肿瘤细胞周期测定方法研究

肿瘤是一类与细胞周期密切相关的疾病。流式细胞术对肿瘤细胞周期的分析可以观察到肿瘤细胞倍体的变化,有助于肿瘤的早期发现和治疗。区分肿瘤细胞所处于的细胞周期,明确地了解每个时相所占细胞周期的比例,对于肿瘤药物的研发具有重要意义。该文着重对G₀期和G₁期,G₂期和M期,以及S期细胞的测定方法进行归纳、总结,并对这些方法的优缺点及适用条件进行阐述和总结,为肿瘤药物的研发及基础研究等方面的科研工作提供参考。     

Cell cycle arrest through inhibition of tubulin polymerization by withaphysalin F,a bioactive compound isolated from <Emphasis Type="Italic">Acnistus arborescens</Emphasis>

Many compounds used in the treatment of cancer possess tubulin-interacting properties that lead to mitotic arrest. Withaphysalins are potent cytotoxic compounds that are commonly found in plants belonging to the Solanaceae family, such as Acnistus arborescens; however, the cytotoxic mechanisms or molecular targets of these compounds remain unknown. Thus, the aim of this study was to evaluate the effects of whitaphysalins on cancer cell cycle progression and tubulin interaction. In this report, we show the antiproliferative activity of withaphysalin F and its effect in arresting cells in the G₂/M phase of the cell cycle. These two effects are the result of the interference of withaphysalin F in the polymerization of microtubules. Withaphysalin F also induced DNA fragmentation, which can be related to an increase in mitochondrial membrane depolarization. These results suggest that interference of withaphysalin F in microtubule polymerization may induce cell cycle arrest in the G₂/M phase and therefore contribute to growth inhibition of tumor cells in vitro. Taken together, these studies indicate that withaphysalin F could potentially be used as an anticancer drug.     

Recent trends in cancer drug resistance reversal strategies using nanoparticles

Introduction: Resistance to chemotherapy is a major obstacle in the successful amelioration of tumors in many cancer patients. Resistance is either intrinsic or acquired, involving mechanisms such as genetic aberrations, decreased influx and increased efflux of drugs. Strategies for the reversal of resistance involve the alteration of enzymes responsible for drug resistance, the modulation of proteins regulating apoptosis mechanisms and improving the uptake of drugs using nanotechnology. Novel strides in the reversal of drug resistance are emerging, involving the use of nanotechnology, targeting stem cells, etc.

Areas covered: This paper reviews the most recent cancer drug reversal strategies involving nanotechnology for targeting cancer cells and cancer stem cells (CSCs), for enhanced uptake of micro- and macromolecular inhibitors.

Expert opinion: Nanotechnology used in conjunction with existing therapies, such as gene therapy and P-glycoprotein inhibition, has been shown to improve the reversal of drug resistance; the mechanisms involved in this include specific targeting of drugs and nucleotide therapeutics, enhanced cellular uptake of drugs and improved bioavailability of drugs with poor physicochemical characteristics. Important strategies in the reversal of drug resistance include: a multifunctional nanoparticulate system housing a targeting moiety; therapeutics to kill resistant cancer cells and CSCs; cytotoxic drugs and a tumor microenvironment stimuli-responsive element, to release the encapsulated therapeutics.     

In vitro screening of synergistic ascorbate–drug combinations for the treatment of malignant mesothelioma

Malignant mesothelioma (MMe) is a lethal tumor arising from the mesothelium of serous cavities as a result of exposure to asbestos. Current clinical standards consist of combined treatments, but an effective therapy has not been established yet and there is an urgent need for new curative approaches. Ascorbate is a nutrient that is also known as a remedy in the treatment of cancer. In the present study, we have tested the cytotoxicity of ascorbate to MMe cells in combination with drugs used in MMe therapy, such as cisplatin, etoposide, gemcitabine, imatinib, paclitaxel, and raltitrexed, as well as with promising antitumor compounds like taurolidine, α-tocopherol succinate, and epigallocatechin-3-gallate (EGCG). Dose–response curves obtained for each compound by applying the neutral red uptake (NRU) assay to MMe cells growing in vitro, allowed to obtain IC₅₀ values for each compound used singularly. Thereafter, NRU data obtained from each ascorbate/drug combination were analyzed through Tallarida’s isobolograms at the IC₅₀ level (Tallarida, 2000), revealing synergistic interactions for ascorbate/gemcitabine and ascorbate/EGCG. These results were further confirmed through comparisons between theoretical additivity IC₅₀ and observed IC₅₀ from fixed-ratio dose–response curves, and over a broad range of IC levels, by using Chou and Talalay’s combination index (Chou and Talalay, 1984). Synergistic interactions were also shown by examining apoptosis and necrosis rates, using the caspase 3 and lactic dehydrogenase assays, respectively. Hence, data indicate that ascorbate/gemcitabine and ascorbate/EGCG affect synergistically the viability of MMe cells and suggest their possible use in the clinical treatment of this problematic cancer.     

