Peroxiredoxin 2 is highly expressed in human oral squamous cell carcinoma cells and is upregulated by human papillomavirus oncoproteins and arecoline, promoting proliferation.

Peroxiredoxin 2 (PRDX2) is upregulated in various cancers including oral squamous cell carcinoma (OSCC). It is a known tumor promoter in some cancers, but its role in OSCC is unclear. This study aimed to investigate the effect of arecoline, an alkaloid of the betel nut, and human papillomavirus type 16 (HPV16) E6/E7 oncoproteins on induction of PRDX2 expression, and also the effects of PRDX2 overexpression in oral cell lines. Levels of PRDX2 protein were determined using western blot analysis of samples of exfoliated normal oral cells (n = 75) and oral lesion cells from OSCC cases (n = 75). Some OSCC cases were positive for HPV infection and some patients had a history of betel quid chewing. To explore the level of PRDX2 by western blot, the proteins were extracted from oral cell lines that were treated with arecoline or retroviruses containing HPV16 E6 gene and HPV16 E6/E7 expressing vector. For analysis of PRDX2 functions, cell proliferation, cell-cycle progression, apoptosis and migration was compared between oral cells overexpressing PRDX2 and cells with PRDX2-knockdown. PRDX2 expression levels tended to be higher in OSCC samples that were positive for HPV infection and had history of betel quid chewing. Arecoline treatment in vitro at low concentrations and overexpression of HPV16 E6 or E6/E7 in oral cells induced PRDX2 overexpression. Interestingly, in oral cells, PRDX2 promoted cell proliferation, cell-cycle progression (G2/M phase), cell migration and inhibited apoptosis. Upregulation of PRDX2 in oral cells was induced by arecoline and HPV16 oncoproteins and promoted growth of OSCC cells

Suppression of miR-22, a tumor suppressor in cervical cancer, by human papillomavirus 16 E6 via a p53/miR-22/HDAC6 pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that function to down-regulate gene expression involving in various cellular processes related to carcinogenesis. Recently, miR-22 was identified as a tumor-suppressing miRNA in many human cancers. However, the regulatory mechanism and the specific function of this miRNA in cervical cancer remain unclear. In the present study, we carried out gene transfection, western blot and quantitative RT-PCR to explore the regulatory mechanism and the functional role of miR-22 in cervical cancer. We verified that miR-22 was down-regulated in cervical cancer tissues and cervical cancer cell lines relative to matched non-tumor tissues and normal human cervical keratinocyte line (HCK1T). By contrast, histone deacetylase 6 (HDAC6) was inversely correlated with miR-22 in both cervical tissues and cancer cell lines. Mechanically, HDAC6 was down-regulated by miR-22 at the post-transcriptional level, via a specific target site within the 3'UTR, identified by a luciferase reporter assay. Moreover, we also showed that the correlation between miR-22 and HDAC6 expression was regulated by an E6/p53 pathway in HCK1Ts expressing HPV16 E6. For functional study, an ectopic expression of miR-22 could inhibit cell proliferation and migration, and could induce apoptosis of cervical cancer cell lines. These findings demonstrated that miR-22 was down-regulated in cervical cancer and inversely collated with its downstream target HDAC6. MiR-22 acts as tumor suppressor by inhibiting proliferation and migration, and by inducing apoptosis of cervical cancer cell lines by targeting the 3'UTR of HDAC6. This newly identified E6/p53/miR-22/HDAC6 regulatory network might be a candidate therapeutic target for cervical cancer

Dinactin: A New Antitumor Antibiotic with Cell Cycle Progression and Cancer Stemness Inhibiting Activities in Lung Cancer

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the most complex diseases, despite the existence of effective treatments such as chemotherapy and immunotherapy. Since cancer stem cells (CSCs) are responsible for chemo- and radio-resistance, metastasis, and cancer recurrence, finding new therapeutic targets for CSCs is critical. Dinactin is a natural secondary metabolite produced by microorganisms. Recently, dinactin has been revealed as a promising antitumor antibiotic via various mechanisms. However, the evidence relating to cell cycle progression regulation is constrained, and effects on cancer stemness have not been elucidated. Therefore, the aim of this study is to evaluate the new function of dinactin in anti-NSCLC proliferation, focusing on cell cycle progression and cancer stemness properties in Lu99 and A549 cells. Flow cytometry and immunoblotting analyses revealed that 0.1–1 µM of dinactin suppresses cell growth through induction of the G0/G1 phase associated with down-regulation of cyclins A, B, and D3, and cdk2 protein expression. The tumor-sphere forming capacity was used to assess the effect of dinactin on the cancer stemness potential in NSCLC cells. At a concentration of 1 nM, dinactin reduced both the number and size of the tumor-spheres. The quantitative RT-PCR analyses indicated that dinactin suppressed sphere formation by significantly reducing expression of CSC markers (i.e., ALDH1A1, Nanog, Oct4, and Sox2) in Lu99 cells. Consequently, dinactin could be a promising strategy for NSCLC therapy targeting CSCs

