Apoptosis Of Oral Squamous Cell Carcinoma Cell Lines Research Articles

Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Ascomycota, Lecanoromycetes), lichenized fungus on oral squamous cell carcinoma cell line; in-vitro study

BackgroundScientists and medical professionals are actively striving to improve the efficacy of treatment methods for oral squamous cell carcinoma (OSCC), the most frequently occurring cancer within the oral cavity, by exploring the potential of natural products. The active pharmacological compounds found in lichenized fungi have shown potential for aiding in cancer treatment. Recent research aims to evaluate the impact of the lichenized fungus Ramalina sinensis (R. sinensis) on the cell viability and apoptosis of OSCC cell lines, considering the anti-inflammatory and anti-cancer capabilities of lichens.MethodsRamalina sinensis (Ascomycota, Lecanoromycetes) was selected for investigation of its effects on a human oral squamous cell carcinoma cell line. Acetone and methanol extracts of R. sinensis on an OSCC cell line (KB cell line, NCBI Code: C152) were investigated. Viability was assessed by MTT assay analysis, and apoptotic cells were measured using flow cytometry analysis. Scratch assay was used to assess cell migration. The chemical composition and metabolic profiling of R. sinensis were investigated.ResultsThe growth and multiplication of KB cells were observed to undergo a gradual but remarkable inhibition when exposed to various concentrations. Specifically, concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg/mL exhibited a significant suppressive effect on the proliferation of KB cells. The inhibition of cell proliferation exhibited a statistically significant difference between the extracts obtained from acetone and methanol. Flow cytometry results show an increase in apoptosis of OSCC cells by acetone extract. R. sinensis exerted a concentration-dependent inhibitory effect on the migration of OSCC cells. The chemical composition of R. sinensis was investigated using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS), and 33 compounds in the acetone and methanol extracts of R. sinensis were detected.ConclusionThe findings provide evidence supporting the beneficial effects of R. sinensis extract on inducing apoptosis in OSCC cells and exerting anti-cancer properties.

Chrysin promotes Cisplatin-induced apoptosis via oxidative DNA damage in oral squamous cell carcinoma

Chrysin is an apigenin analog with high therapeutic potential by modulation of apoptotic signaling pathways, improving conventional chemotherapy's effectiveness. In this study, we introduced novel evidence that the Chrysin effect with Cisplatin intensely induced cell death through DNA damage in SCC-25 and CAL-27 of oral squamous cell carcinoma (OSCC). The result of Chrysin and Cisplatin alone or in combination with cell proliferation was assessed using the MTT assay. The expression levels of 8-Hydroxy-2′-deoxyguanosine (8-OXO-dG) were analyzed using an enzyme-linked immunosorbent assay (ELISA). DCFH-DA fluorescence dye was utilized to measure reactive oxygen species (ROS) formation. ELISA cell death and caspase 8 activity assays were used to measure the apoptosis rate in these cells. Chrysin significantly stimulates the cytotoxic activity of Cisplatin. Besides, the co-treatment of Chrysin plus Cisplatin significantly increased the expression levels γ-H2AX in both cell lines. Besides, this combined treatment considerably improved ROS levels and significantly down-regulated the antioxidant enzyme expression. Moreover, Chrysin treatment led to the enhancement of Cisplatin-induced apoptosis in OSCC cell lines when compared to either Chrysin or Cisplatin treated cells. Chrysin and Cisplatin co-treatment improved cytotoxicity via the increased levels of ROS production, which results in oxidative DNA damage and leads to potent apoptosis induction in tumor cells. Generally, this co-treatment could propose a therapeutic approach that hopefully promotes patients' clinical outcomes of OSCC. These data confirm the chemo-sensitizer effect of Chrysin in OSCC cells.

Exploring the Cytotoxic Effects of the Extracts and Bioactive Triterpenoids from Dillenia indica against Oral Squamous Cell Carcinoma: A Scientific Interpretation and Validation of Indigenous Knowledge.

