Oral Cancer HSC-3 Research Articles

Okanin Inhibits Cell Growth and Induces Apoptosis and Pyroptosis in Oral Cancer.

Okanin, a flavonoid compound derived from Bidens pilosa L., has garnered attention for its anti-inflammatory properties. Although Bidens pilosa is commonly used in healthcare products and functional foods, the anticancer potential of okanin, particularly in oral cancer, remains underexplored. This study aims to investigate the effects of okanin on oral cancer cell lines and its potential as a therapeutic agent. The study involved assessing the cytotoxic effects of okanin on oral cancer cell lines SAS, SCC25, HSC3, and OEC-M1. The IC50 values were determined using methylene blue assays, and the clonogenic capacity was evaluated through colony formation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis. Caspase-3/7 activity assays and annexin V/7-AAD staining confirmed the induction of apoptosis and pyroptosis. In vivo efficacy was assessed using a SAS xenograft model, and immunohistochemical analysis of xenograft tissue was performed to examine pyroptosis-related markers. Okanin exhibited potent cytotoxic effects with IC50 values of 12.0 ± 0.8, 58.9 ± 18.7, 18.1 ± 5.3, and 43.2 ± 6.2 μM in SAS, SCC25, HSC3, and OEC-M1 cells, respectively. It caused dose- and time-dependent reductions in cell viability and significantly impaired clonogenic capacity. Flow cytometry revealed G2/M cell cycle arrest and increased sub-G1 population, indicating cell cycle disruption and death. Okanin induced both apoptosis and pyroptosis, as confirmed by caspase-3/7 activity and annexin V/7-AAD staining. In vivo, okanin reduced tumor growth and involved pyroptosis-related markers such as CASP1, GSDMC, GSDMD, and GSDME. Okanin demonstrates significant anticancer potential, particularly in oral cancer, by inducing both apoptosis and pyroptosis. Its efficacy in reducing tumor growth in vivo further supports its potential as a novel therapeutic option. Further mechanistic studies are needed to elucidate the pathways involved in okanin-mediated cell death and to explore its clinical applications.

Chitinase 3-Like-1 Expression Is Upregulated Under Inflammatory Conditions in Human Oral Epithelial Cells.

Chitinase 3-like-1 (CHI3L1), also known as YKL-40, is a partially secreted glycoprotein and is involved in inflammatory disorders, including inflammatory bowel diseases. CHI3L1 is known to play a role in biological responses such as cell proliferation, tissue remodeling, and inflammation. CHI3L1 forms an immune complex (known as a Chitosome complex) with IL-13 receptor alpha 2 (IL-13 Rα2) and transmembrane protein 219 (TMEM219) to activate the MAPK/ERK and PKB/AKT signaling pathways. The objective of this study is to investigate how the expressions of CHI3L1 and a Chitosome complex in human oral cavity epithelial cells are linked with intraoral inflammatory diseases. CHI3L1 and Chitosome complex mRNA expressions were analyzed using human oral squamous cancer cell lines, HSC3 and HSC4 cells. Signaling activation in HSC4 cells was analyzed by using the western blot technique. Immunohistological analysis was performed using surgical samples obtained from patients with benign oral cavity tumors and cysts. Increased expression of CHI3L1 was observed in both HSC3 and HSC4 cells after TNFα stimulation. The expression of Chitosome complex factors increased as CHI3L1 levels increased, resulting in the activation of a downstream signaling pathway. Among the intraoral tissues, the epithelial cells from inflammatory lesions, but not benign tumors, were found to be intensively stained with the anti-CHI3L1 antibody. It was indicated that the formation of a Chitosome complex is induced during inflammation, leading to the activation of signaling pathways.

Insight into OroxylinA-7-O-β-d-Glucuronide-Enriched Oroxylum indicum Bark Extract in Oral Cancer HSC-3 Cell Apoptotic Mechanism: Role of Mitochondrial Microenvironment.

