Current understanding of genetic associations with delayed hypersensitivity reactions induced by antibiotics and anti-osteoporotic drugs.

Drug-induced delayed hypersensitivity reactions (DHRs) is still a clinical and healthcare burden in every country. Increasing reports of DHRs have caught our attention to explore the genetic relationship, especially life-threatening severe cutaneous adverse drug reactions (SCARs), including acute generalized exanthematous pustulosis (AGEP), drug reactions with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). In recent years, many studies have investigated the immune mechanism and genetic markers of DHRs. Besides, several studies have stated the associations between antibiotics-as well as anti-osteoporotic drugs (AOD)-induced SCARs and specific human leukocyte antigens (HLA) alleles. Strong associations between drugs and HLA alleles such as co-trimoxazole-induced DRESS and HLA-B*13:01 (Odds ratio (OR) = 45), dapsone-DRESS and HLA-B*13:01 (OR = 122.1), vancomycin-DRESS and HLA-A*32:01 (OR = 403), clindamycin-DHRs and HLA-B*15:27 (OR = 55.6), and strontium ranelate (SR)-SJS/TEN and HLA-A*33:03 (OR = 25.97) are listed. We summarized the immune mechanism of SCARs, update the latest knowledge of pharmacogenomics of antibiotics- and AOD-induced SCARs, and indicate the potential clinical use of these genetic markers for SCARs prevention in this mini review article.

IFIT2 Depletion Promotes Cancer Stem Cell-like Phenotypes in Oral Cancer.

(1) Background: Cancer stem cells (CSCs) are a small cell population associated with chemoresistance, metastasis and increased mortality rate in oral cancer. Interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) depletion results in epithelial to mesenchymal transition, invasion, metastasis, and chemoresistance in oral cancer. To date, no study has demonstrated the effect of IFIT2 depletion on the CSC-like phenotype in oral cancer cells. (2) Methods: Q-PCR, sphere formation, Hoechst 33,342 dye exclusion, immunofluorescence staining, and flow cytometry assays were performed to evaluate the expression of the CSC markers in IFIT2-depleted cells. A tumorigenicity assay was adopted to assess the tumor formation ability. Immunohistochemical staining was used to examine the protein levels of IFIT2 and CD24 in oral cancer patients. (3) Results: The cultured IFIT2 knockdown cells exhibited an overexpression of ABCG2 and CD44 and a downregulation of CD24 and gave rise to CSC-like phenotypes. Clinically, there was a positive correlation between IFIT2 and CD24 in the patients. IFIT2high/CD24high/CD44low expression profiles predicted a better prognosis in HNC, including oral cancer. The TNF-α blockade abolished the IFIT2 depletion-induced sphere formation, indicating that TNF-α may be involved in the CSC-like phenotypes in oral cancer. (4) Conclusions: The present study demonstrates that IFIT2 depletion promotes CSC-like phenotypes in oral cancer.

IFIT2-depleted metastatic oral squamous cell carcinoma cells induce muscle atrophy and cancer cachexia in mice.

