Antitumor Alkylating Agents Research Articles

Re: Rapid Acquisition of Multicellular Drug Resistance After a Single Exposure of Mammary Tumor Cells to Antitumor Alkylating Agents

Re: Rapid Acquisition of Multicellular Drug Resistance After a Single Exposure of Mammary Tumor Cells to Antitumor Alkylating Agents

Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents.

Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating agents in vivo after serial passage into syngeneic mice manifest their resistance in vitro only when cultured as three-dimensional multicellular aggregates or spheroids. We examined whether a single exposure of mouse EMT-6 or human MDA-MB-231 breast cancer cells to alkylating agents in vitro is sufficient for the induction of a resistance phenotype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. Mouse EMT-6 and human MDA-MB-231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed after a subsequent alkylating-agent exposure of cells cultured as monolayers or three-dimensional aggregates. A single in vitro exposure to 12.5-microM cisplatin (CDDP) for 5 days or 25 microM 4-hydroperoxycyclophosphamide (4-O2H CTX) for 1 or 3 days without changing the medium was sufficient to induce transient but substantial resistance in EMT-6 cells as determined by clonogenic assays. Such resistance was not detected when monolayer cell cultures were used. The concentration of 4-O2H-CTX and the length of time the cells remained in three-dimensional culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimensional cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architecture of the cell aggregates, which now formed much more compact multicellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enough to bring about resistance in MDA-MB-231 cells detectable only in three-dimensional cultures, as well as the change in three-dimensional architecture. Rapid acquisition of resistance likely represents a physiologic mechanism of adaptation operative at the multicellular level rather than a stable genetic change and may be one of the reasons for the rapid development of drug resistance acquired by tumors in vivo. In vivo drug exposure may result in transient and low levels of drug resistance that may nevertheless be clinically relevant.

Antitumor alkylating agents: in vitro cross-resistance and collateral sensitivity studies.

Cell lines resistant to five antitumor alkylating agents (CDDP, PAM, 4-HC, HN2, and BCNU) were developed from five parental human tumor lines representative of solid tumors with a range of sensitivities to antitumor alkylating agents. The parental cell lines were SCC-25 squamous carcinoma of the head and neck, MCF-7 breast carcinoma, SW2 small-cell lung cancer, SL6 non-small-cell lung carcinoma, and G3361 melanoma. Survival curves using colony formation as the endpoint were generated for each of the 25 cell lines to each of the five alkylating agents. Comparison of the drug concentrations that reduced the survival of the alkylating agent-resistant cell lines by 90% (IC90 values) with the IC90 values obtained for the corresponding parental cell lines was used as a measure of the resistance/sensitivity of the alkylating agent-resistant lines to each drug tested. Although cross-resistance among the alkylating agents was generally uncommon, several patterns of response emerged. Cross-resistance occurred in 27 of the 105 determinations and occurred most frequently in the cell lines in which resistance was developed to PAM (57%) or BCNU (38%). Cross-resistance to HN2 occurred most frequently. Collateral sensitivity was equally as common, occurring in 25 of the 105 determinations. Collateral sensitivity occurred most frequently in the cell lines made resistant to 4-HC. The 4-HC-resistant cell lines were most frequently collaterally sensitive to PAM and to BCNU. Cross-resistance developed most frequently in the MCF-7 breast carcinoma and SCC-25 squamous-cell carcinoma cell lines, whereas collateral sensitivity developed most frequently in the SW2 small-cell lung cancer line and the G3361 melanoma cell line and least frequently in the MCF-7 breast carcinoma cell line and the SL6 non-small-cell lung cancer cell line. The implication of these findings for the development of strategies for clinical treatment are discussed.

Effect of hemoglobin solution on the response of intracranial and subcutaneous 9L tumors to antitumor alkylating agents

The 9L gliosarcoma growing subcutaneously in the hind leg of the Fisher 344 rat contains major areas of severe (< 5 mmHg) hypoxia, making up about 49% of the tumor. Intravenous administration of an ultrapurified polymerized bovine hemoglobin solution (8 ml/kg) along with normal air breathing reduces the percentage of severe hypoxia to about 24% and increases oxygenation throughout the tumor. Coadministration of the hemoglobin solution increased the tumor growth delay of subcutaneously implanted 9L tumors treated with carmustine (BCNU), cyclophosphamide, or ifosfamide but did not significantly change the tumor growth delay produced by cisplatin (CDDP). Coadministration of the hemoglobin solution with each of the four antitumor alkylating agents resulted in a near doubling of the percentage of increase in life span in animals bearing intracranial tumors treated with the combination as compared with animals treated with the drugs alone. Increases in serum blood urea nitrogen (BUN) and creatinine levels in treated animals returned to normal by 11 days posttreatment. Major changes in liver enzymes occurred with the combination of cyclophosphamide and the hemoglobin solution at 4 days posttreatment; however, these values returned to the levels in the untreated control animals within 1 week thereafter. These results indicate that further exploration of the use of hemoglobin solutions in cancer therapy is warranted.

