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* Department of Neuroscience, University of Rome "Tor Vergata," Rome, Italy;
Experimental Clinical Laboratory, Institute for Cancer Research "Regina Elena," Rome, Italy;
Department of Clinical and Experimental Medicine and Pharmacology, University of Messina and "Centro Neurolesi Bonino-Pulejo" (IRCCS), Messina, Italy; and
MGI Pharma, Baltimore, Maryland, USA
1Correspondence: Department of Neuroscience, University of Rome "Tor Vergata," Via Montpellier 1, Rome 00133, Italy. E-mail: graziani{at}uniroma2.it
ABSTRACT
Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the antitumor activity of the topoisomerase I inhibitor irinotecan (CPT-11), which is used to treat advanced colorectal carcinoma. Since PARP inhibitors sensitize tumor cells also to the methylating agent temozolomide (TMZ) and clinical trials are evaluating CPT-11 in combination with TMZ, we tested whether the PARP inhibitor GPI 15427 (10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one) increases the efficacy of CPT-11 + TMZ against colon cancer. Moreover, due to the ability of PARP inhibitors to avoid cell death consequent to PARP-1 overactivation, we evaluated whether oral administration of GPI 15427 provides protection from the dose-limiting intestinal toxicity of CPT-11. The results of colony formation assay indicated that GPI 15427 increased the antiproliferative effects (combination index <1) of TMZ + SN-38 (the active metabolite of CPT-11) against colon cancer cells. Accordingly, GPI 15427 (40 mg/kg/dayx5 days per os) in combination with TMZ (10 mg/kg/dayx5 days) + CPT-11 (4 mg/kg/dayx5 days) significantly reduced the growth of tumor xenografts. Oral administration of GPI 15427 (40 mg/kg/q2x3 days) prevented intestinal injury and diarrhea induced by CPT-11 (30 mg/kg/day x 3 days) reducing inflammation and PARP-1 overactivation, as evidenced by immunohistochemical staining of intestinal tissue with antipoly(ADP-ribose) antibody (Ab). In conclusion, the PARP inhibitor represents a novel strategy to enhance the antitumor efficacy and reduce toxicity of chemotherapy in colon cancer.—Tentori, L., Leonetti, C., Scarsella, M., Muzi, A., Mazzon, E., Vergati, M., Forini, O., Lapidus, R., Xu, W., Dorio, A. S., Zhang, J., Cuzzocrea, S., Graziani, G. Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma.
Key Words: chemotherapy • cancer • diarrhea • mucositis
COLORECTAL CANCER is one of the most common gastrointestinal tract malignancies, and irinotecan (CPT-11) is a cornerstone drug in management of the advanced disease. In fact, CPT-11 in combination with 5-fluorouracil/leucovorin is now considered the standard chemotherapy for metastatic colorectal cancer. Moreover, CPT-11 induces remission in 15–30% of tumors resistant to 5-fluorouracil (1)
.
CPT-11 is a semisynthetic water-soluble derivative of the natural alkaloid camptothecin that requires activation by a carboxylesterase to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. Topoisomerase I cleaves a single strand of DNA and forms a short-lived intermediate with the nick (cleavable complex), allowing rotation of DNA around the noncleaved strand and relaxation of the torsional strain. Religation of strand break restores DNA integrity and is followed by enzyme dissociation from the double helix. Inhibitors of topoisomerase I noncovalently bind to topoisomerase I-DNA, stabilize the cleavable complex and inhibit the religation step. Since this interaction is reversible, cytotoxicity occurs only when the replication fork encounters a cleavable complex, which is converted into a permanent double strand break.
Even though CPT-11 has shown activity also against a number of refractory tumors, resistance to this compound remains a critical problem (2)
. A recent approach to counteract resistance, which is currently evaluated in clinical trials, relies on CPT-11 combination with the anticancer agent temozolomide (TMZ), due to synergistic effects observed in preclinal models (3
4
5
6
7)
.
TMZ is a DNA methylating agent that generates a wide spectrum of base adducts mainly represented by N7-methylguanine, N3-methyladenine, and O6-methylguanine (8)
. Despite being produced in low amounts, O6-methylguanine is generally considered the main cytotoxic lesion produced by TMZ. In fact, resistance to TMZ is due to high levels of O6-alkylguanine DNA alkyltransferase, which transfers the alkyl adduct from the O6 position of guanine to an internal cysteine residue, or due to functional defects of the mismatch repair system (MR) (9)
. If not repaired by O6-alkylguanine DNA alkyltransferase, O6-methylguanine inappropriately pairs with thymine and the resultant mismatches trigger the intervention of MR, which removes the improperly paired pyrimidine to reinsert it again opposite to O6-methylguanine. Repeated cycles of MR-mediated excision/resynthesis eventually provoke DNA nicks with apoptosis induction and growth arrest.
