Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

USP15 plays an essential role for caspase-3 activation during Paclitaxel-induced apoptosis

Paclitaxel (also known as Taxol) is a well-known anticancer agent that blocks cell mitosis and kills tumor cells, and is often used in clinic to treat cancers. Despite the success of Paclitaxel, the development of drug resistance prevents its clinical applicability. Here, we screened an siRNA library against the entire human genomes using HeLa cells, and have find that lack of USP15 (ubiquitin-specific protease 15) causes Paclitaxel resistance. We also observed the decreased expression of USP15 in Paclitaxel-resistant human ovarian cancer samples. In addition, we have demonstrated that USP15 plays an essential role for stability and activity of caspase-3 during Paclitaxel-induced apoptosis. Thus, USP15 may be a candidate diagnostic marker and therapeutic target for Paclitaxel-resistant cancers.     

Molecular evolution of paclitaxel biosynthetic genes <Emphasis Type="Italic">TS</Emphasis> and <Emphasis Type="Italic">DBAT</Emphasis> of <Emphasis Type="Italic">Taxus</Emphasis> species

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.     

Paclitaxel may inhibit autoimmune diseases by sparing or increasing the number of CD4(+) CD25(+) regulatory T cells

Paclitaxel, a representative of taxanes, exhibits cytotoxic effects against a broad range of tumors. Strikingly, an emerging body of data suggests that paclitaxel also exerts effects on immune system by stimulating anti-tumor and anti-autoimmunity effects, supporting the idea that paclitaxel suppresses tumor through several mechanisms and not solely through inhibiting cell division. Based on the accumulating data, we hypothesized that paclitaxel may inhibit autoimmune diseases by sparing or actively increasing the number of CD4(+) CD25(+) Treg cells. The hypothesis, if proved to be correct, will significantly improve our understanding of the tumor immunity, autoimmunity and its related pathological effects. It will influence our choice on immunosuppressive drugs for cancer patients with autoimmune diseases. It will also impact the immunotherapy for tumors.     

Human sperm cryopreservation in cancer patients: Links with deprivation and mortality

Evidence is mounting for a relationship between human semen quality and environmental/lifestyle/socioeconomic factors including long term health outcomes such as mortality. The relationship between pre-freeze and post-thaw semen quality in cancer patients and these factors are unknown. Frozen semen from 217 cancer patients was thawed and analysed using a validated CASA method. Post-thaw quality was matched and compared with WHO semen analysis performed prior to storage. The English Indices of Deprivation 2010 were matched with patients and then examined for relationships with pre-freeze and post-thaw semen quality. There is a relationship between semen quality and deprivation in cancer patients. Compared with pre-freeze semen quality, post-thaw semen quality has a stronger relationship with deprivation. Sperm cryopreservation may have potential as a systemic health diagnostic test and is predictive of cancer patient mortality.     

Adipose-derived stromal cell transplantation for treatment of stress urinary incontinence

We aimed to investigate the application of adipose-derived stromal cells in the treatment of stress urinary incontinence (SUI). Animal models of stress urinary incontinence were established with Sprague-Dawley female rats by complete cutting of the pudendal nerve. Rat adipose-derived stromal cells were isolated, cultured and successfully transplanted into animal models. Effects of stem cell transplantation were evaluated through urodynamic testing and morphologic changes of the urethra and surrounding tissues before and after transplantation. Main urodynamic outcome measures were measured. Intra-bladder pressure and leak point pressure were measured during filling phase. Morphologic examinations were performed. Transplantation of adipose-derived stem cells significantly strengthened local urethral muscle layers and significantly improved the morphology and function of sphincters. Urodynamic testing showed significant improvements in maximum bladder capacity, abdominal leak point pressure, maximum urethral closure pressure, and functional urethral length. Morphologic changes and significant improvement in urination control were consistent over time. It was concluded that periurethral injection of adipose-derived stromal cells improves function of the striated urethral sphincter, resulting in therapeutic effects on SUI. Reconstruction of the pelvic floor through transplantation of adipose-derived cells is a minimally invasive and effective treatment for SUI.     

