Rami G. Azrak

Therapeutic Synergy Between Irinotecan and 5-Fluorouracil against Human Tumor Xenografts

Purpose: Although the combination of irinotecan and 5-Fluorouracil is clinically active, it is associated with significant toxicity and resistance. Studies were carried out to define the optimal dosage, sequence, and timing for the combination in mice bearing xenografted human tumors. Experimental Design: The maximum tolerated dose of irinotecan and 5-Fluorouracil in combination was determined in nude mice. Therapeutic efficacy against established human colon carcinoma xenografts, HCT-8 and HT-29, and human head and neck squamous cell carcinoma xenografts, FaDu and A253, was determined using the rugs individually, simultaneously, and in sequence with various intervals in between. Treatments were i.v. weekly × 4. Immunohistochemical and reverse transcription-PCR measurements of relevant drug-metabolizing enzymes, apoptosis-related proteins, cell cycle distribution, cyclin A, and S phase fraction expression were carried out and compared with the therapeutic outcome. Results: The maximum tolerated dose of irinotecan resulted in cure rates of 30% or less in all xenografts. No cures were achieved with FUra alone. Concurrent administration of irinotecan and FUra, or of FUra 24 h before irinotecan, resulted in cure rates of <20%, except for FaDu (60%). Administration of irinotecan 24 h before FUra resulted in the highest cure rates, 80% in HCT-8, 0% in HT-29, 100% in FaDu, and 10% in A253. Conclusions: The optimal therapeutic synergy was achieved when irinotecan was administered 24 h before 5-Flurouracil. Sensitivity to this combination was associated with poor differentiation status, higher cyclin A index, recruitment of cells into S phase, and induction of Bax expression and apoptosis.

Lack of microvessels in well-differentiated regions of human head and neck squamous cell carcinoma A253 associated with functional magnetic resonance imaging detectable hypoxia, limited drug delivery, and resistance to irinotecan therapy.

Purpose: Combination chemotherapy with irinotecan (CPT-11; 50 mg/kg/week × 4 intravenously), followed 24 hour later by 5-fluorouracil (50 mg/kg/week × 4 intravenously), results in 10 and 100% cure rates of animals bearing human head and neck squamous cell carcinoma xenografts A253 and FaDu, respectively. A253 consists of 30% well-differentiated and avascular and 70% poorly differentiated regions with low microvessel density (10/×400), whereas FaDu is uniformly poorly differentiated with higher microvessel density (19/×400). Studies were carried out for determining the role of well-differentiated and avascular regions in drug resistance in A253 and detection of such regions with noninvasive functional magnetic resonance (fMR) imaging. Experimental Design: Tumors were harvested for histopathologic evaluation and immunohistochemistry (CD31, CD34; differentiation marker: involucrin; hypoxia markers: carbonic anhydrase IX, pimonidazole; vascular endothelial factor (VEGF) and Ki67) immediately after fMR imaging following the 3rd dose of chemotherapy. High-performance liquid chromatography determination of intratumoral drug concentration of 7-ethyl-10-hydroxyl-camptothecin and autoradiography with 14C-labeled CPT-11 was done 2 hours after CPT-11 administration. Results: Although A253 xenografts showed three times higher concentration of 7-ethyl-10-hydroxyl-camptothecin, FaDu was more responsive to therapy. After therapy, A253 tumor consisted mostly (∼80%) of well-differentiated regions (positive for involucrin) lacking microvessels with a hypoxic rim (positive for carbonic anhydrase IX and pimonidazole) containing few proliferating (Ki67 positive) poorly differentiated cells. Autoradiography revealed that well-differentiated A253 tumor regions showed 5-fold lower 14C-labeled CPT-11 concentrations compared with poorly differentiated areas (P < 0.001). Blood oxygen level dependant fMR imaging was able to noninvasively distinguish the hypoxic and well-vascularized regions within the tumors. Conclusion: Avascular-differentiated regions in squamous cell carcinoma offer sanctuary to some hypoxic but viable tumor cells (carbonic anhydrase IX and Ki67 positive) that escape therapy because of limited drug delivery. This study provides direct evidence that because of a specific histologic structure, avascular, well-differentiated hypoxic regions in tumors exhibit low drug uptake and represent a unique form of drug resistance.

