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Uterine fibroids, also known as uterine leiomyoma is the most common benign tumor of the uterus found in women of reproductive age. Uterine fibroids are the cause of major quality-of-life issues for approximately 25% of all women who suffer from clinically significant symptoms of uterine fibroid. Despite the prevalence of fibroid, currently, there are no effective treatment options for fibroid. The lack of understanding of the etiology of fibroid contributes to the scarcity of medical therapies available. Sex steroid hormones, dysregulation of cell signaling pathways, miRNA expression, and cytogenetic abnormalities may all implicate in fibroid etiology. Several herbal medicines have been used as anti-inflammation and antitumor agents. All of them have a common capability to inhibit expression of pro-inflammatory cytokines, proliferative genes, and pro-angiogenetic genes. Exploring herbal medicines as remedies lighten the hope of treatment. In the current review article, we discuss signal transduction pathways activated herbal medicines. We also address the possibility of using herbal medicines for uterine fibroid treatment.

Leiomyomas (myomas) are the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. In the current study, we investigated the inhibitory effect of resveratrol on the proliferation of primary human myoma cell cultures. Resveratrol arrested cell proliferation via integrin αvβ3. It also inhibited integrin αvβ3 expression and protein accumulation. Concurrently, constitutive AKT phosphorylation in myoma cells was inhibited by resveratrol. Expressions of proapoptotic genes, such as cyclooxygenase (COX)-2, p21 and CDKN2, were induced by resveratrol in myoma cells. On the other hand, expressions of proliferative (anti-apoptotic) genes were either inhibited, as in BCL2, or unchanged, as in cyclin D1 and proliferating cell nuclear antigen (PCNA). The accumulation of insulin-like growth factor (IGF)-1 receptor (IGF-1R) was inhibited by resveratrol in primary myoma cells. IGF-1-induced cell proliferation was inhibited by co-incubation with resveratrol. Therefore, growth modulation of myoma cells occurs via mechanisms dependent on cross-talk between integrin αvβ3 and IGF-1R. Our findings suggest that resveratrol can be considered an alternative therapeutic agent for myomas.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth induced by periodontopathic bacteria that results in the progressive destruction of periodontal tissues. Treatment of periodontitis is painful and time-consuming. Recently, herbal medicines have been considered for use in treating inflammation-related diseases, including periodontitis. Resveratrol and its derivative 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucoside (THSG), a polyphenol extracted from Polygonum multiflorum, have anti-inflammatory properties and other medical benefits. Here, we highlight the importance of resveratrol and its glycosylated derivative as possible complementary treatments for periodontitis and their potential for development as innovative therapeutic strategies. In addition, we present evidence and discuss the mechanisms of action of resveratrol and THSG on periodontitis, focusing on Porphyromonas gingivalis-induced inflammatory responses in human gingival fibroblasts and animal modeling of ligature-induced periodontitis. We also illuminate the signal transduction pathways and the cytokines involved.

Nonpeptide hormones, such as thyroid hormone, dihydrotestosterone, and estrogen, have been shown to stimulate cancer proliferation via different mechanisms. Aside from their cytosolic or membrane-bound receptors, there are receptors on integrin αv β3 for nonpeptide hormones. Interaction between hormones and integrin αv β3 can induce signal transduction and eventually stimulate cancer cell proliferation. Resveratrol induces inducible COX-2-dependent antiproliferation via integrin αv β3 . Resveratrol and hormone-induced signals are both transduced by activated extracellular-regulated kinases 1 and 2 (ERK1/2); however, hormones promote cell proliferation, while resveratrol induces antiproliferation in cancer cells. Hormones inhibit resveratrol-stimulated phosphorylation of p53 on Ser15, resveratrol-induced nuclear COX-2 accumulation, and formation of p53-COX-2 nuclear complexes. Subsequently, hormones impair resveratrol-induced COX-2-/p53-dependent gene expression. The inhibitory effects of hormones on resveratrol action can be blocked by different antagonists of specific nonpeptide hormone receptors but not integrin αv β3 blockers. Results suggest that nonpeptide hormones inhibit resveratrol-induced antiproliferation in cancer cells downstream of the interaction between ligand and receptor and ERK1/2 activation to interfere with nuclear COX-2 accumulation. Thus, the surface receptor sites for resveratrol and nonpeptide hormones are distinct and can induce discrete ERK1/2-dependent downstream antiproliferation biological activities. It also indicates the complex pathways by which antiproliferation is induced by resveratrol in various physiological hormonal environments. .

Background: Reverse T3 (rT3; 3,3',5'-triiodo-L-thyronine) is widely regarded as an inactive naturally occurring analog of thyroid hormone. rT3 is known to bind to the thyroid hormone analog receptor on plasma membrane integrin αvβ3. This integrin is generously expressed by tumor cells and is the initiation site for the stimulation by L-thyroxine (T4) at physiological free concentrations on cancer cell proliferation. Results: In the present studies, we show that rT3 caused increases of proliferation in vitro of 50% to 80% (P < 0.05-0.001) of human breast cancer and glioblastoma cells. Conclusion: rT3 may be a host factor supporting cancer growth.

