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Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for P-glycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 +/- 0.1% at 1 min to 11.13 +/- 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.

FOLFOX6 regimen has been used in treating advanced colorectal cancer for a period, but there is no systemic study on FOLFOX6 regimen in treating Chinese colorectal cancer patients with liver metastasis. This study was to evaluate the efficacy of FOLFOX6 regimen on Chinese colorectal cancer patients with liver metastasis, and observe the adverse events.Ninety-one colorectal cancer patients with liver metastasis were treated by FOLFOX6 regimen. FOLFOX6 regimen consisted of 2-hour infusion of oxaliplatin (100 mg/m(2)) and 2-hour infusion of leucovorin (CF) (400 mg/m(2)) on Day l, followed by 5-fluorouracil (5-FU) bolus (400 mg/m(2)) on Day 1 and 46-hour infusion (2.4 g/m(2)). FOLFOX6 regimen was repeated at 2-week intervals. The clinical efficacy and adverse events were evaluated.The objective response rate for all patients was 40.7%, with 4 cases of complete remission (CR), 33 partial remission (PR), 19 stable disease (SD), and 35 progressive disease (PD). There was no significant difference in objective response rate between the patients with and without previous treatment (P0.05). The median survival time was 17.0 months for all patients, 20.0 months for the patients without previous treatment, and 12.0 months for the patients with previous treatment. The time to progress (TTP) was 7.0 months for all patients, 9.0 months for the patients without previous treatment, and 6.0 months for the patients with previous treatment. Peripheral neuritis, gastrointestinal reaction, and myelosuppression were major adverse events. All patients with 5-FU-associated adverse events recovered after treatment.FOLFOX6 regimen can be used in treating colorectal cancer with liver metastasis for its efficacy and less toxicity.

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.

To evaluate the efficacy and safety of imatinib mesylate in the treatment of patients as preoperative supplement, or used alone for unresectable and/or metastatic gastrointestinal stromal tumors (GIST).A total of 51 cases with advanced GIST were proved pathologically. Among them, CD117 was detected positive in 47 patients and negative in 4 patients; 4 patients received imatinib mesylate before operation and 47 patients with unresectable and (or) metastatic GIST received oral imatinib mesylate daily at dose of 300-800 mg. One patient was lost in follow-up and the objective effect was evaluated in 50 patients.3 of the 50 patients (6.0%) achieved complete response (CR), 34 (68%) had partial response (PR), 5 (10.0%) had stable disease (SD) and 8 (20.0%) had progression disease (PD). The median time to progression (mTTP) was 16 months during which most of the patients experienced benefit. 32 patients had been followed up for more then 1 year. The 1-year and 2-year survival rate were 95.3%, 89% respectively. 50 patients were valuable for the toxicity assessment according to the WHO standard. The main toxicity included grade I-II edema of periorbital area and lower limb in 72.0% (36/50) patients, leukopenia was present in 46% (23/50) and intratumoral bleeding in 4.0% (2/50). Other toxicities included mild fatigue (28.0%), abdominal pain (14.0%), efflorescence (18.0%), nausea and vomiting (18.0%).As an inhibitor of tyrosine kinase, imatinib mesylate is generally well tolerated and has been proved to be effective and safe during prolonged treatment of patients with advanced gastrointestinal stromal tumors. Its toxicity is acceptable.

Blood flow patterns with CT perfusion imaging (CTPI) had been innovated to delineate abnormal hemodynamic lesions in the liver, brain, and kidney. This study was to evaluate the blood flow patterns of pulmonary masses by CTPI, and determine the value of CTPI in differential diagnosis among benign, malignant, and inflammatory masses.Fifty-two patients with previously diagnosed pulmonary masses (37 with malignant masses, 7 with benign masses, and 8 with inflammatory masses) underwent dynamic CTPI. Time-density curves (TDC) of artery, vein, and pulmonary masses as well as mass perfusion images and parameters including perfusion volume (PV), peak height (PH), mean transit time (MTT) and blood volume (BV) were obtained by Phlips CT-perfusion software.The values of PV, PH, and BV were significantly higher in malignant masses and active inflammatory masses than in benign masses [(27.63+/-15.06) ml.min(-1).ml(-1) and (30.80+/-20.33) ml.min(-1).ml(-1) vs. (11.81+/-3.74) ml.min(-1).ml(-1), (28.46+/-12.07) Hu and (32.15+/-15.89) Hu vs. (10.41+/-3.77) Hu, (21.64+/-10.97) ml/100 g and (28.38+/-14.55) ml/100 g vs. (10.61+/-5.33) ml/100 g, P0.01]. However, the differences of MTT among malignant, inflammatory, and benign masses were not significant [(28.39+/-21.66) s, (25.91+/-14.57) s, and (29.86+/-13.57) s, P=0.928]. No significant differences in the 4 parameters were found between malignant and inflammatory masses. When PV20 ml.min(-1).ml(-1) and pH15 Hu were set as a diagnostic threshold (excluded active inflammatory masses), the sensitivity, specificity, and accuracy were 91.9%, 100%, and 84.1%, respectively.CT perfusion imaging provides quantitative information about blood flow patterns of pulmonary masses and is an applicable diagnostic method for differentiating pulmonary masses.

