Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy

Cytochrome P450-based gene therapy can substantially increase the sensitivity of tumor cells to P450-activated cancer chemotherapeutic prodrugs such as cyclophosphamide (CPA) without increasing host toxicity. While the role of 4-OH-CPA, the primary active metabolite of CPA, in eliciting tumor cell death is well established, the effect of 4-OH-CPA exposure on the capacity of P450-expressing tumor cells for continued metabolism and activation of CPA has not been investigated. The present study addresses this question and characterizes the impact of CPA dose and treatment schedule on the ability of P450-expressing tumor cells to sustain prodrug activation over time. 9L gliosarcoma cells expressing human P450 2B6 and treated with CPA in a continuous manner exhibited a time- and CPA dose-dependent decrease in P450-catalyzed CPA 4-hydroxylase activity. This decrease reflects a selective, 4-OH-CPA-induced loss of cellular P450 protein content. By contrast, when the P450-expressing tumor cells were treated with CPA as a single 8 hours exposure, cellular CPA 4-hydroxylase activity and P450 protein expression were substantially prolonged when compared to continuous prodrug treatment. This schedule-dependent effect of CPA was influenced by the level of P450 protein expressed in the tumor cells. At high P450 protein and activity levels, which could be achieved by culturing the tumor cells at high cell density, net production and release of 4-OH-CPA into the culture media was increased substantially. This increase fully offset the decline in CPA 4-hydroxylase activity as the tumor cells underwent CPA-induced apoptotic death. These findings demonstrate the impact of CPA dose and treatment schedule on the efficacy of P450 gene-directed enzyme prodrug therapy, with bolus CPA treatment being compatible with sustained expression of P450 protein and maintenance of P450-dependent prodrug activation by the target tumor tissue.     

Enhanced bystander cytotoxicity of P450 gene-directed enzyme prodrug therapy by expression of the antiapoptotic factor p35

Cytochrome P450 gene-directed enzyme prodrug therapy substantially augments intratumoral activation of anticancer prodrugs, such as cyclophosphamide (CPA), leading to a strong increase in antitumor effect without a corresponding increase in host toxicity. Attempts to additionally increase tumor cell kill by enhancing the intrinsic chemosensitivity of P450-expressing tumor cells by chemical means (depletion of cellular glutathione) or by coexpression of proapoptotic factors was shown to result in the desired increase in chemosensitivity, but with a decrease in net production of bystander cytotoxic drug metabolites because of accelerated death of the prodrug-activating tumor cells. Moreover, tumor cell P450 activity declined during the course of apoptosis induced by P450-activated CPA, limiting the potential of the tumor cell for continued production of activated drug metabolites. This limitation could be overcome by retroviral delivery of the baculovirus-encoded caspase inhibitor p35 to P450-expressing tumor cells. p35 substantially prolonged the activation of CPA by P450 "factory cells," leading to an increase in their bystander cytotoxicity toward P450-deficient tumor cells. This effect was greatest in tumor cells treated with CPA for an 8-h period, a schedule designed to model the effective time period of drug exposure in bolus CPA-treated patients in vivo. Notably, retroviral transduction of tumor cells with p35 did not induce drug resistance, as shown by the absence of long-term tumor cell survival or detectable colony formation activity after CPA treatment. These findings demonstrate that antiapoptotic factors, such as p35, can be used in a novel manner to enhance prodrug activation gene therapy by delaying tumor cell death, thereby increasing the net production of bystander cytotoxic metabolites and, hence, the overall effectiveness of the anticancer strategy.     

Combination of the bioreductive drug tirapazamine with the chemotherapeutic prodrug cyclophosphamide for P450/P450-reductase-based cancer gene therapy