MR deficiency has instead been associated with increased sensitivity to CPT-11, probably due to the involvement of MR components in the recombinational repair of DNA double strand breaks generated by the topoisomerase I inhibitor (10
, 11)
.
A novel molecular approach to enhance the antitumor activity of topoisomerase I inhibitors or methylating agents relies on the use of chemical inhibitors of poly(ADP-ribose) polymerase (PARP) (12
13
14
15
16)
. PARP defines a family of enzymes that cleaves NAD+ to nicotinamide and ADP-ribose to form long and branched (ADP-ribose) polymers on glutamic acid residues of a number of proteins. Poly (ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, gene transcription, cell cycle progression, cell death, chromatin functions, and genomic stability. In the case of TMZ, the enhancing effect deriving from inhibition of poly(ADP-ribosyl)ation derives from impairment of the repair of N-methylpurines that generally do not contribute to cytotoxicity. In fact, these methyl adducts are promptly removed by the short-patch base excision repair (BER), which requires PARP-1 and PARP-2 function. During the repair process, PARP-1 and PARP-2 intervention takes place soon after the action of the N-methylpurine DNA glycosylase and apurinic/apyrimidinic endonuclease and is triggered by the DNA nicks resulting from the interruption of sugar-phosphate backbone. Replacement of the damaged nucleotide occurs by means of the coordinate intervention of PARP-1, PARP-2, DNA polymerase ß, XRCC1, and ligase III (17)
. Inhibition of PARP-1 and PARP-2 prevents completion of the repair process with generation of permanent single strand breaks.
The molecular mechanisms underlying tumor chemosensitization to topoisomerase I poisons by PARP inhibitors have been in part clarified by recent findings showing that PARP-1 interacts with and promotes the activity of topoisomerase I and that poly(ADP-ribos)ylated poly (ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks (18
, 19)
. Consequently, PARP inhibition hampers topoisomerase I activity favoring the action of the enzyme poisons.
A major concern deriving from the use of biomodulators of resistance is that they can potentially increase toxicity exerted by chemotherapy toward normal tissues. However, this might not be the case for PARP inhibitors since these compounds have been shown to protect normal tissues from untoward effects induced by certain genotoxic agents that induce PARP-1 overactivation (20)
. In fact, elevated DNA damage, which saturates cell repair ability, triggers PARP-1 overactivation with consequent extensive NAD+ consumption during the synthesis of (ADP-ribose) polymers, which leads to ATP depletion and cell death (21)
. The aim of the present study was to establish whether the orally bioavailable PARP inhibitor GPI 15427 (10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one, MGI Pharma, Baltimore, MD, USA) (13
, 16)
enhances the antitumor efficacy of CPT-11 and TMZ combination against colon cancer and provides protection from the intestinal dose-limiting toxicity of CPT-11.
MATERIALS AND METHODS
Cell lines
Colon cancer cell lines and their MR functional status are indicated in Table 1
. Cultures were maintained in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics.
|
O6-alkylguanine DNA alkyltransferase activity assay
Cells (1x106) were lysed in 0.5 ml of a buffer containing 0.5% 3-[(3-cholamidopropyl) dimethylammonio]propanesulfonate, 50 mM Tris-HCl pH 8, 1 mM EDTA, 3 mM dithiothreitol, 100 mM NaCl, 10% glycerol, protease inhibitors and incubated at 4°C for 30 min (22)
. Various amounts of cell extracts were incubated with 10 µg of calf thymus DNA previously labeled with N-[3H]-methyl-N-nitrosourea (GE Healthcare, Milan, Italy; 18 Ci/mmol). O6-alkylguanine DNA alkyltransferase activity was determined by measuring the transfer of [3H]-methyl groups from methylated DNA to O6-alkylguanine DNA alkyltransferase and expressed as fmol of methyl groups per mg of proteins in cell extract.
N-methylpurine DNA glycosylase activity assay
Cells (1x107) were sonicated at 4°C in 0.1 ml buffer I (50 mM Tris-HCl, 3 mM dithiothreitol, and 2 mM EDTA, pH 8.3), with freshly added 1 mM 4-(2-aminoethyl)-benzene-sulfonyl fluoride hydrochloride. Various amounts of cell extracts were incubated with 15 µg of freshly dissolved calf thymus DNA methylated by N-[3H]methyl-N-nitrosourea in a total volume of 100 µl of buffer II (20 mM Tris-HCl, 1 mM dithiothreitol, 60 mM NaCl, and 1 mM EDTA, pH 8). N-methylpurine DNA glycosylase activity was expressed as fmol of methylpurines released per mg of proteins.
