Abstract
A new platinum derivative of the form H[Pt(IV)(Hdigly)Cl2(OH)2] (Hdigly⩵glycylglycine) damaged the Candida albicans cell membrane and inhibited the growth of the cells. The cytotoxic activity of H[Pt(IV)(Hdigly)Cl2(OH)2] on mammalian cells was 10-fold lower than that of cis-diammine-dichloroplatinum (cisplatin). Substitution of platinum for peptides is effective for enhancement of antifungal activity and reduction of the toxicity to mammalian cells.
The incidence of infection caused by opportunistic fungal pathogens such as Candida albicans has increased markedly with increases in frequency of organ transplantation, cancer chemotherapy, and numbers of patients with human immunodeficiency virus infection (2, 15). cis-diamminedichloroplatinum(II), or cisplatin, is one of the most commonly used anticancer drugs and is effective against sarcoma, leukemia, and mammary carcinoma (7, 8, 13). Since cisplatin is associated with side effects such as nephrotoxicity, nausea, and vomiting, many platinum complexes have been prepared in the hope of developing new derivatives with more potent activity and less toxicity. These trials indicated that the toxicity of platinum could be regulated by addition of imino ether, functional amines, and amino acid esters (5, 6, 14). Conversion of platinum(II) complexes to platinum(IV) analogs is another approach for preparation of new types of cytotoxic platinum complexes and moderating the toxicity of platinum(II) complexes. In the present study, we analyzed the mode of anti-Candida activity of a new platinum derivative, H[Pt(IV)(Hdigly)Cl2(OH)2] (molecular weight, 432.1).
H[Pt(IV)(Hdigly)Cl2(OH)2] was prepared using K2[Pt(II)Cl4] as the starting material, according to a method reported previously (10). Twelve isolates of C. albicans (NIH A207; IFO 0578 and 1060; and JCB 1542, 1621, 2072, 2074, 2076, 2077, 2085, 2374, and 2900) were used for testing the antifungal activity of H[Pt(IV)(Hdigly)Cl2(OH)2]. MIC tests were performed by a modification of the M27-A method of the National Committee for Clinical Laboratory Standards (9). Each test was repeated at least three times, and MICs were constant. The growth of each C. albicans strain was inhibited by addition of H[Pt(IV)(Hdigly)Cl2(OH)2] (Table 1). The mean MIC of H[Pt(IV)(Hdigly)Cl2(OH)2] was threefold lower than that of cisplatin, suggesting that the oxidation of glycylglycine and its substitution with platinum(II) enhanced the anti-Candida activity of the platinum derivative. To analyze the damage to the C. albicans membrane by H[Pt(IV)(Hdigly)Cl2(OH)2], potassium ion content and protein release were measured (3, 12). The potassium ion content in C. albicans (107 cells) treated with the Pt derivative (0.1 mM) decreased to one-third that of an untreated control. When C. albicans protoplasts (106 cells) were exposed to 0.1 mM H[Pt(IV)(Hdigly)Cl2(OH)2], 30% lysis was observed within 4 h. These results suggested that H[Pt(IV)(Hdigly)Cl2(OH)2] might damage the C. albicans cell membrane. In C. albicans protoplasts treated with cisplatin, protein release was not detected (data not shown).
TABLE 1.
MICs of various drugs for C. albicans
| C. albicans strain | MIC (mM) of:
|
||
|---|---|---|---|
| H[Pt(IV)(Hdigly) Cl2(OH)2] | Cisplatin | Fluconazole | |
| NIH A207 | 0.098 | 0.172 | 0.043 |
| IFO 0579 | 0.076 | 0.220 | 0.021 |
| IFO 1060 | 0.066 | 0.320 | 0.023 |
| JCB 2077 | 0.065 | 0.222 | 0.022 |
| JCB 2076 | 0.065 | 0.350 | 0.021 |
| JCB 2074 | 0.067 | 0.304 | 0.021 |
| JCB 2072 | 0.089 | 0.172 | 0.026 |
| JCB 2900 | 0.085 | 0.312 | 0.024 |
| JCB 1542 | 0.077 | 0.264 | 0.021 |
| JCB 1621 | 0.071 | 0.170 | 0.029 |
| JCB 2374 | 0.088 | 0.270 | 0.024 |
| JCB 2085 | 0.072 | 0.240 | 0.048 |
| Mean ± SE | 0.077 ± 0.003 | 0.251 ± 0.018 | 0.027 ± 0.003 |
Cytotoxicity of H[Pt(IV)(Hdigly)Cl2(OH)2] on mammalian cells was measured by the Alamar blue method (1). Briefly, Meth A fibrosarcoma, MH134Y hepatoma, and HEp-2 carcinoma cells were cultured in 10% fetal bovine serum–RPMI 1640. Bone marrow cells from the thigh bone of BALB/c mice (SLC, Shizuoka, Japan) were cultured in 10% fetal bovine serum–RPMI 1640 with recombinant mouse granulocyte-macrophage colony-stimulating factor (10 U/ml; Genzyme Co.). These cells (106 cells/ml) were mixed with cisplatin or H[Pt(IV)(Hdigly)Cl2(OH)2] and then incubated at 37°C for 48 h. After incubation, Alamar blue (Alamar Biosciences Inc.) was added to these cells and the absorbance at 540 nm minus that at 620 nm was measured to estimate the number of viable cells. As shown in Table 2, cisplatin effectively inhibited the growth of these cells, but the activities of H[Pt(IV)(Hdigly)Cl2(OH)2] were significantly lower than those of cisplatin. Cytotoxicity of fluconazole to Meth A fibrosarcoma cells was similar to that of H[Pt(IV)(Hdigly)Cl2(OH)2] (data not shown).
TABLE 2.
MICs of various drugs for mammalian cells
| Drug | Mammalian cells | MIC (mM) |
|---|---|---|
| Cisplatin | Bone marrow | 0.019 |
| Meth A fibrosarcoma | 0.017 | |
| MH134Y hepatoma | 0.010 | |
| HEp-2 carcinoma | 0.033 | |
| H[Pt(IV)(Hdigly)Cl2(OH)2] | Bone marrow | 0.280 |
| Meth A fibrosarcoma | 0.220 | |
| MH134Y hepatoma | 0.175 | |
| HEp-2 carcinoma | >0.333 |
Cisplatin, a platinum-containing anticancer agent, has been used in cancer chemotherapy because of its strong cytotoxicity, but cisplatin is not used as an antifungal agent due to its serious side effects, such as immunosuppression (4). Previously, we reported that the substitution with platinum(II) for peptides down-regulated the cytotoxicity of platinum on mammalian cells (11). The growth of C. albicans was strongly inhibited by the addition of H[Pt(IV)(Hdigly)Cl2(OH)2], and the MIC indicated that this effect was threefold stronger than that of cisplatin (Table 1). The cytotoxic activities of H[Pt(IV)-(Hdigly)Cl2(OH)2] for mammalian cells were significantly lower than those of cisplatin, suggesting that H[Pt(IV)-(Hdigly)Cl2(OH)2] selectively injured Candida but not mammalian cells (Table 2). Because the leakage of potassium ions from C. albicans was increased by treatment with H[Pt(IV)(Hdigly)Cl2(OH)2] and this Pt derivative showed lytic activity against C. albicans protoplasts, it was suggested that destruction of the Candida cell membrane was a cause for the anti-Candida activity of H[Pt(IV)(Hdigly)Cl2(OH)2].
These results suggested that substitution of platinum for peptides may be effective for enhancement of antifungal activity and reduction of the cytotoxicity to mammalian cells.
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