Abstract
Background: Optimal conditions for efficient concomitant chemoradiation treatment of colorectal cancer with cisplatin still need to be better defined. In addition, intolerance of healthy tissue to cisplatin prevents the full exploitation of its radiosensitizing potential. A liposomal formulation of cisplatin, Lipoplatin™, was proposed to overcome its toxicity. Using an animal model of colorectal cancer, we determined the platinum window, defined by studying the pharmacokinetics and time-dependent intracellular distribution of cisplatin and Lipoplatin™. Materials and Methods: In nude mice bearing HCT116 human colorectal carcinoma treated with cisplatin or Lipoplatin™, the platinum accumulation in blood, serum, different normal tissues, tumor and different tumor cell compartments was measured by inductively coupled plasma mass spectrometry. Radiation treatment (15 Gy) was given 4, 24, and 48 h after drug administration and was correlated to the amount of platinum–DNA adducts in the cancer cells. The resulting tumor growth delay is reported and correlated to apoptosis analysis. Results: The greatest effects and highest apoptosis were observed when radiation was given at 4 h or 48 h after drug injection. These times correspond to the times of maximal platinum binding to tumor DNA. An enhancement factor (ratio of group treated by combined treatment compared to chemotherapy alone) of 13.00 was obtained with Lipoplatin™, and 4.09 for cisplatin when tumor irradiation was performed 48 h after drug administration. Conclusion: The most efficient combination treatment of radiation with cisplatin or Lipoplatin™ was observed when binding of platinum to DNA was highest. These results improve our understanding over the mechanisms of platinum-induced radiosensitization and should have significant impact on the design of more efficient treatment protocols.
Cisplatin is a chemotherapeutic agent, which is also widely used as a radiosensitizer (1-6). Addition of cisplatin to radiotherapy has been approved as a standard procedure because improved treatment has been achieved in a number of randomized trials compared with radiotherapy alone. However, in other studies, concurrent radiation treatment with cisplatin administration did not result in better efficacy (3, 6-9). The explanation for the latter results is mainly associated with severe side-effects of either chemotherapy or radiotherapy, which may limit the escalation of anticancer drug concentrations, as well as radiation doses. In addition, there is still no general consensus for the optimal timing between drug administration and radiation treatment. Therefore, the precise determination of this timing appears a promising objective for improving the efficacy of the combined treatment without added toxicity. Optimal timing requires determination of platinum drug concentration in the tumor during the course of radiation treatment in order to assess the time evolution of radiosensitization (10, 11).
DNA damage induced by cisplatin-based radiosensitization has been proposed to be mainly responsible for cytotoxicity (12). This toxicity is related to the formation of cisplatin-DNA adducts, which include intra- and interstrand cross links of two guanosine nucleotides (GG) or adenosine guanosine nucleotides (AG), and monobifunctional binding to guanosine (12). Different mechanisms underlying the antitumor effect of concurrent radiation and cisplatin treatments have been proposed (13). Radiation induces single- and double-strand DNA breaks and DNA base damage, while cisplatin forms adducts with DNA. The presence of cisplatin-DNA adducts can reduce the repair of sublethal and potentially lethal DNA damage (14). Moreover, ionizing radiation preferentially induces DNA damage where cisplatin is located (15). In a previous in vitro study, we showed that a high level of cisplatin-DNA adduct formation appeared to be associated with a better treatment effect in human HCT116 colorectal cancer cells (16). However, there are still some inconsistencies in the literature regarding the relationship between the efficacy of anticancer platinum therapy and cisplatin-DNA adduct formation (17), and it is not yet clear how to assess, in the clinic, the information underlying the efficacy of radiotherapy at maximum cisplatin-DNA adduct concentration in cancer cells.
Lipoplatin™ is a new liposomal formulation of cisplatin. It is currently being developed, and aims to reduce the systemic toxicity of cisplatin, while improving its accumulation in primary tumor tissue (18). A phase I/II study in advanced gastric cancer shows that chemoradiotherapy with Lipoplatin™ is feasible, with minor toxicity (19). This suggests that the therapeutic index of Lipoplatin™ could be larger than that of cisplatin. In our previous report (20), we showed that distribution of cisplatin in different tumor cell compartments was affected by its encapsulation in liposomes. Therefore, studying the mechanisms of its combination with radiotherapy is important in defining the role of Lipoplatin™ as a radiosensitizer and also to confirm our hypothesis for this formulation.
