Asiaticoside Increases Caspase-9 Activity in MCF-7 Cells and Inhibits TNF-α and IL-6 Expression in Nude Mouse Xenografts via the NF-κB Pathway.

Background: We hypothesized that the antitumor effects of asiaticoside on breast cancer are driven by its ability to decrease the expression of tumor inflammation-promoting genes and increase apoptotic signaling. In this study, we aimed to better understand the mechanisms of action of asiaticoside as a chemical modulator or as a chemopreventive agent in breast cancer. Methods: MCF-7 cells were cultured and treated with 0, 20, 40, and 80 µM asiaticoside for 48 h. Fluorometric caspase-9, apoptosis, and gene expression analyses were conducted. For the xenograft experiments, we divided nude mice into the following 5 groups (10 animals per group): group I, control mice; group II, untreated tumor-bearing nude mice; group III, tumor-bearing nude mice treated with asiaticoside at weeks 1-2 and 4-7 and injected with MCF-7 cells at week 3; group IV, tumor-bearing nude mice injected with MCF-7 cells at week 3 and treated with asiaticoside beginning at week 6; and group V, nude mice treated with asiaticoside, as a drug control. After treatment, weight measurements were performed weekly. Tumor growth was determined and analyzed using histology and DNA and RNA isolation. Results: In MCF-7 cells, we found that asiaticoside increased caspase-9 activity. In the xenograft experiment, we found that TNF-α and IL-6 expression decreased (p < 0.001) via the NF-κB pathway. Conclusion: Overall, our data suggest that asiaticoside produces promising effects on tumor growth, progression, and tumor-associated inflammation in MCF-7 cells as well as a nude mouse MCF-7 tumor xenograft model.

Early initiation of insulin attenuates histological and functional changes in the liver of streptozotocin-induced diabetic rats using 99mTc-sulfur colloid functional imaging.

This study aimed to investigate the effect of insulin on the reticuloendothelial system (RES) in the liver and spleen in diabetic rats. Sprague Dawley rats were divided into control, diabetic rats (DM) and diabetic rats treated with insulin (IDM) for 2 weeks. Rats were imaged with technetium-99m-sulfur colloid (99mTc-SC) tracer to determine regional distributions of the tracer for all groups by drawing regions of interest and then obtained the ratios as the cumulative counts of heart, liver, and spleen to the whole body (WB). Liver tissue from sacrificed rats from each group was examined by light and electron microscopy. 99mTc-SC uptake ratios showed a lower liver to WB uptake ratio in the DM rats compared to both controls and IDM rats. Electron microscopy showed severe vacuolization of the hepatocytes of DM rats. The IDM rats show complete resolution of the vacuolization. The early administration of insulin for 2 weeks to diabetic rats could significantly resolve the phagocytic RES function and histological changes in the liver.

Mangiferin protect oxidative stress against deoxynivalenol induced damages through Nrf2 signalling pathways in endothelial cells.

Several cereal grains contain a mycotoxin food contaminant called deoxynivalenol (DON), which presents a significant health risk as it is one of the most commonly found mycotoxins. The current paper examines the ameliorative effect of mangiferin (MAN) in vascular endothelial cells induced through activating the Nrf2 signalling pathway on dietary DON-induced oxidative changes. The study infers that the intercellular reactive oxygen species (ROS) levels and malondialdehyde decrease due to MAN. Other effects include in human umbilical vein endothelial cells (HUVECs), the oxidative stress-induced cell damage is reduced due to protective effects and superoxide dismutase (SOD), and catalase (CAT) activities also reveal an improvement. In HUVECs, the Nrf2-regulated antioxidant enzyme genes' expression is activated by Nrf2 nuclear translocation induction and this activity suppresses the oxidative stress damage. The genes in HUVECs include HO-1 and NQO1. Moreover, in HUVECs, the nucleus translocation of Nrf2 reduces the Nrf2, HO-1, whereas NQO1 expression decreases the cytoprotective effects against oxidative stress reduce with the rejection of Nrf2 with siRNA. This paper pioneers in inferring that oxidative stress-induced HUVECs' cell injury is suppressed by MAN through Nrf2, signalling pathway activation.