Juan-Juan Yin,1,2 Stepan P Shumyak,2 Christopher Burgess,2 Zhi-Wei Zhou,2 Zhi-Xu He,3 Xue-Ji Zhang,4 Shu-Ting Pan,2,5 Tian-Xin Yang,6 Wei Duan,7 Jia-Xuan Qiu,5 Shu-Feng Zhou21Xiaolan People’s Hospital, Southern Medical University, Zhongshan, Guangdong, People’s Republic of China; 2Department of Pharmaceutical Sciences, College ofPharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, 4Research Center forBioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 5Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic ofChina; 6Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 7School of Medicine, Deakin University, Waurn Ponds, VIC, AustraliaAbstract: Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE1), which formed the complex CDE1-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (Kd=1,617 M-1). The structure of the targeting vector CDE1 was fully characterized using 1H- and 13C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE1-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE1-Ada-DOX binds to recombinant human estrogen receptor α fragments with a Kd of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE1-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE1 molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE1-Ada-DOX had an unexpected lower drug uptake (when the host–guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE1-Ada-DOX was significantly increased when the host–guest ratio was adjusted to be less than half at the concentration of CDE1 over 5 µM due to the release of the estrone residues. CDE1 elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism.Keywords: breast cancer, drug vector, functionalized, membrane estrogen receptor, polysaccharide, targeted drug delivery

A number of drugs and herbal compounds have been documented to cause hepatoxicity. Schisandrin B (Sch B) is an active dibenzocyclooctadiene isolated from Schisandrae fructus, with a wide array of pharmacological activities. However, the potential hepatotoxicity of Sch B is a major safety concern, and the underlying mechanism for Sch B-induced liver toxic effects is not fully elucidated. In the present study, we aimed to investigate the liver toxic effects and the molecular mechanisms of Sch B in mouse liver and macrophage cells. The results have shown that Sch B exhibits potent grow inhibitory, proapoptotic, and proautophagic effects in AML-12 and RAW 264.7 cells. Sch B markedly arrested cells in G1 phase in both cell lines, accompanied by the down-regulation of cyclin dependent kinase 2 (CDK2) and cyclin D1 and up-regulation of p27 Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl) B-cell lymphoma 2 (Bcl-2), but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax). Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP) in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Sch B for clinical use., Video abstract

Ning-Kui Niu,1–3 Zi-Li Wang,1 Shu-Ting Pan,2,4 Hui-Qiang Ding,1 Giang HT Au,2 Zhi-Xu He,5 Zhi-Wei Zhou,2,5 Guozhi Xiao,6 Yin-Xue Yang,7 Xueji Zhang,8 Tianxin Yang,9 Xiao-Wu Chen,10Jia-Xuan Qiu,4 Shu-Feng Zhou2 1Department of Spinal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Orthopedics, General Hospital of Tianjin Medical University, Tianjin, 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 6Department of Biochemistry, Medical Center, Rush University, Chicago, IL, USA; 7Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 8Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 10Department of General Surgery, The First People’s Hospital of Shunde affiliated to Southern Medical University, Foshan, Guangdong, People’s Republic of China Abstract: Osteosarcoma (OS) is the most common malignant bone tumor occurring mostly in children and adolescents between 10 and 20 years of age with poor response to current therapeutics. Alisertib (ALS, MLN8237) is a selective Aurora kinase A inhibitor that displays anticancer effects on several types of cancer. However, the role of ALS in the treatment of OS remains unknown. This study aimed to investigate the effects of ALS on the cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK) signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS) generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2) in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy. Keywords: ALS, autophagy, apoptosis, osteosarcoma, PI3K/Akt/mTOR pathway, EMT

Xiang-Dong Han,1 Zhi-WeiZhou,2–4 WeiYang,1 Hang-Cheng Ye,1 Ying-ZiXu,1 Yun-Feng Huang,1 Tong Zhang,1 Shu-Feng Zhou2 1School of Pharmacy, Shanghai University ofTraditional Chinese Medicine, Shanghai, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, 4Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, People’s Republic of China Abstract: Ischemic heart disease (IHD) is the leading cause of death worldwide and remains a major life-threatening factor in humans. Apoptosis has been implicated in the pathogenesis of IHD. The Chinese herbal formula Huo Luo Xiao Ling Dan (HLXLD), one of the commonly used Chinese herbal formulas, consists of Salviae miltiorrhizae, Angelica sinensis, Gummi olibanum, and Commiphora myrrha, with a wide spectrum of pharmacological activity. However, the mechanism of action and molecular targets of HLXLD in the treatment of IHD are unclear. This study aimed to computationally predict the molecular interactions between the major active components of HLXLD and key regulators of apoptosis and then examine the effect of HLXLD on coronary artery ligation-induced acute myocardial ischemia in rats. The molecular interactions between the major active components of HLXLD, including ferulic acid, ligustilide, succinic acid, vanillic acid, tanshinone IIA, tanshinone IIB, danshensu, salvianolic acid A, salvianolic acid C, protocatechuic aldehyde, and β-boswellic acid and human protein molecules including B cell lymphoma-extra large (Bcl-xl), B cell lymphoma 2 antagonist/killer 1 (Bak1), B cell lymphoma 2 (Bcl-2), procaspase 3, and caspase 9 with regard to hydrogen bond formation, charge interaction, and π-π stacking using Discovery Studio® program 3.1. The 12 HLXLD components were predicted by ADMET (absorption, distribution, metabolism, excretion and toxicity) Predictor to have favorable pharmacokinetic and low hepatotoxicity profiles. The acute myocardial ischemia was established by surgical ligation of the left anterior descending coronary artery. The rats were divided into a sham operative group, a model group, a positive control group treated with 0.2 mg/kg isosorbide mononitrate, and groups treated with 2.7, 5.4, or 10.8 g/kg HLXLD. The results showed that administration of HLXLD increased mean arterial pressure, left ventricular systolic pressure, heart rate, and maximal rate of rise/descent of left ventricular pressure levels. Administration of HLXLD significantly ameliorated coronary artery ligation-induced tissue damage in the left ventricle, with restored arrangement of myocardial fibers and recovered myoplasm in rats. Furthermore, HLXLD markedly increased the expression level of Bcl-2 but decreased the level of cleaved caspase 3. Taken together, administration of HLXLD attenuated acute myocardial ischemia-induced damage in cardiomyocytes and inhibited apoptotic death of cardiomyocytes, thereby exerting a cardioprotective effect in rats with IHD. These findings suggest that HLXLD may represent a promising herbal formula for the treatment of cardiovascular disease by counteracting apoptotic cell death via multiple active compounds. More studies are warranted to fully elucidate the mechanisms of action, identify the therapeutic targets, and validate the efficacy and safety of HLXLD in the treatment of IHD. Keywords: Huo Luo Xiao Ling Dan, myocardial ischemia, coronary artery ligation, hemodynamics, apoptosis, molecular docking