In the present study, we determined the correlation between different cellular levels of DNA-platinum adduct as a function of time after administration of the drug and the efficacy of combined treatment with ionizing radiation. The investigations were performed using nude mice implanted with the human colorectal xenografts of HCT116 cancer. We also assessed the variation of platinum concentrations in blood and in different normal tissues. The results of this study should provide useful information enabling suitable scheduling of chemoradiotherapy treatment of colorectal cancer.
Materials and Methods
Cell line. The HCT116 colorectal carcinoma cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were routinely cultured in modified Eagle's medium (MEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin and 100 μM streptomycin in a fully humidified incubator at 37°C and with 5% CO2.
Animal xenograft model. All experiments were performed with outbred male nude mice at 4-6 weeks of age (Charles River Laboratories, Saint-Constant, QC, Canada). The animals were maintained in our animal facility, under specific pathogen-free conditions. Housing and all procedures involving animals were performed according to the protocol approved by the Université de Sherbrooke Animal Care and Use Committee (protocol number: 235-10B). HCT116 tumor cells (2×106, 0.1 ml) were inoculated subcutaneously (s.c.) into each rear flank. Tumor measurements began one week post-injection and continued biweekly. Tumor volumes were calculated with the formula: V (mm3)=π/6 × a (mm) × b2 (mm2), where a and b were the largest and smallest perpendicular tumor diameters, respectively. The pharmacokinetic studies began when tumor volumes reached the range of 30-60 mm3. The tumor-bearing animals were randomized into different groups of three to five animals each. For all procedures (implantation, chemotherapy and radiotherapy), nude mice were anesthetised with an intraperitoneal injection of ketamine/xylazine (87/13 mg/ml) at 1 ml/kg.
Drug preparation. Cisplatin was obtained through the pharmacy of the Centre Hospitalier Universitaire de Sherbrooke. Lipoplatin™ was generously provided by Regulon Inc. (Athens, Greece). All drugs were diluted to the given concentrations in a solution of 5% dextrose immediately before use.
Pharmacokinetic studies. Drugs were dissolved in 5% dextrose for a final concentration of 1 mg/ml to be given as a single dose of 10 mg/kg into the mice tail vein. Three groups of mice were studied with the following treatment: i) cisplatin, ii) Lipoplatin™, and iii) 5% dextrose alone, as a control group. Three to five animals from each group were sacrificed at 4, 24, 48, 72, and 96 h post-injection. Blood samples were obtained via cardiac puncture for whole-blood and serum platinum assessments. Tumors and samples of different tissues were taken to determine platinum accumulation in the cytoplasm, and the nucleus, as well as in DNA extractions.
Determination of cytoplasmic and nuclear platinum accumulation. Samples were processed with the nuclear and cytoplasm extraction kit Activemotif (ActiveMotif, Carlsbad, CA, USA). Briefly, tumors were weighed and diced into very small pieces using a razor blade. Three milliliters of ice-cold hypotonic buffer supplemented with 3 μl of 1 M dithiothreitol and 3 μl of detergent were added per gram of tissue and incubated on ice for 15 min, and then centrifuged for 10 min at 850 ×g and 4°C. The hypotonic buffer and detergent were supplied in the kit. Thereafter, the supernatants corresponding to the cytoplasmic fraction were transferred into pre-chilled microcentrifuge tubes, while the pellets were gently resuspended in 500 μl of hypotonic buffer, and incubated on ice for 15 min. The detergent (25 μl) was added and then tubes vortexed. Samples were centrifuged for 30 s at 14,000 ×g in a microcentrifuge pre-cooled at 4°C and these second supernatants were combined with the first ones. The resulting pellets corresponded to the nuclear fraction.
