Dihydroartemisinin Induces ER Stress-Mediated Apoptosis in Human Tongue Squamous Carcinoma by Regulating ROS Production.

BACKGROUND: Tongue squamous cell carcinoma is a fatal disease characterized by high invasion and early metastasis. Dihydroartemisinin, an antimalarial drug with multiple biological activities, is reported to be a potential anti-cancer agent. OBJECTIVE: This study aimed to evaluate the antitumor effect of Dihydroartemisinin on tongue squamous cell carcinoma cells, and to identify the underlying mechanisms of Dihydroartemisinin-induced cell apoptosis. METHODS: Here, Cell Counting Kit 8 assay and colony formation assay were conducted to study cell proliferation. Annexin V-FITC/propidium iodide staining and western blot analysis were performed to analyze cell apoptosis. DCFHDA probe was used to measure the generation of cellular reactive oxygen species. Endoplasmic reticulum stress activation was also determined via western blot analysis. RESULTS: The results showed that Dihydroartemisinin substantially inhibited cell proliferation and induced cell apoptosis in vivo. Moreover, reactive oxygen species production and endoplasmic reticulum stress activation were both observed after stimulation with Dihydroartemisinin. However, the reactive oxygen species inhibitor N-acetylcysteine significantly alleviated Dihydroartemisinin-induced endoplasmic reticulum stress and apoptosis. CONCLUSION: These results imply that Dihydroartemisinin induced cell apoptosis by triggering reactive oxygen speciesmediated endoplasmic reticulum stress in CAL27 cells. In addition, Dihydroartemisinin might be an effective drug for tongue squamous cell carcinoma therapy.

Identification of biomarkers related to glycolysis with weighted gene co-expression network analysis in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common tumor in the oral cavity and maxillofacial region. Increasing evidence suggests that aerobic glycolysis plays an important role in the occurrence, development, and prognosis of OSCC. Therefore, the identification of biomarkers related to glycolysis in OSCC represents considerable potential for improving its treatment. METHODS: In the present study, a single-sample gene-set enrichment analysis (ssGSEA) algorithm with weighted gene co-expression network analysis (WGCNA) were used to quantify the degree of glycolysis and identify key modules with the greatest correlation with glycolysis. RESULTS: Glycolytic scores significantly correlated with prognosis. In the key module 5 HUB genes were finally selected, which displayed a robust predictive effect. The expressions of key genes were associated with glycolysis. CONCLUSIONS: The research comprehensively analyzed the glycolysis of OSCC and identified several biomarkers related to glycolysis. These biomarkers may represent potential therapeutic targets for future OSCC therapy.

CDC2 Is an Important Driver of Vascular Smooth Muscle Cell Proliferation via FOXM1 and PLK1 in Pulmonary Arterial Hypertension.

A key feature of pulmonary arterial hypertension (PAH) is the hyperplastic proliferation exhibited by the vascular smooth muscle cells from patients (HPASMC). The growth inducers FOXM1 and PLK1 are highly upregulated in these cells. The mechanism by which these two proteins direct aberrant growth in these cells is not clear. Herein, we identify cyclin-dependent kinase 1 (CDK1), also termed cell division cycle protein 2 (CDC2), as having a primary role in promoting progress of the cell cycle leading to proliferation in HPASMC. HPASMC obtained from PAH patients and pulmonary arteries from Sugen/hypoxia rats were investigated for their expression of CDC2. Protein levels of CDC2 were much higher in PAH than in cells from normal donors. Knocking down FOXM1 or PLK1 protein expression with siRNA or pharmacological inhibitors lowered the cellular expression of CDC2 considerably. However, knockdown of CDC2 with siRNA or inhibiting its activity with RO-3306 did not reduce the protein expression of FOXM1 or PLK1. Expression of CDC2 and FOXM1 reached its maximum at G1/S, while PLK1 reached its maximum at G2/M phase of the cell cycle. The expression of other CDKs such as CDK2, CDK4, CDK6, CDK7, and CDK9 did not change in PAH HPASMC. Moreover, inhibition via Wee1 inhibitor adavosertib or siRNAs targeting Wee1, Myt1, CDC25A, CDC25B, or CDC25C led to dramatic decreases in CDC2 protein expression. Lastly, we found CDC2 expression at the RNA and protein level to be upregulated in pulmonary arteries during disease progression Sugen/hypoxia rats. In sum, our present results illustrate that the increased expression of FOXM1 and PLK1 in PAH leads directly to increased expression of CDC2 resulting in potentiated growth hyperactivity of PASMC from patients with pulmonary hypertension. Our results further suggest that the regulation of CDC2, or associated regulatory proteins, will prove beneficial in the treatment of this disease.