PARP activity assay
Cells (5x106) were lysed in 0.5 ml of a buffer containing 0.1% Triton X, 50 mM Tris-HCl pH 8, 0.6 mM EDTA, 14 mM ß-mercaptoethanol, 10 mM MgCl2, and protease inhibitors. Proteins (25 µg) were incubated with 2 µCi 32P-NAD+ (GE Healthcare), 10 µM NAD+, 50 mM Tris-HCl, 10 mM MgCl2, 14 mM ß-mercaptoethanol, 10 µg nuclease-treated salmon testes DNA. PARP activity was expressed as fmol of 32P-NAD+/µg of protein (13)
.
For analysis of GPI 15427 on cellular PARP, intact cells (5x105 tumor cells) were treated with the inhibitor and permeabilized with digitonin (0.1 mg/ml) in the presence of 0.25 µCi 3H-NAD+ (PerkinElmer, Milan, Italy) (23)
.
For in vivo PARP inhibition, peripheral blood lymphocytes PBL, from untreated or GPI 15427 (40 mg/kg per os)-treated mice (8/group), were separated from whole blood by Ficoll-Hypaque density gradient, permeabilized and incubated with 3H-NAD+ in the presence of 5 µg palindromic deoxyoligonucleotide (CGGAATTCCG).
Northern and Western blot analysis
Northern blot analysis was performed using total RNA (15 µg) and cDNA probes corresponding to the DNA binding domain of PARP-1 (kindly provided by Prof. Alexander Burkle, University of Konstanz, Germany) or to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Western blot analysis was performed with monoclonal antibodies directed against PARP-1 (BD Biosciences, Milan, Italy), breast cancer resistance protein (Chemicon, Temecula, CA, USA), and actin (Sigma-Aldrich). Signals were quantified using a Kodak densitometer (Rochester, NY, USA).
Colony formation assay
Cells were seeded in triplicate into 6-well plates (2x102/well) and, after overnight incubation, treated with GPI 15427 (2 µM), TMZ (Schering-Plough, Kenilworth, NJ, USA; 31–1000 µM) or SN-38 (Alexis, Florence, Italy; 0.1–5 nM). When used in combination with TMZ, SN-38 was added to cultures 1 h after TMZ exposure. For experiments to assess the ability of GPI 15427 to enhance the antiproliferative effects of TMZ or SN-38, the PARP inhibitor was added to cell cultures 15 min before the anticancer drugs. Cells were cultured to allow colony formation, and after 10–14 days colonies were fixed and stained with rhodamine B basic violet 10 (Sigma-Aldrich). Only colonies comprising >50 cells were scored as survival colonies. Chemosensitivity was evaluated in terms of IC50, i.e., the concentration of the drug capable of inhibiting colony-forming ability by 50%.
To evaluate whether the combination TMZ+SN-38 or TMZ+SN-38+GPI 15427 was synergic, tumor cells were exposed to TMZ or SN-38 alone or in combination at fixed equipotent ratios (corresponding to 1, 0.5, 0.2, 0.1 x the IC50 for each drug) in the absence or presence of a nontoxic dose of GPI 15427 (2 µM). The dose-effect curves were analyzed by the median-effect method of Chou and Talalay using the CalcuSyn Software (Biosoft, Cambridge, UK). The combination index (CI) indicates a quantitative measure of the degree of drug interaction in terms of synergistic (CI<1), additive (CI=1), or antagonistic effect (CI>1). Graphs were generated plotting CI as a function of the fraction of cells affected (Fa) by the dose of the anticancer drugs.
In vivo studies of antitumor activity
HT-29 or LoVo cells (1x106) were inoculated intramuscle (i.m.) in male athymic cluster of differentiation CD-1 mice (nu/nu genotype, 8/group). Tumors were measured with caliper and treatment started when nodules reached 200–400 mm3. Xenograft growth was monitored by measuring tumor nodules every 2–4 days for 3 wk.
TMZ was dissolved in dimethyl-sulfoxide, diluted in saline (0.5 mg/ml) and administered intraperitoneally (i.p.) at 10 mg/kg/day x 5 days and CPT-11 (Campto®, Aventis, Milan, Italy) i.p at 4 mg/kg/day x 5 days, doses corresponding to 1/15 of the maximum tolerated dose of TMZ + GPI 15427 per os and 1/10 of the maximum tolerated dose of CPT-11 + GPI 15427 per os. GPI 15427 was dissolved in 70 mM PBS without potassium and administered by oral gavage once a day (40 mg/kg) for 5 days, 1 h before TMZ or CPT-11. Since the synergistic effect deriving from TMZ and CPT-11 combination is schedule dependent (4)
, CPT-11 was administered 1 h after TMZ. Treatment was repeated for two cycles. Control mice were always treated with vehicles.
Toxicity was assessed on the basis of apparent drug-related deaths (within 7 days after the last treatment) and net body wt loss [i.e., (initial wt-lowest wt)/initial wt x100%].