Quantification of DNA-bound platinum. DNA was extracted according to a salting-out procedure (21). Briefly, the tumors were weighed and diced into very small pieces, and homogenized with a pre-chilled Dounce homogenizer. On ice, 3 ml of a lysing buffer containing 10 mM Tris-HCl, 400 mM sodium chloride and 2 mM EDTA were added. Subsequently, 0.1 ml of 20% sodium dodecylsulfate and 0.5 ml of proteinase K (10 mg/ml) were added and the samples incubated overnight at 37°C. DNA was precipitated by adding 1.2 ml of 5 M sodium chloride. The tube was gently agitated for 1 min, centrifuged at 2,500 × g for 15 min and then the supernatant was transferred to another tube. The DNA was precipitated with 2.5 vol. of 95% ethanol, spooled out and air-dried briefly. The DNA was then dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and RNase A (0.05 ml of a 10 mg/ml solution) was added and incubated for 1 h at 37°C. The DNA was precipitated a second time with ethanol and re-dissolved in TE buffer. The amount of DNA extracted was evaluated by measuring its absorbance in the solution at 260 nm (A260) with a spectrophotometer (Synergy HT, BIO-TEX, Winooski, VT, USA) and calculated using the following equation: A260×50 (μg/ml).
Platinum quantification using an inductively coupled plasma mass spectrometer (ICP-MS). The quantity of platinum accumulated in tissue samples and in the cytoplasm, nucleus and DNA of tumor were determined with ICP-MS (ELAN DRC-II; PerkinElmer, Waltham, MA, USA) (22). Briefly, samples were treated with 23% nitric acid and 8% H2O2. The solutions were then injected in the ICP-MS to quantify the platinum concentration. As internal controls, platinum and thallium (m/z 195 and 205, respectively) were quantified in untreated animals.
Chemotherapy treatment. The tested drugs were dissolved in 5% dextrose for a final concentration of 1 mg/ml to be given as a single dose of 10 mg/kg into the tail vein. The platinum drugs were injected at 4, 24 and 48 h prior to radiation treatment. The selected drug concentration of cisplatin and Lipoplatin™ did not induce the appearance of any side-effects after combined treatment, as the body weight of all groups of animals significantly increased between initial and final time of combined treatment (Table I).
Tumor irradiation. Mice were anaesthetized and positioned in our in-house constructed stereotactic frame designed for the 4C Gamma Knife (Elekta Instruments AB, Stockholm, Sweden) (23, 24). The radiation treatment (15 Gy, dose rate of 3.6 Gy/min) using 8 mm collimators was delivered at predetermined coordinates targeting the tumor, which had a diameter of about 7 mm. Radiation was applied to one side of the rear flank, whereas the other side was kept as the non-irradiated control tumor.
Apoptosis analysis. Groups of three mice bearing HCT116 tumors were treated with platinum drugs at 24 or 48 h before the radiation treatment, as previously described. Two days after irradiation, tumors were removed, fixed in 10% buffered formalin and embedded in paraffin. Tumor sections of 4 μm were taken at three different levels. Apoptotic cells within tumor tissues were analyzed with a commercially available ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Millipore, Temecula, CA, USA), following the manufacturer's protocol. Briefly, formalin-fixed, paraffin-embedded tissue slides were rehydrated using xylene, rinsed with ethanol, and then incubated with a solution of 3% hydrogen peroxide. The samples were treated with 20 μg/ml of proteinase K for 15 min at room temperature. After washing and incubation with equilibration buffer included in the kit for 5 min, then 33 μl of terminal deoxynucleotidyl transferase (TdT) enzyme in 77 μl of reaction buffer included in the kit was incubated on the slides for 1 h at 37°C. After applying stop solution included in the kit for 10 min and washing, the samples were incubated with anti-digoxigenin peroxidase conjugate (65 μl/cm2 of specimen surface area) for 30 min at room temperature. Slides were developed with a 1:20 dilution of diaminobenzidine (3,3’-diaminobenzidine) substrate, counterstained with methyl green, dehydrated, and coverslipped. Terminal deoxynucleotidyl transferase-mediated dUPT nick end labeling (TUNEL)-positive apoptotic nuclei were detected with a fluorescence microscope. A positive control slide was prepared by nicking DNA with DNaseI and treated similarly (data not shown). Samples treated similarly but without enzyme treatment served as negative controls. To determine the amount of cellular area stained with diaminobenzidine (positive for apoptosis) and the amount of tumor area counterstained with methyl green (negative for apoptosis), the tumor sections were scanned with an Olympus FSX100 microscope (Olympus, Center Valley, PA, USA) and the values were quantified using Adobe Photoshop CS5 (Adobe Systems Canada, Ottawa, Ontario, Canada) analysis software. Briefly, the percentage of apoptotic cells was calculated as the quotient of the diaminobenzidine-positive area over the total area (diaminobenzidine positive + methyl green negative), multiplied by 100.
