Apoptosis induced in advanced CD8F1-murine mammary tumors by the combination of PALA, MMPR and 6AN precedes tumor regression and is preceded by ATP depletion.

The drug combination N-(phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN), referred to as PMA, induces regressions of advanced CD8F1 murine mammary carcinomas in vivo. We demonstrated that CD8F1 tumor regressions were preceded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL endlabeling, and fragmentation of DNA into nucleosomal "ladder" patterns. These indications of apoptosis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h, then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated controls) of the cell population for at least 24 h after MMPR + 6AN administration. The 5'-phosphate derivative of MMRP, MMPR-5P, which inhibits de novo purine biosynthesis, was present at a "steady-state" level, and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition, MMPR-5P was retained in CD8F1 tumors at a high level over a prolonged period (> 96 h) even as tumors were undergoing regression. The prolonged presence of MMPR-5P was important for optimal chemotherapeutic effect, since treatment with iodotubercidin (IodoT), an inhibitor of MMPR/adenosine kinase, 6 h after MMPR+6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CD8F1 tumors. In addition, compared to the PMA-treated group, there was a significant restoration of ATP levels after treatment with IodoT. In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to correlate with the degree of tumor shrinkage at 24 h, after tumors had sufficient time to respond to treatment. These results define the time-course of drug-induced apoptosis in CD8F1 tumors, show that ATP depletion occurs prior to apoptosis and demonstrate that prolonged retention of MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a component of the biochemical apoptotic cascade in the CD8F1 tumor.

Evaluation of chemotherapy and radiation enhancement and 31P NMR spectral changes induced by biochemical modulation.

The combination of N-(phosphonacetyl)-L-aspartate (PALA), 6-methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) has been shown to be an effective antineoplastic regimen and also to enhance the effects of other antineoplastic agents (1-4). To further enhance the effect of this combination, we investigated the effects of adding adriamycin, at its maximally tolerated dose, to this regimen. The response rate (complete regression+partial regression) for the four-drug regimen was higher than for the three-drug regimen, and the tumor growth delay was also significantly higher than for treatment with PALA, MMPR, 6AN, or after treatment with maximally tolerated doses of adriamycin alone (11 mg/kg). The addition of adriamycin to PALA, MMPR, 6AN did not result in enhancement of the effect of radiation, as measured by tumor growth delay studies and tumor control (complete and partial regression rate). The mechanism of action of the combination of PALA, MMPR, and 6AN is not known definitively, but a possible mechanism previously suggested is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Treatment with PALA, MMPR, 6AN, and adriamycin (at 2.5 hr post MMPR, 6AN) resulted in a nadir NTP/Pi value, as determined by 31P NMR spectroscopy, at approximately 10 hr post MMPR + 6AN (7.5 hr post adriamycin), which was not significantly different from the NTP/Pi value determined after treatment with the three-drug combination.

Marked enhancement in vivo of paclitaxel's (taxol's) tumor-regressing activity by ATP-depleting modulation.

Paclitaxel alone is active against the CD8F1 murine spontaneous mammary cancer, and when administered following an ATP-depleting combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6-AN) (PMA) produced significantly enhanced partial tumor regressions over that produced by either paclitaxel alone at the maximal tolerated dose (MTD), or by the PMA drug combination alone, against advanced, first passage spontaneous murine breast tumors. The anticancer activity of paclitaxel is due to enhancement and stabilization of microtubule polymerization. Pertinently, microtubule disassembly (an ATP-dependent process) is known to sharply decrease in the presence of ATP depletion. Thus, the dramatic therapeutic enhancement observed with paclitaxel in combination with PMA is in agreement with biochemical expectations, since PMA has been shown to deplete ATP in CD8F1 tumor cells. The augmented therapeutic results were obtained with approximately one-third the MTD of paclitaxel as a single agent and suggest the potential clinical benefit of more effective treatment with lesser amounts of drug.

Correlation of retention of tumor methylmercaptopurine riboside-5'-phosphate with effectiveness in CD8F1 murine mammary tumor regression.

