Paclitaxel induces autophagy in gastric cancer BGC823 cells.

This paper explores the connection between paclitaxel, a chemotherapeutic agent, and gastric cancer cells. In this experiment, it is demonstrated that paclitaxel triggers autophagy and inhibits proliferation of gastric cancer cells. An 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect cell viability and the IC50 of paclitaxel. Western blot was used to detect the expression levels of P62, and to measure the protein expression of autophagy. Immunofluorescence was used to reveal the appearance of punctate structures in the cytoplasm-this ultrastructure associated with autophagy was observed by microscopy. Electron microscopy revealed the formation of double-membrane autophagosomes, a typical structure of autophagy. In conclusion, our research indicates that paclitaxel may influence gastric cancer BGC823 cells by way of inducing autophagy.

Sodium butyrate-induced apoptosis and ultrastructural changes in MCF-7 breast cancer cells.

This study investigated the effects of sodium butyrate (NaB) on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and analyzed the relevant mechanism. Here, we demonstrated that a certain concentration of NaB effectively induced MCF-7 cell apoptosis. Cell counting kit-8 (CCK-8) assay was used to detect cell viability and the apoptosis rate. Western blotting was used to detect changes in the Bcl-2 expression level. We observed cell shape changes with microscopy. Immunofluorescence revealed some apoptotic nuclei. Electron microscopy revealed thick nucleoli, chromatin margination, reduced mitochondria, and dramatic vacuoles. Collectively, our findings elucidated the morphological mechanism by which NaB changed the ultrastructure of MCF-7 cells.

Ritonavir binds to and downregulates estrogen receptors: molecular mechanism of promoting early atherosclerosis.

Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERß expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17ß-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17ß-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis.

The Ultrastructure of MCF-7 Breast Cancer Cells after Vasodilator-Stimulated Phosphoprotein Knockdown.

Inhibition of vasodilator-stimulated phosphoprotein (VASP) expression could modulate the adhesion and proliferation of breast cancer cells. However, the underlying mechanisms are not well defined. Here, we show that knockdown of the VASP changes the ultrastructure of human MCF-7 breast cancer cells. Transfection of VASP shRNA significantly lowered the expression of VASP protein in MCF-7 cells. In the shRNA-VASP group, immunofluorescence showed diminished presence of F-actin, and it was lower in the nucleus than in the cytoplasm. After VASP was inhibited, the MCF-7 cells were oval in shape with blunt lamellipodium, disappearance of the cristae of mitochondria, decreased microvilli and more vacuoles. Collectively, our findings elucidated the morphological mechanism that knockdown of the VASP changed the ultrastructure of MCF-7 cells.

The protective effect of curcumin on hepatotoxicity and ultrastructural damage induced by cisplatin.

We investigate the protective effect of curcumin (CU) on the hepatic ultrastructural damage induced by cisplatin in mice. 18 adult Kunming mice were randomly divided into normal saline (NS) group, cisplatin treatment group (CP) and CU + CP group (n = 6 for each group). Mice in control group and CP group were administered with NS (20 mL/kg/day) and CU + CP group were i.p injected with CU (200 mg/kg/day) for 10 days. Then cisplatin (50 mg/kg/day) was injected in mice of CP group and CU + CP group, while those in control group were given the same volume of NS. Five days after injection all mice were killed and liver dissected. The hepatic morphological structures were observed under light microscope and transmission electron microscope. The results indicated that CU alleviated the hepatic histopathological damages induced by cisplatin, which included declined body weight, vacuolated cytoplasm and blurred liver trabecular structure. Moreover, no hepatic ultrastructural damages were observed in the CU protective group with condensed and marginated nuclear chromatin, bile canaliculi outstreched and bile deposited.

Ritonavir-induced hepatotoxicity and ultrastructural changes of hepatocytes.

To investigate the effect of ritonavir on hepatocyte proliferation, we detected the change of cleaved caspase-3 expression level in the hepatocytes. Furthermore, the morphological and ultrastructural changes of hepatocytes derived from RTV-treated mice have been observed. The results showed that ritonavir can evidently inhibit hepatocyte proliferation and increase cleaved caspase-3 expression level. Under the electron microscope, chromatin margination, mitochondrial cristae disappearance, karyopyknosis and cytoplasmic vacuolization can be observed in the hepatocytes of mice treated with ritonavir. In conclusion, the mechanism of ritonavir's hepatotoxicity is that it induces apoptosis of hepatocytes via the caspase-cascade system.

Matrine inhibits the adhesion and migration of BCG823 gastric cancer cells by affecting the structure and function of the vasodilator-stimulated phosphoprotein (VASP).