Enhancement of alkylating agent activity by SR-4233 in the FSaIIC murine fibrosarcoma.

The most commonly used antineoplastic drugs are more cytotoxic toward normally oxygenated tumor cells than toward hypoxic tumor cells. To examine the ability of SR-4233, a new cytotoxic agent, to overcome the resistance of hypoxic tumor cells to antitumor alkylating agents, we tested the cytotoxic effect of SR-4233 alone and in combination with varying doses of cisplatin (CDDP), cyclophosphamide (CPM), carmustine (BCNU), or melphalan (L-PAM) on tumor cells and bone marrow cells isolated from C3H/FeJ mice bearing the FSaIIC fibrosarcoma. When SR-4233 alone was given, tumor cell killing was limited. When SR-4233 was administered just before single-dose treatment with CDDP, CPM, BCNU, or L-PAM, however, marked dose enhancement leading to increased cytotoxic effects on tumor cells and on bone marrow cells was observed. Similar experiments with tumor cell subpopulations, selected by Hoechst 33342 dye diffusion, confirmed that while cytotoxicity to both bright (oxygenated) and dim (hypoxic) cells was increased by combining each alkylating agent with SR-4233, the enhancement of the effect was relatively greater in the subpopulation of dim cells. The delay in the growth of tumors in animals treated with the combination of SR-4233 and CDDP, CPM, or L-PAM was 1.6-fold to 5.3-fold greater than that in animals treated with each alkylating agent alone. Our results suggest that SR-4233 may have the potential to improve the clinical efficacy of commonly used antitumor alkylating agents.

Enhancement by perflusion emulsion (Oxygent) and carbogen breathing of the tumor growth delay of the FSaIIC fibrosarcoma after treatment with antitumor alkylating agents.

Many anticancer drugs require oxygen to be cytotoxic, or are selectively cytotoxic toward cells under oxygenated conditions. The effects of the dilute perfluorochemical emulsion Fluosol-DA with a wide variety of chemotherapeutic agents have been explored; however, it has not been possible to determine the optimal level of circulating perfluorochemical emulsion with anticancer drugs because the volume of Fluosol that may be administered in limited. Using a concentrated 90% Perflubron emulsion, Oxygent, a wide range of perfluorochemical emulsion doses have been examined in combination with melphalan, cyclophosphamide and BCNU in a murine solid tumor model. When Oxygent was administered by injection i.v. just prior to the injection of melphalan (10 mg/kg), the greatest tumor growth delays were obtained with Oxygent levels between 4 and 12 g PFC/kg. With each of these drugs the greatest tumor growth delays were obtained when the drug was prepared in the emulsion and the combination injected i.v. In each case, each dose of drug was followed by 6 h. of breathing carbogen.

Use of perfluorochemical emulsions in cancer therapy.

Over the past 10 years, the use of perfluorochemical emulsions (PFCE) and carbogen or oxygen breathing has been explored as an adjuvant to radiation therapy and/or chemotherapy in the treatment of solid tumors. The rationale for the use of PFCE and oxygen breathing in this therapeutic setting is that solid tumor masses contain areas of hypoxia which are therapeutically resistant. Since x-rays and many chemotherapeutic agents require oxygen to be maximally cytotoxic and most normal tissues are well-oxygenated, the additional oxygen put in circulation by the PFCE should not increase the normal tissue toxicities produced by the various therapies. The largest body of preclinical work and all of the clinical studies in cancer conducted with PFCE, thus far, have been done with Fluosol-DA, 20%. Oxygen microelectrode studies have confirmed increased oxygenation in previously hypoxic tumor regions after the administration of Fluosol-DA and carbogen breathing. The preclinical studies have shown very positive effects with single dose and fractionated radiation in several rodent solid tumor models. Many widely used anticancer drugs including antitumor alkylating agents and adriamycin are enhanced by PFCE and carbogen breathing for longer time periods (6 h). More recently, several experimental concentrated PFCE preparations have become available and work with these is actively under way in several laboratories. Clinical studies with radiation and four or five chemotherapeutic drugs as single agents have indicated that Fluosol-DA followed by oxygen breathing can be administered safely in a variety of cancer therapeutic settings. Further clinical studies with Fluosol-DA are planned.

ChemInform Abstract: Syntheses and Stereochemistry of the Antitumor Alkylating Agents Related to 4‐Hydroperoxyisophosphamide.

AbstractDurch Ozonolyse von O‐(3‐Butenyl)‐phospholdiamidaten mit 2‐Äthylaminogruppen, die in 3‐Position unsymmetrisch mit Cl, Br, CH3SO2 und p‐Tosyl substituiert sind, werden 4‐Hydroperoxy‐1,3,2‐oxazaphosphorinan‐2‐oxide synthetisiert.

Syntheses and Stereochemistry of the Antitumor Alkylating Agents Related to 4-Hydroperoxyisophosphamide

Syntheses and Stereochemistry of the Antitumor Alkylating Agents Related to 4-Hydroperoxyisophosphamide