Colorectal cancer is a serious medical problem in Taiwan. New, effective therapeutic approaches are needed. The selection of promising anticancer drugs and the transition from pre-clinical investigations to clinical trials are often challenging. The deaminated thyroid hormone analog (tetraiodothyroacetic acid, tetrac) and its nanoparticulate analog (NDAT) have been shown to have anti-proliferative activity in vitro and in xenograft model of different neoplasms, including colorectal cancers. However, mechanisms involved in tetrac- and NDAT-induced anti-proliferation in colorectal cancers are incompletely understood. We have investigated possible mechanisms of tetrac and NDAT action in colorectal cancer cells, using a perfusion bellows cell culture system that allows efficient, large-scale screening for mechanisms of drug actions on tumor cells. Although integrin αvβ3 in K-RAS wild type colorectal cancer HT-29 cells was far less than that in K-RAS mutant HCT116 cells, HT-29 was more sensitive to both tetrac and NDAT. Results also indicate that both tetrac and NDAT bind to tumor cell surface integrin αvβ3, and the agents may have different mechanisms of anti-proliferation in colorectal cancer cells. K-RAS status appears to play an important role in drug resistance that may be encountered in treatment with this drug combination.

A marine sesterterpenoid-type natural product, heteronemin, retains anticancer effects. In the current study, we investigate the antitumor mechanism of heteronemin in cholangiocarcinoma cells and further explore its molecular targets. Initially, heteronemin exhibited potent cytotoxic effects against cholangiocarcinoma HuccT1 and SSP-25 cells. In vitro, heteronemin altered the abilities of cell adhesion and cell migration in HuccT1 and SSP-25 cell lines. It repressed messenger ribonucleic acid (mRNA) expression levels of transforming growth factor (TGF)-β, mothers against decapentaplegic homolog (SMAD) and Myc,whose protein products play important roles in regulating cell growth, angiogenesis, and metastasis. In addition, heteronemin altered several signaling pathways. The results indicate that heteronemin was able to modulate cell adhesion, the expression of extracellular matrix (ECM) receptors, the TGF-β pathway, cell motility, the membrane integration, metastasis response, matrix metalloproteinase (MMP) remodeling, the regulation of metabolism, sprouting angiogenesis, transcription factors, and vasculogenesis in cholangiocarcinoma cell lines. The results also suggest that it activated multiple signal transduction pathways to induce an anti-proliferation effect and anti-metastasis in cholangiocarcinoma. In conclusion, heteronemin may be used as a potential medicine for anticancer therapy.

// Yu-Chen SH Yang 1, * , Yu-Tang Chin 2, * , Meng-Ti Hsieh 2, 3 , Hsuan-Yu Lai 2 , Chien-Chih Ke 4 , Dana R. Crawford 5 , Oscar K. Lee 6 , Earl Fu 7 , Shaker A. Mousa 8 , Patricia Grasso 9 , Leroy F. Liu 2 , Heng-Yu Chang 10, 11 , Heng-Yuan Tang 8 , Hung-Yun Lin 2, 3 , Paul J. Davis 8, 9 1 Joint Biobank, Office of Human Research, Taipei Medical University, Taipei, Taiwan 2 Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan 3 PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 4 Biomedical Imaging Research Center, National Yang-Ming University, Taipei, Taiwan 5 Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, USA 6 Stem Cell Research Center, National Yang-Ming University, Taipei, Taiwan 7 Department of Periodontology, School of Dentistry, National Defense Medical College, Taipei, Taiwan 8 Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, New York, USA 9 Department of Medicine, Albany Medical College, Albany, New York, USA 10 Department of Biochemistry and Molecular Cell Biology, College of Medicine, Taipei Medical University, Taipei, Taiwan 11 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan * These authors have contributed equally to this work Correspondence to: Heng-Yu Chang, e-mail: hychang@tmu.edu.tw Hung-Yun Lin, e-mail: linhy@tmu.edu.tw Keywords: obesity, leptin, OB3-leptin peptide, cancer cell invasion Received: September 15, 2015 Accepted: March 18, 2016 Published: March 30, 2016 ABSTRACT Obesity results in increased secretion of cytokines from adipose tissue and is a risk factor for various cancers. Leptin is largely produced by adipose tissue and cancer cells. It induces cell proliferation and may serve to induce various cancers. OB3-leptin peptide (OB3) is a new class of functional leptin peptide. However, its mitogenic effect has not been determined. In the present study, because of a close link between leptin and the hypothalamic-pituitary-thyroid axis, OB3 was compared with leptin in different thyroid cancer cells for gene expression, proliferation and invasion. Neither agent stimulated cell proliferation. Leptin stimulated cell invasion, but reduced adhesion in anaplastic thyroid cancer cells. Activated ERK1/2 and STAT3 contributed to leptin-induced invasion. In contrast, OB3 did not affect expression of genes involved in proliferation and invasion. In vivo studies in the mouse showed that leptin, but not OB3, significantly increased circulating levels of thyrotropin (TSH), a growth factor for thyroid cancer. In summary, OB3 is a derivative of leptin that importantly lacks the mitogenic effects of leptin on thyroid cancer cells.

