In this study, we demonstrated synergism between the effects of Gemcitabine and Cisplatin in several ovarian cancer cell lines. The most pronounced effects were observed in the Cisplatin-resistant cell lines. The management of platinum-resistant ovarian cancer remains unsatisfactory. Resistance to Cisplatin is a multifactorial phenomenon, the elements of which may be placed in three general categories: (a) reduced intracellular accumulation of Cisplatin, (b) elevated levels of glutathione and metallothionein, and (c) increased tolerance or repair of DNA damage[
25‐
27]. Because Cisplatin acts by forming intrastrand and interstrand DNA cross-links and DNA protein cross-links, thus resulting in DNA damage, overcoming these lesions with heightened repair mechanisms is an important factor for Cisplatin resistance[
28]. We previously reported that the PI3K/Akt cascade plays a role in Cisplatin resistance[
12‐
14]. It has been demonstrated that Cisplatin resistance is primarily due to the reduction of DNA damage and the evasion of apoptosis, the latter of which includes the loss of damage recognition and activation of the PI3K/Akt pathway[
29,
30]. Although it is well known that Gemcitabine is one of the most active drugs in patients with platinum-resistant ovarian carcinoma, the mechanisms underlying these phenomena have not yet been characterized. In previous studies, Peters G.J., et al. showed that the combination of Cisplatin and Gemcitabine produces synergistic effects in platinum-resistant ovarian cancer cells[
28]. Moufarij M.A., et al. and Ledermann J.A., et al. demonstrated the inhibitory effects of Gemcitabine on the repair of Cisplatin-induced intrastrand adduction and interstand cross-linking in platinum-resistant ovarian cancer cells[
31,
32]. However, these authors did not indicate either the mechanisms or the signal cascades underlying the synergistic effects of such combination treatment with Cisplatin and Gemcitabine in platinum-resistant ovarian cancer cells. In this study, we found that combination treatment with Cisplatin and Gemcitabine significantly inhibits the level of the Cisplatin-induced Akt activity in Cisplatin-resistant cell lines (Caov-3 and A2780CP cells). We clarified that Gemcitabine exerts its cytotoxic effects by interfering with the antiapoptotic machinery and significantly enhancing PARP cleavage. Moreover, we found that combination treatment with Cisplatin and Gemcitabine significantly inhibits both the levels of invasive activity and the Cisplatin-induced MMP9 activity in Caov-3 and A2780CP cells; however, these effects are not achieved with treatment with either Cisplatin or Gemcitabine alone. We found that Cisplatin induced the VEGF expression in the Cisplatin-resistant cell lines. Combined treatment with Cisplatin and Gemcitabine significantly inhibits the Cisplatin-induced VEGF expression in platinum-resistant ovarian cancer cells, although no such effects are observed after treatment with either Cisplatin or Gemcitabine alone. Moreover, we found that combined treatment consisting of Cisplatin and Gemcitabine significantly inhibits intra-abdominal tumor cell dissemination and ascites production compared to that observed following treatment with Cisplatin or Gemcitabine alone. Whether these phenomena are represented by other anticancer agents is unclear. It has been reported that Topotecan, a topoisomerase-1 inhibitor, inhibits the Akt and VEGF cascade in platinum-resistant ovarian cancers[
16]. However, 5-fluorouracil (5-FU), which exhibits similar
in vitro findings to the thymidylate synthase inhibitor, has no effect on the synergistic inhibition of cell viability in Caov-3 cells after combined treatment with Cisplatin and 5-FU, and does not inhibit the Cisplatin-induced Akt activity in Caov-3 cells (Additional file
4: Figure S4A, S4B). We found that Gemcitabine is a most effective molecular targeting agent with the ability to suppress the Akt kinase activity, the ability to induce cellular apoptosis and an anti-angiogenic activity in platinum-resistant ovarian cancer cell lines.
In the current clinical trial, the response rate to treatment with Gemcitabine and Cisplatin ranged from 16% to 64% in the Cisplatin-resistant ovarian carcinomas with measurable disease, although the frequency of grade 3 and 4 neutrophil toxicity ranged from 20% to 81.5% and the platelet toxicity ranged from 36% to 96.5%[
19,
20,
33‐
35]. It is said that one must take into consideration that the use of less toxic and single agent drugs may be worthwhile in a treatment process that may span many years. However, previous clinical studies have shown that Gemcitabine and Cisplatin can be used to treat patients who have developed platinum-resistance and failed to respond to other second-line therapies over time, using a combination therapy that may result in an appropriate response[
20]. In the present study, we were unable to show whether other factors, such as a reduced accumulation of Cisplatin or elevated levels of glutathione and metallothionein, affect the resistance of Cisplatin-resistant ovarian cancer. Such knowledge may be helpful for developing future strategies to more effectively circumvent the multifactorial mechanisms of platinum resistance. We believe that our data provide scientific justification for both previous and future trials of combination treatment with Gemcitabine and Cisplatin in patients with platinum-resistant ovarian cancer.
In conclusion, we herein demonstrated that Gemcitabine inhibits the Akt kinase activity and angiogenetic activity following treatment with Cisplatin in platinum-resistant ovarian cancer cells. These results provide a rationale for using Gemcitabine in clinical regimens containing molecular targeting agents against platinum-resistant ovarian cancers.