Nanoparticle albumin bound-paclitaxel for treatment of advanced non-small cell lung cancer: an evaluation of the clinical evidence

Introduction: Nanoparticle albumin-bound paclitaxel (nab-paclitaxel), a microtubule inhibitor, has demonstrated clinical efficacy in the treatment of advanced non-small cell lung cancer (NSCLC) either as monotherapy or in combination. Nab-paclitaxel was developed to reduce the toxicities associated with solvent-bound paclitaxel (sb-paclitaxel).

Areas covered: This review first focuses on the clinical trials evaluating the efficacy and tolerability of nab-paclitaxel in NSCLC at different settings. The approval of nab-paclitaxel in combination with carboplatin at the front-line setting for advanced NSCLC was based on the key phase III study, which showed that nab-paclitaxel/carboplatin was associated with superior overall response rate and favorable toxicity profile compared to sb-paclitaxel/carboplatin. The review also addresses the nab-paclitaxel pharmacology, other combinations (e.g. immunotherapy with PD-1/PD-L1 inhibitors), potential biomarkers (e.g. caveolin-1), and special subgroups (e.g. the elderly, squamous histology).

Expert opinion: Existing data has established the role of nab-paclitaxel in the management of advanced NSCLC. Emerging evidence, such as preliminary results from Keynote-407 and IMpower 131 studies, indicates that novel combinations of nab-paclitaxel/carboplatin and PD-1/PD-L1 inhibitors could further improve clinical benefits with manageable toxicity. Nevertheless, in order to better position nab-paclitaxel and to improve patient selection, future studies are warranted to further understand its mechanism of action, predictive biomarkers, and potential synergism with other agents.     

Novel histone deacetylase inhibitors bearing a 4-piperidin-4-yl-triazole scaffold as antitumor agents

Histone deacetylases have proven to be promising targets for the development of anticancer drugs. In this work, we reported the design and synthesis of a series of 20 novel hydroxamic acid-based histone deacetylase inhibitors with 4-piperidin-4-yl-triazole as the core structure. Five newly obtained compounds displayed excellent HDAC6 inhibitory activities. Among them, compounds WY-12 and WY-15 also exhibited excellent antiproliferative activities against six human tumor cell lines. WY-15 could increase the level of acetylated histone H3 in a dose-dependent manner. Furthermore, WY-15 remarkably induced cell cycle arrest of Sy5y cancer cells in G₀/G₁ phase. Finally, the high potency of compound WY-15 toward HDAC6 was rationalized by molecular docking study.     

Triphendiol (NV-196), development of a novel therapy for pancreatic cancer

Despite incremental progress in the treatment of pancreatic adenocarcinoma, the prognosis of patients remains poor. Here, we report the preclinical studies in pancreatic cancer cells that demonstrate the efficacy of triphendiol (NV-196, a synthetic isoflavene) both as a monotherapy and as a gemcitabine sensitizer. The in-vitro effects of triphendiol on the pancreatic cancer cell lines HPAC and MIAPaCa-2 were determined using cell proliferation, flow cytometry, and western blot analysis. The antiproliferative activity of triphendiol was also investigated in two xenograft models of pancreatic cancer (HPAC and MIAPaCa-2). As a monotherapy, triphendiol-inhibited cell proliferation-induced p53-independent G2/M cell cycle arrest and activation of the intrinsic (mitochondrial) apoptosis pathway. Triphendiol-induced apoptosis was caspase independent and death receptor independent, whereas cell necrosis was caspase mediated. Using combination index analysis, we have shown that pretreatment of pancreatic cancer cells with triphendiol enhanced the cytotoxic effect of gemcitabine, the standard of care used to treat advanced pancreatic cancer. In xenograft models of pancreatic cancer, the rate of tumor proliferation on mice coadministered with triphendiol and gemcitabine was significantly reduced when compared with the corresponding tumor proliferation rates from the respective monotherapy-control and vehicle-control groups. Triphendiol was recently granted Investigational New Drug status by the US Food and Drug Administration. These data justify the commencement of clinical studies investigating the utility of combining triphendiol and gemcitabine in patients with early-stage and late-stage pancreatic cancer.     

