stained cells indicated that the growth inhibiting effect of goniothalamin was consistent with a strong induction of apoptosis at late stage. This is because the cellular membrane integrity was lost, so the cells exhibited annexin V- and propidium iodidedouble positive up to 85.87 ± 0.78 and 57.69 ± 1.12 in HepG2 and Chang cells after 24 hours, respectively. In order to confirm apoptotic mechanism in the goniothalamintreated cells, caspase 3 activity upon the same treatment conditions was carried out. The results indicate that caspase 3 activity was significantly elevated early in IC50 treated Chang cells (574% of control) after 24 hours and late in IC50 treated cells after 72 hours in HepG2 cells (879% of control). Our findings suggest a potential mechanism for the strong growth inhibitory effect of goniothalamin on this HepG2 liver cancer cells. However, less sensitivity to normal liver Chang cell line was observed by this compound. An important feature of the cytotoxicity by goniothalamin is that it is mediated through apoptosis

The inhibitory activity of cocoa phenolic extract against pro-inflammatory mediators secretion induced by lipopolysaccharide in RAW 264.7 cells

Cocoa is a rich source of polyphenols that has been traditionally used as the treatment of several types of inflammation related disease. The response to inflammation comprises the consecutive release of mediators and the enlistment of circulating leukocytes, such as macrophages. Currently, Cocoa-derived polyphenolics have shown anti-inflammatory effects in vivo, but the therapeutic benefits in vitro remain unclear. Therefore, in this study, the effect of cocoa polyphenolic extract (CPE) on RAW 264.7 macrophage cells sensitized by lipopolysaccharide as in vitro inflammatory model was investigated. The anti-inflammatory activity of CPE was assessed by measuring its ability to inhibit the pro-inflammatory enzyme 5-lipoxygenase (5-LOX) and the pro-inflammatory mediators prostaglandin E2 (PGE2), reactive oxygen species (ROS), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). The results show that CPE significantly inhibits 5-LOX activity (p < 0.01). In addition, CPE dose-dependently suppressed the production of PGE2, ROS, NO and TNF-α in RAW 264.7 cells. These data suggest that CPE may be used for the treatment of inflammation and it’s related-diseases

Hippurate-Zinc layered Hydroxide Nanohybrid and its synergistic effect with Tamoxifen on the HepG2 cell lines.

A new simple preparation method for a hippurate intercalated zinc-layered hydroxide (ZLH) nanohybrid has been established, which does not need an anion-exchange procedure to intercalate the hippurate anion into ZLH interlayers. Methods: The hippuric acid nanohybrid (HAN) was prepared by direct reaction of an aqueous suspension of zinc oxide with a solution of hippuric acid via a one-step method. Results: The basal spacing of the nanohybrid was 21.3 Å, indicating that the hippurate anion was successfully intercalated into the interlayer space of ZLH, and arranged in a monolayer fashion with the carboxylate group pointing toward the ZLH inorganic interlayers. A Fourier transform infrared study confirmed the formation of the nanohybrid, while thermogravimetry and differential thermogravimetry analyses showed that the thermal stability of the nanohybrid was markedly enhanced. The loading of hippurate in the nanohybrid was estimated to be about 38.7% (w/w), and the release of hippurate from the nanohybrid was of a controlled manner, and therefore the resulting material was suitable for use as a controlled-release formulation. HAN has synergistic properties with tamoxifen toward a HepG2 cell line, with an IC 50 value of 0.35 compared with hippurate. In the antiproliferative assay, the ratio of viable cells account for cells treated by the combination tamoxifen with HAN to untreated cells was sharply reduced from 66% to 13% after 24 and 72 hours, respectively. The release of hippuric acid anions from HAN occurred in a controlled manner, and the resulting material is suitable for a controlled-release formulation

Hippuric acid nanocomposite enhances doxorubicin and oxaliplatin-induced cytotoxicity in MDA-MB231, MCF-7 and Caco2 cell lines.

Background: The aim of the current study is to design a new nanocomposite for inducing cytotoxicity of doxorubicin and oxaliplatin toward MDA-MB231, MCF-7, and Caco2 cell lines. A hippuric acid (HA) zinc layered hydroxide (ZLH) nanocomposite was synthesized under an aqueous environment using HA and zinc oxide (ZnO) as the precursors. Methods: The hippuric acid nanocomposite (HAN) was prepared by the direct reaction of a HA solution with an aqueous suspension of ZnO. Results: The basal spacing of the nanocomposite was 21.3 Å, which is average of four harmonics at 2θ = 8.32°, 12.50°, 16.68°, and 20.84°. This result indicates that the hippurate anion was successfully intercalated into the interlayer space of ZLH. The combinations of HAN with chemotherapy (drugs) has inhibited the cell growth of the MDA-MB231, MCF-7, and Caco2 cancer cells when compared to drugs alone. An IC50 value for the combination of HAN with doxorubicin toward MCF-7 is 0.19 ± 0.15 µg/mL and toward MDA-MB231 is 0.13 ± 0.10 µg/mL. Similarly, the IC50 for the combination of HAN with oxaliplatin toward Caco2 is 0.24 ± 0.11 µg/mL. In the antiproliferative results, the equal combination of HAN (0.5 µg/mL) with doxorubicin (0.5 µg/mL) has reduced the cell proliferation in MCF-7 and MDA-MB- 231 cells into 37.3% and 17.6%, respectively after 24 hours. Similarly, the antiproliferation percentage for equal combination HAN with oxaliplatin (5.00 µg/mL) toward Caco2 is 72.7% after 24 hours. Conclusion: The resulting combination HAN with drugs has exhibited higher inhibition in cells growth in all cancer cell line

