Enhancing the Effective Chemotherapy: The Combined Inhibition of Rhinacanthin-C, 5-Fluorouracil, and Etoposide on Oral Cancer Cells.

OBJECTIVE: To investigate the effects of rhinacanthin-C (Rh-C), 5-FU, and etoposide on growth inhibition, as well as the effects of a combination of these inhibitors on the oral cell lines SCC9 and HSC4. METHODS: Cancer cell growth inhibition and inhibition combination were determined using the SRB assay. Cell viability and early apoptosis were determined using flow cytometry on cells stained with Annexin 5 and PI. Western blotting was performed to study the molecular mechanism of these inhibitors on oral cancer cells. RESULTS: The results showed that etoposide, 5-FU, and Rh-C exhibited more potent anti-proliferative effects on HSC4 cells compared to SCC9 cells in a time- and concentration-dependent manner. The combination of Rh-C and 5-FU was more effective in inhibiting cell growth than the drugs used alone. The combination of 5-FU and Rh-C resulted in a decrease in live HSC4 cells, with the highest percentage of cell death observed at a ratio of 40:6 µM. Furthermore, the combination of 5-FU and Rh-C reduced P-Akt levels leading to a decrease in cell survival. CONCLUSIONS: HSC4 cells were found to be more sensitive to the inhibitory effect of these drugs compared to SCC9 cells. These findings suggest that the use of Rh-C as a complementary therapy with 5-FU may have the potential for the treatment of oral cancer. the underlying mechanisms responsible for this difference in sensitivity between the two cell lines need to be further investigated.

Induction of S arrest and apoptosis in human oral cancer cells by Rhinacanthin-C extracted from Rhinacanthus nasutus via modulating Akt and p38 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The search for effective herbal medicines for complementary treatments is on the rise due to the high incidence of recurrence and mortality rate in human oral cancer. Rhinacanthus nasutus KURZ., an annual herb found mostly in Southeast Asia including Thailand, has been wildly used as a traditional folk medicine for the treatment of several diseases including cancer. However, the anti-cancer effect of Rhinacanthin-C (Rh-C) as a major naphthoquinone compound found in R. nasutus and the underlying mechanism of its action on human oral cancer cells remain unknown. AIM OF THE STUDY: To investigate the anti-cancer mechanism of Rh-C extracted from R. nasutus in human oral cancer cells. MATERIALS AND METHODS: The anti-proliferative effect of Rh-C on human oral squamous cell carcinoma (HSC4) was determined and compared to normal oral cells (human gingival fibroblasts, HGF, and normal oral keratinocytes, NOK) using the SRB colorimetric method. The molecular mechanism of Rh-C was explored using flow cytometry, colorimetric assay, in vitro human topoisomerase II assay, and Western blotting. RESULTS: Rh-C displayed a time- and concentration-dependent growth inhibition on HSC4 and was much less effective on both tested normal oral cells. Rh-C inhibited Akt phosphorylation whereas over-activated p38 MAPK phosphorylation in HSC4 but not in HGF. Rh-C also inhibited topoisomerase II activity. As a result, the cell cycle was arrested in S-phase as the expression of CDK1/2 and Cyclin A2 was decreased. Eventually, the induction of HSC4 cell apoptosis was mediated by increased caspase 3 activity. CONCLUSIONS: Rh-C isolated from R. nasutus possesses anti-cancer properties on human oral cancer cells by causing the S arrest and the apoptotic induction via modulating Akt/p38 signaling pathways. The results provide molecular bases for further developing Rh-C as a potential drug candidate or a complementary treatment for oral cancer.

Inactivation of AKT/NF­κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin.

