Oral Squamous Cell Carcinoma HSC-3 Research Articles

Effect of Agkistrodon acutus venom (AAVC-I) on apoptosis through modulation of the Keap1/Nrf2 pathway in HSC-3 oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial regions. Patients with OSCC exhibit a poor response to conventional chemoradiotherapies, which are associated with severe side effects. Therefore, it is essential to identify an effective therapeutic method to treat patients with OSCC. An anti-tumor compound, Agkistrodon acutus venom component I (AAVC-I), purified from Agkistrodon acutus venom, has demonstrated anticancer activity both in vitro and in vivo. However, the mechanism of AAVC-I's anticancer activity in cancer cells has yet to be established. This study aimed to investigate the mechanism of AAVC-I-induced apoptosis in HSC-3 OSCC cells and explore its regulatory effect on oxidative stress. Survival rates of human OSCC cell HSC-3 were detected by Cell Counting Kit-8 (CCK-8). The reactive oxygen species (ROS) level was analyzed by flow cytometry and fluorescence microscopy. The mitochondrial membrane potential was analyzed by cytometry and fluorescent microplate reader. Apoptosis of HSC-3 cells was analyzed using flow cytometry. The oxidative stress level was evaluated using glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits. In addition, the target proteins were analyzed by western blot. AAVC-I reduced HSC-3 cells' survival rates in a dose-dependent manner with a 50% inhibiting concentration (IC50) of 8.86 µg/mL. It induced apoptosis of HSC-3 cells and the expression of cleaved caspase-3, cleaved caspase-9, and Cyt-c increased significantly, whereas the expression level of Bcl-2 decreased in AAVC-I-treated HSC-3 cells. Thus, AAVC-I caused apoptosis of HSC-3 via the activation of the intrinsic apoptotic pathway. In addition, AAVC-I reduced the mitochondrial membrane potential in HSC-3, enhanced intracellular ROS, and increased intracellular oxidative stress levels in comparison to that of untreated control cells. Furthermore, AAVC-I increased the expression of Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) levels. These findings demonstrate the inhibitory effects and associated mechanisms of AAVC-I on the HSC-3 OSCC cell line. This insight could be valuable for investigating AAVC-I as a potential therapeutic option for patients with OSCC.

Cationic liposomes as a drug-free system for efficient anticancer therapy by intracytoplasmic delivery of sodium bicarbonate

Developing the delivery systems with high therapeutic efficacy and low side effects is of great interest and significance for anticancer therapy. Compared to the high cost in synthesizing new chemotherapeutic drugs, exploring the anticancer potentials of existing chemicals is more convenient and efficient. Sodium bicarbonate (BC), a simple inorganic salt, has shown its tumor inhibition capacity via regulating the acidity of tumor microenvironment. However, the effects of intracytoplasmic BC on tumor growth and the potentials of BC to serve as an anticancer agent are still unknown. Herein, we developed a BC-loaded cationic liposome system (BC-CLP) to deliver BC into the cytosol of cancer cells. The in vitro studies showed that the BC-CLP containing 1% BC (w/v) had a size of 112.9 nm and a zeta potential of 19.1 mV, which reduced the viability of the model cancer cells (human oral squamous cell carcinoma HSC-3 cells) to 13.7%. In contrast, the neutral BC-LP caused less than 50% viability reduction. We further found that BC-CLP released BC directly into cytoplasm via membrane fusion pathway rather than endocytosis, leading to the remarkable increase of cytosolic pH, which may contribute to the anticancer effect of BC-CLP. Our findings indicate that BC-CLP is a potential system for high-efficiency cancer therapy without causing drug-related side effects or resistance.

Human artificial chromosome carrying 3p21.3-p22.2 region suppresses hTERT transcription in oral cancer cells

Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been observed to be reactivated in immortalized cells. Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of hTERT transcription compared with the parental HSC3 cells. This study examined whether hTERT transcription regulators are present in the 3p21.3 region. We constructed a human artificial chromosome (HAC) vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.

A PKM2 inhibitor induces apoptosis and autophagy through JAK2 in human oral squamous cell carcinoma cells

The enzyme pyruvate kinase M2 (PKM2) is involved in glycolysis, which plays an important role in the regulation of tumor progression. In this study, we investigated the anti-tumor activity of N-(4-(3-(3-(methylamino)-3-oxopropyl)-5-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-1H-pyrazol-1-yl)phenyl)propiolamide (MTP), a PKM2 inhibitor, in oral squamous cell carcinoma (OSCC) cells. Our results showed that MTP inhibited cell growth with IC50 values of 0.59 μM and 0.78 μM in SCC2095 and HSC-3 OSCC cells, respectively. MTP induced caspase-dependent apoptosis, which was associated with the modulation of PKM2 and oncogenic biomarkers epidermal growth factor receptor and β-catenin. In addition, MTP increased the generation of reactive oxygen species (ROS) and modulated the expression of autophagic gene products, including LC3B-II and p62. Western blotting showed that MTP inhibited Janus kinase 2 (JAK2) signaling, and JAK2 overexpression partially reversed MTP-mediated cytotoxicity. Taken together, these data indicate the potential use of MTP as a therapeutic agent for OSCC.

