Correction to: Gamma synuclein is a novel nicotine responsive protein in oral cancer malignancy.

[This corrects the article DOI: 10.1186/s12935-020-01401-w.].

Gamma synuclein is a novel nicotine responsive protein in oral cancer malignancy.

BACKGROUND: The mechanisms of neuronal protein γ-synuclein (SNCG) in the malignancy of oral squamous cell carcinoma (OSCC) are not clear. This study tested the hypothesis that SNCG is involved in nicotine-induced malignant behaviors of OSCC. The effect of nicotine on SNCG expression and epithelial-to-mesenchymal transition (EMT) markers were examined. METHODS: Short hairpin RNA (shRNA) and an antagonist specific for α7-nicotine acetylcholine receptors (α7-nAChRs) were used to examine the role of α7-nAChRs in mediating the effects of nicotine. Knockdown of SNCG in nicotine-treated cells was performed to investigate the role of SNCG in cancer malignancy. The in vivo effect of nicotine was examined using a nude mouse xenotransplantation model. RESULTS: Nicotine increased SNCG expression in a time- and dose-dependent manner. Nicotine treatment also increased E-cadherin and ZO-1 and decreased fibronectin and vimentin expression. After specific knockdown of α7-nAChRs and inhibition of the PI3/AKT signal, the effect of nicotine on SNCG expression was attenuated. Silencing of SNCG abolished nicotine-induced invasion and migration of OSCC cells. The xenotransplantation model revealed that nicotine augmented tumor growth and SNCG expression. CONCLUSION: Nicotine upregulated SNCG expression by activating the α7-nAChRs/PI3/AKT signaling that are participated in nicotine-induced oral cancer malignancy.

Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.

Most cases of oral squamous cell carcinoma (OSCC) develop from visible oral potentially malignant disorders (OPMDs). The latter exhibit heterogeneous subtypes with different transformation potentials, complicating the early detection of OSCC during routine visual oral cancer screenings. To develop clinically applicable biomarkers, we collected saliva samples from 96 healthy controls, 103 low-risk OPMDs, 130 high-risk OPMDs, and 131 OSCC subjects. These individuals were enrolled in Taiwan's Oral Cancer Screening Program. We identified 302 protein biomarkers reported in the literature and/or through in-house studies and prioritized 49 proteins for quantification in the saliva samples using multiple reaction monitoring-MS. Twenty-eight proteins were successfully quantified with high confidence. The quantification data from non-OSCC subjects (healthy controls + low-risk OPMDs) and OSCC subjects in the training set were subjected to classification and regression tree analyses, through which we generated a four-protein panel consisting of MMP1, KNG1, ANXA2, and HSPA5. A risk-score scheme was established, and the panel showed high sensitivity (87.5%) and specificity (80.5%) in the test set to distinguish OSCC samples from non-OSCC samples. The risk score >0.4 detected 84% (42/50) of the stage I OSCCs and a significant portion (42%) of the high-risk OPMDs. Moreover, among 88 high-risk OPMD patients with available follow-up results, 18 developed OSCC within 5 y; of them, 77.8% (14/18) had risk scores >0.4. Our four-protein panel may therefore offer a clinically effective tool for detecting OSCC and monitoring high-risk OPMDs through a readily available biofluid.

Clathrin-mediated endocytosis is required for ANE 30-100K-induced autophagy.

BACKGROUND: We identified an autophagy-inducing areca nut (AN) ingredient (AIAI) in the 30-100 kDa fraction of AN extract (ANE 30-100K). This study was to analyze the role of endocytosis in ANE 30-100K-induced autophagy. METHODS: We used benzyl alcohol, dynasore, and shRNA of clathrin and dynamin to assess whether ANE 30-100K-induced cytotoxicity and accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II were affected in oral (OECM-1) and esophageal (CE81T/VGH) carcinoma cells. RESULTS: Both benzyl alcohol and dynasore effectively reduced ANE 30-100K-induced cytotoxicity and LC3-II accumulation in OECM-1 and CE81T/VGH cells. Downregulated protein expression of both clathrin and dynamin by their shRNA also significantly attenuated ANE 30-100K-induced elevation of LC3-II levels in CE81T/VGH cells. CONCLUSIONS: These results indicate that AIAI may be engulfed by cells through clathrin-mediated endocytosis, which promotes the execution of the following autophagy program.