The high activation of protein kinase B (AKT)/nuclear factor­κB (NF­κB) signaling has often been associated with the induction of non­small cell lung cancer (NSCLC) cell survival and resistance to cisplatin, which is one of the most widely used chemotherapeutic drugs in the treatment of NSCLC. The inhibition of AKT/NF­κB can potentially be used as a molecular target for cancer therapy. Eurycomalactone (ECL), a quassinoid from Eurycoma longifolia Jack, has previously been revealed to exhibit strong cytotoxic activity against the human NSCLC A549 cell line, and can inhibit NF­κB activity in TNF­α­activated 293 cells stably transfected with an NF­κB luciferase reporter. The present study was the first to investigate whether ECL inhibits the activation of AKT/NF­κB signaling, induces apoptosis and enhances chemosensitivity to cisplatin in human NSCLC cells. The anticancer activity of ECL was evaluated in two NSCLC cell lines, A549 and Calu­1. ECL decreased the viability and colony formation ability of both cell lines by inducing cell cycle arrest and apoptosis through the activation of pro­apoptotic caspase­3 and poly (ADP­ribose) polymerase, as well as the reduction of anti­apoptotic proteins Bcl­xL and survivin. In addition, ECL treatment suppressed the levels of AKT (phospho Ser473) and NF­κB (phospho Ser536). Notably, ECL significantly enhanced cisplatin sensitivity in both assessed NSCLC cell lines. The combination treatment of cisplatin and ECL promoted cell apoptosis more effectively than cisplatin alone, as revealed by the increased cleaved caspase­3, but decreased Bcl­xL and survivin levels. Exposure to cisplatin alone induced the levels of phosphorylated­AKT and phosphorylated­NF­κB, whereas co­treatment with ECL inhibited the cisplatin­induced phosphorylation of AKT and NF­κB, leading to an increased sensitization effect on cisplatin­induced apoptosis. In conclusion, ECL exhibited an anticancer effect and sensitized NSCLC cells to cisplatin through the inactivation of AKT/NF­κB signaling. This finding provides a rationale for the combined use of chemotherapy drugs with ECL to improve their efficacy in NSCLC treatment.

Salivary and serum interleukin-17A and interleukin-18 levels in patients with type 2 diabetes mellitus with and without periodontitis.

OBJECTIVE: Interleukin (IL)-17A and IL-18 have been proposed to play important roles in periodontitis and type 2 diabetes mellitus (DM), but human data are conflicting. The present study aimed to investigate the roles of IL-17A and IL-18 in periodontitis and DM by measuring salivary and serum levels, respectively. MATERIALS AND METHODS: A total of 49 participants with type 2 DM and 25 control subjects without type 2 DM were recruited. A periodontal screening and recording (PSR) index (0, 1-2, 3, and 4) was used to classify whether these subjects had periodontitis. Salivary and serum IL-17A and IL-18 levels were measured by enzyme-linked immunosorbent assay. Multiple linear regression analyses were used to evaluate the associations between these cytokines and clinical parameters. RESULTS: Salivary IL-17A levels were not significantly different between patients with DM and controls, however, the levels were significantly higher in controls with periodontitis than those without periodontitis (p = 0.031). Salivary IL-17A levels were significantly associated with the PSR index (ß = 0.369, p = 0.011). Multiple linear regression analyses revealed the association of salivary IL-18 levels and fasting plasma glucose (ß = 0.270, p = 0.022) whereas serum IL-18 levels were associated with HbA1C (ß = 0.293, p = 0.017). No correlation between salivary and serum levels of IL-17A and IL-18 was found. CONCLUSION: Salivary IL-17A was strongly associated with periodontitis, whereas salivary IL-18 was associated with FPG and serum IL-18 was associated with HbA1C. These results suggest the role of these cytokines in periodontal inflammation and DM.

Enhancement of Radiosensitivity by Eurycomalactone in Human NSCLC Cells Through G2/M Cell Cycle Arrest and Delayed DNA Double-Strand Break Repair.

