nitrophenols and Colorectal-Neoplasms

Induction of mitochondria-controlled (intrinsic) apoptosis is a mainstay of current anti-neoplastic chemotherapies. Activation of this death pathway is counteracted by BCL-2-like proteins, which functionally set the threshold for apoptosis and determine whether malignant cells are sensitive or resistant to anti-cancer treatments. Hence, unlocking the intrinsic apoptotic cascade and promoting the cell's commitment to undergo apoptosis concordantly promotes efficacy of anti-cancer treatments. Here, we show that hyperosmotic stress enforces addiction of colorectal cancer cells to BCL-XL, thereby exhausting the protective capacity of BCL-2-like proteins and priming mitochondria for death. Our work identifies osmotic pressure as a cell extrinsic factor that modulates responsiveness of colorectal cancer cells to therapy.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; HCT116 Cells; HT29 Cells; Humans; Mitochondria; Nitrophenols; Osmotic Pressure; Piperazines; RNA Interference; Signal Transduction; Sulfonamides

Colorectal carcinoma (CRC) that represents one of the major causes for cancer-related death in humans is often associated with over-expression of anti-apoptotic proteins of Bcl-2 family. The aim of presented study was to determine the effect of ABT-737 inhibitor of anti-apoptotic proteins Bcl-2, Bcl-XL and Bcl-w as well as cyclin-dependent kinase 2 (CDK2) inhibitor SU9516 alone and in combination with ABT-737 on survival of colorectal cell lines HT29 and Caco-2. We have shown that both Caco-2 and HT29 cells that are relatively resistant to ABT-737 are also partially sensitive to SU9516, which increased sensitivity of Caco-2 but not HT29 cells to ABT-737. Increased sensitivity of Caco-2 cells to ABT-737 after addition of SU9516 correlated well with SU9516-induced decrease of Mcl-1 expression while we have not observed downregulation of Mcl-1 after the treatment of HT29 cells with SU9516. Instead of this, we have shown that treatment of HT29 cells with SU9516 is associated with decreased expression of tumour suppressor protein p53. Our findings provide a rationale for clinical use of Bcl-2 family inhibitors in combination with CDK2 inhibitors for treatment of Mcl-1-dependent colorectal tumours associated with expression of Bcl-2, Bcl-XL and Bcl-w proteins. In addition, we have shown potential of CDK2 inhibitors for treatment of tumours expressing R273H mutant p53.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Caco-2 Cells; Cell Survival; Colorectal Neoplasms; Cyclin-Dependent Kinase 2; Dose-Response Relationship, Drug; Drug Synergism; HT29 Cells; Humans; Imidazoles; Indoles; Nitrophenols; Piperazines; Sulfonamides; Treatment Outcome

The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)-collectively referred to as combo-Rx, in hypoxic CRC cell lines.. Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines.. In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses.. The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinases; Morpholines; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Signal Transduction; Sulfonamides; TOR Serine-Threonine Kinases; Tumor Hypoxia; Up-Regulation

Colorectal cancer (CRC) is the second most common malignant neoplasia in women and men worldwide. The B-cell lymphoma 2 (Bcl-2) protein family is mainly known for its pivotal role in the regulation of the mitochondrial death pathway. Anti-apoptotic Bcl-2 proteins may provide survival benefits and induce therapy resistance in cancer cells. Among anti-apoptotic Bcl-2 proteins, we found solely Bcl-xL strongly upregulated in human CRC specimens. In order to study protein function in the context of tumor initiation and progression in vivo, we generated a mouse model lacking Bcl-xL in intestinal epithelial cells (Bcl-xL(IEC-KO)). If challenged in an inflammation-driven tumor model, Bcl-xL(IEC-KO) mice showed a significantly reduced tumor burden with lower tumor numbers per animal and decreased tumor sizes. Analysis of cell death events by immunohistochemistry and immunoblotting revealed a striking increase of apoptosis in Bcl-xL-negative tumors. qRT-PCR and immunohistochemistry excluded changes in proliferative capacity and immune cell infiltration as reasons for the reduced tumor load and thereby identify apoptosis as key mechanism. Human CRC tissue was cultured ex vivo and treated with the small molecule compound ABT-737, which inhibits Bcl-xL and Bcl-2. Under ABT-737 treatment, the amount of apoptotic tumor cells significantly increased compared with controls, whereas proliferation levels remained unaltered. In summary, our findings identify Bcl-xL as a driver in colorectal tumorigenesis and cancer progression, making it a valuable target for clinical application.