Oroxylum indicum, of the Bignoniaceae family, has various ethnomedical uses such as an astringent, anti-inflammatory, anti-bronchitis, anti-helminthic and anti-microbial, including anticancer properties. The druggability of OI stem bark extract was determined by its molecular docking interactions with PARP and Caspase-3, two proteins involved in cell survival and death. Note that 50 µg/mL of Oroxylum indicum extract (OIE) showed a significant (p < 0.05%) toxicity to HSC-3 cells. MTT aided cell viability and proliferation assay demonstrated that 50 µg/mL of OIE displayed significant (p < 0.5%) reduction in cell number at 4 h of incubation time. Cell elongation and spindle formation was noticed when HSC-3 cells were treated with 50 µg/mL of OIE. OIE initiated DNA breakage and apoptosis in HSC-3 cells, as evident from DNA ladder assay and calcein/EB staining. Apoptosis potential of OIE is confirmed by flow cytometer and triple-staining (live cell/apoptosis/necrosis) assay. Caspase-3/7 fluorescence quenching (LANCE) assay demonstrated that 50 µg/mL of OIE significantly enhanced the RFU of caspases-3/7, indicating that the apoptosis potential of OIE is probably through the activation of caspases. Immuno-cytochemistry of HSC-3 cells treated with 50 µg/mL of OIE showed a significant reduction in mitochondrial bodies as well as a reduction in RFU in 60 min of incubation time. Immunoblotting studies clearly showed that treatment of HSC-3 cells with OI extract caused caspase-3 activation and PARP deactivation, resulting in apoptotic cell death. Overall, our data indicate that OIE is an effective apoptotic agent for human squamous carcinoma cells and it could be a future cancer chemotherapeutic target.

Enhancement of Anticancer Potential of Pterostilbene Derivative by Chalcone Hybridization.

Pterostilbene, a natural metabolite of resveratrol, has been indicated as a potent anticancer molecule. Recently, several pterostilbene derivatives have been reported to exhibit better anticancer activities than that of the parent pterostilbene molecule. In the present study, a series of pterostilbene derivatives were designed and synthesized by the hybridization of pterostilbene, chalcone, and cinnamic acid. The cytotoxic effect of these hybrid molecules was determined using two oral cancer cell lines, HSC-3 and OECM-1. (E)-3-(2-((E)-4-Hydroxystyryl)-4,6-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (4d), with IC50 of 16.38 and 18.06 μM against OECM-1 and HSC-3, respectively, was selected for further anticancer mechanism studies. Results indicated that compound 4d effectively inhibited cell proliferation and induced G2/M cell cycle arrest via modulating p21, cyclin B1, and cyclin A2. Compound 4d ultimately induced cell apoptosis by reducing the expression of Bcl-2 and surviving. In addition, cleavage of PARP and caspase-3 were enhanced following the treatment of compound 4d with increased dose. To conclude, a number of pterostilbene derivatives were discovered to possess potent anticancer potentials. Among them, compound 4d was the most active, more active than the parent pterostilbene.

&lt;i&gt;In Vitro&lt;/i&gt; Anti-Cancer Activities of &lt;i&gt;Luvunga scandens &lt;/i&gt;(‘Mengkurat Jakun’) on Oral Squamous Cell Carcinoma Cells

Luvunga scandens (“Mengkurat Jakun”) is one of the medicinal plant that produce triterpenoid compounds. A number of studies have reported that the compounds possess anti-proliferative activities against various type of cancer cells. The present study aims for anticancer potential of two triterpenoids derived from L. scandens namely flindissol (compound-1) and 3-oxotirucalla-7,24-dien-21-oic-acid (compound-2) on human oral cancer HSC-3 cells. Cytotoxic activities of the triterpenoids were assessed by MTT assay. Apoptosis activities were determined by flow cytometry and caspase 3/7 assay. The MTT assay showed that compounds-1 and -2 markedly induced cytotoxicity on HSC-3 cells with IC50 10.7 μM and 8.3 μM, respectively. Flow-cytometry analysis demonstrated that both compounds increase the percentage of apoptotic cells by 18.2 % and 16.6 % respectively. Moreover, the caspase 3/7 assay confirmed that compounds-1 and -2 markedly induced caspase 3/7 activities in HSC-3 cells. These results suggest that triterpenoids extracted from L. scandens could be a potential candidate for oral cancer treatment.