Triterpenoids are ubiquitously distributed secondary metabolites, primarily scrutinized as a source of medication and preventive measures for various chronic diseases. The ease of isolation and excellent pharmacological properties of triterpenoids are notable reasons behind the exponential rise of extensive research on the bioactive triterpenoids over the past few decades. Herein, we attempted to explore the anticancer potential of the fruit extract of the ethnomedicinal plant Dillenia indica against oral squamous cell carcinoma (OSCC) and have exclusively attributed the efficacy of the extracts to the presence of two triterpenoids, namely, betulinic acid (BA) and koetjapic acid (KA). Preliminary in vitro screening of both BA and KA unveiled that the entities could impart cytotoxicity and induce apoptosis in OSCC cell lines, which were further well-supported by virtual screening based on ligand binding affinity and molecular dynamic simulations. Additionally, the aforementioned metabolites could significantly modulate the critical players such as Akt/mTOR, NF-κB, and JAK/STAT3 signaling pathways involved in the regulation of important hallmarks of cancer like cell survival, proliferation, invasion, angiogenesis, and metastasis. The present findings provide insight and immense scientific support and integrity to a piece of indigenous knowledge. However, in vivo validation is a requisite for moving to clinical trials and developing it as a commercial drug.

Long noncoding RNA PVT1 promotes tumor cell proliferation, invasion, migration and inhibits apoptosis in oral squamous cell carcinoma by regulating miR‑150‑5p/GLUT‑1.

Oral squamous cell carcinoma (OSCC) is a cancer with high morbidity and mortality. Research has demonstrated that long non-coding RNAs (lncRNAs) are critical for tumor initiation and development. In the present study, we aimed to ascertain the functions and potential mechanisms of lncRNA plasmacytoma variant translocation 1 (PVT1) in OSCC. Firstly, we found that the expression of PVT1 was increased in human OSCC tumor tissues and it was related to reduced survival of the patients. Furthermore, miR-150-5p expression was downregulated in OSCC tumor tissues and it was negatively related with PVT1. Moreover, GLUT-1 protein expression was upregulated in human OSCC tumor tissues. In addition, cell proliferation capacity was measured by CCK-8 assay and cell invasion and migration were measured by Transwell assay. PVT1 overexpression promoted cell proliferation, invasion and migration, while these effects were abrogated by PVT1 downregulation. In addition, luciferase gene reporter assay verified the miR-150-5p directly binds with PVT1, which regulates the biological functions of OSCC. Additionally, luciferase gene reporter assay confirmed that GLUT-1 was a target for miR-150-5p. The promotion of cell proliferation, invasion and migration in LV-PVT1-transfected cells was eliminated following miR-150-5p overexpression. Finally, in vivo nude mouse xenograft model further verified that PVT1 knockdown inhibited tumor growth, formation, invasion and migration. According to the results, PVT1 is increased in human OSCC tumor tissues, and is related to the poor prognosis of human OSCC patients. We uncovered a previously unappreciated PVT1/miR-150-5p/GLUT-1 signaling axis that promotes cell proliferation, invasion, migration and inhibits apoptosis in OSCC cell lines and in vivo, which suggests that this axis could be a target for the treatment of OSCC.

CCHE1 accelerated the initiation of oral squamous cell carcinoma through enhancing PAK2 expression by sponging miR-922.

