Hepatic lipid homeostasis is crucial for overall liver health, necessitating an understanding of its underlying physiological mechanisms. While G protein-coupled receptor 119 (GPR119) has emerged as a key receptor in lipid metabolism, its physiological role in the liver remains poorly explored. We investigated the role of Gpr119 in hepatic lipid homeostasis using the zebrafish hepatocyte cell line (ZFL), alongside both high-fat diet (HFD)-induced and genetic knockout (gpr119-/-) zebrafish models. We demonstrated that pharmacological activation of Gpr119 by the agonist MBX-2982 attenuated HFD-induced hepatic steatosis and liver injury. Conversely, endogenous gpr119 deficiency induced spontaneous hepatic lipid accumulation and elevated serum transaminase activities under basal physiological conditions. Mechanistically, Gpr119 signaling limits lipid accumulation by coordinately downregulating de novo lipogenesis while promoting lipolysis and very-low-density lipoprotein (VLDL)-dependent lipid clearance. Collectively, our findings indicate that Gpr119 is a regulator of hepatic lipid homeostasis, functioning to restrict lipid supply and accelerate lipid clearance. This study provides in vivo genetic evidence highlighting the role of Gpr119 in hepatic lipid homeostasis, thereby advancing our physiological understanding of liver function and lipid handling.

To investigate whether the combination of chemotherapy with staged Chinese herbal medicine (CHM) therapy could enhance health-related quality of life (QoL) in non-small-cell lung cancer (NSCLC) patients and prolong the time before deterioration of lung cancer symptoms, in comparison to chemotherapy alone. A prospective, double-blind, randomized, controlled trial was conducted from December 14, 2017 to August 28, 2020. A total of 180 patients with stage I B–IIIA NSCLC from 5 hospitals in Shanghai were randomly divided into chemotherapy combined with CHM (chemo+CHM) group (120 cases) or chemotherapy combined with placebo (chemo+placebo) group (60 cases) using stratified blocking randomization. The European Organization for Research and Treatment of Cancer (EORTC) Quality-of-Life-Core 30 Scale (QLQ-C30) was used to evaluate the patient-reported outcomes (PROs) during postoperative adjuvant chemotherapy in patients with early-stage NSCLC. Adverse events (AEs) were assessed in the safety analysis. Out of the total 180 patients, 173 patients (116 in the chemo+CHM group and 57 in the chemo+placebo group) were included in the PRO analyses. The initial mean QLQ-C30 Global Health Status (GHS)/QoL scores at baseline were 57.16 ± 1.64 and 57.67 ± 2.25 for the two respective groups (P>0.05). Compared with baseline, the chemo+CHM group had an improvement in EORTC QLQ-C30 GHS/QoL score at week 18 [least squares mean (LSM) change 17.83, 95

Epithelial-to-mesenchymal transition (EMT) is a developmental program that plays a vital role in gastric cancer, including aspects of tumor progression, the metastatic process, and resistance to treatment. Here, we have designed an in vitro model that mimics the features of EMT as observed in gastric cancer. The results showed that both migration and invasion were enhanced in gastric cancer cells with Brachyury overexpression. Additionally, the expression of IL-8 increased, while IL-8RA and IL-8RB levels significantly decreased in the in vitro model. Overall, the in vitro model offers an opportunity to study these phenomena relevant to EMT as they may occur in vivo in gastric cancer, as well as potential drug interactions that could interfere with these processes.

The burden of colorectal cancer (CRC) varies substantially across different geographical locations. However, there was no further quantitative analysis of regional social development and the disease burden of CRC. In addition, the incidence of early- and late-onset CRC has increased rapidly in developed and developing regions. The main purpose of this study was to investigate the trends in CRC burden across different regions, in addition to the epidemiological differences between early and late-onset CRC and their risk factors. In this study, estimated annual percentage change (EAPC) was employed to quantify trends in age-standardized incidence rate (ASIR), mortality rate, and disability-adjusted life-years. Restricted cubic spline models were fitted to quantitatively analyze the relationship between trends in ASIR and Human Development Index (HDI). In addition, the epidemiological characteristics of early- and late-onset CRC were investigated using analyses stratified by age groups and regions. Specifically, meat consumption and antibiotic use were included to explore the differences in the risk factors for early- and late-onset CRC. The quantitative analysis showed that the ASIR of CRC was exponentially and positively correlated with the 2019 HDI in different regions. In addition, the growing trend of ASIR in recent years varied substantially across HDI regions. Specifically, the ASIR of CRC showed a significant increase in developing countries, while it remained stable or decreased in developed countries. Moreover, a linear correlation was found between the ASIR of CRC and meat consumption in different regions, especially in developing countries. Furthermore, a similar correlation was found between the ASIR and antibiotic use in all age groups, with different correlation coefficients for early-onset and late-onset CRC. It is worth mentioning that the early onset of CRC could be attributable to the unrestrained use of antibiotics among young people in developed countries. In summary, for better prevention and control of CRC, governments should pay attention to advocate self-testing and hospital visits among all age groups, especially among young people at high risk of CRC, and strictly control meat consumption and the usage of antibiotics.