The application of data mining techniques to oral cancer prognosis.

This study adopted an integrated procedure that combines the clustering and classification features of data mining technology to determine the differences between the symptoms shown in past cases where patients died from or survived oral cancer. Two data mining tools, namely decision tree and artificial neural network, were used to analyze the historical cases of oral cancer, and their performance was compared with that of logistic regression, the popular statistical analysis tool. Both decision tree and artificial neural network models showed superiority to the traditional statistical model. However, as to clinician, the trees created by the decision tree models are relatively easier to interpret compared to that of the artificial neural network models. Cluster analysis also discovers that those stage 4 patients whose also possess the following four characteristics are having an extremely low survival rate: pN is N2b, level of RLNM is level I-III, AJCC-T is T4, and cells mutate situation (G) is moderate.

Tumor-associated macrophages promote oral cancer progression through activation of the Axl signaling pathway.

BACKGROUND: Recent studies suggest that tumor-associated macrophages (TAMs) promote tumor growth and metastasis. Our previous report demonstrated that Axl signaling promotes carcinogenesis and progression of oral squamous cell carcinoma (OSCC). This study aims to test the potential involvement of growth arrest-specific gene 6 (Gas6)/Axl signaling in the protumoral effect of TAMs. METHODS: Co-culture experiments by incubation of OSCC cells (YD38 and OE) and macrophages (THP-1) were performed. The expression of Gas6/Axl and epithelial-mesenchymal transition (EMT) genes were examined in YD38 and OE cells. The effect of Gas6/Axl signaling on co-cultured cancer cells was further investigated by knocking down Axl expression and neutralizing Gas6. Axl and TAM distribution were analyzed by immunohistochemistry in OSCC tissues. RESULTS: Activation of Axl signaling and increased expression of mesenchymal markers, along with increased invasion/migration ability of OSCC cells, was noted upon co-culture with THP-1. Neutralization of Gas6 in the co-culture system or knockdown of Axl in YD38 caused the co-culture effects to be diminished. Co-culture with THP-1 increased nuclear factor (NF)-κB nuclear translocation and transcription activity in YD38 cells. A significant association between the TAM count and expression of phosphorylated Axl (P = 0.004) was found in vivo cancer tissues. CONCLUSIONS: TAMs play a protumor role in OSCC and likely promote tumor progression through activation of the Gas6/Axl-NF-κB signaling pathway. Therefore, Gas6/Axl and NF-κB signaling in OSCC cells may be a putative target for therapeutic intervention.

Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity.

BACKGROUND: We previously demonstrated the autophagy-inducing activity in the crude extract of areca nut (ANE) and its 30-100 kDa fraction (ANE 30-100 K). This study aimed to analyze whether chronic ANE and ANE 30-100 K stimulations lead to higher stress resistance and autophagic activity in oral cells, and whether the resulting autophagic status in stimulated cells correlates with stress resistance. MATERIALS AND METHODS: Malignant cells from the mouth oral epidermoid carcinoma Meng-1 (OECM-1) and blood (Jurkat T) origins were stimulated with non-cytotoxic ANE and ANE 30-100 K for 3 months. Sensitivity to anticancer drugs of and autophagy status in stimulated cells, analyzed respectively by XTT assay and calculating microtubule-associated protein 1 light chain 3-II LC3-II/ß-actin ratios from Western blot, were compared to non-treated cells. Autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), were used to assess whether autophagy inhibition interferes the altered chemoresistance. RESULTS: Areca nut extract-stimulated (ANE-s) and ANE 30-100 K-stimulated (30-100 K-s) OECM-1 and Jurkat T cells generally exhibited higher cisplatin and 5-fluorouracil (5-FU) resistances, compared to non-stimulated cells. Most stimulated cells expressed significantly higher levels of LC3-II and Atg4B proteins. Interestingly, these cells also showed stronger tolerances against hypoxia environment and expressed higher LC3-II levels under glucose-deprived and hypoxia conditions. Finally, both 3-MA and CQ alleviated, albeit to different degrees, the increased chemoresistance in ANE-s and/or 30-100 K-s cells. CONCLUSIONS: Chronic stimulations of ANE or ANE 30-100 K may increase tolerance of oral cancer and leukemia T cells to anticancer drugs, as well as to glucose deprivation and hypoxia conditions, and cause an elevation of autophagy activity responsible for increased drug resistance.

