Analysis of the intra- and intertumoral heterogeneity of hypoxia in pancreatic cancer patients receiving the nitroimidazole tracer pimonidazole.

BACKGROUND: Hypoxia is thought to be an adverse feature of pancreatic cancer, but direct measurement in patients is technically challenging. To address this, we characterised the intra/interpatient heterogeneity of hypoxia in surgical specimens from patients who received the 2-nitroimidazole tracer pimonidazole pre-operatively. METHODS: Pimondazole was given intravenously 16-20 h before pancreatectomy, and the extent and intratumoral heterogeneity of hypoxia determined by image analysis applied to multiple tissue blocks stained by immunohistochemistry. Intra/interpatient heterogeneity was estimated by variance component analysis. RESULTS: Pimonidazole staining was analysed in 10 tumours. The extent of labelling varied amongst patients (0-26%), with a broader range of hypoxia in the epithelial (1-39%) compared with the stromal (1-13%) compartments. Variance component analysis demonstrated greater inter- than intrapatient variability of hypoxia, and that multiple (4-5) tumour sections are required to provide a consistent evaluation of its extent in individual tumours. CONCLUSIONS: There is significant intra- and intertumoral heterogeneity of hypoxia in pancreatic cancers, and these do not appear to be generally more hypoxic than other cancer types. This study establishes the feasibility to assess hypoxia in pancreatic cancer patients using pimonidazole, but questions the reliability of measurements made using a single tissue section.

BRCA1 and BRCA2 mutations sensitize to chemotherapy in patient-derived pancreatic cancer xenografts.

BACKGROUND: Germline mutations of the BRCA tumour suppressors have been associated with increased risk of pancreatic cancer. Clinical evidence suggests that these patients may be more sensitive to treatment with cisplatin. As the frequency of germline BRCA mutations is low, definitive experimental data to support the clinical observations are still missing. METHODS: We tested gemcitabine and cisplatin sensitivity of four BRCA1 and BRCA2 mutant and three BRCA1 and BRCA2 wild-type (WT) patient-derived pancreatic cancer xenografts. RESULTS: We observed treatment sensitivity to gemcitabine and cisplatin in the BRCA WT and mutant models. The BRCA1 and BRCA2 mutant xenografts were significantly more sensitive to cisplatin although these models also showed sensitivity to gemcitabine. The BRCA1 and BRCA2 WT models showed sensitivity to gemcitabine but not cisplatin. Treatment sensitivity in the xenograft models closely resembled treatment response in the corresponding patients. DISCUSSION: We have characterised a panel of xenografts derived from pancreatic cancer patients carrying germline BRCA mutations, and shown that their genetic features resemble the patient donor. Our results support further clinical testing of treatment regimens combining gemcitabine and platinum drugs in this patient population, as well as preclinical research aiming to identify mechanisms of cisplatin resistance in BRCA mutant pancreatic cancers.

A phase I/II study of imatinib plus reinduction therapy for c-kit-positive relapsed/refractory acute myeloid leukemia: inhibition of Akt activation correlates with complete response.

This phase I/II study evaluated imatinib as a c-kit inhibitor combined with mitoxantrone, etoposide and cytarabine therapy for patients with primary refractory or relapsed c-kit+ acute myeloid leukemia (AML). Imatinib was escalated through three dose levels in successive six patient cohorts. The combination was well tolerated up to 400 mg/day imatinib. Of 21 patients treated at this dose, 13 (62%) achieved complete response (CR), 7 (33%) were non-responders and one died during induction. The CR rate was 80% in patients with standard-risk karyotype versus 33% in patients with adverse karyotype. The CR rate for primary non-responders was 6/14 (43%) versus 7/7 (100%) for relapsed patients. AML blasts from peripheral blood were assayed for phosphorylated Akt (pAkt) and phosphorylated ERK (pERK) by flow cytometry before to and after imatinib dosing. Of eight patients achieving CR with reinduction, seven demonstrated marked (≥60%) pAkt inhibition with imatinib therapy. In contrast, all the six non-responders to reinduction demonstrated <60% pAkt inhibition (P=0.005). There was no correlation between pERK inhibition and response to therapy. These results indicate that lack of pAkt inhibition in vivo is associated with resistance to reinduction therapy using this regimen. Further studies using agents that are able to inhibit Akt more effectively are warranted.