Cytotoxicity mechanisms of plumbagin in drug-resistant tongue squamous cell carcinoma.

OBJECTIVES: To evaluate the inhibitory effect and mechanism of plumbagin (PLB) against drug-resistant tongue squamous cell carcinoma (TSCC), and whether its antitumour effect is not affected by tumour drug resistance. METHODS: TSCC sensitive CAL27 cells and drug-resistant CAL27/RE cells were used to study the cytotoxicity and mechanism of PLB in vitro, including CCK-8 analysis, colony formation, DAPI staining, flow cytometry assay, transmission electron microscopy, western blotting assay, autophagy, apoptosis and ROS fluorescent probes. BALB/c nude mice xenograft models were used to study the growth inhibitory effect of PLB in vivo. KEY FINDINGS: The results showed that the cell viability and proliferation inhibition and apoptosis induction abilities of PLB on drug-resistant cells were more obvious than that on sensitive cells. And PLB induced protective autophagy in TSCC cells. Mechanistically, PLB induced apoptosis and autophagy by generating reactive oxygen species to mediate JNK and AKT/mTOR pathways. Finally, the growth inhibitory effect of PLB against drug-resistant TSCC was also confirmed in vivo. CONCLUSIONS: PLB will be a promising anticancer agent to overcome drug-resistant TSCC without being affected by its drug resistance properties.

Glaucocalyxin A induces apoptosis and autophagy in tongue squamous cell carcinoma cells by regulating ROS.

PURPOSE: Tongue squamous cell carcinoma (TSCC) is the most common highly invasive oral cancer. Glaucocalyxin A (GLA) is a diterpenoid component isolated from Rabdosia japonica var. with anti-bacterial and anti-cancer biological properties. However, the role of GLA in human TSCC remains uncertain. The aim of this paper was to investigate the anti-cancer effect of GLA on TSCC cells as well as its underlying mechanism. METHODS: Cell viability and growth were analyzed by CCK-8 assay and colony formation, respectively. DAPI staining and flow cytometry assay were used to detect the cell apoptosis. Lysotracker Red staining was used to observe the lysosomes and autolysosomes of TSCC cells. ROS fluorescent probe was used to test the intracellular ROS levels. Western blotting was used to detect the expression levels of apoptosis- and autophagy-related proteins. RESULTS: GLA inhibits the cell viability and growth in TSCC cells. GLA induces TSCC cells apoptosis, autophagy and ROS production in a time- and concentration-dependent manner. In addition, GLA inhibits the viability of TSCC cells by inducing intracellular ROS production. Finally, GLA triggers ROS-dependent apoptosis and autophagy in TSCC cells. CONCLUSION: Our results consistently suggested that GLA can induce apoptosis and autophagy in TSCC cells by generating ROS. GLA may serve as a promising therapeutic drug for overcoming TSCC.

Prognostic significance of an autophagy-related long non-coding RNA signature in patients with oral and oropharyngeal squamous cell carcinoma.