We have previously reported that recombinant adeno-associated virus serotype 3 (AAV3) vectors transduce human liver tumors more efficiently in a mouse xenograft model following systemic administration. Others have utilized AAV8 vectors expressing miR-26a and miR-122 to achieve near total inhibition of growth of mouse liver tumors. Since AAV3 vectors transduce human hepatic cells more efficiently than AAV8 vectors, in the present studies, we wished to evaluate the efficacy of AAV3-miR-26a/122 vectors in suppressing the growth of human hepatocellular carcinoma (HCC) cells in vitro, and human liver tumors in a mouse model in vivo. To this end, a human HCC cell line, Huh7, was transduced with various multiplicities of infection (MOIs) of AAV3-miR-26a or scAAV3-miR-122 vectors, or both, which also co-expressed a Gaussia luciferase (GLuc) reporter gene. Only a modest level of dose-dependent growth inhibition of Huh7 cells (~12–13%) was observed at the highest MOI (1 × 105 vgs/cell) with each vector. When Huh7 cells were co-transduced with both vectors, the extent of growth inhibition was additive (~26%). However, AAV3-miR-26a and scAAV3-miR-122 vectors led to ~70% inhibition of growth of Huh-derived human liver tumors in a mouse xenograft model in vivo. Thus, the combined use of miR-26a and scAAV3-miR-122 delivered by AAV3 vectors offers a potentially useful approach to target human liver tumors.

目的 研究基于高通量测序技术的胰腺癌湿热证患者肠道菌群结构与功能的变化情况,探讨肠道菌群与胰腺癌发病的关联,以期为临床诊治提供新思路.方法 将2018年1月至2018年12月期间复旦大学附属肿瘤医院和上海中医药大学附属岳阳中西医结合医院收治的31例胰腺癌湿热证患者作为观察组,另选择同期接受健康体检的8例正常人员作为对照组.采集两组人员的粪便样本,提取DNA,建库,经Illumina HiSeq PE150平台实施高通量测序,获得样本的生物学信息,对组间样本进行物种注释分析比较及功能注释分析比较.结果 ①经去冗余后,得到328 787条线粒体开放阅读框(ORFs);两组共有基因为137 849个,观察组独有基因为114 439个,对照组独有基因为60 473个.②在门水平及属水平上,两组间的物种丰度均存在明显差异.在门水平上,观察组菌群丰度中变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)、互养菌门(Synergistetes)、纤维杆菌门(Fibrobacteres)的丰度高于对照组,而拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、螺旋体门(Spirochaetes)、念珠菌门(Candidatus)、衣原体门(Chlamydiae)的丰度低于对照组.在属水平上,观察组菌群丰度中艾克曼菌(Akkermansia)、柯林斯氏菌(Collinsella)、链球菌(Streptococcus)、沃氏嗜胆菌(Bilophila)的丰度高于对照组,而双歧杆菌(Bifidobacterium)、普氏菌(Prevotella)、拟杆菌(Bacteroides)、粪杆菌(Faecalibacterium)、梭状杆菌(Clostridium)、罗斯氏菌(Roseburia)的丰度低于对照组.③基于距离矩阵的主坐标分析(PCoA)显示,两组样本的菌群组成结构差异明显.④KEGG数据库功能注释及分析结果显示,观察组在新陈代谢、环境信息处理、细胞加工相关通路上的基因表达水平高于对照组;观察组与对照组的功能丰度分布可以明显区分开.结论 相较于正常人,胰腺癌湿热证患者体内存在肠道菌群结构失调及功能变异,故监测肠道菌群变化可为临床防治胰腺癌提供有价值的信息.