Jin-Ping Li,1,2 Yin-Xue Yang,3 Qi-Lun Liu,1 Zhi-Wei Zhou,2,4 Shu-Ting Pan,2,5 Zhi-Xu He,4 Xueji Zhang,6 Tianxin Yang,7 Si-Yuan Pan,8 Wei Duan,9 Shu-Ming He,10 Xiao-Wu Chen,11 Jia-Xuan Qiu,5 Shu-Feng Zhou2,41Department of Surgical Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 4Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 5Department of Oral and Maxillofacial Surgery, TheFirst Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 6Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 7Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 8Department of Pharmacology, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 9School of Medicine, Deakin University, Waurn Ponds, VIC, Australia; 10Department of Obstetrics and Gynecology, Xiaolan People’s Hospital affiliated to Southern Medical University, Zhongshan, Guangdong, People’s Republic of China; 11Department of General Surgery, The First People’s Hospital of Shunde affiliated to Southern Medical University, Shunde, Foshan, Guangdong, People’s Republic of China Abstract: Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and ß-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy. Keywords: Danusertib, breast cancer, cell cycle, apoptosis, autophagy, EMT&nbsp

Shu-Ting Pan,1,* Zhi-Wei Zhou,2,3,* Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Jia-Xuan Qiu,1 Shu-Feng Zhou2 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Research Center forBioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of ColorectalSurgery, General Hospital of Ningxia Medical University, Yinchuan, 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China *These two authors contributed equally tothis work Abstract: 5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS generation in A549 cells. Collectively, this SILAC study quantitatively evaluates the proteomic response to treatment with DMXAA that helps to globally identify the potential molecular targets and elucidate the underlying mechanism of DMXAA in the treatment of NSCLC. Keywords: DMXAA, non-small cell lung cancer, cell cycle, apoptosis, autophagy, SILAC

Jia-Xuan Qiu,1,2 Zhi-Wei Zhou,3 Zhi-Xu He,4 Xueji Zhang,5 Shu-Feng Zhou,3 Shengrong Zhu11Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 4Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 5Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of ChinaAbstract: Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, ß-bisabolene, ß-sesquiphelandrene, 6-gingerdione, (-)-zingiberene, and methyl-6-isogingerol) and human cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A2, 2C9, 2C19, 2D6, and 3A4 mainly through hydrogen bond formation, to a lesser extent, via π–π stacking. The pharmacokinetic simulation studies showed that the [I]/[Ki] value for CYP2C9, 2C19, and 3A4 ranged from 0.0002 to 19.6 and the R value ranged from 1.0002 to 20.6 and that ginger might exhibit a high risk of drug interaction via inhibition of the activity of human CYP2C9 and CYP3A4, but a low risk of drug interaction toward CYP2C19-mediated drug metabolism. Furthermore, it has been evaluated that the 12 ginger components possessed a favorable ADMET profiles with regard to the solubility, absorption, permeability across the blood–brain barrier, interactions with CYP2D6, hepatotoxicity, and plasma protein binding. The validation results showed that there was no remarkable effect of ginger on the metabolism of warfarin in humans, whereas concurrent use of ginger and nifedipine exhibited a synergistic effect on platelet aggregation in humans. Moreover, ginger components showed a rapid half-life and no to low toxicity in humans. Taken together, this study shows that ginger components may regulate the activity and expression of various human CYPs, probably resulting in alterations in drug clearance and response. More studies are warranted to identify and confirm potential ginger–drug interactions and explore possible interactions of ginger with human CYPs and other functionally important proteins, to reduce and avoid side effects induced by unfavorable ginger–drug interactions.Keywords: CYP, drug metabolism, ginger, drug interaction, docking

Yong-Hui Ding,1,2 Zhi-Wei Zhou,2,3 Chun-Fang Ha,1 Xue-Yu Zhang,1 Shu-Ting Pan,4 Zhi-Xu He,3 Jeffrey L Edelman,2 Dong Wang,5 Yin-Xue Yang,6 Xueji Zhang,7 Wei Duan,8 Tianxin Yang,9 Jia-Xuan Qiu,4 Shu-Feng Zhou2,3 1Department of Gynecology, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 5Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 8School of Medicine, Deakin University, Waurn Ponds, Australia; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA Abstract: Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5′-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells. Further studies are warranted to validate the efficacy and safety of ALS in the treatment of ovarian cancer. Keywords: alisertib, Aurora kinase A, epithelial ovarian cancer, cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition, sirtuin 1

Tanshinone IIB (TSB) is a major active constituent of the root of Salvia miltiorrhiza (Danshen) used in the treatment of acute stroke. Danshen extracts and TSB have shown marked neuron-protective effects in mouse studies but there is a lack of clinical evidence for the neuron-protective effects of Danshen and its active ingredients. This study investigated the neuron-protective effects of TSB in experimentally stroked rats. TSB at 5 and 25 mg/kg by intraperitoneal injection significantly reduced the focal infarct volume, cerebral histological damage and apoptosis in rats subjected to middle cerebral artery occlusion (MCAO) compared to MCAO rats receiving vehicle. This study demonstrated that TSB was effective in reducing stroke-induced brain damage and may represent a novel drug candidate for further development. Further mechanistic studies are needed for the neuron-protective activity of TSB.

To investigate the clinical features and treatment of anal canal adenocarcinoma.Clinical data of 49 patients with anal canal adenocarcinoma treated in our hospital from January 1965 to March 2002 were analyzed retrospectively.The ratio of male to female was 1.3. The median age was 56 years old. Anal bleeding, tapering stool and anal lump were the most common symptoms. Chronic perianal diseases were complicated in 36.7% of the cases. The median follow-up was 66 months. Local recurrence and inguinal lymph node metastasis were found in 7 cases respectively, lung metastasis in 2, supraclavicular and mediastinal metastasis in 1 respectively. The 3-year survival rates in the patients with resection alone, radiochemotherapy alone, resection combined with radiochemotherapy, and without any treatment were 41.3%, 20.0%, 56.3% and 15.0%, respectively, and the 5-year survival rates were 34.4%, 0, 37.5%, 0, respectively.Anal canal adenocarcinoma is a rare and fatal malignancy. Abdomino-perineal resection combined with postoperative radiochemotherapy is the principal treatment.