BACKGROUND: Interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) is a reported metastasis suppressor in oral squamous cell carcinoma (OSCC). Metastases and cachexia may coexist. The effect of cancer metastasis on cancer cachexia is largely unknown. We aimed to address this gap in knowledge by characterizing the cachectic phenotype of an IFIT2-depleted metastatic OSCC mouse model. METHODS: Genetically engineered and xenograft tumour models were used to explore the effect of IFIT2-depleted metastatic OSCC on cancer cachexia. Muscle and organ weight changes, tumour burden, inflammatory cytokine profiles, body composition, food intake, serum albumin and C-reactive protein (CRP) levels, and survival were assessed. The activation of the IL6/p38 pathway in atrophied muscle was measured. RESULTS: IFIT2-depleted metastatic tumours caused marked body weight loss (-18.2% vs. initial body weight, P < 0.001) and a poor survival rate (P < 0.01). Skeletal muscles were markedly smaller in IFIT2-depleted metastatic tumour-bearing mice (quadriceps: -28.7%, gastrocnemius: -29.4%, and tibialis: -24.3%, all P < 0.001). Tumour-derived circulating granulocyte-macrophage colony-stimulating factor (+772.2-fold, P < 0.05), GROα (+1283.7-fold, P < 0.05), IL6 (+245.8-fold, P < 0.001), IL8 (+616.9-fold, P < 0.001), IL18 (+24-fold, P < 0.05), IP10 (+18.8-fold, P < 0.001), CCL2 (+439.2-fold, P < 0.001), CCL22 (+9.1-fold, P < 0.01) and tumour necrosis factor α (+196.8-fold, P < 0.05) were elevated in IFIT2-depleted metastatic tumour-bearing mice. Murine granulocyte colony-stimulating factor (+61.4-fold, P < 0.001) and IL6 (+110.9-fold, P < 0.01) levels were significantly increased in IFIT2-depleted metastatic tumour-bearing mice. Serum CRP level (+82.1%, P < 0.05) was significantly increased in cachectic shIFIT2 mice. Serum albumin level (-26.7%, P < 0.01) was significantly decreased in cachectic shIFIT2 mice. An assessment of body composition revealed decreased fat (-81%, P < 0.001) and lean tissue (-21.7%, P < 0.01), which was consistent with the reduced food intake (-19.3%, P < 0.05). Muscle loss was accompanied by a smaller muscle cross-sectional area (-23.3%, P < 0.05). Muscle atrophy of cachectic IFIT2-depleted metastatic tumour-bearing mice (i.v.-shIFIT2 group) was associated with elevated IL6 (+2.7-fold, P < 0.05), phospho-p38 (+2.8-fold, P < 0.05), and atrogin-1 levels (+2.3-fold, P < 0.05) in the skeletal muscle. Neutralization of IL6 rescued shIFIT2 conditioned medium-induced myotube atrophy (+24.6%, P < 0.01). CONCLUSIONS: Our results suggest that the development of shIFIT2 metastatic OSCC lesions promotes IL6 production and is accompanied by the loss of fat and lean tissue, anorexia, and muscle atrophy. This model is appropriate for the study of OSCC cachexia, especially in linking metastasis with cachexia.

Antitumor Effects of the Novel Quinazolinone Holu-12: Induction of Mitotic Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma CAL27 Cells.

BACKGROUND/AIM: Quinazolinone is a privileged chemical structure employed for targeting various types of cancer. This study aimed to demonstrate the antitumor activity of synthesized 6,7-disubstituted-2-(3-fluorophenyl) quinazolines (HoLu-11 to HoLu-14). MATERIALS AND METHODS: The cytotoxicity was assessed by the sulforhodamine B (SRB) assay. The cell cycle was examined by flow cytometry. The expression levels of cell cycle- and apoptosis-related proteins were estimated by western blotting. A xenograft animal model was used to explore the antitumor effects of HoLu-12. RESULTS: Among four synthetic quinazolinone derivatives, HoLu-12 significantly reduced the viability of oral squamous cell carcinoma (OSCC) cells. HoLu-12 induced G2/M arrest and increased the expression of cyclin B, histone H3 (Ser10) phosphorylation, and cleaved PARP, indicating that HoLu-12 could induce mitotic arrest and then apoptosis. Moreover, the combination of HoLu-12 and 5-fluorouracil (5-FU) displayed synergistic toxic effect on OSCC cells. HoLu-12 significantly inhibited tumor growth in vivo. CONCLUSION: HoLu-12 induces mitotic arrest and leads to apoptosis of OSCC cells. Furthermore, HoLu-12 alone or in combination with 5-FU is a potential therapeutic agent for OSCC.

SAHA Overcomes 5-FU Resistance in IFIT2-Depleted Oral Squamous Cell Carcinoma Cells.

Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) is a member of the interferon-stimulated gene family that contains tetratricopeptide repeats (TPRs), which mediate protein-protein interactions in various biological systems. We previously showed the depletion of IFIT2 enhanced cell migration and metastatic activity in oral squamous cell carcinoma (OSCC) cells via the activation of atypical PKC signaling. In this study, we found that IFIT2-knockdown cells displayed higher resistance to 5-fluorouracil (5-FU) than control cells. The comet assay and annexin V analysis showed decreased DNA damage and cell death in IFIT2-knockdown cells compared to control cells treated with 5-FU. Cell cycle progression was also perturbed by 5-FU treatment, with the accumulation of IFIT2-depleted cells in S phase in a time-dependent manner. We further observed the overexpression of thymidylate synthase (TS) and thymidine kinase (TK) in IFIT2-knockdown cells. Inhibition of TS alone or double inhibition of TS and TK1 using the siRNA technique increased susceptibility to 5-FU in IFIT2-knockdown cells. We further identified that suberanilohydroxamic acid (SAHA) treatment decreased the expression of TS in IFIT2-knockdown cells and demonstrated that pretreatment with SAHA sensitized IFIT2-knockdown cells to 5-FU in vitro and in vivo. In conclusion, IFIT2 knockdown enhances TS expression, which mediates 5-FU resistance, and SAHA pretreatment suppresses TS expression and hence sensitizes cells to 5-FU. SAHA will be an effective strategy for the treatment of OSCC patients with 5-FU resistance.