Effects of arecoline on proliferation of oral squamous cell carcinoma cells by dysregulating c-Myc and miR-22, directly targeting oncostatin M

<div><p>Arecoline, the major alkaloid of areca nut, is known to induce oral carcinogenesis, however, its mechanism is still needed to elucidate. This study investigated the effects of arecoline on cell viability and cell-cycle progression of oral squamous cell carcinoma (OSCC) cells as well as a relevant cellular gene expression. The results showed that a low concentration of arecoline (0.025 μg/ml) increased OSCC cell viability, proportion of cells in G2/M phase and cell proliferation. Simultaneously, it induced IL-6, STAT3 and c-Myc expression. Interestingly, c-<i>myc</i> promoter activity was also induced by arecoline. MiR-22 expression in arecoline-treated OSCC cells was suppressed and comparable to an upregulated c-Myc expression. In arecoline-treated OSCC cells, oncostatin M (OSM) expression was significantly upregulated and inversely correlated with miR-22 expression. Likewise, OSM expression and its post-transcriptional activity were significantly decreased in miR-22-transfected OSCC and 293FT cells. This result demonstrated that miR-22 directly targeted OSM. Interestingly, miR-22 played an important role as a tumor suppresser on suppressing cell proliferation, migration and cell-cycle progression of OSCC cells. This result suggested the effect of arecoline to promote cell proliferation and cell-cycle progression of OSCC cells might be involved in induction of c-Myc expression and reduction of miR-22 resulting in OSM upregulation.</p></div

The effects of arecoline on cell viability and cell-cycle progression.

<p>Cytotoxicity (A and B) and cell proliferation (C and D) were determined in arecoline-untreated or treated OSCC cell lines at various concentrations for 24 hours using the MTT assay. Statistical significance of the differences of cell viability (%) was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001). Cell-cycle phase distribution (E and F) in ORL-48(T) cells treated with 0 and 0.025 μg/ml of arecoline in synchronized condition was analyzed by flow cytometry. The percentages of G0/G1, S and G2/M population (G) of arecoline-treated cells were compared to untreated ORL-48(T) cells as control. Statistical significance of the differences of G2/M population was analyzed using Paired <i>t</i>-test (*<i>P</i> < 0.05).</p

Relative expression levels of miR-22 and OSM.

<p>100 and 500 ng/well of mock control (pIRES2-EGFP vector) and pIRES-miR-22 vectors were transfected into ORL-48(T) and ORL-136(T) cells. At 24 and 48 hours post-transfection, pri-miR-22 (A-B) and OSM (C-D) expression was determined by RT-PCR. Protein levels of OSM (E) were determined in ORL-48(T) and ORL-136(T) cells at 48 hours after transfection with 100 ng of mock control and pIRES-miR-22 vectors. Relative intensity of OSM protein band (F-G) was calculated using ImageJ 1.49v software. Statistical significance of the differences was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001).</p

Effect of arecoline on IL-6 and STAT3 in ORL-48(T) and ORL-136(T) cells.

<p>ORL-48(T) and ORL-136(T) cells were treated with 0, 0.025 and 25 μg/ml arecoline for 24 hours. Expression of IL-6 (A and D) and STAT3 (B and E) were investigated by RT-PCR and their amplicons were visualized by 2% agarose gel electrophoresis (C and F). Statistical significance of the differences of relative expression was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05 and **<i>P</i> < 0.01).</p

MiR-22 targets OSM and miR-22 functions in cell proliferation, migration and cell-cycle assay.

<p>The construct of the miR-22 targets sequence within the OSM 3′UTR WT and Mut in pGL3-Control vector. The luciferase gene was linked to the 3′UTR WT and Mut of OSM. 293FT cells were co-transfected with 250 ng pIRES-miR-22 and 100 ng pGL3-OSM 3′UTR WT or Mut vectors (A). The normalized luciferase activity in pIRES-miR-22 and pGL3-OSM 3′UTR WT or Mut co-transfected cells was relative to normalized luciferase activity of pIRES2-EGFP and OSM 3′UTR WT or Mut co-transfected cells (B). A green fluorescence expression vector (pEGFP-N3) was transfected for monitoring transfection efficiency. Statistical significance of the differences of luciferase activity was analyzed using Two-way ANOVA (*<i>P</i> < 0.05). Cell proliferation and migration in pIRES-miR-22-transfected ORL-48(T) cells were measured by a hemocytometer and wound healing assay at different incubation time points (C-E). The photograph was taken under 4X objective lens NIS-Elements Advanced Research Imaging Software version 3.0. Statistical significance of the differences of cell viability and wound closure was analyzed using Student's <i>t</i>-test (*<i>P</i> < 0.05 and ***<i>P</i> < 0.001). Cell-cycle assay in miR-22 or mock-transfected ORL-48(T) for 48 hours post-transfection was performed by flow cytometry (F). Statistical significance of the differences of G2/M and G0/G1 population was analyzed using Paired <i>t</i>-test (*<i>P</i> < 0.05 and (**<i>P</i> < 0.01, respectively).</p

Primer sequences.

<p>Primer sequences.</p