Thidiazuron decreases epithelial-mesenchymal transition activity through the NF-kB and PI3K/AKT signalling pathways in breast cancer.

Breast cancer is the major type among the women population globally. The treatment of cancer metastasis has made modest progress due to multiple factors. Thidiazuron (TDZ) is a novel plant growth regulator that has been shown to have anticancer effects. Therefore, we explored the anti-metastatic potentials of TDZ in cell lines by assessing its potential to suppress the epithelial-mesenchymal transition (EMT). We pretreated the BEAS-2B and breast cancer (MDA-MB-231) cells with TDZ and deliberated alteration in a cell viability, mammosphere, migration, NF-кB signalling, PI3K/AKT signalling and matrix metalloproteinase (MMP) expression and analysed the EMT induction by TGF-ß/TNF-α-stimulated BEAS-2B cells. Treatment with TDZ (5-50 µmol) diminished the migration and invasion of the extremely metastatic MDA-MB-231 cells. Additionally, TDZ treatment led to down-regulation of uPAR, uPA, VEGF and MMP-2/-9 expression and up-regulation of TIMP-1/2 expression in these cells. Furthermore, TDZ treatment blocked invasion and EMT in non-tumorigenic BEAS-2B epithelial cells stimulated with TGF-ß/TNF-α.TDZ prevents EMT and may thus block metastasis of breast cancer cells.

Kaempferol Inhibits Zearalenone-Induced Oxidative Stress and Apoptosis via the PI3K/Akt-Mediated Nrf2 Signaling Pathway: In Vitro and In Vivo Studies.

In this study, kaempferol (KFL) shows hepatoprotective activity against zearalenone (ZEA)-induced oxidative stress and its underlying mechanisms in in vitro and in vivo models were investigated. Oxidative stress plays a critical role in the pathophysiology of various hepatic ailments and is normally regulated by reactive oxygen species (ROS). ZEA is a mycotoxin known to exert toxicity via inflammation and ROS accumulation. This study aims to explore the protective role of KFL against ZEA-triggered hepatic injury via the PI3K/Akt-regulated Nrf2 pathway. KFL augmented the phosphorylation of PI3K and Akt, which may stimulate antioxidative and antiapoptotic signaling in hepatic cells. KFL upregulated Nrf2 phosphorylation and the expression of antioxidant genes HO-1 and NQO-1 in a dose-dependent manner under ZEA-induced oxidative stress. Nrf2 knockdown via small-interfering RNA (siRNA) inhibited the KFL-mediated defence against ZEA-induced hepatotoxicity. In vivo studies showed that KFL decreased inflammation and lipid peroxidation and increased H2O2 scavenging and biochemical marker enzyme expression. KFL was able to normalize the expression of liver antioxidant enzymes SOD, CAT and GSH and showed a protective effect against ZEA-induced pathophysiology in the livers of mice. These outcomes demonstrate that KFL possesses notable hepatoprotective roles against ZEA-induced damage in vivo and in vitro. These protective properties of KFL may occur through the stimulation of Nrf2/HO-1 cascades and PI3K/Akt signaling.

Study of the cytotoxicity of asiaticoside on rats and tumour cells.

