Research Paper
In vitro toxicity to breast cancer cells of microsphere-delivered mitomycin C and its combination with doxorubicin

https://doi.org/10.1016/j.ejpb.2005.09.011Get rights and content

Abstract

To better understand and design microsphere systems for the locoregional delivery of anticancer drug combinations to solid tumors, (1) the cytotoxicity of microsphere-delivered mitomycin C (MMC) was evaluated and (2) various schedules of MMC and doxorubicin (Dox) were tested for their toxicity in vitro towards a murine breast cancer cell-line, EMT6. To accomplish the former MMC was loaded onto oxidized sulfopropyl dextran microspheres, released in a pH 7.4 buffer solution and tested for its potency against EMT6 cells versus a standard MMC solution. For the latter EMT6 cells were exposed to MMC or Dox as single agents or together using various drug concentrations and schedules. The efficacy of the treatments was measured using a clonogenic assay. MMC released from the microspheres showed similar activity against EMT6 cells to freshly prepared MMC solutions. Greater-than-additive toxicity was observed when MMC was given either simultaneously or after Dox exposure. In contrast, administration of MMC before Dox exposure resulted in toxicity that ranged from additive to sub-additive; this reduced toxicity was mainly due to increasing cell density arising from the design of the assay. These results help explain our previous in vivo investigations using microsphere-delivered combinations of the same agents in EMT6 solid tumors.

Introduction

The efficacy of conventional chemotherapy in the treatment of solid tumors can be limited by several factors including poor drug penetration into the tumor, systemic toxicity of chemotherapeutic agents, and the development of multidrug resistance [1], [2], [3], [4]. Although doxorubicin (Dox) is active against many solid tumors, and is one of the most widely used anticancer drugs in current clinical practice [5], [6], it causes many acute toxicities (e.g. hair loss, nausea, and vomiting) as well as irreversible cardiac toxicity, which restricts the repeated administration of the drug. Mitomycin C (MMC) is also a potent anticancer agent but is currently used mainly as a second line or adjunctive agent [5]. Its bioreductive activation mechanism (i.e. activation by cellular enzymes) has suggested its usefulness in targeting cells in the hypoxic regions of solid tumors [7]. Like Dox, the use of MMC is associated with a number of acute toxicities; however, it is the chronic toxicities (e.g. irreversible myelosuppression and hemolytic-uremic syndrome) that limit the clinical applications of the drug [5], [8].

In contrast to conventional systemic drug administration, locoregional delivery of anticancer agents to solid tumors by a slow-release system, such as microspheres (MS), does not rely on the tumor vasculature to deliver drug to target sites. As a result, it is possible to achieve both high local drug concentrations in tumor tissues for extended periods of time and reduced systemic circulation of the drug leading to higher therapeutic efficacy and lower systemic toxicity [9], [10], [11], [12], [13]. Locoregional delivery also has the potential to minimize the development of cellular drug resistance mechanisms (e.g. drug efflux transporters) that may be induced by continued exposure to non-lethal doses of anticancer drugs.

A MS system has previously been developed in our laboratory for the locoregional delivery of Dox to solid tumors [14], [15], [16]. Although the Dox-loaded MS (Dox-MS) demonstrated reduced systemic toxicity following intratumoral injection, the observed therapeutic efficacy was lower than expected based on the results of in vitro studies using cells in monolayer [14], [15], [16]. Solid tumors may contain hypoxic regions that can contribute to both chemotherapeutic and radiotherapeutic resistance [17]. It has been speculated that bioreductive agents like MMC may be more effective than Dox for locoregional delivery [17], [18], [19]. This speculation led us to develop a MS system using oxidized-dextran microspheres (Ox-MS) for the delivery of MMC [20]. This previous work has demonstrated that MMC-loaded Ox-MS (MMC-Ox-MS) is a better candidate for intratumoral administration to solid tumors than Dox-MS. Moreover, combining Dox-MS and MMC-Ox-MS for locoregional (intratumoral injection) yielded at least an additive toxicity in the EMT6 model tumor system without evidence of additional toxicity [21]. This previous in vivo work, which is summarized in Fig. 1, was only done with Dox-MS and MMC-Ox-MS being injected intratumorally at the same time. The effectiveness of drug combinations has been shown to be dependent on the timing of drug administration [22], [23]. Therefore, it was of interest to determine if the scheduling of one drug relative to the other was important for their increased activity.

While in vivo preclinical tests are critical for determining the efficacy of microsphere-delivered drugs and drug combinations, the accuracy, simplicity and sensitivity of in vitro cell culture assays allows for more rapid evaluation of the developed microsphere systems and various drug combinations. Thus, the present work was designed to investigate—[1] the in vitro cytotoxicity of microsphere-delivered MMC and [2] the ability of MMC/Dox combinations to inhibit the clonal expansion of murine breast cancer cells. That is, to define effective schedules for possible future in vivo studies using the developed MMC-Ox-MS and Dox-MS systems. A monolayer-cell system was employed to test the nature of the interaction of MMC and Dox at various concentrations and schedules to eliminate the interference of Dox binding in a multilayer-cell model [24], [25], which affects the available drug concentrations thus complicating the analysis of the concentration effect.

Section snippets

Materials

Sulfopropyl dextran MS (SP-MS; Sephadex SP C-25) and Dox were purchased from Sigma Chemical Company (St Louis, MO, USA). MMC was obtained from Faulding (Canada), Inc. (Kirkland, QUE, Canada). All cell culture plastic-ware was purchased from Sarstedt (Montreal, QUE, Canada). Cell culture media, α-Minimum Essential Medium (α-MEM), was acquired from the Ontario Cancer Institute (Toronto, Ont., Canada) and fetal bovine serum (FBS) was purchased from Cansera International Inc. (Etobicoke, Ont.,

Cytotoxicity of blank Ox-MS or SP-MS

The surviving fraction of EMT6/WT cells directly incubated with up to 10 mg/ml of blank Ox-MS or SP-MS for 7 days was measured (Test #1, Table 1). Using a clonogenic assay method, no change in cell survival was observed indicating that the MS themselves, or any materials released from the blank MS, are not toxic to the cells under the conditions studied (data not shown).

In vitro activity of MMC released from Ox-MS

As shown in Fig. 3, the in vitro activity of released MMC in the EMT6/WT cell-line (Test #3, Table 1) is similar to that of a

Discussion

We have previously studied the effectiveness of intratumorally injected microspheres containing Dox or MMC given alone or together in the in vivo EMT6 murine solid tumor system [13], [21]. Results indicated a greater than simple addition of individual drug toxicities when the drugs were administered together when percent tumor growth delay was used as an endpoint (Fig. 1). The present in vitro studies using the same tumor cell line yielded additional information regarding [1] the biological

Conclusion

MMC released from Ox-MS retains its activity in vitro when compared to fresh drug solutions. An increasing level of resistance is observed when EMT6/WT cells are treated with a toxic dose of Dox up to 72 h after MMC. This is due to both a cell density effect, whereby cells initially exposed to MMC undergo increased proliferation and a rapid loss in toxicity of the drug combination over the first 24 h between MMC and Dox administration. Conversely, a toxic dose of MMC applied simultaneously or up

Acknowledgements

This work is supported by the Canadian Institutes of Health Research (MOP53165, MOP63005). The authors would like to thank Dr I.F. Tannock for providing the cells and Ms G. Lim for statistical consultation. The Ontario Graduate Scholarship, University of Toronto Open Fellowship, Ben Cohen Bursary, and Pharmacy Alumni Graduate Student Award to RC are also gratefully acknowledged.

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