Protumoral effect of macrophage through Axl activation on mucoepidermoid carcinoma.

OBJECTIVE: This study aims to test the potential involvement of Axl signaling in the protumoral effect of tumor-associated macrophages (TAMs) in mucoepidermoid carcinoma (MEC). MATERIALS AND METHODS: We carried out cocultured experiments by incubation of MEC cells (UTMUC-1) and macrophages (THP-1) and examined Axl activation status. The expression of MMPs and behavior change were examined in UT-MUC-1 cells. The effect of Axl signaling on co-cultured cancer cells was further investigated by knockdown Axl expression and suppression by Axl-specific inhibitor R428. RESULTS: Activation of Axl signaling and increased expression and activity of MMP-2 and MMP-9 along with increased invasion/migration ability in MEC cells were observed when co-cultured with TAMs. Upon knockdown of Axl in MEC or addition of R428 in the co-cultured system, these co-cultured effects were diminished. CONCLUSION: TAMs play a protumoral role in MEC via activation of the Axl signaling pathway, up-regulating MMPs expression, and increasing invasion/migration ability.

Role of SIRT3 in the regulation of redox balance during oral carcinogenesis.

BACKGROUND: Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases, which play an important role in regulating cancer tumorigenesis; however, their role in oral cancer has been controversial. SIRT3 is localized in the mitochondria, where it deacetylates and activates several enzymes involved in cellular redox balance and defense against oxidative damage. RESULTS: We found that compared with normal human oral keratinocytes (HOK), SIRT3 is highly expressed in oral squamous cell carcinoma (OSCC) cell lines, but the enzymatic deacetylation is significantly reduced. We also sequenced the entire coding region of SIRT3 and found the same mutation in 2 different OSCC cell lines. This point mutation is located in close proximity to the active site of deacetylase in the SIRT3 protein, and reduces the overall enzymatic efficiency of deacetylation. Furthermore, up-regulation of SIRT3 inhibited the cell growth of OSCCs and decreased the levels of basal reactive oxygen species (ROS) in both OSCC lines. To verify that the SIRT3 sequence variation was associated with oral carcinogenesis, we sequenced the SIRT3 gene from 21 OSCC patients, and 5 of the 21 patients (23.8%) carried the heterozygous missense mutation, p.Val208Ile. The heterozygous missense mutation in these patients was present in gremlin DNA isolated from both normal and tumor tissues. CONCLUSIONS: Our findings provide a valuable insight into the potential role of SIRT3 in the development of oral squamous cell carcinoma, by showing that a non-synonymous point mutation in SIRT3 contributes to reduced catalytic activity of the protein and affects redox balance in OSCCs.

Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance.

BACKGROUND: Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting. RESULTS: A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants. CONCLUSIONS: Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome.

Axl is a prognostic marker in oral squamous cell carcinoma.