A novel biocoagulant agent from mushroom chitosan as water and wastewater therapy

A new commercial cationic polyelectrolyte chitosan (CM), obtained from the waste of mushroom production, was examined using models of water and wastewater namely kaolin and palm oil mill effluent (pome). As it is biocompatible, widely available, and economically feasible, chitosan mushroom has high potential to be a suitable replacement for alum. Also, it can be a promising alternative to chitosan obtained traditionally from Crustaceans due to its higher zeta potential and homogeneity based on the raw material required for its production. A wide range of coagulant dose (5-60 mg l-1) and wastewater pH (2-12) were taken into account to find the optimal conditions of coagulation. The optimal doses are 10 and 20 mg l-1 at best pH (11 and 3) when treated with kaolin and palm oil mill effluent, respectively, while 1200 mg l-1 of alum was not enough to reach the efficiency of chitosan mushroom. On the other hand, the optimum dose of chitosan mushroom (20 mg l-1) at pH 3 of pome produced (75, 73, and 98%) removal of chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solids (TSS), respectively. The significant potential of chitosan mushroom was proved by zeta potential measurement. Indeed, it possesses the highest zeta potential (+70 mV) as compared to the traditional chitosan produced from crustaceans. In short, chitosan mushroom as a biocoagulant is eco-friendly and it enhances water quality that meets the requirements of environmental conservatives

Cytotoxic effects of Mangifera indica L. kernel extract on human breast cancer (MCF-7 and MDA-MB-231 cell lines) and bioactive constituents in the crude extract

Background: Waterlily Mango (Mangifera indica L.) is thought to be antioxidant-rich, conferred by its functional phytochemicals. Methods: The potential anticancer effects of the ethanolic kernel extract on breast cancer cells (MDA-MB-231 and MCF-7) using MTT, anti-proliferation, neutral red (NR) uptake and lactate dehydrogenase (LDH) release assays were evaluated. Cytological studies on the breast cancer cells were also conducted, and phytochemical analyses of the extract were carried out to determine the likely bioactive compounds responsible for such effects. Results: Results showed the extract induced cytotoxicity in MDA-MB-231 cells and MCF-7 cells with IC50 values of 30 and 15 μg/mL, respectively. The extract showed significant toxicity towards both cell lines, with low toxicity to normal breast cells (MCF-10A). The cytotoxic effects on the cells were further confirmed by the NR uptake, antiproliferative and LDH release assays. Bioactive analyses revealed that many bioactives were present in the extract although butylated hydroxytoluene, a potent antioxidant, was the most abundant with 44.65%. Conclusions: M. indica extract appears to be more cytoxic to both estrogen positive and negative breast cancer cell lines than to normal breast cells. Synergistic effects of its antioxidant bioactives could have contributed to the cytotoxic effects of the extract. The extract of M. indica, therefore, has potential anticancer activity against breast cancer cells. This potential is worth studying further, and could have implications on future studies and eventually management of human breast cancers

The effect of sulfate contents on the surface properties of iron–manganese doped sulfated zirconia catalysts

The iron–manganese doped sulfated zirconia catalysts were prepared via precipitation method; the sulfation was carried out by impregnation with different amounts of sulfate (4%, 10% and 16% SO4− 2 by weight) with the addition of Fe–Mn doped and calcined at 600 °C for 3 h. The prepared catalysts were characterized by TGA-DTA, XRD, BET, FT-IR, TEM, TPD-NH3 and XPS. XRD and BET results revealed that the addition of sulfate imparts special stabilization to the catalytically active tetragonal phase of zirconia. All the iron–manganese doped sulfated zirconia catalysts were found to have strong acid sites, high surface area and small crystallite size

Controlled release and angiotensin-converting enzyme inhibition properties of an antihypertensive drug based on a perindopril erbumine-layered double hydroxide nanocomposite.