Abstract PO-050: Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis

Abstract The impact of peripheral neurons on carcinogenesis is an understudied process in head and neck cancer. Peripheral nerve innervation is increased in preclinical models of oral cancer; sympathectomy resulted in smaller, less invasive cancers. Local neurotransmitter release from peripheral neurons innervating cancer has been linked to cancer growth and immune suppression. We hypothesize that local neurotransmitter release from trigeminal sensory neurons (TGNs) and sympathetic postganglionic neurons promotes cancer proliferation and suppresses the immune response. We used real-time PCR to determine RNA expression in human oral cancer cell lines and a colorimetric cell proliferation assay to quantify cancer cell growth in response to the sympathetic and sensory neurotransmitters. Oral cancer cell lines, HSC-3 and SCC-4, overexpress the norepinephrine (NE) receptor, adrenergic receptor beta 2 (ADRβ2) and sensory neurotransmitter receptors, receptor activity modifying protein1 (RAMP1) and neurokinin1 receptor (NK1R) compared to the non-tumorigenic cell line, HaCaT. Treatment with sympathetic (NE) and sensory (CGRP, substance p) neurotransmitters for 48 hours significantly increased cell proliferation in HSC-3 (&amp;gt;2-fold) and SCC-4 (&amp;gt;1.5-fold) cell lines, but had no effect on HaCaT cell proliferation. Using optical clearing and immunohistochemistry, we observed increased PGP9.5+ neuronal fiber density in the microenvironment using high-resolution microscopy in a tongue oral cancer xenograft mouse model. In vitro we measured a 44.6 ± 1.14% increase in TGN sprouting after 48-hour exposure to conditioned media from the HSC-3, compared to conditioned media from HaCaT. Lastly, HSC-3 cell culture supernatant injection (50 µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10 ± 1.2 % of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50 µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) compared to HSC-3 supernatant without pharmacologic nerve block. These results together suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction might improve strategies to treat head ad neck cancer by targeting peripheral neurons in the cancer microenvironment. Citation Format: Megan A. Atherton, Marci L. Nilsen, Nicole N. Scheff. Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-050.

Antimetastatic Effects of Sesamin on Human Head and Neck Squamous Cell Carcinoma through Regulation of Matrix Metalloproteinase-2.

Background: Sesamin is a lignin present in sesame oil from the bark of Zanthoxylum spp. Sesamin reportedly has anticarcinogenic potential and exerts anti-inflammatory effects on several tumors. Hypothesis/Purpose: However, the effect of sesamin on metastatic progression in human head and neck squamous carcinoma (HNSCC) remains unknown in vitro and in vivo; hence, we investigated the effect of sesamin on HNSCC cells in vitro. Methods and Results: Sesamin-treated human oral cancer cell lines FaDu, HSC-3, and Ca9-22 were subjected to a wound-healing assay. Furthermore, Western blotting was performed to assess the effect of sesamin on the expression levels of matrix metalloproteinase (MMP)-2 and proteins of the MAPK signaling pathway, including p-ERK1/2, P-p38, and p-JNK1/2. In addition, we investigated the association between MMP-2 expression and the MAPK pathway in sesamin-treated oral cancer cells. Sesamin inhibited cell migration and invasion in FaDu, Ca9-22, and HSC-3 cells and suppressed MMP-2 at noncytotoxic concentrations (0 to 40 μM). Furthermore, sesamin significantly reduced p38 MAPK and JNK phosphorylation in a dose-dependent manner in FaDu and HSC-3 cells. Conclusions: These results indicate that sesamin suppresses the migration and invasion of HNSCC cells by regulating MMP-2 and is thus a potential antimetastatic agent for treating HNSCC.

Pomegranate extract inhibits migration and invasion of oral cancer cells by downregulating matrix metalloproteinase-2/9 and epithelial-mesenchymal transition.