Anlotinib Suppresses Oral Squamous Cell Carcinoma Growth and Metastasis by Targeting the RAS Protein to Inhibit the PI3K/Akt Signalling Pathway.

Oral squamous cell carcinoma (OSCC) is a malignant tumour originating from the mucosal lining of the oral cavity. Its characteristics include hidden onset, high recurrence, and distant metastasis after operation. At present, clinical treatment usually includes surgery, chemotherapy, radiotherapy, or the joint use of these modalities. Unfortunately, multidrug resistant is one of the important obstacles that causes cancer chemotherapy failure. Anlotinib, which has recently been proven to have good antitumour effects, is a novel multitargeted tyrosine kinase inhibitor. However, there are few studies of the anlotinib-associated mechanism in OSCC and its underlying molecular mechanism. In our study, in vitro models of human oral squamous cell carcinoma HSC-3 cells were used to determine the efficacy of anlotinib. On the one hand, we showed that anlotinib treatment significantly reduced the viability and proliferation of HSC-3 cells and decreased cell migration by inhibiting the activation of the Akt phosphorylation pathway. On the other side, anlotinib inhibited PI3K/Akt/Bad phosphorylation and promoted apoptosis of HSC-3 cells by activating RAS protein expression. In brief, these results indicated that anlotinib had prominent antitumour activity in OSCC, mainly by inhibiting the PI3K/Akt phosphorylation pathway. This work provides evidences and a basic principle for using anlotinib to treat patients with OSCC for clinical research.

Circular RNA Circ_0008450 Regulates the Invasion, Migration, and Proliferation of Oral Squamous Carcinoma Human Oral Squamous Cell 3 Cells by Targeting miR-1294

To determine the role of the circ_0008450/miR-1294 molecular axis in the proliferation, migration, and invasion of oral squamous cell carcinoma (OSCC) cells, the expression levels of circ_0008450 and miR-1294 in OSCC and neighboring non-cancerous tissues were detected by real-time quantitative polymerase chain reaction. Human OSCC HSC3 cells were transfected with negative control (NC) small interfering (Si) RNA (Si-NC), Si-circ_0008450, micro RNA (miR)-NC, miR-1294 mimics, anti-miR-1294, anti-Si-circ_0008450, or anti-miR-NC. The activity of HSC3 cells was detected with the CCK-8 assay. The transwell assay was used to assess the invasion and migration of HSC3 cells. The targeted relationship between miR-1294 and CIRc_0008450 was detected with the dualluciferase report assay. Western blot analysis was performed to measure the protein expression levels of matrix metalloproteinase (MMP)-2, MMP-9, and Ki-67 (also known as MKI67). As compared to neighboring non-cancerous tissues, the expression levels of circ_0008450 in OSCC tissues were significantly increased (P < 0.05), while miR-1294 levels were significantly decreased (P < 0.05). As compared to the Si-NC group, cell activity was significantly decreased (P < 0.05), the numbers of migrating and invading cells were significantly decreased (P < 0.05), and the protein levels of Ki-67, MMP-2, and MMP-9 were significantly decreased (P < 0.05) in the Si-circ_0008450 group. The results of the double luciferase assay confirmed targeted binding of circ_0008450 to miR-1294. As compared to the miR-NC group, cell activity was significantly decreased (P < 0.05) in the miR-1294 group, as were the numbers of migrating and invading cells (P < 0.05), and the protein levels of Ki-67, MMP-2, and MMP-9 (P < 0.05). As compared to the Si-circ_0008450+anti-miR-NC group, cell activity was significantly increased (P < 0.05) in the Si-circ_0008450+anti-miR-1294 group, as were the numbers of migrating and invading cells (P < 0.05), and the protein levels (P < 0.05) of Ki-67, MMP-2, and MMP-9. Interference with circ_0008450 reduced the invasion, migration, and proliferation of OSCC cells on account of upregulation via binding with miR-1294.