BACKGROUND: Overexpression of the receptor tyrosine kinase Axl is implicated in several diseases. The present study was conducted to determine the biologic and clinical significance of Axl in oral squamous cell carcinoma (OSCC). METHODS: The expression of Axl was examined in a panel of OSCC cell lines. Activation of Axl by Gas6 treatment and silencing of Axl via Axl shRNA were used to examine the effect of Axl on OSCC cell line. Expression of Axl in cancer tissues were examined by immunohistochemical staining. The associations between Axl expression and clinicopathologic features and prognosis were analyzed. RESULTS: Varied Axl expression was noted in OSCC cell lines. Compared with control cells, modulated Axl signal affected epithelial-mesenchymal gene expression and cell invasion and migration. The immunoreactivity of Axl was low in normal epithelium, and a progressively increased positive percentage was noted, from normal/hyperplastic epithelium (10.9%) to dysplasia (30.8%) to cancer tissue (54.5%). Axl expression correlated with lymph node status (P = .001) and clinical stage (P = .014) of OSCC. Patients with high expression of Axl showed poor prognosis compared with those with low Axl expression patients (P < .001). In multivariate prognostic analysis according to the Cox proportional hazard regression model, Axl expression remained as an independent prognostic factor (P = .037; CI, 1.042-3.839). CONCLUSIONS: Our data indicated that Axl signal promotes OSCC carcinogenesis and progression. The expression of Axl is a valuable marker for OSCC aggressiveness and clinical outcome.

Psychoactive substance use and the risk of motor vehicle crash injuries in southern Taiwan.

The purpose of this study was to examine the association between psychoactive drug use and motor vehicle crash (MVC) injuries requiring hospitalization in southern Taiwan. A case-control study was conducted in southern Taiwan from January 2009 to December 2009. The cases included car or van drivers who were involved in MVCs and required hospitalization. Demographic and trauma-related data were collected from questionnaires and hospital and ambulance records. Urine and/or blood samples were collected on admission. The controls consisted of drivers who were randomly recruited while driving on public roads. Study subjects were interviewed and asked to provide urine samples. All blood and urine samples were tested for alcohol and a number of other legal and illegal drugs. Only those subjects who provided urine and/or blood specimens were included in the study. During the study period, 254 case patients and 254 control drivers were enrolled. The analysis showed an odds ratio (OR) of 3.41 (95% confidence intervals (95% CI), 1.76-6.70; p < 0.001) for persons taking benzodiazepines, and an OR of 3.50 (95% CI, 1.81-6.85; p < 0.001) for those taking alcohol (blood alcohol concentrations (BAC) ≥ 0.8 g/l) with regard to hospitalizations due to MVCs. For persons taking combinations of benzodiazepines and alcohol, the OR increased to 5.12 (95% CI: 1.77-15.91, p < 0.001). This study concluded that drug use among motor vehicle drivers increases the risk of MVCs that require hospitalization. From a public health perspective, the high risk ratios are concerning, and preventive measures are warranted.

Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation.

Obesity results from an imbalance between caloric intake and energy expenditure, and it is highly associated with colorectal carcinogenesis and therapeutic resistance in patients with colorectal cancer (CRC). Dysregulation of adipokine production in obesity has been reported to cause malignant behaviors in CRC. Leptin, which is the principal hormone secreted by adipocytes and an obesity-associated adipokine, is significantly overexpressed in CRC tissues. However, the effect of leptin on chemoresistance in CRC is unclear. Therefore, the aim of this study was to clarify the role of leptin and the underlying mechanisms in mediating 5-fluorouracil (5-FU) resistance in CRC. We used palmitate to artificially generate obese adipocytes. As expected, lipid accumulation was significantly increased in obese adipocytes. We demonstrated that CRC cells incubated with conditioned media (CM) harvested from obese adipocytes were associated with increased resistance to 5-FU. Notably, this increase in resistance to 5-FU was through the elevated production and secretion of leptin. Leptin could further stimulate the expression of AXL and activate its downstream signaling molecule, PLCγ, thereby resulting in an increased expression of p-glycoprotein (P-gp) in CRC cells. Mechanistically, leptin induced AXL expression via the inhibition of AMPK and subsequent increase in YAP activation and nuclear translocation. In addition, nuclear YAP interacted with TEAD and promoted the occupancy of TEAD on the AXL promoter, thereby stimulating AXL promoter activity after leptin treatment. Furthermore, leptin neutralization rescued the sensitivity of CRC tumors to 5-FU in mice fed on a high-fat diet (HFD). These results indicated that leptin mediated 5-FU resistance through YAP-dependent AXL overexpression in CRC.

