Search

The large and rapid increase in the incidence and mortality of colorectal cancer (CRC) demonstrates the urgent need for new drugs with higher efficacy to treat CRC. However, the lack of applicable and reliable preclinical models significantly hinders the progress of drug development. Patient-derived xenograft (PDX) models are currently considered reliable in vivo preclinical models for predicting drug efficacy in cancer patients. This study successfully uses the CRC PDX model to develop clinical indications for the new multi-target kinase inhibitor BPR1J481 and demonstrated the anti-cancer mechanism and competitive advantages of this drug candidate. The results demonstrate that BPR1J481 exhibits significant anticancer efficacy by inducing apoptosis in CRC PDX tumor tissues and corresponding PDX-derived CRC cells. Through kinase competitive binding and kinase activity assays, we discover that BPR1J481 effectively inhibits SRC kinase activity by directly binding to its active site. The reduction in SRC phosphorylation observed in CRC PDX tumor tissues and derived cells upon treatment with BPR1J481 further validates its inhibitory potential. Furthermore, the decrease in viable cells after SRC knockout and the poorer prognosis observed in patients with higher SRC expression, emphasizes the critical significance and clinical relevance of SRC in CRC. Additionally, BPR1J481 exhibits robust anti-angiogenic effects by suppressing VEGF- and PDGF-induced endothelial cell proliferation, migration, and capillary-like tube formation through inhibition of VEGFR2 and PDGFRβ phosphorylation. Remarkably, BPR1J481 appears to demonstrate greater efficacy against CRC compared to regorafenib. These findings highlight the therapeutic potential of BPR1J481 for patients with CRC.

Abstract Background Cathepsin S (CTSS) is a cysteine protease that played diverse roles in immunity, tumor metastasis, aging and other pathological alterations. At the cellular level, increased CTSS levels have been associated with the secretion of pro-inflammatory cytokines and disrupted the homeostasis of Ca2+ flux. Once CTSS was suppressed, elevated levels of anti-inflammatory cytokines and changes of Ca2+ influx were observed. These findings have inspired us to explore the potential role of CTSS on cognitive functions. Methods We conducted classic Y-maze and Barnes Maze tests to assess the spatial and working memory of Ctss −/− mice, Ctss +/+ mice and Ctss +/+ mice injected with the CTSS inhibitor (RJW-58). Ex vivo analyses including long-term potentiation (LTP), Golgi staining, immunofluorescence staining of sectioned whole brain tissues obtained from experimental animals were conducted. Furthermore, molecular studies were carried out using cultured HT-22 cell line and primary cortical neurons that treated with RJW-58 to comprehensively assess the gene and protein expressions. Results Our findings reported that targeting cathepsin S (CTSS) yields improvements in cognitive function, enhancing both working and spatial memory in behavior models. Ex vivo studies showed elevated levels of long-term potentiation levels and increased synaptic complexity. Microarray analysis demonstrated that brain-derived neurotrophic factor (BDNF) was upregulated when CTSS was knocked down by using siRNA. Moreover, the pharmacological blockade of the CTSS enzymatic activity promoted BDNF expression in a dose- and time-dependent manner. Notably, the inhibition of CTSS was associated with increased neurogenesis in the murine dentate gyrus. These results suggested a promising role of CTSS modulation in cognitive enhancement and neurogenesis. Conclusion Our findings suggest a critical role of CTSS in the regulation of cognitive function by modulating the Ca2+ influx, leading to enhanced activation of the BDNF/TrkB axis. Our study may provide a novel strategy for improving cognitive function by targeting CTSS.

Multidrug resistance (MDR) is a major challenge in cancer chemotherapy. Nanoparticles as drug delivery systems (DDSs) show promise for MDR cancer therapy. However, current DDSs require sophisticated design and construction based on xenogeneic nanomaterials, evoking feasibility and biocompatibility concerns. Herein, a simple but versatile biological DDS (bDDS) composed of human red blood cell (RBC)-derived vesicles (RDVs) with excellent biocompatibility was surface-linked with doxorubicin (Dox) using glutaraldehyde (glu) to form Dox-gluRDVs that remarkably suppressed MDR in uterine sarcoma through a lysosomal-mitochondrial axis-dependent cell death mechanism. Dox-gluRDVs can efficiently deliver and accumulate Dox in lysosomes, bypassing drug efflux transporters and facilitating cellular uptake and retention of Dox in drug-resistant MES-SA/Dx5 cells. The transfer of lysosomal calcium to the mitochondria during mitochondria-lysosome contact due to lysosomal Dox accumulation may result in mitochondrial ROS overproduction, mitochondrial membrane potential loss, and activation of apoptotic signaling for the superior anti-MDR activity of Dox-gluRDVs in vitro and in vivo. This work highlights the great promise of RDVs to serve as a bDDS of Dox to overcome MDR cancers but also opens up a reliable strategy for lysosomal-mitochondrial axis-dependent cell death for fighting against other inoperable cancers.