All procedures involving animal care were performed in compliance with national and international guidelines (European Economy Community Council Directive 86/109, OLJ318, Dec. 1, 1987).
Myelosuppression assay
Athymic nu/nu mice (6/group) received vehicle, TMZ (100 mg/kg/dayx5 days), GPI 15427 (40 mg/kg/dayx5 days) or GPI 15427 + TMZ. Three mice per group were sacrificed on day 8 or 15 and whole blood was analyzed for complete blood count.
Intestinal toxicity assay
Wistar rats (10/group) were treated i.p. with CPT-11 (30 mg/kg/dayx3 days) (24)
and received vehicle or two oral doses of GPI 15427 (40 mg/kg) 30 min before and 1 h after each CPT-11 treatment. The day after the last treatment, rats were anesthetized with urethane (1.4 g/kg/i.p.) and jejunum was collected. A 0.5 cm segment of intestine was fixed in 10% buffered formalin and embedded into paraffin wax. Serial 7 µm sections were mounted on silane-coated glass slides. Sections were dewaxed in xylene, rehydrated in graded ethanol, stained with hematoxylin/eosin, and analyzed using light microscopy (Zeiss, Milan Italy).
For analysis of (ADP-ribose) polymers by immunohistochemistry, sections were dewaxed, permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min, and incubated overnight with an anti-(ADP-ribose) polymers Ab (Alexis, 1:50 in PBS, v/v). Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA, Milan, Italy). Densitometry of (ADP-ribose) polymers staining was performed as described previously using an Imaging Densitometer (AxioVision, Zeiss, Milan, Italy) and a computer program (25)
. In particular, the densitometry analysis of immunocytochemistry photographs (n=5) was carried out in section in which the jejunum was oriented in order to observe all the mucosa villi. In this type of section it is possible to evaluate the presence/absence of the positive staining.
Terminal deoxynucleotidyltransferase (TdT)-mediated uridine triphosphate (UTP) end labeling (TUNEL) assay was performed using a commercially available kit (Apotag, HRP kit, DBA).
For diarrhea assessment all animals were checked three times daily and diarrhea was recorded. The severity of diarrhea was scored as follows: 0, no diarrhea; 1, mild diarrhea (staining of anus); 2, moderate diarrhea (staining over top of the legs and lower abdomen); 3, severe diarrhea (staining over legs and higher abdomen, often associated with continual oozing).
Statistical analysis
For tumor xenografts, the results were analyzed by 1-way ANOVA for multiple comparisons. Residuals were examined for normality and homogeneity of variance. Because the P value for the ANOVA F statistic was <0.001, we calculated the 95% confidence intervals for the differences between the groups using the post-test Bonferroni multiple comparisons method (Primer of Biostatistics Statistical Software Program, McGraw-Hill Medical). A P value of <0.05 was considered significant.
Student’s t tests was used for the other statistical analyses. All statistical analyses were two-sided and results were considered to be statistically significant at P < 0.05.
RESULTS
GPI 15427 inhibits the activity of colon cancers with different levels of PARP-1
Cell lines were analyzed for PARP-1 activity (which accounts for >90% of the cellular poly(ADP-ribosyl)ation activity) and for the expression of the corresponding protein or transcript. The results indicate that activity correlated with expression of PARP-1 protein and that LoVo and HCT-116 showed the highest level of PARP-1, whereas HT-29 and HCT-116 Chr3 the lowest (Fig. 1
). HT-29 and HCT-116 Chr3 lines showed also the lowest levels of PARP-1 transcript (Fig. 2
). Cells were then exposed to graded concentrations of the PARP inhibitor GPI 15427 and assayed for PARP-1 activity 1 h later. The results show that GPI 15427 inhibited PARP-1 activity in all cell lines, with comparable IC50s (range of mean values: 74–87 nM, Table 2
), indicating high and uniform penetration of the drug.
|
|
|
Recovery of PARP-1 activity on GPI 15427 treatment was assessed exposing LoVo cells, which express high levels of PARP-1, to 2 µM GPI 15427 (a concentration equivalent to the peak plasma concentration reached in animals treated per os with 40 mg/kg) (16)
. The results of three independent experiments indicate that a 15 min treatment with GPI 15427 induced 90 ± 2% inhibition of PARP-1 activity, which persisted unchanged 24 h later when GPI 15427 was not removed from culture medium (90±5% inhibition). On the other hand, activity largely recovered within 24 h when the inhibitor was removed from culture (23±4% inhibition). Comparable results were obtained in HT-29 cells, characterized by low levels of PARP-1 (data not shown).