The intercalation of perindopril erbumine into Zn/Al-NO3-layered double hydroxide resulted in the formation of a host-guest type of material. By virtue of the ionexchange properties of layered double hydroxide, perindopril erbumine was released in a sustained manner. Therefore, this intercalated material can be used as a controlled-release formulation. Results: Perindopril was intercalated into the interlayers and formed a well ordered, layered organic-inorganic nanocomposite. The basal spacing of the products was expanded to 21.7 Å and 19.9 Å by the ion-exchange and coprecipitation methods, respectively, in a bilayer and a monolayer arrangement, respectively. The release of perindopril from the nanocomposite synthesized by the coprecipitation method was slower than that of its counterpart synthesized by the ion-exchange method. The rate of release was governed by pseudo-second order kinetics. An in vitro antihypertensive assay showed that the intercalation process results in effectiveness similar to that of the antihypertensive properties of perindopril. Conclusion: Intercalated perindopril showed better thermal stability than its free counterpart. The resulting material showed sustained-release properties and can therefore be used as a controlled-release formulation

Preparation of hippurate-zinc layered hydroxide nanohybrid and its synergistic effect with tamoxifen on HepG2 cell lines

Samer Hasan Hussein Al Ali1, Mothanna Al-Qubaisi2, Mohd Zobir Hussein1,3, Zulkarnain Zainal1, Muhammad Nazrul Hakim41Department of Chemistry, Faculty of Science; 2Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science; 3Advanced Materials and Nanotechnology Laboratory, Institute of Advanced Technology; 4Department of Biomedical Science, Faculty of Medicine and Health Science, Universiti Putra Malaysia, Serdang, Selangor, MalaysiaBackground: A new simple preparation method for a hippurate-intercalated zinc-layered hydroxide (ZLH) nanohybrid has been established, which does not need an anion-exchange procedure to intercalate the hippurate anion into ZLH interlayers.Methods: The hippuric acid nanohybrid (HAN) was prepared by direct reaction of an aqueous suspension of zinc oxide with a solution of hippuric acid via a one-step method.Results: The basal spacing of the nanohybrid was 21.3 &amp;Aring;, indicating that the hippurate anion was successfully intercalated into the interlayer space of ZLH, and arranged in a monolayer fashion with the carboxylate group pointing toward the ZLH inorganic interlayers. A Fourier transform infrared study confirmed the formation of the nanohybrid, while thermogravimetry and differential thermogravimetry analyses showed that the thermal stability of the nanohybrid was markedly enhanced. The loading of hippurate in the nanohybrid was estimated to be about 38.7% (w/w), and the release of hippurate from the nanohybrid was of a controlled manner, and therefore the resulting material was suitable for use as a controlled-release formulation. HAN has synergistic properties with tamoxifen toward a HepG2 cell line, with an IC50 value of 0.35 compared with hippurate. In the antiproliferative assay, the ratio of viable cells account for cells treated by the combination tamoxifen with HAN to untreated cells was sharply reduced from 66% to 13% after 24 and 72 hours, respectively.Conclusion: The release of hippuric acid anions from HAN occurred in a controlled manner, and the resulting material is suitable for a controlled-release formulation.Keywords: hippuric acid, nanohybrids, zinc oxide, zinc-layered hydroxide, synergistic effec

Selective cytotoxicity of goniothalamin against hepatoblastoma HepG2 cells.

Liver cancer has become one of the major types of cancer with high mortality and liver cancer is not responsive to the current cytotoxic agents used in chemotherapy. The purpose of this study was to examine the in vitro cytotoxicity of goniothalamin on human hepatoblastoma HepG2 cells and normal liver Chang cells. The cytotoxicity of goniothalamin against HepG2 and liver Chang cell was tested using MTT cell viability assay, LDH leakage assay, cell cycle flow cytometry PI analysis, BrdU proliferation ELISA assay and trypan blue dye exclusion assay. Goniothalamin selectively inhibited HepG2 cells [IC 50 = 4.6 (±0.23) μM in the MTT assay; IC 50 = 5.20 (±0.01) μM for LDH assay at 72 hours], with less sensitivity in Chang cells [IC 50 = 35.0 (±0.09) μM for MTT assay; IC 50 = 32.5 (±0.04) μM for LDH assay at 72 hours]. In the trypan blue dye exclusion assay, the Viability Indexes were 52 ± 1.73% for HepG2 cells and 62 ± 4.36% for Chang cells at IC 50 after 72 hours. Cytotoxicity of goniothalamin was related to inhibition of DNA synthesis, as revealed by the reduction of BrdU incorporation. At 72 hours, the lowest concentration of goniothalamin (2.3 μL) retained 97.6% of normal liver Chang cells proliferation while it reduced HepG2 cell proliferation to 19.8% as compared to control. Besides, goniothalamin caused accumulation of hypodiploid apoptosis and different degree of G2/M arrested as shown in cell cycle analysis by flow cytometry. Goniothalamin selectively killed liver cancer cell through suppression of proliferation and induction of apoptosis. These results suggest that goniothalamin shows potential cytotoxicity against hepatoblastoma HepG2 cells