Traditional clinicopathological indices are insufficient in predicting the prognosis of patients diagnosed with oral and oropharyngeal squamous cell carcinoma (OSCC/OPSCC). Notably, autophagy and long non-coding RNAs (lncRNAs) regulate the development and progression of various types of cancer. The present study aimed to assess the association between autophagy-related lncRNAs and the prognosis of patients diagnosed with OSCC/OPSCC. Gene sequencing and clinicopathological data of patients with OSCC/OPSCC were downloaded from The Cancer Genome Atlas database, while gene set functional classification was downloaded from the Gene Set Enrichment Analysis database. Out of the 413 transcriptome data samples and 402 clinicopathological data samples retrieved, a total of nine autophagy-related lncRNAs, including PTCSC2, AC099850.3, LINC01963, RTCA-AS1, AP002884.1, UBAC2-AS1, AL512274.1, MIR600HG and AL354733.3, were screened. This was geared towards establishing a signature through gene co-expression network, univariate and Least Absolute Shrinkage and Selection Operator Cox regression analyses. Based on this signature, the patients were subdivided into a high-risk group and a low-risk group. Kaplan-Meier survival analysis revealed that the overall survival of the high-risk group was significantly lower than that of the low-risk group. Furthermore, principal components analysis demonstrated that the patients diagnosed with OSCC/OPSCC could be distinguished into low-survival and high-survival groups according to the signature. Univariate and multivariate Cox regression analyses of clinicopathological data and the signature revealed that the signature could potentially be used as an independent prognostic factor for OSCC/OPSCC. In addition, reverse transcription-quantitative PCR analysis of clinical samples demonstrated the validity of the signature. In summary, the present study revealed that the signature based on autophagy-related lncRNAs potentially acts as an independent prognostic indicator for patients with OSCC/OPSCC. Furthermore, it promotes research on targeted diagnosis and treatment of patients diagnosed with OSCC/OPSCC.

A competing risk nomogram for predicting cancer-specific death of patients with buccal mucosa cancer.

OBJECTIVES: Our aim was to develop and validate a competing risk nomogram to determine the probability of cancer-specific death in buccal mucosa cancer (BMC) patients. MATERIALS AND METHODS: We examined the records of BMC patients in the Surveillance, Epidemiology, and End Results (SEER) Program and First Affiliated Hospital of Nanchang University (China). We adopted the cumulative incidence function and Fine-Gray proportional hazards model based on univariate and multivariate analyses by R-software to identify the risk factors associated with cancer-specific death. Subsequently, a nomogram was developed and validated to predict the 3- and 5-year probability of cancer-specific death. RESULTS: In 1,286 BMC patients identified from SEER database, cumulative incidences of cancer-specific death after diagnosis were 33.4% and 35.5% for 3 and 5 years, respectively. In the training cohort (n = 902) from SEER database, the Fine-Gray model indicated that age, Tumor Node Metastasis (TNM) stages, grade, surgery, and histological type were independent risk factors associated with cancer-specific death, based on which a prognostic nomogram was developed. In the internal validation cohort from SEER database (n = 384) and the external validation cohort from our medical center (n = 174), the nomogram was well calibrated and showed remarkable prediction performance. CONCLUSION: The nomogram created herein may prove to be a good assistant tool for assessing the prognosis of BMC patients.

Emerging role of NRF2 in ROS-mediated tumor chemoresistance.

Chemoresistance is a central cause for the tumor management failure. Cancer cells disrupt the redox homeostasis through reactive oxygen species (ROS) regulatory mechanisms, leading to tumor progression and chemoresistance. The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of neutralizing cellular ROS and restoring redox balance. Understanding the role of NRF2 in ROS-mediated chemoresistance can be helpful in the development of chemotherapy strategies with better efficiency. In this review, we sum up the roles of ROS in the development of chemoresistance to classical chemotherapy agents including cisplatin, 5-fluorouracil, gemcitabine, oxaliplatin, paclitaxel, and doxorubicin, and how to overcome ROS-mediated tumor chemoresistance by targeting NRF2. Finally, we propose that targeting NRF2 might be a promising strategy to resist ROS-driven chemoresistance and acquire better efficacy in cancer treatment.

Plumbagin Enhances the Anticancer Efficacy of Cisplatin by Increasing Intracellular ROS in Human Tongue Squamous Cell Carcinoma.

