Current Molecular Pharmacology - Volume 12, Issue 4, 2019

Background: Aggressive behavior of tumor metastasis comes from certain mutations, changes in cellular metabolic and signaling pathways that are majorly altered by tumor microenvironment (TME), its other components and growth factors like transforming growth factor-β1 (TGF-β1) which is chiefly known for its epithelial to mesenchymal transformation (EMT). EMT is a critical step of metastasis cascade in actual human lung cancer scenario. Objective: Our present study is focused on unveiling the in-vivo metastatic behavior of TGF-β1 treated lung cancer cells that undergo EMT. Methods: The lung cancer epithelial A549 cells were treated in-vitro with TGF-β1 (3-5ng/ml for 72 h) for EMT. After confirming the transformation of cells by phenotype modifications, wound healing and cell migration assay and qRT-PCR analyses of EMT biomarkers including E. Cadherin, Vimentin, Snail, Slug, MMP2 and MMP9; those TGF-β1 modified cells were probed with fluorescent trackers and were injected into the tail vein of BALB/c nude mice for metastatic dissemination studies. Results: Our findings indicate that the distribution of TGF-β1 treated A549 cells as compared to W.T A549 towards lungs is less in terms of total relative fluorescent cluster count, however, the difference is insignificant (52±4, 60±5 respectively). Additionally, we show that TGF-β1 treated cells tend to metastasize almost 2, 3, 1.5, 2 and 1.7 times more than W.T towards liver, brain, ovaries, bones and adrenal gland, respectively, which is very much like human lung cancer metastasis. Conclusion: Conclusively, it is the first study ever reporting that a pre-treatment of cells with TGF-β1 for experimental lung cancer metastasis mouse model may portray a more precise approach for the development of potential therapeutic treatments. Additional pre-treatment studies with the application of other TME conditions like hypoxia and factors like NFΚB, VEGF etc. may be a future prospect to develop a better understanding.

Objective: Bafilomycin-A1 and ML9 are lysosomotropic agents, irrespective of cell types. However, the mechanisms of lysosome targeting either bafilomycin-A1 or ML9 are unclear. Methods: The present research has been carried out by different molecular and biochemical analyses like western blot, confocal imaging and FACS studies, as well as molecular docking. Results: Our data shows that pre-incubation of neonatal cardiomyocytes with ML9 for 4h induced cell death, whereas a longer period of time (24h) with bafilomycin-A1 was required to induce an equivalent effect. Neither changes in ROS nor ATP production is associated with such death mechanisms. Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9. Conclusion: Bafilomycin-A1 and ML9 are lysosomotropic agents, involved in cell death events. But such death events are not associated with ATP and ROS production. Furthermore, both the drugs target lysosomes through different mechanisms. For the latter, cell death is likely due to lysosomal membrane permeabilization and release of lysosomal proteases into the cytosol.

Objective: Mesalazine, 5-aminosalicylic acid (5-ASA), is an anti-inflammatory drug that is most widely used for the treatment of Inflammatory Bowel Disease (IBD). Despite extensive clinical use, the exact pharmacological mechanism underlying the anti-colitic effects of 5-ASA has not yet been elucidated. A potential molecular mechanism underlying 5-ASA-mediated anti-colitic activity was investigated. Methods: An anti-inflammatory pharmacology of 5-ASA was scrutinized in human colon carcinoma cells and murine macrophages and in a TNBS-induced rat colitis model. Results: 5-ASA induced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrate acetyl-CoA carboxylase in cells. 5-ASA activation of AMPK occurred regardless of the presence of the pro-inflammatory mediators, Tumor Necrosis Factor Alpha (TNF-α) and lipopolysaccharide. 5-ASA inhibits TNF-α-dependent Nuclear Factor-Kappa B (NF-ΚB) activation, which was dampened by AMPK inhibition. Oral gavage of sulfasalazine (a colon-specific prodrug of 5- ASA) or rectal administration of 5-ASA ameliorated 2,4,6-trinitrobenzene sulfonic acid (TNBS)- induced rat colitis and activated AMPK in the inflamed colonic tissues while markedly diminishing the levels of NF-ΚB-regulated pro-inflammatory mediators cyclooxygenase-2, inducible nitric oxide synthase, and cytokine-induced neutrophil chemoattractant-3, elevated by the induction of inflammation. Rectal co-administration of 5-ASA and an AMPK inhibitor undermined 5-ASA-mediated activation of AMPK and its anti-colitic effects. Conclusion: These findings suggest that the activation of AMPK is involved in 5-ASA-mediated anticolitic effects at least partly via interference with pro-inflammatory NF-ΚB signaling.