A sensitive and accurate high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detector was developed and validated for simultaneous determination of benazepril (BZL) and its active metabolite, benazeprilat (BZT), in human plasma. The plasma sample, after spiked with riluzole as an internal standard (IS), was subjected to a solid-phase extraction (SPE) prior to a HPLC analysis. Chromatographic separations were achieved on a Hypersil BDS C(18) (300 mm x 4.6mm, 5 microm). The mobile phase consisted of phosphate buffer (pH 2.6; 10mM) and acetonitrile mixture in a gradient mode. Detection was carried out at a wavelength of 237 nm. The retention times of BZL, BZT and IS were at about 6.2, 15.4 and 16.2 min, respectively. The calibration curve was linear in the range of 20-2000 ng/mL for both BZL and BZT (r(2)>0.997). At three quality control concentrations of 100, 500, and 1500 ng/mL, the intra-day and inter-day relative standard deviation ranged from 2.8 to 8.6% for BZL and from 2.2 to 8.5% for BZT, while the mean absolute percentage error ranged from -7.5 to 6.7% for BZL and from -6.0 to 3.2% for BZT. The limit of detection (LOD) was 10 ng/mL and the limit of quantification (LOQ) was 20 ng/mL for both BZL and BZT in human plasma. The method was successfully applied to bioequivalence evaluation of benazepril hydrochloride formulations in healthy Chinese.

It is not very clear about the factors that affect the prognosis of liver metastases from colorectal cancer. This study was to investigate the clinicopathologic factors related to the prognosis of liver metastases from colorectal cancer.The clinicopathologic factors and follow-up data of 197 patients with liver metastases from colorectal cancer, treated from Jan. 1996 to Dec. 2000, were analyzed retrospectively. The prognostic index (PI) of patients was calculated based on the results of multivariate analysis and patients were classified into different hazard groups accordingly.The overall 1-, 3-, and 5-year survival rates were 59.04%, 17.73%, and 11.48%. Univariate analysis revealed that extrahepatic invasion, primary tumor resection, liver metastasis resection, type of primary tumor, serum CEA concentration, number and size of liver metastases, and distribution of liver metastases were associated with prognosis. Multivariate analysis identified that the resection of liver metastases, serum CEA concentration, number and size of liver metastases were prognostic factors. The patients were classified into high risk, moderate risk, and low risk groups according to the PI value, and there was significant difference in survival rates between each two groups.Liver metastasis resection, serum CEA concentration, number and size of liver metastases are important prognostic factors for liver metastases from colorectal cancer. In order to improve the survival rate, liver metastases should be resected for suitable patients. Moreover, PI value could be used to predict the prognosis of patients with liver metastases from colorectal cancer.

Colorectal cancer is one of the most common malignant tumors in China. Synchronous liver metastases occur in 10%-25% colorectal cancer patients. This study was to elucidate the prognostic factors and treatment choices for colorectal cancer patients with synchronous liver metastases.Records of 220 colorectal cancer patients with synchronous liver metastases initially treated at Cancer Center of Sun Yat-sen University from Dec. 1995 to Dec. 2002 were reviewed. Prognostic factors were analyzed by Kaplan-Meier method and Cox regression model with SPSS12.0 software.The 5-year overall survival rate was 5.52%, the median survival time of the patients was 12.93 months. Univariate analysis of clinical characteristics revealed that the number, size and distribution of liver metastases, extrahepatic disease, serum CEA level at diagnosis, N status, and histology were prognostic factors. Univariate analysis of clinical treatment factors showed that treatment modality, primary site resection, and chemotherapy regimen were prognostic factors. Multivariate analysis showed that the number and size of liver metastases, extrahepatic disease, serum CEA value at diagnosis, treatment modality, primary tumor resection, and chemotherapy regimen were independent prognostic factors of colorectal cancer patients with synchronous liver metastases.Liver metastases larger than 5 cm in diameter, bilobar liver metastases, extrahepatic invasion or metastases, and CEA level more than 200 microg/L at diagnosis are adverse prognostic factors. Primary tumor resection and liver resection should be considered for all suitable patients with colorectal metastases to the liver alone. Liver-target intervention and/or systemic chemotherapy might be a good choice for inoperable patients. If possible, systemic chemotherapy containing oxaliplatin is preferred.

The incidence of colorectal cancer is increasing in China, of which rectal cancer accounts for over 50%. Because of the limitation of pelvic cavity, the selection of surgical procedures, the proportion of sphincter saving and prognosis of male patients are different from those of female patients. This study was to analyze the correlations of clinicopathologic features of rectal cancer in 384 male patients to their prognosis.Correlations of 12 clinicopathologic factors of 384 male patients with rectal cancer to the prognosis were studied with univariate and multivariate analysis.Univariate analysis revealed that tumor site, gross type, histologic type, infiltrative extent to bowel wall, site of lymphatic metastasis and Dukes' stage were prognostic factors of male patients with rectal cancer. Multivariate analysis revealed that histologic type and site of lymphatic metastasis were independent prognostic factors.Histologic type and site of lymphatic metastasis are independent prognostic factors of male patients with rectal cancer.