Tirapazamine (TPZ) is a bioreductive drug that exhibits greatly enhanced cytotoxicity in hypoxic tumor cells, which are frequently radiation-resistant and chemoresistant. TPZ exhibits particularly good activity when combined with alkylating agents such as cyclophosphamide (CPA). The present study examines the potential of combining TPZ with CPA in a cytochrome P450-based prodrug activation gene therapy strategy. Recombinant retroviruses were used to transduce 9L gliosarcoma cells with the genes encoding P450 2B6 and NADPH-P450 reductase. Intratumoral coexpression of P450 2B6 with P450 reductase sensitized 9L tumor cells to CPA equally well under normoxic (19.6% O2) and hypoxic (1% O2) conditions. The P450 2B6/P450 reductase combination also sensitized 9L tumor cells to TPZ under both culture conditions. Interestingly, bystander cytotoxic effects were observed for both CPA and TPZ under hypoxia. Furthermore, TPZ exerted a striking growth-inhibitory effect on CPA-treated 9L/2B6/P450 reductase cells under both normoxia and hypoxia, which suggests the utility of this drug combination for P450-based gene therapy. To evaluate this possibility, 9L tumor cells were transduced in culture with P450 2B6 and P450 reductase and grown as solid tumors in severe combined immune deficient mice in vivo. Although these tumors showed little response to TPZ treatment alone, tumor growth was significantly delayed, by up to approximately four doubling times, when TPZ was combined with CPA. Some toxicity from the drug combination was apparent, however, as indicated by body weight profiles. These findings suggest the potential benefit of incorporating TPZ, and perhaps other bioreductive drugs, into a P450/P450 reductase-based gene therapy strategy for cancer treatment.     

Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide<Superscript>+</Superscript>

Background  

Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37).     

肿瘤微环境乏氧对化疗敏感性的影响

肿瘤微环境乏氧不仅使肿瘤自身更具侵袭性,容易发生远处转移,而且能使肿瘤对放化疗的抗拒性增加,从而降低其疗效.肿瘤微环境乏氧影响化疗敏感性的机制可分为直接耐药和间接耐药两种,现简要综述此方面的研究进展.     

改善肿瘤细胞乏氧状态对非小细胞肺癌放疗效果的影响

 目的 为了观察改善肿瘤细胞的乏氧状态对非小细胞肺癌（NSCLC）放疗效果的影响。方法 随机选择Ⅱ~Ⅳ期的NSCLC患者42例，资料完整者38例，在放射治疗前30 min内，采用舒氧康静脉内给氧的方法，提高血液中的氧分压，改善肿瘤细胞的乏氧状态；给氧前、后分别作血气分析，然后及时给予放射治疗，每次2 Gy，每日1次，每周5 d，总剂量60 ~ 70 Gy，随机选取同期的常规普放NSCLC患者37例作对照，按WHO疗效评价标准评价疗效，并作统计学处理。结果 试验组内给氧前后的动脉氧分压（PO2）分别为（85.6±7.5）mmHg，（103±9.7）mmHg；内给氧前后的血氧饱和度（SaO2）分别为（89.5±6.1）%和（94.4±5.2）%；放疗有效率为63.16 %（24／38）。对照组的有效率为43.24 %（16／37），试验组的疗效优于对照组（0.05＜P＜0.1），而两组之间毒副反应的发生率差异无统计学意义（P）。结论 放疗前静脉内给氧可改善肿瘤细胞的乏氧状态，提高放射治疗效果。     

基因导向性酶前体药物疗法在肿瘤治疗中的研究进展

基因导向性酶前体药物疗法(GDEPT)为肿瘤生物治疗最引人注目的研究领域,是继手术、放疗和化疗等传统疗法之后,一种具有良好应用前景的肿瘤治疗新措施,主要包括HSV-tk-GCV系统疗法和CD-5-FC系统疗法.为克服传统GDEPT疗法的不足,目前多主张联合基因治疗肿瘤,有人还提出一种全新的GDEPT疗法.现综述近几年来GDEPT在肿瘤基因治疗中的研究进展.     

肿瘤微环境在肿瘤细胞有氧糖酵解中的作用

肿瘤的发生发展和细胞代谢异常密切相关。肿瘤细胞摄入大量的葡萄糖,即使有足够的氧份供应,也主要借助糖酵解途径产生能量满足快速生长的需求。肿瘤细胞糖酵解受多方面因素影响,肿瘤微环境即为其中之一。近年来,有证据表明在肿瘤细胞糖代谢中,肿瘤微环境的免疫/炎症因子,转化生长因子β(Transforming growth factor-β,TGF-β)、肿瘤坏死因子α(Tumor necrosis factor alpha,TNF-α)、白细胞介素-4(Interleukin,IL-4)和白细胞介素-6(Interleukin,IL-6)等通过调控肿瘤细胞有氧糖酵解产生的能量与物质使细胞快速增殖,在肿瘤细胞糖代谢中起着至关重要的作用。因此,从肿瘤微环境中的免疫/炎症因子调控肿瘤细胞糖酵解的角度关注肿瘤的发生发展,可能为肿瘤的控制及临床治疗提供新思路。     