YAP-Dependent BiP Induction Is Involved in Nicotine-Mediated Oral Cancer Malignancy.

Cigarette smoking is a significant risk factor for the development and progression of oral cancer. Previous studies have reported an association between nicotine and malignancy in oral cancer. Recent studies have also demonstrated that nicotine can induce endoplasmic reticulum (ER) stress in tumor cells. Binding immunoglobulin protein (BiP) acts as a master regulator of ER stress and is frequently overexpressed in oral cancer cell lines and tissues. However, the effect of nicotine on BiP in oral cancer is unknown. Therefore, this study aimed to evaluate the role of BiP and its underlying regulatory mechanisms in nicotine-induced oral cancer progression. Our results showed that nicotine significantly induced the expression of BiP in time- and dose-dependent manners in oral squamous cell carcinoma (OSCC) cells. In addition, BiP was involved in nicotine-mediated OSCC malignancy, and depletion of BiP expression remarkably suppressed nicotine-induced malignant behaviors, including epithelial-mesenchymal transition (EMT) change, migration, and invasion. In vivo, BiP silencing abrogated nicotine-induced tumor growth and EMT switch in nude mice. Moreover, nicotine stimulated BiP expression through the activation of the YAP-TEAD transcriptional complex. Mechanistically, we observed that nicotine regulated YAP nuclear translocation and its interaction with TEAD through α7-nAChR-Akt signaling, subsequently resulting in increased TEAD occupancy on the HSPA5 promoter and elevated promoter activity. These observations suggest that BiP is involved in nicotine-induced oral cancer malignancy and may have therapeutic potential in tobacco-related oral cancer.

Betel quid extract promotes oral cancer cell migration by activating a muscarinic M4 receptor-mediated signaling cascade involving SFKs and ERK1/2.

Betel quid (BQ) is a widely accepted etiological factor for oral squamous cell carcinoma (OSCC) in Southeast Asia, but how BQ chewing leads to oral carcinogenesis remains to be elucidated. We have previously demonstrated that the activation of Src family kinases (SFKs) is critical for BQ-induced oral cancer cell motility. Here we investigate whether this biological effect is mediated by specific membrane receptors in oral cancer cells. We found that BQ-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cell migration could be inhibited by atropine, suggesting the involvement of the muscarinic receptor family. The enhanced activities of ERK1/2 and cell migration were significantly counteracted by PD102807, the selective antagonist of muscarinic M4 receptor. Moreover, cold BQ extract effectively competed with a known ligand, [(3)H]-N-methyl scopolamine, for binding to muscarinic M4 receptor in vitro, thereby implying that BQ could activate motility-promoting signaling pathways through direct interaction with the receptor. The requirement of muscarinic M4 receptor for BQ-induced oral cancer cell migration was demonstrated by knockdown of the receptor using RNA interference (RNAi). Remarkably, ectopic expression of muscarinic M4 receptor in two oral cancer cell lines, Ca9-22 and SCC-9, further augmented BQ-induced cell migration by 83% and 99%, respectively. Finally, we verified that BQ-induced oral cancer cell migration was mediated through a muscarinic M4 receptor-->SFKs-->ERK1/2 signaling pathway. Thus, our findings have identified a novel signaling cascade mediating BQ-induced oral cancer cell motility, which could be a therapeutic target for BQ-related oral malignancies.