YH-16, a new recombinant angiogenesis inhibitor, has demonstrated synergetic effects with chemotherapy in non-small-cell lung cancer treatment in stage II clinical trial. This study was to investigate the effect of YH-16 on liver metastases from colon cancer.Inhibitory concentration 50% (IC(50)) of YH-16 on vascular endothelial cells and colon cancer cell line CT26 was determined by MTT assay. Furthermore, the mouse mode of colon cancer liver metastases was established by inoculating CT26 cells into the subcapsule of spleen. 60 mice were randomly divided into four groups: control group (0 mg/kg), low-dose YH-16 group (0.40 mg/kg), medium-dose YH-16 group (0.75 mg/kg) and high-dose YH-16 group (1.5 mg/kg). The numbers of liver metastases were examined 2 weeks after drug injection. The expression of vascular endothelial growth factor (VEGF) was detected by immunohistochemical method in liver metastases, and tumor microvessel density (MVD) was measured by immunostaining using factor VIII monocolonal antibody.Proliferation of CT26 and vascular endothelial cells was inhibited by YH-16, which the IC(50) was (2.16+/-0.28) microg/ml and (0.64+/-0.10) microg /ml, respectively. In vivo, the liver metastasis rates in control, low-dose, medium-dose and high-dose groups were 100%, 92.3%, 80% and 73.3%, respectively (P0.05). However, YH-16 did not inhibit the growth of spleen tumors of which median volumes were 1.180 cm(3), 1.201 cm(3), 0.887 cm(3) and 0.781 cm(3), respectively (P0.05). There was no difference of VEGF expression in liver metastases among the four groups. Moreover, MVD was 65.00+/-9.58, 58.15+/-8.81, 51.60+/-7.10 and 44.53+/-11.47 in the four groups. MVD in medium-dose and high-dose YH-16 groups was lower than that in control group and MVD was lower in high-dose group than that in medium-dose and low-dose groups (P0.05).Angiogenesis inhibitor YH-16 can inhibit liver metastases from colorectal cancer.

The prognosis of patients diagnosed with Dukes' B rectal carcinoma varies largely. Micrometastasis via lymphatic vessel may have occurred in Dukes' B rectal carcinoma although no clinical pathologic evidence of lymph node metastasis was found. This study was to investigate the correlation of lymphatic vessel density (LVD) to the prognosis of Dukes' B rectal carcinoma.Immunohistochemistry was used to display the lymphatic vessels with lymphatic endothelium-specific marker, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), in 54 specimens from Dukes' B rectal carcinoma. LVD in tumor, peri-tumor and normal tissues was calculated. All patients had complete clinical and follow-up data.Of the 54 specimens, 11 (20.4%) were LYVE-1-negative in tumor, peri-tumor, and normal tissues, 43 (79.6%) were LYVE-1-positive. LVD was significantly higher in peri-tumor than in tumor and normal tissues (6.05+/-5.61 vs. 0.89+/-0.66 and 4.11+/-3.29, P0.05), and was significantly lower in tumor than in normal tissues (P0.05). No significant correlation was found between LVD and recurrence rate (P0.05), or overall survival (P0.05).Lymphangiogenesis may occur in peri-tumor tissues. LVD may not be a prognostic factor of Dukes' B rectal carcinoma.

The prognosis of colon cancer after radical resection has seldom been reported, and the results are different. This study aimed to investigate the clinicopathologic factors related to recurrence and metastasis of colon cancer after radical resection.The clinicopathologic and follow-up data of 152 patients with colon cancer, treated with radical resection from Jan. 1999 to Dec. 2000, were analyzed retrospectively.The overall recurrence and metastasis rate was 19.74%, and the liver metastasis rate was 9.87%. Univariate analysis showed that blood transfusion, disease duration, tumor size, tumor movement, histological differentiation, Dukesostage, and lymph node involvement were correlated to recurrence and metastasis of colon cancer after operation; blood transfusion, serum concentration of carcinoembryonic antigen (CEA), tumor movement, histological differentiation, Dukesostage, and lymph node involvement were correlated to liver metastasis. Multivariate analysis showed that tumor movement, histological differentiation, and lymph node involvement were prognostic factors for recurrence and metastasis, while serum concentration of CEA, histological differentiation, and lymph node involvement were prognostic factors for liver metastasis.Tumor movement, histological differentiation, and lymph node involvement are important prognostic factors for recurrence and metastasis of colon cancer after radical resection. Patients with high serum concentration of CEA, poor histological differentiation, and lymph node involvement have increased risk of liver metastasis.

The incidence of low and middle rectal cancer is high in China. Researches on improving treatment efficacy of this disease have constantly been concerned. This study was to evaluate the correlation of clinicopathologic features to the prognosis in low and middle rectal cancer.The clinicopathologic data of 599 patients with low and middle rectal cancer, treated from 1990 to 1999 in Cancer Center of Sun Yat-sen University, were analyzed retrospectively. Curative resection was performed for all patients, including abdominoperineal resection (ARP, 355 cases) and low anterior resection (LAR, 244 cases). Survival rate was calculated using life table method, and differences between survival curves were tested by log-rank test. Cox regression model was used for multivariate prognostic analysis.The overall 5-year survival rate was 70.7%; it was significantly lower in APR group than in LAR group (67.5% vs. 75.2%, P=0.026). Univariate analysis showed that local recurrence, perioperative blood transfusion, lymph node metastasis, T stage, histology, macropathology, operation pattern, and distance from anal margin were correlated to prognosis (P0.05). Multivariate analysis showed that local recurrence, perioperative blood transfusion, lymph node metastasis, and T stage were independent prognostic factors (P0.01).Local recurrence, perioperative blood transfusion, lymph node metastasis, and T stage are important prognostic factors of low and middle rectal cancer. LAR has become the preferred option in curative surgery for low and middle rectal cancer.