A phase I and pharmacokinetic study of fixed-dose selenomethionine and irinotecan in solid tumors.

PURPOSE: We conducted a phase I study to determine the maximum tolerated dose (MTD) of irinotecan with fixed, nontoxic high dose of selenomethionine. EXPERIMENTAL DESIGN: Selenomethionine was given orally as a single daily dose containing 2,200 mug of elemental selenium (Se) starting 1 week before the first dose of irinotecan. Irinotecan was given i.v. once weekly x 4 every 6 weeks (one cycle). The starting dose of irinotecan was 125 mg/m(2)/wk. Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for selenium and irinotecan and its metabolites. RESULTS: Three of four evaluable patients at dose level 2 of irinotecan (160 mg/m(2)/wk) had a dose-limiting diarrhea. None of the six evaluable patients at dose level 1 (125 mg/m(2)/wk irinotecan) had a dose-limiting toxicity. One patient with history of irinotecan-refractory colon cancer achieved a partial response. The long half-life of selenium resulted in a prolonged accumulation towards steady-state concentrations. No significant changes in the pharmacokinetics of CPT-11, SN-38, or SN-38G were identified; however, the coadministration of selenomethionine significantly reduced the irinotecan biliary index, which has been associated with gastrointestinal toxicity. CONCLUSIONS: Selenomethionine at 2,200 mug/d did not allow the safe escalation of irinotecan beyond the previously defined MTD of 125 mg/m(2). None of the patients receiving 125 mg/m(2) of irinotecan had grade >2 diarrhea. Unexpected responses and disease stabilizations were noted in a highly refractory population. Further escalation of selenomethionine is recommended in future trials to achieve defined protective serum concentrations of selenium.

Prostate derived Ets transcription factor (PDEF) is a potential prognostic marker in patients with prostate cancer

Reduced expression of prostate‐derived Ets transcription factor (PDEF) leads to morphologic change as well as increased migration and invasiveness of prostate cancer cells. However, the clinical relevance of PDEF expression and its relationship to anti‐apoptotic protein survivin is yet to be determined.

Prostate‐derived Ets transcription factor as a favorable prognostic marker in ovarian cancer patients

We have previously shown that ovarian tumors express prostate‐derived Ets transcription factor (PDEF). However, the precise role of PDEF in the prognosis of ovarian cancer is unknown. In our study, we report for the first time that expression of PDEF in tumor lesions of patients with ovarian cancer is associated with favorable prognosis. Evaluation of samples from 40 patients with ovarian cancer showed that early stage (IA) and borderline (IIB, III) ovarian tumors expressed higher levels of PDEF mRNA and protein and lower levels of survivin compared to late stage ovarian tumors (IIIC and IV, p < 0.05). Normal ovarian tissues expressed the highest levels of PDEF mRNA and protein when compared to tumor tissues (p < 0.05). A Log‐Rank test showed that overall survival of patients with PDEF‐positive and survivin‐negative ovarian tumors was significantly longer than those with PDEF‐negative and survivin‐positive tumors (p < 0.01). Forced expression of PDEF in PDEF‐negative ovarian tumor cells inhibited tumor cell growth, induced apoptosis, downregulated survivin expression and its promoter activity. Furthermore, treatment of ovarian cancer cells with vitamin D or a selenium compound resulted in re‐expression of PDEF, downregulation of survivin, induction of apoptosis and inhibition of tumor cell growth when compared to untreated controls (p < 0.05). Together, these observations showed an inverse correlation between PDEF and survivin expression and suggested that increased PDEF expression along with reduced survivin was associated with prolonged survival of patients with ovarian cancer.