Design, synthesis and antitumour evaluation of pyrrolo[1,2-f]-phenanthridine and dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives.

A series of 1,2-bis(hydroxymethyl)pyrrolo[1,2-f]phenanthridine derivatives and their alkyl (ethyl and isopropyl) carbamates and 12,13-bis(hydroxymethyl)-9,14-dihydro-dibenzo[f,h]pyrrolo[1,2-b]isoquinoline derivatives were synthesized for antiproliferative evaluation. The preliminary antitumour studies revealed that these two types of bis(hydroxymethyl) derivatives showed significant antitumour activities and were able to inhibit the growth of various human tumour cell lines in vitro. Several of the derivatives were demonstrated to cause DNA interstrand cross-links by an alkaline agarose gel shifting assay. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, delaying cell cycle progression in the G2/M phase and triggering apoptosis. Compound 21a, dissolved in a vehicle suitable for intravenous administration, was selected for antitumour studies in animal models. We demonstrated that at a dose that did not cause body weight loss in mice, compound 21a could significantly suppress the growth of tumour xenografts of human lung cancer H460 and colorectal cancer HCT-116 cells in nude mice. Our present results confirm the antitumour activities of these conjugates.

Novel Antrodia cinnamomea Extract Reduced Cancer Stem-Like Phenotype Changes and Resensitized KRAS-Mutant Colorectal Cancer via a MicroRNA-27a Pathway.

Colorectal cancer (CRC) is one of the most common causes of death in Taiwan. Previous studies showed that Antrodia cinnamomea (AC) can treat poisoning, diarrhea, and various types of cancer. Therefore, we purified a novel ubiquinone derivative, AC009, and investigated its antitumor effects. Cell viability assays revealed that AC009 reduced the viability of several human CRC cell lines. AC009 treatment resulted in cell-cycle arrest/apoptosis, and these effects may occur via caspase and Bcl-2 signaling pathways. We demonstrated that AC009 could significantly inhibit in vivo tumor growth in xenograft mouse models. Using messenger RNA (mRNA) and microRNA (miRNA) microarrays, we found that KRAS gene expression was also regulated by AC009, possibly through specific miRNAs. AC009 also reduced cancer stem-cell marker CD44+/CD24+ expression and restored the tumor inhibition effect of cetuximab in KRAS-mutant CRC. Moreover, we found that miRNA-27a could restore the tumor inhibition effect of cetuximab in KRAS-mutant CRC cells. Taken together, our results suggest that AC009 has therapeutic potential against human wild-type and KRAS-mutant CRC.

Long-Term Exposure to Oroxylin A Inhibits Metastasis by Suppressing CCL2 in Oral Squamous Cell Carcinoma Cells.

Oroxylin A (Oro-A), the main bioactive flavonoid extracted from Scutellaria radix, has been reported to inhibit migration in various human cancer cell models. In this study, we further explored the anti-migration effects of Oro-A on oral squamous cell carcinoma (OSCC) cells and investigated the underlying mechanisms. A 24-h (short-term) exposure of OSCC cells to non-cytotoxic concentrations (5⁻20 µM) of Oro-A significantly suppressed cell migration according to a wound-healing assay. Furthermore, a 30-day exposure (long-term) to Oro-A (20 µM), which did not exhibit a cytotoxic effect on OSCC cells, significantly suppressed cell migration more than short-term Oro-A exposure. To uncover the molecular mechanisms underlying the inhibitory effect of long-term Oro-A exposure on OSCC migration, a cDNA microarray and the Ingenuity software were used. Overall, 112 upregulated and 356 downregulated genes were identified in long-term Oro-A-exposed cells compared with untreated OSCC cells. Among them, 75 genes were reported to be associated with cancer cell migration. Consistent with the cDNA microarray results, we found that the expression levels of several cell migration-related genes, such as LCN2, ID-1, MDK, S100A9 and CCL2, were significantly decreased in long-term Oro-A-exposed OSCC cells using a quantitative real-time polymerase chain reaction (Q-PCR) assay. The Western blotting and enzyme-linked immunosorbent assay (ELISA) results also demonstrated that CCL2 expression at the mRNA and protein levels was significantly decreased in long-term Oro-A-exposed OSCC cells compared with untreated OSCC cells. Moreover, the expression levels of downstream CCL2 targets, including p-ERK1/2, NFκB, MMP2, and MMP9, were also decreased in long-term Oro-A-exposed OSCC cells. Further, Oro-A treatment suppressed in vivo metastasis. These results suggest that long-term Oro-A treatment inhibits metastasis via CCL2 signaling in OSCC cells.