肿瘤缺氧及其靶向治疗研究进展

缺氧微环境是多数实体瘤的固有特征之一,其主要机制在于肿瘤血管组织分布紊乱和结构异常。缺氧可引起细胞发生一系列适应性改变,包括无氧糖酵解的增强、保护性应激蛋白的表达提高等。研究证实,缺氧不仅导致辐射抗拒,尤其低传能线密度（LET）的照射,也是引起耐药及肿瘤扩散转移的重要因素。同时,缺氧正逐渐成为肿瘤治疗的一个特异性靶点,如缺氧特异性细胞毒药物、乏氧细胞放射增敏剂,以及利用缺氧诱导目的基因靶向肿瘤组织表达等均展现出可喜的应用前景。     

Effects of hypoxia on tumor metabolism

Rapidly growing tumors invariably contain hypoxic regions. Adaptive response to hypoxia through angiogenesis, enhanced glucose metabolism and diminished but optimized mitochondrial respiration confers survival and growth advantage to hypoxic tumor cells. In this review, the roles of hypoxia, the hypoxia inducible factors, oncogenes and tumor suppressors in metabolic adaptation of tumors are discussed. These new insights into hypoxic metabolic alterations in tumors will hopefully lead us to target tumor bioenergetics for the treatment of cancers.     

The inhibitory effect of TU-100 on hepatic stellate cell activation in the tumor microenvironment

Introduction: The tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation.Materials and Methods: The HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 μg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC’s culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression.Results: Colon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death.Conclusions: TU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.     

Differential cytotoxicity and bystander effect of the rabbit cytochrome P450 4B1 enzyme gene by two different prodrugs: implications for pharmacogene therapy

The time course of cytotoxicity induction and the bystander effect of the rabbit cytochrome P450 4B1 (cyp4B1)/4-ipomeanol (4-IM) or 2-aminoanthracene (2-AA) pharmacogene therapy systems were investigated and compared with the herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV-tk/GCV) system. Experiments were performed in rat 9L gliosarcoma cells stably expressing cyp4B1 (9L-4B1), HSV-tk (9L-tk), or their egfp (enhanced green fluorescent protein) fusion genes. Cyp4B1-mediated activation of 2-AA showed a high cell killing efficiency within only 48 hours with an onset after already 15 minutes of prodrug exposure. Residual 9L-4B1 cells were mostly damaged sublethally upon 2-AA treatment showing an S phase arrest by cell cycle analysis. 4-IM treatment of 9L-4B1 cells generated an overall weaker cell killing, especially after prodrug exposure times of less than 48 hours. Residual cells surviving 4-IM treatment showed a G2/M arrest and restarted proliferation after prodrug treatment was stopped. HSV-tk/GCV pharmacogene therapy resulted in a slower cytotoxicity induction than cyp4B1/2-AA treatment with a significantly lower cell killing efficiency after 24 and 48 hours. HSV-tk/GCV-mediated cytotoxicity was widely similar to the cytotoxicity induced by cyp4B1/4-IM with the exception of a continuous 48-hour prodrug exposure where 4-IM treatment showed a significantly higher cell killing rate. Cells surviving HSV-tk/GCV suicide gene therapy were not viable and showed an S-phase arrest. Whereas HSV-tk/GCV induced a strong bystander effect, only moderate bystander cell death depending on cell-to-cell contact was demonstrated in 9L/9L-4B1 cocultures upon 2-AA treatment and was even absent with 4-IM, thereby contrasting with earlier reports. The absence of a strong bystander effect may limit, on one hand, the overall utility of the cyp4B1 systems for cancer gene therapy. On the other hand, the weak bystander effect together with the fast induction of cytotoxicity may provide marked advantages for the use of the cyp4B1 systems as biosafety enhancers for gene marking or replacement studies and donor lymphocyte infusions after allogeneic bone marrow transplantation.     

Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer

Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo.     

肿瘤干细胞及其微环境与肿瘤的耐药、复发和转移

肿瘤干细胞(NSC)是肿瘤发生发展的根源,其特殊的微环境可通过调控NSC或者其自身变化参与肿瘤的耐药、复发和转移.根除NSC和干扰其特殊微环境可能是治疗肿瘤的新策略.     