BACKGROUND: Cancer chemoprevention is considered one of the most promising areas in current cancer research, and asiaticoside, which is derived from the plant Centella asiatica, has a relative lack of systemic toxicity. The purpose of this study was to investigate whether asiaticoside is effective against 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinogenicity in vitro (MCF-7 and other cells) and in vivo (DMBA-induced rat cancer). METHODS: An MTT assay was performed involving the treatment of MCF-7 cells for 48 h with H2O2 alone and H2O2 + different asiaticoside concentrations. Flow cytometry was performed, and the level of caspase 3, tumour necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1) were quantified. Adult female Sprague-Dawley (SD) rats were divided into five groups designated I (control), II (DMBA-induced cancer), III (pre- and post-treatment with asiaticoside (200 µg/animal) in DMBA-induced cancer), IV (post-treatment with asiaticoside in DMBA-induced cancer), and V (treated with asiaticoside alone, drug control). Twelve weeks post-DMBA, rats developed mammary tumours. Rats either were sacrificed or imaged with MIBI. Histological examination of tumour tissues was performed. Tumour MIBI uptake ratios were determined. The data are expressed as the means ± standard deviation. Appropriate t-test and ANOVA statistical methods were used to compare data. RESULTS: The IC50 of asiaticoside for MCF-7 cells was determined to be 40 µM. Asiaticoside has potential for hydrogen peroxide cytotoxicity, and the caspase-3 activity increased with increasing asiaticoside dose in MCF-7 cells treated for 48 h. The expression of the cytokines TNF-α and IL-1ß was significantly decreased and correlated with MIBI uptake ratios in vitro and in vivo after asiaticoside administration. CONCLUSION: This study demonstrates that asiaticoside is effective in vitro and in vivo in inducing apoptosis and enhancing anti-tumour activity.

Geraniol attenuates oxidative stress and neuroinflammation-mediated cognitive impairment in D galactose-induced mouse aging model.

D-galactose (D-gal) administration was proven to induce cognitive impairment and aging in rodents' models. Geraniol (GNL) belongs to the acyclic isoprenoid monoterpenes. GNL reduces inflammation by changing important signaling pathways and cytokines, and thus it is plausible to be used as a medicine for treating disorders linked to inflammation. Herein, we examined the therapeutic effects of GNL on D-gal-induced oxidative stress and neuroinflammation-mediated memory loss in mice. The study was conducted using six groups of mice (6 mice per group). The first group received normal saline, then D-gal (150 mg/wt) dissolved in normal saline solution (0.9%, w/v) was given orally for 9 weeks to the second group. In the III group, from the second week until the 10th week, mice were treated orally (without anesthesia) with D-gal (150 mg/kg body wt) and GNL weekly twice (40 mg/kg body wt) four hours later. Mice in Group IV were treated with GNL from the second week up until the end of the experiment. For comparison of young versus elderly mice, 4 month old (Group V) and 16-month-old (Group VI) control mice were used. We evaluated the changes in antioxidant levels, PI3K/Akt levels, and Nrf2 levels. We also examined how D-gal and GNL treated pathological aging changes. Administration of GNL induced a significant increase in spatial learning and memory with spontaneously altered behavior. Enhancing anti-oxidant and anti-inflammatory effects and activating PI3K/Akt were the mechanisms that mediated this effect. Further, GNL treatment upregulated Nrf2 and HO-1 to reduce oxidative stress and apoptosis. This was confirmed using 99mTc-HMPAO brain flow gamma bioassays. Thus, our data suggested GNL as a promising agent for treating neuroinflammation-induced cognitive impairment.

Intracellular localization of (99m)Tc-d,l-HMPAO in rat blood.

