Decytabine enhances cytotoxicity induced by oxaliplatin and 5-fluorouracil in the colorectal cancer cell line Colo-205

<p>Abstract</p> <p>Background</p> <p>DNA methylation is an epigenetic phenomenon known to play an important role in the development of cancers, including colorectal cancer (CRC). Aberrant methylation of promoter regions of genes is potentially reversible, and if methylation is important for cancer survival, demethylation should do the opposite. To test this we have addressed the hypothesis that DNA methyltransferase inhibitors (DNMTi), decytabine and zebularine, potentiate inhibitory effects of classical anti-CRC cytostatics, oxaliplatin and 5-fluorouracil (5-FU), on survival of CRC cells <it>in vitro</it>.</p> <p>Results</p> <p>Isobole and median effect analysis revealed that decytabine shows potent synergistic interaction with oxaliplatin and 5-FU and that this is probably not the class effect of DNMTi as zebularine shows strong antagonistic interaction with oxaliplatin. The synergistic combination treatment was also applied to the cultures to investigate their mechanisms of action. We have shown that combinations of decytabine with cytostatics produced dose-dependent growth inhibition and treatment-induced apoptosis.</p> <p>Conclusion</p> <p>The observed synergism between decytabine and cytostatics is most probably related to the augmented apoptotic signal and allowed for significant (both biologically and statistically) reduction of the cytotoxic doses of cytostatics used.</p

Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors

Chemotherapeutic agents, DNMTi and their combinations influence the cell cycle progression of colorectal cancer cells.

<p><b>A.</b> Changes in the cell cycle distribution of SW48 and HT-29 cells after 72 h of treatment with the evaluated agents. The cells were stained with propidium iodide (PI) and then analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle was determined using ModFit LT™ (version 3.0). Each bar represents the mean ±S.D. (n≥4). Significant difference at <i>P</i><0.05 are indicated by asterisk (*). <b>B.</b> Analysis of <i>CCNE1</i> and <i>ATM</i> mRNA levels by semi-quantitative RT-PCR method after 72 h incubation of CRC cells with chemotherapeutic agents, DNMTi and their combinations at concentrations as indicated. M, marker [bp]; <i>GAPDH</i>, transcript encoding glyceraldehyde-3-phosphate dehydrogenase, a constitutively expressed gene, used as an internal control. <b>C.</b> Western blotting analysis of the cell cycle regulatory proteins. The β-actin was used as a gel loading control. OXA, oxaliplatin; 5-FU, 5-fluorouracil; DAC, decitabine; ZEB, zebularine.</p

Combinations of chemotherapeutics with DNMTi agents induce major signaling checkpoints in response to DNA damage.

<p><b>A.</b> Quantification of ATR and ATM phosphorylation level following 72(MFI) was used. Data represented the mean ±SD (n = 3). Significant difference at <i>P</i>≤0.05 is indicated by an asterisk (*). <b>B.</b> Analysis of protein phosphorylation levels using Western blotting method. The detection of β-actin was used as a gel loading control. OXA, oxaliplatin; 5-FU, 5-fluorouracil; DAC, decitabine; ZEB, zebularine; a.u., arbitrary units.</p

Induction of apoptosis in colorectal cancer cells following 72 h incubation with chemotherapeutic agents, DNMT inhibitors and their combinations.

<p><b>A.</b> Detection of apoptosis by the annexin V-fluorescein isothiocyanate (FITC)/porpidium iodide (PI) analysis. Data are expressed as the means ±S.D. (n≥4). An asterisk (*) indicates that the induction of apoptosis by the evaluated agents was significant in comparison with the combination of chemotherapeutic agents and DNMT inhibitors <i>versus</i> chemotherapeutic agents applied alone (<i>P</i><0.05). <b>B.</b> Western blotting analysis of pro-apoptotic protein levels. The β-actin was used as a gel loading control. <b>C.</b> Representative cytograms of flow cytometry experiments demonstrating changes in Δ<i>Ψ_m</i> in CRC cells lines after 72 h of incubation with chemotherapeutic agents, DNMT inhibitors and their combinations. Cells were stained with JC-1. Cells in the R2 quadrant were counted as cells deprived of mitochondrial membrane potential. An asterisk (*) indicates a significant difference between experimental groups and control group at <i>P</i><0.05. OXA, oxaliplatin; 5-FU, 5-fluorouracil; DAC, decitabine; ZEB, zebularine.</p

Effects of chemotherapeutic agents and DNMT inhibitors on cell viability of colorectal cancer cell lines.

<p>The cells were treated singly or in combinations with the indicted doses of the agents for 72; 5-FU, 5-fluorouracil; DAC, decitabine; and ZEB, zebularine. Each point represents the mean ±SD (n = 5), asterisks indicate a significance at <i>P</i><0.05 for comparison with oxaliplatin or 5-FU alone.</p

Interactions of standard chemotherapeutics with DNMT inhibitors in the human colorectal cancer cell lines SW48 and HT-29.

<p><b>A.</b> Isobolograms at a 50% effect level. Concentrations of particular drugs are indicated on x and y axis. The isobolograms were constructed by connecting the IC₅₀ values of demethylating agents (on the ordinate) with the IC₅₀ of oxaliplatin or 5-FU plotted on the abscissa. When the doses of two agents in combination are lower or higher than the additive doses, the synergy or antagonism is present, respectively. <b>B.</b> Combination index values (CI) with a 95% confidence interval at all effect levels as calculated by the CalcuSyn software. A CI value significantly less than 1 indicates synergy, a CI value insignificantly different from 1 indicates addition, and a CI significantly higher than 1 indicates antagonism.</p

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinase Exerts Synergistic Effect against Chronic Myeloid Leukemia Cells

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely &ldquo;cure&rdquo; the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1-transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells

Gain of Function (GOF) Mutant p53 in Cancer&mdash;Current Therapeutic Approaches

Continuous development of personalized treatments is undoubtedly beneficial for oncogenic patients&rsquo; comfort and survival rate. Mutant TP53 is associated with a worse prognosis due to the occurrence of metastases, increased chemoresistance, and tumor growth. Currently, numerous compounds capable of p53 reactivation or the destabilization of mutant p53 are being investigated. Several of them, APR-246, COTI-2, SAHA, and PEITC, were approved for clinical trials. This review focuses on these novel therapeutic opportunities, their mechanisms of action, and their significance for potential medical application