Discovering drug candidates for the modulation of metastasis is of great importance in inhibiting oral cancer malignancy. Although most pomegranate extract applications aim at the antiproliferation of cancer cells, its antimetastatic effects remain unclear, especially for oral cancer cells. The aim of this study is to evaluate the change of two main metastasis characters, migration and invasion of oral cancer cells. Further, we want to explore the molecular mechanisms of action of pomegranate extract (POMx) at low cytotoxic concentration. We found that POMx ranged from 0 to 50 μg/mL showing low cytotoxicity to oral cancer cells. In the case of oral cancer HSC-3 and Ca9-22 cells, POMx inhibits wound healing migration, transwell migration, and matrix gel invasion. Mechanistically, POMx downregulates matrix metalloproteinase (MMP)-2 and MMP-9 activities and expressions as well as epithelial-mesenchymal transition (EMT) signaling. POMx upregulates extracellular signal-regulated kinases 1/2 (ERK1/2), but not c-Jun N-terminal kinase (JNK) and p38 expression. Addition of ERK1/2 inhibitor (PD98059) significantly recovered the POMx-suppressed transwell migration and MMP-2/-9 activities in HSC-3 cells. Taken together, these findings suggest to further test low cytotoxic concentrations of POMx as a potential antimetastatic therapy against oral cancer cells.

Differential expression of claudin‑4, occludin, SOX2 and proliferating cell nuclear antigen between basaloid squamous cell carcinoma and squamous cell carcinoma.

Basaloid squamous cell carcinomas (BSCCs) in oral lesions are extremely rare, and the histology is not well understood. Histologically, they are often similar to conventional squamous cell carcinoma (SCC). The present study was designed with an aim to distinguish BSCC from SCC using claudin‑4, occludin, SRY‑box2 (SOX2) and proliferating cell nuclear antigen (PCNA) immunoreactivities and staining patterns. Three BSCCs (with abundant, with moderate, and without squamous components) specimens and 20SCC specimens were selected for comparison of their immunoreactivity. These specimens were stained with claudin‑4, occludin, SOX2 and PCNA. In addition to histological analysis, the expression of claudin‑4, occludin and PCNA was determined in oral cancer HSC2 and HSC3cells with or without SOX2 overexpression, and cell proliferation was determined by XTT assay. Claudin‑4 had strong and occludin had weak immunoreactivity as detected in the membrane of squamous components of BSCC but not in cancer cells. No obvious detection of squamous components and cancer cells were observed in SCC. SOX2 and PCNA immunoreactivities in SCC had dot‑like staining patterns in the nuclei of partial and marginal cancer cells. In contrast, in BSCCs, SOX2 and PCNA had diffuse staining patterns in almost all cancer cells. SOX2 overexpression had little effect on the expression levels of claudin‑4, occludin and PCNA. It also had little effect on the cell proliferation of HSC2 and HSC3cells. Differences in immunoreactivity and staining pattern may be valuable to distinguish between BSCC and SCC in diagnosis.

Morphine inhibits cell viability and growth via suppression ofvascular endothelial growth factor in human oral cancer HSC-3 cells.

Although many oral cancer patients require opioids, the effects of morphine and related drugs on oral cancer progression have not been well established. Thus, we examined the effects of morphine exposure on the viability of human oral squamous carcinoma HSC-3 cells and aimed to identify the underlying mechanism. We exposed HSC-3 cells to the various concentrations of morphine (0, 0.1, 1, 10, 100, or 1000μmol/L) for 48h and, subsequently, evaluated cell viability using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and cytotoxicity using the lactate dehydrogenase (LDH) assay. To explore the effects of morphine on cell proliferation further, colony formation assay and cell cycle analysis were performed. Additionally, the intracellular expression of nuclear factor kappa B (NF-κB) was analyzed using flow cytometry, and vascular endothelial growth factor (VEGF)-A was evaluated using human VEGF assay. Morphine exposure reduced cell viability and enhanced cytotoxicity in HSC-3 cells in a concentration-dependent manner. The number of colonies in the morphine-treated groups was significantly lower than that in the control group. Consistent with these results, morphine exposure significantly reduced the concentration of VEGF in the cell culture medium in a concentration-dependent manner. However, our data show that morphine at clinical concentrations (0.1-10μmol/L) does not affect cell cycle and apoptosis. Our results suggest that in human oral cancer HSC-3 cells, morphine exposure inhibits cell viability and growth via suppression of VEGF in clinical conditions.