GPI 15427 increases the antiproliferative activity of CPT-11 and TMZ combination in colon cancer cells with different chemosensitivity profiles
It has been demonstrated that MR deficiency is associated with resistance to TMZ and increased sensitivity to CPT-11 (9
10
11)
. All the cell lines used for this study, with the exception of HT-29 and HCT-116 Chr3, are characterized by MR functional defects (Table 1)
. Analysis of the expression of breast cancer resistance protein, an ATP binding cassette transporter, which is regarded as an important determinant of resistance to camptothecins (26)
, reveals that HT-29 expressed higher levels of this protein with respect to the other cell lines. Analysis of O6-alkylguanine DNA alkyltransferase, as an indicator of cell ability to repair O6-methylguanine, and of N-methylpurine DNA glycosylase, which repairs N-methylpurines, indicates that HT-29 and LoVo cells showed the highest levels of O6-alkylguanine DNA alkyltransferase and N-methylpurine DNA glycosylase activity (Fig. 3
).
|
Evaluation of sensitivity to SN-38 reveals that HT-29 was the most resistant, in accordance with breast cancer resistance protein expression and MR proficiency. The MR-proficient HCT-116 Chr3 was more resistant than its MR-deficient counterpart, consistent with a protective role of MR against camptothecin cytotoxicity. Most cell lines were resistant to TMZ either due to MR deficiency (HCT-15, HCT-116, LoVo) or to elevated O6-alkylguanine DNA alkyltransferase activity (HT-29). The MR-proficient HCT-116 Chr3 line was 3-fold more sensitive than its MR-deficient counterpart. In all cell lines, 2 µM GPI 15427 significantly enhanced the antiproliferative effects of SN-38 (2–5-fold) or TMZ (5- to 16-fold) (Fig. 4
).
|
Since TMZ and CPT-11 combination is currently evaluated in clinical trials, we then investigated the influence on tumor cell growth of the combination TMZ + SN-38 in the presence or not of the PARP inhibitor GPI 15427. Cells were treated with TMZ and SN-38 in combination at fixed equipotent ratios selected on the basis of the IC50 values of each cell line, as described in Materials and Methods. Figure 5
shows the results of the median effect analysis using Calcusyn Software. The CI values indicate that the combination TMZ + SN-38 was highly synergistic in LoVo cells and antagonistic in HCT-116 Chr3 cells; the addition of GPI 15427 always potentiated the antiproliferative effect of the drug combination.
|
Oral administration of GPI 15427 enhances the antitumor efficacy of TMZ+CPT-11 in HT-29 and LoVo xenografts
To examine whether GPI 15427 was capable of inhibiting PARP in vivo, mice were treated with 40 mg/kg GPI 15427 per os, a dose capable of increasing the antitumor efficacy of TMZ (16)
, and 1 h or 24 h later PBL were analyzed for PARP activity. The results indicate that GPI 15427 inhibited PARP activity of PBL by 
60% [untreated control: fmol 23,3 (95% confidence intervals: 17–28), GPI 15427: fmol 9,7 (95% confidence intervals: 5–10), P=0.0007], suggesting that the compound was bioavailable and pharmacologically active. At 24 h activity completely recovered.
Toxicity studies determined the five daily administrations of 40 mg/kg/day GPI 15427 per os +10 mg/kg/day/i.p. TMZ + 4 mg/kg/day/i.p. CPT-11 as the maximum tolerated dose of the 3 drug combination. Among the colon cancer cell lines analyzed in vitro, HT-29 and LoVo were selected for the in vivo studies. HT-29 is a highly aggressive tumor with a doubling time of 9 days and is the most resistant to SN-38 in vitro. From colon cell lines with higher PARP activities, LoVo was chosen because of its high resistance to SN-38. Moreover, both lines are resistant to TMZ due to high O6-alkylguanine DNA alkyltransferase levels (HT-29) or to MR deficiency (LoVo). In HT-29 xenograft model, TMZ + CPT-11 did not inhibit tumor growth. It is noteworthy that GPI 15427 significantly enhanced the antitumor effect of TMZ + CPT-11 combination (Fig. 6
and Table 3
). On the other hand, treatment with GPI 15427 did not significantly affect tumor growth inhibition in the groups treated with TMZ or CPT-11. In LoVo xenografts the combination of TMZ + CPT-11 was more effective than in HT-29 xenografts, resulting in a statistically significant delay in tumor growth. Also in this model, GPI 15427 significantly enhanced growth inhibition induced by CPT-11 + TMZ, increasing the growth delay by 12 days with respect to treatment with CPT-11 + TMZ and causing tumor regression in all animals (Table 3)
.
