Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

Clinico-pathological studies on the effect of different anti-neoplastic chemotherapy regimens on transmissible venereal tumours in dogs

Thirty-two dogs affected with transmissible veneral tumour (TVT) were divided into three treatment groups. In group I vincristine sulphate at 0.025 mg/kg body weight, in group II vinblastine sulphate at 0.150 mg/kg body weight, and in group III vinblastine sulphate at 0.100 mg/kg body weight plus methotrexate at 0.35 mg/kg body weight were given intravenously at weekly intervals. Biopsies were performed on days 0, 3, 7 and 14. The tissues were preserved in 10% neutral buffered formalin and processed routinely for haematoxylin and eosin staining. Histopathologically, the untreated TVT was characterized by sheets or bundles of mostly rounded cells having a large, highly basophilic nucleus with a prominent, highly basophilic nucleolus. Both vincristine and vinblastine primarily affected the nuclei of neoplastic cells, causing condensation, karyorrhexis and karyolysis within 3 days of chemotherapy. The regressing tumour mass showed marked infiltration by lymphocytes, lymphoblasts and macrophages by day 7. There was nearly complete regression of the tumour by day 14, as shown by the almost complete loss of neoplastic cells, with fibrous tissue substitution. However, in group III, the changes occurred more slowly and more injections were needed for complete regression. In both groups I and II, 11/12 of the animals responded completely to the chemotherapy within 3 weeks, while in group III, 6/8 of the dogs responded to the treatment by 21–28 days.Abbreviations SD standard deviation - TVT transmissible venereal tumour     

Fasting reduces the incidence of vincristine‐associated adverse events in dogs

Fasting has been shown to decrease chemotherapy‐associated adverse events (AEs), in part through insulin‐like growth factor (IGF‐1) reduction, and may induce a protective effect on normal cells during chemotherapy treatment in mice and people. The purpose of this study was to evaluate the effect of fasting on constitutional, bone marrow and gastrointestinal (GI) AEs, and serum glucose, IGF‐1 and insulin levels in dogs receiving vincristine. The study was a prospective, crossover clinical trial in tumour‐bearing dogs. Dogs were randomized to be fasted for 24 to 28 hours prior to and 6 hours following their first or second vincristine treatment, and fed normally for the alternate dose. A significant reduction in nausea, anorexia, lethargy and serum insulin was observed when dogs were fasted; however, no significant differences were found in other GI symptoms, neutrophil count, serum glucose or IGF‐1. Fasting prior to vincristine therapy is a safe and effective treatment modality that helped mitigate constitutional and GI AEs in tumour‐bearing dogs.     

P‐Glycoprotein Mediated Drug Interactions in Animals and Humans with Cancer

Drug–drug interactions can cause unanticipated patient morbidity and mortality. The consequences of drug–drug interactions can be especially severe when anticancer drugs are involved because of their narrow therapeutic index. Veterinary clinicians have traditionally been taught that drug–drug interactions result from alterations in drug metabolism, renal excretion or protein binding. More recently, drug–drug interactions resulting from inhibition of P‐glycoprotein‐mediated drug transport have been identified in both human and veterinary patients. Many drugs commonly used in veterinary patients are capable of inhibiting P‐glycoprotein function and thereby causing an interaction that results in severe chemotherapeutic drug toxicity. The intent of this review is to describe the mechanism and clinical implications of drug–drug interactions involving P‐glycoprotein and anticancer drugs. Equipped with this information, veterinarians can prevent serious drug–drug interactions by selecting alternate drugs or adjusting the dose of interacting drugs.     

Clinical Pharmacokinetics and Effects of Vincristine Sulfate in Dogs with Transmissible Venereal Tumor (TVT)

This study was conducted to evaluate the pharmacokinetic characteristics of vincristine and their correlation with its clinical effects in dogs with transmissible venereal tumor (TVT). Dogs with TVT were intravenously administered vincristine sulfate at a dose of 0.7 mg/m² of body surface area. Blood samples were collected starting from 5 min to 48 hr after drug administration. The plasma concentration of vincristine was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters of vincristine were characterized using a two-compartmental pharmacokinetic model. The volume of distribution, distribution half-life, elimination half-life and plasma clearance were 0.660 ± 0.210 l/kg, 21.5 ± 6.90 min, 47.6 ± 14.2 min and 0.010 ± 0.001 l/min/kg, respectively. Tumor regression was determined at weekly interval by a physical examination and histopathological analysis. In our study, three to eight administrations of vincristine at a dose of 0.7 mg/m² were able to induce a complete tumor regression without any evidence of gross lesion of disease. Therefore, this investigation provides the pharmacokinetic characteristics of vincristine in dogs with TVT, which may be used as an integration tool to gain a better understanding of the disposition properties of the drug and the correlation of these properties with the drug’s clinical effects. In addition, we validated the LC-MS/MS method and found that it is suitable for the pharmacokinetic study of vincristine in dog plasma.     