Fenofibrate induces human hepatoma Hep3B cells apoptosis and necroptosis through inhibition of thioesterase domain of fatty acid synthase

This study demonstrated that fenofibrate, a lipid-lowering drug, induced a significant time-dependent cytotoxicity of hepatoma Hep3B cells. Hep3B cells are significantly more sensitive to cell killing by fenofibrate than hepatoma HepG2, lung cancer CH27 and oral cancer HSC-3 cells. From the result of docking simulation, fenofibrate can bind excellently to the thioesterase domain of fatty acid synthase (FASN) binding site as orlistat, a FASN inhibitor, acts. The fenofibrate-induced cell cytotoxicity was protected by addition of palmitate, indicating that the cytotoxic effect of fenofibrate is due to starvation of Hep3B cells by inhibiting the formation of end product in the FASN reaction. Inhibition of lipid metabolism-related proteins expression, such as proteins containing thioesterase domain and fatty acid transport proteins, was involved in the fenofibrate-induced Hep3B cell death. Fenofibrate caused S and G2/M cell cycle arrest by inducing cyclin A/Cdk2 and reducing cyclin D1 and E protein levels in Hep3B cells. The anti-tumor roles of fenofibrate on Hep3B cells by inducing apoptosis and necroptosis were dependent on the expression of Bcl-2/caspase family members and RIP1/RIP3 proteins, respectively. These results suggest that fenofibrate has an anti-cancer effect in Hep3B cells and inhibition of lipid metabolism may be involved in fenofibrate-induced Hep3B cells apoptosis and necroptosis.

Comparison of the in vitro and in vivo toxic effects of thymoquinone using oral cancer HSC-3 and HSC-4 cell lines, oral fibroblasts, HACAT cell line, and Zebrafish embryos

Abstract The discovery and development of anticancer agents were made through long processes from screening new compounds in various cell lines, animal experimental studies, until clinical trials Majority of cancer chemotherapeutic agents have been initially screened in an in vitro cell-based system and then subsequently studied in an in vivo model using small animals destined for pharmacokinetic properties. Zebrafish (Danio rerio) has become one of the most popular animal model systems used for chemical toxicity tests and drug discovery. Thymoquinone (TQ) is the main constituent of black seed (Nigella sativa, spp) essential oil which shows anti-neoplastic activities in various tumor cells. However, to date, studies on the toxicity of TQ on normal cells or animal model are still insufficient. Here we report that TQ is selective toxic to different human cell lines and show negligible effect on the Zebrafish. The MTT assay demonstrated that TQ induced cytotoxicity on oral cancer cells (HSC-3 and HSC-4) but not on the normal cells (human oral fibroblast and HACAT cells). Moreover, no significant morphological change was observed in the Zebrafish model following TQ exposure at the concentration (0.003 mg/mL), similar to those in the cancer cells. The study suggests that TQ as a cancer chemotherapeutic candidate without apparent adverse effects.

Preliminary investigations of anti-cancer properties of Myrmecodia Pendens on human oral cancer HSC-3 cell lines

Abstract Myrmecodia pendens (M. pendens) or also known as Sarang semut is an epiphytic plant which might have potential as a new source of alternative therapeutic agent. Recently, it is reported that M. pendens can inhibit the proliferation of HeLa and MCM-B2 cell lines. We report here for the first time the anti-cancer potential of M. pendens on oral cancer cells derived from human oral cavity, HSC-3 cell lines. MTT assay results showed that M. pendens crude extract markedly induced cytotoxicity on HSC-3 cell lines after 24 h (IC50 = 5 mg/mL) and 48 h (IC50 = 3 mg/ml). The apoptotic activity induced by M. pendens was detected in HSC-3 cell lines by flow cytometry within 24 h. These results suggest that M. pendens may be a potential candidate for oral cancer treatment.