Activity of a novel, dual PI3-kinase/mTor inhibitor NVP-BEZ235 against primary human pancreatic cancers grown as orthotopic xenografts.

The phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway is frequently deregulated in pancreatic cancers, and is believed to be an important determinant of their biological aggression and drug resistance. NVP-BEZ235 is a novel, dual class I PI3K/mammalian target of rapamycin (mTor) inhibitor undergoing phase I human clinical trials. To simulate clinical testing, the effects of NVP-BEZ235 were studied in five early passage primary pancreatic cancer xenografts, grown orthotopically. These tumours showed activated PKB/Akt, and increased levels of at least one of the receptor tyrosine kinases that are commonly activated in pancreatic cancers. Pharmacodynamic effects were measured following acute single doses, and anticancer effects were determined in separate groups following chronic drug exposure. Acute oral dosing with NVP-BEZ235 strongly suppressed the phosphorylation of PKB/Akt, followed by recovery over 24 h. There was also inhibition of Ser235/236 S6 ribosomal protein and Thr37/46 4E-BP1, consistent with the effects of NVP-BEZ235 as a dual PI3K/mTor inhibitor. Chronic dosing with 45 mg kg(-1) of NVP-BEZ235 was well tolerated, and produced significant tumour growth inhibition in three models. These results predict that agents targeting the PI3K/Akt/mTor pathway might have anticancer activity in pancreatic cancer patients, and support the testing of combination studies involving chemotherapy or other molecular targeted agents.

Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer.

Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.

Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.

Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.

Inhibition of gamma-glutamyl transpeptidase activity decreases intracellular cysteine levels in cervical carcinoma.

PURPOSE: To determine whether gamma-glutamyl transpeptidase (gamma-GT) is involved in the maintenance of elevated cysteine levels in cervical carcinoma. METHODS: Four cervical carcinoma cell lines were tested in vitro for cysteine accumulation and gamma-GT levels. The highest and lowest gamma-GT-expressing cell lines were used in in vivo experiments to determine the effect of gamma-GT inhibition on cysteine levels. RESULTS: Treatment of a series of cervical carcinoma cell lines with acivicin decreased intracellular cysteine concentrations. Cysteine depletion was evident in Me180 cells which had the greatest levels of gamma-GT activity, and had a more pronounced cysteine decrease in medium with glutathione and cysteine concentrations simulating the in vivo situation. Also investigated were the effects of inhibition of gamma-GT activity on intracellular cysteine levels in xenografts grown in severe combined immunodeficient (SCID) mice. With the use of 35 mg/kg of acivicin, gamma-GT activity decreased to basal levels of detection in both tumour types and significant decreases in cysteine levels were seen in the high gamma-GT-expressing tumours (Me180). Thus, inhibition of gamma-GT activity may have therapeutic potential in high-expressing cancers. CONCLUSIONS: In tumours and cell lines with elevated levels of gamma-GT activity, inhibition of this enzyme led to decreases of cysteine levels.

Assessment of tumor cell repopulation after chemotherapy for advanced ovarian cancer: pilot study.

BACKGROUND: Repopulation of clonogenic tumor cells appears to increase during fractionated radiation treatment and is recognized as an important factor affecting local control. Given the longer intervals between cycles and longer total duration of treatment, the impact of repopulation is likely to be greater after chemotherapy. METHODS: We assessed tumor cell repopulation with the proliferative marker Ki-67 in 21 patients with ovarian carcinoma who received initial chemotherapy. Paraffin slides were evaluated from the diagnostic biopsy and from tumor obtained at debulking surgery after chemotherapy. Immunohistochemistry using the MIB-1 antibody was performed on the paired samples and analyzed with a digital imaging device linked to a color camera mounted on a transmitted-light microscope. The ratio of Ki-67 positive to all nuclei was used as a proliferative index and compared for pre- and postchemotherapy specimens. RESULTS: All patients received platinum-based chemotherapy and most showed a response to treatment. The median duration between last chemotherapy and debulking surgery was 33 days (range, 22-50 days). Four (19%) of 21 patients showed an increased proliferative index after chemotherapy, and the remainder showed a decrease (n = 12) or no significant change (n = 5). CONCLUSIONS: Our results did not suggest an increase in proliferation of tumor cells after this type of chemotherapy in the majority of patients with ovarian cancer.