Modulation of cell death in the tumor microenvironment

The microenvironment of solid human tumors is characterized by heterogeneity in oxygenation. Hypoxia arises early in the process of tumor development because rapidly proliferating tumor cells outgrow the capacity of the host vasculature. Formation of solid tumors thus requires coordination of angiogenesis with continued tumor cell proliferation. However, despite such neovascularization, hypoxia is persistent and frequently found in tumors at the time of diagnosis. Tumors with low oxygenation have a poor prognosis, and strong evidence suggests this is because of the effects of hypoxia on malignant progression, angiogenesis, metastasis, and therapy resistance. The presence of viable hypoxic cells is likely a reflection of the development of hypoxia tolerance resulting from modulation of cell death in the microenvironment. This acquired feature has been explained on the basis of clonal selection-the hypoxic microenvironment selects cells capable of surviving in the absence of normal oxygen availability. However, the persistence and frequency of hypoxia in solid tumors raises a second potential explanation. We suggest that stable microregions of hypoxia may play a positive role in tumor growth. Although hypoxia inhibits cell proliferation and in tumor cells will eventually induce cell death, hypoxia also provides angiogenic and metastatic signals. The development of hypoxia tolerance will thus allow prolonged survival in the absence of oxygen and generation of a persistent angiogenic signal. We will discuss the concept of hypoxia tolerance and review mechanisms used by cancer cells to acquire this phenotype. The concept of hypoxia tolerance has important implications for current and future therapeutic approaches. Most therapeutic efforts to combat hypoxia have focused on targeting the presence of hypoxia itself. Our hypothesis predicts that targeting the biological responses to hypoxia and the pathways leading to hypoxia tolerance may also be attractive therapeutic strategies. Copyright 2003, Elsevier Science (USA). All rights reserved.     

Cancer gene therapy: Combination with radiation therapy and the role of bystander cell killing in the anti-tumor effect

Current anti-cancer modalities such as surgery, chemo- and radiation therapies have only limited success in cancer treatment. Gene therapy is a promising new tool to improve outcomes. In this review, first we summarize the various strategies to kill tumor cells, and then focus on the bystander effect of gene therapy. A variety of strategies, such as gene-directed enzyme pro-drug therapy, activation of an anti-tumor immune attack, application of replication-competent and oncolytic viral vectors, tumor-specific as well as radiation and hypoxiainduced gene expression, might be applied to target tumor cells. We put special emphasis on the combination of these approaches with local tumor irradiation. Using the available vector systems, only a small portion of cancer cells contains the therapeutic genes under clinical situations. However, cells directly targeted by gene therapy will transfer death signals to neighboring cancer cells. This bystander cell killing improves the efficiency of cancer gene therapy. Death signals are delivered by cell-to-cell communication through gap junction intercellular contacts, release of toxic metabolites into the neighborhood or to larger distances, phagocytosis of apoptotic bodies, and the activation of the immune system. Bystander cell killing can be enhanced by the introduction of gap junction proteins into cells, by further activating the immune system with immune-stimulatory molecules, or by introducing genes that help the transfer of cytotoxic genes and/or metabolites into bystander cells. In conclusion, although bystander cell killing can improve therapeutic effects, there should be additional developments in cancer gene therapy for a more efficient clinical application.(Pathology Oncology Research Vol 12, No 2, 118–124)     