Body weight of male nude mice bearing HCT116 colorectal tumor after combined treatment of cisplatin or Lipoplatin™ and Gamma Knife irradiation.
Statistical analysis. The mean±SD were calculated. p<0.05 was considered statistically significant (two-tailed paired Student's t-test).
Results
Platinum concentration in whole blood, serum, tumor and normal tissues. The time-concentration profiles of platinum in the blood and serum are shown in Figures 1a and b. After i.v. administration (10 mg/kg) of cisplatin or Lipoplatin™, the initial maximum of platinum concentration in whole blood and serum was reached at the first assessment time point (4 h), followed by a rapid decline over the next 24 h. Thereafter, the decline was very slow. The platinum concentration in whole blood was about three times higher than that in the serum. While free cisplatin reached a higher peak level in kidney tissues than observed with the liposomal formulation (Figure 1c), both drugs exhibited similar levels of platinum accumulation in liver tissues at different time points, with no well-defined initial peak (Figure 1d). A higher level of platinum accumulation was observed in muscle tissues with cisplatin than with Lipoplatin™ (Figure 1e). Accumulation of platinum in the tumor tissue after administration of cisplatin, and Lipoplatin™ are shown in Figure 1f. Over a sampling period of 96 h, the platinum concentration in tumor was about five-times higher in the cisplatin-treated group than in the Lipoplatin™-treated group. For both drugs, the total tumor platinum content declined gradually over time.
Platinum accumulation in the cytoplasm and nucleus, and platinum-DNA adducts in tumor tissue. The time profiles of platinum accumulation in the cytoplasm and nucleus of HCT116 tumor cells are shown in Figures 2a and b. The amount of platinum in the cytoplasm and the nucleus declined gradually over time for both cisplatin and Lipoplatin™. Cisplatin administration led to about 3- to 5-fold more platinum accumulation in the cytoplasm and the nuclei of cells of tumor tissues, respectively, than did Lipoplatin™. Regarding the level of DNA-bound platinum in the nuclei of tumor cells, for both drugs, the maximum was measured at 4 h, declining to reach a first nadir at 24 h, increasing again to reach a second peak at 48 h before declining again (Figure 2c). While the initial and later peak levels of DNA-bound platinum were much higher in the case of cisplatin, for both drugs, the level of DNA-bound platinum declined to a similar nadir level at 24 h.
Pharmacokinetic profiles of platinum concentration in a) whole blood, b) serum, c) kidney, d) liver, e) muscle, and f) tumor obtained after a single i.v. injection of 10 mg/kg cisplatin or Lipoplatin™, in nude mice bearing HCT116 colorectal tumor. The amount of platinum was measured by an inductively coupled plasma mass spectrometer. Each point represents the mean±SD of the concentration in 3-5 mice.
Time dependence of platinum concentration in a) cytoplasm, b) nucleus and c) platinum-DNA adducts obtained after a single i.v. injection of 10 mg/kg cisplatin, or Lipoplatin™, in nude mice bearing HCT116 colorectal tumor. The amount of platinum bound was measured by inductively coupled plasma mass spectrometer. Each point represents the mean±SD of the concentration in 3-5 mice.
Efficacy of platinum-based chemotherapy alone and in combination with radiation treatment (IR).
Tumor growth delay. Figure 3 shows the tumor response when the drugs were injected before radiation treatment. Results in term of the time necessary to reach five-times the initial tumor volume (5Td) and the tumor growth delays (TGD=5Td value for a treated group minus 5Td for the control group) are reported in the Table II. A single cisplatin administration significantly induced a tumor growth delay compared to non-treated animals (p<0.05), whereas no significant improvement was observed when Lipoplatin™ was given alone. Treatment with radiation-alone (15 Gy) led to a good tumor response, with a 5Td of 19±1.0 days compared to 10.5±0.71 days for the non-treated control group (p<0.01). The tumor response was further improved when cisplatin or Lipoplatin™ were combined with tumor irradiation (Figures 3a and b). A significant difference in TGD between the groups of single-drug treatments and combined modalities was initially observed after eight and six days for cisplatin and Lipoplatin™, respectively. With both drugs, the TGD was better when the irradiation was delivered at 4 h or 48 h after drug administration compared to 24 h after drug administration (p<0.01). It is noteworthy that no improvement was observed when cisplatin was administered 24 h prior the radiation treatment, compared to the irradiated control. Indeed, the TGDs were similar for the group treated with radiation alone (8.5±1.00 days), and the group treated with cisplatin 24h prior the radiation treatment (10.5±1.41 days, p=0.19) (Table II).