Cisplatin is widely used in the treatment of tongue squamous cell carcinoma (TSCC), but its clinical efficacy is limited by drug resistance and toxic side effects. Hence, a novel compound capable of enhancing the anticancer effect of cisplatin while reducing the side effects is urgently needed. We have previously shown that plumbagin (PLB), an anticancer phytochemical, is able to inhibit the growth of TSCC in vitro and in vivo. The objective of this study was to investigate the effect of PLB in reversing the resistance of TSCC to cisplatin as well as its molecular mechanisms. Here, we found that PLB enhances cisplatin-induced cytotoxicity, apoptosis, and autophagy in CAL27 and cisplatin-resistant CAL27/CDDP cells. PLB could inhibit the viability and growth of TSCC cells by increasing the production of intracellular reactive oxygen species (ROS). In addition, the combination treatment of PLB and cisplatin resulted in a synergistic inhibition of TSCC viability, apoptosis, and autophagy by increasing intracellular ROS, which may be achieved by activating JNK and inhibiting AKT/mTOR signaling pathways. Finally, the synergistic treatment was also demonstrated in vivo. Therefore, PLB combined with cisplatin is a potential therapeutic strategy against therapy TSCC cisplatin resistance.

ROS enhances the cytotoxicity of cisplatin by inducing apoptosis and autophagy in tongue squamous cell carcinoma cells.

Cisplatin is one of the most widely used anticancer agents for patients with tongue squamous cell carcinoma (TSCC), but its efficacy is limited by chemoresistance. Accumulated evidence has demonstrated that reactive oxygen species (ROS) plays a critical role in multiple tumor chemotherapy resistance. In the present study, we aimed to investigate the role of ROS in cisplatin resistance of TSCC and explore its underlying molecular mechanism in vitro. Our results showed that pre-treatment with ROS scavenger N-acetylcysteine reduced cisplatin-induced cytotoxicity, autophagy, and apoptosis in TSCC cells. Down-regulation of intracellular ROS attenuated apoptosis and autophagy of TSCC cisplatin-resistant CAL27/CDDP cells by reversing the inhibition of p38MAPK/mTOR pathway. Taken together, these findings suggest that down-regulation of intracellular ROS reduces the cytotoxicity of cisplatin by inhibiting apoptosis and autophagy in TSCC cells involving p38MAPK/mTOR mediated pathway. Low intracellular ROS levels may be one of the main mechanisms of cisplatin resistance in TSCC.

Deep-eutectic-solvent synthesis of N/O self-doped hollow carbon nanorods for efficient energy storage.

N/O self-doped hollow carbon nanorods (HCNs) with micro/mesoporous walls are fabricated based on a new deep-eutectic-solvent that serves as an all-in-one precursor, self-template, and self-dopant agent. The carbon-based supercapacitor using an ionic liquid electrolyte exhibits a high energy density of 116.5 W h kg-1 with excellent long-term cycling performance.

Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery.

The human cytochrome P450 (CYP) superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA ("Orthologous MAtrix") Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery.

3-Anhydro-6-hydroxy-ophiobolin A, a fungal sesterterpene from Bipolaris oryzae induced autophagy and promoted the degradation of α-synuclein in PC12 cells.

Autophagy is defined as an evolutionarily conserved process responsible for degradation of the cytoplasmic components including protein aggregates via the lysosomal machinery. Increasing evidence has linked defective autophagic degradation of protein aggregates with the pathogenesis of neurodegenerative disorders, and it is suggested that promotion of autophagy is regarded as a potential therapeutic for these diseases including Parkinson's disease (PD). Here we identified, 3-anhydro-6-hydroxy-ophiobolin A (X15-2), an ophiobolin derivative from Bipolaris oryzae that can strongly induce autophagic degradation of α-synuclein, the major constituent of Lewy bodies. We showed that X15-2 induced autophagy is dependent on both Beclin1 and Beclin2. Knockout of ATG5 by CRISPER/Cas9 prevented X15-2 induced autophagy and degradation of α-synuclein. Mechanistically, we showed that X15-2 induces ROS and the activation of JNK signaling for the autophagic degradation of α-synuclein in PC12 cells.

The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy.