OBJECTIVE: The objective of this study was to investigate the intracellular localization of technetium 99m-hexamethylpropylene amine oxime ((99m)Tc-d,l-HMPAO) in rat red blood cells (RBCs) and plasma. MATERIALS AND METHODS: Sprague-Dawley rats (n = 36) were injected with 129.5 MBq (99m)Tc-d,l-HMPAO. Using a heparinized syringe, 2-ml blood samples were withdrawn from the rats 10, 20, 30, 40, 50 and 60 min after (99m)Tc-d,l-HMPAO injection. Blood was centrifuged for 10 min at 2,240 rpm to separate RBCs from plasma. A chloroform/methanol/distilled water mixture was added to the RBCs and plasma in separate tubes to extract (99m)Tc-d,l- HMPAO into aqueous (Aq) and lipid (Lp) phases. The (99m)Tc-d,l-HMPAO activities in Aq and Lp phases were measured separately using a dose calibrator. The data were analyzed using Student's t test and a one-way ANOVA. RESULTS: More than 70% of the injected dose of (99m)Tc-d,l-HMPAO was retained in the RBCs for up to 60 min, compared to less than 20% in plasma. The uptakes in the Aq for plasma were 88.77 +/- 1.04, 94.23 +/- 2.42, 90.15 +/- 1.88, 98.59 +/- 0.89, 96.42 +/- 1.50 and 97.59 +/- 1.92% at 10, 20, 30, 40, 50 and 60 min after injection, respectively. The corresponding uptakes in the Lp phases were: 11.23 +/- 0.04, 5.77 +/- 0.07, 9.85 +/- 0.06, 1.41 +/- 0.08, 3.58 +/- 0.04 and 2.41 +/- 0.06, respectively. Similar values (% +/- SE) and trends were obtained in the Aq and Lp phases for RBCs. A highly significant difference between the Aq and Lp phases at each time point studied was observed (p < 0.0001). There was no statistically significant difference between the time points in the Aq phase, but there was a significant difference (p < 0.001) between time points in the Lp phase. CONCLUSION: This study demonstrated that most of the intracellular localization of (99m)Tc-d,l-HMPAO was in the Aq phase compared to Lp phase in both RBCs and plasma up to 60 min.

Choline treatment affects the liver reticuloendothelial system and plasma fatty acid composition in diabetic rats.

PURPOSE: This study investigated effects of choline treatment on hepatic reticuloendothelial and biliary functions and plasma fatty acid composition in diabetic rats. METHODS: Diabetes was induced by streptozotocin (STZ). Choline was administered to untreated rats and a portion of STZ-treated rats for two sequences of five consecutive days, separated by a 2-day interval. Hepatic functions were studied using (99m) Tc Tin (II) colloid (TIN) and 99 mTc mebrofenin [bromo-iminodiacetic acid (BrIDA)] imaging. The TIN-uptake ratios (organ/whole body) of heart, liver and spleen, and the BrIDA-uptake ratios (organ or tissue/whole body) of liver, biliary tree and abdomen were obtained following imaging studies. Fatty acids were analysed by GC/MS. RESULTS: Choline treatment did not attenuate hyperglycaemic development. Diabetic rats showed (i) a decreased TIN-uptake ratio in liver with co-increased ratios in heart and spleen; choline treatment diminished these changes, (ii) elevated BrIDA-uptake ratios in biliary tree and abdomen but not in liver; choline treatment did not attenuate the elevations and (iii) decreases in plasma palmitoleic acid and oleic acid, reflecting an impaired stearoyl-CoA desaturase function; choline treatment did not affect the diminutions, but caused a decrease in arachidonic acid with a co-increase in linoleic acid. Some rats developed hypoproteinemia (HPO). HPO rats also exhibited decreases in plasma palmitoleic acid and oleic acid. Diabetes caused almost absence of palmitoleic acid in HPO rats. Choline treatment exerted no effect on the plasma fatty acid composition of diabetic HPO rats. CONCLUSIONS: Choline treatment affected hepatic reticuloendothelial function and plasma fatty acid composition, but not hepatobiliary function, in diabetic rats. Whether choline treatment is beneficial requires further studies.

Assessment of tracer 99mTc(V)-DMSA uptake as a measure of tumor cell proliferation in vitro.

PURPOSE: To examine whether (99m)Tc(V)-DMSA could be used as a non-invasive measure of cancer cell proliferation. METHODS: Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with (99m)Tc(V)-DMSA in media for 60 min at 37°C. DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-(3)H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups. RESULTS: In all cell lines rates of (99m)Tc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-(3)H]thymidine incorporation and expression of PCNA and Ki-67. CONCLUSION: Rates of (99m)Tc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo.

Effect of asiaticoside on 99mTc-tetrofosmin and 99mTc-sestamibi uptake in MCF-7 cells.

