Combined Treatment of Heteronemin and Tetrac Induces Antiproliferation in Oral Cancer Cells.

BACKGROUND: Heteronemin, a marine sesterterpenoid-type natural product, possesses an antiproliferative effect in cancer cells. In addition, heteronemin has been shown to inhibit p53 expression. Our laboratory has demonstrated that the thyroid hormone deaminated analogue, tetrac, activates p53 and induces antiproliferation in colorectal cancer. However, such drug mechanisms are still to be studied in oral cancer cells. METHODS: We investigated the antiproliferative effects by Cell Counting Kit-8 and flow cytometry. The signal transduction pathway was measured by Western blotting analyses. Quantitative PCR was used to evaluate gene expression regulated by heteronemin, 3,3',5,5'-tetraiodothyroacetic acid (tetrac), or their combined treatment in oral cancer cells. RESULTS: Heteronemin inhibited not only expression of proliferative genes and Homo Sapiens Thrombospondin 1 (THBS-1) but also cell proliferation in both OEC-M1 and SCC-25 cells. Remarkably, heteronemin increased TGF-ß1 expression in SCC-25 cells. Tetrac suppressed expression of THBS-1 but not p53 expression in both cancer cell lines. Furthermore, the synergistic effect of tetrac and heteronemin inhibited ERK1/2 activation and heteronemin also blocked STAT3 signaling. Combined treatment increased p53 protein and p53 activation accumulation although heteronemin inhibited p53 expression in both cancer cell lines. The combined treatment induced antiproliferation synergistically more than a single agent. CONCLUSIONS: Both heteronemin and tetrac inhibited ERK1/2 activation and increased p53 phosphorylation. They also inhibited THBS-1 expression. Moreover, tetrac suppressed TGF-ß expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

Role of Integrin αvß3 in Doxycycline-Induced Anti-Proliferation in Breast Cancer Cells.

Doxycycline, an antibiotic, displays the inhibition of different signal transduction pathways, such as anti-inflammation and anti-proliferation, in different types of cancers. However, the anti-cancer mechanisms of doxycycline via integrin αvß3 are incompletely understood. Integrin αvß3 is a cell-surface anchor protein. It is the target for estrogen, androgen, and thyroid hormone and plays a pivotal role in the proliferation, migration, and angiogenic process in cancer cells. In our previous study, thyroxine hormones can interact with integrin αvß3 to activate the extracellular signal-regulated kinase 1/2 (ERK1/2), and upregulate programmed death-ligand 1 (PD-L1) expression. In the current study, we investigated the inhibitory effects of doxycycline on proliferation in two breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Doxycycline induces concentration-dependent anti-proliferation in both breast cancer cell lines. It regulates gene expressions involved in proliferation, pro-apoptosis, and angiogenesis. Doxycycline suppresses cell cyclin D1 (CCND1) and c-Myc which play crucial roles in proliferation. It also inhibits PD-L1 gene expression. Our findings show that modulation on integrin αvß3 binding activities changed both thyroxine- and doxycycline-induced signal transductions by an integrin αvß3 inhibitor (HSDVHK-NH2). Doxycycline activates phosphorylation of focal adhesion kinase (FAK), a downstream of integrin, but inhibits the ERK1/2 phosphorylation. Regardless, doxycycline-induced FAK phosphorylation is blocked by HSDVHK-NH2. In addition, the specific mechanism of action associated with pERK1/2 inhibition via integrin αvß3 is unknown for doxycycline treatment. On the other hand, our findings indicated that inhibiting ERK1/2 activation leads to suppression of PD-L1 expression by doxycycline treatment. Furthermore, doxycycline-induced gene expressions are disturbed by a specific integrin αvß3 inhibitor (HSDVHK-NH2) or a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) kinase (MAPK/ERK, MEK) inhibitor (PD98059). The results imply that doxycycline may interact with integrin αvß3 and inhibits ERK1/2 activation, thereby regulating cell proliferation and downregulating PD-L1 gene expression in estrogen receptor (ER)-negative breast cancer MDA-MB-231 cells.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside improves female ovarian aging.