Abstract Background Lymph node and distant metastasis contribute to poor outcomes in patients with oral squamous cell carcinoma (OSCC). The mechanisms regulating cancer migration and invasion play a key role in OSCC. Methods We determined migration and invasion ability of OSCC by wound-healing assay, two-chamber transwell invasion assay and cell mobility tracking and evaluated tumor metastasis in vivo. Western blot (WB), qRT-PCR, RNA-seq, dual-luciferase reporter assays and nuclear/cytoplasmic fractionation were performed to investigate the potential mechanism. Immunohistochimical (IHC) staining determined vimentin and PDZK1IP1 expression in OSCC tissues. Results and conclusion In this study, we determined that miR-455-5p was associated with lymph node metastasis and clinical invasion, leading to poor outcomes in patients with OSCC. MiR-455-5p promoted oral cancer cell migration and invasion and induced epithelial-to-mesenchymal transition (EMT). We also identified a new biomarker, PDZK1IP1 (MAP17), that was targeted by miR-455-5p. PDZK1IP1 knockdown led to migration, metastasis, EMT, and increased transforming growth factor-β signaling in OSCC. In addition, miR-455-5p overexpression and PDZK1IP1 inhibition promoted collective OSCC cell migration. According to data from the Cancer Genome Atlas database and the NCKU-OrCA-40TN data set, miR-455-5p and PDZK1IP1 are positively and negatively correlated, respectively, with partial EMT score. High miR-455-5p expression was associated with high vimentin levels and low MAP17 H-scores. The patients with low MAP17 expression had higher rates of disease recurrence than did patients with high MAP17 expression, especially for patients with clinical invasion risk factors and low MAP17 expression. These results suggest that miR-455-5p suppresses PDZK1IP1 expression and mediates OSCC progression. MiR-455-5p and PDZK1IP1 may therefore serve as key biomarkers and be involved in regulating partial EMT in OSCC cells. PDZK1IP1 expression may also serve as an independent factor that impacts outcomes in patients with clinical risk factors for recurrence.

Abstract Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca2+) via NCLX, a mitochondrial Na+/Ca2+ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.

ABSTRACT Human papillomavirus (HPV) is recognized as a major cause of oropharyngeal cancer (OPC) in Western countries. Less is known regarding its contribution to the OPC occurring in Asia. The current study aimed to investigate the association between antibody responses to HPV16 E7 and head and neck cancer (HNC) risk in a hospital‐based case–control study conducted in Taiwan with 693 HNC cases and 1,035 controls. A positive association was observed between seropositivity to HPV16 E7 and OPC risk, whereas no significant association was found in the non‐OPC cases. The increased OPC risk associated with seropositivity to HPV16 E7 was more significant among nonbetel quid or noncigarette users. Seropositivity to HPV16 E7 showed moderate agreement with P16 expression in OPC. OPC patients that were seropositive to HPV16 E7 or p16 positive were more highly educated and less likely to use alcohol, betel quids, and cigarettes compared to HPV16 E7 seronegative or p16 negative OPC patients. Furthermore, patients with p16 positive OPC were more likely to be women compared to patients with p16 negative OPC, likely owing to the low prevalence of alcohol, betel quid, and cigarette users among women. Overall, this study suggested that similar to Western countries, HPV may also be an important risk factor of OPC in Taiwan. With the declining consumption of betel quids and cigarettes in Taiwan, a higher percentage of OPC cases in Taiwan will be attributed to HPV in the future. Public health measures, including HPV vaccination, need to be implemented to prevent the occurrence of HPV‐positive OPC.