Enhancement factor (EF). The EF, which is the ratio of TGD measured with a group treated by radiation and chemotherapy compared to TGD obtained with chemotherapy-alone, was calculated to better assess the effect obtained by radiation with cisplatin, and Lipoplatin™ at 4, 24 and 48 h after drug administration (Table II). When radiation was given at 4 h and 48 h after cisplatin administration, the EF was 3.73 and 4.09 respectively, suggesting an important improvement, while a modest EF of 1.91 was measured when radiation treatment was given 24 h after cisplatin administration. The encapsulation of cisplatin in a liposome, i.e. Lipoplatin™, resulted in a much more important effect when combined with tumor irradiation: the EF was 13.67, 10.33 and 13.00 at time intervals of 4, 24 and 48 h when tumor irradiation was combined with Lipoplatin™ after drug administration, respectively. As for cisplatin, the lowest EF with Lipoplatin™ was measured at 24 h post drug administration.
Induction of apoptosis. The number of apoptotic cells was scored in tumor sections after treatment (Figure 4). The percentage of apoptotic cells was very low in the untreated control group (0.94±0.38%) and the group treated with radiation-alone (2.86±0.89%). When the tumor was irradiated 24 h after cisplatin administration, a modest but significant increase of apoptotic cells (3.48±0.69%) was measured compared to tumors treated with cisplatin-alone (1.97±0.77%) (p<0.05). Incubation with cisplatin-alone required 48 h before a significant increase in the number of apoptotic cells (13.40±2.81%) was observed compared to treatment with cisplatin-alone or radiation-alone (p<0.01). With a similar trend, a higher percentage (p<0.05) of apoptotic cells was observed when radiation was given 48 h after Lipoplatin™ administration (15.29±2.90%) compared to when given 24 h after drug administration (5.72±1.23%).
Discussion
For most clinical chemoradiotherapy protocols of weekly cisplatin plus five fractionated doses of radiation, time between the administration of cisplatin and tumor irradiation can change the anti-tumor response obtained by combining them. The present experiments were performed with the chemotherapeutic agents cisplatin and Lipoplatin™ to investigate the maximum concomitant effect between these agents and radiation at different times after drug administration. We also assessed the level of platinum in blood, serum, and different normal tissues with the aim to facilitate the transition to human trials where extraction of cancerous tissue samples is more difficult. To correlate the efficacy of combined treatments, we defined the platinum window of the maximum concomitant effect, which was associated with the maximum level of platinum- DNA adducts in tumor cells. Supporting the importance of platinum-DNA adducts, in clinical study with cisplatin chemotherapy Los and co-workers reported that cisplatin-induced DNA modifications were observed in human tumor biopsies, and were positively correlated with tumor remission (25).
The tumor growth delay after initial treatment of nude mice bearing HCT116 colorectal tumor with a) cisplatin and b) Lipoplatin™ followed by radiotherapy. Tumor growth delay is reported as Vt/V0 ratio where Vt is the mean tumor volume on a given day during the treatment and V0 is the mean tumor volume at the beginning of the treatment. A single dose of platinum drug of 10 mg/kg and a single dose of radiation of 15 Gy were administered. Each symbol represents the mean±S.D of the results obtained with five mice.
It was clear from the EF values that cisplatin and its liposomal formulation (Lipoplatin™) can act as radiosensitizers. The EFs, the results of apoptotic analysis and the pharmacokinetics data showed that cisplatin and Lipoplatin™ were most efficient radiosensitizers when their binding to tumoral DNA was maximized. In contrast, we found less or even nearly no improvement in the combined effect of cisplatin and radiotherapy, when binding of cisplatin to tumoral DNA was minimal (i.e. 24 h after drug injection).