Topics: Animals; Apoptosis; bcl-X Protein; Biphenyl Compounds; Carcinogenesis; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Enterocytes; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Inflammation; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Knockout; Nitrophenols; Oncogenes; Organ Specificity; Phenotype; Piperazines; Sulfonamides; Up-Regulation

Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials.. Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux.. This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12.. The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC.

Topics: Antineoplastic Agents; Autophagy; Biphenyl Compounds; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Indoles; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Signal Transduction; Sulfonamides

Evasion of apoptosis is a hallmark of cancer, and reversing this process by inhibition of survival signaling pathways is a potential therapeutic strategy. Phosphoinositide 3-kinase (PI3K) signaling can promote cell survival and is upregulated in solid tumor types, including colorectal cancer (CRC), although these effects are context dependent. The role of PI3K in tumorigenesis combined with their amenability to specific inhibition makes them attractive drug targets. However, we observed that inhibition of PI3K in HCT116, DLD-1, and SW620 CRC cells did not induce apoptotic cell death. Moreover, these cells were relatively resistant to the Bcl-2 homology domain 3 (BH3) mimetic ABT-737, which directly targets the Bcl-2 family of apoptosis regulators. To test the hypothesis that PI3K inhibition lowers the apoptotic threshold without causing apoptosis per se, PI3K inhibitors were combined with ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis by 2.3- to 4.5-fold and reduced expression levels of MCL-1, the resistance biomarker for ABT-737. PI3K inhibition enhanced ABT-737-induced apoptosis a further 1.4- to 2.4-fold in CRC cells with small interfering RNA-depleted MCL-1, indicative of additional sensitizing mechanisms. The observation that ABT-737-induced apoptosis was unaffected by inhibition of PI3K downstream effectors AKT and mTOR, implicated a novel PI3K-dependant pathway. To elucidate this, an RNA interference (RNAi) screen of potential downstream effectors of PI3K signaling was conducted, which demonstrated that knockdown of the TEC kinase BMX sensitized to ABT-737. This suggests that BMX is an antiapoptotic downstream effector of PI3K, independent of AKT.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Survival; Colorectal Neoplasms; Drug Synergism; Furans; HCT116 Cells; Humans; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Signal Transduction; Sulfonamides; TOR Serine-Threonine Kinases

Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC), prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-xL or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cadherins; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Indoles; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides

High levels of BCL-2 family members in colorectal carcinoma cause resistance to treatment. Inhibition of NANOG or its paralog NANOGP8 reduces the proliferation, stemness, and tumorigenicity of colorectal carcinoma cells. Our hypothesis was that inhibition of NANOG/NANOGP8 enhances the cytotoxic effect of BH3 mimetics targeting BCL-2 family members in colorectal carcinoma cells through reducing expression of MCL-1, a prosurvival BCL-2 protein.. Lentiviral vector (LV) shRNA to NANOG (shNG-1) or NANOGP8 (shNp8-1) transduced colorectal carcinoma cells that were also exposed to the BH3 mimetics ABT-737 or ABT-199 in vivo in colorectal carcinoma xenografts and in vitro where proliferation, protein and gene expression, and apoptosis were measured.. Clone A and CX-1 were sensitive to ABT-737 and ABT-199 at IC50s of 2 to 9 μmol/L but LS174T was resistant with IC50s of 18 to 30 μmol/L. Resistance was associated with high MCL-1 expression in LS174T. LVshNG-1 or LVshNp8-1 decreased MCL-1 expression, increased apoptosis, and decreased replating efficiency in colorectal carcinoma cells treated with either ABT-737 or ABT-199 compared with the effects of either BH3 mimetic alone. Inhibition or overexpression of MCL-1 alone replicated the effects of LVshNG-1 or LVshNp8-1 in increasing or decreasing the apoptosis caused with the BH3 mimetic. The combination therapy inhibited the growth of LS174T xenografts in vivo compared with untreated controls or treatment with only LV shRNA or ABT-737.. Inhibition of NANOGP8 or NANOG enhances the cytotoxicity of BH3 mimetics that target BCL-2 family members. Gene therapy targeting the NANOGs may increase the efficacy of BH3 mimetics in colorectal carcinoma.

