Human Oral Squamous Carcinoma HSC-3 Research Articles

Gene expression of selected apoptotic markers in human oral squamous carcinoma HSC-3 cell line treated with Myrmecodia pendans plant extract

Introduction: Myrmecodia pendans (M. pendans), also known as Sarang Semut is an epiphyte with potential as a new therapeutic agent. Recently, we reported that M. pendans induced apoptotic activity in human oral squamous carcinoma (HSC-3) cell line. This study aimed to study how M. pendans extract affected apoptotic markers BAX/BCL-2 expression. Materials and methods: M. pendans was purchased from West Papua, Indonesia. The hypocotyl was allowed to dry thoroughly, then extracted aqueously. The extract was kept at -80 °C before freeze drying. The crude extract was used in the experiments. HSC-3 cells were cultured in Dulbecco’s Modified Essential Medium, then exposed to M. pendans at 2.5, 5.0 and 7.5 mg/ml for 24 and 48 h. Doxorubicin (0.005 mg/ml) was used as positive control. BAX and BCL-2 expression was analysed by Quantitative Real Time Polymerase Chain Reaction. Results: BAX and BCL-2 expression in HSC-3 cells was affected by M. pendans. At 24 h, highest levels of BAX and BCL-2 were observed with M. pendans at 2.5 mg/ml, while downregulation was observed at 5 mg/ml. However, after 48 h, BAX and BCL-2 were downregulated at all concentrations. The ratio of BAX/ BCL-2 in HSC-3 cells at 24 h showed significant upregulation at all treatment concentrations, but was downregulated after 48 h. Conclusion: M. pendans extract induced apoptotic activity in HSC-3 cells. This study suggests that induction of apoptosis in HSC-3 cell line is regulated via the pro-apoptotic (BAX) and anti- apoptotic (BCL -2) pathways.

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.

Lactobacillus casei Strain Shirota Enhances the In Vitro Antiproliferative Effect of Geniposide in Human Oral Squamous Carcinoma HSC-3 Cells.

This study investigated the enhanced antiproliferative effect of Lactobacillus casei strain Shirota (LcS) on geniposide actions in human oral squamous carcinoma HSC-3 cells. An MTT assay, flow cytometry, qPCR assay, western blot and HPLC were used for this study. The concentration of 1.0 × 106 CFU/mL of LcS had no effect on the HOK normal oral epithelial cells and HSC-3 cancer cells. The 25 and 50 µg/mL geniposide concentrations also had no impact on HOK normal oral epithelial cells, but they had remarkable inhibitory effects on the growth of HSC-3 cancer cells, which are enhanced in the presence of LcS. By the flow cytometry assay, the LcS-geniposide-H (1.0 × 106 CFU/mL LcS and 50 µg/mL geniposide)-treated HSC-3 cancer cells had the largest number of cells undergoing apoptosis compared to cells treated with other combinationsand obviously more than cells treated with only geniposide-H (50 µg/mL geniposide). Geniposide-H could increase the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, p53, p21, IκB-α, Fas, FasL, TIMP-1, and TIMP-2 as well as decrease those of Bcl-2, Bcl-xL, HIAP-1, HIAP-2, NF-κB, COX-2, iNOS, MMP-2, and MMP-9 compared to other groups of cells, and LcS further enhanced these changes, with results that are greater than for the cells treated with only a high concentration of geniposide. The results of this study show thatLcS enhanced the antiproliferative effect of geniposide in HSC-3 cancer cells.

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases.

Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA) is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, DAPI (4’,6-diamidino-2-phenylindole) staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction) assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL) and 22.4% (200 μg/mL). After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), DR4 (death receptor 4) and DR5 (death receptor 5) were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), XIAP (X-linked inhibitor of apoptosis protein), cIAP-1 (cellular inhibitor of apoptosis 1), cIAP-2 (cellular inhibitor of apoptosis 2) and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and IκB-α expression levels were the highest, and Bcl-2, Bcl-xL, XIAP, cIAP-1, cIAP-2, survival, and NF-κB expression levels were the lowest. These results proved that UDCA could induce apoptosis of HSC-3 cancer cells through caspase activation, and the higher concentration of UDCA had stronger effects in vitro. UDCA might be a good nutrient for oral cancer prevention.

TWEAK Induces Apoptosis through a Death-signaling Complex Comprising Receptor-interacting Protein 1 (RIP1), Fas-associated Death Domain (FADD), and Caspase-8

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.

Berberine induces cell cycle arrest and apoptosis in human HSC‐3 oral cancer cells

Evidence has accumulated that berberine induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address berberine affecting human oral quamous carcinoma cancer HSC-3 cells. In this study, we have examined the effects of berberine on cell growth and apoptosis and cell cycle regulation in human oral squamous carcinoma HSC-3 cells. Berberine induced dose- and time-dependent of irreversible inhibition of cell growth and cellular DNA synthesis. It was also confirmed by phase-light microscopy which showed that berberine induced morphological changes in HSC-3 cells. Propidium iodide/annexin V staining for flow cytometric analysis showed that berberine induced apoptosis correlating with caspase-3 activation in examined cells. Flow cytometry studies of the cell cycle distribution showed that berberine induced mainly G0/G1-phase arrest. Flow cytometry examinations also showed that berberine induced reactive oxygen species (ROS) and Ca2+ production and dysfunction of mitochondrial membrane potential (MMP) which are correlated with apoptosis. In conclusion, our data supports a view that berberine initially induces an endoplasmic reticulum stress response based on ROS and Ca2+ production then followed by dysfunction mitochondria, resulting in apoptosis of these oral cancer HSC-3 cells. Prolonged exposure of the HSC-3 cells to berberine causes increased percentage of apoptosis through decreased levels of MMP, cytochrome c release and activation of caspase-3.