吉西他滨的药效学与药动学及其节拍化疗研究进展

目的综述吉西他滨的药效动力学(简称药效学)与药物代谢动力学(简称药动学)及其在节拍化疗中的研究进展,为临床制定安全有效的给药方案提供借鉴。方法以"gemcitabine""pharmacodynamics""pharmacokinetics""metronomic chemotherapy"为关键词,查询PubMed及Web of Science等数据库中收录的相关文献,并从药效学作用、药动学过程、节拍化疗中的研究进展3个方面进行综述。结果与结论吉西他滨属胞嘧啶核苷类抗肿瘤药物,用于多种实体肿瘤尤其是胰腺癌与肺癌的治疗。吉西他滨是最早应用于节拍化疗的抗肿瘤药物之一,单用或与顺铂、紫杉醇等多种细胞毒类药物联用化疗效果良好,临床疗效显著。     

Albumin-bound Paclitaxel: in metastatic breast cancer

A new formulation of paclitaxel, 130-nanometre albumin-bound paclitaxel (nab-paclitaxel), solubilises hydrophobic paclitaxel and may increase paclitaxel delivery to tumour cells. Intravenous nab-paclitaxel 260 mg/m(2) had a higher maximum whole-blood concentration, shorter time to peak concentration, larger distribution volume and greater clearance than a 175 mg/m(2) dose of a conventional polyoxyethylated castor oil (Cremophor EL) solublised paclitaxel (CrEL-paclitaxel). The reconciled target-lesion response rate was significantly higher in patients receiving intravenous nab-paclitaxel 260 mg/m(2) once every 3 weeks than in those receiving CrEL-paclitaxel 175 mg/m(2) once every 3 weeks (21.5% vs 11.1%) in a randomised, nonblind, phase III trial in 454 patients with metastatic breast cancer. The objective response rate (ORR) was also significantly greater in nab-paclitaxel than in CrEL-paclitaxel recipients (33% vs 19%). In noncomparative phase II trials, ORRs of 48% and 51% were observed in patients receiving nab-paclitaxel 175 or 300 mg/m(2) once every 3 weeks. nab-Paclitaxel 260 mg/m(2) caused less grade 4 neutropenia than CrEL-paclitaxel 175 mg/m(2). The incidence of grade 3 sensory neuropathy was higher in nab-paclitaxel recipients, reflecting the higher dosage of nab-paclitaxel, and improved with treatment interruption. Despite the absence of corticosteroid and antihistamine premedication, no severe hypersensitivity reactions were reported.     

吉西他滨对胰腺癌外周血调节性T细胞影响的研究

目的 探讨吉西他滨对胰腺癌外周血调节性T细胞的影响,为进一步提高过继免疫治疗的疗效提供依据和参考。方法 本研究入选2012年1月至2014年10月收治的32例胰腺癌患者,分为吉西他滨组(吉西他滨+过继免疫治疗)和对照组(单纯过继免疫治疗),观察两组患者治疗前后外周血调节性T细胞水平、化疗不良反应,并对两组患者的生存时间进行分析比较。结果 吉西他滨组患者治疗后外周血调节性T细胞水平较治疗前显著降低,与对照组相比亦显著降低,两组比较有统计学差异(P<0.05),吉西他滨组中位生存时间较对照组延长1.3个月(10.0个月与8.7个月比较)。结论 吉西他滨化疗可以显著降低胰腺癌患者外周血调节性T细胞水平,有效调节患者的肿瘤免疫耐受,提高过继免疫治疗的疗效。     

Emerging protein kinase inhibitors for treating pancreatic cancer

Introduction: Pancreatic cancer, the incidence and mortality of which are increasing around the world, has the most dismal prognosis among the commonly encountered cancers. Systemic chemotherapy plays an important role in the treatment of patients with pancreatic cancer, and development of more effective chemotherapies is being sought.