Radiotherapy (RT) is an important treatment for non-small cell lung cancer (NSCLC). However, the major obstacles to successful RT include the low radiosensitivity of cancer cells and the restricted radiation dose, which is given without damaging normal tissues. Therefore, the sensitizer that increases RT efficacy without dose escalation will be beneficial for NSCLC treatment. Eurycomalactone (ECL), an active quassinoid isolated from Eurycoma longifolia Jack, has been demonstrated to possess anticancer activity. In this study, we aimed to investigate the effect of ECL on sensitizing NSCLC cells to X-radiation (X-ray) as well as the underlying mechanisms. The results showed that ECL exhibited selective cytotoxicity against the NSCLC cells A549 and COR-L23 compared to the normal lung fibroblast. Clonogenic survival results indicated that ECL treatment prior to irradiation synergistically decreased the A549 and COR-L23 colony number. ECL treatment reduced the expression of cyclin B1 and CDK1/2 leading to induce cell cycle arrest at the radiosensitive G2/M phase. Moreover, ECL markedly delayed the repair of radiation-induced DNA double-strand breaks (DSBs). In A549 cells, pretreatment with ECL not only delayed the resolving of radiation-induced γ-H2AX foci but also blocked the formation of 53BP1 foci at the DSB sites. In addition, ECL pretreatment attenuated the expression of DNA repair proteins Ku-80 and KDM4D in both NSCLC cells. Consequently, these effects led to an increase in apoptosis in irradiated cells. Thus, ECL radiosensitized the NSCLC cells to X-ray via G2/M arrest induction and delayed the repair of X-ray-induced DSBs. This study offers a great potential for ECL as an alternative safer radiosensitizer for increasing the RT efficiency against NSCLC.

Up-regulation of interferon-stimulated gene 15 and its conjugation machinery, UbE1L and UbcH8 expression by tumor necrosis factor-α through p38 MAPK and JNK signaling pathways in human lung carcinoma.

Interferon-stimulated gene 15 (ISG15) is a member of the family of ubiquitin-like proteins. Similar to ubiquitin, conjugation of ISG15 to cellular proteins requires cascade reactions catalyzed by at least 2 enzymes, UbE1L and UbcH8. Expression of ISG15 and its conjugates is up-regulated in many cancer cells, yet the underlying mechanism of up-regulation is still unclear. In this study, we showed that TNF-α, similar to the response by IFN-ß, could directly induce expression of ISG15 and its conjugation machinery, UbE1L and UbcH8, in human lung carcinoma, A549. The early response of their expression was effectively blocked by specific inhibitors of p38 MAPK (SB202190) and JNK (SP600125), but not by B18R, a soluble type-I IFN receptor. In addition, luciferase reporter assay together with serial deletions and site-directed mutagenesis identified a putative C/EBPß binding element in the ISG15 promoter, which is necessary to the response by TNF-α. Taken together, expression of ISG15 and ISG15 conjugation machinery in cancer cells is directly up-regulated by TNF-α via p38 MAPK and JNK pathways through the activation of C/EBPß binding element in the ISG15 promoter. This study provides a new insight toward understanding the molecular mechanism of ISG15 system and inflammatory response in cancer progression.

Salivary and serum cystatin SA levels in patients with type 2 diabetes mellitus or diabetic nephropathy.

OBJECTIVE: To investigate putative salivary biomarkers for screening and diagnosis of type 2 diabetes mellitus and diabetic nephropathy. DESIGN: Saliva and serum samples were collected from 29 patients with type 2 diabetes, 20 patients with diabetic nephropathy, eight patients with non-diabetic induced nephropathy, and 25 healthy subjects. Initially, pooled unstimulated saliva samples from six sex- and age-matched healthy subjects and six patients with type 2 diabetes were subjected to two-dimensional gel electrophoresis, followed by mass spectrometry. Protein expression of cystatin SA in the saliva of patients with type 2 diabetes was further examined in saliva and serum using enzyme-linked immunosorbent assay (ELISA). RESULTS: Two-dimensional gel electrophoresis revealed upregulation of salivary cystatin SA in patients with type 2 diabetes. ELISA showed a weak trend of increasing salivary cystatin SA levels in patients with type 2 diabetes, compared with those levels in healthy subjects. When patients were stratified according to periodontal status, linear regression analyses revealed that salivary cystatin SA levels were associated with Periodontal Screening and Recording (PSR) index (ß = 0.297, p < 0.05) when the analysis was adjusted for age, sex, HbA1C, estimated glomerular filtration rate (eGFR), and number of teeth. Serum cystatin SA levels were negatively associated with eGFR (ß = -0.534, p < 0.0001) when the analysis was adjusted for age, sex, HbA1C, number of teeth, and PSR index. CONCLUSIONS: Salivary cystatin SA was associated with periodontal disease severity; moreover, serum cystatin SA levels could reflect kidney function.

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.

