Yue Pan

Hepatoprotective effects of berberine on liver fibrosis via activation of AMP-activated protein kinase

AIMS We investigated the protective effect of berberine (BBR) on chronic liver fibrosis in mice and the potential mechanism underlying the activation of AMP-activated protein kinase (AMPK) pathway. MAIN METHODS CCl4-induced chronic liver fibrosis model in mice was established and activated rat hepatic stellate cell was treated with BBR. Cell viability was evaluated by SRB assay and protein expressions were detected by Western blot. KEY FINDINGS Our results showed that BBR ameliorated the liver fibrosis in mice with CCl4-induced liver injury and inhibited the proliferation of hepatic stellate cell in dose- and time-dependent manner. BBR decreased the enzyme release of ALT, AST, and ALP in serum, elevated SOD and reduced MDA content of liver tissue in CCl4-induced liver fibrosis model. BBR delayed the formation of regenerative nodules and reduced necrotic areas compared to CCl4 group. Moreover, BBR treatment activated AMPK, decreased the protein expression of Nox4, TGF-β1 and the phosphorylated Akt. The expression of smooth muscle actin (α-SMA), the marker of activated hepatic stellate cell, was also reduced by BBR treatment. SIGNIFICANCE Our studies firstly demonstrated that BBR exerted hepatoprotective effects possibly via activation of AMPK, blocking Nox4 and Akt expression. Our findings may benefit the development of new strategies in the prevention of chronic liver disease.

The shape effect of magnetic mesoporous silica nanoparticles on endocytosis, biocompatibility and biodistribution

Although the aspect ratio (AR) play a crucial role in determining biological effects of homogeneous nanomaterials, studies available concerning how the shape contributes to biological effect of heterogeneous nanomaterials is limited. To systematically clarify the shape influence on the endocytosis, biocompatibility and biodistribution of magnetic mesoporous silica nanoparticles (M-MSNPs), three FITC-labeled M-MSNPs with different aspect ratio (AR=1, 2, and 4) were specifically designed and constructed through altering the ratios of CTAB/TEOS in a modified so-gel method. We have demonstrated that long-rod M-MSNP2 possessed higher intracellular internalization amount than the short-rod M-MSNP1 and the sphere-like M-MSNP0 in both cancer cells and normal cells due to the difference in the endocytosis pathways. However, there are no significant shape effects on biocompatibility including cytotoxicity and hemolytic rate. Moreover, biodistribution in HepG2 tumor-bearing mice showed that M-MSNPs administrated intravenously were mainly presented in reticuloendothelial system (RES) organs including liver, spleen and kidney. In particular, sphere-like M-MSNP0 were easily trapped in the liver, while long-rod M-MSP2 exhibited more retention in the spleen. It is worth noting that rod-like M-MSNPs are preferentially accumulated in tumor sites than sphere-like M-MSNPs, indicating an improved drug delivery efficacy in cancer therapy. Our findings may provide useful data for deeply understanding the interaction between the different shapes and biological behavior of M-MSNPs, which is expected to give rise to a new generation of heterogeneous M-MSNPs with significantly enhanced efficacy and safety for the cancer theranostics. STATEMENT OF SIGNIFICANCE In this work, we systematically clarified the shape influence on the endocytosis, biocompatibility and biodistribution of homogeneous nanomaterials. We have demonstrated that rod-like magnetic mesoporous silica nanoparticles (M-MSNPs) were capable of higher intracellular internalization and tumor accumulation than sphere-like M-MSNPs, which was expected to give rise to a new generation of heterogeneous M-MSNPs with significantly enhanced efficacy and safety for the cancer theranostics.

Real-Time Visualizing and Tracing of HSV-TK/GCV Suicide Gene Therapy by Near-Infrared Fluorescent Quantum Dots

Exploring intracellular behavior of suicide gene is significant for improving the efficacy and safety of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-TK/GCV) system in cancer therapy. Molecular imaging represents a powerful tool to understand gene transportation and function dynamics. In this work, we reported a quantum-dot-based technique for revealing the procedure of HSV-TK/GCV suicide gene therapy by constructing covalent linkage between near-infrared fluorescent quantum dots (QDs) and TK gene. This stable QD labeling did not influence either the QDs fluorescence or the biological activity of TK gene. Furthermore, we visualized and dynamically traced the intracellular behavior antitumor effect of TK gene in vitro and in vivo. It is demonstrated that TK gene was shuttled to the nucleus after a-24 h treatment; at that time the single dose of GCV administration exerts the gradually increasing lethal effect until to 72 h. Real-time tracing the formation of hepatocellular carcinoma treated with HSV-TK/GCV suicide gene system in vivo by QD-based NIR fluorescence imaging provides useful insight toward QD-based theranostics in future cancer therapy.