YAP/TAZ activation, induced by disruption of E-cadherin-mediated cell-to-cell contact, promotes the cadherin switch by facilitating nuclear translocation of Slug in human oral squamous cell carcinoma HSC-4 cells

YAP/TAZ activation, induced by disruption of E-cadherin-mediated cell-to-cell contact, promotes the cadherin switch by facilitating nuclear translocation of Slug in human oral squamous cell carcinoma HSC-4 cells

Antitumor Effects of Fucoidan Via Apoptotic and Autophagic Induction on HSC-3 Oral Squamous CellCarcinoma.

Objective:Many studies suggested that fucoidan has anticancer potential. The objective of the present study was to determine the cytotoxic effects and mechanism of cell death induced by fucoidan extracted from Fucus vesiculosus on HSC-3 oral squamous cell carcinoma. Methods:HSC-3 cells were treated with 0, 100, 200, and 400 μg/mL of fucoidan. Cell viability was measured using MTT assay. Apoptosis and cell cycle were measured with a flow cytometry-based assay. Chromatin condensation and nuclear fragmentation were determined using Hoechst 33342 staining. Mitochondrial membrane potential (ΔΨm) was determined using the JC-1 kit. The apoptotic, anti-apoptotic, and autophagic markers study were done by western blot analysis. Results:the viable cell number of treated HSC-3 cells was decreased. Moreover, treated cells were arrested in the G0/G1 phase. Annexin V/PI staining revealed that fucoidan could induce apoptosis in HSC-3 cells. Western blot analysis suggested the up-regulation of apoptotic markers including cleaved caspase-3, cleaved PARP, Bax, and autophagic markers including LC3-II and Beclin-1 but down-regulation of anti-apoptotic markers, Bcl-2. Fucoidan could disturb ΔΨm and induce chromatin condensation with nuclear fragmentation. Conclusion:fucoidan has potential in anticancer properties against HSC-3 cells manifested by the induction of apoptosis, cell cycle arrest, and autophagy.

AIRE is induced in oral squamous cell carcinoma and promotes cancer gene expression.

Autoimmune regulator (AIRE) is a transcriptional regulator that is primarily expressed in medullary epithelial cells, where it induces tissue-specific antigen expression. Under pathological conditions, AIRE expression is induced in epidermal cells and promotes skin tumor development. This study aimed to clarify the role of AIRE in the pathogenesis of oral squamous cell carcinoma (OSCC). AIRE expression was evaluated in six OSCC cell lines and in OSCC tissue specimens. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was elevated in 293A cells stably expressing AIRE, and conversely, was decreased in AIRE-knockout HSC3 OSCC cells when compared to the respective controls. Upregulation of STAT1, and ICAM in OSCC cells was confirmed in tissue specimens by immunohistochemistry. We provide evidence that AIRE exerts transcriptional control in cooperation with ETS1. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was increased in 293A cells upon Ets1 transfection, and coexpression of AIRE further increased the expression of these proteins. AIRE coprecipitated with ETS1 in a modified immunoprecipitation assay using formaldehyde crosslinking. Chromatin immunoprecipitation and quantitative PCR analysis revealed that promoter fragments of STAT1, ICAM1, CXCL10, and MMP9 were enriched in the AIRE precipitates. These results indicate that AIRE is induced in OSCC and supports cancer-related gene expression in cooperation with ETS1. This is a novel function of AIRE in extrathymic tissues under the pathological condition.

Protein kinase D1 regulates the growth and metabolism of oral squamous carcinoma cells in tumor microenvironment

To observe the effect of protein kinase D1 (PKD1) on the growth and metabolism of oral squamous cell carcinoma HSC-4 cells and related molecular mechanisms in the tumor microenvironment. HSC-4 cell lines were transfected with shRNA plasmids. Three groups (Wild, control-shRNA, and PKD1-shRNA) were cultured under acidic or hypoxic environment for a certain time. Western blot was used to detect the expression of autophagy-related and glycolytic-related proteins. The proliferation changes were detected by CCK-8 kits. The PKD1-knockdown HSC-4 cell line was established. PKD1 silencing increased autophagy activity. Under hypoxic and acidic conditions, the PKD1-knockdown HSC-4 cells showed lower proliferation than the parental cells. PKD1-knockdown also decreased the expression of hypoxia induciblefactor 1α (HIF-1α) and pyruvate kinase M2 (PKM2). Under hypoxic and acidic conditions, PKD1 gene silencing can increase apoptotic autophagy activity. Downregulated PKD1 gene expression can reduce the glycolysis of oral squamous cell carcinoma cells and inhibit tumor cell proliferation. This study revealed the important role of PKD1 in the metabolism and growth of oral squamous cell carcinoma, making it a possible target for the treatment of oral squamous cell carcinoma.