|
|
Protective effect of PARP inhibitor on CPT-11-induced jejunum damage and delayed diarrhea
We then investigated whether oral administration of the PARP inhibitor GPI 15427 might attenuate CPT-11 toxicity, using a well-established rat model for evaluating CPT-11-induced intestinal mucosa damage (24)
. Rats were treated with GPI 15427 (40 mg/kg/q2x3 days) together with 30 mg/kg/q x 3 days CPT-11, a dose previously used to assess the efficacy of other compounds in preventing CPT-11 intestinal damage (24)
. Histological analysis of jejunum sections from rats treated with CPT-11 showed severe intestinal injury (Fig. 7
A). Necro-inflammatory debris in small foci was detected within the epithelial cell layer together with focal loss of cells. Numerous neutrophils were observed among epithelial cells and edematous lamina propria. Additionally, some areas with an increased inflammatory reaction characterized by lymphatic infiltration were detected (Fig. 7B
). The severity of mucosa damage was also demonstrated by the presence of apoptotic cells (Fig. 7C
) and mitotic figures (Fig. 7D
). Conversely, the sections of jejunum from rats treated with GPI 15427 + CPT-11 or with vehicle showed no significant histological alterations (Fig. 7E, F
).
|
To assess whether intestinal damage was associated with PARP-1 activation, jejunum sections were analyzed for the presence of poly(ADP-ribosyl)ated proteins by immunohistochemical staining with an anti-(ADP-ribose) polymers Ab. Jejunum sections from CPT-11-treated rats showed positive staining for (ADP-ribose) polymers in the damaged areas (Fig. 8
A) mainly localized in epithelial cells (Fig. 8B
), in inflammatory cells (Fig. 8C
), and in the surrounding area (Fig. 8D
). Densitometry analysis indicated that 6.1 ± 0.21% of total jejunum tissue area positively stained with anti-(ADP-ribose) polymers Ab (Fig. 8D
), whereas only 0.5 ± 0.09% of total jejunum tissue area was found to be positive in the jejunum of rats treated with GPI 15427 + CPT-11 (Fig. 8E
). No positive (ADP-ribose) polymers staining was found in the jejunum of rats treated with vehicle (Fig. 8F
).
|
To test whether the tissue damage was associated with apoptosis, we measured TUNEL-like staining in the jejunum tissue. Almost no apoptotic cells were detectable in the jejunum tissue of vehicle-treated rats (Fig. 9
A), while sections from CPT-11-treated rats showed a marked appearance of dark brown apoptotic cells (Fig. 9B
, B1), characterized by chromatin compaction into uniformly dense masses in perinuclear membrane, formation of apoptotic bodies and membrane blebbing (see particles B1). No apoptotic cells or fragments were found in the jejunum of rats treated with GPI 15427 + CPT-11 (Fig. 9C
).
|
The ability of GPI 15427 to protect animals from delayed diarrhea was also investigated. In animals that received CPT-11 mild to moderate diarrhea developed after 24 h. Oral administration of GPI 15427 reduced severity of delayed diarrhea (Fig. 10
). No diarrhea was recorded in control animals.
|
GPI 15427 did not exacerbate myelotoxicity induced by full dose of TMZ
We evaluated whether PARP inhibition exacerbates myelosuppression induced by TMZ, the dose-limiting side effect of this drug in patients. Mice were treated with GPI 15427 per os 40 mg/kg/day x 5 days ± 100 mg/kg TMZ and complete blood count analyzed on day 8 and 15 post-initiation of treatment. TMZ treatment caused a 78% and 60% decline of WBC on days 8 and 15, respectively (Table 4
), whereas GPI 15427 had no effect on WBC. Coadministration of TMZ and GPI 15427 did not enhance the myelosuppressive effects of TMZ. No changes in red blood cells, hemoglobin (Hb), or platelets were observed (data not shown).
|
DISCUSSION
In the present study we demonstrate for the first time that oral administration of GPI 15427, a recently developed PARP inhibitor, enhances the antitumor activity of CPT-11 and TMZ used in combination against colon cancer. It is noteworthy that GPI 15427 provides protection from intestinal damage induced by CPT-11 and did not exacerbate myelotoxicity of TMZ.
The cell lines tested showed different pattern of susceptibility to CPT-11 and TMZ, mainly due to breast cancer resistance protein expression, O6-alkylguanine DNA alkyltransferase levels, or MR functional status. MR plays an important role in the maintenance of genomic stability due to its involvement in the postreplicative repair. Germline mutations of MR genes cause susceptibility to hereditary nonpolyposis colorectal cancer, which accounts for 5% of colorectal cancers, and MR defects or microsatellite instability occur in 
15% of sporadic colorectal tumors (27
,28)
. Besides being involved in genomic surveillance, MR strongly influences tumor susceptibility or resistance to a number of anticancer drugs (29)
. All MR-deficient colon cancer lines were resistant to TMZ, with IC50s 7- to 15-fold higher than the plasma peak concentration reached in patients (60 µM). Resistance to TMZ of the MR-proficient HT-29 line is instead due to high O6-alkylguanine DNA alkyltransferase activity. In regard to N-methylpurine DNA glycosylase levels and sensitivity to methylating agents, conflicting results have been reported. In fact, embryonic stem cells derived from N-methylpurine DNA glycosylase knockout mice were more sensitive than wild-type (WT) cells, whereas N-methylpurine DNA glycosylase-deficient bone marrow cells were more resistant (30
,31)
. Moreover, overexpression of N-methylpurine DNA glycosylase was associated with increased sensitivity to TMZ, suggesting that imbalanced BER process might generate toxic intermediates (32)
. Our results indicate that the ability to repair N-methylpurines does not seem to substantially affect susceptibility to TMZ. In fact, HCT-15 and HCT-116, which are both MR deficient and tolerant to O6-methylguanine, possess different sensitivity to TMZ even though they have similar N-methylpurine DNA glycosylase activity.