Treatment with a combination (PMA) of (N-phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) induced partial regressions of CD8F1 murine mammary tumors and provided for tumor growth inhibition without regression of Colon 38 tumors. HPLC-nucleotide pool analysis of CD8 mammary tumors obtained at various times after treatment with PMA revealed that MMPR-5'-phosphate, which inhibits de novo purine nucleotide biosynthesis, was constant at levels of approximately 2.5 nmol/mg protein for 72 hr after treatment. In contrast, the MMPR-5'-phosphate levels of C38 tumors decreased from 24-hr levels at 1.5 nmol/mg protein with a half-time of about 24 hr. Treatment of CD8 tumor-bearing mice with iodotubercidin, a potent inhibitor of adenosine/MMPR kinase, at various times after PMA, reversed both the accumulation of high levels of MMPR-5'-phosphate and the number of partial tumor regressions. These data demonstrate that a cycle of MMPR rephosphorylation is active in the CD8 mammary tumor and suggest that this recycling of MMPR is important for the optimal effect of PMA treatment.

Enhanced antitumor activity of an adriamycin + 5-fluorouracil combination when preceded by biochemical modulation.

A three-drug combination, PMA, consisting of (phosphonacetyl)-L-aspartic acid + 6-methylmercaptopurine riboside + 5-aminonicotinamide, preceding either 5-fluorouracil (5-FU) or adriamycin (Adr), produced tumor-regressing activity in a murine advanced breast tumor model not attainable with either 5-FU or Adr as single agents, or with any lesser combination of these drugs administered at maximally tolerated doses. Marked tumor-regressing activity was further increased significantly by using 5-FU and Adr together in conjunction with the modulatory biochemical conditioning (particularly ATP depletion) provided by pretreatment with PMA.

Enhancement of chemotherapeutically-induced apoptosis in vivo by biochemical modulation of poly (ADP-ribose) polymerase.

An inhibitor of poly (ADP-ribose) polymerase, 1,5-dihydroxyisoquinoline (DHIQ), evaluated in vivo against a murine advanced breast cancer, significantly improved by 20% the PR rate of tumor-regressing chemotherapy. A detailed sequential biochemical cascade is proposed for chemotherapy-induced apoptosis, and the rationale for the utilization of the inhibitor is explained.

Biochemical modulation of tumor cell energy. IV. Evidence for the contribution of adenosine triphosphate (ATP) depletion to chemotherapeutically-induced tumor regression.

DNA-damaging agents, e.g. Adriamycin (ADR), are reported to cause tumor regression by induction of apoptosis. A reduction in the intracellular content of ATP is part of the biochemical cascade of events that ultimately results in programmed death of the cell, or apoptosis. A chemotherapeutic three-drug combination (PMA) consisting of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6AN) significantly lowers levels of ATP in CD8F1 murine breast tumors in vivo and produces tumor regression by apoptosis. Addition of the DNA-damaging antitumor agent ADR to PMA was found to further significantly deplete ATP in CD8F1 murine breast tumors in vivo with a concomitant significant increase in the number of tumor regressions. The correlative biochemical and therapeutic results are consistent with, and support, the hypothesis that ATP depletion is a significant factor and, therefore, is a worthy therapeutic target in the production of apoptosis.

On the relationship of ATP depletion to chemotherapeutically-induced tumor-regression.

A positive correlation was found between increasing ATP depletion and enhanced tumor-regressing activity by combining PMA, a three drug combination (PALA, MMPR+6-AN), with FUra, or Adria, or FUra+Adria. For example, at 48 h post treatment, ATP was 68% of saline controls with a 10% tumor regression rate (PMA); ATP was 55% with a 60% regression rate (PMA+FUra); ATP was 54% with an 80% regression rate (PMA+Adria); and ATP was 30% with a;100% regression rate (PMA+FUra+Adria). The results give support to the suggestion that ATP depletion may be a significant factor in the production of chemotherapeutically-induced apoptosis.

Biochemical modulation of tumor cell energy in vivo: II. A lower dose of adriamycin is required and a greater antitumor activity is induced when cellular energy is depressed.

A quadruple drug combination--consisting of a triple-drug combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-amino-nicotinamide (6-AN), designed to primarily deplete cellular energy in tumor cells, + Adriamycin (Adria)--yielded significantly enhanced anticancer activity (i.e., tumor regressions) over that produced by either Adria alone at maximum tolerated dose (MTD) or by the triple-drug combination, against large, spontaneous, autochthonous murine breast tumors. The adenosine triphosphate (ATP)-depleting triple-drug combination administered prior to Adria resulted in a 100% tumor regression rate (12% complete regression; 88% partial regression) of spontaneous tumors. Histological examination of treated tumors demonstrated that the treatment-induced mechanism of cancer cell death was by apoptosis. The augmented therapeutic results (100% tumor regressions) were obtained with approximately one-half the MTD of Adria as a single agent and suggest the potential clinical benefit of longer, more effective, and safer treatment by low doses of Adria when combined with the triple-drug combination. Two likely mechanisms of action are discussed: (1) prevention of DNA repair; (2) complementary disruption of biochemical pathways by both the triple-drug combination and the biochemical cascade of apoptosis that is induced by a DNA-damaging anticancer agents such as Adria.