Abstract S-1, an oral pyrimidine fluoride-derived agent, is effective against various cancers. Sorafenib, an oral multikinase inhibitor, was found to prolong the survival of various cancers and enhance the cytotoxicity of chemotherapeutic agents. We conducted a phase I dose escalation study to determine dose-limiting toxicity (DLT) and maximal tolerated dose (MTD) of S-1 when combined with sorafenib for refractory solid tumors. Eligible patients received escalating doses (30, 35, and 40 mg/m2 bid) of S-1 Day 1 (D1)–D14 and continuous sorafenib 400 mg bid from cycle 1 D8 every 21 days in a standard 3 + 3 study design. Primary endpoint was MTD. Thirteen patients were enrolled between May 2010 and Feb 2012. DLT developed in two (one grade 3 erythema and one prolonged grade 2 hand-foot-skin reaction) of the 6 patients at 35 mg/m2 dose level. One pancreatic neuroendocrine tumor (pNET) patient achieved a durable partial response (27.9 months). Four colon cancer patients had stable disease and 3 of them had progression-free survival greater than 6 months. This study determined the recommended (MTD) S-1 dose of 30 mg/m2 bid for this regimen. This result warrants further phase II studies for advanced pNET and colon cancer to evaluate the efficacy of this combination.

Abstract Ephrin type-A receptor 10 (EPHA10) has been implicated as a potential target for breast and prostate cancer therapy. However, its involvement in oral squamous cell carcinoma (OSCC) remains unclear. We demonstrated that EPHA10 supports in vivo tumor growth and lymphatic metastasis of OSCC cells. OSCC cell migration, epithelial mesenchymal transition (EMT), and sphere formation were found to be regulated by EPHA10, and EPHA10 was found to drive expression of some EMT- and stemness-associated transcription factors. Among EPHA10 ligands, exogenous ephrin A4 (EFNA4) induced the most OSCC cell migration and sphere formation, as well as up-regulation of SNAIL, NANOG, and OCT4. These effects were abolished by extracellular signal-regulated kinase (ERK) inhibition and NANOG knockdown. Also, EPHA10 was required for EFNA4-induced cell migration, sphere formation, and expression of NANOG and OCT4 mRNA. Our microarray dataset revealed that EFNA4 mRNA expression was associated with expression of NANOG and OCT4 mRNA, and OSCC patients showing high co-expression of EFNA4 with NANOG or OCT4 mRNA demonstrated poor recurrence-free survival rates. Targeting forward signaling of the EFNA4-EPHA10 axis may be a promising therapeutic approach for oral malignancies, and the combination of EFNA4 mRNA and downstream gene expression may be a useful prognostic biomarker for OSCC.

Abstract Background Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. Methods Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. Results We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. Conclusions Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.

BackgroundPartial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging.MethodsGene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan–Meier curves of estimated overall survival were compared for statistical difference using the log-rank test.ResultsCompared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types.ConclusionsAs the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

In the original publication [...]

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC50 values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC50 values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC50 values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

Abstract Poor oral hygiene is an established risk factor of head and neck cancer (HNC); however, its role in the survival of HNC patients is unclear. This study evaluated the association between oral hygiene habits, including regular dental visits, frequency of tooth brushing, and use of dental floss, and the overall survival (OS) of HNC patients using interview data collected from 740 HNC patients. In addition, the interactions between oral hygiene and the polymorphisms of TLR2 and TLR4 on the OS of HNC patients were assessed. The analysis indicated that poor oral hygiene was significantly associated with poorer OS of HNC patients (hazard ratio (HR) = 1.38, 95% confidence interval (CI): 1.03‐1.86). This association was modified by a single nucleotide polymorphism, rs11536889, of TLR4. A significant association between poor oral hygiene and worse survival of HNC was observed among those with the CG or CC genotype (HR = 2.32, 95% CI: 1.41‐3.82) but not among those with the GG genotype (HR = 0.95, 95% CI: 0.65‐1.40). Our results suggested that poor oral hygiene is not only a risk factor but may also be a prognostic factor of HNC.

Ciclesonide is an inhaled corticosteroid used to treat asthma and has been repurposed as a treatment for mildly ill COVID-19 patients, but its precise mechanism of action is unclear. Herein, we report that ciclesonide blocks the coronavirus-induced production of the cytokines IL-6, IL-8, and MCP-1 by increasing IκBα protein levels and significantly decreasing p65 nuclear translocation. Furthermore, we found that the combination of ciclesonide and dbq33b, a potent tylophorine-based coronavirus inhibitor that affects coronavirus-induced NF-κB activation a little, additively and synergistically decreased coronavirus-induced IL-6, IL-8, and MCP-1 cytokine levels, and synergistically inhibited the replication of both HCoV-OC43 and SARS-CoV-2. Collectively, the combination of ciclesonide and dbq33b merits consideration as a treatment for COVID-19 patients who may otherwise be overwhelmed by high viral loads and an NF-κB-mediated cytokine storm.