Reduced fertility associated with normal aging may reflect the over-maturity of oocytes. It is increasingly important to reduce aging-induced infertility since recent trends show people marrying at later ages. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), a polyphenol extracted from Polygonum multiflorum, has been reported to have anti-inflammatory and anti-aging properties. To evaluate whether THSG can reduce aging-related ovarian damage in a female mouse model of aging, THSG was administered by gavage at a dose of 10 mg/kg twice weekly, starting at 4 weeks of age in a group of young mice. In addition, the effect of THSG in a group of aged mice was also studied in mice starting at 24 weeks of age. The number of oocytes in the THSG-fed group was higher than in the untreated control group. Although the percentage of secondary polar bodies (PB2) decreased during aging in the THSG-fed group, it decreased much more slowly than in the age-matched control group. THSG administration increased the quality of ovaries in young mice becoming aged. Western blotting analyses also indicated that CYP19, PR-B, and ER-ß expressions were significantly increased in 36-week-old mice. THSG also increased oocyte numbers in aged mice compared to mice without THSG fed. Studies of qPCR and immunohistochemistry (IHC) analyses of ovaries in the aged mice groups were conducted. THSG increased gene expression of anti-Müllerian hormone (AMH), a biomarker of oocyte number, and protein accumulation in 40-week-old mice. THSG increased the expression of pgc1α and atp6, mitochondrial biogenesis-related genes, and their protein expression. THSG also attenuated the fading rate of CYP11a and CYP19 associated with sex hormone synthesis. And THSG maintains a high level of ER-ß expression, thereby enhancing the sensitivity of estrogen. Our findings indicated that THSG increased or extended gene expression involved in ovarian maintenance and rejuvenation in young and aged mice. On the other hand, THSG treatments significantly maintained oocyte quantity and quality in both groups of young and aged mice compared to each age-matched control group. In conclusion, THSG can delay aging-related menopause, and the antioxidant properties of THSG may make it suitable for preventing aging-induced infertility.

Heteronemin and tetrac derivatives suppress non-small cell lung cancer growth via ERK1/2 inhibition.

The most common cancer, lung cancer, causes deaths worldwide. Most lung cancer patients have non-small cell lung carcinomas (NSCLCs) with a poor prognosis. The chemotherapies frequently cause resistance therefore search for new effective drugs for NSCLC patients is an urgent and essential issue. Deaminated thyroxine, tetraiodothyroacetic acid (tetrac), and its nano-analogue (NDAT) exhibit antiproliferative properties in several types of cancers. On the other hand, the most abundant secondary metabolite in the sponge Hippospongia sp., heteronemin, shows effective cytotoxic activity against different types of cancer cells. In the current study, we investigated the anticancer effects of heteronemin against two NSCLC cell lines, A549 and H1299 cells in vitro. Combined treatment with heteronemin and tetrac derivatives synergistically inhibited cancer cell growth and significantly modulated the ERK1/2 and STAT3 pathways in A549 cells but only ERK1/2 in H1299 cells. The combination treatments induce apoptosis via the caspases pathway in A549 cells but promote cell cycle arrest via CCND1 and PCNA inhibition in H1299 cells. In summary, these results suggest that combined treatment with heteronemin and tetrac derivatives could suppress signal transduction pathways essential for NSCLC cell growth. The synergetic effects can be used potentially as a therapeutic procedure for NSCLC patients.

Effect of Estrogen on Heteronemin-Induced Anti-proliferative Effect in Breast Cancer Cells With Different Estrogen Receptor Status.

Estrogen (E2) has multiple functions in breast cancers including stimulating cancer growth and interfering with chemotherapeutic efficacy. Heteronemin, a marine sesterterpenoid-type natural product, has cytotoxicity on cancer cells. Breast cancer cell lines, MCF-7 and MDA-MB-231, were used for investigating mechanisms involved in inhibitory effect of E2 on heteronemin-induced anti-proliferation in breast cancer cells with different estrogen receptor (ER) status. Cytotoxicity was detected by cell proliferation assay and flow cytometry, gene expressions were determined by qPCR, mechanisms were investigated by Western blot and Mitochondrial ROS assay. Heteronemin exhibited potent cytotoxic effects against both ER-positive and ER-negative breast cancer cells. E2 stimulated cell growth in ER-positive breast cancer cells. Heteronemin induced anti-proliferation via suppressing activation of ERK1/2 and STAT3. Heteronemin suppressed E2-induced proliferation in both breast cancer cells although some gene expressions and anti-proliferative effects were inhibited in the presence of E2 in MCF-7 and MDA-MB-231 cells with a higher concentration of heteronemin. Heteromenin decreased the Bcl-2/Bax ratio to inhibit proliferation in MDA-MB-231 but not in MCF-7 cells. Both heteronemin and E2 increased mitochondrial reactive oxygen species but combined treatment reversed superoxide dismutase (SOD)s accumulation in MCF-7 cells. Heteronemin caused G0/G1 phase arrest and reduced the percentage of cells in the S phase to suppress cancer cell growth. In conclusion, Heteronemin suppressed both ER-positive and ER-negative breast cancer cell proliferation. Interactions between E2 and heteronemin in signal transduction, gene expressions, and biological activities provide insights into the complex pathways by which anti-proliferation is induced by heteronemin in E2-replete environments.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

NDAT suppresses pro-inflammatory gene expression to enhance resveratrol-induced anti-proliferation in oral cancer cells.