Potentiation of a three drug chemotherapy regimen by radiation.

The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.

Biochemical modulation of tumor cell energy: regression of advanced spontaneous murine breast tumors with a 5-fluorouracil-containing drug combination.

This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide plus 5-fluorouracil, in CD8F1 mice bearing spontaneous, autochthonous, breast tumors or first-passage advanced transplants of these spontaneous tumors. The combination and sequence of administration of these drugs were selected on the basis of known potentiating biochemical interactions. High performance liquid chromatography and nuclear magnetic resonance spectroscopy measurements of biochemical changes resulting from treatment with N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide indicated a severe depletion of cellular energy levels in the treated tumors. 6-Aminonicotinamide produced a severe block of the pentose shunt, and 5-fluorouracil severely inhibited both thymidylate synthase and thymidine kinase in the treated tumors. This quadruple drug combination, administered on a 10-11-day schedule, produced an impressive partial tumor regression rate of 67% of large, spontaneous, autochthonous, murine breast tumors and a tumor regression rate of 74% of first-passage transplants of the spontaneous breast tumors.

Biochemical modulation of 5-fluorouracil with leucovorin or delayed uridine rescue. Correlation of antitumor activity with dosage and FUra incorporation into RNA.

Two strategies for modulation of 5-fluorouracil (FUra) activity were compared in vivo in advanced murine CD8F1 breast tumors with regard to three parameters: chemotherapeutic activity, inhibition of thymidylate synthase (TSase) activity, and incorporation of FUra into RNA, (FU)RNA. Inhibition of TSase by FUra was modulated by leucovorin (LV), and the incorporation of FUra into RNA was increased by the administration of otherwise lethal doses of FUra followed by uridine "rescue". Thymidylate synthase activity was inhibited substantially (49%) by low-dose FUra at 25 mg/kg, but was not further enhanced (48%) by repeated daily treatments at the same dose (FUra25 x 4). Inhibition of TSase was somewhat enhanced (55%) by the addition of LV to FUra25 x 4, and a greater therapeutic effect was obtained with FUra25 x 4 + LV over FUra25 x 4 alone. FUra as a single agent at the maximum tolerated weekly dose of 100 mg/kg inhibited TSase activity 66-73%. This inhibition was further enhanced slightly by the addition of LV (71-82%), and its therapeutic efficacy was greater than with FUra25 x 4 with or without LV. However, in contrast to low dose FUra25 x 4, the antitumor effect of FUra100 was not enhanced by LV. (FU)RNA increased with FUra dose from 0.4 (FUra25) to 2.2 nmol/mg DNA (FUra100). At a very-high-dose of FUra (200-225 mg/kg) followed by uridine "rescue", TSase inhibition was not further enhanced, but both (FU)RNA (4.8 nmol/mg DNA) and the therapeutic efficacy were increased. Since TSase could not be further inhibited at doses above FUra100, the increased chemotherapeutic efficacy correlated with increased (FU)RNA.

In vivo monitoring of changes in 5-fluorouracil metabolism induced by methotrexate measured by 19F NMR spectroscopy.

We have used in vivo 19F NMR spectroscopy to study the metabolism of 5-fluorouracil (FUra) in tumors with and without pretreatment with methotrexate (MTX). Using the CD8F1 murine mammary tumor as an in vivo model, we observed signals from FUra, alpha-fluoro-beta-alanine (F beta ALA), alpha-fluoro-beta-ureidopropionic acid (FUPA), and 5-fluorouracil-nucleotides (FUN) after intravenous or intraperitoneal injection of 150 mg/kg FUra. Formation of FUN was increased about 1.7-fold in CD8F1 tumor with methotrexate pretreatment as determined by acid extraction and HPLC analysis. A comparison of in vivo NMR spectra from FUra and sequential MTX + FUra-treated tumors showed a significantly higher ratio of the FUN signal to the initial total 19F signal in the MTX + FUra-treated tumors (p less than 0.001) for animals receiving FUra either intravenously or intraperitoneally. In addition, tumors treated with MTX + FUra had significantly longer time durations during which FUN was detected, independent of the mode of administration. These experiments indicate that in vivo 19F NMR spectroscopy can be used to noninvasively monitor alterations of 5-fluorouracil metabolism that occur with administration of modulating agents such as methotrexate. Further studies, in both murine tumor models and patients, are indicated to determine if these results can be correlated with tumor response.

