Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive ovarian carcinoma cell lines

Abstract

Although epithelial ovarian cancers (EOCs) are initially treated with platinum-based chemotherapy, EOCs vary in platinum responsiveness. Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive EOC cell lines is valuable for development of therapeutic strategies to avoid platinum inefficacy and to exploit platinum sensitivity. TOV-21G devoid of FANCF expression, OV-90 and SKOV-3 were employed as examples of platinum-sensitive, platinum-intermediate and platinum-resistant cell lines, respectively. Antineoplastic agents examined included mitomycin C, doxorubicin, etoposide, gemcitabine, chlorambucil, paclitaxel, triapine and X-rays. Their effectiveness against cell lines was analyzed by clonogenic assays. Cytotoxic profiles of mitomycin C and carboplatin were similar, with mitomycin C exhibiting greater potency and selectivity against TOV-21G than carboplatin. Cytotoxic profiles of doxorubicin, etoposide and X-rays overlapped with that of carboplatin, while OV-90 overexpressing Rad51 was more resistant to chlorambucil than SKOV-3. The efficacy of paclitaxel and triapine was independent of platinum sensitivity or resistance. Consistent with these cytotoxic profiles, cisplatin/mitomycin C, triapine, and paclitaxel differed in the capacity to induce phosphorylation of H2AX, and produced unique inhibitory patterns of DNA/RNA syntheses in HL-60 human leukemia cells. Paclitaxel and triapine in combination produced additive antitumor effects in M109 murine lung carcinoma. In conclusion, mitomycin C is potentially more effective against Fanconi anemia pathway-deficient EOCs than carboplatin. Doxorubicin and etoposide, because of their overlapping cytotoxic properties with carboplatin, are unlikely to be efficacious against platinum-refractory EOCs. Paclitaxel and triapine are effective regardless of platinum sensitivity status, and promising in combination for both platinum-sensitive and platinum-refractory EOCs.

Keywords: Fanconi anemia pathway; carboplatin; mitomycin C; paclitaxel; platinum responsiveness; triapine.

Figure 1

Diversity in carboplatin sensitivity among EOC cell lines, and characteristics of SKOV-3, OV-90 and TOV-21G cells employed in the study. Panel A, the bar graph was generated using the reported IC₅₀ values for carboplatin in a panel of 39 EOC cell lines [26]. Panel B, the information about the origin, phenotypes and mutations in each cell line were collected from ATCC catalog and earlier reports [–25].

Figure 2

Cytotoxic profiles of various antineoplastic agents against SKOV-3, OV-90 and TOV-21G cells. Panel A, cytotoxicity was measured using clonogenic assays. For the guanine O⁶-chloroethylating agent 90CE, cells were pretreated with 10 µM O⁶-benzylguanine for 2 h to abrogate activity of O⁶-methylguanine-DNA methyltransferase (MGMT). O⁶-Benzylguanine was present in the medium throughout treatment and colony propagation. Panel B, for clarification, values of LC₉₀ (concentration giving 90% lethality) and fold resistance (degrees of resistance in OV-90 and SKOV-3 relative to TOV-21G) are tabulated.

Figure 3

Western analyses of relevant DNA damage response proteins following treatment with carboplatin or triapine. Whole cell extracts were prepared from SKOV-3, OV-90 and TOV-21G cells untreated, or treated with 10 µM carboplatin or 1 µM triapine for 24 h. BRCA2, p53, and RPA70 were resolved by 6% PAGE. Rad51, PCNA, γH2AX, DCK and MGMT were resolved by 15% PAGE. RPA70, replication protein A 70 kDa DNA-binding subunit; PCNA, proliferating cell nuclear antigen; DCK, deoxycytidine kinase; MGMT, O⁶-methylguanine-DNA methyltransferase.

Figure 4

Inhibition of DNA/RNA syntheses and induction of γH2AX by cisplatin, mitomycin C, triapine or paclitaxel. Panels A, for short-term treatment, HL-60 cells (2 × 10⁶ cells/ml) were treated with various agents for 2 h, and labeled with ³H-thymidine or ³H-uridine for 1 h. For long-term treatment, HL-60 cells (4 × 10⁵ cells/ml) were treated with various agents for 18 h, condensed to 2 × 10⁶ cells/ml and labeled with ³H-thymidine or 3H-uridine for 1 h. Panels B, HL-60 cells (4 × 10⁵ cells/ml) were treated with various agents for 18 h. Histones extracted from intact cells were subjected to 15% PAGE and western analyses using an anti-γH2AX antibody. For a loading control, duplicate gels were used to stain core histones with GelCode Blue Safe Protein Stain (Thermo Scientific).

Figure 5

Antineoplastic agents cataloged according to their effectiveness against TOV-21G, OV-90 and SKOV-3 (A) and distinguishable cytotoxic mechanisms of carboplatin/mitomycin C, triapine and paclitaxel (B).

Figure 6

Antitumor activity of paclitaxel and triapine, alone or in combination, against Madison 109 murine lung carcinoma. Tumor-bearing mice (5 mice/group) were treated with i.p. injection of paclitaxel at a dose of 10 mg/kg once a day for 4 days, triapine isethionate salt (triapine ITS) at a dose of 10 mg/kg (equivalent to 6 mg/kg of triapine) twice a day, 8 h apart, for 4 days, or combination of both. Paclitaxel dissolved in DMSO at 10 mg/ml was injected in a volume of 10 µl/10 g weight using insulin syringes. Triapine ITS dissolved in water at 1 mg/ml (pH 4.5) was neutralized with 2 molar equivalents of NaHCO₃ immediately before injection.