细胞色素P450花生四烯酸表氧化酶对肿瘤细胞增殖的影响

背景与目的：细胞色素P450（cytochromeP450,CYP）花生四烯酸表氧化酶对内皮细胞具有促进增殖、抑制凋亡的作用。本研究主要探讨CYP表氧化酶对肿瘤细胞增殖的影响,并初步探讨其影响细胞增殖的相关信号转导机制。方法：用重组腺相关病毒（recombinant adeno-associatedvirus,rAAV）介导CYP花生四烯酸表氧化酶基因CYP2J2（cytochromeP4502J2）、CYPF87V（cytochromeP450F87V）和反义CYP2J2基因分别转染A549、Tea-8113、HepG2、Ncl-H4464种肿瘤细胞．采用MTT法、细胞计数法和流式细胞术分析CYP表氧化酶对肿瘤细胞增殖的影响：并用Westernblot法检测转染前后Tca-8113细胞中表皮生长因子受体（epidermal growth factor receptor,EGFR）、ERKI／2和Akt磷酸化水平：最后将转染rAAV-CYP2J2、rAAV-CYPF87V、rAAV-antiCYP2J2和rAAV-GFP的Tca-8113细胞分别接种至裸鼠皮下,观察皮下移植瘤的牛长情况。结果：转染CYP2J2和CYPF87V后,A549、Tca-8113、HepG2和Ncl-H446细胞的细胞增殖数分别是相应未转染细胞的1．7倍和2．0倍、1．4倍和1．5倍、1．6倍和1．8倍、2．2倍和2．0倍;转染反义CYP2J2阻断细胞自身CYP2J2表达后,上述4种细胞增殖显著减低：流式细胞术结果显示转染CYP表氧化酶基fj;I的肿瘤细胞中S／G2／M期细胞增加了210％。Westernblot结果显示,转染CYP2J2基因后,EGFR、ERK1／2和Akt磷酸化水平分别为未转染细胞的2倍、2．3倍和2．4倍,同时P13K表达水平也L调为未转染细胞的1．9倍：相反．转入反义CYP2J2基因明显抑制这些蛋白的磷酸化和表达。在体皮下移植瘤实验结果显示,转染rAAV-CYP2J2、rAAV—CYPF87V组裸鼠皮下移植瘤出现的时间（分别为5．8d和6．0d）明显较对照组和转染rAAV—GFP组（分别为8．4d和8．6d）短：而转染反义rAAV-CYP2J2组移植瘤生长缓慢,出现时间也明显较长（约10d）。结论：CYP花生四烯酸表氧化酶能促进肿瘤细胞     

Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N,a hypoxia activated anthraquinone DI-N-oxide prodrug

Purpose: To establish the role of the human cytochromes P450 (CYPs) in the reductive metabolism of the novel anthraquinone di-N-oxide prodrug AQ4N.Methods and Materials: Metabolism of AQ4N was conducted in a panel of 17 human phenotyped liver microsomes. AQ4N and metabolites were detected by reverse phase isocratic HPLC. CYP inhibitors and Spearman rank correlation were used to determine the significance of AQ4N metabolism versus specific CYP activity and/or expression.Results: Anaerobic metabolism of AQ4N to the 2-electron reduction product, AQM, and the 4-electron reduced tertiary amine, AQ4, occurred in all 17 human liver microsome preparations. The range (± SE) for total AQ4N turnover was 14.26 ± 1.43 nmol/incubate (highest) to 3.65 ± 1.05 nmol/incubate (lowest). Metabolism was not detected in the absence of NADPH or microsomes. In aerobic incubates, AQM was less than 4% of anaerobic values whereas AQ4 was undetectable. CYP-mediated metabolism of AQ4N was inhibited completely by ketoconazole (KET) and carbon monoxide (CO), two global inhibitors of CYP-mediated metabolism. AQ4N metabolism correlated significantly with probes for CYP 3A, specifically benzoxylresorufin O-dealkylation [r(s) = 0.70, p <0.01] and tamoxifen N-demethylation (r(s) = 0.85, p < 0.01), but not with probes for CYPs 2C, 2D, and 1A. CYP 3A involvement was confirmed by the use of the CYP 3A specific inhibitor, triacetyloleandomycin (TAO), which repressed the formation of AQM to 13% of the uninhibited value and abolished completely the formation of AQ4. Alpha-naphthoflavone (ANF), an inhibitor of CYP 2C and 1A, had no significant effect on AQ4N metabolism.Conclusions: These data suggest that the human CYP 3A enzymes can contribute to the reductive metabolism of AQ4N. CYP 3A enzymes are highly expressed in a broad spectrum of human cancers. The results show that AQ4N requires anaerobic conditions for CYP 3A-mediated reduction and hence this subfamily of enzymes is likely to selectively activate AQ4N in hypoxic tumors.     

CAR-T细胞治疗突破实体肿瘤微环境新策略的研究进展

免疫治疗可通过提高患者自身免疫力以达到抗肿瘤目的,其中嵌合型抗原受体修饰T细胞(chimeric antigen receptor modified T cells,CAR-T)在血液系统肿瘤中已显现出良好疗效。实体肿瘤微环境(tumor microenvironment,TME)中免疫抑制细胞及分子等可限制CAR-T细胞在肿瘤部位浸润及其在浸润部位产生细胞毒作用。因此CAR-T细胞治疗在血液系统肿瘤中的显著疗效未在实体肿瘤中呈现。本文就如何突破TME限制,提高CAR-T细胞归巢能力及细胞毒作用,从而提高其在实体瘤中的疗效做一综述。