Prognostic Values of EPDR1 Hypermethylation and Its Inhibitory Function on Tumor Invasion in Colorectal Cancer.

Aberrant DNA methylation is a potential mechanism underlying the development of colorectal cancer (CRC). Thus, identification of prognostic DNA methylation markers and understanding the related molecular functions may offer a new perspective on CRC pathogenesis. To that end, we explored DNA methylation profile changes in CRC subtypes based on the microsatellite instability (MSI) status through genome-wide DNA methylation profiling analysis. Of 34 altered genes, three hypermethylated (epidermal growth factor, EGF; carbohydrate sulfotransferase 10, CHST10; ependymin related 1, EPDR1) and two hypomethylated (bone marrow stromal antigen 2, BST2; Rac family small GTPase 3, RAC3) candidates were further validated in CRC patients. Based on quantitative methylation-specific polymerase chain reaction (Q-MSP), EGF, CHST10 and EPDR1 showed higher hypermethylated levels in CRC tissues than those in adjacent normal tissues, whereas BST2 showed hypomethylation in CRC tissues relative to adjacent normal tissues. Additionally, among 75 CRC patients, hypermethylation of CHST10 and EPDR1 was significantly correlated with the MSI status and a better prognosis. Moreover, EPDR1 hypermethylation was significantly correlated with node negativity and a lower tumor stage as well as with mutations in B-Raf proto-oncogene serine/threonine kinase (BRAF) and human transforming growth factor beta receptor 2 (TGFßR2). Conversely, a negative correlation between the mRNA expression and methylation levels of EPDR1 in CRC tissues and cell lines was observed, revealing that DNA methylation has a crucial function in modulating EPDR1 expression in CRC cells. EPDR1 knockdown by a transient small interfering RNA significantly suppressed invasion by CRC cells, suggesting that decreased EPDR1 levels may attenuate CRC cell invasion. These results suggest that DNA methylation-mediated EPDR1 epigenetic silencing may play an important role in preventing CRC progression.

Blocking TNF-α inhibits angiogenesis and growth of IFIT2-depleted metastatic oral squamous cell carcinoma cells.

Our previous study demonstrated that the depletion of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) promoted metastasis and was associated with a poor prognosis in patients with oral squamous cell carcinoma (OSCC). Our current study explores the major downstream signaling involved in IFIT2 depletion-induced OSCC metastasis. To this end, we used two cell lines (designated sh-control-xeno and sh-IFIT2-xeno) derived from human OSCC xenografts expressing sh-control and sh-IFIT2, respectively, and one metastatic OSCC subline (sh-IFIT2-meta) from an IFIT2-depleted metastatic tumor. We found that the sh-IFIT2-meta cells proliferated more slowly than the sh-control-xeno cells but exhibited higher migration and chemoresistance. Using microarray technology and Ingenuity Pathway Analysis, we found that TNF-α was one of the major downstream targets in IFIT2-depleted OSCC cells. Quantitative real-time PCR, western blotting, and ELISA results confirmed that TNF-α was upregulated in the sh-IFIT2-meta cells. Blocking TNF-α abolished the angiogenic activity induced by the sh-IFIT2-meta cells. Furthermore, the human-specific TNF-α antibody golimumab significantly inhibited in vivo angiogenesis, tumor growth and metastasis of sh-IFIT2-meta cells. These results demonstrate that IFIT2 depletion results in TNF-α upregulation, leading to angiogenesis and metastasis of OSCC cells.

HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.

Heat shock protein 70 (HSP70) has been shown to be a substrate of Polo-like kinase 1 (PLK1), and it prevents cells arrested in mitosis by arsenic trioxide (ATO) from dying. Here, we report that HSP70 participates in ATO-induced spindle elongation, which interferes with mitosis progression. Our results demonstrate that HSP70 and PLK1 colocalize at the centrosome in ATO-arrested mitotic cells. HSP70 located at the centrosome was found to be phosphorylated by PLK1 at Ser6³¹ and Ser6³³. Moreover, unlike wild-type HSP70 (HSP70(wt)) and its phosphomimetic mutant (HSP70(SS631,633DD)), a phosphorylation-resistant mutant of HSP70 (HSP70(SS631,633AA)) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70(SS631,633AA). Conversely, mitotic spindles in cells ectopically expressing HSP70(SS631,633DD) were more resistant to nocodazole-induced depolymerization than in those expressing HSP70(wt) or HSP70(SS631,633AA). In addition, inhibition of PLK1 significantly reduced HSP70 phosphorylation and induced early onset of apoptosis in ATO-arrested mitotic cells. Taken together, our results indicate that PLK1-mediated phosphorylation and centrosomal localization of HSP70 may interfere with spindle dynamics and prevent apoptosis of ATO-arrested mitotic cells.