Estrogen, 17β-estradiol (E2) has multiple functions in breast cancers including stimulating cancer growth and interfering with chemotherapeutic efficacy. Heteronemin, a marine sesterterpenoid-type natural product, processes anti-proliferative effect in cancer cells. In the current study, we investigate the antitumor mechanism of heteronemin in breast cancer cells and further explore its molecular targets. Heteronemin exhibited potent cytotoxic effects against breast cancer cells. On the other hand, E2 stimulated cancer cell growth. Heteronemin and E2 showed different effects on gene expression of proliferation, angiogenesis or growth factor receptors in breast cancer cells. NanoString® analysis was further used to detect mRNAs, heteronemin repressed gene expression of transforming growth factor (TGF)-β, mothers against decapentaplegic homolog (SMAD) and PCNA, whose protein products play important roles in regulating cell growth, angiogenesis, and metastasis. The results indicate that heteronemin was able to modulate cell adhesion, expression of extracellular matrix (ECM) receptors, TGF-β pathway, cell motility, membrane integration, metastasis response, matrix metalloproteinase (MMP) remodeling, regulation of metabolism, sprouting angiogenesis, transcription factors, and vasculogenesis in breast cancer cells. However, those effects were partially reversed by E2. Furthermore, Heteronemin and E2 altered several signaling transduction pathways. In summary, this study provides insight into the complex pathways by which anti-proliferation is induced by heteronemin in E2-depleted and -repleted environments. Citation Format: Zi-Lin Li, Yi-shin Pan, Tung-Yung Huang, Ya-Jung Shih, Yi-Ru Chen, Heng-Yuan Tang, Jacqueline Whang-Peng, Hung-Yun Lin, Paul J. Davis, Kuan Wang. Effect of estrogen on heteronemin-induced anti-proliferation in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5034.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth induced by periodontopathic bacteria that results in the progressive destruction of periodontal tissues. Treatment of periodontitis is painful and time-consuming. Recently, herbal medicines have been considered for use in treating inflammation-related diseases, including periodontitis. Resveratrol and its derivative 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucoside (THSG), a polyphenol extracted from Polygonum multiflorum, have anti-inflammatory properties and other medical benefits. Here, we highlight the importance of resveratrol and its glycosylated derivative as possible complementary treatments for periodontitis and their potential for development as innovative therapeutic strategies. In addition, we present evidence and discuss the mechanisms of action of resveratrol and THSG on periodontitis, focusing on Porphyromonas gingivalis-induced inflammatory responses in human gingival fibroblasts and animal modeling of ligature-induced periodontitis. We also illuminate the signal transduction pathways and the cytokines involved.

Nonpeptide hormones, such as thyroid hormone, dihydrotestosterone, and estrogen, have been shown to stimulate cancer proliferation via different mechanisms. Aside from their cytosolic or membrane-bound receptors, there are receptors on integrin α

// Yu-Tang Chin 1, * , Meng-Ti Hsieh 1, * , Sheng-Huei Yang 2 , Po-Wei Tsai 3 , Shwu-Huey Wang 4 , Ching-Chiung Wang 3 , Yee-Shin Lee 2 , Guei-Yun Cheng 1 , Wei-Chun HuangFu 1, 2 , David London 5 , Heng-Yuan Tang 5 , Earl Fu 6 , Yun Yen 2, 7 , Leroy F. Liu 1 , Hung-Yun Lin 1, 2 , Paul J. Davis 5, 8 1 Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan 2 PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 3 School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan 4 Core Facility, Taipei Medical University, Taipei, Taiwan 5 Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, New York, USA 6 Department of Periodontology, School of Dentistry, National Defense Medical Center and Tri-Service General Hospital, Taipei, Taiwan 7 Department of Molecular Pharmacology, City of Hope National Medical Center and Beckman Research Center, Duarte, California, USA 8 Albany Medical College, Albany, New York, USA * These authors contributed equally to this work Correspondence to: Hung-Yun Lin, e-mail: linhy@tmu.edu.tw Keywords: stilbene, integrin αvβ3, apoptosis, anti-proliferation, breast cancer Received: July 21, 2014 Accepted: October 23, 2014 Published: November 08, 2014 ABSTRACT We have used a perfusion bellows cell culture system to investigate resveratrolinduced anti-proliferation/apoptosis in a human estrogen receptor (ER)-negative breast cancer cell line (MDA-MB-231). Using an injection system to perfuse media with stilbene, we showed resveratrol (0.5 – 100 μM) to decrease cell proliferation in a concentration-dependent manner. Comparison of influx and medium efflux resveratrol concentrations revealed rapid disappearance of the stilbene, consistent with cell uptake and metabolism of the agent reported by others. Exposure of cells to 10 μM resveratrol for 4 h daily × 6 d inhibited cell proliferation by more than 60%. Variable extracellular acid-alkaline conditions (pH 6.8 – 8.6) affected basal cell proliferation rate, but did not alter anti-proliferation induced by resveratrol. Resveratrol-induced gene expression, including transcription of the most up-regulated genes and pro-apoptotic p53-dependent genes, was not affected by culture pH changes. The microarray findings in the context of induction of anti-proliferation with brief daily exposure of cells to resveratrol—and rapid disappearance of the compound in the perfusion system—are consistent with existence of an accessible initiation site for resveratrol actions on tumor cells, e.g., the cell surface receptor for resveratrol described on integrin αvβ3.

