Synergistic Inhibition Of Proliferation Research Articles

Dual inhibition of αV integrins and Src kinase activity in colon cancer cells

e14609 Background: Integrins are commonly upregulated in tumor cells and are important regulators of invasion and metastasis. Integrin signaling is initiated upon engagement of ECM and requires Src as well as various other proteins including ILK, FAK, and paxillin. Methods: Presence of αv integrins on CRC cell lines was established by flow cytometry. CNTO 95 (10μg/ml, a monoclonal anti-αv integrin antibody; Ortho Biotech), and dasatinib (<200nM; BMS, src small molecule inhibitor) were used to study the effects of combined integrin and src inhibition on proliferation and serum-induced migration of colon cancer cell lines in vitro. Downstream signaling changes were assessed by immunoblotting for phosphorylated forms of src, FAK (Y397, Y576/577 and Y925), paxillin, GSK3β and AKT (S473) in HT29, HCT116 and RKO. Results: Proliferation was inhibited by dasatinib in HT29, HCT116, DLD1 and HCT15 in a dose-dependent manner, while RKO and SW48 were resistant. Combining CNTO 95 with dasatinib further inhibited proliferation in all dasatinib-sensitive cells, yet resistant cells were unaffected. Migration was blocked by both CNTO 95 and dasatinib in all cell lines, and combining the two drugs produced augmented effect. Src activation and src- dependent FAK phosphorylation at sites 576 and 925 were blocked by dasatinib; CNTO 95 had little effect alone, but potentiated the effect of dasatinib. Paxillin phosphorylation was modestly blocked by both compounds, but the combination produced significantly augmented inhibition in the three cell lines tested. The phosphorylation status of AKT and GSK-3β, which are downstream of ILK, was inhibited by both drugs as single agents. The combination of dasatinib and CNTO 95 produced further inhibition. Conclusions: Dual inhibition of Src by dasatinib and αv integrins by CNTO 95 produced additive to synergistic inhibition of proliferation in dasatinib sensitive CRC cell lines, and inhibition of migration in all cell lines tested. Decreased phospho-paxillin levels may be responsible for the pronounced inhibition of migration observed after dual treatment with dasatinib and CNTO 95 in these CRC cell lines. These data support the rationale for combined Src/integrin inhibition in colon cancer, and further suggest approaches to patient selection strategies. [Table: see text]

The histone deacetylase inhibitor PXD101 synergises with 5-fluorouracil to inhibit colon cancer cell growth in vitro and in vivo

Histone deacetylase inhibitors (HDACi) inhibit the growth of cancer cells, and combinations of HDACi with established chemotherapeutics can lead to synergistic effects. We have investigated effects of PXD101 (HDACi in phase II clinical trials) in combination with 5-fluorouracil, on tumour cell proliferation and apoptosis both in vitro and in vivo. HCT116 cells were studied using proliferation and clonogenic assays. Synergistic inhibition of proliferation and clonogenicity was determined by incubation with PXD101 and 5-fluorouracil, and analysis using CalcuSyn software. The effect of combining PXD101 and 5-fluorouracil on apoptosis was examined in vitro using PARP-cleavage and TUNEL. Finally, the effectiveness of combining PXD101 and 5-fluorouracil in vivo was tested using both HT-29 and HCT116 xenograft models. Synergistic inhibition of proliferation and clonogenicity was obtained when HCT116 cells were incubated with PXD101 and 5-fluorouracil. 5-fluorouracil combined with PXD101 also increased DNA fragmentation and PARP cleavage in HCT116 cells. Incubation with PXD101 down regulated thymidylate synthase expression in HCT116 cells. In vivo studies, using mouse HT29 and HCT116 xenograft models, showed improved reductions in tumour volume compared to single compound, when PXD101 and 5-fluorouracil were combined. PXD101 and 5-fluorouracil synergistically combine in their anti-tumour effects against colon cancer cells in vitro and show enhanced activity when combined in vivo. Based on the results presented herein, a rationale for the use of PXD101 and 5-fluorouracil in combination in the clinic has been demonstrated.