BAD induces apoptosis in cells over-expressing Bcl-2 or Bcl-xL without loss of mitochondrial membrane potential.

Inhibitors of Bcl-2 may be useful therapeutic agents for the treatment of a wide variety of malignancies including leukemia. A potential prototype of such a compound is the endogenous Bcl-2 and Bcl-xL binding protein BAD. Previous reports indicate that BAD can overcome the anti-apoptotic effect of Bcl-xL but not Bcl-2. If BAD cannot induce apoptosis in cells over-expressing Bcl-2, it would limit the application of molecules like BAD as novel anti-tumor agents. We report that transient transfection of BAD induced cell death in cells with and without over-expression of Bcl-2 or Bcl-xL. Forty-eight hours after transfection, BAD increased cell death in COS, COS Bcl-2, and COS Bcl-xL cells as demonstrated by decreased GFP expression, and an increase in the number of number of floating cells. In addition, BAD induced cell death in leukemic cell lines over-expressing Bcl-2 and Bcl-xL as determined by changes in luciferase activity. BAD-induced apoptosis was not accompanied by loss of mitochondrial membrane potential. Therefore, we conclude that transient transfection of BAD directly induces apoptosis in cells over-expressing Bcl-2 or Bcl-xL and validates the pursuit of molecules like BAD as novel therapeutic agents.

Wortmannin inhibits pkb/akt phosphorylation and promotes gemcitabine antitumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice.

Pancreatic cancer is resistant to almost all classes of cytotoxic agents. Gemcitabine seems to be the current drug of choice. We have recently reported that inhibition of the phosphatidylinositide 3-kinase-protein kinase B (PKB/Akt) cell survival pathway by wortmannin enhances gemcitabine-induced apoptosis in cultured human pancreatic cancer cells (1). The present study investigated the effects of wortmannin on orthotopic human pancreatic cancer xenografts implanted in severe combined immunodeficient mice. Animals were given single i.v. bolus injections of 0.175, 0.35, or 0.7 mg/kg of wortmannin and killed at 0.5, 1, 2, or 4 h after treatment. Phosphorylated PKB/Akt levels in tumor tissues were measured by fluorescence image analysis. Wortmannin was found to inhibit PKB/Akt phosphorylation in a time- and dose-dependent manner, reaching a plateau at 4 h and at 0.7 mg/kg. The levels of phosphorylated PKB/Akt were maximally decreased by approximately 50% relative to the vehicle control. Subsequently, the extent of apoptosis in tumors treated with gemcitabine or wortmannin alone or in combination was determined using terminal deoxynucleotidyl transferase-mediated nick end labeling assay and computerized image analysis. Orthotopic tumors exposed to 80 mg/kg gemcitabine for 48 h and then 0.7 mg/kg wortmannin for 4 h showed a 5-fold increase (P = 0.002) in apoptosis compared with those treated with each agent alone and with the vehicle control. The combination treatment also significantly (P < 0.001) inhibited tumor growth. Taken together, our findings support the potential of phosphatidylinositide 3-kinase inhibitors as adjuncts to conventional chemotherapy in the treatment of pancreatic cancer.

Hypoxia-inducible factor-1alpha is an intrinsic marker for hypoxia in cervical cancer xenografts.