The obesity-regulated gene, leptin, is essential for diet. Leptin resistance causes obesity and related diseases. Certain types of diet are able to decrease leptin resistance. However, leptin has been shown to be correlated with inflammation and stimulate proliferation of various cancers. Two synthetic leptin derivatives (mimetics), OB3 and [D-Leu-4]-OB3, show more effective than leptin in reducing obesity and diabetes in mouse models. OB3 inhibits leptin-induced proliferation in ovarian cancer cells. However, effects of these mimetics in hepatocellular carcinoma (HCC) have not been investigated. In the present study, we examined the effects of OB3 and [D-Leu-4]-OB3 on cell proliferation and gene expressions in human HCC cell cultures. In contrast to what was reported for leptin, OB3 and [D-Leu-4]-OB3 reduced cell proliferation in hepatomas. Both OB3 and [D-Leu-4]-OB3 stimulated expression of pro-apoptotic genes. Both compounds also inhibited expressions of pro-inflammatory, proliferative and metastatic genes and PD-L1 expression. In combination with leptin, OB3 inhibited leptin-induced cell proliferation and expressions of pro-inflammation-, and proliferation-related genes. Furthermore, the OB3 peptide inhibited phosphoinositide 3-kinase (PI3K) activation which is essential for leptin-induced proliferation in HCC. These results indicate that OB3 and [D-Leu-4]-OB3 may have the potential to reduce leptin-related inflammation and proliferation in HCC cells.

Thyroid hormone induces tumor cell and blood vessel cell proliferation via a cell surface receptor on heterodimeric integrin αvβ3. We investigated the role of thyroid hormone-induced internalization of nuclear integrin αv monomer. Physiological concentration of thyroxine (free T4, 10(-10) M), but not 3,5,3'-triiodo-l-thyronine (T3), induced cellular internalization and nuclear translocation of integrin αv monomer in human non-small-cell lung cancer (H522) and ovarian carcinoma (OVCAR-3) cells. T4 did not complex with integrin αv monomer during its internalization. The αv monomer was phosphorylated by activated ERK1/2 when it heterodimerized with integrin β3 in vitro. Nuclear αv complexed with transcriptional coactivator proteins, p300 and STAT1, and with corepressor proteins, NCoR and SMRT. Nuclear αv monomer in T4-exposed cells, but not integrin β3, bound to promoters of specific genes that have important roles in cancer cells, including estrogen receptor-α, cyclooxygenase-2, hypoxia-inducible factor-1α, and thyroid hormone receptor β1 in chromatin immunoprecipitation assay. In summary, monomeric αv is a novel coactivator regulated from the cell surface by thyroid hormone for the expression of genes involved in tumorigenesis and angiogenesis. This study also offers a mechanism for modulation of gene expression by thyroid hormone that is adjunctive to the nuclear hormone receptor (TR)-T3 pathway.

BACKGROUND: Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation (Nanotetrac) act at a cell surface receptor to block angiogenesis and tumor cell proliferation. OBJECTIVE: The complex anti-angiogenic properties of tetrac and Nanotetrac caused us to search in the literature and in certain of our unpublished mRNA experiments for evidence that these agents affect the early inflammatory response, perhaps through actions on specific cytokines and chemokines. RESULTS AND DISCUSSION: Tetrac and Nanotetrac inhibit expression in tumor cells of cytokine genes, e.g., specific interleukins, and chemokine genes, such as fractalkine (CX3CL1), and chemokine receptor genes (CX3CR1) that have been identified as high priority targets in the development of inflammation-suppressant drugs. The possibility is also examined that tetrac formulations have an effect on the function of inflammatory cells.

Periodontitis, a chronic infection by periodontopathic bacteria, induces uncontrolled inflammation, which leads to periodontal tissue destruction. 2,3,5,4′-Tetrahydroxystilbene-2-O-beta-glucoside (THSG), a polyphenol extracted from Polygoni Multiflori, reportedly has anti-inflammatory properties. In this study, we investigated the mechanisms of THSG on thePorphyromonas gingivalis-induced inflammatory responses in human gingival fibroblasts and animal modeling of ligature-induced periodontitis. Human gingival fibroblast cells were treated with lipopolysaccharide (LPS) extracted fromP. gingivalisin the presence of resveratrol or THSG to analyze the expression of TNF-α, IL-1β, and IL-6 genes. Increased AMP-activated protein kinase (AMPK) activation and SirT1 expression were induced by THSG. Treatment of THSG decreased the expression of LPS-induced inflammatory cytokines, enhanced AMPK activation, and increased the expression of SirT1. In addition, it suppressed the activation of NF-κB when cells were stimulated withP. gingivalisLPS. The anti-inflammatory effect of THSG and P. Multiflori crude extracts was reproduced in ligature-induced periodontitis animal modeling. In conclusion, THSG inhibited the inflammatory responses ofP. gingivalis-stimulated human gingival fibroblasts and ameliorated ligature-induced periodontitis in animal model.