Oral squamous cell carcinoma (OSCC) is a normal form of mouth cancer, comprising the majority of oral cancers. A large number of long non-coding RNAs (lncRNAs) have been reported due to their oncogenic function in cancers. Recent studies show that lncRNA CCHE1 is an oncogene in a wide range of cancers. Whether CCHE1 accelerates the progression of OSCC is still undiscovered. The qRT-PCR analysis was used to determine CCHE1, miR-922, and PAK2 expression levels. CCK8 and colony formation assays were applied to evaluate OSCC cell proliferative ability. Transwell assay was performed to investigate the capability of cell migration and invasion. Cell apoptosis was assessed by flow cytometry analysis. The distribution of CCHE1 in OSCC cells was confirmed via subcellular fractionation assay. Luciferase reporter assay was used to verify the connection between miR-922 and CCHE1 or PAK2. qRT-PCR analysis identified the upregulation of CCHE1 in OSCC cells. Knockdown of CCHE1 curbed the proliferation, migration, and invasion and hastened the apoptosis in OSCC cell lines. Moreover, it was found that miR-922 could interact with CCHE1. Besides, PAK2 was identified as the target gene of miR-922 and its expression was negatively modulated by miR-922 and positively regulated by CCHE1. Restoration assay manifested that the suppressing influence of CCHE1 depletion on OSCC progression was rescued by amplified PAK2. CCHE1 increases the expression of PAK2 to promote the progression of OSCC by competitively binding to miR-922 in OSCC cells.

Hsa_circRNA_100533 regulates GNAS by sponging hsa_miR_933 to prevent oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck region. Circular RNA (circRNA), as one kind of noncoding RNA, involves in biological processes in diverse cancers. circRNA functions mainly as the microRNA (miRNA) sponge, competitively binding to miRNAs to regulate target gene expressions. However, the expression profiles and roles of circRNAs in OSCC are still unexplored. circRNA microarrays and quantitative real-time polymerase chain reaction was used to identify the hsa_circRNA_100533 downregulated in OSCC tissues and cell lines. Bioinformatics methods were used to predict the interactions among circRNAs, miRNA, and target genes. Based on the luciferase reporter assay and AGO2 RIP assay, we found that hsa_circRNA_100533 binds to miRNAs as a miRNA sponge. hsa_circRNA_100533 inhibited cell proliferation, migration, and promoted cell apoptosis in OSCC cell lines, which could be blocked by hsa-miR-933 overexpression. hsa_circRNA_100533 binds to hsa-miR-933 as a miRNA sponge to regulate GNAS expression, and to modulate cell proliferation, migration, and apoptosis. In summary, the hsa_circRNA_100533-miR-933-GNAS axis affect the proliferation and apoptosis of OSCC cells through the mechanism of competing endogenous RNAs. hsa_circRNA_100533 may function as promising diagnostic biomarkers and effective therapeutic targets for OSCC.

LncRNA MEG3 suppresses migration and promotes apoptosis by sponging miR-548d-3p to modulate JAK-STAT pathway in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a lethal malignancy and its prognosis remains dismal. Thus, a deeper understanding of the mechanisms is needed to provide a new insight for new therapies. It has been reported that long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) was downregulated in OSCC tissues, however, its functional mechanism remains uncertain. Here, we found that the overexpression of MEG3 suppressed migration and promoted apoptosis in OSCC cell lines, while inhibition of MEG3 exhibited opposite effect. We also found that MEG3 could effectively sponge miR-548d-3p and decrease its expression level. Moreover, miR-548d-3p repressed the expression of SOCS5 and SOCS6 through binding their 3'UTR, thereby modulating the JAK-STAT signaling pathway and functioning as an oncogene in OSCC cells. Importantly, overexpression of MEG3 enhanced the expression of SOCS5 and SOCS6 to regulate JAK-STAT pathway, whereas miR-548d-3p overexpression decreased the effects of MEG3 on levels of SOCS5/SOCS6. Furthermore, upregulated expression of miR-548d-3p could abrogate the effect of MEG3 overexpression on migration and apoptosis in OSCC cell lines. In addition, the overexpression of MEG3 inhibited tumor migration and facilitated apoptosis in vivo. Together, our results revealed that MEG3 could modulate JAK-STAT pathway via miR-548d-3p/SOCS5/SOCS6 to suppresses migration and promote apoptosis in OSCC. Our research indexed a new functional mechanism of MEG3 in OSCC, and this mechanism may be a potential prognostic factor and therapeutic target. © 2019 IUBMB Life, 2019.

Cell division cycle 7 is a potential therapeutic target in oral squamous cell carcinoma and is regulated by E2F1.