Traditional Chinese Medicine (TCM) has been practiced in China for thousands of years. As a complementary and alternative treatment, herbal medicines that are frequently used in the TCM are the most accepted in the Western world. However, animal materials, which are equally important in the TCM practice, are not well-known in other countries. On the other hand, the Chinese doctors had documented the toxic profiles of hundreds of animals and plants thousand years ago. Furthermore, they saw the potential benefits of these materials and used their toxic properties to treat a wide variety of diseases, such as heavy pain and cancer. Since the 50s of the last century, efforts of the Chinese government and societies to modernize TCM have achieved tremendous scientific results in both laboratory and clinic. A number of toxic proteins have been isolated and their functions identified. Although most of the literature was written in Chinese, this review provide a summary, in English, regarding our knowledge of the clinical use of the toxic proteins isolated from a plant, Tian Hua Fen, and an animal, scorpion, both of which are famous toxic prescriptions in TCM.

Non-alcoholic fatty liver disease (NAFLD) is a major public health concern worldwide. The aim of the present study was to observe the effect of diosgenin on NAFLD and investigate the underlying mechanisms. Diosgenin treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Diosgenin significantly inhibited high glucose (HG)-induced triglyceride (TG) accumulation and sterol regulatory element‑binding protein-1c (SREBP-1c) mRNA increase in HepG2 cells, which were partially abolished by the AMPK inhibitor compound C. Diosgenin also significantly inhibited the increase of liver X receptor (LXR) α mRNA induced by HG or T0901317. However, T0901317‑induced upregulation of LXRα and SREBP-1c mRNA was not blocked by compound C. Following a high-fat diet for 16 weeks, the body and liver weights of the experimental rats were significantly increased, but this effect was significantly suppressed by diosgenin. Diosgenin and fenofibrate ameliorated lipid deposition in the liver and reduced the increase of hepatic TG content. Diosgenin significantly decreased the alanine aminotransferase (ALT) level, whereas fenofibrate significantly increased the ALT and aspartate aminotransferase levels. Diosgenin also increased AMPK and ACC phosphorylation and suppressed LXRα in the liver. In conclusion, the results of the present study suggested that diosgenin is a potential agent for preventing the development of NAFLD through the AMPK and LXR signaling pathways.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). However, the transduction efficiency of these vectors ranged between ~6-87% in HSPCs obtained from several different donors (n=11). Such a wide range of transduction efficiency of AAV6 vectors is presumably due to different levels of expression of the putative receptors and/or co-receptors on these cells. In our more recent studies, we observed that the transduction efficiency of AAV2 vectors could be augmented both by performing transduction of HSPCs with the wild-type (wt)-AAV2 vectors at high cell density, or by using capsid-modified Y444F+Y500F+Y731F+T491V-mutant AAV2 vectors. In the present studies, we examined whether similar strategies could also be employed to increase the transduction efficiency of HSPCs from donors that are not transduced efficiently by wt-AAV6 vectors. Primary human HSPCs were transduced with either 3×103 or 3×104 vgs/cell of scAAV6-EGFP vectors either at low-density (20,000 cells in 50 ml) or at high-density (200,000 cells in 50 ml). scAAV2-EGFP vectors were also used for comparison. Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results showed that whereas only ~14% of the cells transduced at low-density with high moi expressed the transgene, the transduction efficiency at high-density increased up to ~20% and 25%, at low, and high mois, respectively, also with a significant increase in the mean fluorescence intensity, thus corroborating that the initial cell-cell contact was a critical factor in achieving increased transduction. Next, the transduction efficiencies of the wt- and the capsid-modified triple-mutant (Y705F+Y731F+T492V) AAV6 vectors were compared. Again, the wt- and the capsid-modified quadruple-mutant (y444F+Y500f+Y731F+T491V) AAV2 vectors were used for comparison. HSPCs were transduced with 1×104 vgs/cell at high-density (200,000 cells in 50 ml), and transgene expression was evaluated by flow cytometry 48 hrs post-transduction. These results also clearly documented that the ~27% transduction efficiency of the wt-AAV6 vectors was increased by up to ~45% with the capsid-modified AAV6 vectors, with a concomitant increase in the mean fluorescence intensity. Taken together, these studies further corroborate the novel mechanism of "cross-transduction" by recombinant AAV vectors of human cells in general, and HSPCs in particular. Additional human HSPC transplant studies in a murine xenograft model in vivo are currently underway, the successful completion of which is likely to have significant implications of these strategies in the optimal use of AAV6 vectors in the potential gene therapy of human disorders involving the hematopoietic system.