Resveratrol is a naturally occurring stilbene with desirable cardioprotective and anti-cancer properties. We have demonstrated the existence of a plasma membrane receptor for resveratrol near the arginine-glycine-aspartate (RGD) recognition site on integrin α(v)β₃ that is involved in stilbene-induced apoptosis of cancer cells. Resveratrol treatment in vitro causes activation and nuclear translocation of mitogen-activated protein kinase (ERK1/2), consequent phosphorylation of Ser-15 of p53, and apoptosis. An RGD peptide blocks these actions of resveratrol. By a PD98059-inhibitable process, resveratrol causes inducible COX-2 to accumulate in the nucleus where it complexes with pERK1/2 and p53. Chromatin immunoprecipitation reveals binding of nuclear COX-2 to promoters of certain p53-responsive genes, including PIG3 and Bax. NS-398, a specific pharmacologic inhibitor of COX-2, prevents resveratrol-induced complexing of nuclear ERK1/2 with COX-2 and with pSer-15-p53 and subsequent apoptosis; cyclooxygenase enzyme activity is not involved. Molecular steps in the pro-apoptotic action of resveratrol in cancer cells include induction of intranuclear COX-2 accumulation relevant to activation of p53. Epidermal growth factor, estrogen, and thyroid hormone act downstream of ERK1/2 to prevent resveratrol-induced apoptosis.

Resveratrol is a naturally occurring trihydroxyl-diphenylethylene compound that has been shown experimentally to have beneficial effects in the treatment of cancer and cardiovascular disease. Resveratrol induces programmed cell death (apoptosis) in these cells and activates important signal transducing proteins including extracellular signal-regulated kinases (ERKs) 1 and 2 in cancer cells. Resveratrol also causes nuclear accumulation of the enzyme cyclooxygenase (COX)-2 and of the oncogene suppressor protein, p53. We have studied the molecular basis of the anticancer actions of resveratrol using human ovarian carcinoma (OVCAR-3) cells. Our findings include the following: (i) nuclear accumulation of COX-2 in resveratrol-treated cells is blocked by the ERK1/2 inhibitor, PD98059; (ii) an inhibitor of COX-2 activity, NS398, prevents accumulation of ERK1/2, COX-2, activated p53 and small ubiquitin-like modifier (SUMO-1) in the nucleus; (iii) apoptosis, quantitated by nucleosome enzyme-linked immunosorbent assay and the nuclear abundance of the pro-apoptotic protein, BcL-xs, were inhibited by NS398. This finding implicates nuclear COX-2 in p53-mediated apoptosis induced by resveratrol. Sumoylation is important to stabilization of p53 and a COX-2-SUMO-1 interaction suggests sumoylation of COX-2 in resveratrol-treated cells and (iv) chromatin immunoprecipitation studies showed binding of induced nuclear COX-2 to the promoter region of PIG3 and Bax, pro-apoptotic gene targets of transcriptionally active p53. Nuclear accumulation of activated ERK1/2 and sumolyated COX-2 are essential to resveratrol-induced pSer-15-p53-mediated apoptosis in human ovarian cancer cells.

A uterine fibroid is the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. We aimed to determine the expression of growth factor molecules for growth inhibition after treatment of resveratrol in the primary cell lines from human uterine fibroid. In terms of growth rate, of the 24 uterine fibroid primary cell culture cells, 5 were mildly sensitive, 13 were medium-, and 6 were sensitive to resveratrol treatment. 19 out of the 24 primary cell lines responded to treatment of resveratrol from middle to sensitive ranges. Resveratrol arrested cell proliferation and also induced apoptosis, indicating that an intact apoptotic pathway was present in these cells by resveratrol. Reduction of integrin αvβ3 expression diminished resveratrol-induced antiproliferative effect in uterine fibroid cells. Resveratrol induced the expression of proapoptotic genes such as COX-2 and p21. On the other hand, the expression of proliferative (antiapoptotic) genes was either inhibited in such as BCL2 and CDKN2 or not changed in Cyclin D1 and PCNA. The expression of mRNA and protein of insulin-like growth factor-I (IGF-I) was inhibited by resveratrol in primary uterine fibroid cells. The proapoptotic proteins such as caspase 3 and caspase 9 were activated after resveratrol treatment and demonstrated that resveratrol induced apoptosis in the resveratrol-sensitive primary cell cultures, which was consistent with studies of the flow cytometry. In addition, resveratrol blocked the expression of integrin αvβ3 and protein. Concurrently, constitutive AKT phosphorylation in uterine fibroid cells was inhibited by resveratrol. Therefore, growth modulation of uterine fibroid cells occurs via mechanisms dependent on crosstalk between integrin αvβ3 and IGF-1R. Our findings suggest that resveratrol can be considered as an alternative therapeutic agent for uterine fibroid. Citation Format: Yi-Ru Chen, Ya-Jung Shih, Heng-Yuan Tang Tang, Hung-Yun Lin, Paul J. Davis. Resveratrol inhibits cell proliferation via crosstalk between integrin αvβ3 and IGF-1R in primary cell cultures of human uterine fibroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5465.