The hypoxia-inducible factor 1 (HIF-1) is known to induce the expression of several proteins linked to the maintenance of oxygen homeostasis, cellular energy metabolism, and tumor progression. Its alpha subunit (HIF-1alpha) is stabilized under hypoxic conditions and, therefore, might represent an intrinsic marker for tissue hypoxia. Here we report on the spatial relationship between HIF-1alpha and the nitroimidazole hypoxia marker EF5 in cervical carcinoma xenografts, and on their spatial relationship to tumor blood vessels. EF5 was administered to mice bearing ME180 and SiHa cervical cancer xenografts. Frozen tumor tissue sections, triple-stained for HIF-1alpha, the endothelial cell marker CD31, and EF5, were imaged using wide-field multiparameter immunofluorescence microscopy. Expression levels of EF5 and HIF-1alpha were similar in ME180 xenografts, but the percentage of tumor area stained with EF5 was significantly smaller than the percentage of HIF-1alpha-positive area in SiHa tumors. In both tumor types the EF5-HIF-1alpha overlap was statistically significant, thus confirming their spatial and temporal colocalization. Spatial distribution analysis of EF5 and HIF-1alpha is consistent with different pO2 value "thresholds" for EF5 binding and HIF-1alpha expression. Summarized, our results indicate that HIF-1alpha is a useful intrinsic marker for hypoxia in cervical carcinoma xenografts.

Imaging of whole tumor cut sections using a novel scanning beam confocal fluorescence MACROscope.

Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at microm resolutions. We have used a novel confocal scanning laser MACROscope (CSLM) capable of acquiring images over fields of view up to 2cm x 2cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxia-inducible transcription factor 1 alpha (HIF-1alpha), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helical structure and independent of Bcl-2.

Since the over-expression of Bcl-2 is a common cause of multi-drug resistance, cytotoxic peptides that overcome the effects of Bcl-2 may be clinically useful. We harnessed the death-promoting alpha helical properties of the BH3 domain of BAD by fusing it to the Antennapedia (ANT) domain, which allows for cell entry (ANTBH3BAD). Treatment of 32D cells with the ANTBH3BAD peptide results in a 99% inhibition of colony formation. No significant toxicity is observed after treatment with ANT or BH3BAD alone. A mutant fusion peptide unable to bind Bcl-2 induces cell death as effectively as the wild-type ANTBH3BAD. Furthermore, 32D cells over-expressing Bcl-2 show no resistance to the ANTBH3BAD peptide. Therefore, the toxicity of the peptide was independent of the Bcl-2 pathway. We demonstrate that the toxicity of the peptide is due to its alpha helicity that disrupts mitochondrial function. Since this peptide overcomes major forms of drug resistance, it may be therapeutically useful if appropriately targeted to malignant cells.

Differential effects of buthionine sulphoximine in hypoxic and non-hypoxic regions of human cervical carcinoma xenografts.

BACKGROUND AND PURPOSE: Recently we reported increased glutathione (GSH) levels in hypoxic regions of ME 180 and SiHa cervical cancer xenografts. Since this association might act synergistically to protect from radiotherapy, we examined the differential effects of the GSH depleting agent buthionine suiphoximine (BSO) in relation to tumor oxygenation. MATERIALS AND METHODS: The nitroimidazole EF5 was used to label tumor hypoxia. GSH levels were determined in cryostat sections using a sensitive HPLC assay and in parallel sections using fluorescence image analysis. Using a dual-labeling method, GSH levels were determined selectively in hypoxic and non-hypoxic tumor regions. RESULTS: GSH levels were higher in hypoxic than in non-hypoxic regions of cervical carcinoma xenografts. Treatment with BSO produced a more pronounced GSH depletion in regions of hypoxia, resulting in similar post-treatment levels in hypoxic and non-hypoxic areas. CONCLUSIONS: BSO effectively depletes GSH in hypoxic microregions of tumors. These findings suggest a potential role for BSO as an adjunct to radiotherapy in cervical cancer patients.

Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors.