Cell division cycle 7 (Cdc7) plays important roles in the regulation of the initiation of DNA replication throughout S phase. Whether inhibition of Cdc7 has a direct antitumour effect in oral squamous cell carcinoma (OSCC) remains unclear. In this study, XL413, a novel Cdc7 inhibitor, markedly inhibited the viability of OSCC cells but not that of non-tumour primary cells. There was a synergistic effect between XL413 and DNA-damaging agents (e.g. cisplatin and 5-fluorouracil) on OSCC in vitro and in vivo. Moreover, XL413 exhibited a notable antitumour effect on OSCC patients with high Cdc7 expression in mini patient-derived xenografts model. The proliferation was significantly inhibited in OSCC cells after Cdc7 silencing. Cdc7 knockdown significantly induced apoptosis in OSCC cell lines. Furthermore, we demonstrated that Cdc7 was overexpressed and transcriptionally regulated by E2F1 in OSCC by using chromatin immunoprecipitation and luciferase assays. Our results reveal that XL413 has an excellent antitumour effect in OSCC. Importantly, it does not inhibit the proliferation of non-tumour cells. These findings suggest that the overexpression of Cdc7 promotes progression in OSCC and that inhibition of Cdc7 is a very promising therapy for OSCC patients.

Role of c-Met in the progression of human oral squamous cell carcinoma and its potential as a therapeutic target.

Mesenchymal-epithelial transition factor (c-Met) is the only high affinity receptor for hepatocyte growth factor (HGF), and is frequently activated in many human cancers. However, little is known about the role of the HGF/c-Met signaling pathway in the progression of human oral squamous cell carcinoma (OSCC). This study evaluated the role of the HGF/c-Met signaling pathway in the progression of human OSCC. We found that the expression of c-Met was significantly increased in human OSCC tissues than in normal mucosa adjacent to the tumor (P<0.05), but was not correlated with clinicopathological parameters. Additionally, the selective c-Met inhibitor JNJ was found to inhibit cell viability and migration and promote apoptosis in OSCC cell lines, and also blocked the activation AKT, ERK1/2, and NF-κB p65; thus, suggesting that HGF/c-Met signaling may play an important role in the tumorigenic properties of OSCC cells via the AKT, ERK1/2, and NF-κB pathways. Collectively, these results indicated that HGF/c-Met signaling may serve essential roles in the progression of human OSCC, and may thus be a basis for the development of novel therapeutic approaches in the treatment of OSCC.

Long non-coding RNA UCA1 contributes to the progression of oral squamous cell carcinoma by regulating the WNT/β-catenin signaling pathway.

With the development of functional genomics studies, a mass of long non‐coding RNAs (LncRNA) were discovered from the human genome. Long non‐coding RNAs serve as pivotal regulators of genes that are able to generate LncRNA–binding protein complexes to modulate a great number of genes. Recently, the LncRNA urothelial carcinoma‐associated 1 (UCA1) has been revealed to be dysregulated, which plays a critical role in the development of a few cancers. However, the role of the biology and clinical significance of UCA1 in the tumorigenesis of oral squamous cell carcinoma (OSCC) remain unknown. We found that UCA1 expression levels were upregulated aberrantly in tongue squamous cell carcinoma tissues and associated with lymph node metastasis and TNM stage. We explored the expression, function, and molecular mechanism of LncRNA UCA1 in OSCC. In the present work, we revealed that UCA1 silencing suppressed proliferation and metastasis and induced apoptosis of OSCC cell lines in vitro and in vivo, which might be related to the activation level of the WNT/β‐catenin signaling pathway. Our research results emphasize the pivotal role of UCA1 in the oncogenesis of OSCC and reveal a novel LncRNA UCA1–β‐catenin–WNT signaling pathway regulatory network that could contribute to our understanding in the pathogenesis of OSCC and assist in the discovery of a viable LncRNA‐directed diagnostic and therapeutic strategy for this fatal disease.

