When cancer chemotherapeutic drugs are administered on a traditional MTD schedule, an obligatory rest period between treatment cycles allows for the recovery of sensitive host tissues (e.g., bone marrow) but also provides an opportunity for recovery and regrowth of tumor cells and tumor-associated endothelial cells. This problem can be circumvented by administration of CPA using a regular, repeating (metronomic) schedule, which is associated with anti-angiogenic activity (12
) and has received much attention as a possible alternative to traditional MTD regimens. When metronomic CPA is combined with P450 gene-directed enzyme-prodrug therapy, tumor cell expression of a CPA-activating P450 enzyme further enhances activity and leads to a sustained anti-tumor response (13
). Presently, we investigate the impact of intratumoral P450-catalyzed CPA activation on tumor-associated endothelial cells and the role of liver vs. tumor cell P450 metabolism in the enhanced anti-tumor response. Our findings lead us to conclude that a close collaboration between hepatic P450 prodrug activation and intratumoral prodrug activation catalyzed by the P450 transgene is required to maximize the therapeutic response in this gene therapy model. Thus, while liver P450 prodrug activation alone is sufficient to induce the anti-angiogenic effect that is a hallmark of metronomic CPA, the small tumors that result retain strong proliferative potential and often become angiogenesis-independent. Moreover, the anti-angiogenic action of metronomic CPA suppresses the uptake of liver-derived 4-OH-CPA (J Ma & DJ Waxman, unpublished data)
, thereby rendering the tumor cells resistant to direct cytotoxicity. However, when metronomic CPA is combined with intratumoral expression of a CPA-activating P450 enzyme, the anti-angiogenic effect of liver-activated CPA (‘killing from the outside’) collaborates with intratumoral P450-dependent cytotoxicity (‘killing from the inside’) to enhance overall tumor cell killing, substantially prolonging the tumor-free period after cessation of drug treatment.
An unanticipated finding of the present study was the absence of an effect of intratumoral P450 expression on the anti-angiogenic action of metronomic CPA. Although this finding could be viewed as supporting the hypothesis that the direct anti-tumor actions of metronomic CPA, rather than its anti-angiogenic effects, are responsible for the anti-tumor response seen in the context of P450 GDEPT, that interpretation is not consistent with our earlier finding that regression of 9L/P450 tumors is induced by a metronomic, anti-angiogenic CPA schedule but not by MTD CPA treatment (13
). Moreover, various drug-resistant tumor models used in earlier studies confirm the critical role of anti-angiogenesis in the anti-tumor activity of metronomic CPA (12
). An important feature of P450-based GDEPT using CPA is that the active metabolite, 4-OH-CPA, readily diffuses across cell membranes and shows a strong bystander cytotoxic effect on surrounding P450-deficient tumor cells, both in vitro
) and in vivo
). However, this bystander cytotoxicity does not extend to include tumor-associated endothelial cells (). This discrepancy could relate to the location of endothelial cells within the tumor. The anatomical structure of the liver determines that hepatocytes have direct access to CPA in the blood, enabling liver P450 to dominate CPA metabolism following i.p.
drug administration. The activated metabolite, 4-OH-CPA, enters systemic circulation and reaches the tumor vasculature, where tumor-associated endothelial cells are the first cells exposed to 4-OH-CPA and its DNA cross-linking, cytotoxic decomposition product phosphoramide mustard. In contrast, 9L tumor cells have more limited access to circulating 4-OH-CPA (23
), presumably reflecting the dysfunctional tumor vasculature. Moreover, in the case of 9L/2B11 tumors, 4-OH-CPA formed intratumorally needs to cross through both tumor stroma and endothelial basal membranes before it can reach tumor-associated endothelial cells. Thus, the extensive exposure of these endothelial cells to liver-derived 4-OH-CPA, coupled with their restricted access to 4-OH-CPA formed intratumorally, effectively limits the bystander killing of tumor-associated endothelial cells by tumor cell P450-derived 4-OH-CPA.