Enhancement of anticancer agent activity by selective inhibition of rapidly proliferating tissues of the host.

Most cytotoxic drugs used in cancer therapy do not discriminate between neoplastic and normal proliferating cells. To avoid irreversible damage to vital host tissues, such as bone marrow and intestine, drugs must be administered at dosages which usually prove insufficient to eradicate all of the neoplastic cells present. This review focuses on an approach to improve cancer chemotherapy by selectively protecting normal, proliferating cells during treatment, thereby permitting the administration of otherwise lethal doses of drug. Preclinical in vivo studies of cytokinetic modulation with interferon, or L-histidinol, as well as recent clinical studies of interferon modulation of the activity of 5-fluorouracil are reviewed.

Hematologic effects of interleukin-1 beta, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor in tumor-bearing mice treated with fluorouracil.

Myelosuppression following intensive chemotherapy in cancer patients is associated with increased morbidity and mortality. Hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), alone or in combination with interleukin-1 (IL-1), have been shown to counteract myelosuppression resulting from some, but not all, chemotherapeutic regimens. In an attempt to apply these findings to intensive therapy with proliferation-dependent chemotherapeutic drugs such as fluorouracil (5-FU), we investigated combination biochemotherapy in a murine model. Female CD8F1 [(BALB/c X DBA/8)F1] mice bearing first-passage transplants of spontaneous CD8F1 breast tumors were treated intraperitoneally once a week for 3 successive weeks with a course of 5-FU alone or with a course of 5-FU in combination with recombinant human interleukin-1 beta (rHuIL-1 beta) alone or in combination with CSFs. rHuIL-1 beta alone or in combination with rHuG-CSF or recombinant murine GM-CSF significantly improved tumor growth inhibition (60% vs. 90%) and survival (20% vs. 90%-100%), increased the maximally tolerated dose of 5-FU, accelerated recovery of neutrophil counts in peripheral blood, and reduced duration of significant neutropenia and loss of body weight (29% vs. 10% loss). Clinical trials of IL-1 have been initiated in patients with advanced cancer receiving multiple courses of high-dose 5-FU.

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones.

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth in vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogen- or prolactin-dependent growth. Primary tumors still displayed a constitutional expression of alpha-, beta-, and gamma-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in alpha- and beta-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of alpha-, beta-, and gamma-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10-14 days. The accumulation of beta-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance beta-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.

Chemotherapeutic activity of L-histidinol against spontaneous, autochthonous murine breast tumors.

In experiments designed to investigate the biochemical basis for the diminution of the antitumor activity of 5-fluorouracil (FUra) by L-histidinol in CD8FI breast tumors, it was discovered that L-histidinol inhibits RNA and DNA synthesis in these tumors. This finding suggested the possibility that L-histidinol might have antiproliferative activity in the CD8FI breast tumor, and on that basis we evaluated different administration schedules of L-histidinol for antitumor activity in vivo. The present report describes a schedule of L-histidinol administration which yielded significant activity against spontaneous, autochthonous CD8FI breast tumors consistently at tolerable doses. To our knowledge, there are no previous reports of in vivo antitumor activity associated with the administration of L-histidinol as a single agent.

Advanced breast cancer: response to somatostatin.

It has previously been reported that a somatostatin analogue has a direct antiproliferative effect on human breast cancer cells in vitro. Here we report preliminary data on the effects of the in vivo administration of SMS in patients with advanced breast cancer. The regimen consisted of iv infusion of 750 micrograms SMS t.i.d. for 10 days followed by 5 days at 500 micrograms im b.i.d. A partial response was observed in 3 out of 10 patients treated. Moreover, a marked reduction of oedema, cyanosis and bleeding from ulcerated tumor lesions was noted in most of the treated patients. Administration of SMS was devoid of toxic side effects. It is suggested that SMS may be of potential value in the therapeutic approach to advanced breast cancer.