Cancer cells frequently show abnormal signaling via the mitogen activated protein kinase (MAP kinase) pathway due to increased activity of surface receptors for growth factors, or as a result of ras mutations. The development of potent anti-cancer agents that target this pathway prompts the need for analytical methods that allow pharmacodynamic monitoring of drug effects in patients during early phase clinical trial. We describe such a method, based on the activation of T-lymphocytes in undiluted peripheral blood using phorbol myristate acetate (PMA). Following rapid hypotonic lysis and formaldehyde fixation, activation of the MAP kinase pathway can then be demonstrated using phospho-specific antibodies that recognize the activated mediators MEK or ERK, followed by surface labeling with anti-CD3 to identify T-lymphocytes. This method was used to investigate the effects of a MEK inhibitor, U0126, and a new raf kinase inhibitor BAY 37-9751 in blood samples from normal donors. Dose-dependent inhibition of pERK activation was demonstrated for both agents. Furthermore, differential effects on pMEK activation allowed the molecular targets of the two inhibitors to be distinguished. In addition to monitoring drug effects in patients during treatment with inhibitors of the MAP kinase pathway, the general methodology described in this paper has the potential for wide application to the study of signal transduction at the single cell level using flow cytometry.

Respiratory chain-generated oxidative stress following treatment of leukemic blasts with DNA-damaging agents.

Oxidative stress occurs in diverse life forms during programmed cell death and appears to be a significant mediator since a wide range of manipulations that enhance cellular antioxidant systems are protective. Using a recently developed flow cytometry technique to assess respiratory chain function, we have investigated the mechanism of reactive oxygen generation in OCI/AML-2 leukemic blasts following treatment with cytosine arabinoside, etoposide, and gamma-irradiation. Increases in mitochondrially generated reactive oxygen were seen using all three agents, in association with hyperpolarization of the mitochondrial inner membrane. Increased reactive oxygen occurred when mitochondria were energized using substrates for either complex I or complex II, indicating that the likely source is complex III (cytochrome c reductase). These findings are consistent with impaired adenine nucleotide exchange across the mitochondrial membrane, recently proposed to be an important event during the early stages of apoptosis induction (M. G. Vander Heiden et al., 1999, Mol. Cell 3, 159-167). Elevations of the antioxidants glutathione and thioredoxin occurred in association with this oxidative stress, likely the result of feedback mechanisms based on redox-sensitive transcription factors. Since glutathione and thioredoxin can protect from drug-induced apoptosis, their upregulation in response to respiratory chain-generated reactive oxygen might represent a cellular adaptation to DNA damage that promotes cell survival.

Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry.

BACKGROUND: Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin. METHODS: Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy. RESULTS: Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy. CONCLUSIONS: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.

Equilibrative-sensitive nucleoside transporter and its role in gemcitabine sensitivity.

Salvage of preformed nucleosides requires transport across the plasma membrane by sodium-dependent (concentrative) and sodium-independent (equilibrative) mechanisms. These transport systems are also the route of cellular uptake for nucleoside analogues, including gemcitabine (2',2'-difluorodeoxycytidine), a deoxycytidine analogue used in the treatment of pancreatic cancer. To determine whether gemcitabine cytotoxicity is influenced by the equilibrative-sensitive nucleoside transporter (es-NT), basal levels of the es-NT were quantified in three human pancreatic cancer cell lines (PANC-1, HS-766T, and PK-8) and one human bladder cancer cell line (MGH-U1) by flow cytometric analysis, and the results were compared with gemcitabine cytotoxicity assessed by clonogenic assay. To determine whether the salvage pathway of DNA synthesis can be up-regulated by inhibiting de novo DNA synthesis, combination experiments were carried out using the thymidylate synthase (TS) inhibitors 5-fluorouracil or raltitrexed with gemcitabine in a concurrent and sequential fashion. No relationship between basal es-NT and gemcitabine cytotoxicity was demonstrated. For two pancreatic cell lines, sequence-dependent effects of the combination of TS inhibitors and gemcitabine were seen with maximum effect when the TS inhibitors preceded gemcitabine. This was also associated with a significant increase in es-NT levels caused by the TS inhibitors. Thus, modulation of the es-NT by pretreatment with TS inhibitors may have the potential to improve the therapeutic benefit of gemcitabine.