To study the effect of angiogenesis inhibitor YH-16 in combination with 5-FU on liver metastasis of colorectal cancer.In vitro, the inhibitory effects of YH-16 and 5-FU on the growth of vascular endothelial cells and colorectal cancer cells were examined by MTT assay. In vivo, colorectal cancer cells were transplanted into BALB/c mice, and the mice were divided into six groups randomly:control group, low-dose YH-16 group, middle-dose YH-16 group, high-dose YH-16 group, 5-FU group and combination group. The number of liver metastases, the size of primary tumor and the toxicity were examined after 2 weeks postoperatively. The expression of vascular endothelial growth factor (VEGF) in liver metastases was detected by immunohistochemistry, and tumor microvessel density (MVD) was measured by immunostaining with CD34 and factor VIII (monoclonal antibodies.In vitro, YH-16 inhibited the growth of colon cancer cells and vascular endothelial cells, with the IC50 at (2.16+/-0.28) microg/ml and (0.64+/-0.10) microg/ml respectively. In vivo high-dose YH-16 and 5-FU had a remarkable inhibitory effect on liver metastasis, and the combination group showed significant enhancement on this effect (P0.05). The combination group and 5-FU group could inhibit the growth of primary tumor, but not found in YH-16 group. The toxicity of YH-16 was lower than that of 5-FU (P0.05), and the difference was not found in the toxicity between combination group and 5-FU group (P0.05). Expression of VEGF in liver metastases was clearly inhibited by YH-16 in combination with 5-FU or 5-FU alone compared to the control group, and MVD in middle-dose and high-dose YH-16 group, 5-FU group and combination group was lower than that in control group (P0.05).The angiogenesis inhibitor YH-16 can inhibit liver metastasis of colorectal cancer through inhibiting the growth of vascular endothelial cells. YH-16 in combination with 5-FU has additive effect on inhibitory activity against liver metastasis.

Jin-Lian He,1 Zhi-Wei Zhou,2,3 Juan-Juan Yin,2 Chang-Qiang He,1 Shu-Feng Zhou,2,3 Yang Yu1 1College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China Abstract: Drug metabolizing enzymes (DMEs) and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC) is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE) on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2) cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(P)H: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to the nuclei. Additionally, SCE significantly suppressed the expression of cytosolic Kelch-like ECH-associated protein 1 (the repressor of Nrf2) and remarkably increased Nrf2 stability in HepG2 cells. Taken together, our findings suggest that the hepatoprotective effects of SCE may be partially ascribed to the modulation of DMEs and drug transporters via Nrf2-mediated signaling pathway. SCE may alter the pharmacokinetics of other coadministered drugs that are substrates of these DMEs and transporters and thus cause unfavorable herb–drug interactions. Keywords: Nrf2, Keap1, HepG2 cell, drug metabolizing enzyme, drug transporter, P-gp, MRP, OATP, Schisandra chinensis

Li-Fang Zhang,1,2 Zhi-Wei Zhou,2 Zhen-Hai Wang,1 Yan-Hui Du,1 Zhi-Xu He,3 Chuanhai Cao,2 Shu-Feng Zhou2,31Department of Neurology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Department ofPharmaceutical Science, College ofPharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory forRegenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of ChinaAbstract: There is an increasing prevalence of Alzheimer’s disease (AD), which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff), caffeine (Caff), and melatonin (Mel) have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-ß (Aß) generation in Neuro-2a (N2a)/amyloid precursor protein (APP) cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aβ40/42 and Aβ oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. Theexpression and/or phosphorylation levels of glycogen synthase kinase 3β (GSK3β), Erk1/2, PI3K, Akt, Tau, Wnt3α, β-catenin, and Nrf2 were detected by Western blot assay. The results showed that regimen 1 produced an additive antiamyloidogenic effect with significantly reduced extracellular levels of Aβ40/42 and Aβ42 oligomers. Regimen 2 did not result in remarkable effects, and regimen 3 showed a less antiamyloidogenic effect compared to regimen 1. Coff or Caff, plus Mel reduced oxidative stress in N2a/APP cells via the Nrf2 pathway. Coff or Caff, plus Mel inhibited GSK3β, Akt, PI3K p55, and Tau phosphorylation but enhanced PI3K p85 and Erk1/2 phosphorylation in N2a/APP cells. Coff or Caff, plus Mel downregulated Wnt3α expression but upregulated β-catenin. However, Coff or Caff plus Mel did not significantly alter the production of T helper cell (Th)1-related interleukin (IL)-12 and interferon (IFN)-γ and Th2-related IL-4 and IL-10 in N2a/APP cells. The autophagy of cells was not affected by the combinations. Taken together, combination of Caff or Coff, before treatment with Mel elicits an additive antiamyloidogenic effects in N2a/APP cells, probably through inhibition of Aβ oligomerization and modulation of the Akt/GSK3β/Tau signaling pathway.Keywords: Alzheimer’s disease, Aβ oligomer, coffee, caffeine, melatonin, Tau, Nrf2, chronotherapy, Akt

Toxicities and response are different when patients with advanced colorectal cancer were treated with standard FOLFOX6 regimen. Serum level of dihydropyrimidine dehydrogenase (DPD) relates to efficacy and toxicities of chemotherapy containing 5-fluorouracil (5-FU). This study was to explore relationship of DPD to serum concentration of 5-FU in colorectal cancer patients treated with FOLFOX6 regimen, and their correlation to treatment response and adverse events.Serum level of DPD in 72 patients with colorectal cancer was detected by high-performance liquid chromatography (HPLC) before chemotherapy. Serum concentration of 5-FU at steady state was detected by HPLC after patients received FOLFOX6 regimen. Treatment response and adverse events in the patients were assessed.Serum levels of DPD were normally distributed in 72 patients (ranged 1.55-5.94), while serum concentrations of 5-FU at steady state were not (ranged 141.1-1 741.9 microg/L). Serum level of DPD was negatively correlated with serum concentration of 5-FU (r=-0.460, P0.01). Occurrence of adverse events was lower when 5-FU concentration was less than 600 microg/L than when 5-FU concentration was more than 600 microg/L (P0.05). The mean serum concentration of 5-FU was significantly higher in patients with complete response and partial response than in patients with steady disease, and progressive disease (513.9 microg/L vs. 409.8 microg/L, and 259.3 microg/L, P0.05). Serum level of DPD was lower in patients suffered oral mucositis and diarrhea of grade II-IV than in patients suffered oral mucositis and diarrhea of grade 0-I (P=0.016, P=0.047). Serum level of DPD had no relation with treatment response of the patients (r=0.312, P=0.078).DPD level and serum 5-FU concentration vary a lot among patients with colorectal cancer. DPD level negatively correlates with serum 5-FU concentration. Serum concentration of 5-FU correlates with treatment effect and toxicities.

