John Mendoza

Dependence of Anticancer Activity of Camptothecins on Maintaining Their Lactone Function

Abstract: Camptothecins contain a lactone ring that exists in the closed form below ph 7. Above 7, the open (CPT+) and the closed (CPT) form coexist in a 50–50 ratio in mouse plasma and in a 90–10 ratio in human plasma due to the high affinity of human serum albumin (HSA) for CPT+. CPT+ is much less toxic than CPT and it is excreted much faster. In complete RPMI 1640 culture medium, the equilibrium CPT+‐CPT is 50–50. If 4% HSA is added, it moves to 90–10 modeling for the human physiological situation.

Conversion of 9-nitro-camptothecin to 9-amino-camptothecin by human blood cells in vitro.

To the Editor: In recent reports, we have shown that the compound 9-nitro-camptothecin (9NC) is cytostatic or cytotoxic for human cells in vitro, depending on the ability of these cells to induce tumors when xenografted into nude mice (1-5). Specifically, cells that are not tumorigenic in nude mice accumulate at late-S/G2 phase of the cell cycle, in presence of 9NC, and remain arrested for a prolonged period of time or re-enter cell cycling upon cessation of exposure to 9NC. In contrast, tumorigenic cells die by apoptosis while they traverse the S phase in the presence of 9NC, and cell death continues regardless of discontinuation of exposure to the drug. Also, we have shown that 9NC suppresses growth of and induces regression of human tumors established in nude mice

9-nitro-camptothecin delays growth of U-937 leukemia tumors in nude mice and is cytotoxic or cytostatic for human myelomonocytic leukemia lines in vitro

Abstract:  The camptothecin derivatives 9‐nitro‐camptothecin (9NC) and 9‐amino‐camptothecin (9AC) inhibit similarly growth of HL‐60, KG‐1, and U‐937 cells in vitro, whereas growth of THP‐1 cells is inhibited by 9AC, but not by 9NC. Growth inhibition is accompanied by enlargement of cells which contain one (HL‐60, THP‐1) or more (KG‐1, U‐937) nuclei. Flow cytometry studies showed that 9NC‐treated HL‐60 and U‐937 cells accumulate in the S and G2 phases of the cell cycle; then they die by apoptosis, with the HL‐60 cells being more sensitive than U‐937 cells to 9NC. In contrast, 9NC‐treated KG‐1 and THP‐1 cells accumulate in S and G2 phases, but resist death by apoptosis. Of the cell lines tested, only U‐937 cells xenografted in nude mice generated subcutaneous myeloid tumors, which exhibited a delayed growth in the presence of 9NC. Further, 9NC‐treated advanced U‐937 tumors regressed temporarily, indicating that U‐937 cells consist of 9NC‐sensitive and 9NC‐resistant populations.

Structure-activity relationship of alkyl camptothecin esters.

Abstract: The cytotoxicity of camptothecin (CPT) esters 1–6 was measured. Like parental camptothecin, esters 2 and 3, but not 1, 4, 5, and 6, inhibited proliferation of human leukemia cells in culture and induced programmed cell death as assessed by flow cytometry studies. Exhibition of similar levels of antiproliferative activities of CPT 2 and 3 required different incubation time periods in cell cultures, with CPT and 3 requiring the shortest and longest periods, respectively. Both 2 and 3 were inactive against cells resistant to the semisynthetic CPT derivative 9‐nitrocamptothecin and unable to stabilize DNA‐topoisomerase I (Topo I) “cleavable complexes” in a cell‐free system, suggesting that Topo I activity was required but insufficient for the mechanism of action of 2 and 3. Mouse liver homogenate converted esters to parental CPT, but the conversion rates were different with different esters. Of four tested esters in this experiment, ester 2 had the fastest conversion rate. In vivo studies showed that ester 2 had an exceptional lack of toxicity in nude mice, even at enormous doses, and demonstrated extensive activity against human breast and colon tumors grown as xenografts in immunodeficient nude mice, whereas no antitumor activity was observed for the other esters. In conclusion, ester 2 is a prodrug of the antitumor compound CPT, and it can be administered at very high doses in mice with no appearance of toxicity. This study provides a basis for further evaluation of CPT ester 2 as an investigational anticancer agent.

Pharmacology of Camptothecin Esters

Abstract: An intact lactone ring of camptothecins is a structural requirement for their anticancer activity. Propionate esters of camptothecin (CPT) and 9‐nitrocamptothecin (9NC), CZ48 and CZ112, respectively, have been synthesized as derivatives resistant to lactone hydrolysis and are chemotherapeutically active. In this study, we have examined the mechanism of action of CZ48 and CZ112 and their distribution, metabolism, and toxicity. CZ112 incubated in human plasma retained its lactone structure longer than 9NC (t1/2: 10.5 and <1 hr for CZ112 and 9NC, respectively). This resistance to lactone hydrolysis was also observed in mouse plasma or albumin solutions. Neither CZ48 nor CZ112 inhibit topoisomerase I and thus are prodrugs dependent on hydrolysis to CPT or 9NC, respectively. Rates of hydrolysis of CZ48 to CPT are higher by homogenates of mouse liver, spleen, lung, and kidney than by plasma. Rates of hydrolysis by tumor cells in culture vary and were higher by breast cancer and melanoma cells than by colon cancer cells. On the basis of these and other data, it is proposed that CZ48 and CZ112 may act as anticancer agents by resisting hydrolysis to camptothecins while in circulation. Hydrolysis in tissues may release intact lactone in target tissues.