Possible mechanism of betel-quid-extract-induced expression of matrix metalloproteinase-2.

BACKGROUND/PURPOSE: Betel quid extract (BQE) has been demonstrated to induce matrix metalloproteinase (MMP)-2 expression. This study aimed to establish the possible mechanism involved in this event. METHODS: Western blotting, reverse-transcription polymerase chain reaction, and gelatin zymography were used to study the expression level of MMP-2. LY294002, PD98059, U0126, N-acetyl-L-cysteine, SB203580, SP600125, and Bay 11-7082 were used to pretreat OECM-1 cells before BQE treatment and MMP-2 detection. RESULTS: OECM-1 cells were subjected to short-term (10 minutes) or long-term (24 hours) BQE treatment (designated as SBT and LBT, respectively), and we found that both treatments increased MMP-2 protein and extracellular signal-regulated kinase (ERK) phosphorylation levels in a concentration- and time-dependent manner. LBT also increased MMP-2 mRNA level. LBT-induced MMP-2 secretion was not inhibited by PD98059 (up to 50 µM) when ERK was effectively blocked, but was attenuated by LY294002 (0-10 µM) in a concentration-dependent manner. This LBT effect was inhibited strongly by SB203580 (10 µM), SP600125 (10 µM), and Bay 11-7082 (10 µM) and mildly by N-acetyl-L-cysteine (5 mM), but not by U0126 (10 µM). CONCLUSION: Both SBT and LBT upregulate MMP-2 expression, and LBT-induced MMP-2 expression might be mediated by phosphoinositide 3-kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB, and to a lesser extent, by reactive oxygen species, rather than by ERK.

Malignant development in patients with oral potentially malignant disorders detected through nationwide screening: Outcomes of 5-year follow-up at a single hospital.

BACKGROUND: Although survival rate and quality of life are improved if patients with oral carcinoma can be detected early, however, such lesions are usually asymptomatic; therefore, it is hard to raise awareness. Screening has proved to be cost-effective for early detection. METHODS: Sixty-two patients with oral carcinomas and 555 patients with oral potentially malignant disorders (OPMDs) who were detected through screening were examined the relationship between clinicopathological features and follow-up outcomes. RESULTS: The 5-year cumulative cancer-free interval rate was 94.1%, and the annual malignant transformation rate was 1.16%. The rate of interval carcinoma development from Candida hyperplasia, oral submucous fibrosis, homogeneous leukoplakia, non-homogenous leukoplakia, and verrucous hyperplasia, was 13.6%, 5.7%, 4.6%, 12.1%, and 21.3%, respectively. Significant independent risk factors for interval carcinoma development were heavy betel quid chewing, verrucous hyperplasia, and surgery refusal. CONCLUSIONS: Well-designed risk assessment, treatment, and surveillance program could lead to earlier cancer detection and thereby reduce mortality and morbidity.

α7-Nicotine acetylcholine receptor mediated nicotine induced cell survival and cisplatin resistance in oral cancer.

OBJECTIVE: To investigate the effect of nicotine on cell survival and cisplatin resistance in oral cancer and the possible involvement of α7-nicotinic acetylcholine receptors (α7-nAChRs). DESIGN: The effects of nicotine on cell survival and cisplatin-induced apoptosis were assessed. Knockdown of α7-nAChRs by short hairpin RNA and the specific antagonist methyllycaconitine (MLA) was used to examine the involvement of α7-nAChRs in modulating the effects of nicotine. Apoptosis signal molecules were examined in nicotine- and cisplatin-treated cells. RESULTS: Nicotine increased the survival of the oral cancer cells YD8 and OEC-M1 in a dose- and time-dependent manner. Nicotine treatment accelerated cell cycle progression in the oral cancer cells, and significantly reduced cisplatin-induced cell apoptosis. In the α7-nAChR-silenced cells, the prosurvival effect of nicotine in the cisplatin-treated cells was attenuated. Co-treatment of cisplatin and nicotine attenuated the effect of cisplatin on Bcl-2 expression. In addition, the effect of nicotine on cell survival under cisplatin treatment was attenuated with the addition of the Bcl-2 inhibitor ABT-737. CONCLUSIONS: Treating oral cancer cells with nicotine increased cell survival and cisplatin resistance, in which α7-nAChRs were involved.