DNA damage response following X-irradiation in oral cancer cell lines HSC3 and HSC4

ObjectiveThe objective of this study was to characterize the DNA damage response in two human oral cancer cell lines following X-irradiation. DesignTo visualize radiation-induced cell cycle alterations, two human oral cancer cell lines, HSC3 and HSC4, expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were established in this study. G2 arrest kinetics following irradiation were obtained from two-color flow cytometric analysis and pedigrees of Fucci fluorescence. DNA double strand break repair kinetics were obtained from immunofluorescence staining for phosphorylated histone H2AX, p53-binding protein 1, phosphorylated DNA-dependent protein kinase catalytic subunit, and breast cancer susceptibility gene 1. ResultsBoth cell lines showed apparent G2 arrest after 10 Gy of irradiation, but it was more enhanced in the HSC3-Fucci cells. Radiosensitivity was higher in the HSC3-Fucci cells than in HSC4-Fucci cells. Pedigree analysis of Fucci fluorescence revealed that the HSC3-Fucci cells exhibited a significantly longer green phase (normally indicating S/G2/M phases, but here reflective of G2 arrest) when irradiated in the red phase (G1 phase) than HSC4-Fucci cells irradiated in either red or green phases. Non-homologous end joining was marginally suppressed during the G1 phase and markedly more likely to be impaired during the S/G2 phases in HSC3-Fucci cells. When G2 arrest was abrogated by checkpoint kinase 1 or Wee1 inhibitors, only HSC4-Fucci cells exhibited radiosensitization. ConclusionsWe characterized DNA damage response in HSC3-Fucci and HSC4-Fucci cells following irradiation and the former demonstrated inefficient non-homologous end joining, especially during the S/G2 phases, resulting in enhanced G2 arrest. These findings may have clinical implications for oral cancer.

Gypenosides induce cell death and alter gene expression in human oral cancer HSC-3 cells.

Gypenosides (Gyp), the primary components of Gynostemma pentaphyllum Makino, have long been used as a Chinese herbal medicine. In the present study, the effects of Gyp on cell viability, the cell cycle, cell apoptosis, DNA damage and chromatin condensation were investigated in vitro using human oral cancer HSC-3 cells. The results of the present study indicated that Gyp induces cell death, G2/M phase arrest and apoptosis in HSC-3 cells in a dose-dependent manner. It was also demonstrated that Gyp decreased the depolarization of mitochondrial membrane potential in a time-dependent manner. A cDNA microarray assay was performed and the results indicated that a number of genes were upregulated following Gyp treatment. The greatest increase was a 75.42-fold increase in the expression of GTP binding protein in skeletal muscle. Levels of the following proteins were also increased by Gyp: Serpine peptidase inhibitor, clade E, member 1 by 20.25-fold; ras homolog family member B by 18.04-fold, kelch repeat and BTB domain containing 8 by 15.22-fold; interleukin 11 by 14.96-fold; activating transcription factor 3 by 14.49-fold; cytochrome P450, family 1 by 14.44-fold; ADP-ribosylation factor-like 14 by 13.88-fold; transfer RNA selenocysteine 2 by 13.23-fold; and syntaxin 11 by 13.08-fold. However, the following genes were downregulated by GYP: Six-transmembrane epithelial antigen of prostate family member 4, 14.19-fold; γ-aminobutyric acid A receptor by 14.58-fold; transcriptional-regulating factor 1 by 14.69-fold; serpin peptidase inhibitor, clade B, member 13 by 14.71-fold; apolipoprotein L 1 by 14.85-fold; follistatin by 15.22-fold; uncharacterized LOC100506718; fibronectin leucine rich transmembrane protein 2 by 15.61-fold; microRNA 205 by 16.38-fold; neuregulin 1 by 19.69-fold; and G protein-coupled receptor 110 by 22.05-fold. These changes in gene expression illustrate the effects of Gyp at the genetic level and identify potential targets for oral cancer therapy.

Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation.