Currently, to preserve the anal function and improve the patients' quality of life, low anterior resection has become the preferred option in curative rectal cancer surgery. As the use of stapling instruments provides more reliable anastomoses in low anterior resection for rectal cancer, it enlarges the indication of this procedure. The aim of this study was to review the operation results and their outcomes of 449 rectal cancer patients who recieved of curative low anterior resections with stapling devices, and intent to find some measures that can reduce complications and improve long-term effects of this procedure.The study included 449 patients who had a potentially curative anterior resection with stapled anastomosis in rectal cancer between Jan.1990 and Sept. 2002 at Sun Yat-sen University Cancer Center. All patients had complete follow-up data. All data were analyzed by SPSS8.0 software, risk factors for anastomotic leakage and recurrence were analyzed by Logistic regression, survival was analyzed by life table, and prognostic factors were screened by multivariate COX model.There were 11 cases of anastomotic leakage and 23 cases of anastomotic recurrence after operation. The 5-year survival rate was 78.4%. Age of/= 65 years, and tumor involvement of more than half circumference were risk factors for anastomotic leakage, blood transfusion during operation was the risk factor for anastomotic recurrence. The independent factors for poor survival were stage of disease and tumor differentiation.Stapling devices can improve the anal reservation rate in low rectal cancer surgery, and stapled anastomoses is safe and feasible. Adequate preparation of bowel ends, a tension-free anastomosis with excellent blood supply and skilled stapled anastomoses were key measures to reduce anastomotic leakage, While TME, multidisciplinary therapy and the principle of avoiding medical spread, were key measures to improve treatment effect of rectal cancer.

The efficacy and toxicity of 5-FU was various in different patients. It was reported that they were correlated to the activity of dihydropyrimidine dehydrogenase (DPD). This study is to measure DPD activity in blood and to analyze the relationship among DPD activity, the toxicity of 5-FU based adjuvant chemotherapy and the 5-FU plasma concentration in colorectal cancer patients.30 colorectal cancer patients were enrolled into the study to receive adjuvant chemotherapy 2 weeks after cured resection. The regimen was 5-FU 425 mg/m(2) plus CF 60 mg/m(2) continuous infused for 2 hours, daily for 5 days. The concentration of endogenous uracil (U) and dihydrouracil (UH(2)) were assayed by high performance liquid chromatography (HPLC). The UH2-U ratio in plasma was used to represent DPD activity in blood. The plasma samples were collected before chemotherapy in all patients to detect the DPD activity in blood, and after 5-FU infusion at day 1 and day 5 to measure 5-FU plasma concentration. The relationship among the DPD activity in blood, the toxicity of chemotherapy and the plasma concentration of 5-FU in all patients were analyzed.30 colorectal cancer patients have received adjuvant chemotherapy. The DPD activity in the blood of 30 colorectal cancer patients before chemotherapy was 4.09+/-1.21 (2.14-6.7), showed a trend of normal distribution. The 5-FU plasma concentration after 5-FU infusion was (2 079.12+/-621.41) microg/L (1 200.10-3 554.80 microg/L) at the first day, and (2 197.64+/-606.78) microg/L at the fifth day (1 259.00-3 441.03 microg/L). There was a negative relationship between the DPD activity in blood and the 5-FU plasma concentration at day 1 (r=-0.773, P=0.00), and day 5 after 5-FU infusion(r = -0.833, P = 0.00). No significant difference between the 5-FU plasma concentration of day 1 and day 5 was found (P=0.458). The 5-Fu associated toxicities had a negative relationship with DPD activity in blood, and had a positive relationship with 5-FU plasma concentration (P0.05).The results indicated that the DPD activity in blood can be used to predict the toxicity and the 5-FU plasma concentration in patients with 5-FU based chemotherapy.

The optimal distal molecular clearance margin of rectal cancer hasn't been confirmed. This study was designed to explore the molecular margin of distal intramural spread (DIS)in rectal cancer, and its prognostic value, and to further clarify the required distal margin of radical surgery for rectal cancer.Sixty-one P53 positive specimens,resected from patients with rectal cancer from Aug.1996 to Oct. 1997, were collected. Microscopic DIS was examined by P53-immunohistochemistry (P53-IHC),comparing with conventional hematoxylineosin (HE)staining in consecutive large slice. Tissue shrinkage ratio,comparing the distal clearance margin measured in fresh specimens to that measured in large slice after fixed in each case,was used to convert macroscopically measured extent of distal spread to its actual extent. After long-term follow-up, the survival curves of 4 DIS groups were estimated by Life-table method.With P53-IHC,DIS was observed in 50 cases (82.0%), DIS extents were 0.11-3.50 cm with the mean of 0.59 cm, DIS extent of3.00 cm was detected in 1 case only. Meanwhile,DIS was observed in 29 cases (47.5%)by HE staining, DIS extents were 0.10-1.39 cm with the mean of 0.13 cm. There was significant difference between the 2 means (P0.0001). The long-term result indicated that the survival rate of DIS extent of1.00 cm group was lower than those of non-DIS group,and DIS extent of0.50 cm group (P0.05).DIS was more exactly detected by P53-IHC than by HE. Most of DIS extents were less than 1 cm in rectal cancer. For over 95% cases, 3 cm distal to the rectal cancers was relatively safe in radical operations. The poor prognosis can be predicted in cases with DIS extent of1 cm.

Tumor microvessel density (MDV)partly indicates tumor angiogenesis,and it is a hot topic in the study of oncology. However, the relationship between tumor MVD and the clinicopathological characteristics of rectal cancer are unclear. This study was to explore the correlation of tumor MVD to metastasis and recurrence of rectal cancer.Records of 97 rectal cancer patients who had undergone radical resection in the Department of Abdominal Surgery, Cancer Center,Sun Yat-sen University from 1996 to 1997 were retrospectively analyzed. The paraffin-embedded tissues of resected tumor and para-tumor tissues were collected,microvessels were detected by immunohistochemistry using mouse anti-human CD31 antibody. The MVD was then calculated microscopically, its relations with clinicopathological parameters were analyzed with software package of SPSS 9.0.The values of MVD in tumor tissues,and non-cancerous para-tumor mucosa were 45.26+/-19.52,and 11.82+/-3.48 per high magnification field (x400)(P0.001). The MVD in the treatment-failed patients (recurrence and/or metastasis) was 62.72+/-21.75, while it was 41.23+/-16.68 in the patients with disease-free survival, the difference was significant (P0.001).The tumors with higher MVD are more likely to relapse or metastasize after operation. Tumor tissue MVD can be used as a biological indicator for post-operative metastasis and recurrence in rectal cancer.

