Metastatic melanoma has poor prognosis and is usually refractory to most

Metastatic melanoma has poor prognosis and is usually refractory to most standard chemotherapies. for TMZ/ABT-737 synergy as exhibited by a p53-null collection, indicating that TMZ and ABT-737 together induce Noxa in a p53-impartial fashion. These results demonstrate that targeting anti-apoptotic Bcl-2 users is usually a encouraging method for treating metastatic melanoma, and that clinical trials with TMZ and Bcl-2 inhibitors are warranted. Introduction The incidence of metastatic melanoma has increased rapidly in Rabbit Polyclonal to PTRF recent decades, but regrettably there has been little improvement in therapeutic efficacy [1]. Dacarbazine is usually the standard first-line treatment for 520-34-3 supplier advanced melanoma, but its response rate is usually poor, averaging around 15% with no improvement in survival period [1], [2]. Temozolomide (TMZ), which spontaneously decomposes into the active metabolite of dacarbazine [3], is usually frequently used off-label in place of dacarbazine because of its ease of use and bioavailability; however, its response rate is usually equally poor. Chemotherapeutic brokers 520-34-3 supplier that can be combined with TMZ to increase its response rate are therefore highly sought after, as an effective combination would have immediate clinical application. Brokers that have been combined with TMZ in clinical trials include arsenic trioxide and ascorbic acid [4], cisplatin [5], and thalidomide [6]; regrettably, they were found to have no benefit. Whether and to what degree TMZ induces apoptosis in melanoma cells is usually a subject of argument. Some studies have shown that clinically relevant doses of TMZ do not induce 520-34-3 supplier significant levels of apoptosis in melanoma cells studies for both melanoma and other malignancy cells have shown that the Mcl-1/Noxa ratio is usually crucial for determining resistance or sensitivity to ABT-737 [13], [14], [15], [16], [17], [18]. In the present study, we tested the BH3-only mimetic ABT-737 in combination with the generally used alkylating agent TMZ, and found 520-34-3 supplier strong synergistic induction of apoptosis in several melanoma cell lines within a short time period, and a significant reduction in tumor growth in a mouse xenograft model. We found that Noxa was induced by the combination treatment, but not by single drug treatments, and that knockdown of Noxa almost completely abrogated cell death induced by the combination. Although induction of p53 was sufficient to cause Noxa-mediated cell death, it was not necessary, indicating that the ABT-737/TMZ combination induces Noxa through a p53-impartial pathway. Results ABT-737 synergistically induces apoptosis in melanoma cells when combined with temozolomide MTS experiments (Fig. 1A and data not shown) showed that TMZ alone reduced total viability, and that this was reduced further in the presence of ABT-737. IC50 values for each drug at 72 h are outlined in Table H1, and time-course data are shown in Fig. S1. Median effect analysis showed that the combination was synergistic over a wide range of drug concentrations at 72 h (Fig. S2), with combination index (CI) values ranging from 0.1 to 0.4 for 1205Lu and 0.3 to 0.8 for A375. The visual appearance of the cells (Fig. 1B) made it obvious that the combination of ABT-737 and TMZ induced cell death whereas TMZ alone primarily reduced cell proliferation by 72 h. To quantify the level of apoptosis in combination treatments compared to single agent treatments, we performed Annexin V assays after cells were uncovered to 400 M TMZ alone, 3.3 M ABT-737 alone, or both brokers in combination for 72 h for several cell lines. Physique 1C shows that ABT-737 and TMZ alone induced little cell death above the controls treated with vehicle. For combination treatments however, high levels of cell death were found for all cell lines, indicating a synergistic effect between TMZ and ABT-737. Physique 1 TMZ and ABT-737 cause strong, synergistic cell death in.