Preclinical development of a novel inhibitor of oncogenic B-Raf

13056 Background: The discovery of oncogenic B-Raf mutations in a majority of patients with metastatic melanoma, and in many tumors from patients with colorectal cancer and other cancers, presents the opportunity to develop oncogene-selective inhibitors with a favorable safety profile. Methods: Guided by co-crystallography, a novel chemical scaffold has been developed into a series of potent inhibitors of oncogenic B-Raf with selectivity versus wild-type B-Raf. From this series, a potent inhibitor has been chosen as a candidate for development. Results: Consistent with the structure-guided approach, this compound shows pronounced selectivity versus a wide array of over 70 other kinases covering all branches of the kinome. This selectivity translates to a wide cellular therapeutic index: inhibition of proliferation of a panel of cell lines bearing the V600E oncogenic B-Raf mutation occurs at IC50s ranging from 40–400 nM while inhibition of cell lines lacking oncogenic B-Raf occurs at IC50s greater than 6500 nM. This cell-based selectivity for the oncogenic B-Raf is greater than the selectivity shown in biochemical assays, supporting that tumor cells bearing the oncogenic B-Raf protein are more dependent on the MAP kinase pathway. Combination experiments with a series of cytotoxic and targeted clinical anti-cancer agents reveal multiple examples of synergistic inhibition of proliferation in vitro, and this synergy generally appears selectively in oncogenic B-Raf-bearing cells. The good oral bioavailability (F > 70%) allows for prolonged exposure in both rodents and non-rodents. Robust efficacy is evident in a murine COLO205 tumor xenograft model, with once-daily oral dosing at 20 mg/kg over 14 days resulting in > 80% inhibition of tumor growth with no effect on body weight. Conclusions: Since this compound is highly selective and targets a B-Raf variant that is absent in all non-transformed cells, it may have a broad therapeutic index, alone or in combination, for the treatment of patients with V600E oncogenic B-Raf tumors. [Table: see text]

A study of cytotoxic synergy of UCN-01 and flavopiridol in syngeneic pair of cell lines

Flavopiridol and UCN-01 are two novel protein kinase inhibitors with diverse cellular effects that may complement each other with regards to induction of apoptosis. HeLa cells engineered to overexpress human survivin (HeLa-S) were at least approximately 4.8-fold resistant to UCN-01 relative to proliferation observed in control HeLa cells (HeLa-V). Flavopiridol cytotoxicity as measured using the MTT assay was unaffected in HeLa-S cells when compared with HeLa-V cells. Similarly, simultaneous treatment of HeLa-V cells with flavopiridol and UCN-01 for 72 hours did not result in synergistic inhibition of proliferation; however, in HeLa-S cells, this combination resulted in synergistic inhibition of cell proliferation. Flavopiridol and UCN-01 augmented apoptosis in HeLa-S cells (as compared with HeLa-V cells) as measured by caspase-3 cellular activity assay, DNA fragmentation and PARP cleavage by western blot. In HeLa-V and -S cells, combination treatment resulted in caspase-8 cleavage. Caspase-9 was expressed in HeLa-V cells; however, there was a marked reduction of caspase-9 content in HeLa-S cells only. Combination treatment resulted in a significant reduction in survivin abundance in HeLa-S and SKBR3-UR cells, but not in their respective parental lines. The synergy of Flavopiridol and UCN-01 are selectively toxic to survivin-overexpressing cell lines and the mechanism of toxicity involves caspase-dependent cell death.

Co-targeting IGF-1R and c-kit: synergistic inhibition of proliferation and induction of apoptosis in H 209 small cell lung cancer cells.