Melatonin exerts antimetastatic effects on liver and breast cancer and also inhibits matrix metalloproteinase (MMP) activity. However, the detailed impacts and underlying mechanisms of melatonin on oral cancer cell metastasis are still unclear. This study showed that melatonin attenuated the 12-O-tetradecanoylphorbol-13-acetate-induced migration of oral cancer cell lines, HSC-3 and OECM-1. Zymography, quantitative real-time PCR, and Western blotting analyses revealed that melatonin lessened MMP-9 enzyme activity as well as the expression of MMP-9 mRNA and protein. Furthermore, melatonin suppressed the phosphorylation of the ERK1/2 signalling pathway, which dampened MMP-9 gene transcription by affecting the expression of transcriptional coactivators, such as CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300), and decreasing histone acetylation in HSC-3 and OECM-1 cells. Examinations on clinical samples exhibited that MMP-9, CREBBP, and EP300 were significantly increased in oral cancer tissues. Moreover, the relative level of CREBBP was positively correlated with the expression of MMP-9 and EP300. In conclusion, we demonstrated that melatonin inhibits the motility of HSC-3 and OECM-1 cells in vitro through a molecular mechanism that involves attenuation of MMP-9 expression and activity mediated by decreased histone acetylation.

Tetrandrine induces apoptosis Via caspase-8, -9, and -3 and poly (ADP ribose) polymerase dependent pathways and autophagy through beclin-1/ LC3-I, II signaling pathways in human oral cancer HSC-3 cells.

Tetrandrine is a bisbenzylisoquinoline alkaloid that was found in the Radix Stephania tetrandra S Moore. It had been reported to induce cytotoxic effects on many human cancer cells. In this study, we investigated the cytotoxic effects of tetrandrine on human oral cancer HSC-3 cells in vitro. Treatments of HSC-3 cells with tetrandrine significantly decreased the percentage of viable cells through the induction of autophagy and apoptosis and these effects are in concentration-dependent manner. To define the mechanism underlying the cytotoxic effects of tetrandrine, we investigated the critical molecular events known to regulate the apoptotic and autophagic machinery. Tetrandrine induced chromatin condensation, internucleosomal DNA fragmentation, activation of caspases-3, -8, and -9, and cleavage of poly (ADP ribose) polymerase (PARP) that were associated with apoptosis, and it also enhanced the expression of LC3-I and -II that were associated with the induction of autophagy in human squamous carcinoma cell line (HSC-3) cells. Tetrandrine induced autophagy in HSC-3 cells was significantly attenuated by bafilomycin A1 (inhibitor of autophagy) pre-treatment that confirmed tetrandrine induced cell death may be associated with the autophagy. In conclusion, we suggest that tetrandrine induced cell death may be through the induction of apoptosis as well as autophagy in human oral cancer HSC-3 cells via PARP, caspases/Becline I/LC3-I/II signaling pathways.

Molecular modelling, synthesis, cytotoxicity and anti-tumour mechanisms of 2-aryl-6-substituted quinazolinones as dual-targeted anti-cancer agents

Our previous study demonstrated that 6-(pyrrolidin-1-yl)-2-(3-methoxyphenyl)quinazolin-4-one (HMJ38) was a potent anti-tubulin agent. Here, HMJ38 was used as a lead compound to develop more potent anti-cancer agents and to examine the anti-cancer mechanisms. Using computer-aided drug design, 2-aryl-6-substituted quinazolinones (MJ compounds) were designed and synthesized by introducing substituents at C-2 and C-6 positions of HMJ38. The cytotoxicity of MJ compounds towards human cancer cells was examined by Trypan blue exclusion assay. Microtubule distribution was visualized using TubulinTracker(TM) Green reagent. Protein expression of cell cycle regulators and JNK was assessed by Western blot analysis. Compounds MJ65-70 exhibited strong anti-proliferative effects towards melanoma M21, lung squamous carcinoma CH27, lung non-small carcinoma H460, hepatoma Hep3B and oral cancer HSC-3 cells, with one compund MJ66 (6-(pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one) highly active against M21 cells (IC50 about 0.033 μM). Treatment of CH27 or HSC-3 cells with MJ65-70 resulted in significant mitotic arrest accompanied by increasing multiple asters of microtubules. JNK protein expression was involved in the MJ65-70-induced CH27 and M21 cell death. Consistent with the cell cycle arrest at G2/M phase, marked increases in cyclin B1 and Bcl-2 phosphorylation were also observed, after treatment with MJ65-70. MJ65-70 are dual-targeted, tubulin- and JNK-binding, anti-cancer agents and induce cancer cell death through up-regulation of JNK and interfering in the dynamics of tubulin. Our work provides a new strategy and mechanism for developing dual-targeted anti-cancer drugs, contributing to clinical anti-cancer drug discovery and application.