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin alphavbeta3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-alpha (ER-alpha)-positive MCF-7 cells and ER-alpha-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-alpha transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3alpha, 17beta-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin alphavbeta3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin alphavbeta3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-alpha-positive and ER-alpha-negative breast cancer cells, but by discrete mechanisms.

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.

The stilbene resveratrol (RV) initiates p53-dependent apoptosis via plasma membrane integrin alphaVbeta3 in human cancer cells. A thyroid hormone (L-thyroxine, T(4)) membrane receptor also exists on alphaVbeta3. Stilbene and T(4) signals are both transduced by extracellular-regulated kinases 1 and 2 (ERK1/2); however, T(4) promotes cell proliferation in cancer cells, whereas RV is pro-apoptotic. Thyroid hormone has been shown to interfere with RV-induced apoptosis. However, the mechanisms involved are not fully understood. In this study, we examined the mechanism whereby T(4) inhibits RV-induced apoptosis in glioma cells. RV activated conventional protein kinase C and ERK1/2 and caused nuclear localization of cyclooxygenase-2 (COX-2), consequent p53 phosphorylation and apoptosis. RV-induced ERK1/2 activation is involved in not only COX-2 expression but also nuclear COX-2 accumulation. NS-398, a COX-2 inhibitor, did not affect ERK1/2 activation, but reduced the nuclear abundance of COX-2 protein and the formation of complexes of nuclear COX-2 and activated ERK1/2 that are required for p53-dependent apoptosis in RV-treated cells. T(4) inhibited RV-induced nuclear COX-2 and cytosolic pro-apoptotic protein, BcLx-s, accumulation. Furthermore, T(4) inhibited RV-induced apoptosis by interfering with the interaction of nuclear COX-2 and ERK1/2. This effect of T(4) was prevented by tetraiodothyroacetic acid (tetrac), an inhibitor of the binding of thyroid hormone to its integrin receptor. Tetrac did not, in the absence of T(4), affect induction of apoptosis by RV. Thus, the receptor sites on alphaVbeta3 for RV and thyroid hormone are discrete and activate ERK1/2-dependent downstream effects on apoptosis that are distinctive.

Recent evidence suggests that the thyroid hormone l-thyroxine (T4) stimulates growth of cancer cells via a plasma membrane receptor on integrin αVβ3. The contribution of this recently described receptor for thyroid hormone and receptor-based stimulation of cellular mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)] activity, to enhancement of cell proliferation by thyroid hormone was quantitated functionally and by immunologic means in three glioma cell lines exposed to T4. At concentrations of 1 to 100 nmol/L, T4 caused proliferation of C6, F98, and GL261 cells, measured by accumulation of proliferating cell nuclear antigen (PCNA) and radiolabeled thymidine incorporation. This effect was inhibited by the T4 analogue, tetraiodothyroacetic acid, and by an αVβ3 RGD recognition site peptide, both of which block T4 binding to integrin αVβ3 but are not agonists. Activation of MAPK by T4 was similarly inhibited by tetraiodothyroacetic acid and the RGD peptide. The thyroid hormone 3,5,3′-triiodo-l-thyronine (T3) and T4 were equipotent stimulators of PCNA accumulation in C6, F98, and GL261 cells, but physiologic concentrations of T3 are 50-fold lower than those of T4. In conclusion, our studies suggest that glioblastoma cells are thyroid hormone dependent and provide a molecular basis for recent clinical observations that induction of mild hypothyroidism may improve duration of survival in glioblastoma patients. The present experiments infer a novel cell membrane receptor-mediated basis for the growth-promoting activity of thyroid hormone in such tumors and suggest new therapeutic approaches to the treatment of patients with glioblastoma. (Cancer Res 2006; 66(14): 7270-5)

Because the androgen and estrogen nuclear hormone receptors are subject to acetylation, we speculated that the nuclear thyroid hormone receptor-beta1 (TRbeta1), another superfamily member, was also subject to this posttranslational modification. Treatment of 293T cells that contain TRbeta1(wt) with l-thyroxine (T4)(10(-7)M, total concentration) resulted in the accumulation of acetylated TR in nuclear fractions at 30-45 min and a decrease in signal by 60 min. A similar time course characterized recruitment by TR of p300, a coactivator protein with intrinsic transacetylase activity. Recruitment by the receptor of SRC-1, a TR coactivator that also acetylates nucleoproteins, was also demonstrated. Inhibition of the MAPK (ERK1/2) signal transduction cascade by PD 98059 blocked the acetylation of TR caused by T4. Tetraiodothyroacetic acid (tetrac) decreased T4-induced acetylation of TR. At 10(-7)M, 3,5,3'-triiodo-l-thyronine (T3) was comparably effective to T4 in causing acetylation of TR. We studied acetylation in TR that contained mutations in the DNA-binding domain (DBD) (residues 128-142) that are known to be relevant to recruitment of coactivators and to include the MAPK docking site. In response to T4 treatment, the K128A TR mutant transfected into CV-1 cells recruited p300, but not SRC-1, and was subject to acetylation. R132A complexed with SRC-1, but not p300; it was acetylated equally well in both the absence and presence of T4. S142E was acetylated in the absence and presence of T4 and bound SRC-1 under both conditions; this mutant was also capable of binding p300 in the presence of T4. There was no serine phosphorylation of TR in any of these mutants. We conclude that (1) TRbeta1, like AR and ER, is subject to acetylation; (2) the process of acetylation of TR requires thyroid hormone-directed MAPK activity, but not serine phosphorylation of TR by MAPK, suggesting that the contribution of MAPK is upstream in the activation of the acetylase; (3) the amino acid residue 128-142 region of the DBD of TR is important to thyroid hormone-associated recruitment of p300 and SRC-1; (4) acetylation of TR DBD mutants that is directed by T4 appears to be associated with recruitment of p300.