Potentiation of irinotecan sensitivity by Se-methylselenocysteine in an in vivo tumor model is associated with downregulation of cyclooxygenase-2, inducible nitric oxide synthase, and hypoxia-inducible factor 1α expression, resulting in reduced angiogenesis

Until recently, the use of Se-methylselenocysteine (MSC) as selective modulator of the antitumor activity and selectivity of anticancer drugs including irinotecan, a topoisomerase I poison, had not been evaluated. Therapeutic synergy between MSC and irinotecan was demonstrated by our laboratory in mice bearing human squamous cell carcinoma of the head and neck tumors. In FaDu xenografts, a poorly differentiated tumor-expressing mutant p53, the cure rate was increased from 30% with irinotecan alone to 100% with the combination of irinotecan and MSC. Cellular exposure to cytotoxic concentration of SN-38, the active metabolite of irinotecan (0.1 μM) alone and in combination with noncytotoxic concentration of MSC (10 μM) did not result in additional enhancement of chk2 phosphorylation and downregulation of specific DNA replication-associated proteins, cdc6, MCM2, cdc25A, nor increase in PARP cleavage, caspase activation and the 30–300 kb DNA fragmentation induced by SN-38 treatment. MSC did not alter significantly markers associated with apoptosis, nor potentiate irinotecan-induced apoptosis. These results indicate that apoptosis is unlikely to be one of the main mechanism associated with the observed in vivo therapeutic synergy. In contrast, significant downregulation of cyclooxygenase-2 (COX-2) expression and activity was observed in the cells exposed to SN-38 in combination with MSC compared to SN-38 alone. Moreover, the inhibition of PGE2 production was also observed in the cells treated with the combination as compared with SN-38 alone. Analysis of tumor tissues at 24 h after treatment with synergistic modality of irinotecan and MSC revealed significant downregulation of COX-2, inducible nitric oxide synthase (iNOS) and hypoxia-induced factor-1α expression (HIF 1α). Moreover, decreased microvessel density was observed after irinotecan treatment with the addition of MSC. These results suggest that observed therapeutic synergy correlates with the inhibition of neoangiogenesis through the downregulation of COX-2, iNOS and HIF-1α expression.

Targeting the oncogenic protein beta-catenin to enhance chemotherapy outcome against solid human cancers

BackgroundBeta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of β-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3β) through phosphorylation. Cell growth and proliferation is associated with β-catenin translocation from the cytoplasm into the nucleus.This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on β-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of β-catenin, (2) investigate the kinetics and the mechanism of β-catenin inhibition, and (3) confirm that inhibition of β-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs.ResultsIn six human cancer cell lines, the inhibition of total and nuclear expression of β-catenin by MSeA was dose and time dependent. The involvement of GSK-3β in the degradation of β-catenin was cell type dependent (GSK-3β-dependent in HT-29, whereas GSK-3β-independent in HCT-8). However, the pronounced inhibition of β-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.Knockout of β-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.Collectively, the generated data demonstrate that β-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs.ConclusionsThis study demonstrates that β-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of β-catenin by GSK-3β is not a general mechanism but is cell type dependent.

The mechanism of methylselenocysteine and docetaxel synergistic activity in prostate cancer cells

The study was designed to evaluate the combination treatment of methylselenocysteine (MSeC) and docetaxel and to delineate the underlying mechanism associated with observed in vitro synergy between MSeC and docetaxel in prostate cancer cells. Cells were treated with different concentrations and schedules (concurrent or sequential) of MSeC and docetaxel alone or in combination. Cell growth/death was assessed with sulforhodamine B assay, trypan blue assay, and time-lapse video. Loewe synergism/antagonism model was used to determine whether the combination effect was additive, synergistic, or antagonistic. Apoptosis and caspase-3 activity were evaluated with cell death ELISA assay and caspase activity assay, respectively. Synergy between MSeC and docetaxel was further assessed in the presence and absence of z-VAD-fmk, a pan-caspase inhibitor. Effect of MSeC and docetaxel alone or in combination on the cellular expression of the antiapoptotic protein survivin was measured with Western blot analyses. Pretreatment with MSeC was crucial to enhance docetaxel antitumor activity. The enhanced antitumor activity of the sequential combination treatment of MSeC and docetaxel (MSeC/docetaxel) was highly synergistic. Apoptosis increased after MSeC/docetaxel, compared with each drug alone or concurrent treatment. Pretreatment with z-VAD-fmk converted the synergy into antagonism, suggesting that the synergy is caspase-dependent apoptosis. The survivin level was down-regulated following MSeC/docetaxel treatment when compared with each drug alone. In conclusion, pretreatment with MSeC was essential to markedly sensitize cells to docetaxel. The synergy between MSeC and docetaxel in C2G prostate cancer cells is associated with increased level of caspase-dependent apoptosis and decreased level of survivin. [Mol Cancer Ther 2006;5(10):2540–8]