Abstract Traditional Chinese medicine (TCM) has held, and still holds, an important position in primary health care in China, especially in the rural areas. However, scientific evidence of its mechanism is, for the most part, limited and additional better-designed laboratory research is needed. To date, numerous leading chemical compounds from TCM drugs have been identified and evaluated for their anti-tumor effects. However, little effort has been made on the protein-encoding genes isolated from TCM drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated viral (rAAV) vectors has not been attempted. For example, Trichosanthin (TCS), an antiviral plant defense protein found in the root tuber of a TCM herb, Trichosanthes kirilowii, has been used in anti-HIV clinical trials in the USA. Its enzymatic activities were recognized as type I ribosome-inactivating protein. Although the nucleotide sequence of the TCS gene was determined more than 20 years ago, to date, there was little report to deliver TCS-encoding gene into malignant cells. In the present studies, we synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The expression of mRNA was confirmed by RT-PCR and protein expression by Western blot assays against the FLAG tag, respectively. Among these genes, TCS gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Next, rAAV vectors containing the TCS gene driven by a liver cancer specific promoter, α-fetoprotein promoter (AFPp) were generated and tested. The results indicated that rAAV-AFPp-TCS vectors significantly inhibited the growth of hHCC cell lines in vitro as well as hHCC tumor growth in vivo following intra-tumor injections in a murine xenograft model. These studies suggest that the use of TCM cytotoxic genes may prove to be a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular. Note: This abstract was not presented at the meeting. Citation Format: Yuanhui Zhang, Yuan Wang, Lina Wang, George Aslanidi, Arun Srivastava, Changquan Ling, Chen Ling. Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5339. doi:10.1158/1538-7445.AM2015-5339

The human embryonic kidney cell line, HEK293, is readily transduced by the most commonly used AAV2 serotype vectors because these adherent cells abundantly express the cell surface receptor, heparan sulfate proteoglycan (HSPG), and one of the co-receptors, human fibroblast growth factor receptor 1 (FGFR1) for AAV2. However, a human erythroleukemia cell line, K562, commonly used as a model for human hematopoietic cell transduction, is not transduced as efficiently, although these cells grown in suspension, express both HSPG and FGFR1 only at modestly lower levels. We reasoned that the lack of proximity of HSPG and FGFR1 on K562 cells might account for the suboptimal transduction of these cells, and hypothesized that if the transduction was performed at high cell density, presumably allowing for HSPG on one cell to come in close proximity to FGFR1 on the neighboring cell, then AAV2 bound to HSPG on one cell could utilize FGFR1 on the neighboring cell to gain entry in the latter, and vice versa, thus leading to increased transduction. To test this hypothesis, HEK293 and K562 cells were transduced with 3×103 vgs/cell of scAAV2-EGFP vectors either at low-density (60,000 cells in 50 ml) or at high-density (480,000 cells in 50 ml). Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. Whereas ~90% of HEK293 cells expressed the transgene at low-density, the transduction efficiency at high-density increased up to ~95%. On the other hand, only ~35% of K562 cells were transduced at low-density, but interestingly, the transduction efficiency at high-density increased to ~65%, thus corroborating that close cell-cell contact facilitated vector entry into these cells. That the initial cell-cell contact was critical in achieving improved transduction, was further corroborated by experiments in which cells were transduced at low-density, and subsequently pooled together to reach high-density, and conversely, cells were transduced at high-density, and soon after transduction, were diluted to low-density. The increased transduction was observed only under the latter condition. Similar results were obtained with two additional human cell lines, M07e and Raji, which express low to extremely low levels of HSPG and FGFR1, respectively, and consequently, are transduced extremely poorly by AAV2 vectors. These studies were also extended to include primary human CD34+ hematopoietic stem/progenitor cells (HSPCs), also grown in suspension, and known to be transduced sub-optimally by AAV2 vectors. In these studies also, whereas only~5% of these cells were transduced at low-density, the transduction efficiency increased up to ~20% at high-density. Taken together, our studies have revealed a novel mechanism, which we have termed "cross-transduction", which AAV vectors exploit to gain entry into target cells, and which may have implications in the optimal use of AAV vectors in human gene therapy applications.