Receptor-mediated mitophagy is one of the major mechanisms of mitochondrial quality control essential for cell survival. We previously have identified FUNDC1 as a mitophagy receptor for selectively removing damaged mitochondria in mammalian systems. A critical unanswered question is how receptor-mediated mitophagy is regulated in response to cellular and environmental cues. Here, we report the striking finding that BCL2L1/Bcl-xL, but not BCL2, suppresses mitophagy mediated by FUNDC1 through its BH3 domain. Mechanistically, we demonstrate that BCL2L1, but not BCL2, interacts with and inhibits PGAM5, a mitochondrially localized phosphatase, to prevent the dephosphorylation of FUNDC1 at serine 13 (Ser13), which activates hypoxia-induced mitophagy. Our results showed that the BCL2L1-PGAM5-FUNDC1 axis is critical for receptor-mediated mitophagy in response to hypoxia and that BCL2L1 possesses unique functions distinct from BCL2.

Reciprocal interactions between tumor-associated macrophages and CD44-positive cancer cells via osteopontin/CD44 promote tumorigenicity in colorectal cancer.

PURPOSE: CD44 is of functional importance for tumor initiation and progression in colorectal cancer, but how this molecule benefits cancer cells from the tumor microenvironment, especially tumor-associated macrophages (TAM), remains poorly defined. EXPERIMENTAL DESIGN: In vivo tumorigenic assays were conducted to assess the role of murine TAMs in the tumorigenesis of human colorectal cancer cells. Both in vitro and in vivo osteopontin (OPN) expression levels in TAMs were examined by immunohistochemistry, quantitative PCR, and Western blotting. Soft agar colony formation assays were used to estimate the clonogenicity of colorectal cancer cells that had received different treatments. The relationships between the expression levels of OPN, CD44v6, and CD68 and clinical prognosis were evaluated by tissue microarray analysis. RESULTS: We found that macrophages, when coinjected or cocultured with CD44-positive colorectal cancer cells, were able to produce higher levels of OPN, which in turn facilitated the tumorigenicity and clonogenicity of the colorectal cancer cells. The knockdown of CD44 or treatment with blocking antibodies to CD44 attenuated OPN secretion. OPN, through binding to its receptor CD44, activated c-jun-NH(2)-kinase signaling and promoted the clonogenicity of colorectal cancer cells. Moreover, tissue microarray data have shown that OPN expression, in combination with CD44v6, has a negative correlation with colorectal cancer patient survival. CONCLUSIONS: These results suggest that the OPN-CD44 interaction is important for colorectal cancer progression and could serve as a potential therapeutic target for the treatment of colorectal cancer.

EGFR and HER2 expression in primary cervical cancers and corresponding lymph node metastases: implications for targeted radiotherapy.

BACKGROUND: Proteins overexpressed on the surface of tumor cells can be selectively targeted. Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are among the most often targeted proteins. The level and stability of expression in both primary tumors and corresponding metastases is crucial in the assessment of a receptor as target for imaging in nuclear medicine and for various forms of therapy. So far, the expression of EGFR and HER2 has only been determined in primary cervical cancers, and we have not found published data regarding the receptor status in corresponding metastatic lesions. The goal of this study was to evaluate whether any of these receptors are suitable as target for clinical diagnosis and therapy. METHODS: Expression of EGFR and HER2 was investigated immunohistochemically in both lymph node metastases and corresponding primary cervical cancers (n = 53). HER2 and EGFR expression was scored using HercepTest criteria (0, 1+, 2+ or 3+). RESULTS: EGFR overexpression (2+ or 3+) was found in 64% (35/53) of the primary cervical tumors and 60% (32/53) of the corresponding lymph node metastases. There was a good concordance between the primary tumors and the paired metastases regarding EGFR expression. Only four patients who had 2+ or 3+ in the primary tumors changed to 0 or 1+ in lymph node metastases, and another two cases changed the other way around. None of the primary tumors or the lymph node metastases expressed HER2 protein. CONCLUSION: The EGFR expression seems to be common and stable during cervical cancer metastasis, which is encouraging for testing of EGFR targeted radiotherapy. HER2 appears to be of poor interest as a potential target in the treatment of cervical cancer.