Chronic Stimulation of the Autophagy-inducing Ingredient of Areca Nut Promotes Tumor Growth In Vivo Through Up-regulation of Tumoral Autophagy.

BACKGROUND/AIM: Autophagy can be either tumor promotive or suppressive. We previously identified an autophagy-inducing activity in the 30-100 kDa fraction of areca-nut-extract (ANE 30-100K) and showed that several tumor cells subjected to chronic ANE 30-100K stimulation (CAS) exhibited higher resistance against stressed environments including serum-free (SF) conditions in vitro. Herein, we aimed to assess whether CAS can also provide growth advantages for tumor cells in vivo and the therapeutic effect of autophagy inhibition on CAS-treated tumors. MATERIALS AND METHODS: Esophageal CE81T/VGH cells and nude mice were used as experimental models. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ), as well as another anticancer drug cisplatin (DDP), were chosen to challenge CAS-treated CE81T/VGH cells in vitro and in vivo. RESULTS: CAS-treated CE81T/VGH cells expressed higher levels of microtubule-associated protein 1 light chain 3A/B-II (LC3-II) and beclin 1 proteins, and showed stronger resistance to SF and hypoxia conditions, that were mitigated by CQ or 3-MA in vitro. Furthermore, CAS-treated CE81T/VGH cells induced significantly larger tumors in mice, which were also attenuated by single 3-MA or CQ treatment. Finally, the combined treatment of 3-MA or CQ with DDP further up-regulated DDP-induced caspase-3 activity in vitro and exhibited synergistic anti-tumor effects on mice. CONCLUSION: CAS may up-regulate tumoral autophagy and provide growth advantage for tumors both in vitro and in vivo. Furthermore, autophagy inhibition alone or in combination with DDP may achieve positive therapy for tumors encountered with CAS.

Tumor-associated macrophage correlated with angiogenesis and progression of mucoepidermoid carcinoma of salivary glands.

BACKGROUND: There is considerable controversy about whether tumor-associated macrophages (TAMs) promote or inhibit tumor progression. The present study examined the clinicopathologic significance of TAMs and their association with tumor angiogenesis, cell proliferation, and apoptosis in mucoepidermoid carcinoma (MEC). The potential effect of TAMs on cancer cells was also investigated. METHODS: CD68, CD34, Ki-67, and vascular endothelial growth factor (VEGF)-A immunohistochemical staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) were applied to samples from 41 MEC patients. The biologic effect of macrophages on MEC cancer cells was examined in a co-culture system. RESULTS: The proliferation index (PI) was 11.7+/-5.9%, and the apoptotic index (AI) was 4.1+/-2.3% in cancer patients. PI was significantly correlated with tumor grade, and the PI/AI ratio was significantly correlated with tumor size and stage. The distributions of intratumoral TAMs and microvessel density (MVD) were heterogeneous. TAM count associated strongly with tumor size, grading, and MEC staging. A greater intratumoral MVD was observed frequently in patients with large, intermediate/high-grade, and advanced-stage tumors. VEGF-A expression correlated significantly with tumor size and stage. MVD count was closely associated with TAM count and VEGF-A expression. Co-cultured cancer cells with macrophages increased migration and invasion ability of cancer cells. Co-cultured endothelial cells with cancer cells elevated VEGF-A expression, proliferation, and migration, and tube formation of endothelial cells. CONCLUSION: Our data suggest that TAMs play a tumor-promoting role in MEC. The TAM count, intratumoral MVD, and PI/AI ratio are potentially useful markers of progression in patients with MEC.