Areas covered: This review article provides a summary about protein kinase inhibitors that have been investigated for the treatment of pancreatic cancer, not only existing agents targeting RAS, EGFR, VEGFR, MEK, etc., but also various compounds targeting, including the MAPK, PI3 K/Akt/mTOR, and JAK/STAT signaling pathways, trials of which are currently ongoing. To date, none has shown sufficient efficacy as to merit becoming established as a standard treatment agent for pancreatic cancer.

Expert opinion: As the toxicities of protein kinase inhibitors usually differ from those of cytotoxic agents, it could be of value to use these agents in combination with gemcitabine plus nab-paclitaxel. It may be reasonable to identify a suitable disease and/or predictive markers for new compounds in proof of concept trials. It is an urgent need to conduct phase III trials, on the basis of the results obtained, in subpopulations with biomarkers to predict the efficacy of these drugs.     

Development of nanoliposomal irinotecan (nal-IRI,MM-398, PEP02) in the management of metastatic pancreatic cancer

Introduction: Systemic chemotherapy remains the standard of care for patients with advanced pancreatic ductal adenocarcinoma (PDAC). The introductions of FOLFIRINOX and nab-paclitaxel/gemcitabine combinations have improved the first-line treatment outcomes of patients with metastatic PDAC; while second-line therapy options are limited. Based on the results of pivotal NAPOLI-1 study, nanoliposomal irinotecan (nal-IRI) plus 5-fluorouracil and leucovorin (5-FU/LV) became the first US Food and Drug Administration (FDA) approved regimen for patients with metastatic PDAC with previous gemcitabine-based chemotherapy in November 2015.

Areas covered: We reviewed and summarized the rationale, pharmacokinetics, therapeutic efficacy and adverse events of nal-IRI alone or combined with 5-FU/LV for metastatic PDAC with previous gemcitabine-based chemotherapy.

Expert opinion: In the NAPOLI study, nal-IRI plus 5-FU/LV significantly improved the overall survival, progression-free survival and objective response rate compared to 5-FU/LV alone. The nal-IRI plus 5-FU/LV treatment was associated with a manageable toxicity profile and comparable outcomes in patients with negative demographic characteristics. The relatively sparse of neurotoxicity makes nal-IRI plus 5-FU/LV as a more favorable option than oxaliplatin-containing regimens and the current recommended standard treatment for patients with metastatic PDAC after frontline nab-paclitaxel/gemcitabine treatment. The front-line therapeutic role of nal-IRI is currently under investigation.     

Cell-cycle analysis and micronuclei frequency reveals G0/G1 blockers as weak micronuclei inducers

Micronuclei (MN) formation is generally attributed to error in DNA synthesis or mitosis, which are represented by the S or G₂/M phase respectively, in the cell-cycle histogram. Interestingly, many of the known anticancer drugs target these cell-cycle phases to elicit cytotoxicity. Here, we attempted to identify whether any correlation exists between the cell-cycle effect and MN induction potential using various treatments. In addition, we tracked down MN in cycling cells to assess its final fate. We treated SiHa cells with various known drugs and correlated their effects on cell-cycle and MN frequency. MN-tracking studies were performed in peripheral mononuclear and siHa cells upon staining with Giemsa and ethidium bromide respectively. We observed MN induction by all the tested drugs irrespective of their basic effect on cell cycle. However, MN induction was more with drugs which interfere with the S or G₂/M than the G₀/G₁ phase. Our results indicate G₀/G₁ blockers to be comparatively safer drugs. Additionally, our results show that expulsion out of cells may be one of the main fates of drug-induced MN.     

Overcoming drug resistance in pancreatic cancer

Introduction: Pancreatic cancer has the worst survival rate of all cancers. The current standard care for metastatic pancreatic cancer is gemcitabine, however, the success of this treatment is poor and overall survival has not improved for decades. Drug resistance (both intrinsic and acquired) is thought to be a major reason for the limited benefit of most pancreatic cancer therapies.

Areas covered: Previous studies have indicated various mechanisms of drug resistance in pancreatic cancer, including changes in individual genes or signaling pathways, the influence of the tumor microenvironment, and the presence of highly resistant stem cells. This review summarizes recent advances in the mechanisms of drug resistance in pancreatic cancer and potential strategies to overcome this.