We have previously reported the development of capsid-modified next generation (NextGen) AAV serotype vectors that transduce cells and tissues more efficiently at reduced vector doses (Proc. Natl. Acad. Sci., USA, 105: 7827-7832, 2008). More recently, we have also described the development of genome-modified generation X (GenX) AAV vectors that also transduce cells and tissues more efficiently (J. Virol, 89: 952-961, 2015). The recombinant AAV genome contains inverted terminal repeats (ITRs) of 145 nucleotides at both ends. In these studies, a 20-nucleotide sequence, termed the D-sequence, was replaced with a substitute sequence, which led to enhanced transgene expression in human cell lines in vitro an in murine hepatocytes in vivo (J. Virol., 89: 952-961, 2015). In our present studies, we observed that a sequence, GGTTCCT, at the end of the D-sequence, shares partial homology to the consensus glucocorticoid receptor-binding element (GRE) site, 5'-GGTACANNNTGTT/CCT-3'. The TGTTCT half-site is an essential core element, which has been reported to be sufficient to relay glucocorticoid signaling. In electrophoretic mobility-shift assays (EMSAs), we documented that purified GR protein could specifically bind to double-stranded D-sequence oligonucleotides, suggesting that the D-sequence potentially functions as a ½ GRE site. Based on these results, we hypothesized that replacement of the D-sequence with a full GRE binding-site in the ITR might further increase transgene expression from these GenX AAV genomes. To this end, recombinant AAV vectors were generated in which the D-sequence was replaced with a fully functional GRE site. Insertion of a full GRE binding-site in the ITR significantly increased the transgene expression from these GenX AAV genomes following encapsidation in the wild-type (WT) AAV2 capsid vectors in human cell lines in vitro, and the extent of the transgene expression was further increased by dexamethasone-treatment. When 1×1010 vgs of recombinant AAV2 vectors containing the Gaussia luciferase (Gluc) reporter gene in the unmodified AAV genome, or those containing the GRE sequence, were administered via tail-vein into C57BL6/J mice, the transduction efficiency of the AAV-GRE vectors was ~8-fold higher in murine hepatocytes in vivo up to 9-weeks post-vector administration. More interestingly, when AAV-GRE genomes containing the enhanced green fluorescence protein (EGFP) reporter gene were encapsidated in the optimal NextGen AAV capsid-modified quadruple-mutant (Y444F+Y500F+Y730F+T491V) AAV2 vectors, the transduction efficiency of these vectors was further increased by ~8-fold in murine hepatocytes in vivo at a dose as low as 5×108 vgs/mouse. Taken together, the availability of these novel GenX AAV vectors containing the D-sequence substitution, and the fully functional GRE site insertion, to achieve high-efficiency transgene expression, has implications in the use of these vectors in human gene therapy.

Although recombinant AAV3 serotype vectors were largely ignored previously, owing to their poor transduction efficiency in all cells and tissues examined, our initial observation of their selective tropism for human liver cancer cell lines and primary human hepatocytes (Mol Genet Metabol., 98: 289-299, 2009; Hum Gene Ther., 21: 1741-1747, 2010; Gene Ther., 19: 375-84, 2012), has led to renewed interest in this serotype, since AAV3 vectors and their variants have now proven to be extremely efficient in targeting human and non-human primate hepatocytes in vitro as well as in vivo (Mol Ther., 22: S2, 2014; Mol Ther., 22: S91, 2014; Nature, 506: 382-386, 2014; Hum Gene Ther., 25: 1023-1034, 2014). Previously, it wasreported that the combination of ITR8 with AAV8 capsids (AAV8/8) resulted in vectors that led to at least 2-fold increase in transgene expression in mouse liver, compared with AAV8 capsids pseudotyped with ITR2 (AAV2/8) vectors (Mol. Ther. 11: S156, 2005), and that the ITRs from AAV serotypes 1-6 were interchangeable when they are packaged into AAV8 capsids (AAV1/8 – AAV6/8), but played no role in transgene expression in murine hepatocytes in vivo (J. Virol., 80: 426-439, 2006). In our present studies, we wished to evaluate the relative contribution of the cis-acting ITR from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the recombinant AAV3 vector production and transduction. To this end, we utilized two helper plasmid: pAAVr2c3, which carries rep2 and cap3 genes and pAAVr3c3, which carries rep3 and cap3 genes. Plasmid transfection assays revealed that both AAV2 and AAV3 Rep proteins were expressed at similar levels in the presence of adenoviral helper genes. We also generated two sets of single-stranded AAV vector constructs carrying an expression cassette containing the EGFP reporter gene flanked by either ITR2 or ITR3. Plasmid transfections of the ITR2- or the ITR3-containing expression cassettes into cultured cells also revealed no difference in the extent of transgene expression. The combination of ITR3 and Rep3 resulted in ~5-fold higher rAAV3 vector titers, as determined by quantitative PCR assays. Next, purified viral stocks of AAV3 vectors were generated and designated as Rep2-ITR2 and Rep3-ITR3, respectively. Interestingly, the transduction efficiency of Rep3-ITR3 AAV3 vectors was ~4-fold higher than that of Rep2-ITR2 AAV3 vectors in a human hepatocellular carcinoma cell line, Huh7, under identical conditions. Additional studies are currently underway to evaluate the efficacy of the Rep2-ITR2 and Rep3-ITR3 vectors in a murine xenograft model in vivo. In summary, our studies document that the combined use of the AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids lead to the production of recombinant AAV3 vectors with higher titers and with higher transduction efficiency, and suggest that the use of homologous ITRs, Rep proteins, and capsids are similarly likely to be applicable to other AAV serotypes vectors for their optimal use in human gene therapy.