Molecular characterization of canine BCR‐ABL–positive chronic myelomonocytic leukemia before and after chemotherapy

Genetic aberrations linked to tumorigenesis have been identified in both canine and human hematopoietic malignancies. While the response of human patients to cancer treatments is often evaluated using cytogenetic techniques, this approach has not been used for dogs with comparable neoplasias. The aim of this study was to demonstrate the applicability of cytogenetic techniques to evaluate the cytogenetic response of canine leukemia to chemotherapy. Cytology and flow cytometric techniques were used to diagnose chronic myelomonocytic leukemia in a dog. High‐resolution oligonucleotide array comparative genomic hybridization (oaCGH) and multicolor fluorescence in situ hybridization (FISH) were performed to identify and characterize DNA copy number aberrations (CNAs) and targeted structural chromosome aberrations in peripheral blood WBC at the time of diagnosis and following one week of chemotherapy. At the time of diagnosis, oaCGH indicated the presence of 22 distinct CNAs, of which trisomy of dog chromosome 7 (CFA 7) was the most evident. FISH analysis revealed that this CNA was present in 42% of leukemic cells; in addition, a breakpoint cluster region‐Abelson murine leukemia viral oncogene homolog (BCR‐ABL) translocation was evident in 17.3% of cells. After one week of treatment, the percentage of cells affected by trisomy of CFA7 and BCR‐ABL translocation was reduced to 2% and 3.3%, respectively. Chromosome aberrations in canine leukemic cells may be monitored by molecular cytogenetic techniques to demonstrate cytogenetic remission following treatment. Further understanding of the genetic aberrations involved in canine leukemia may be crucial to improve treatment protocols.     

The morphological effects of vincristine sulfate on canine bone marrow cells

This study documents the morphologic changes observed in the bone marrow aspirate biopsies from dogs 6 and 24 hours after receiving a single therapeutic dose (0.025 mg/kg) of vincristine sulfate (Oncovin: Eli Lilly & Co., Indianapolis, Ind.) intravenously. The most striking cytologic changes were observed in the erythroid cell line. Abnormalities included increased numbers of mitotic figures, abnormal nuclear configurations, and fragmented nuclei. Erythroid cells in metaphase were prominent in marrow samples collected 6 hours post-vincristine, accounting for a mean of 27% of all erythroid precursors. Fragmented nuclei and atypical nuclear configurations were seen in low numbers (mean = 7%) of erythroid cells from these animals. In contrast, marrow collected from dogs 24 hours post-vincristine exhibited low numbers (mean = 1%) of erythroid cells in metaphase, but erythroid cells with atypical nuclear configurations and fragmented nuctei accounted for a mean of 41% of the erythroid cells present. Less dramatic increases in the number of mitotic non-erythroid cells were seen 6 hours post-vincristine (mean = 5% of non-erythroid cells) and 24 hours post-vincristine (mean = 1% of non-erythroid cells). Only rare nuclear fragmentation was observed in these cell lines. Significant alterations in megakaryocytes and myeloid to erythroid (M:E) ratios were not observed in samples taken 6 hours post-vincristine; however, M:E ratios were considerably higher in three of the four samples taken from dogs 24 hours post-vincristine. Similar time-related changes were observed in four clinical cases in which bone marrow aspirates were performed after vincristine administration.     

长春新碱对天祝白牦牛胚胎成纤维细胞的作用

目的：探讨长春新碱对天祝白牦牛胚胎成纤维细胞生长的抑制作用,并用碱性磷酸酶指标判断天祝白牦牛胚胎成纤维细胞在长春新碱作用下去分化的可能性。方法：选取白牦牛胚胎成纤维细胞生长良好的24孔培养板,分别滴加浓度依次为0.1、0.5、1.0、1.5、2.0、2.5μg/mL长春新碱,每天在倒置相差显微镜下观察不同浓度的长春新碱对细胞形态的作用,并进行碱性磷酸酶染色检测。结果：适量浓度的长春新碱可以使细胞的形态发生变化（细胞变圆）,而且在去除长春新碱后,细胞能正常生长。高浓度的长春新碱会使细胞死亡崩解。长春新碱并不能使白牦牛胚胎成纤维细胞中的碱性磷酸酶浓度增高。结论：长春新碱浓度为0.5μg/mL时,对白牦牛胚胎成纤维细胞的抑制作用最佳,但是它并不能使细胞中碱性磷酸酶的含量增高。     

含硒化合物联合医用长春新碱应用对艾氏腹水癌的抑制机制

为寻找医治艾氏腹水癌的新型制剂,并评价药效,对小鼠腹腔注射肿瘤细胞,建立荷艾氏腹水癌细胞(EAC)的小鼠模型;并于接种后第7天开始用抗癌药,观察荷瘤小鼠(接种EAC后产生血性腹水的小鼠)的生存周期,通过对动物体内抗氧化水平指标(SOD、GSH、MDA)的检测,判定硒化合物的保护作用;用流式细胞术及对细胞进行DNA梯检测,评估药物对肿瘤细胞调亡的影响。结果表明含硒化合物,尤其是有机硒化合物,甲基硒代半胱氨酸(SeMC)与抗癌药长春新碱联合应用可使荷瘤小鼠的生存期延长、机体的抗氧化水平升高及肿瘤细胞凋亡率增多,较对照组有显著差异(P<0.01)。研究认为,含硒化合物与医用长春新碱联用对荷瘤小鼠的保护性,主要是出于硒的抗氧化能力,以及使长春新碱促进肿瘤细胞的凋亡来实现的。