Assessment of apoptosis. a) Terminal deoxynucleotidyl transferase-mediated dUPT nick end labeling (TUNEL)-positive apoptotic nuclei assay after chemoradiotherapy. Animals were irradiated at 24 or 48 h after drug administration. A single dose of cisplatin or Lipoplatin™ of 10 mg/kg and a single dose of radiation (15 Gy) were administered. b) Percentage of apoptotic cells after chemoradiotherapy. Apoptotic cells were counted in 10 random fields per tumor (n=3) (mean±S.D.). Magnification, ×40. Statistically significant differences: p<0.05 *from the control group; **from both radiation-treated and the control group; ***from radiation alone, chemotherapy alone and the control group.
While the initial high DNA-platinum peak at 4 h is explained by drug influx into tumor cells, the reasons for the low level of adduct at 24 h and the second DNA-platinum peak at 48 h are unclear. However, this observation leads to the following hypothesis. At 4 h, the majority of DNA-platinum adducts are intrastrand cross-links binding as monoadducts. These monoadducts are rapidly excised by the nucleotide excision repair system, leading to the reduction of DNA-platinum binding (26). Twenty-four hours after administration of cisplatin, most of the DNA monoadducts disappear, while the repair system becomes more saturated with repair-resistant DNA-platinum interstrand cross links, which would maximize at 48 h.
There are many mechanisms that can enhance the effects of radiation (27, 28). Among these, essentially two mechanisms may explain the radiosensitizing properties of cisplatin. Previous in vitro studies (29-31) and some experiments with tumor-bearing mice (32-34) suggested the inhibition of repair of radiation damage to DNA. Whereas from other investigations, it was postulated that owing to the binding of cisplatin to DNA, there is an increase in the immediate species created by the primary ionizing events in cells which causes the additional damage (35). Zheng et al. (15) measured DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) induced by the impact of 1, 10, 100 and 60,000 eV electrons on thin solid films of DNA with and without cisplatin bonded to two adjacent guanine bases. From a comparison of their results obtained at low energy (1-100 eV) with those obtained at 60 keV, they found that production of SSBs and DSBs induced by low-energy secondary electrons, created by the primary radiation, was substantially enhanced when cisplatin was covalently bonded to DNA. Such damage is known to promote cell death. The latter hypothesis implies that the effect of platinum drugs and radiation should be maximal when binding of platinum to DNA of cancer cells is maximized. This is precisely the result obtained in the present study. Determination of such fundamental mechanisms of the radiosensitizing action of platinum drugs may have implications in the design of new chemotherapeutic agents, as well as in the development of more efficient protocols for chemoradiation therapy. In addition, since there was no correlation in the effect of combined therapies and cisplatin found either in the blood or in normal tissues, this support the notion that the best combined effect can only be determined by quantifying the level of cisplatin–DNA adducts in tumor cells.
We reported previously that the intracellular platinum distribution in tumor from human HCT116 colorectal cancer cells for encapsulated cisplatin may be different than that of cisplatin alone (20). In this in vivo study, identical concentrations of platinum drugs (10 mg/kg) were administered to the animals. Lipoplatin™ administration alone led to similar results in TGD to that of untreated animals; the liposomal formulation was therefore less effective than cisplatin administered alone. Indeed, most previous animal and human studies have administered 3-6 times higher doses of Lipoplatin™ than cisplatin (36). For example, Ravaioli and co-workers used Lipoplatin™ at 100 mg/m2 every two weeks as second-line chemotherapy in heavily pre-treated patients with advanced non-small cell lung cancer (NSCLC) and obtained 5% partial remission and 16% stable disease (36). On the other hand, Stathopoulos and co-workers used 400 mg/m2 every two weeks (200 mg/m2 D1 and 2 every 14 days) on a similar group of patients, and obtained 38% partial remission and 43% stable disease, with only minor toxicities of grade I (37). The dose of cisplatin in 14-day schedules was 75 mg/m2 compared to 200 mg/m2 for Lipoplatin™ in randomized phase III study in combination with paclitaxel in non-squamous NSCLC had shown a much lower toxicity profile in the Lipoplatin™ arm (38). Furthermore, the study of non-squamous NSCLC demonstrated a statistically significantly higher response rate with the Lipoplatin™ treatment. Consequently, Lipoplatin™ should achieve a higher level of tumor accumulation of platinum and DNA-platinum adducts.