Targeting FAT1 Inhibits Carcinogenesis, Induces Oxidative Stress and Enhances Cisplatin Sensitivity through Deregulation of LRP5/WNT2/GSS Signaling Axis in Oral Squamous Cell Carcinoma.

FAT atypical cadherin 1 (FAT1) regulates cell-cell adhesion and extracellular matrix architecture, while acting as tumor suppressor or oncogene, context-dependently. Despite implication of FAT1 in several malignancies, its role in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we document the driver-oncogene role of FAT1, and its mediation of cell-death evasion, proliferation, oncogenicity, and chemoresistance in OSCC. In-silica analyses indicate FAT1 mutations are frequent and drive head-neck SCC, with enhanced expression defining high-risk population and poor prognosis. We demonstrated aberrant FAT1 mRNA and protein expression in OSCC compared with non-cancer tissues, whereas loss-of-FAT1-function attenuates human primary SAS and metastatic HSC-3 OSCC cell viability, without affecting normal primary human gingival fibroblast cells. shFAT1 suppressed PCNA and upregulated BAX/BCL2 ratio in SAS and HSC-3 cells. Moreover, compared with wild-type cells, shFAT1 concomitantly impaired HSC-3 cell migration, invasion, and clonogenicity. Interestingly, while over-expressed FAT1 characterized cisplatin-resistance (CispR), shFAT1 synchronously re-sensitized CispR cells to cisplatin, enhanced glutathione (GSH)/GSH synthetase (GSS)-mediated oxidative stress and deregulated LRP5/WNT2 signaling. Concisely, FAT1 is an actionable driver-oncogene in OSCC and targeting FAT1 in patients with erstwhile cisplatin-resistant OSCC is therapeutically promising.

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.

Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Ouabain signaling in oral squamous cell carcinoma cells.

Accurate evaluation of the anti-cancer effects of ouabain, a cardiac glycoside, requires an understanding of its signaling pathway. This study examined the effects of ouabain stimulation on spontaneous interleukin (IL)-8 and IL-1α secretion in the HSC3 oral squamous cell carcinoma cell line. IL-8 secretion was reduced and IL-1α secretion was increased in the cells. Real-time polymerase chain reaction confirmed that these changes were regulated at the transcriptional level. Further analysis revealed that ouabain stimulation induced phosphorylation of activator protein (AP)-1 components (c-Jun and c-Fos) but not nuclear factor kappa B (NF-κB) components (p65 and p50). A luciferase assay demonstrated that the NF-κB-binding site located at 1 kb upstream of the TATA box in the IL-8 gene contributed to the reduction in IL-8 secretion. Pre-incubation of the cells with BAPTA-AM and L-glutathione increased IL-8 secretion, which indicates that Ca2+ ions and reactive oxygen species are associated with the ouabain-mediated reduction in IL-8 levels. The inhibitory effect of ouabain was attributed to reduced nuclear translocation of the NF-κB p65 subunit. Taken together, these findings indicate that ouabain exerts opposing effects on transcription factors NF-κB and AP-1.

Anti-invasive and Anti-tumor Effects of Dryopteris crassirhizoma Extract by Disturbing Actin Polymerization.

Aim. To evaluate the anti-invasive effect of ethanol extracts of rhizome of Dryopteris crassirhizoma (EEDC) in matrix invasion and formation of functional invadopodia and to determine the anti-tumor effect of EEDC in a mouse model of mandibular invasion by gingival squamous cell carcinoma (SCC). Methods. The rhizome of D crassirhizoma was extracted in ethanol. The anti-invasive effect of EEDC was analyzed with a Matrigel-coated transwell invasion and 3D culture system. Crucial factors related to the control of cancer cell invasion by EEDC were determined using a human protease array. Molecular evidence supporting the anti-invasive effect of EEDC in oral SCC (OSCC) cells used an invadopodia-mediated extracellular matrix (ECM) degradation; an in vivo athymic mouse model was also provided. Results. EEDC treatment (10 µg/mL) suppressed transwell migration and invasion of HSC-3 OSCC cells without cytotoxicity. Decreased levels of matrix metalloprotease (MMP)-7, kalikrein 10, cathepsin V, MMP-2, and cathepsin D were also found in EEDC-treated HSC-3 cells based on human protease array. The anti-invasive effects of EEDC involved the suppression of invadopodia-mediated ECM degradation via inhibition of globular-actin elongation. The anti-invasive effect resulting from disturbance of functional invadopodia formation by EEDC was observed even at a low concentration of 5 µg/mL. The phosphorylation of cortactin involved in functional invadopodia formation was decreased at EEDC concentrations that inhibited invadopodia formation. The anti-tumor effect of EEDC was also observed in a mouse xenograft model. Administration of EEDC resulted in inhibition of tumor growth and progression. Conclusions. EEDC represents a potential anti-invasive and anti-tumor agent in cancer control.