Resveratrol, a naturally occurring stilbene with antitumor properties, caused mitogen-activated protein kinase [MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2)] activation, nuclear translocation of Ser15-phosphorylated p53, and p53-dependent apoptosis in hormone-insensitive DU145 prostate cancer cells. Exposure of these cells to epidermal growth factor (EGF) for up to 4 hours resulted in brief activation of MAPK followed by inhibition of resveratrol-induced signal transduction, p53 phosphorylation, and apoptosis. Resveratrol stimulated c-fos and c-jun expression in DU145 cells, an effect also suppressed by EGF. An inhibitor of protein kinase C (PKC)-α, -β, and -γ (CGP41251) enhanced Ser15 phosphorylation of p53 by resveratrol in the absence of EGF and blocked EGF inhibition of the resveratrol effect. EGF caused PKC-α/β phosphorylation in DU145 cells, an effect reversed by CGP41251. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate) enhanced EGF action on ERK1/2 phosphorylation without significantly altering p53 phosphorylation by resveratrol. DU145 cells transfected with a dominant-negative PKC-α construct showed resveratrol-induced ERK1/2 phosphorylation and Ser15 phosphorylation of p53 but were unresponsive to EGF. Thus, resveratrol and EGF activate MAPK by discrete mechanisms in DU145 cells. The stilbene promoted p53-dependent apoptosis, whereas EGF opposed induction of apoptosis by resveratrol via a PKC-α-mediated mechanism. Resveratrol also induced p53 phosphorylation in LNCaP prostate cancer cells, an effect also inhibited by EGF. Inhibition of PKC activation in LNCaP cells, however, resulted in a reduction, rather than increase, in p53 activation and apoptosis, suggesting that resveratrol-induced apoptosis in these two cell lines occurs through different PKC-mediated and MAPK-dependent pathways.

In CV-1 cells transfected with wild-type (wt) nuclear thyroid hormone receptor TRbeta1 (TR), L-thyroxine (T(4)) causes activation and nuclear translocation of mitogen-activated protein kinase (MAPK, ERK1/2), co-immunoprecipitation of MAPK and TR, and MAPK-dependent serine phosphorylation of TR. In the present studies, we have identified (1) the likely site of TR serine phosphorylation in the TR DNA-binding domain (DBD) by T(4)-activated MAPK, (2) the site of MAPK docking on TR induced by T(4), and (3) functional consequences of TR docking site and serine phosphorylation site mutations on co-repressor and co-activator binding and on transcriptional activation by wt and mutant receptors in T(4)-treated cells. Plasmids containing TR(wt), serine 142-substituted TR (TR(S142A) or TR(S142E)), TR(K128A), TR(R132A), or TR(R133A) were transfected into CV-1 cells, and the cells were treated with 10(-7) M T(4) for 30 min. Activated MAPK was present in nuclear fractions of all T(4)-treated cells and co-immunoprecipitated prominently with TR(wt), TR(S142A), and TR(S142E). TR(K128A) complexing with activated MAPK was minimally detectable, but no association of MAPK with TR(R132A) or TR(R133A) was seen in cells treated with T(4). Serine phosphorylation of TR(wt), but not of any mutants, occurred with T(4). In in vitro phosphorylation studies, constitutively activated MAPK phosphorylated only TR(wt). We concluded that serine 142 of the TR DBD is the likely site of phosphorylation by T(4)-activated MAPK and that the docking site on TR for activated MAPK includes residues 128-133 (KGFFRR), a basic amino acid-enriched motif novel for MAPK substrates. TR mutations in the proposed MAPK docking domain and at residue 142 modulated T(4)-conditioned shedding of co-repressor and recruitment of co-activator proteins by the receptor, and they altered transcriptional activity of TR in a thyroid hormone response element-luciferase reporter assay.