Background: Colorectal Cancer (CRC) is a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape constitute a well-documented hallmark of CRC phenotype. The accumulation of aberrant DNA methylation and histone acetylation plays a major role in altering gene activity and driving tumor onset, progression and metastasis. Objective: In this study, we evaluated the effect of Suberoylanilide Hydroxamic Acid (SAHA), a panhistone deacetylase inhibitor, and Decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco-2 human colon cancer cell line in vitro. Results: Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco-2 cell line. On the other hand, the sequential treatment of Caco-2 cells, first with DAC and then with SAHA, induced a synergistic anti-tumor effect with a significant enhancement of growth inhibition and apoptosis induction in Caco-2 cell line as compared to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro-apoptotic proteins Bax, p53 and cytochrome c, downregulates the expression of antiapoptotic Bcl-2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti-neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β-catenin signaling. Conclusion: In conclusion, our results provide evidence for the profound anti-tumorigenic effect of sequentially combined SAHA and DAC in the CRC cell line and offer new insights into the corresponding underlined molecular mechanism.

Background: Breast cancer is the most common cancer among women. Radiation therapy is used for treating almost every stage of breast cancer. A strategy to reduce irradiation side effects and to decrease the recurrence of cancer is concurrent use of radiation and radiosensitizers. We studied the effect of the antimanic drug lithium on radiosensitivity of estrogen-receptor (ER)-positive MCF-7 and ER-negative, invasive, and radioresistant MDA-MB-231 breast cancer cell lines. Methods: MCF-7 and MDA-MB-231 breast cancer cell lines were treated with 30 mM and 20 mM concentrations of lithium chloride (LiCl), respectively. These concentrations were determined by MTT viability assay. Growth curves were depicted and comet assay was performed for control and LiCl-treated cells after exposure to X-ray. Total and phosphorylated inactive levels of glycogen synthase kinase-3beta (GSK-3β) protein were determined by ELISA assay for control and treated cells. Results: Treatment with LiCl decreased cell proliferation after exposure to X-ray as indicated by growth curves of MCF-7 and MDA-MB-231 cell lines within six days following radiation. Such treatment increased the amount of DNA damages represented by percent DNA in Tails of comets at 0, 1, 4, and even 24 hours after radiation in both studied cell lines. The amount of active GSK-3β was increased in LiCl-treated cells in ER-positive and ER-negative breast cancer cell lines. Conclusion: Treatment with LiCl that increased the active GSK-3β protein, increased DNA damages and decreased survival independent of estrogen receptor status in breast cancer cells exposed to ionizing radiation.

Background: Cadmium (Cd) impairs gametogenesis and damages the blood-testis barrier. Objective: As the primary mechanism of Cd-induced damage is oxidative stress, the effects of two natural antioxidants, myo-inositol (MI) and seleno-L-methionine (Se), were evaluated in mice testes. Methods: Eighty-four male C57 BL/6J mice were divided into twelve groups: 0.9% NaCl (vehicle; 1 ml/kg/day i.p.); Se (0.2 mg/kg/day per os); Se (0.4 mg/kg/day per os); MI (360 mg/kg/day per os); MI plus Se (0.2 mg/kg/day); MI plus Se (0.4 mg/kg/day); CdCl2 (2 mg/kg/day i.p.) plus vehicle; CdCl2 plus MI; CdCl2 plus Se (0.2 mg/kg/day); CdCl2 plus Se (0.4 mg/kg/day); CdCl2 plus MI plus Se (0.2 mg/kg/day); and CdCl2 plus MI plus Se (0.4 mg/kg/day). After 14 days, testes were processed for biochemical, structural and immunohistochemical analyses. Results: CdCl2 increased iNOS and TNF-α expression and Malondialdehyde (MDA) levels, lowered glutathione (GSH) and testosterone, induced testicular lesions, and almost eliminated claudin-11 immunoreactivity. Se administration at 0.2 or 0.4 mg/kg significantly reduced iNOS and TNF-α expression, maintained GSH, MDA and testosterone levels, structural changes and low claudin-11 immunoreactivity. MI alone or associated with Se at 0.2 or 0.4 mg/kg significantly reduced iNOS and TNF-α expression and MDA levels, increased GSH and testosterone levels, ameliorated structural organization and increased claudin-11 patches number. Conclusion: We demonstrated a protective effect of MI, a minor role of Se and an evident positive role of the association between MI and Se on Cd-induced damages of the testis. MI alone or associated with Se might protect testes in subjects exposed to toxicants, at least to those with behavior similar to Cd.