<b>Circadian Expression of Differentiated Embryonic Chondrocytes Expressed Genes 1 </b><b>and 2 in Human Oral Squamous Cell Carcinoma HSC-3 Cells </b>

<b>Circadian Expression of Differentiated Embryonic Chondrocytes Expressed Genes 1 </b><b>and 2 in Human Oral Squamous Cell Carcinoma HSC-3 Cells </b>

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.

Mecambridine induces potent cytotoxic effects, autophagic cell death and modulation of the mTOR/PI3K/Akt signaling pathway in HSC-3 oral squamous cell carcinoma cells.

Plant secondary metabolites including alkaloids, demonstrate a complex diversity in their molecular scaffolds and exhibit tremendous pharmacological potential as anti-cancerous therapeutics. The present study aimed to evaluate the anticancer activity of a natural alkaloid, mecambridine, against human oral squamous cell carcinoma (OSCC). An MTT assay was used to evaluate cytotoxic effects of mecambridine on HSC-3 oral squamous cell carcinoma cells. Effects of mecambridine on autophagy-associated proteins were analyzed by western blotting. Effects on reactive oxygen species (ROS) and mitochondrial membrane potential were assessed by flow cytometry. Results indicated that mecambridine exhibited an IC50 value of 50 µM and exerted its cytotoxic effects in a dose dependent manner on OSCC HSC-3 cells. Furthermore, it was observed that mecambridine decreases cell viability and induces autophagy in a dose-dependent manner. The underlying mechanism for the induction of autophagy was demonstrated to be associated with ROS-mediated alterations in mitochondrial membrane potential and modulation of the mechanistic target of rapamycin/phosphoinositide 3-kinase/protein kinase B (m-TOR/PI3K/Akt) signaling pathway in HSC-3 at the IC50. In conclusion, the present study suggests that mecambridine exhibits substantial anticancer activity against OSCC HSC-3 cells by induction of autophagy and modulates the expression of the mTOR/PI3K/Akt signaling cascade which is considered a potential target pathway for anti-cancer agents.

Pipoxolan Exhibits Antitumor Activity Toward Oral Squamous Cell Carcinoma Through Reactive Oxygen Species-mediated Apoptosis.

Pipoxolan is frequently prescribed as a smooth muscle relaxant. Pipoxolan has also been shown to have anticancer activity. Our study investigated whether pipoxolan induced apoptosis in oral squamous cell carcinoma (OSCC). Cell cytotoxicity was evaluated by the MTT assay. Cell apoptosis and cell-cycle distribution were measured by annexin V/propidium iodide (PI) double staining and flow cytometry, respectively. Apoptotic-related proteins were assessed by western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Following exposure of TW206 OSCC cells to pipoxolan, a time-dependently decrease in MMP and an increase in ROS were observed. However, these effects were significantly abrogated by the free radical scavenger N-acetyl-L-cysteine. Since high levels of ROS were produced early in the treatment, intracellular ROS seemed to play a key role in pipoxolan-induced apoptosis. In HSC-3 OSCC cells, our results demonstrated that pipoxolan treatment caused a time-dependent increase of protein expression of active caspase-3 and -9, cytosolic cytochrome c, cleavage of poly (ADP-ribose) polymerase, and B-cell lymphoma 2 (BCL2)-like protein 4 (BAX). However, expression of BCL2 itself was reduced. Clearly, such an increase in BAX/BCL2 ratio would be associated with apoptosis. In addition, pipoxolan markedly suppressed the protein expression of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and phosphorylation of protein kinase B (AKT). These data suggest that pipoxolan acts against HSC-3 in vitro via intrinsic apoptotic signaling pathways, and inhibition of PI3K/AKT signaling.

Abstract 803: Targeting β-catenin/CBP signaling in OSCC

Abstract Objectives: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype. Methods: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24hiCD29hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24lowCD29low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice. Results: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24hiCD2hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24lowCD29low. Treatment of CD44+CD24hiCD29hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy. Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000. Citation Format: Khalid Alamoud, Khikmet Sadykov, Vinay Kartha, Stefano Monti, Anna Belkina, Jennifer Snyder-Cappione, Sara Pai, Maria Kukuruzinska. Targeting β-catenin/CBP signaling in OSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2017-803