Topics: Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Line, Tumor; Colorectal Neoplasms; Down-Regulation; HEK293 Cells; Homeodomain Proteins; HT29 Cells; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Myeloid Cell Leukemia Sequence 1 Protein; Nanog Homeobox Protein; Nitrophenols; Piperazines; RNA, Small Interfering; Sulfonamides; Xenograft Model Antitumor Assays

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Camptothecin; Cell Line, Tumor; Colorectal Neoplasms; Drug Compounding; Drug Synergism; Humans; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Nitrophenols; Piperazines; Sulfonamides; Thrombocytopenia

Oxaliplatin is frequently used in the treatment of metastatic colorectal cancer (CRC). Our previous work shows that oxaliplatin induces the pro-apoptotic protein Noxa in CRC cells. The Bcl2-inhibitor ABT-737 is particularly effective in cells with high Noxa levels. Therefore, we tested whether oxaliplatin and ABT-737 display synergy in killing CRC cells.. A panel of CRC cell lines was treated with oxaliplatin and ABT-737, either alone or in combination. Apoptosis was measured by FACS analysis of sub-G1 DNA content and by Western blot analysis of caspase-3 processing. Noxa expression was suppressed by lentiviral RNA interference.. Oxaliplatin and ABT-737 displayed a strong synergistic apoptotic response, which was dependent on wildtype TP53 and oncogenic KRAS. TP53 and KRAS were required for drug-induced Noxa expression and this was essential for tumor cell apoptosis. Oxaliplatin, but not ABT-737, induced p53 accumulation, but both drugs stimulated Noxa expression. Combination treatment of mice with subcutaneous tumor xenografts drastically reduced tumor volume, while single drug treatment had no effect.. ABT-737 synergizes with oxaliplatin to kill colorectal cancer cells. This requires induction of Noxa by wildtype TP53 and oncogenic KRAS. Future studies should explore the anti-tumor efficacy of this drug combination in mouse models for spontaneous CRC development and in patient-derived tumor cell cultures and xenografts.

Topics: Animals; Biphenyl Compounds; Caspase 3; Cell Death; Cell Line, Tumor; Colorectal Neoplasms; Drug Synergism; Mice; Models, Biological; Nitrophenols; Organoplatinum Compounds; Oxaliplatin; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Apoptosis and autophagy have been shown to be negatively regulated by prosurvival Bcl-2 proteins. We determined whether the anticancer agent celecoxib, alone or combined with a small molecule Bcl-2/Bcl-x(L) antagonist (ABT-737), can induce autophagy in colon cancer cells. Furthermore, we determined whether inhibition of autophagy can drive colon cancer cells into apoptosis. Celecoxib was shown to induce apoptosis that was attenuated by ectopic Bcl-2 or Bax knockout. ABT-737 synergistically enhanced celecoxib-induced cytotoxicity that was primarily due to apoptosis as shown by caspase cleavage and Annexin V labeling that was attenuated by a pan caspase inhibitor (z-VAD-fmk). Celecoxib triggered conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form, as shown by immunoblotting and a punctate fluorescence pattern of an ectopic GFP-LC3 protein. Celecoxib-induced conversion of LC3 was due to autophagy induction, as supported using the lysosome inhibitor, bafilomycin A1, which produced an accumulation of LC3II. ABT-737 enhanced celecoxib-induced LC3 conversion and p62/SQSTM1 degradation. Inhibition of autophagy was then studied in an effort to drive cells into apoptosis. 3-methyladenine (3-MA) blocked LC3 conversion, and 3-MA and wortmannin significantly enhanced apoptotic signaling in cells treated with celecoxib plus ABT-737. Furthermore, knockdown of Atg8/LC3B or Vps34 using siRNA attenuated p62 degradation and enhanced apoptotic signaling; Vps34 siRNA potentiated annexin V(+), PI(-) labeled cells induced by celecoxib + ABT-737. In conclusion, celecoxib induces apoptosis and autophagy that can both be potentiated by ABT-737. Inhibition of autophagy was shown to enhance apoptosis, suggesting a novel therapeutic strategy against colon cancer.

Topics: Apoptosis; Autophagy; bcl-2-Associated X Protein; Biphenyl Compounds; Caspases; Celecoxib; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cyclooxygenase 2 Inhibitors; Dose-Response Relationship, Drug; Humans; Nitrophenols; Phosphatidylinositol 3-Kinases; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; RNA, Small Interfering; Signal Transduction; Sulfonamides