Several groups have demonstrated that a number of naturally occurring compounds can significantly enhance the therapeutic efficacy of viral vectors, especially recombinant AAV vectors (Ren et al., Neurosci Lett. 479(3): 187-91, 2010; Zhang et al., Gene Ther. 18(2): 128-34, 2011; Mitchell et al., J Virol. 87(8): 4571-83, 2013; Wang et al., J Integr Med. 12(1): 20-34, 2014). In our recently published studies (Ling et al., Hum Gene Ther. 25(12): 1023-34, 2014), we provided further evidence of enhanced efficacy of a combinatorial approach involving drug-mediated chemotherapy and rAAV vector-mediated cancer gene therapy to target human hepatocellular carcinoma. In these studies, we identified that shikonin, a naphthoquinone, inhibits human liver tumor growth as well as significantly enhances the efficacy of rAAV vectors carrying a therapeutic gene, tricosanthin (TCS), in a murine xenograft model in vivo. Although our initial mechanistic studies suggested that shikonin-mediated enhancement of rAAV vector transduction was through its proteasome inhibitory activity, which was similar to that of MG132, a specific proteasome inhibitor. However, no further increase was observed when cells were co-treated with both shikonin and MG132. Since shikonin is a multifunctional compound, we wished to evaluate its effect on rAAV vector-mediated transgene expression in additional human cell types. In a human erythroleukemia cell line, K562, frequently used as a model for hematopoietic cell transduction studies, shikonin enhanced the transduction efficiency of various rAAV serotype vectors by up to 10-fold. Surprisingly, however, MG132 had no effect in K562 cells, even at the highest concentration that did not induce cytotoxicity. Thus, in a systematic study, various proteasome and protease inhibitors were evaluated. Western blot assays for the detection of polyubiquitinated total cellular proteins were performed to confirm the effectiveness of these drugs in K562 cells. Whereas carfilzomib only had a modest effect (<2-fold enhancement), surprisingly, bortezomib significantly reduced the AAV vector-mediated transgene expression in K562 cells. E-64 and PMSF, a cysteine and a serine protease inhibitor, respectively, also had no effect. Additional studies revealed that a specific inhibitor of the reactive oxygen species (ROS) formation, blocked the enhancing effect of shikonin on rAAV vector-mediated transgene expression in K562 cells but not other cell types. Furthermore, shikonin treatment did augment rAAV vector-mediated transduction ex vivo in both mouse and human primary hematopoietic stem cells. Taken together, our studies also emphasize the need to exercise caution in interpreting rAAV vector-mediated transgene expression data among different cell types involving the use of multifunctional drugs.

OBJECTIVE: Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine（TCM） drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus（r AAV） vectors has not been attempted. METHODS: We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS: The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin（TCS） gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION: Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

OBJECTIVE: In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo.METHODS: Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time.RESULTS: We observed that treatment with pristimerin, at as low as 1 mu mol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg center dot d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes.CONCLUSION: These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.