Partial characterization of human leukemia U-937 cell sublines resistant to 9-nitrocamptothecin P

Abstract: Human leukemia U‐937 cell sublines exhibiting various levels of resistance to 9‐nitrocamptothecin (9NC) were developed after exposure to progressively increased 9NC concentrations. Increases in 9NC resistance of the cells were accompanied by decreases in proliferation rate; appearance of morphological and functional features that correlate with granulocytic maturation; decreased synthesis of topoisomerase I; increased synthesis of topoisomerase II; and inability or decreased ability to induce tumors when xenografted in nude mice. 9NC‐resistant cells, transferred and propagated in 9NC‐free media for 6 months, continue to exhibit resistance and other features similar to cells propagated in continual presence of 9NC. Finally, 9NC‐resistant U‐937 cells respond to physiological and non‐physiological agents of cell differentiation, indicating that alternative treatments can be successfully used to inhibit growth of 9NC‐resistant U‐937 cells and tumors.

Sensitivity of camptothecin-resistant human leukemia cells and tumors to anticancer drugs with diverse mechanisms of action.

Human leukemia U-937 cell clones resistant to 9-nitrocamptothecin (9NC) appear after exposure to increase 9NC-concentrations. Drug resistance is irreversible, regardless of whether the 9NC-resistant (U-937/CR150) cells grow in media with or without 9NC. U-937/CR150 cells are more sensitive than wild type U-937 (U-937/wt) cells to topoisomerase II-directed drugs, amsacrine, daunorubicin, and etoposide. The mitotic inhibitor, vincristine, induces hyperdiploidy in U-937/wt, but not in U-937/CR150 cells, whereas the antimetabolites, cytarabine and methotrexate, and the nitrosourea, carmustine, elicit similar responses in both U-937/wt and U-937/CR150 cells. U-937/CR150-generated tumors in nude mice are sensitive to etoposide. The clinical implications of increased sensitivity of 9NC-resistant tumors to some anticancer drugs are discussed.

Anticancer Activity of New Haloalkyl Camptothecin Esters against Human Cancer Cell Lines and Human Tumor Xenografts Grown in Nude Mice

All chemotherapeutic agents currently in use have a narrow window of therapeutic index of 1 to 1.2. Camptothecin ester compounds are reported to have a wider therapeutic index when being used to treat human xenografts in nude mice. As a continuous effort in searching for better chemotherapeutic agents for treating cancers, new haloalkyl camptothecin and 9-nitrocamptothecin ester derivatives 2a-b and 3a-d were prepared by respective acylation of camptothecin 1a and 9-nitrocamptothecin 1b with the corresponding acylating agents. These new derivatives were tested in vitro against 8 human cancer cell lines using 7 different concentrations ranging from 5 to 300 nM and also in vivo against various types of human tumor xenografts grown in nude mice. Most of these new compounds started showing inhibitory effects on the growth of 8 cancer cell lines at concentration of 80 nM and achieved greater than 70% inhibitions against these cell lines when the concentration increased to 300 nM. Compound 2a and 3a showed good activity against human tumor xenografts in nude mice. Compared to mother compound camptothecin, 3a was much less toxic in mice with a better therapeutic index, having the potential to be further developed as a safer treatment for cancers.

Acquisition of cellular resistance to 9-nitro-camptothecin correlates with suppression of transcription factor NF-κB activation and potentiation of cytotoxicity by tumor necrosis factor in human histiocytic lymphoma U-937 cells

Resistance of tumor cells to chemotherapeutic agents is a major problem in cancer therapy. Continuous exposure of human histiocytic lymphoma U-937 cells to 9-nitro-camptothecin (9NC), an inhibitor of the nuclear DNA topoisomerase I, induces resistance to this drug. Because of the involvement of the nuclear factor NF-kappa B in the expression of several growth regulatory genes, we examined the activation of this transcription factor in 9NC-resistant U-937 cells. We found that resistance to increasing concentrations of 9NC correlated with resistance to tumor necrosis factor (TNF)-dependent activation of NF-kappa B. However, the constitutive synthesis of NF-kappa B proteins remained unaffected. Cellular resistance was not unique to TNF, as other activators of NF-kappa B, including interleukin-1, phorbol ester and hydrogen peroxide, also had no effect. There was no difference between 9NC-sensitive and -resistant cells in the activation of NF-kappa B by okadaic acid. Other transcription factors, including AP-1 and Oct-1, were not affected in the resistant cells. When examined for the inhibitory subunit of NF-kappa B (I kappa B alpha), resistant cells showed a faster rate of resynthesis than the control. Interestingly, although 9NC resistance correlated with resistance to TNF-dependent NF-kappa B activation, TNF-dependent cytotoxicity in these cells was enhanced by several hundred fold despite a significant decrease in the number of TNF receptors. In conclusion, our results suggest that NF-kappa B activation may play a role in tumor cell killing by 9NC but not by TNF.

Synthesis and anti-tumor activity of alkenyl camptothecin esters

AbstractAim:To study the degrees of influence of changing side ester chains at position C20 of camptothecin on the anti-tumor activity of the molecules.Methods:The esterification reaction of camptothecin 1 and 9-nitrocamptothecin 2 with crotonic anhydride in pyridine gave the corresponding esters 3 and 4, respectively. The acylation of 1 and 2 with cinnamoyl chloride gave products 7 and 8. Epoxidation reaction of 3 and 4 with m-chloroperoxybenzoic acid in benzene solvent gave the products 5 and 6. Esters 3, 4, and 5 were tested for anti-tumor activity against 14 human cancer cell lines.Results:Both in vitro and in vivo anti-tumor activity studies for these esters were conducted and the data demonstrated positive results, that is, these esters were active against the tested tumor lines.Conclusion:Alkenyl esters 3 and 4 showed strong anti-tumor activity in vitro against 14 different cancer cell lines. Ester 3 was active against human breast carcinoma in mice and the toxicity of the agent was not observed in mice during the treatment, implying that this agent is effective for treatment with low toxicity.