Purpose: Resveratrol (Calbiochem, La Jolla, California) is a naturally occurring stilbene reported to cause apoptosis in various cultured cancer cells. In the current study the effect of resveratrol was determined in the androgen insensitive DU 145 prostate cancer cell line. Induction of apoptosis and activation of apoptosis related signal transduction pathways were measured.Materials and Methods: DU 145 cells were treated with resveratrol and apoptosis was measured by determining nucleosome content. Activation of mitogen activated protein kinase (MAPK) (extracellular signal-regulated kinase 1/2), p53 content and serine-15 phosphorylation of p53 were measured by immunoblot. Electrophoretic mobility shift assay of p53 binding to DNA, and measurement of p21 and glyceraldehyde-3-phosphate dehydrogenase messenger RNA were also done.Results: Resveratrol induced apoptosis in DU 145 cells. The stilbene activated MAPK and caused increased abundance of p53 and serine-15 phosphorylated p53. Resveratrol induced seri...

Integrin αvβ3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvβ3 that specifically binds thyroid hormone and thyroid hormone analogues. From this receptor, thyroid hormone (L-thyroxine, T4; 3,5,3’-triiodo-L-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated nongenomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvβ3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvβ3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intracellular kinase-mediated regulation of gene expression, hormone analogues at αvβ3 have additional effects on intracellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvβ3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.

The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3'-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.

P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrinαvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4and of 3,5,3′-triiodo-L-thyronine (T3) atαvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently viaαvβ3. Intracellular acidification and decreased H+efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

Resveratrol is a naturally occurring polyphenol, which causes apoptosis in cultured cancer cells. We describe a cell surface resveratrol receptor on the extracellular domain of hetero-dimeric αVβ3 integrin in MCF-7 human breast cancer cells. This receptor is linked to induction by resveratrol of extracellular-regulated kinases 1 and 2 (ERK1/2)- and serine-15-p53-dependent phosphorylation leading to apoptosis. The integrin receptor is near the Arg-Gly-Asp (RGD) recognition site on the integrin; an integrin-binding RGD peptide inhibits induction by resveratrol of ERK1/2- and p53-dependent apoptosis. Antibody (Ab) to integrin αVβ3, but not to αVβ5, inhibits activation by resveratrol of ERK1/2 and p53 and consequent apoptosis in estrogen receptor-α (ERα) positive MCF-7, and ERα-negative MDA-MB231 cells. Resveratrol is displaced from the purified integrin by an RGD, but not RGE, peptide, and by αVβ3 integrin-specific Ab. Resveratrol action is blocked by siRNAβ3, but not by siRNAαV. [14C]-Resveratrol binds to c...

At thyroid hormone response elements on specific genes, complexes of nuclear thyroid hormone receptors (TRs) and 3,5,3'-triiodo-L-thyronine (T(3)), coactivator or corepressor nucleoproteins, and histone acetylases or deacetylases mediate genomic effects of the hormone. Nongenomic effects of the hormone are those whose initiation does not primarily depend upon formation of the TR-T(3) complex. Among the nongenomic effects of thyroid hormone are a set of actions initiated at a cell surface receptor on integrin αvβ3 that are relevant to a) intracellular trafficking of proteins, including TRβ1, b) serine phosphorylation and acetylation of this nuclear receptor, c) assembly within the nucleus of complexes of coactivators and corepressor, and d) transcription of specific genes, including that for TRβ1. These actions initiated at αvβ3 are reviewed here and appear to be adjunctive to the genomic actions of the TR-T(3) complex.

Integrins are heterodimeric structural components of the plasma membrane whose ligands include a large number of extracellular matrix (ECM) proteins. The ligands contain Arg–Gly–Asp (RGD) sequences that enable recognition of ECM proteins by as many as eight integrins, but other distinguishing features of the proteins permit the integrins to generate intracellular signals specific to the ECM molecules. Recently, integrin αvβ3 has been shown to have a panel of previously unappreciated small molecule receptor sites for thyroid hormone and hormone analogues, for dihydrotestosterone, and for resveratrol, a polyphenol that has certain estrogen-like features. These binding sites are close to the RGD recognition site of αvβ3. The thyroid hormone receptor site on the extracellular domain of αvβ3 contains two domains with discrete functions in terms of intracellular protein trafficking and gene expression. Occupancy of the receptor by a deaminated thyroid hormone analogue, tetraiodothyroacetic acid (tetrac), prevents cell responses to agonist thyroid hormones (l-thyroxine; 3, 5, 3′-triiodo-l-thyronine) and modulates expression of a number of cancer cell survival pathway genes in an up- or downregulation pattern coherent to induction of cell death. The small molecule thyroid hormone receptor on the integrin also regulates activity of five vascular growth factor receptors and/or their ligands, providing control of angiogenesis via specific pharmacologic regulation of this thyroid hormone receptor. The resveratrol receptor induces programmed cancer cell death via p53, even when the latter has undergone specific mutations. There is also evidence for the presence of several receptors on integrin αvβ3 for authentic steroids, including a dihydrotestosterone site that supports proliferation of breast cancer cells that lack nuclear androgen and estrogen receptors. The existence of these small molecule hormone receptors on an integrin with a remarkably complex functional profile defines novel pharmacologic options via individual small molecule receptor manipulation for control of cancer cell behavior. This refinement of up-down control at the level of discrete receptors is not a function of the use of αvβ3 antibody or RGD peptides that occlude regions of the integrin.