Aims: The aim is to study the role of miR-675-5p coded by long non-coding RNA H19 in the development of Nasopharyngeal Cancer (NPC) and whether miR-675-5p regulates the invasion and metastasis of NPC through targeting SFN (14-3-3σ). The study further validated the relationship between H19, miR-675-5p and SFN in NPC and their relationship with the invasion and metastasis of NPC. Methods: Western blot was used to detect the expression of 14-3-3σ protein in immortalized normal nasopharyngeal epithelial cells NP69 and different metastatic potential NPC cells, 6-10B and 5-8F. At the same time, to find out the relationship between 14-3-3σ protein and the expression of H19 and miR-675-5p, the expression of H19 and miR-675-5p in normal nasopharynx epithelial cells NP69 and varied nasopharyngeal carcinoma cells 6-10B and 5-8F were quantified by real-time PCR. MiR-675-5p mimic and inhibitor were transfected into NPC 6-10B to over-express and down-express miR-675-5p; miR-675-5p mimic negative control and inhibitor negative control were transfected into NPC 6-10B as control groups. The effect of over-expression and down-expression by miR-675-5p on the expression of 14-3-3σ protein was detected by Western blotting. The 3’-UTR segments of SFN, containing miR-675-5p binding sites were amplified by PCR and the luciferase activity in the transfected cells was assayed to detect whether SFN is the direct target of miR-675-5p. Transwell and scratch assays were used to verify the changes in NPC invasion and metastasis ability of mimics and inhibitors transfected with miR-675-5p. Results: The expression of 14-3-3σ protein in normal nasopharynx epithelial cells NP69 is significantly higher than in varied nasopharyngeal carcinoma cells, 6-10B and 5-8F (P<0.05), and the 14-3-3σ protein levels in low-metastatic nasopharyngeal carcinoma cell 6-10B is higher than in high-metastatic nasopharyngeal carcinoma cell 5-8F. The expression of H19 and miR-675-5p are significantly higher in NPC cells than in NP69 cell (P<0.05). The expression of H19 and miR-675-5p in high-Metastatic nasopharyngeal carcinoma cell 5-8F was higher than in low-Metastatic nasopharyngeal carcinoma cell 6-10B. The expression of 14-3-3σ protein in miR-675-5p mimic cells was significantly lower than in mimic NC (negative control) group and blank control group. However, compared with the blank control group, mimic NC showed no significant difference in 14-3-3σ protein between the two groups. The miR-675-5p inhibitor group was significantly higher than the inhibitor NC group and the blank control group (p<0.05), but there was no significant difference in the expression of 14-3-3σ protein in the inhibitor NC group and the blank control group (p>0.05). Dual-luciferase reporter assay system shows the 3’-UTR segments of SFN containing miR-675-5p binding sites. SFN was the target gene of miR-675-5p. Conclusion: 14-3-3σ is downregulated in NPC and is involved in the development of NPC. H19 and miR- 675-5p are upregulated in NPC, which is related to the development of NPC. The over-expression of miR- 675-5p inhibits the expression of 14-3-3σ protein. SFN is the target gene of miR-675-5p. MiR-675-5p targets SFN, downregulates its protein expression and promotes the invasion and metastasis of NPC.