MR-deficient lines were more sensitive to SN-38 than the MR-proficient HT-29 line and HCT-116 was more sensitive than the MR-proficient counterpart HCT-116 Chr3. Hypersensitivity of MR-deficient tumors to topoisomerase I inhibitor has been attributed to reduced double-strand break repair by nonhomologous end-joining (10
, 11)
. Moreover, the endogenous expression of breast cancer resistance protein, a membrane transporter known to confer resistance to camptothecins, contributes to the resistant phenotype of HT-29 (26
, 33)
.
It has been reported that PARP-1 might participate in colorectal carcinogenesis, since its expression was found to be increased in carcinomas with respect to the corresponding normal intestinal epithelium. Moreover, PARP-1 expression was highest in undifferentiated normal cells of intestinal crypts (34)
. The cell lines tested in the present study differed in PARP-1 levels, with the lowest level being expressed by the moderately differentiated HT-29 (35)
. In all cell lines the results of colony formation assay indicated that inhibition of PARP by GPI 15427 significantly increased sensitivity to both SN-38 and TMZ. The entity of the chemosensitizing effect was not substantially influenced by endogenous PARP-1 levels. In fact, GPI 15427 induced a similar sensitization to TMZ or SN-38 in LoVo and HT-29 cells, which possess different PARP-1 levels. In regard to TMZ, the most pronounced effect was observed in HCT-15 cells, which showed the highest level of resistance to the drug. In HT-29 cells, GPI 15427 enhanced TMZ antiproliferative activity by 5-fold, whereas O6-benzylguanine, a selective inhibitor of O6-alkylguanine DNA alkyltransferase currently under investigation as a chemosensitizer in combination with TMZ, increased the cytostatic effect of TMZ barely by 1.2-fold (36)
. In addition, O6-alkylguanine DNA alkyltransferase inhibitors cannot be utilized for chemosensitization to TMZ of MR-deficient tumors.
Treatment with TMZ, followed by SN-38, induced synergistic antiproliferative effects mainly in LoVo cells; this effect was confirmed in vivo, since the anticancer drug combination significantly delayed cell growth with respect to treatment with the single agents. It has been reported that O6-methylguanine induced by N-methyl-N'-nitro-N-nitrosoguanidine increases topoisomerase I cleavage complexes (37)
. Our data indicate that the synergistic antiproliferative effect exerted by TMZ + CPT-11 seems to be independent of O6-methylguanine, since all cell lines express moderate/high levels of O6-alkylguanine DNA alkyltransferase and are consistent with those previously reported in rhabdomyosarcoma and neuroblastoma models (3)
. In HCT-116 Chr3, the TMZ and SN-38 combination resulted in antagonist effect likely due to tumor cell sensitivity to TMZ. It can be hypothesized that TMZ-induced growth arrest would prevent cells from entering S phase, thus counteracting the toxicity of topoisomerase I inhibitors, which requires DNA duplication and occurs when replication fork encounters a cleavable complex.
Inhibition of PARP markedly increased the antiproliferative effects of the combination TMZ + SN-38 in all cell lines. Moreover, the in vivo results indicate that this potentiation was particularly evident with the highly aggressive HT-29 line, which possesses a doubling time 2-fold shorter than LoVo and was less responsive to TMZ + CPT-11. The results obtained in vivo also indicate that oral administration of GPI 15427 significantly inhibited PARP activity of PBL, suggesting that the compound is well absorbed and pharmacologically active.
In the experimental in vivo model used in the present study, TMZ or CPT-11 did not inhibit tumor growth nor was their activity significantly potentiated when used in combination with the PARP inhibitor. The apparent discrepancy with the in vitro results showing the ability of GPI 15427 to enhance the antiproliferative activity of TMZ or CPT-11 combined with the PARP inhibitor is likely due to the low doses of chemotherapeutic agents used in vivo.