Chk1 signaling pathways that mediated G2M checkpoint in relation to the cellular resistance to the novel topoisomerase I poison BNP1350

A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity in vitro and in vivo. A BNP1350-resistant human head and neck carcinoma A253 cell line, denoted A253/BNPR, was developed. The A253/BNPR cell line was approximately 9-fold resistant to BNP1350 and 4-fold cross-resistant to another topoisomerase I inhibitor SN-38, the active metabolite of irinotecan. After drug treatment with equimolar concentrations of BNP1350 (0.7 microM) for 2h, activation of the DNA double-strand break repair protein complexes was similar in the two cell lines, suggesting that DNA dsb repair is not attributable to resistance to BNP1350 in the A253/BNPR cells. Cell cycle analysis indicates that the A253 cell line accumulated primarily in S phase, but G(2) phase accumulation was observed in the A253/BNPR cell line at 48 h after drug removal. Elevated chk1 phosphorylation at Ser(345) following DNA damage induced by BNP1350 was accompanied by G(2) accumulation in the A253/BNPR cell line, while exposure to equimolar concentrations of BNP1350 (0.7 microM) induced S-phase arrest and no increased phosphorylation of chk1 at Ser(345) in the A253 cell line. Under the same conditions, increased chk1 activity was observed in the A253/BNPR cell line, but not in the A253 cell line. Moreover, stimulated binding of 14-3-3 proteins to chk1 was observed in BNP1350-treated A253/BNPR cells. To confirm relationship between chk1 expression/phosphorylation and drug resistance to topo I poisons, we examined the effects of chk1 or chk2 antisense oligonucleotides on the cellular growth inhibition. Chk1 antisense oligonucleotide can sensitize the A253/BNPR cells to killing by topo I inhibitor BNP1350, but no significant sensitization of BNP1350-induced growth inhibition was observed in the drug-sensitive cell line. Chk2 antisense oligonucleotide has only a small sensitization effect on BNP1350-induced growth inhibition in both cell lines. The data indicate that the chk1 signaling pathways that mediate cell cycle checkpoint are associated with cellular resistance to BNP1350 in the A253/BNPR cell line.

Irinotecan pharmacokinetic and pharmacogenomic alterations induced by methylselenocysteine in human head and neck xenograft tumors

The combination of methylselenocysteine and irinotecan (CPT-11) is synergistic against FaDu and A253 xenografts. Methylselenocysteine/CPT-11 increased tumor cure rate to 100% in FaDu and to 60% in A253. In this study, the effect of methylselenocysteine on pharmacokinetic and pharmacogenetic profiles of genes relevant to CPT-11 metabolic pathway was evaluated to identify possible mechanisms associated with the observed combinational synergy. Nude mice bearing tumors (FaDu and A253) were treated with methylselenocysteine, CPT-11, and a combination of methylselenocysteine/CPT-11. Samples were collected and analyzed for plasma and intratumor concentration of CPT-11 and 7-ethyl-10-hydroxyl-camptothecin (SN-38) by high-performance liquid chromatography. The intratumor relative expression of genes related to the CPT-11 metabolic pathway was measured by real-time PCR. After methylselenocysteine treatment, the intratumor area under the concentration-time curve of SN-38 increased to a significantly higher level in A253 than in FaDu and was associated with increased expression of CES1 in both tumors. Methylselenocysteine/CPT-11 treatment, compared with CPT-11 alone, resulted in a significant decrease in levels of ABCC1 and DRG1 in FaDu tumors and an increase in levels of CYP3A5 and TNFSF6 in A253 tumors. No statistically significant changes induced by methylselenocysteine/CPT-11 were observed in the levels of other investigated variables. In conclusion, the significant increase in the cure rate after methylselenocysteine/CPT-11 could be related to increased drug delivery into both tumors (CES1), reduced resistance to SN-38 (ABCC1 and DRG1) in FaDu, and induced Fas ligand apoptosis (TNFSF6) in A253. No correlation was observed between cure rate and other investigated variables (transporters, degradation enzymes, DNA repair, and cell survival/death genes) in either tumor.