HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5916fjev1 20/10/1709    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tentori, L. Articles by Graziani, G. Search for Related Content PubMed PubMed Citation Articles by Tentori, L. Articles by Graziani, G.

Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma

Lucio Tentori^*, Carlo Leonetti, Marco Scarsella, Alessia Muzi^*, Emanuela Mazzon, Matteo Vergati^*, Olindo Forini^*, Rena Lapidus, Weizheng Xu, Annalisa Susanna Dorio^*, Jie Zhang, Salvatore Cuzzocrea and Grazia Graziani^*,1

^* 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

SPECIFIC AIMS

Poly(ADP-ribose) polymerase (PARP) inhibitors recently entered into clinical trials to enhance the efficacy of cancer treatment and to counteract ischemic tissue damage. The dual effect of PARP inhibitors is attributed to the double-edged role of PARP-1, which may act as a survival factor, favoring DNA repair when the damage is moderate or as cell death mediator when DNA damage is extensive.

As chemosensitizers, PARP inhibitors have been shown to enhance the antitumor activity of the methylating agent temozolomide (TMZ) and of the topoisomerase I inhibitor irinotecan (CPT-11), which is used to treat advanced colorectal carcinoma. As an anti-ischemia agent, PARP inhibitors have been shown to reduce tissue damage in various pathological conditions, including intestinal ischemia.

The aims of the present study were to investigate whether 1) PARP inhibitor enhances the antitumor efficacy of CPT-11 and TMZ combination against colon cancer, and 2) PARP inhibition modulates the dose-limiting toxicities of the chemotherapeutic agents toward normal tissues.

PRINCIPAL FINDINGS

1. GPI 15427 inhibits the PARP activity of various colon cancers with different levels of PARP-1
To determine whether PARP-1 levels might influence the response to the PARP inhibitor GPI 15427, colon cancer cell lines were analyzed for PARP-1 activity and expression. The results indicate that 1) LoVo and HCT-116 showed the highest levels of PARP-1, while HT-29 and HCT-116Chr3 the lowest; 2) GPI 15427 inhibited PARP-1 activity in all cell lines regardless of the enzyme levels, with similar IC₅₀s (74–87 nM), indicating a uniform penetration of the drug into nuclei.

2. GPI 15427 increases the antiproliferative activity of CPT-11 and TMZ combination in colon cancer cells with different chemosensitivities
Most colon cancer cell lines were resistant to TMZ either due to mismatch repair (MR) deficiency (HCT-15, HCT-116, LoVo) or to elevated O⁶-alkylguanine DNA alkyltransferase activity (HT-29). HT-29 was the most resistant to SN-38, the active metabolite of CPT-11, due to MR proficiency and high expression of breast cancer resistance protein, a membrane transporter known to confer resistance to topoisomerase I inhibitors. The combination TMZ+SN-38 was highly synergistic (combination index<1) in most cell lines and the addition of GPI 15427 universally potentiated the antiproliferative effect of the drug combination.

3. 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 and peripheral blood lymphocytes (PBL) were analyzed for PARP activity at 1 h or 24 h after treatment. GPI 15427 inhibited PARP activity in PBL by 60% [untreated control: 23.3 fmol (95% confidence intervals: 17–28), GPI 15427: 9.7 fmol (95% confidence intervals: 5–10), P=0.0007], consistent with the compound’s oral bioavailability. 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, even though HT-29 has the lowest PARP activity. From colon cell lines with higher PARP activities, LoVo was chosen because of its high resistance to SN-38. Moreover, both cell 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, while treatment with GPI 15427 did not affect tumor growth in the groups treated with either TMZ or CPT-11 (Fig. 1 ). In the LoVo xenograft model, 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.

View larger version (18K): [in this window] [in a new window]   Figure 1. GPI 15427 per os increases the antitumor activity of CPT-11 and TMZ combination in resistant and highly proliferating HT-29 colon cancer cells. Symbols represent the means of tumor nodule volumes determined in 8 animals for each group every 3 days. Bars: ± SD. The tumor growth curve corresponding to the group treated with GPI 15427 as single agent was omitted since it overlaps that of the control group. Differences between the 3 drug combination and all the other groups were statistically significant starting from day 10 onward (P<0.05, using the post-test Bonferroni multiple comparisons method). Treatment produced animal body wt loss <10% of original wt. All mice recovered the initial body wt 2 wk after treatment.

4. PARP inhibitor provides protection from CPT-11-induced intestinal damage and does not exacerbate TMZ-induced myelotoxicity
We then assessed whether oral administration of the PARP inhibitor GPI 15427 might attenuate CPT-11-induced intestinal toxicity, as delayed diarrhea is the dose-limiting side effect of this family of chemotherapeutics in clinics. To this end, Wistar rats were treated with GPI 15427 (40 mg/kg/q2x3d) together with 30 mg/kg/day x 3 days CPT-11, a dose known to induce mucosa damage. Histological analysis of jejunum sections from animals treated with CPT-11 showed severe intestinal injury (Fig. 2 A–D). Conversely, the sections of jejunum from rats receiving GPI 15427 + CPT-11 or vehicle showed no significant histological alterations (Fig. 2E, F ).