A major concern with the use of biomodulators of resistance is the increase of toxicity of chemotherapy toward normal tissues. In regard to CPT-11, its dose-limiting toxicity is diarrhea, which is of two types: acute and delayed-onset. Acute diarrhea occurs within 24 h, is the result of the cholinergic activity of CPT-11, and is prevented or rapidly suppressed by atropine. Delayed-onset diarrhea occurs >24 h after drug administration and can be grade 3 (severe) or grade 4 (life-threatening) in up to 40% of the patients (38)
. The current strategy to treat delayed diarrhea consists in a high dose of the mu-opiate receptor agonist loperamide or somatostatin analog octreotide, but the success of these approaches is often limited (39)
. Delayed diarrhea has been attributed to SN-38, generated from CPT-11 by intestinal carboxylesterases or from the SN-38 glucuronide present in bile by mucosal and bacterial ß-glucoronidase. However, the mechanism by which this molecule provokes mucosa injury in the small intestine is still uncertain (24)
. In the present study we demonstrate that CPT-11 doses that generate high SN-38 concentrations induce PARP-1 overactivation with ADP-ribose polymer formation, which contributes to intestinal damage. Oral administration of GPI 15427 prevents ADP-ribose polymer accumulation and protects epithelial cells from cell death. In fact, an extensive synthesis of (ADP-ribose) polymers is known to deplete the cellular pools of NAD+ and ATP leading to cell death. PARP inhibition would spare cells from energy loss, preventing metabolic failure and providing cytoprotection (40)
. Indeed, GPI 15427 reduced inflammation of jejunum and severity of delayed diarrhea in animals receiving CPT-11 at doses higher than those used in combination with TMZ. The involvement of PARP-1 in inflammatory diseases, including acute and chronic inflammation of the gut, and the protective effect of PARP inhibitors have been demonstrated in various experimental models (41)
. Besides the involvement in PARP-mediated cellular suicidal pathway, PARP-1 enhances the activities of NF-
B and activating protein AP-1, key transcription factors regulating the expression of inflammatory mediators and adhesion molecules (41)
. In the present model, the PARP inhibitor also prevents inflammation as indicated by the preservation of the intestinal architecture, with the absence of edema and lymphatic infiltration.
Reduction of CPT-11 toxicity by PARP inhibitor appears in some ways in contrast to the enhancement of antitumor efficacy exerted by GPI 15427 in combination with TMZ + CPT-11. This dual role of PARP inhibitor is due to the fact that PARP-1 may act as a survival factor, favoring DNA repair when the damage is moderate, or as a cell death mediator in the presence of extensive DNA damage that triggers PARP-1 overactivation. In this case, oral doses of GPI 15427 allow immediate achievement of sufficient PARP inhibition in the gut wall just where it is required to prevent mucosal damage. On the other hand, chemosensitization induced at the tumor level by GPI 15427 in combination with low doses of CPT-11 + TMZ relies on inhibition of the PARP activity that is needed for DNA repair.
GPI 15427 did not exacerbate myelotoxicity induced by full doses of TMZ. This might be due to the complete recovery of PARP function when GPI 15427 is no longer available. On the contrary, when TMZ is combined in vivo with O6-benzylguanine, an increase of myelotoxicity was reported, which required a marked reduction of TMZ dosing (42)
. Indeed, when HT-29 or LoVo cells were treated with O6-benzylguanine O6-alkylguanine DNA alkyltransferase activity did not recover after 24 h (data not shown). Upon interaction with O6-benzylguanine, O6-alkylguanine DNA alkyltransferase is rapidly degraded by the proteasome, and this contributes to induce a long-lasting depletion of O6-alkylguanine DNA alkyltransferase since the protein has to be resynthesized (43)
. The ability of GPI 15427 to prevent CPT-11 intestinal toxicity but not TMZ-induced myelotoxicity might be attributed to the higher concentrations of the PARP inhibitor reached in the gut by means of oral administration with respect to those reached in bone marrow following absorption and distribution of the compound.
In conclusion, these data indicate that GPI 15427 sensitizes tumors to methylating agents and topoisomerase I poison combination, representing a novel strategy to enhance the efficacy and reduce toxicity of chemotherapy in colon cancer. Since PARP inhibitors have recently entered phase I-II clinical trials in combination with TMZ, our findings on the combination of PARP inhibitor with TMZ + CPT-11 will provide the rational basis for the development of new clinical protocols.
ACKNOWLEDGMENTS
This work was supported by grants from the Italian Ministry of Education and Research "Fondo per gli Investimenti della Ricerca di Base" (FIRB) to G.G. and "Programmi di Ricerca scientifica di rilevante Interesse Nazionale" (PRIN) projects to G.G. and L.T. J.Z., W.X., and R.L. work for MGI PHARMA, which is developing PARP inhibitors for cancer treatment.
Received for publication February 16, 2006. Accepted for publication March 31, 2006.
REFERENCES
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