Adipose tissue-secreted miR-27a promotes liver cancer by targeting FOXO1 in obese individuals

The current notion that obesity is a major risk factor for the development of and the mortality associated with a subset of liver cancer is well appreciated. However, detailed mechanistic insights underlying this relationship are lacking. Better understanding of the adipose tissue-secreted miRNAs that play a potential role in defining primary liver cancer development and mediating the obesity-cancer communication offers the potential for new insights into tumor growth and interventions to modulate tumor formation and progression. In this study, we clearly demonstrated that miR-27a is more highly upregulated in cancer, plasma, and adipose samples from obese liver cancer cases, and therefore reasoned that miR-27a excreted from adipose tissue leads to liver cancer development. To address this idea, we prepared miR-27a-overexpressing 3T3-L1 adipocytes and cocultured them with HepG2 liver cancer cells. Our results demonstrated that secretory miR-27a promoted liver cancer cell proliferation through the downregulation of the transcription factor FOXO1 and promoted the G1/S cell cycle transition by decreasing the cell cycle inhibitors p21 and p27 and increasing the cell cycle regulator cyclin D1. These findings improve our understanding of the involvement of miR-27a in obesity-liver cancer communication and might provide a novel putative target for obesity-driven primary liver cancer diagnosis and therapy.

Selective inhibition of liver cancer growth realized by the intrinsic toxicity of a quantum dot-lipid complex

Using the intrinsic toxicity of nanomaterials for anticancer therapy is an emerging concept. In this work, we discovered that CdTe/CdS quantum dots, when coated with lipids (QD-LC) instead of popular liposomes, polymers, or dendrimers, demonstrated extraordinarily high specificity for cancer cells, which was due to the difference in the macropinocytosis uptake pathways of QD-LC between the cancer cells and the normal cells. QD-LC-induced HepG2 cell apoptosis was concomitant with the activation of the JNK/caspase-3 signaling pathway. Moreover, QD-LC treatment resulted in a delay in the latent period for microtumor formation of mouse hepatocarcinoma H22 cells and inhibited tumor growth, with a reduction of 53.2% in tumor volume without toxicity in major organs after intratumoral administrations to tumor-bearing mice. Our results demonstrate that QD-LC could be a very promising theranostic agent against liver cancer.

MiR-27a promotes hepatocellular carcinoma cell proliferation through suppression of its target gene peroxisome proliferator-activated receptor γ.

Background: MicroRNAs (miRNAs) function as essential posttranscriptional modulators of gene expression, and are involved in a wide range of physiologic and pathologic states, including cancer. Numerous miRNAs are deregulated in hepatocellular carcinoma (HCC). This study aimed to investigate the role of miR-27a in the development of HCC. Methods: The expression of MiR-27a was measured by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to examine changes in the viability of HepG2, Bel-7402, Bel-7404 hepatoma cell lines associated with up-regulation or down-regulation of miR-27a. A dual-luciferase activity assay was used to verify a target gene of miR-27a. Immunohistochemistry, qRT-PCR, Western blotting analysis, and cell cycle and apoptosis flow cytometric assays were used to elucidate the mechanism by which miR-27a modulates liver cancer cell proliferation. Results: The expression of miR-27a was significantly increased in HCC tissues and HepG2, Bel-7402, Bel-7404 hepatoma cell lines (P < 0.05). We also found that the down-regulation of miR-27a in HepG2 cells dramatically inhibited proliferation, blocked the G1 to S cell cycle transition and induced apoptosis (P < 0.05). In addition, miR-27a directly targeted the 3’-untranslated region of peroxisome proliferator-activated receptor &ggr; (PPAR-&ggr;), and ectopic miR-27a expression suppressed PPAR-&ggr; expression on the mRNA and protein levels. The rosiglitazone-induced overexpression of PPAR-&ggr; attenuated the effect of miR-27a in HCC cells. Conclusions: Our findings suggested that miRNA-27a promoted HCC cell proliferation by regulating PPAR-&ggr; expression. MiR-27a may provide a potential therapeutic strategy for HCC treatment.

Berberine induces apoptosis by suppressing the arachidonic acid metabolic pathway in hepatocellular carcinoma

Berberine (BBR) has been suggested as a potential candidate anticancer agent due to its high anticancer activity and multiple mechanisms. In the present study, the inhibitory effect of BBR on hepatocellular carcinoma (HCC) via the suppression of the arachidonic acid (AA) metabolic pathway was investigated. BBR was demonstrated to reduce the viabilities of H22, HepG2 and Bel‑7404 cells, in a dose‑ and time‑dependent manner, and increase the number of apoptotic cells. BBR induced the translocation of apoptosis‑inducing factor between the mitochondria and the nucleus, and had no effects on the protein expression levels of caspase‑3 or ‑9. In addition, BBR significantly suppressed the protein expression levels of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)‑2 and elevated the content ratio of AA to prostaglandin E2 (PGE2). Furthermore, BBR reduced the volume and weight of tumors in a H22 transplanted tumor model in mice. The results of the present study demonstrated that elevation in the ratio of AA to PGE2 via suppression of the protein expression of cPLA2 and COX‑2 in the AA metabolic pathway is involved in the inhibitory effect of BBR in HCC.