Expert opinion: Increasing drug delivery efficiency and decreasing drug resistance is the current aim in pancreatic cancer treatment, and will also benefit the treatment of other cancers. Understanding the molecular and cellular basis of drug resistance in pancreatic cancer will lead to the development of novel therapeutic strategies with the potential to sensitize pancreatic cancer to chemotherapy, and to increase the efficacy of current treatments in a wide variety of human cancers.     

Enhanced antiproliferative activity of Herceptin (HER2)-conjugated gemcitabine-loaded chitosan nanoparticle in pancreatic cancer therapy

Currently, effective drug delivery in pancreatic cancer treatment is a major challenge. Nanomedicine plays an essential role by delivering anticancer drugs in a targeted manner to the malignant tumor cells, leading to increased efficacy by reducing the toxicity of anticancer drugs to normal, sensitive sites. This study investigated the preparation and characterization of a targeted system represented by Herceptin-conjugated gemcitabine-loaded chitosan nanoparticles (HER2-Gem-CS-NPs) for pancreatic cancer therapy. The targeted NPs displayed superior antiproliferative activity along with an enhanced S-phase arrest, leading to apoptosis in comparison with unconjugated gemcitabine-loaded nanoparticles and free gemcitabine due to higher cellular binding with eventual uptake and prolonged intracellular retention. Thus, HER2-Gem-CS-NPs are able to provide an efficient and targeted delivery of gemcitabine for pancreatic cancer treatment. FROM THE CLINICAL EDITOR: This study investigated the preparation and characterization of a targeted drug delivery system consisting of Herceptin-conjugated gemcitabine-loaded chitosan nanoparticles for pancreatic cancer therapy.     

小白菊内酯抑制紫杉醇诱导的肿瘤细胞凋亡

目的　探讨NF κB在紫杉醇诱导肿瘤细胞凋亡中的作用及小白菊内酯对紫杉醇诱导凋亡的影响。方法　以人乳癌BCap37细胞和人表皮KB细胞为研究对象 ,用 5,1 0和 2 0 μmol·L-1 小白菊内酯预处理细胞 ,以DNA凋亡梯状条带、DNA含量、MTT、细胞甩片及电泳迁移率变动分析 (EMSA)法检测它对紫杉醇诱导细胞凋亡的影响并探索其作用靶点。结果　通过检测DNA凋亡梯状条带、DNA含量和细胞生存率 ,2 0μmol·L-1 小白菊内酯能显著抑制紫杉醇所诱导的BCap37和KB细胞凋亡 ,但不影响紫杉醇诱导肿瘤细胞G2 /M期阻滞。EMSA实验表明小白菊内酯能抑制紫杉醇诱导激活NF κB。结论　小白菊内酯通过抑制NF κB的激活来抑制紫杉醇所诱导的肿瘤细胞凋亡 ,而紫杉醇诱导肿瘤细胞凋亡的过程可能与G2 /M期阻滞无关     

Adenoviral-mediated overexpression of human equilibrative nucleoside transporter 1 (hENT1) enhances gemcitabine response in human pancreatic cancer

Nucleoside-derived anticancer agents must be transported across the plasma membrane as a preliminary step to their conversion into active drugs. Hence, modulation of a specific nucleoside transporter may affect bioavailability and contribute significantly to sensitizing tumor cells to these anticancer agents. We have generated and functionally characterized a new recombinant adenovirus (Ad-hENT1) that has allowed us to overexpress the equilibrative nucleoside transporter hENT1 and to analyze its effects in human pancreatic tumor cells. Overexpression of hENT1 is associated with changes in cell cycle profile, in a variable manner depending on the particular cell type, thus suggesting a metabolic link between hENT1-mediated transport processes and the enzymatic machinery responsible for intracellular nucleoside metabolism. When assayed in vivo in a human pancreatic adenocarcinoma xenograft, intratumoral Ad-hENT1 injection improved the therapeutic response to gemcitabine. In summary, hENT1 overexpression is associated with alterations in nucleoside enzymatic machinery and cell cycle progression in cultured cells and enhances gemcitabine action in vivo.     

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization,induces G2/M arrest,and triggers apoptosis in human leukemia HL-60 cells

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC₅₀ values in the nanomolar range. Cell cycle arrest in G₂/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G₂/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential.