Platinum (Pt) drugs cisplatin and oxaliplatin play a central role in cancer chemotherapy. The prototype drug cisplatin is used in ovarian, testicular, bladder, small cell lung and head and neck cancers among many others. Oxaliplatin while active in some of the above cancers is uniquely active in colon carcinoma when administered in combination with 5-fluorouracil and has been approved for clinical use for this indication. It is widely believed that Pt–DNA adduct formation is the key lesion for the activity of these agents preventing DNA replication and transcription and the resulting DNA damage itself inducing down-stream signaling leading to apoptotic cell death [⁴⁵]. However, all platinum drugs also bind to RNA and protein within the cell and these interactions may also play a role in their mechanism of action. Mechanisms of resistance to platinum agents are multifactorial including impaired drug accumulation, inactivation by thiols, enhanced DNA repair, replicative bypass, tolerance to damage and altered down-stream signaling [¹⁷, ³⁶, ³⁷, ³⁹].

Microarray studies on oxaliplatin- [⁴, ⁴²] or cisplatin [⁴²]-treated cells have suggested that platinum drugs can affect the expression of genes in many other pathways and identified spermidine/spermine N¹ acetyl transferase (SSAT) a key polyamine catabolic pathway enzyme as a prominently up-regulated gene. Other microarray studies have identified SSAT up-regulation by oxaliplatin in HCT-116 colon carcinoma cells [¹, ⁴] and MCF-7 cells and by 5-fluorouracil (5-FU) in HCT-116 cells [¹, ⁴⁷] and MCF-7 cells [³¹]. Our own gene expression profiling studies of oxaliplatin- or cisplatin-treated A2780 ovarian carcinoma cells indicated that both drugs not only up-regulate SSAT gene expression but they also down-regulate the polyamine biosynthetic pathway enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) [⁴²]. We and others have found that oxaliplatin up-regulated SSAT expression is not translated into protein and/or activity to any significant extent [¹, ²³, ²⁴]; but when combined with the polyamine analog N¹,N¹¹diethylnorspermine (DENSPM), a synergistic induction of both SSAT mRNA [¹, ²³, ²⁴], protein [¹] and activity occurs [²³, ²⁴]. This is not surprising because both SSAT mRNA and protein have short half-lives and polyamine analogs have been shown to facilitate increased transcription and the stability of the transcript [⁷] as well as increase the translation and stability of the SSAT protein [⁷].

Polyamines, small cationic molecules which are essential for cell proliferation and survival, are a well-recognized therapeutic target. SSAT is the key enzyme in polyamine catabolism and its induction leads to inhibition of cell growth and apoptosis [⁹, ²⁵]. Because polyamine levels and metabolic rates are higher in tumor than in normal cells [³⁴], drugs have been designed to either up-regulate polyamine catabolism or down-regulate their biosynthesis. The clinically tested polyamine analog DENSPM targets the polyamine pathway by up-regulating catabolism via SSAT induction and decreasing their biosynthesis via down-regulation of the biosynthetic enzymes ODC and SAMDC [⁴¹]. DENSPM has been tested in Phase I and Phase II clinical trials [¹¹, ¹⁹, ⁴⁰, ⁴⁶], but with no demonstrated single agent activity. A few studies, including our own, have shown that oxaliplatin and DENSPM combination synergistically up-regulates the expression of SSAT in A2780 [²³] and HCT-116 cells [¹, ¹⁰, ²⁴] with concurrent decline in polyamine pools [¹⁰, ²³, ²⁴]. Others have shown that DENSPM combined with paclitaxel or 5-FU produces a synergistic induction of SSAT mRNA and activity in MDA-MB-231 cells [³³]. The synergistic induction of SSAT protein and activity by oxaliplatin/DENSPM combinations in HCT-116 and other colon carcinoma cell lines and A2780 ovarian carcinoma cells result in synergistic cell kill [¹, ²⁴] or enhancement of oxaliplatin cytotoxicity [²³]. While exhibiting lower levels of synergy in SSAT induction after oxaliplatin/DENSPM combination, the oxaliplatin-resistant HCT-116 cells showed an enhanced cell death following treatment with this drug [¹]. In addition, siRNA-mediated down-regulation of SSAT resulted in decreased cell kill [¹]. Cisplatin, DENSPM combinations have been shown to produce synergistic cell death in MDA-MB-231 breast cancer cells [³³]. Mareverti et al., have shown that another polyamine analog N¹, N¹²-bis(ethyl)spermine (BESPM) induced SSAT at much lower levels in cisplatin-resistant C13* ovarian carcinoma cells than the parental cells but the combinations of cisplatin and BESPM still produced synergistic cell kill [²⁹]. All these lines of evidence suggest SSAT induction to be a potential therapeutic target in cancer chemotherapy.

While recent studies focused on combining the clinically relevant DENSPM with oxaliplatin, detailed studies of cisplatin combinations with DENSPM on polyamine pathway and growth are lacking in ovarian cancer cells. The aim of the present work has been to conduct a simultaneous comparison of the interactions of DENSPM with cisplatin and oxaliplatin under identical conditions at the level of SSAT mRNA, activity, polyamine pools and the effect of the combination on inhibition of cell growth in A2780 ovarian carcinoma cells and their corresponding oxaliplatin and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively. The mechanisms of resistance for both A2780/CP and A2780/C10B have been described previously and include decreased drug accumulation and increased glutathione for both cell lines and in addition increased repair of DNA damage in cisplatin-resistant A2780/CP [²¹, ²²].

The polyamine analog DENSPM is a proven inducer of SSAT in many cell types and has also been shown to down-regulate the key biosynthetic pathway enzymes ODC and SAMDC. Induction of SSAT activity and depletion of polyamine pools especially that of spermidine have been shown to be related to inhibition of cancer cell growth [⁸, ²⁷, ³⁵, ⁴⁴]. Therefore, this agent has great potential as a means of targeting the polyamine pathway as a mechanism to kill cancer cells and has accordingly been tested clinically [¹¹, ¹⁹, ⁴⁰, ⁴⁶]. While this drug showed no single agent activity in the clinic, combination studies have yet to be reported. Several in vitro studies of synergistic cell growth inhibition of DENSPM combinations with various chemotherapeutic agents such as oxaliplatin [¹, ²⁴] and 5-fluorouracil [¹] in colon carcinoma cell model, paclitaxel [³³] and to a limited extent of cisplatin [³³] in breast cancer cell models have been reported. Cisplatin is routinely used in ovarian cancer therapy and oxaliplatin has activity in this indication. Due to the importance of platinum agents in the treatment of this malignancy, it is imperative that we gain a better understanding of platinum agent/DENSPM combinations in ovarian cancer models and how this may eventually be brought to the clinical setting to improve treatment. While we noted from our earlier microarray studies that both platinum drugs impact on the polyamine pathway in A2780 ovarian carcinoma cells [⁴²] and that oxaliplatin/DENSPM combinations synergize to produce high SSAT activity and lowered polyamines with enhancement of oxaliplatin cytotoxicity [²³], a comparison of oxaliplatin and cisplatin with DENSPM combinations in the context of SSAT activity, polyamine pools and inhibition of cell growth has not been performed. To our knowledge, there were also no published reports on how cisplatin or oxaliplatin resistance may affect or modulate SSAT activity, polyamine pools and cell growth when platinum agents are combined with DENSPM in comparison with the parental cells in ovarian carcinoma cells.

We show here for the first time that when tested concurrently at a variety of concentrations and combinations with DENSPM, oxaliplatin synergizes with DENSPM in inhibiting cell growth better than cisplatin in this ovarian cancer cell model. It is intriguing why this is the case, as both agents produced high levels of SSAT activity and polyamine pool depletion when combined with DENSPM. It should, however, be emphasized that N¹acetylspermidine considered a good indicator of SSAT activity was detectable only in oxaliplatin/DENSPM combinations. Further, the measured fold increases in SSAT activity levels were higher and both spermine and spermidine were depleted highly in oxaliplatin/DENSPM combinations. In the current study, we observed that platinum agent/DENSPM combinations not only induced highly significant depletion of spermidine and spermine levels, but also a significant increase in putrescine levels. Whether this is due to back conversion of acetylated spermidine or increase in ornithine decarboxylase activity is unclear at this time.

We previously demonstrated that both oxaliplatin and cisplatin up-regulate spermine oxidase (SMO) and polyamine oxidase (PAO) in a concentration-dependent fashion in A2780 cells [²³]. Synergy between oxaliplatin and DENSPM was apparent for SMO up-regulation but not for PAO in A2780 cells [²³]. We also demonstrated that oxaliplatin/DENSPM induced both mRNA and higher protein levels for SMO in human colon carcinoma HCT-116 cells [²⁴]. Spermine oxidase (SMO) oxidizes spermine directly to spermidine; PAO preferentially oxidizes N¹acetylated polyamines to convert acetylated spermine and spermidine to spermidine and putrescine, respectively [⁶]. Oxidation of spermine to spermidine by SMO produces reactive oxygen species (ROS) down-stream to H₂O₂ generated as a reaction product [⁶]. Thus, one cannot rule out the possibility that additional factors such as increased production of highly cytotoxic ROS through SMO reaction may have contributed to the increased cytotoxicity of oxaliplatin/DENSPM.

Previous studies have shown synergism between oxaliplatin/DENSPM combinations on cell death in HCT-116 colon carcinoma cells resistant to oxaliplatin [¹] and that for cisplatin and another polyamine analog BESPM in 2008 ovarian carcinoma cells resistant to cisplatin [²⁹]. The detailed studies presented here exploring a variety of concentrations and ratios of the two drugs indicate that resistance to either oxaliplatin or cisplatin curtails the induction of SSAT, polyamine pool depletion and cell growth inhibition in A2780 cells. Thus, studies presented here comparing the parental with both the oxaliplatin-resistant and cisplatin-resistant cells indicate that the SSAT induction and polyamine pool depletion play a significant role in cell growth inhibition.

While the mechanisms of the platinum drug-polyamine interactions are not completely understood, based on the mechanism of action of polyamine analogs with respect to SSAT mRNA and protein [⁷] it seems likely that the synergistic increases in SSAT activity in the parental A2780 cells in our studies may likely be due to the enhanced stability and translation of platinum drug-induced SSAT mRNA and protein by DENSPM. SSAT activity is post-transcriptionally regulated such that SSAT mRNA requires a translational trigger by addition of a natural polyamine or analog [¹⁴, ¹⁵]. The evidence presented here indicates that cells with acquired resistance to platinum agents are deficient in their ability to up-regulate SSAT gene expression. It is important to note that lack of SSAT induction in the resistant cells in platinum agent/DENSPM combinations is not due to a DENSPM uptake deficiency. In fact, co-treatment with either of the platinum agents consistently increased the DENSPM levels in all parental and resistant cells under these conditions. This effect of the co-treatment with platinum drugs on DENSPM levels in cells has not been reported previously and appears to be a potentially significant contributing factor in the observed synergy in induction of SSAT activity in A2780 cells.

Whether increased DENSPM levels are a reflection of an increased uptake of DENSPM or decreased metabolism is unclear at this time as the experimental conditions consisted of 24 h incubation in drug-free medium following the 20-h combination treatment. DENSPM is known to be metabolized extensively in cells [², ³]. Our previous studies showed that both the oxaliplatin-resistant A2780/C10B cells and the cisplatin-resistant A2780/CP cells are deficient in platinum uptake by ~50% relative to the parental cells under very similar conditions of testing as we described before [²¹, ²²]. It is conceivable that the reduced platinum levels in the resistant cells have affected the SSAT up-regulation which appears to be the key determinant in platinum drug-DENSPM combination induced synergy at the SSAT activity level. We have previously reported the concentration-dependent up-regulation of SSAT gene expression for both oxaliplatin and cisplatin in A2780 ovarian carcinoma cells [²³].

A synergistic interaction of cisplatin and DENSPM has been reported on growth inhibition in murine cell lines L-1210 leukemia and B16F1 melanoma both in vitro and in vivo [²⁰]. Another study reported that pretreatment of cells with certain polyamine analogs (DEHSPM and DENSPM) increased the cytotoxicity of cisplatin in U-251 MG human malignant glioblastoma cells [³²]. This later study suggested that pretreatment of cells with polyamine analogs increased the cisplatin DNA-adduct formation in the linker regions of DNA. Although synergy was noted in these earlier studies the rationale for combining the drugs was not explored with respect to SSAT induction and polyamine pool depletion in these studies. Since polyamines are cationic molecules that interact with the DNA and provide stability [⁴¹] it is highly likely that when polyamines are depleted there may be more interaction between platinum agents and DNA. Previous studies from our laboratory [²¹] and others have established that oxaliplatin is more potent than cisplatin [³⁷]. Lower intracellular Pt-concentrations and fewer DNA-Pt adducts are sufficient for oxaliplatin-mediated cytotoxicity compared to cisplatin [²¹, ³⁷]. It is possible that the observed superiority of oxaliplatin-DENSPM combination in cell growth inhibition in the present study may be related to potency of DACH Pt–DNA adducts relative to CisPt–DNA adduct in the cellular environment after polyamine pool depletion.

Marverti et al., reported that BESPM induces significantly lower levels of SSAT activity in C13* cells relative to sensitive 2008 cells due to decreased SSAT enzyme synthesis and high turnover in resistant cells [²⁸]. Their work further suggested that during selection for cisplatin resistance a labile repressor protein was produced that regulates SSAT gene expression [³⁰]. Using protein synthesis inhibitors, they observed enhanced transcription and stabilization of SSAT mRNA, as well as increased SSAT activity following BESPM treatment of C13* cells [³⁰]. While their focus has been the polyamine analog-induced SSAT activity in cisplatin-resistant cells, it is possible that the highly curtailed SSAT gene expression in response to the corresponding platinum drugs in oxaliplatin and cisplatin-resistant cells in our study may be mediated through similar mechanisms, in addition to the effect of reduced platinum levels.

In conclusion, the studies presented here indicate that the synergy displayed by platinum drug/DENSPM combinations in inhibition of cell growth is a blend of a multitude of events. First, the platinum agents by themselves are potent inducers of SSAT mRNA in parental cells. Co-treatment with platinum agents increases the cellular levels of DENSPM which in turn facilitate increased translation and stability of SSAT protein and depletion of polyamine pools. In the cellular environment with decreased polyamine pools, Pt–DNA adducts may be more frequent. Fewer DACH Pt–DNA adducts being more cytotoxic than cisplatin–DNA adducts, oxaliplatin/DENSPM combination translates into better synergy in cell growth inhibition than cisplatin/DENSPM combination. The curtailed induction of SSAT gene expression in platinum resistance may be related at least partly to the defective platinum uptake in resistant cells. Further studies to define the molecular mechanisms behind platinum drug/DENSPM synergy will pave the way for realization of the full translational potential of these drugs.

This work was supported by RO1CA109619 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute and the National Institutes of Health. We acknowledge the support of NCI Comprehensive Cancer Center grant CA10656 for the PK/PD Core Facility usage.

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