Salvianolic acid A reverses paclitaxel resistance in human breast cancer MCF-7 cells via targeting the expression of transgelin 2 and attenuating PI3 K/Akt pathway

Chemotherapy resistance represents a major problem for the treatment of patients with breast cancer and greatly restricts the use of first-line chemotherapeutics paclitaxel. The purpose of this study was to investigate the role of transgelin 2 in human breast cancer paclitaxel resistance cell line (MCF-7/PTX) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) indicated that transgelin 2 may mediate paclitaxel resistance by activating the phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway to suppress MCF-7/PTX cells apoptosis. The reversal ability of SAA was confirmed by MTT assay and flow cytometry, with a superior 9.1-fold reversal index and enhancement of the apoptotic cytotoxicity induced by paclitaxel. In addition, SAA effectively prevented transgelin 2 and adenosine-triphosphate binding cassette transporter (ABC transporter) including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) up-regulation and exhibited inhibitory effect on PI3 K/Akt signaling pathway in MCF-7/PTX cells. Taken together, SAA can reverse paclitaxel resistance through suppressing transgelin 2 expression by mechanisms involving attenuation of PI3 K/Akt pathway activation and ABC transporter up-regulation. These results not only provide insight into the potential application of SAA in reversing paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer.     

Improved fractional precipitation method for purification of paclitaxel

This study investigated changing the methanol/water ratio during fractional precipitation of paclitaxel, and adding all the distilled water at room temperature, followed mixing for an additional 10 min. When the methanol/water ratio was 50:50, 40:60, and 30:70 (v/v), the paclitaxel yield was 42.0%, 84.3%, and 92.0%, respectively. When using a methanol/water ratio of 50:50 (v/v), a similar high purity and yield of paclitaxel to the case of storing at a low temperature was achieved when adding all the distilled water at room temperature, followed by additional mixing for 10 min and further mixing at room temperature during fractional precipitation. Thus, additional mixing after adding all the distilled water is confirmed as important during fractional precipitation. Furthermore, the present results show that a high yield of high-purity paclitaxel is possible with additional mixing at room temperature after adding all the distilled water, which is significantly more economical than the existing method of storing at a low temperature for a long time after adding all the distilled water during fractional precipitation.     

紫杉醇联合卡铂治疗卵巢癌的临床疗效及对患者血清CA125、CA199、CEA水平的影响

目的:分析紫杉醇联合卡铂治疗卵巢癌的临床疗效及对患者血清糖类抗原125(CA125)、糖类抗原199(CA199)、癌胚抗原(CEA)水平的影响。方法:选择我院2014年1月～2016年12月收治的41例卵巢癌患者,按随机数字表法分为对照组(n=20)和研究组(n=21)。对照组给予紫杉醇联合顺铂治疗,研究组给予紫杉醇联合卡铂治疗。比较两组临床疗效,治疗前后血清CA125、CA199、CEA水平、卡氏评分的变化,不良反应的发生情况和生存情况。结果:治疗后,研究组总有效率显著高于对照组(P0.05);两组血清CA125、CA199及CEA水平均较治疗前明显下降,且研究组低于对照组(P0.05);研究组卡氏评分改善率高于对照组(P0.05),胃肠道反应、神经毒性损伤、骨髓抑制及血液系统毒性率反应发生率低于对照组(P0.05);两组1年生存率及中位生存期比较差异无统计学意义(P0.05)。结论:紫杉醇联合卡铂治疗卵巢癌的疗效明显优于紫杉醇联合顺铂治疗,其能够降低患者血清CA125、CA199及CEA水平,改善患者生活质量。     

地喹氯铵联合紫杉醇注射液对神经胶质瘤患者血清IL-6, Th17 细胞水平 及临床疗效的影响

目的:探讨地喹氯铵联合紫杉醇注射液对神经胶质瘤患者血清IL-6、Th17细胞水平及临床疗效的影响。方法:回顾性研究我院神经胶质瘤患者60例,根据电脑生成的随机数字表将所有患者随机分为实验组与对照组,每组各30例,对照组患者给予地喹氯铵进行治疗,实验组患者在对照组的基础上给予紫杉醇注射液。比较治疗前后两组患者血清IL-6,Th17细胞水平,并对两组患者不良反应发生率及临床总有效率进行统计。结果:与治疗前相比,两组患者治疗后的血清IL-6、Th17细胞水平均显著降低(P0.05);且与对照组相比,实验组患者血清IL-6、Th17细胞水平均较低(P0.05)。与对照组相比,实验组患者的不良反应发生率较低(P0.05);临床总有效率较高(P0.05)。结论:地喹氯铵联合紫杉醇注射液能够显著提高神经胶质瘤患者临床疗效,降低不良反应发生率,其机制可能与降低血清IL-6及Th17细胞水平有关。