Single and repeated dose toxicity of citric acid-based carbon dots and a derivative in mice

Carbon dots (CDs) have recently emerged as a new class of fluorescent nanomaterials and are already competitive in many respects to conventional semiconductor quantum dots (QDs). Despite CDs remarkable advantages in bioimaging, biosensing and other biomedical applications, their biosafety is still unclear. In the present study, the systematic single and repeated dose toxicity and biodistribution of two citric acid-based probes (CDs and a derivative, Et-IPCA) were investigated in vivo. For the single dose toxicity studies, the lethal dose 50 (LD50) of CDs was 391.615 mg kg−1 and 357.771 mg kg−1 for female and male mice, respectively, while Et-IPCA reached its saturation point in solution at 25 mg kg−1 and did not lead to the death of any mice at that concentration. For the repeated dose toxicity studies, although there were temporary changes in hematological parameters suggesting an acute inflammatory response after seven doses, the data on the body weight, organ coefficients, hematological parameters, blood biochemistry, and organ histopathology at the end of the 90 day recovery period suggested that both the CDs and Et-IPCA had low toxicity over the 90 day period. These findings will be useful for the future development of CDs-based drug delivery systems and for the development of clinical applications of these systems.

Hepatic IGF-1R overexpression combined with the activation of GSK-3β and FOXO3a in the development of liver cirrhosis.

AIMS Liver cirrhosis is the common pathological histology manifest among a number of chronic liver diseases and liver cancer. Circulating levels of insulin growth factor-1 (IGF-1) have been recently linked to liver cirrhosis and the development of liver cancer. Herein, we hypothesized that IGF-1R overexpression combining the activation of GSK-3β and FOXO3a were involved in the development of human and murine chronic liver cirrhosis. METHODS Liver samples of patients were screened from the Tissue Bank of the China-Japan Union Hospital of Jilin University. Mice liver fibrosis model was performed using intraperitoneal injection of carbon tetrachloride (CCl4) for 12weeks. Serum IGF-1 levels were detected by enzyme-linked immunosorbent assays (ELISA). Microscopical examination of liver parenchyma was performed using conventional H&E and Massons staining. Moreover, we investigated the IGF-1 receptor (IGF-1R) signaling pathway at different period after CCl4 administration. RESULTS Serum IGF-1 levels were significantly decreased in patients with liver cirrhosis, which is concomitant with the declined circulating levels of IGF-1 in 8 to 12weeks CCl4-treated mice. Furthermore, the expression of IGF-1R was significantly higher at 12w compared with control group. In addition, activation of the GSK-3β and FOXO3a were activated during the process of murine chronic liver injury. CONCLUSION The present study demonstrates that decreased circulating IGF-1 levels are involved in human and murine chronic liver disease. Interestingly, overexpression of the IGF-1R, and activation of GSK3β and FOXO3a might be the molecular mechanisms underlying the development of liver cirrhosis.

Berberine Enhances Chemosensitivity and Induces Apoptosis Through Dose-orchestrated AMPK Signaling in Breast Cancer

Breast cancer is the most common malignancy in women. Although personalized or targeting molecular cancer therapy is more popular up to now, the cytotoxicity chemotherapy for patients with advanced breast cancer is considered as the alternative option. However, chemoresistance is still the common and critical limitation for breast cancer treatment. Berberine, known as AMPK activator, has shown multiple activities including antitumor effect. In this study, we investigate the chemosensitive effect of different dosages berberine on drug-resistant human breast cancer MCF-7/MDR cell in vitro and in vivo, and the mechanisms underlying AMPK activation on Doxorubicin (DOX) chemosensitivity. Our results showed that berberine could overcome DOX resistance in dose-orchestrated manner: On one hand, low-dose berberine can enhance DOX sensitivity in drug-resistance breast cancer cells through AMPK-HIF-1α-P-gp pathway. On the other hand, high-dose berberine alone directly induces apoptosis through the AMPK-p53 pathway with the independence of HIF-1α expression. Taken together, our findings demonstrate that berberine sensitizes drug-resistant breast cancer to DOX chemotherapy and directly induces apoptosis through the dose-orchestrated AMPK signaling pathway in vitro and in vivo. Berberine appears to be a promising chemosensitizer and chemotherapeutic drug for breast cancer treatment.