Comparative proteomic study of dog and human saliva.

Saliva contains many proteins that have an important role in biological process of the oral cavity and is closely associated with many diseases. Although the dog is a common companion animal, the composition of salivary proteome and its relationship with that of human are unclear. In this study, shotgun proteomics was used to compare the salivary proteomes of 7 Thai village dogs and 7 human subjects. Salivary proteomes revealed 2,532 differentially expressed proteins in dogs and humans, representing various functions including cellular component organization or biogenesis, cellular process, localization, biological regulation, response to stimulus, developmental process, multicellular organismal process, metabolic process, immune system process, apoptosis and biological adhesion. The oral proteomes of dogs and humans were appreciably different. Proteins related to apoptosis processes and biological adhesion were predominated in dog saliva. Drug-target network predictions by STITCH Version 5.0 showed that dog salivary proteins were found to have potential roles in tumorigenesis, anti-inflammation and antimicrobial processes. In addition, proteins related to regeneration and healing processes such as fibroblast growth factor and epidermal growth factor were also up-regulated in dogs. These findings provide new information on dog saliva composition and will be beneficial for the study of dog saliva in diseased and health conditions in the future.

Putative salivary protein biomarkers for the diagnosis of oral lichen planus: a case-control study.

BACKGROUND: Salivary protein biomarkers for screening and diagnosis of oral lichen planus (OLP) are not well-defined. The objective of this study was to identify putative protein biomarkers for OLP using proteomic approaches. METHODS: Pooled unstimulated whole saliva was collected from five OLP patients and five healthy control participants. Saliva samples were then subjected to two-dimensional gel electrophoresis, followed by mass spectrometry to identify putative protein biomarkers. Subsequently, a subset of these putative biomarkers were validated in 24 OLP patients and 24 age-matched healthy control subjects, using an enzyme-linked immunosorbent assay (ELISA). Immunoblotting analyses were then performed in 3 pairs of age- and sex-matched OLP patients and healthy controls to confirm results from the ELISA study. RESULTS: Thirty-one protein spots were identified, corresponding to 20 unique proteins. Notably, fibrinogen fragment D and complement component C3c exhibited increased expression in OLP patients, while cystatin SA exhibited decreased expression in OLP patients, compared with healthy control subjects. ELISA analyses indicated increased expression of fibrinogen fragment D and complement component C3c, and decreased expression of cystatin SA, in the saliva of OLP patients. Statistical differences in the expression of salivary complement C3c were observed between OLP patients and healthy control subjects. Immunoblotting analyses confirmed the results of our ELISA study. CONCLUSION: Complement C3c, fibrinogen fragment D and cystatin SA may serve as salivary biomarkers for screening and/or diagnosis of OLP.

Upregulation of maspin expression in human cervical carcinoma cells by transforming growth factor ß1 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 ß1 (TGF-ß1) 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-ß1 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-ß1 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-ß1-induced phosphorylation of Smad2. In addition, mutations at p53-binding sites in the maspin promoter suppressed TGF-ß1-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-ß1-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 maspin's 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.

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/ß), 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-ß 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-ß, 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-ß 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.

Control of cell proliferation via elevated NEDD8 conjugation in oral squamous cell carcinoma.

A prominent feature of all cancers including oral carcinoma is an abnormality in cellular proliferation. Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like protein exerting its biological function through covalent ligation to a family of cullin proteins, which act as a component of the Skp1, cullin, and F-box protein complex that plays a critical role in the regulation of cell cycle. Using Western blot analysis, an increase in NEDD8 conjugation was observed in all highly proliferative cells, including immortalized normal oral keratinocyte (NOK), oral-derived carcinoma (KB, HSC4), and several types of human carcinoma cell lines as compared to normal human gingival fibroblast. The level of NEDD8 conjugation was proportionally dependent on the concentration of serum in culture medium, which also affected proliferation status of oral squamous cell carcinoma. NEDD8 conjugation was required for proliferation of the carcinoma cell since the transfection of dominant negative Ubc12 (Ubc12C111S) could prolong the rate of proliferation. Taken together, up-regulation of the NEDD8 conjugation is an essential requirement for the enhancement of proliferation of oral carcinoma cells.