Although the radiosensitizing role of Lipoplatin™ has not yet been determined in clinical practice, the wider therapeutic index of this formulation suggests such potential (36-38). In addition, our previous study on the concurrent administration of Lipoplatin™ and radiation showed the high potential of concomitant effect in the HCT116 cells when radiation is applied at maximum formation of DNA-platinum adducts (16). In the present study, combined treatment of radiation and Lipoplatin™ led to the highest increase in antitumoral activity. An enhancement factor of 13.00 was obtained with Lipoplatin™, compared to only 4.09 for cisplatin, when tumor irradiation was performed 48 h after drug administration. The effect after treatment with Lipoplatin™ and radiation still correlated with maximum tumoral DNA-platinum levels (4 h and 48 h after drug administration). Administration of Lipoplatin™ also led to lower accumulation of platinum in the normal tissues tested, except for the liver. Considering the lower toxicity of Lipoplatin™ compared to that of cisplatin reported in clinical trials (37-38), the therapeutic index should be larger for Lipoplatin™ than for cisplatin. It remains to be determined whether a larger dose of Lipoplatin™ administered in this pre-clinical model of cancer would result in an improvement of the antitumoral effect while maintaining an acceptable level of toxicity. Nevertheless, these data are valuable to highlight the potential of Lipoplatin™ as a radiosensitizer.
Although similar levels of Lipoplatin™ and cisplatin bound to the DNA 24 h after drug administration were observed, superior treatment occurred with combined administration of Lipoplatin™ and radiation. Only a weak correlation between the cytotoxicity of liposomal platinum drugs and the degree of DNA platination was found. This observation might imply that platinum-based drugs have important targets other than nuclear DNA (e.g. cytoplasmic compartments) (39). For example, it has been proposed that Lipoplatin™ can release cisplatin molecules into the cytoplasm of the tumor cell, which then activate the mitochondrial apoptotic cascade (40). In addition, any released cisplatin molecules may i) undergo reaction with phospholipids; ii) inhibit amino acid transport, protein synthesis and ATPases; and iii) uncouple oxidative phosphorylation (41-43). However, the importance of targets other than DNA in relation to cytotoxicity is still unclear and remains a subject of future study.
It has been proposed that the binding affinity of drugs to the mitochondria is regulated by lipid solubility (44). Hori and co-workers reported that highly lipophilic drugs are more rapidly accumulated in the mitochondria than are non- or poorly-lipophilic drugs (45). The improvement in terms of lipophilicity for Lipoplatin™ compared to free-platinum forms could indicate a higher efficacy for binding to the mitochondrial membrane that would lead to a greater accumulation of drug within the mitochondria. Thus, promotion of liposomal platinum drug accumulation in the mitochondria may contribute to the enhancement of apoptotic cancer cell death. Although this mechanism may in part explain the anticancer effect of Lipoplatin™ for some cancer types, we observed similar induction of apoptosis with cisplatin and Lipoplatin™ in the HCT116 colorectal carcinoma model used in this study. However, the fact remains that Lipoplatin™ was also associated with a large improvement of the radiosensitizing effect.
In conclusion, the narrow therapeutic index of cisplatin excludes dose escalation to improve its radiosensitizing action in the treatment of various types of cancers. There exists a general impression that the maximum potential of this drug may have been reached. However, our current report shows that the radiosensitizing effect of platinum drugs varies considerably according to the time after exposure to the drugs and to the platinum level in different tumor cellular compartments. The efficacy of combined treatments essentially correlates with the amount of DNA-bound platinum in tumor cells: it reaches a maximum at the times corresponding to the highest DNA-platinum concentration in the nucleus (4 h and 48 h); furthermore, efficacy reaches a minimum at low DNA-platinum levels (24 h). The best combined effect was observed with Lipoplatin™ and was also correlated with the maximum DNA-platinum levels in the tumor cells.
Acknowledgements
This research was financed by the Canadian Institute of Health Research (grant #81356). Léon Sanche, Benoit Paquette and Rami Kotb are membres of the Centre de recherche Clinique-Étienne Lebel supported by the Fonds de la Recherche en Santé du Québec. Sanofi-Aventis Canada also provided a partial unrestricted grant to support this project.
- Received May 31, 2013.
- Revision received June 27, 2013.
- Accepted July 1, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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