It is still unclear whether the BH3-only protein Puma (p53 up-regulated modulator of apoptosis) can prime cells to death and render antiapoptotic BH3-binding Bcl-2 homologues necessary for survival through its ability to directly interact with proapoptotic Bax and activate it. In this study, we provide further evidence, using cell-free assays, that the BH3 domain of Puma binds Bax at an activation site that comprises the first helix of Bax. We also show that, in yeast, Puma interacts with Bax and triggers its killing activity when Bcl-2 homologues are absent but not when Bcl-xL is expressed. Finally, endogenous Puma is involved in the apoptotic response of human colorectal cancer cells to the Bcl-2/Bcl-xL inhibitor ABT-737, even in conditions where the expression of Mcl-1 is down-regulated. Thus, Puma is competent to trigger Bax activity by itself, thereby promoting cellular dependence on prosurvival Bcl-2 family members.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Line, Tumor; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Gene Knockdown Techniques; Humans; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides; Yeasts

The intrinsic drug resistance of colorectal cancers is related in part to overexpression of prosurvival Bcl-2 family proteins. We determined the effects of ABT-737, a small-molecule inhibitor of Bcl-2/Bcl-xL but not Mcl-1, on apoptosis induction alone and in combination with CPT-11 and explored mechanisms underlying their cooperativity.. Human colorectal carcinoma cell lines (HCT116 wild-type and Bax(-/-), HT-29, and RKO) were incubated with ABT-737 alone and combined with CPT-11 or bortezomib, and cell viability, caspase cleavage, and Annexin V labeling were measured. In drug-treated cell lines, protein-protein interactions were analyzed by immunoprecipitation. Lentiviral short hairpin RNA was used to knockdown Noxa expression.. ABT-737 induced apoptosis in a dose-dependent manner and its coadministration with the topoisomerase I inhibitor, CPT-11, resulted in a synergistic cytotoxic effect. Apoptosis induction by the drug combination was associated with enhanced caspase-8, caspase-9, and caspase-3 activation and poly(ADP-ribose) polymerase cleavage that were completely abrogated in Bax knockout cells. ABT-737 unsequestered the BH3-only protein Bim from its complex with Bcl-xL or Bcl-2 and disrupted the interaction of Bcl-xL with Bak. CPT-11 treatment up-regulated Noxa expression, as did bortezomib, and enhanced Noxa/Mcl-1 complexes. CPT-11 also disrupted the Mcl-1/Bak interaction. Knockdown of Noxa using short hairpin RNA lentiviral constructs was shown to significantly attenuate the cytotoxic effect of CPT-11 or bortezomib combined with ABT-737 and inhibited caspase-3 cleavage.. Induction of Noxa by CPT-11 or bortezomib can sensitize colorectal cancer cells expressing Mcl-1 to ABT-737. Up-regulation of Noxa may therefore represent an important strategy to enhance the therapeutic efficacy of ABT-737 against colorectal cancer and other solid tumors.

Topics: Apoptosis; bcl-X Protein; Biphenyl Compounds; Camptothecin; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Humans; Irinotecan; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is metabolized to its inactive glucuronide, SN-38 glucuronide. The aim of this study was to determine, the reactivation of SN-38 from SN-38 glucuronide by beta-glucuronidase may represent a significant pathway of SN-38 formation.. The production of SN-38 from irinotecan and SN-38 glucuronide (2.4, 9.6 and 19.2 microm) was measured in homogenates of human colorectal tumour, and matched normal colon mucosa from 21 patients).. The rate of conversion of irinotecan (9.6 microm) was lower in tumour tissue than matched normal colon mucosa samples (0.30+/-0.14 pmol min-1 mg-1 protein and 0.77+/-0.59 pmol min-1 mg-1 protein, respectively; P<0.005). In contrast, no significant difference was observed in beta-glucuronidase activity between tumour and matched normal colon samples (4.56+/-6.9 pmol min-1 mg-1 protein and 3.62+/-2.95 pmol min-1 mg-1 protein, respectively, using 9.6 microm SN-38 glucuronide; P>0.05). beta-Glucuronidase activity in tumour correlated to that observed in matched normal tissue (r2>0.23, P<0.05), whereas this was not the case for carboxylesterase activity. At equal concentrations of irinotecan and SN-38 glucuronide, the rate of beta-glucuronidase-mediated SN-38 production was higher than that formed from irinotecan in both tumour and normal tissue (P<0.05). However, at concentrations that reflect the relative plasma concentrations observed in patients, the rate of SN-38 production via these two pathways was comparable.. Tumour beta-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo.

Topics: Antineoplastic Agents, Phytogenic; Camptothecin; Carboxylesterase; Colorectal Neoplasms; Glucuronidase; Humans; Hydroxyquinolines; Irinotecan; Nitrophenols; Tumor Cells, Cultured