AIM: Vasodilator-stimulated phosphoprotein (VASP) expression is upregulated in human cancers and correlates with more invasive advanced tumor stages. The aim of this study was to elucidate the mechanisms by which matrine, an alkaloid derived from Sophora species plants, acted on the VASP protein in human gastric cancer cells in vitro. METHODS: VASP was expressed and purified. Intrinsic fluorescence spectroscopy was used to study the binding of matrine to VASP. CD spectroscopy was used to examine the changes in the VASP protein secondary structure. Human gastric carcinoma cell line BGC823 was tested. Scratch wound and cell adhesion assays were used to detect the cell migration and adhesion, respectively. Real-time PCR and Western blotting assays were used to measure mRNA and protein expression of VASP. RESULTS: In the fluorescence assay, the dissociation constant for binding of matrine to VASP protein was 0.86 mmol/L, thus the direct binding between the two molecules was weak. However, matrine (50 µg/mL) caused obvious change in the secondary structure of VASP protein shown in CD spectrum. Treatments of BGC823 cells with matrine (50 µg/mL) significantly inhibited the cell migration and adhesion. The alkaloid changed the subcellular distribution of VASP and formation of actin stress fibers in BGC823 cells. The alkaloid caused small but statistically significant decreases in VASP protein expression and phosphorylation, but had no significant effect on VASP mRNA expression. CONCLUSION: Matrine modulates the structure, subcellular distribution, expression and phosphorylation of VASP in human gastric cancer cells, thus inhibiting the cancer cell adhesion and migration.

Echinocystic acid alleviated hypoxic-ischemic brain damage in neonatal mice by activating the PI3K/Akt/Nrf2 signaling pathway.

Neonatal hypoxic-ischemic encephalopathy (HIE) is considered a major cause of death and long-term neurological injury in newborns. Studies have demonstrated that oxidative stress and apoptosis play a major role in the progression of neonatal HIE. Echinocystic acid (EA), a natural plant extract, shows great antioxidant and antiapoptotic activities in various diseases. However, it has not yet been reported whether EA exerts a neuroprotective effect against neonatal HIE. Therefore, this study was undertaken to explore the neuroprotective effects and potential mechanisms of EA in neonatal HIE using in vivo and in vitro experiments. In the in vivo study, a hypoxic-ischemic brain damage (HIBD) model was established in neonatal mice, and EA was administered immediately after HIBD. Cerebral infarction, brain atrophy and long-term neurobehavioral deficits were measured. Hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and dihydroethidium (DHE) staining were performed, and the contents of malondialdehyde (MDA) and glutathione (GSH) were detected. In the in vitro study, an oxygen-glucose deprivation/reperfusion (OGD/R) model was employed in primary cortical neurons, and EA was introduced during OGD/R. Cell death and cellular ROS levels were determined. To illustrate the mechanism, the PI3K inhibitor LY294002 and Nrf2 inhibitor ML385 were used. The protein expression levels of p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, and HO-1 were measured by western blotting. The results showed that EA treatment significantly reduced cerebral infarction, attenuated neuronal injury, and improved brain atrophy and long-term neurobehavioral deficits in neonatal mice subjected to HIBD. Meanwhile, EA effectively increased the survival rate in neurons exposed to OGD/R and inhibited oxidative stress and apoptosis in both in vivo and in vitro studies. Moreover, EA activated the PI3K/Akt/Nrf2 pathway in neonatal mice following HIBD and in neurons after OGD/R. In conclusion, these results suggested that EA alleviated HIBD by ameliorating oxidative stress and apoptosis via activation of the PI3K/Akt/Nrf2 signaling pathway.

Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway.

