Ariyaphong Wongnoppavich

Up-regulation of interferon-stimulated gene15 and its conjugates by tumor necrosis factor-α via type I interferon-dependent and -independent pathways

Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is strongly induced by type I interferons (IFN-α/β), bacterial endotoxin, and cellular stress. Up-regulation of ISG15 is observed in several cancer cell types and is associated with cancer progression. As many cytokines can influence all stages of tumorigenesis, the elevated expression of ISG15 system may be regulated in cancer cells by inflammatory cytokines. In this study, we showed that TNF-α, but not TGF-β and IL-6, up-regulates levels of both ISG15 and its conjugates in human lung carcinoma A549 and human squamous carcinoma HSC4 cell lines. Induction of ISG15 and its conjugates by TNF-α was dose-dependent and required mediation of p38 MAP kinase and Jak1 through up-regulation of endogenous type I interferon expression. SB202190 (p38 MAPK inhibitor) and Jak1 inhibitor suppressed TNF-α-induced expression of ISG15 and its conjugates. However, only SB202190 inhibited the expression of type I interferons by TNF-α. Although B18R, a soluble type I interferon receptor, totally abolished the effect of exogenous IFN-β, it was unable to inhibit completely the TNF-α-induced ISG15 production. In addition, the initiation of ISG15 induction by TNF-α was detected earlier than that of IFN-β induction. Taken together, TNF-α elicits the induction of ISG15 and ISG15 conjugates not only via the autocrine stimulation of type I interferon expression, but also through a type I interferon-independent pathway. These data provide a possible link between inflammatory response and cancer progression.

The effect of purple rice (Oryza sativa L. indica) extract on the inflammatory response in a colon cancer cell line and dextran sulfate-induced tumor promotion in the rat colon

This study investigated the effects of purple rice extract (PRE ) on inflammation-related colon carcinogenesis. Administration of PRE 1 g/kg bodyweight reduced both the number (39.05%) and size of aberrant crypt foci (ACF) (14.28%) in the colons of rats that received dimethylhydrazine (DMH) initiation with promotion by dextran sulfate sodium (DSS) (P<0.05) with the reduction of TNF-α expression in colonic epithelial cells. On the other hand, PRE could suppress the expression of both TNF-α and IL-6 in RAW 264.7 cells activated by lipopolysaccharide (LPS) at both the mRNA and protein levels. Moreover it could reduce the expression of TNF-α and IL-1β mRNA in inflamed HT-29. These findings suggested that PRE is able to modulate the inflammatory process in both activated macrophages and colon cancer cells. Therefore, PRE suppressed macrophage activation and colon cancer cell line response to inflammation and modulated of inflammation induced ACF progression in the rat colon.

Anti-proliferative effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) isolated from Vernonia cinerea on oral squamous cell carcinoma through inhibition of STAT3 and STAT2 phosphorylation

BACKGROUND The high mortality rate of oral cancers has stimulated the search for effective herbal medicines and their pharmacological targets. Vernonia cinerea, a perennial tropical herb, is wildly used as a traditional folk medicine for treatment of intestinal diseases and various skin diseases in addition to possessing anti-cancer activity. However, the effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) as a major sesquiterpene lactone compound found in V. cinerea and the underlying mechanism of its action on oral cancer cells remains unknown. PURPOSE To investigate the anti-cancer activity of 8αTGH extracted from V. cinerea and the underlying mechanism of its action in oral cancer cells. METHODS The anti-proliferative effect of 8αTGH on oral squamous cell carcinoma (HSC4) and lung carcinoma (A549) was determined using the SRB colorimetric method. The molecular mechanism of 8αTGH was explored using kinase inhibitors, followed by Western blotting or RT-qPCR. Flow cytometry and Western blotting were used to assess cell cycle arrest. RESULTS 8αTGH inhibited cancer cell growth more effectively on HSC4 than A549 and was much less effective on tested normal oral cells. 8αTGH inhibited STAT3 phosphorylation on both cancer cells. Notably, 8αTGH was able to suppress the constantly activated STAT2 found only in HSC4. The STAT2 inhibition by 8αTGH consequently caused down-regulation of ISG15 and ISG15 conjugates. As a result, decreased expression of CDK1/2 and Cyclin B1 was detected leading to G2/M cell cycle arrest. CONCLUSION 8αTGH isolated from V. cinerea preferentially inhibits the proliferation of oral cancer cells by causing G2/M cell cycle arrest via inhibition of both STAT3 and STAT2 phosphorylation. The results provide molecular bases for developing 8αTGH as a drug candidate or a complementary treatment of oral cancer.

Marcanine G, a new cytotoxic 1-azaanthraquinone from the stem bark of Goniothalamus marcanii Craib

Abstract The ethanolic extract from the stem bark of Goniothalamus marcanii Craib was shown in preliminary brine shrimp lethality data having good cytotoxic activity. Further bioassay guided isolation was done by means of solvent partition, chromatography and precipitation to provide four isolated compounds: a novel compound 1 with the core structure of 1-azaanthraquinone moiety referred as marcanine G; as well as compounds 2–4 with known aristolactam structures namely, piperolactam C, cepharanone B and taliscanine. These compounds were characterised by spectroscopic techniques. The assessment of cytotoxicity was established on an SRB assay using doxorubicin as a positive control. Marcanine G (1) was considered the most active compound indicating the IC50 values of 14.87 and 15.18 μM against human lung cancer cells (A549) and human breast cancer cells (MCF7), respectively. However, 2 showed mild activity with the IC50 values of 83.72 and 82.32 μM against A549 and MCF7 cells, respectively.

Upregulation of maspin expression in human cervical carcinoma cells by transforming growth factor β1 through the convergence of Smad and non‑Smad signaling pathways

Mammary serine protease inhibitor (maspin), encoded by the serpin family B member 5 gene, serves as a tumor suppressor through the inhibition of cancer cell invasion and metastasis. Paradoxically, maspin levels are upregulated in a number of types of malignant cells. Therefore, the regulation of maspin expression may depend on the genetic or epigenetic background and the specific microenvironment of carcinoma cells. In the present study, it was demonstrated that transforming growth factor β1 (TGF-β1) induced maspin expression at the transcript and protein levels in the human cervical carcinoma HeLa and human oral squamous carcinoma HSC4 cell lines. The inhibition of the mothers against decapentaplegic homolog (Smad)-dependent pathway by a Smad3-specific inhibitor suppressed maspin induction by TGF-β1 in HeLa cells. Inhibition of the non-Smad pathway by pretreatment with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126, or the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB202190, attenuated the effect of TGF-β1 on maspin upregulation, whereas pretreatment with pyrrolidine dithiocarbamate (a nuclear factor κB inhibitor), wortmannin (a phosphoinositide 3-kinase inhibitor) or SP600125 (a c-Jun N-terminal kinase inhibitor) did not. Notably, none of these inhibitors eliminated the TGF-β1-induced phosphorylation of Smad2. In addition, mutations at p53-binding sites in the maspin promoter suppressed TGF-β1-induced maspin expression, indicating the necessity of intact p53-binding sites on the maspin promoter. In summary, the induction of maspin expression in HeLa cells requires the convergence of TGF-β1-induced Smad and non-Smad signaling pathways, in which the latter acts via the intermediate signaling molecules MEK1/2 and p38 MAPK.

Reactive center loop moiety is essential for the maspin activity on cellular invasion and ubiquitin-proteasome level

Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspins action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.