Nano-diamino-tetrac (NDAT), a tetraiodothyroxine deaminated nano-particulated analog, has shown to inhibit expression of pro-inflammatory genes. NDAT inhibits expression of programmed death-ligand 1 (PD-L1). On the other hand, in addition to inhibiting inflammatory effect, the stilbene, resveratrol induces expression of cyclooxygenase-2 (COX-2) and its accumulation. Sequentially, inducible COX-2 complexes with p53 and induces p53-dependent anti-proliferation. In current study, we investigated mechanisms involved in combined treatment of NDAT and resveratrol on anti-proliferation in human oral cancer cells. Both resveratrol and NDAT inhibited expression of pro-inflammatory IL-1ß and TNF-α. They also inhibited expression of CCND1 and PD-L1. Both resveratrol and NDAT induced BAD expression but only resveratrol induced COX-2 expression in both OEC-M1 and SCC-25 cells. Combined treatment attenuated gene expression significantly compared with resveratrol treatment in both cancer cell lines. Resveratrol reduced nuclear PD-L1 accumulation which was enhanced by a STAT3 inhibitor, S31-201 or NDAT suggesting that NDAT may inactivate STAT3 to inhibit PD-L1 accumulation. In the presence of T4, NDAT further enhanced resveratrol-induced anti-proliferation in both cancer cell lines. These findings provide a novel understanding of the inhibition of NDAT in thyroxine-induced pro-inflammatory effect on resveratrol-induced anticancer properties.

Size-exclusion chromatography using reverse-phase columns for protein separation.

Reverse-phase (RP) liquid chromatography (RPLC) and size-exclusion chromatography (SEC) are methods commonly used for protein/peptide separation, and they are based on distinct principles. This study develops a method using RP columns for size-based separation of protein mixtures. Results show that high concentrations of acetonitrile with trifluoroacetic acid as an acid modifier successfully suppressed interactions between proteins and the stationary phase and allowed the RP column to act as a SEC column to separate proteins based on their molecular weight. The reduction of protein disulfide bonds resulted in an improved correlation between the retention time and molecular weight in the RP-based SEC, which indicates that conformation-dependent SEC retention is less important for disulfide-reduced proteins using a current mobile phase. Importantly, the employed salt-free mobile phase allowed the RP-based SEC system to be directly coupled with online mass spectrometry (MS) analysis. Furthermore, by reducing the flow rate and increasing the column length, the separation efficiency was further improved and no adverse effects due to the prolonged separation were observed, which indicates the potential of this strategy to serve as a first-dimensional separation method for constructing an online multi-dimensional LC system. The size-based separation phenomenon with RP columns was further evaluated using a complex protein mixture (a cell lysate), and compared to conventional SEC, more proteins of the cell lysate were observed following the SEC separation principle, which implies the better generality of usage of the RP-based SEC method for protein separation. In summary, results show that the RP-based SEC is highly efficient in achieving protein fractionation. In addition, the employed salt-free mobile phase provides excellent compatibility of the RP-based SEC with other separation strategies or online mass spectrometric analysis. We anticipate that laboratories using RP-HPLC for protein separation will easily be able to move to the size-based separation mode using the same RP-HPLC system.

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach.

In Photofrin-mediated photodynamic therapy (PDT), cell fate can be modulated by the subcellular location of Photofrin. PDT triggers oxidative damage to target cells, including the methionine (Met) oxidation of proteins. Here, we developed a new Met-containing peptide enrichment protocol combined with SILAC-based quantitative proteomics, and used this approach to explore the global Met oxidation changes of proteins in PDT-treated epidermoid carcinoma A431 cells preloaded with Photofrin at the plasma membrane, ER/Golgi, or ubiquitously. We identified 431 Met-peptides corresponding to 302 proteins that underwent severe oxidation upon PDT and observed overrepresentation of proteins related to the cell surface, plasma membrane, ER, Golgi, and endosome under all three conditions. The most frequently oxidized Met-peptide sequence was "QAMXXMM-E/G/M-S/G-A/G/F-XG". We also identified several hundred potential Photofrin-binding proteins using affinity purification coupled with LC-MS/MS, and confirmed the bindings of EGFR and cathepsin D with Photofrin. The enzyme activities of both proteins were significantly reduced by Photofrin-PDT. Our results shed light on the global and site-specific changes in Met-peptide oxidation among cells undergoing Photofrin-PDT-mediated oxidative stress originating from distinct subcellular sites, and suggest numerous potential Photofrin-binding proteins. These findings provide new insight into the molecular targets through which Photofrin-PDT has diverse effects on target cells.

Design and development of solar power-assisted manual/electric wheelchair.

Wheelchairs are an essential assistive device for many individuals with injury or disability. Manual wheelchairs provide a relatively low-cost solution to the mobility needs of such individuals. Furthermore, they provide an effective means of improving the user's cardiopulmonary function and upper-limb muscle strength. However, manual wheelchairs have a loss gross mechanical efficiency, and thus the risk of user fatigue and upper-limb injury is increased. Electric-powered wheelchairs reduce the risk of injury and provide a more convenient means of transportation. However, they have a large physical size and are relatively expensive. Accordingly, the present study utilizes a quality function deployment method to develop a wheelchair with a user-selectable manual/electric propulsion mode and an auxiliary solar power supply system. The auxiliary solar power supply increased the travel range of the wheelchair by approximately 26% compared with that of a wheelchair powered by battery alone. Moreover, the wheelchair has a modular design and can be disassembled and folded for ease of transportation or storage. Overall, the present results suggest that the proposed wheelchair provides an effective and convenient means of meeting the mobility needs of individuals with mobility difficulties.