In addition to its anti-angiogenic activity, metronomic CPA shows direct cytotoxicity toward 9L tumor cells, particularly in tumors that express the canine P450 enzyme 2B11, which catalyzes CPA activation (4-hydroxylation) with high efficiency due to an atypically low Km
of ~70 µM (20
). In 9L tumors, apoptosis induced by the first CPA treatment cycle was transient, while in 9L/2B11 tumors, tumor cell apoptosis increased substantially until day 3. Moreover, only a limited increase in 9L tumor cell apoptosis was observed following the second CPA treatment cycle, suggesting that those tumor cells that are in close proximity to the tumor vasculature and thus are readily accessible to liver-derived 4-OH-CPA were already removed in the first CPA cycle. Direct killing of the surviving tumor cells may thus occur less frequently in the subsequent cycles of CPA treatment. In the case of 9L/2B11 tumors, however, localized prodrug activation was associated with a significant increase in tumor cell apoptosis after the second CPA treatment. Three days after the second CPA injection, when tumor microvessel density and endothelial cell counts both dropped significantly, anti-angiogenesis likely begins to play an important role in the overall anti-tumor effect. An alternative way to induce strong tumor cell cytotoxicity while retaining the anti-angiogenic activity of CPA is to combine metronomic treatment with a traditional MTD or bolus schedule (24
Once activated by P450 enzymes, CPA metabolites generated intratumorally kill P450 2B11-positive tumor cells as well as bystander tumor cells. The bystander killing of P450-negative tumor cells is supported by the fact that more complete overall tumor regression is achieved in 9L/2B11 tumors than 9L tumors (20
), despite the fact that only 29–68% of the tumor cells express P450 2B11 at the time of the initial CPA treatment (). Tumor cell P450 2B11 protein, but not RNA, was substantially decreased beginning with the second CPA treatment cycle, whereas in the case of another P450 tumor model, 9L/2B6, P450 protein and RNA levels were both unchanged after two CPA cycles (unpublished data). This difference may reflect the 20-fold lower Km
(CPA) and 28-fold higher Vmax
ratio exhibited by P450 2B11 compared to P450 2B6 (20
), which can result in high intracellular levels of acrolein or other P450 protein-binding metabolites derived from 4-OH-CPA (26
) and, consequently, P450 protein degradation. The gradual loss of intratumoral P450 protein following metronomic CPA treatment indicates that repeated delivery of the therapeutic P450 gene will likely be required to optimize P450-based GDEPT treatment strategies. The death of P450-expressing 4-OH-CPA-producing ‘factory’ tumor cells can be delayed, however, by introduction of anti-apoptotic factors, which increase the net production of cytotoxic drug metabolites without conferring drug resistance (22
The decline of P450-expressing tumor cells during the course of metronomic CPA treatment was accompanied by a significant enlargement of many of the remaining P450-positive cells, both in the case of 9L/2B11 tumors and 9L/2B6 tumors. Alkylating reagents such as CPA induce DNA crosslinks which eventually leads to programmed cell death (29
). Cell cycle check-points are often perturbed in tumor cells, which can lead to delayed apoptosis and the induction of mitotic catastrophe, as observed in some cytotoxin-treated tumors (30
). The enlarged 9L/P450 cells seen in the metronomic CPA-treated tumors may thus reflect CPA-induced DNA damage culminating in mitotic catastrophe. It is unclear whether the P450 protein that accumulates in these enlarged cells retains CPA metabolic activity.
The endogenous angiogenesis inhibitor TSP-1 is expressed in both host tissues (32
) and tumor cells (33
). Its deficiency leads to increased tumor growth and enhanced tumor angiogenesis (37
). The anti-angiogenic activity of TSP-1 involves multiple mechanisms, including suppression of endothelial cell migration, induction of apoptosis with increased expression of Fas ligand in proliferating endothelial cells (37
), inhibition of VEGF mobilization in the extracellular matrix, and reduction of blood flow by blocking nitric oxide/cGMP-induced relaxation of vascular smooth muscle cells (39
). The function of 9L cell-derived TSP-1, presently shown to comprise ~95% of the tumor-associated TSP-1 RNA in untreated tumors, is unclear, but it may inhibit the growth of tumor metastases as suggested for other tumors (41
). Metronomic CPA was presently shown to decrease tumor cell (rat) TSP-1 expression while increasing host (mouse) TSP-1 such that a substantial fraction of the 9L tumor-associated TSP-1 is of mouse origin after two CPA treatment cycles. CD31/TSP-1 double staining revealed the expression of TSP-1 in host-derived perivascular cells, which is likely an important source of the host TSP-1 detected by qPCR. Metronomic CPA induction of TSP-1 has been also observed in perivascular cells associated with Lewis lung cancer and B16F10 melanoma tumors, where either host cell- or tumor cell-derived TSP-1 may augment the anti-tumor effect of metronomic CPA (35
). Although the number of TSP-1-positive blood vessels is low in 9L tumors (2–8 vessels/section), the close spatial association between perivascular cells and endothelial cells may facilitate interactions between TSP-1 and its endothelial cell membrane receptor, CD36.
In conclusion, while anti-angiogenesis clearly contributes to the overall anti-tumor effect of metronomic CPA, the anti-angiogenic response is very similar for 9L and 9L/2B11 tumors. It is also apparent, however, that the tumor cell population has poor access to liver-derived 4-OH-CPA, especially in the later cycles of metronomic CPA treatment, hence the requirement of intratumoral P450 gene delivery and intratumoral prodrug activation for tumor cell elimination leading to a sustained anti-tumor response. Further increases in intratumoral CPA activation could potentially be achieved by direct intratumoral delivery of CPA (23
) or by inhibition or down-regulation of liver P450 or P450 reductase (44
), both of which would be expected to increase CPA access to tumor-expressed P450 enzymes and enhance intratumoral prodrug activation. Whether the resultant increase in tumor cell-derived 4-OH-CPA at the expense of liver-derived 4-OH-CPA is consistent with maintenance of the overall anti-tumor effect, requires further investigation.