Asb6 upregulation by Areca nut extracts is associated with betel quid-induced oral carcinogenesis.

Betel quit (BQ) chewing is a popular habit, especially in southern and southeastern Asia. Areca nut extracts (ANE), the major components of BQ, have been documented to induce reactive oxygen species, and consequently to cause genetic damage. ANE usage is tightly linked to oral cancer; however, the details of the molecular mechanism that results in carcinogenesis remain unclear. Previously, we successfully established HaCaT cells surviving from the long-term exposure of sublethal doses of ANE (Lai KC, Lee TC. Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts. Mutat Res 2006;599:66-75). Here, we identified the upregulation of Asb6, a coupling protein to the APS adapter protein, which is involved in insulin signaling for glucose transportation, of normal keratinocytes and oral cancer cells under ANE treatment. Immunohistochemical analyses of Asb6 on oral squamous cell carcinoma (OSCC) tissues (n=57) demonstrated the positive correlation between Asb6 upregulation (cancerous tissues versus adjacent normal tissues) and clinicopathological features. We showed that the combination of ANE-enhanced Asb6 expression in vitro and Asb6 upregulation in OSCC patients leads to poor survival status. In conclusion, our results suggest that upregulated Asb6 could act as a prognostic marker for oral cancer.

IFN-induced protein with tetratricopeptide repeats 2 inhibits migration activity and increases survival of oral squamous cell carcinoma.

The function of the IFN-stimulated gene family protein, IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is poorly understood. Here, we report that IFIT2 colocalizes with cytokeratin 18 in oral squamous cell carcinoma (OSCC) cells. Treatment of OSCC cells with IFN-beta significantly increased the expression of IFIT2 and remarkably inhibited cell migration. To further explore the effect of IFIT2 on cell migration, IFIT2 expression was either silenced with a small interfering RNA or increased by ectopic expression. IFIT2 knockdown in OSCC cells led to a significantly higher level of migration in vitro (P < 0.05) compared with control cells; by contrast, IFIT2 overexpression led to a significantly lower level of migration in vitro (P < 0.05). Immunohistochemically, 71.4% of OSCC tissues had elevated IFIT2 protein levels compared with noncancerous matched tissues. Elevated IFIT2 protein expression was positively associated with tumor differentiation status and inversely associated with nodal stage in OSCC specimens (P < 0.05). Higher IFIT2 protein levels in tumor tissues were also associated with better patient survival (P < 0.01). Our present study shows an inverse correlation between IFIT2 expression and cell migration, suggesting that IFIT2 plays an important role in inhibiting this process and that its expression may be associated with better prognosis in patients with OSCC.

Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts.

Chewing betel quid (BQ) is a popular habit worldwide. A causal association between BQ chewing and oral cancer has been well documented. Emerging evidence indicates that sustained exposure to stress induces epigenetic reprogramming of some mammalian cells and increases the mutation rate to accelerate adaptation to stressful environments. In this study, we first confirmed that 24-h treatment with areca nut extracts (ANE; a major component of BQ) at doses over 40 microg/ml induced mutations at the hypoxanthine phosphoribisyltransferase (HPRT) locus in human keratinocytes (HaCaT cells). We then investigated whether the stress of long-term exposure to sublethal doses of ANE (0, 5 and 20 microg/ml for 35 passages) could enhance genetic damage to HaCaT cells. Compared to cells exposed to 0 or 5 microg/ml ANE, cells exposed to 20 microg/ml ANE were slightly but significantly more resistant to a 72-h treatment with ANE and its major ingredients, arecoline and arecaidine, but did not develop cross-resistance to other BQ ingredients or alcohol. The cells that received 20 microg/ml ANE for 35 passages also had a significantly increased mutation frequency at the HPRT locus and an increased frequency in the appearance of micronuclei compared to lower doses. Moreover, increased intracellular levels of reactive oxygen species and 8-hydroxyguanosine in cells exposed to 20 microg/ml ANE suggested that long-term ANE exposure results in the accumulation of oxidative damage. However, cells subjected to long-term treatment of 20 microg/ml ANE contained higher levels of glutathione than unexposed cells. Therefore, after long-term exposure to sublethal doses of ANE, intracellular antioxidative activity may also be enhanced in response to increased oxidative stress. These results suggest that stress caused by long-term ANE exposure enhances oxidative stress and genetic damage in human keratinocytes.