Abstract Background Tumor hypoxia-induced epithelial–mesenchymal transition (EMT) is critical in promoting cancer metastasis. We recently discovered a novel microtubule inhibitor, MPT0B098, that employs a novel antitumor mechanism. It destabilizes hypoxia-inducible factor (HIF)-1α mRNA by blocking the function of human antigen R. Thus, we proposed that MPT0B098 modulates hypoxia-induced EMT. Methods In vitro IC50 values were determined through the methylene blue dye assay. To investigate molecular events, reverse transcriptase-polymerase chain reaction, Western blotting, immunofluorescence staining, and wound healing assay were employed. Results MPT0B098 significantly inhibited HIF-1α expression, epithelial-to-mesenchymal morphology changes, and migratory ability in the human head and neck squamous cell carcinoma cell line OEC-M1. Furthermore, after MPT0B098 treatment, the expression of two mesenchymal markers, vimentin and N-cadherin, was downregulated under hypoxic conditions. Moreover, MPT0B098 suppressed hypoxia-induced EMT in part by inhibiting EMT-activating transcription factors, Twist and SNAI2/Slug. In addition, the inhibition of hypoxia-induced F-actin rearrangement and focal adhesion kinase phosphorylation may have contributed to suppression of EMT by MPT0B098in OEC-M1 cells. MPT0B098 significantly inhibited transforming growth factor(TGF)-β-induced phosphorylation of receptor-associated Smad2/3 by downregulating TGF-β mRNA and protein expression. Conclusions Taken together, this study provides a novel insight into the role of MPT0B098 in inhibiting hypoxia-induced EMT, suggesting its potential use for treating head and neck cancers.

Clec4a has been reported to be an immune suppressor of dendritic cells (DCs), but its potential role in cancer therapy remains to be elucidated. The present study investigated whether downregulating the expression of Clec4a via skin delivery of small hairpin RNA (shRNA) using a gene gun produced stronger host immunity and inhibited tumor progression in animal models. Administration of Clec4a2 shRNA delayed tumor growth in both mouse bladder and lung tumor-bearing mouse models. The result was further confirmed with a compensation experiment showing that the antitumor effects induced by Clec4a2 shRNA were restored by co-injection of a plasmid expressing exogenous Clec4a2. Increased numbers of infiltrating CD4+ and CD8+ T cells at tumor sites were observed in mice treated with Clec4a2 shRNA. Splenocytes from mice with Clec4a2 shRNA administration exhibited stronger cytotoxic activity compared with splenocytes from control mice. CD8-deletion in vivo abrogated the antitumor effects elicited by Clec4a2 shRNA. Additionally, shClec4a enhanced the antitumor effects of the Neu DNA vaccine in the MBT-2 tumor model. In summary, the findings provide evidence that silencing of Clec4a2 expression via skin delivery of shRNA produces an effective antitumor response and that Clec4a2 shRNA may have therapeutic potential as an adjuvant for cancer immunotherapy.

Abstract The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Abstract Background Although substantial evidence supports a 20–30% risk reduction of colon cancer, breast cancer, and endometrial cancer by physical activity (PA), the evidence for head and neck cancer (HNC) is limited. Three published studies on the association between PA and HNC have generated inconsistent results. The current study examined the association between recreational PA (RPA) and HNC risk with a more detailed assessment on the intensity, frequency, duration, and total years of RPA. Methods Data on RPA were collected from 623 HNC cases and 731 controls by in-person interview using a standardized questionnaire. The association between RPA and HNC risk was assessed using unconditional logistic regression, adjusted for sex, age, educational level, use of alcohol, betel quid, and cigarette, and consumption of vegetables and fruits. Results A significant inverse association between RPA and HNC risk was observed in a logistic regression model that adjusted for sex, age, and education (odds ratio (OR) = 0.65, 95% confidence interval (CI): 0.51-0.82). However, after further adjustment for the use of alcohol, betel quid, and cigarette, and consumption of vegetables and fruits, RPA was no longer associated with HNC risk (OR =0.97, 95% CI: 0.73-1.28). No significant inverse association between RPA and HNC risk was observed in the analysis stratified by HNC sites or by the use of alcohol, betel quid, or cigarette. Conclusion Results from our study did not support an inverse association between RPA and HNC risk. The major focus of HNC prevention should be on cessation of cigarette smoking and betel chewing, reduction of alcohol drinking, and promotion of healthy diet that contains plenty of fruits and vegetables.