Most small cell lung cancers (SCLC) coexpress the c-kit protein tyrosine receptor kinase and its ligand stem cell factor, resulting in an autocrine loop. As SCLC growth is also driven by insulin-like growth factor-1 receptor (IGF-1R) signalling, tyrphostins AG 1024 and 1296 (inhibitors of IGF-1R and c-kit activity, respectively) were used to co-target these receptors in H 209 SCLC cells. Combination treatment caused synergy in proliferation inhibition and in apoptosis induction, and also enhanced reduction in phosphorylation of Erk1/Erk2, suggesting that co-targeting IGF-1R and c-kit in SCLC may be more effective than single-agent therapies.

Synergistic interaction of retinoids and interferons in breast cancer

Combination of all-trans-retinoic acid (RA) with either interferon (IFN)-ct or -), results in a synergistic amplification of the anti-proliferative effect on cultured breast cancer cells. RA could be replaced by other biologically active retinoids. The synergism is also observed for the induction of 2'-5'-oligoadenylate synthetase, an enzyme which is involved in anti-viral activity of interferons and possibly in growth regulation of tumor cells. Comparing all-transwith 9-cis-RA, the latter is more effective inhibiting tumor cell growth and in inducing synergism with IFN-y. IFN-y increases retinoic acid receptor-y (RAR-y) expression but had no influence on KAR-~t, whereas RAR-13 was not detectable in any of the examined breast cancer cell lines. RA-mediated increase of the cellular retinoic acid receptor binding protein-II (CRABP-II) was suppressed by IFN-y. This observation indicates that IFN-y-mediated increase in RAR-y and suppression of RA-mediated CRABP-I! activation may play a role in synergistic inhibition of proliferation in breast cancer cell lines. 24

Inhibition of B cell proliferation by antisense DNA to both α and β forms of FcϵR II

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is FcϵR II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, FcϵR II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of FcϵR II; FcϵR IIa (α) and IIb (β) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 μ M, significantly decreased cellular proliferation as measured by the incorporation of [ 3H]thymidine (inhibition range 8–88%). Optimum inhibition of cellular proliferation was apparent at 15 μ M concentration of both anti-sense FcϵR IIa and IIb (FcϵR IIa, mean ± SE = 75 ± 7% inhibition, p < 0.001; FcϵR IIb, mean ± SE = 71 ±7% inhibition, p < 0.001). Anti-sense oligonucleotides complementary to the common part of FcϵR II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a FcϵR II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble FcϵR II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble FcϵR II was important for the induced proliferation. Analysis of cell surface expression for FcϵR II indicated that while there was a pronounced effect on cell number following incubation with anti-sense oligonucleotides, surface expression of FcϵR II was consistent as measured over different time points. PCR analysis revealed that while most cells expressed either the α or the β form of FcϵR II, EBV-transformed cell lines, particularly RPMI 8866, were found to express both α and β forms simultaneously. This may constitute a mechanism whereby EBV infection confers an immortal state to the cell, resulting in its uncontrolled proliferation. Cell lines expressing only one receptor form, either α or β, were unaffected after incubation with anti-sense oligonucleotides. This suggests a possible interplay of both α and β forms in the regulation of normal growth processes.

Modulation of Methotrexate Cytotoxicity with Natural Interferon upon Human Leukemia Cell Line HL-60

The effect of alpha- and gamma-interferon on methotrexate cytotoxicity against human promyelocytic cell line HL-60 has been evaluated. Synergistic inhibition of proliferation is observed with the combination of methotrexate and gamma-interferon. Enhanced cytotoxic effect of methotrexate with alpha- or gamma-interferon is removed by adding thymidine to the growth medium. The depletion of folate pools caused by methotrexate is enhanced in presence of interferons, this depletion is also removed by adding thymidine to the growth media. Synchronization of HL-60 cells in 'S' phase of cell cycle caused by methotrexate is enhanced in the presence of interferons. These results suggest that specially gamma-interferon changes the cellular environment of HL-60 cells in such a way as to make methotrexate more potent cytotoxic agent to HL-60 cells causing cell death. This study also clearly indicates the biochemical role of thymidine in modulating the activity of methotrexate in combination with interferons.