Glucose transporter 1 (GLUT1) plays a primary role in the glucose metabolism of cancer cells. However, to the best of our knowledge, there are currently no anticancer drugs that inhibit GLUT1 function. The present study aimed to investigate the antineoplastic activity of berberine (BBR), the main active ingredient in numerous Traditional Chinese medicinal herbs, on HepG2 and MCF7 cells. The results of Cell Counting Kit­8 assay, colony formation assay and flow cytometry revealed that BBR effectively inhibited the proliferation of tumor cells, and induced G2/M cell cycle arrest and apoptosis. Notably, the results of luminescence ATP detection assay and glucose uptake assay showed that BBR also significantly inhibited ATP synthesis and markedly decreased the glucose uptake ability, which suggested that the antitumor effect of BBR may occur via reversal of the Warburg effect. In addition, the results of reverse transcription­quantitative PCR, western blotting and immunofluorescence staining indicated that BBR downregulated the protein expression levels of GLUT1, maintained the cytoplasmic internalization of GLUT1 and suppressed the Akt/mTOR signaling pathway in both HepG2 and MCF7 cell lines. Augmentation of Akt phosphorylation levels by the Akt activator, SC79, abolished the BBR­induced decrease in ATP synthesis, glucose uptake, GLUT1 expression and cell proliferation, and reversed the proapoptotic effect of BBR. These findings indicated that the antineoplastic effect of BBR may involve the reversal of the Warburg effect by downregulating the Akt/mTOR/GLUT1 signaling pathway. Furthermore, the results of the co­immunoprecipitation assay demonstrated that BBR increased the interaction between ubiquitin conjugating enzyme E2 I (Ubc9) and GLUT1, which suggested that Ubc9 may mediate the proteasomal degradation of GLUT1. On the other hand, BBR decreased the interaction between Gα­interacting protein­interacting protein at the C­terminus (GIPC) and GLUT1, which suggested that the retention of GLUT1 in the cytoplasm may be achieved by inhibiting the interaction between GLUT1 and GIPC, thereby suppressing the glucose transporter function of GLUT1. The results of the present study provided a theoretical basis for the application of the Traditional Chinese medicine component, BBR, for cancer treatment.

CREB1/Lin28/miR-638/VASP Interactive Network Drives the Development of Breast Cancer.

Breast cancer is one of the most common malignant tumors worldwide. Metastasis remains the leading cause of death in breast cancer patients. Research on the mechanism of breast cancer metastasis has become a core issue in breast cancer research. Our previous series of studies have shown that VASP, as a key oncogene, plays an important role in the development of various tumors such as breast cancer. In this study, we find that miR-638 can target to inhibit VASP expression, and Lin28 acts as an RNA-binding protein to regulate the processing of miR-638, which inhibits its maturation and promotes the expression of VASP. In addition, we also find that CREB1 acts as a transcription factor that binds to the promoter of Lin28 gene and activates the Lin28/miR-638/VASP pathway. Furthermore, CREB1 can also directly bind to the promoter of VASP, and activate VASP expression, forming a CREB/Lin28/miR-638/VASP interactive network, which plays an important role in promoting cell proliferation and migration in breast cancer. Our study explained the mechanism of CREB1/Lin28/miR-638/VASP network promoting the development of breast cancer, which further elucidated the mechanism of VASP as a key oncogene, and also provided a theoretical basis for expanding new approaches to tumor biotherapy.

Chlorotoxin targets ERα/VASP signaling pathway to combat breast cancer.

Breast cancer is one of the most common malignant tumors among women worldwide. About 70-75% of primary breast cancers belong to estrogen receptor (ER)-positive breast cancer. In the development of ER-positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti-ER-positive breast cancer, which also provides an important reference for the study of CTX anti-ER-related tumors.

Betulinic acid protects mice from cadmium chloride-induced toxicity by inhibiting cadmium-induced apoptosis in kidney and liver.

Cadmium exposure is closely associated with a variety of diseases including cancers and the accumulation of cadmium has been long recognized as a public health problem. It is therefore of high importance to find methods to reduce cadmium accumulation in the human body. Herein, we report that administration of betulinic acid (BA) protects mice from cadmium chloride (CdCl2)-induced toxicity by inhibiting cadmium-induced apoptosis in both kidney and liver. Mice were given oral doses of 3 mg/kg, 10 mg/kg and 30 mg/kg of BA daily for ten consecutive days, and were injected with one dose of 1 mg/kg CdCl2 after one hour of BA administration every day. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) were assessed by ELISA. Residual cadmium was determined by atomic absorption analysis. Protein expression was evaluated by western blotting. Pretreatment with BA significantly reduced residual cadmium levels in the liver, kidney and testis, increased the cadmium output in urine, and reduced tissue damage induced by CdCl2. Moreover, BA prevented body weight loss by CdCl2 in a dose dependent manner. Furthermore, BA treatment increased the expression levels of B-cell lymphoma 2 (Bcl-2), decreased Bcl-2-associated X (Bax), and inhibited the levels of active caspase-3. Importantly, BA within a dose of 30 mg/kg did not induce any signs of toxicity, and protected mice from the toxicity induced by CdCl2 in a dose-dependent manner. Our findings suggest that BA inhibits CdCl2 induced apoptosis in the kidney and liver, and BA may be an effective agent for the prevention and treatment of cadmium-induced diseases in humans.

Glycine confers neuroprotection through microRNA-301a/PTEN signaling.