It was indicated that there was relationship between estrogen and colorectal cancer. Estrogen receptor (ER) and progesterone receptor (PR) were expressed in colorectal cancer tissue. They were measured using immunohistochemistry with various results,but there was little quantitative study. This study was designed to quantitatively measure the expression of ER and PR in tumor tissues and normal mucosas and analyze its relationship with clinicopathological parameters in colorectal cancer.ER and PR expression in cytoplasm and nucleus of tumor tissues and normal mucosas from 45 colorectal cancer patients were quantitatively measured using radioligand binding assay (RBA).ER and PR were expressed in all tumor tissues and normal mucosas. In cytoplasm, the ER expression level of tumor tissues was higher than that of normal mucosas; they were 7.96+/-3.69 fmol/mg protein and 4.34+/-2.84 fmol/mg protein (P0.01) respectively. And that was the same for PR expression; they were 3.89+/-2.64 fmol/mg protein and 2.50+/-1.73 fmol/mg protein(P0.01)respectively. In nucleus, the ER expression level of tumor tissues was higher than that of normal mucosas; they were 18.42+/-8.30 fmol/mg protein and 11.24+/-5.44 fmol/mg protein (P0.01)respectively. And that was the same for PR expression, they were 9.36+/-5.90 fmol/mg protein and 7.84+/-7.41 fmol/mg protein (P0.05) respectively. There was positive correlation between ER and PR expression in cytoplasm and nucleus of tumor tissues (P0.01) respectively,so was in cytoplasm in normal mucosas (P0.01) respectively, but not in nucleus (P0.05). There was correlation between ER expression in tumor tissues and patients' age,and the expression in these people over 45 years old was higher than those less 45 years old (P0.05), but no correlation between tumor tissues PR expression and age (P0.05). There was no correlation between tumor tissues ER, PR expression and sex, staging, tumor location, size, gross and histological type, invasive depth, lymph nodes metastasis, or tumor tissue CEA expression (P0.05).ER and PR expression in colorectal tumor tissues were higher than those in normal mucosas, and there was positive correlation between ER and PR expression in tumor tissues. All these indicate that ER in colorectal cancer tissues has some activity. The level of the ER, PR expression in tumor tissue could not predict the malignant biological behaviors of colorectal cancer.

Recurrence is a very important prognostic factor for colorectal cancer patients after operation. The selection of patients for individualized follow-up and adjuvant therapy after curative resection of colorectal carcinoma depends on prognostic factors for recurrence. The objective of this study was to investigate the clinicopathologic factors related to recurrence following curative surgery for colorectal cancer.The clinicopathologic factors and follow-up data of 692 cases of colorectal cancers after surgical treatment from 1991 to 1999 were retrospectively analyzed by univariate and multivariate methods.The overall 3-year and 5-year survival rates were 33.1% and 19.7% in recurrent group, and 92.8% and 86.1% in nonrecurrent group, respectively. Univariate analysis showed that Dukes' stage, lymph node metastasis, tumor location, histological differentiation, gross findings and depth of bowel wall invasion were significantly associated with recurrence after operation. Multivariate analysis showed that lymph node metastasis and tumor location were prognostic factors for recurrence after operation. In separate analysis of distant metastasis and local recurrence with multivariate method, lymph node metastasis was an prognostic variable for both distant metastasis and local recurrence. Depth of bowel wall invasion was associated with distant metastasis, and tumor location was associated with local recurrence.Lymph node metastasis is the most important prognostic factor for recurrence or distant metastasis and local recurrence after operation for the patients with colorectal carcinoma. Depth of bowel wall invasion is an important prognostic factor for distant metastasis. Rectal cancer patients should be considered to have additional risks for local recurrence.

Accumulating evidence suggests that intestinal epithelial barrier dysfunction plays an important role in the pathogenesis of hepatic cirrhosis and its complications such as gastrointestinal injury and hepatic encephalopathy. To date, there is no cure for cirrhosis-associated intestinal mucosal lesion and ulcer. This study aimed to investigate the effect of oxymatrine on intestinal epithelial barrier function and the underlying mechanism in carbon tetrachloride (CCl4)-induced cirrhotic rats. Thirty CCl4-induced cirrhotic rats were randomly divided into treatment group, which received oxymatrine treatment (63 mg/kg), and non-treatment group, which received the same dose of 5% glucose solution (vehicle). The blank group (n = 10 healthy rats) received no treatment. Terminal ileal samples were collected for histopathological examination. The expression level of nuclear factor-κB (NF-κB) p65 in ileal tissue was evaluated by immunohistochemistry. The gene and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in ileal tissues were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Additionally, plasma endotoxin level was determined. In comparison to the blank group, a significant alteration in the morphology of intestinal mucosal villi in the non-treatment group was observed. The intestinal mucosal villi were atrophic, shorter, and fractured, and inflammatory cells were infiltrated into the lamina propria and muscular layer. Besides, serious swell of villi and loose structure of mucous membrane were observed. Oxymatrine reversed the CCl4-induced histological changes and restored intestinal barrier integrity. Moreover, oxymatrine reduced the protein expression level of NF-κB p65, TNF-α, and IL-6, which were elevated in the vehicle-treated group. In addition, the serum endotoxin level was significantly decreased after oxymatrine treatment in CCl4-induced cirrhotic rats. The results indicate that oxymatrine improves intestinal barrier function via NF-κB-mediated signaling pathway and may be used as a new protecting agent for cirrhosis-associated intestinal mucosal damage.

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant K a was 1,639 M(-1) as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.