Metformin in combination with 5-fluorouracil suppresses tumor growth by inhibiting the Warburg effect in human oral squamous cell carcinoma.

Cancer cells show enhanced glucose consumption and lactate production even in the presence of abundant oxygen, a phenomenon known as the Warburg effect, which is related to tumor proliferation, progression and drug-resistance in cancers. Hypoxia-inducible factor-1 (HIF-1) and several members of Phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway positively contribute to the Warburg effect, whereas AMP activated protein Kinase (AMPK) acts as a negative regulator. Targeting the regulator molecules of Warburg effect might be a useful strategy to effectively kill cancer cells. Metformin was reported to be effective against various cancers as it inhibits cell proliferation by activating AMPK, and inhibiting mTOR and HIF-1α. Several studies suggested the efficacy of metformin with 5-fluorouracil (5-FU) against esophageal and colon cancer. In this study, we evaluated the efficacy of metformin and 5-FU combined therapy against human oral squamous cell carcinoma (OSCC) invitro and invivo. MTT assay and TUNEL assay revealed that metformin (4 mg/ml) and 5-FU (2.5µg/ml) combination treatment effectively inhibited cell growth and induced apoptosis in OSCC cell lines (HSC2, HSC3 and HSC4) compared to either agent alone. Lactate colorimetric assay detected decreased level of lactate in the supernatants of metformin and 5-FU treated cells compared to cells treated with metformin or 5-FU. Western blot analysis showed marked downregulation of HIF-1α and mTOR expression, and upregulation of AMPKα in cells treated with metformin and 5-FU combination treatment. Combination therapy with metformin (200mg/kg, i.p.) and 5-FU (10mg/kg, i.p.) for 4weeks (5days/week) effectively reduced HSC2 tumor growth (77.6%) compared to metformin (59.9%) or 5-FU (52%) alone in nude mice. These findings suggest that metformin and 5-FU combined therapy could exert strong antitumor effect against OSCC through the inhibition of Warburg phenomenon in tumor cells.

Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs.

Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines.

Plumbagin (PL), a naturally occurring quinoid, exerts antitumoral effects in diverse types of cancer cells. However, the effect of PL on tumor cell proliferation in oral squamous cell carcinoma (OSCC) remains poorly understood. In this study, we assessed the efficacy of PL, in human OSCC cells. The effect of PL on the cell growth and apoptosis of OSCC cell lines was evaluated using MTT and Annexin V assays, respectively. The effect of PL on mitochondrial membrane potential loss and reactive oxygen species (ROS) generation was evaluated using flow cytometry analysis. MTT assay showed that PL dose-dependently suppressed OSCC cell growth, with IC50 values ranging from 3.87 to 14.6 μM. Flow cytometry analysis revealed that PL treatment resulted in a significant decrease in mitochondrial membrane potential and an increase in the number of apoptotic cells. Notably, ROS generation was significantly elevated after PL treatment. Furthermore, a ROS scavenger, N-acetylcysteine (NAC), clearly suppressed the decrease in mitochondrial membrane potential, increase of caspase-3/7 activity, and apoptosis after PL treatment. This study provides the considerable evidence of the tumor-suppressive effect of PL, thereby highlighting its therapeutic potential for OSCC treatment.

Tivantinib (ARQ-197) exhibits anti-tumor activity with down-regulation of FAK in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and the 5 years survival rate of the patients is about 60% in the USA, due to acquired chemotherapeutic resistance and metastasis of the disease. In this study, we found that tivantinib, a selective MET inhibitor, suppresses OCSS cell proliferation and colony formation, however, anti-tumor activities induced by tivantinib are independent of the inhibition of MET signaling pathway. In addition, tivantinib cause G2/M cell cycle arrest and caspases-dependent apoptosis in OSCC cell lines. We also found that tivantinib dose-dependently suppressed the activation and expression of FAK. In all, these data suggested that tivantinib may be developed as a chemotherapeutic agent to effectively treat certain cancers including OSCC.