UNLABELLED: This study was done to examine the effect of asiaticoside on MCF-7 cell uptake of (99m)Tc-tetrofosmin ((99m)Tc-Tfos) and (99m)Tc-sestamibi ((99m)Tc-MIBI). METHODS: The 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the effect of a 50% inhibitory concentration of asiaticoside on MCF-7 cell proliferation. MCF-7 cells were treated with 10, 20, 30, 40, and 50 µM asiaticoside for 48 h and then incubated with 59.2 MBq of either (99m)Tc-Tfos or (99m)Tc-MIBI tracer for 60 min. The uptake of the tracers was measured with a dose calibrator. RESULTS: The 50% inhibitory concentration of asiaticoside for MCF-7 cells was determined with the MTT assay to be 40 µM. The uptake results were expressed as the mean ± SE radioactivity in MBq/mg of protein, and P values were also calculated (P values of 0.03 indicated significant differences). In the control (no asiaticoside) and at 10, 20, 30, 40, and 50 µM asiaticoside, the mean levels of (99m)Tc-Tfos uptake were 0.79 (SE, 0.059) (P = 0.14), 0.84 (SE, 0.057) (P = 0.60), 0.47 (SE, 0.034) (P = 0.03), 0.40 (SE, 0.050) (P = 0.03), 0.37 (SE, 0.050) (P = 0.03), and 0.15 (SE, 0.023) (P = 0.03), respectively; the mean levels of (99m)Tc-MIBI uptake were 0.95 (SE, 0.007) (P = 0.14), 0.81 (SE, 0.009) (P = 0.60), 0.79 (SE, 0.019) (P = 0.03), 0.63 (SE, 0.004) (P = 0.03), 0.13 (SE, 0.006) (P = 0.03), and 0.07 (SE, 0.008) (P = 0.03), respectively. Asiaticoside concentrations of 10, 20, 30, 40, and 50 µM revealed the uptake kinetics for both (99m)Tc-Tfos and (99m)Tc-MIBI in MCF-7 cells. (99m)Tc-Tfos and (99m)Tc-MIBI showed similar trends; the radioactivity uptake was dose dependent, and asiaticoside inhibited 16% and 47% of (99m)Tc-Tfos uptake and (99m)Tc-MIBI uptake in MCF-7 cells, respectively. CONCLUSION: This study showed that asiaticoside, acting as a biochemical modulator, may induce apoptosis and enhance antitumor activity in MCF-7 cells, as determined by (99m)Tc-Tfos and (99m)Tc-MIBI uptake. These findings are promising for cancer chemotherapy. Future studies should be performed to confirm our findings and to further delineate the clinical role of asiaticoside.

Perfusion scanning using 99mTc-HMPAO detects early cerebrovascular changes in the diabetic rat.

BACKGROUND: 99mTc-HMPAO is a well-established isotope useful in the detection of regional cerebral blood flow. Diabetes gives rise to arterial atherosclerotic changes that can lead to significant end organ dysfunction, prominently affecting perfusion to the heart, kidneys, eyes and brain. In the current study, we investigated the role of 99mTc-HMPAO cerebral perfusion scans in detecting early vascular changes in the diabetic brain. METHODS: Cerebral perfusion studies were performed on both control and streptozotocin-(STZ) induced diabetic male Wistar rats. Rat brain imaging using a gamma camera was performed for each group 0.5, 2, 4, and 24 hours post 99mTc-HMPAO injection. Data processing for each cerebral perfusion scan was performed by drawing a region of interest (ROI) circumferentially around the brain (B). Background (BKG) due to signal from the soft tissue of each rat was subtracted. Brain 99mTc-HMPAO uptake minus background counts (net brain counts; NBC) were then compared between the two groups. RESULTS: The NBC (mean +/- SD) for the STZ group were statistically significantly higher (p = 0.0004) than those of the control group at each of the time points studied. CONCLUSION: 99mTc-HMPAO brain scan may be useful in the detection of early atherosclerotic changes in the diabetic rat brain.