Abstract 5129: AGR2 is a potential marker for metastatic human oral squamous cell carcinoma.

Abstract Purpose: The mortality of oral cancer, which is a common subtype of human and neck squamous cell carcinomas (HNSCC), is increasing in Taiwan, Southeast Asia, and eastern countries during the last twenty years. Averagely, the 5-year survival rate of distant metastasis of oral carcinoma is less than 30-40%. Prevention of cancer metastasis to distant organs is one of practical approaches that may reduce the mortality of patients. This study aims to identify novel metastatic tumor markers for oral cancer. Experimental Design and Results: A high metastatic sub-population, HSC-3-5 cells, was divided from human oral cancer HSC-3 by Transwell invasion assay. Our results indicated that HSC-3-5 cells exhibited higher metastatic capacities due to re-arrangement cytoskeleton and subsequently epithelial mesenchymal transition transformation in vitro. In addition, metastatic and tumorigenetic capacities of HSC-3-5 cells were characterized by spontaneous and experimental metastasis experiments in vivo. The microarray-based gene expression profiles of HSC-3/HSC-3-5 cells and their corresponded xenografts (orthotropic and heterotopic) were then performed. Anterior gradient 2 (AGR2), a pro-oncogenic signaling intermediate in various human cancers, was the most significantly up-regulated gene in HSC-3-5 cells compared with HSC-3. Furthermore, overexpression of AGR2 protein was verified in cells, xenograft animal models, and clinical patient tissues, and it encompassed positive relation with OSCC metastasis. Conclusions: A high metastatic sub-population HSC-3-5 cell line derived from human oral cancer HSC-3 cells was divided. HSC-3-5 cells represented higher capacities not only in metastasis but also in tumorigenesis. According to microarray gene profiles and immunohistochemistry analysis, the expression of identified potential biomarker, AGR2, showed positive relationship with tumor metastasis during oral carcinogenesis. Citation Format: Yi-Ting Chen, Chung-Liang Ho, Po-Ku Chen, Yuh-Ling Chen, Chung-Fa Chang. AGR2 is a potential marker for metastatic human oral squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5129. doi:10.1158/1538-7445.AM2013-5129

A novel antitubulin agent, DPQZ, induces cell apoptosis in human oral cancer cells through Ras/Raf inhibition and MAP kinases activation

6-(N,N-Dimethylamino)-2-(naphthalene-1-yl)-4-quinazolinone (DPQZ)-induced apoptosis was accompanied by the characteristics of DNA fragmentation and phosphatidylserine externalization in human oral cancer HSC-3 cells. The IC50 (half maximal inhibitory concentration) value of DPQZ is about 0.25μM at 24h. The interference in the dynamics of tubulin and cell division of DPQZ, like vinblastine (0.01μM), has been proven in this study. Treatment of HSC-3 cells with DPQZ resulted in many of mitotic cells with multipolar spindles. Up-regulation of MAP kinases, such as ERK, JNK, and p38, mediated by DPQZ appears to be involved in DPQZ-induced apoptosis in HSC-3 cells. It is worthy of note that the expression of Ras and c-Raf that lie upstream of ERK were inhibited by DPQZ. In addition, the DPQZ-induced cell death was attenuated by JNK inhibitor SP600125 (3 or 10μM), not by the ERK or p38 inhibitors. JNK inhibitor abolished the DPQZ-induced increase in the phosphorylation of Bcl-2 and the protein levels of proform caspase-3, caspase-8, and caspase-9, indicating that JNK is an upstream activator of Bcl-2 and caspase family members and plays a key role in DPQZ-induced HSC-3 cell apoptosis. We also attempted to develop an anticancer drug that is designed to kill rapidly dividing cancer cells while causing less damage to normal cells. The DPQZ-induced cytotoxicity against human gingival fibroblasts was less than that against HSC-3 cells. Our work provides a new strategy and mechanism for developing anticancer drug and may contribute to clinical anticancer drug discovery and application.