Exploration of novel heterofused 1,2,4-triazine derivative in colorectal cancer


Abstract

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in men and in women. The impact of the new pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide (MM-129) was evaluated against human colon cancer in vitro and in zebrafish xenografts. Our results show that this new synthesised compound effectively inhibits cell survival in BTK-dependent mechanism. Its effectiveness is much higher at a relatively low concentration as compared with the standard chemotherapy used for CRC, i.e. 5-fluorouracil (5-FU). Flow cytometry analysis after annexin V-FITC and propidium iodide staining revealed that apoptosis was the main response of CRC cells to MM-129 treatment. We also found that MM-129 effectively inhibits tumour development in zebrafish embryo xenograft model, where it showed a markedly synergistic anticancer effect when used in combination with 5-FU. The above results suggest that this novel heterofused 1,2,4-triazine derivative may be a promising candidate for further evaluation as chemotherapeutic agent against CRC.

Keywords: 1,2,4-Triazine derivative; apoptosis; colon cancer; zebrafish.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1.
Figure 1.
Chemical structure of MM-129.
Scheme 1.
Scheme 1.
Synthetic pathway for production of new sulphonamide MM-129.
Figure 2.
Figure 2.
Schematic of xenograft assay and analysis of tumour development (a). Site-specific injection (yolk sac) of transfected (red) colon cancer cells (DLD-1 and HT-29) into 48?hpf zebrafish embryos and imaging analysis of tumour growth after 48?h of incubation with MM-129 (50?μM), 5-FU (50?μM), or a combination of these agents (b). Quantification of total fluorescence by colon cancer cells three?days after injection (c) n?=?8, *p?<?.05, **p?<?.01, ***<.001 vs. CON; ^p?<?.05 vs. MM-129, #p?<?.05, ##p?<?.01 vs. 5-FU. Data were presented as mean?±?standard deviation (SD), and analysed using one-way analysis of variance (ANOVA). p?<?.05 was considered statistically significant.
Figure 3.
Figure 3.
Effect of MM-129, 5-fluorouracil (5-FU), and roscovitine (RSC) on cell division in the zebrafish embryo. Zebrafish eggs after 0, 1, 1.25, and 2?h of exposure to MM-129, 5-FU and RSC; n?=?10. hpf: hours post fertilisation; hpt: hours post treatment.
Figure 4.
Figure 4.
Viability of DLD-1 (a) and HT-29 (b) colon cancer cells treated for 24?h with different concentrations of MM-129, roscovitine (RSC) and 5-fluorouracil (5-FU), and the antiproliferative effects of the studied compounds in DLD-1 (c) and HT-29 (d) colon cancer cells measured by the inhibition of [3H]thymidine incorporation into DNA. Mean values?±?SD from three independent experiments (n?=?3) done in duplicate are presented.
Figure 5.
Figure 5.
The down-regulation of Bruton’s kinase (BTK) expression by MM-129. Phosphorylated BTK (pBTK) expression was determined by Western blot (a) and confocal microscopy (d) in DLD-1 and HT-29 cells treated with MM-129, roscovitine and 5-flurouracil in two concentrations: 1?μM and 3?μM for 24?h. The band staining was quantified by densitometry (b, c). Samples used for electrophoresis consisted of 20?μg of protein from six pooled cell extracts (n?=?6). Data were presented as mean?±?standard deviation (SD), and analysed using one-way analysis of variance (ANOVA). ***p<.001 vs. CON intensity of cytoplasmic/nuclear fluorescence was analysed in cell populations. Images of cells labelled with FITC were obtained using a 515LP emission laser and a 488/10 excitation laser (d).
Figure 6.
Figure 6.
Representative flow cytometry dot-plots for annexin V‐FITC-propidium iodide assay of DLD-1 (a) and HT-29 (b) colon cancer cells after 24?h of incubation with roscovitine (RSC), 5-fluorouracil (5-FU), or MM-129 (1?μM and 3?μM).
Figure 7.
Figure 7.
Representative dot-plots presenting the loss of mitochondrial membrane potential (ΔΨm) of DLD-1 (a) and HT-29 (b) colon cancer cells treated for 24?h with roscovitine (RSC), 5-fluorouracil (5-FU), and MM-129 (1?μM and 3?μM).
Figure 8.
Figure 8.
Flow cytometric analysis of caspase-9, caspase-8, caspase-10, and caspases-3/7 activation in the populations of DLD-1 (a, c, e, g) and HT-29 (b, d, f, h) colon cancer cells treated for 24?h with roscovitine (RSC), 5-fluorouracil (5-FU), and MM-129 (1?μM and 3?μM). Mean percentage values from three independent experiments done in duplicate (N?=?6) were presented as mean?±?standard deviation (SD), and analysed using one-way analysis of variance (ANOVA). ***p<.001 vs. CON.
Figure 9.
Figure 9.
Schematic representation of possible anticancer mechanisms of MM-129. 1: BTK inhibition; 2: phosphatidylserine (PS) externalisation; 3: loss of mitochondrial membrane potential; 4: activation of extrinsic pathway of apoptosis; 5: the activation of internal (mitochondrial) apoptosis; 6: activation of executive caspases. The schematic illustration was created in Adobe Photoshop and Photophea software. Akt: protein kinase B; Apaf-1: apoptotic protease activating factor 1; β-catenin: protein responsible for transduction; Bak: Bcl-2 homologous antagonist/killer; Bax: Bcl-2-associated X protein; Bcl-2: antiapoptotic protein; Bid: Bax-like BH3 protein; tBid: truncated BID; BTK: Bruton’s tyrosine kinase; EGF: epidermal growth factor; FADD: Fas-associated death domain protein; IFNγ: interferon gamma; JAK2: non-receptor tyrosine kinase; mTOR: mammalian target of rapamycin; PDGF: platelet-derived growth factor; PI3K: phosphoinositide 3-kinases; PIP3: phosphatidylinositol-3,4,5-triphosphate; PH: pleckstrin homology domain; STAT: signal transducer and activator of transcription; Wnt: family of secreted lipid-modified signalling glycoproteins.
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