View larger version (123K): [in this window] [in a new window]   Figure 2. Protective effects of GPI 15427 on CPT-11-induced jejunum damage. The histological analysis of jejunum sections stained with hematoxylin/eosin from animals treated with CPT-11 showed severe intestinal injury (A). In particular, there was necro-inflammatory debris in small foci within the epithelial cell layer with focal loss of cells; numerous neutrophils were observed among epithelial cells and edematous lamina propria as well as lymphatic infiltration was detected (B). The severity of mucosa damage was also demonstrated by the presence of apoptotic cells (C) and mitotic figures (D). Conversely, the sections of jejunum from rats treated with GPI 15427 + CPT-11 (E) demonstrate neither histological alterations nor the presence of apoptotic cells or/and mitotic figures. No histological changes were found in the tissue from vehicle-treated rats (F). Figures are representative of at least 3 experiments performed on different experimental days.

To assess whether intestinal damage by CPT-11 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 antibody (Ab). Jejunum sections from CPT-11-treated rats showed positive staining for (ADP-ribose) polymers in the damaged areas, whereas no positive staining was found in the jejunum of animals treated with GPI 15427 + CPT-11 or with vehicle.

The ability of GPI 15427 to protect animals from delayed diarrhea was also investigated. In animals that received CPT-11, no diarrhea was observed in the first 24 h. However, mild to moderate diarrhea developed after this time point. When the anticancer drug was administered in combination with oral GPI 15427, a reduction in the severity of delayed diarrhea was observed.

Finally we evaluated whether PARP inhibition might influence myelosuppression induced by TMZ. Mice were treated with GPI 15427 per os 40 mg/kg/day x 5 days ± 100 mg/kg x 5days TMZ and cell blood counts were analyzed on day 8 and 15. TMZ treatment caused a 78% and 60% decline of white blood cells on days 8 and 15, respectively, whereas GPI 15427 had no effect. Coadministration of TMZ and GPI 15427 did not enhance the myelosuppressive effects of TMZ.

CONCLUSIONS AND SIGNIFICANCE

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 xenograft colon cancers. It is noteworthy that GPI 15427 provides protection against intestinal damage induced by CPT-11 and does not exacerbate myelotoxicity of TMZ.

Many cancer cell lines showed different susceptibility to CPT-11 and TMZ, mainly due to differences in breast cancer resistance protein expression, O⁶-alkylguanine DNA alkyltransferase levels, or MR functional status, and possibly different levels of PARP-1. Inhibition of PARP markedly increased the antiproliferative effects of the combination TMZ + SN-38 in all cell lines. Moreover, the in vivo results indicated that this potentiation was particularly evident with the highly aggressive HT-29 line, which possesses a doubling time 2-fold shorter than LoVo. HT-29 was less responsive to TMZ + CPT-11. The results obtained in vivo also indicated that oral administration of GPI 15427 significantly inhibited PARP activity of PBL, suggesting that the compound is well absorbed and pharmacologically active.

A major concern with chemosensitizers is the increase of toxicity of chemotherapy toward normal tissues. In regard to CPT-11, its dose-limiting toxicity is delayed diarrhea, which 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 ßbeta;-glucoronidase. Oral administration of GPI 15427 reduced damage of jejunum and severity of delayed diarrhea in animals after CPT-11 treatment. The amelioration of intestinal damage was associated with reduced (ADP-ribose) polymers formation in the intestinal epithelium, suggesting a role for PARP-1 overactivation in the pathogenesis of CPT-11 toxicity. The ability of GPI 15427 to prevent CPT-11 intestinal toxicity might be attributed to the higher concentrations of the PARP inhibitor reached in the gut by means of oral administration.

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 into 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.

View larger version (30K): [in this window] [in a new window]   Figure 3. A scheme depicting the dual effect of PARP inhibitor as enhancer of TMZ and CPT-11 antitumor activity and as protective agent against CPT-11 untoward effects. In the case of TMZ, chemosensitization derives from impairment of the repair of N-methylpurines (N3-methyladenine and N7-methylguanine), which generally do not contribute to cytotoxicity. In the absence of PARP inhibitors, these methyl adducts are promptly removed by the base excision repair (BER), which requires PARP-1 and PARP-2 functions. PARP inhibition also hampers topoisomerase I activity and favors the toxic effects of CPT-11. Therefore, PARP inhibitors potentiate the antitumor activity of low doses TMZ and CPT-11 used in combination by inhibiting the repair of moderate DNA damage. When DNA damage is severe PARP-1 overactivation causes extensive NAD+ consumption during the synthesis of (ADP-ribose) polymers, which eventually leads to ATP depletion and cell death. Our findings demonstrate that PARP-1 overactivation is involved in intestinal toxicity induced by CPT-11 and that PARP inhibition provides protection from such untoward effect.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5916fje

This article has been cited by other articles:

				R. J. Kinders, M. Hollingshead, S. Khin, L. Rubinstein, J. E. Tomaszewski, J. H. Doroshow, R. E. Parchment, and the National Cancer Institute Phase 0 Clinical Tri Preclinical Modeling of a Phase 0 Clinical Trial: Qualification of a Pharmacodynamic Assay of Poly (ADP-Ribose) Polymerase in Tumor Biopsies of Mouse Xenografts Clin. Cancer Res., November 1, 2008; 14(21): 6877 - 6885. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5916fjev1 20/10/1709    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tentori, L. Articles by Graziani, G. Search for Related Content PubMed PubMed Citation Articles by Tentori, L. Articles by Graziani, G.