BACKGROUND: Glycine is known to protect against neuronal death. However, the underlying mechanism remains to be elucidated. The microRNA-301a is involved in both biological and pathological processes. But it is not known whether microRNA-301a has a neuroprotective property. In this study, we aimed to determine whether glycine-induced neuroprotection requires microRNA-301a-dependent signaling. RESULTS: We provided the first evidence that glycine increased the expression of microRNA-301a in cultured rat cortical neurons and protected against cortical neuronal death through up-regulation of microRNA-301a after oxygen-glucose deprivation. MicroRNA-301a directly bound the predicted 3'UTR target sites of PTEN and reduced PTEN expression in cortical neurons. We revealed that PTEN down-regulation by microRNA-301a mediated glycine-induced neuroprotective effect following oxygen-glucose deprivation. CONCLUSIONS: Our results suggest that 1) microRNA-301a is neuroprotective in oxygen-glucose deprivation-induced neuronal injury; 2) glycine is an upstream regulator of microRNA-301a; 3) glycine confers neuroprotection through microRNA-301a/PTEN signal pathway.

HOXB7-S3 inhibits the proliferation and invasion of MCF-7 human breast cancer cells.

Homeobox B7 (HOXB7) has been found to be overexpressed in numerous types of human cancer. However, the role of HOXB7 in breast cancer remains to be elucidated. The aim of the present study was to investigate the effects of HOXB7 on the proliferation and invasion of breast cancer cells. Initially, reverse transcription quantitative polymerase chain reaction and western blotting were respectively employed to detect the mRNA and protein expression levels of the HOXB7 gene in the MDA­MB­231 and MCF­7 human breast cancer cell lines. Subsequently, small interfering RNAs designed to interfere with the expression of HOXB7 were used to knockdown the expression of HOXB7 in the MCF­7 cell line, the effects of which on cell proliferation, the apoptotic rate and invasion capacity were measured using a Cell Counting kit­8 assay, flow cytometry and transwell chambers, respectively. The results demonstrated that HOXB7 mRNA and protein were all overexpressed in MDA­MB­231 and MCF­7 breast cancer cell lines. Furthermore, HOXB7­S3 effectively inhibited the proliferation and invasion of MCF­7 breast cancer cells. In conclusion, these results demonstrated that HOXB7 may be a potential therapeutic target in human breast cancer.

Protective effect of astragalus polysaccharides on liver injury induced by several different chemotherapeutics in mice.

Side effects are an unavoidable consequence of chemotherapy drugs, during which liver injury often takes place. The current study was designed to investigate the protective effect of Astragalus polysaccharides (APS) against the hepatotoxicity induced by frequently-used chemical therapy agents, cyclophosphamide (CTX), docetaxel (DTX) and epirubicin (EPI)) in mice. Mice were divided into five groups, controls, low or high dose groups (DTXL, CTXL, EPIL or DTXH, CTXH, EPIH), and low or high dose chemotherapeutics+APS groups (DTXL+APS, CTXL+APS, EPIL+APS or DTXH+APS, CTXH+APS, EPIH+APS). Controls were treated with equivalent normal saline for 28 days every other day; low or high dose group were intraperitoneal (i.p) injected with low or high doses of CTX, DTX and EPI for 28 days every other day; low or high dose chemotherapeutics+APS group were separately intraperitoneal (i.p) injected with chemotherapeutics for 28 days every other day and i.p with APS (100 mg/kg) for 7 days continually from the 22th to the 28th days. The body weight, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), histopathological features, and ultrastructure morphological change of liver tissues, protein expression level of caspase-3 were estimated at different time points. With high dose treatment of DTX, CTX and EPI, weight gain was inhibited and serum levels of ALT and AST were significantly increased. Sections of liver tissue showed massive hepatotoxicity in CTXH group compared to the control group, including hepatic lobule disorder, granular and vacuolar degeneration and necrosis in hepatic cells. These changes were confirmed at ultrastructural level, including obvious pyknosis, heterochromatin aggregation, nuclear membrane resolution, and chondrosome crystal decrease. Western blotting revealed that the protein levels of caspase-3 increased in CTXH group. The low dose groups exhibited trivial hepatotoxicity. More interestingly, after 100 mg/kg APS, liver injury was redecued not only regarding serum transaminase activities (low or high dose chemotherapeutics+APS group), but also from pathological and ultrastructural changes and the protein levels of caspase-3 (CTXH+APS group). In conclusion, DTX, CTX and EPI induce liver damage in a dose dependent manner, whereas APS exerted protective effects.