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1.  Orosphere Assay: A method for propagation of head and neck cancer stem cells 
Head & neck  2012;35(7):10.1002/hed.23076.
Background
Recent evidence suggests that head and neck squamous cell carcinomas (HNSCC) harbor a small sub-population of highly tumorigenic cells, named cancer stem cells. A limiting factor in cancer stem cell research is the intrinsic difficulty of expanding cells in an undifferentiated state in vitro.
Methods
Here, we describe the development of the orosphere assay, a method for the study of putative head and neck cancer stem cells. An orosphere is defined as a non-adherent colony of cells sorted from primary HNSCC or from HNSCC cell lines and cultured in 3-D soft agar or ultra-low attachment plates. Aldehyde dehydrogenase (ALDH) activity and CD44 expression were used here as stem cell markers.
Results
This assay allowed for the propagation of head and neck cancer cells that retained stemness and self-renewal.
Conclusion
The orosphere assay is well suited for studies designed to understand the pathobiology of head and neck cancer stem cells.
doi:10.1002/hed.23076
PMCID: PMC3887391  PMID: 22791367
Squamous cell carcinoma; suspension culture; sphere; self-renewal; stemness
2.  Synergistic Combination of Small Molecule Inhibitor and RNA interference Against Anti-apoptotic Bcl-2 Protein in Head and Neck Cancer Cells 
Molecular pharmaceutics  2013;10(7):2730-2738.
B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein that is over-expressed in head and neck squamous cell carcinomas, which has been implicated in development of radio- and chemo-resistance. Small molecule inhibitors such as AT-101 (a BH3-mimetic drug) have been developed to inhibit the anti-apoptotic activity of Bcl-2 proteins, which proved effective in restoring radio- and chemo-sensitivity in head and neck cancer cells. However, high doses of AT-101 are associated with gastrointestinal, hepatic, and fertility side effects, which prompted the search for other Bcl-2 inhibitors. Short interfering RNA (siRNA) proved to inhibit anti-apoptotic Bcl-2 protein expression and trigger cancer cell death. However, transforming siRNA molecules into a viable therapy remains a challenge due to the lack of efficient and biocompatible carriers. We report the development of degradable star-shaped polymers that proved to condense anti-Bcl-2 siRNA into “smart” pH-sensitive and membrane-destabilizing particles that shuttle their cargo past the endosomal membrane and into the cytoplasm of head and neck cancer cells. Results show that “smart” anti-Bcl-2 particles reduced the mRNA and protein levels of anti-apoptotic Bcl-2 protein in UM-SCC-17B cancer cells by 50-60% and 65-75%, respectively. Results also show that combining “smart” anti-Bcl-2 particles with the IC25 of AT-101 (inhibitory concentration responsible for killing 25% of the cells) synergistically inhibit cancer cell proliferation and increase cell apoptosis, which reduced the survival of UM-SCC-17B cancer cells compared to treatment with AT-101 alone. Results indicate the therapeutic benefit of combining siRNA-mediated knockdown of anti-apoptotic Bcl-2 protein expression with low doses of AT-101 for inhibiting the growth of head and neck cancer cells.
doi:10.1021/mp4001662
PMCID: PMC4043997  PMID: 23734725
“Smart” particles; Bcl-2 knockdown; AT-101 inhibitor; Drug delivery
3.  Glucose-Regulated Protein 78 (Grp78) Confers Chemoresistance to Tumor Endothelial Cells under Acidic Stress 
PLoS ONE  2014;9(6):e101053.
Objectives
This study was designed to investigate the activation of the unfolded protein response (UPR) in tumor associated endothelial cells (TECs) and its association with chemoresistance during acidic pH stress.
Materials and Methods
Endothelial cells from human oral squamous cell carcinomas (OSCC) were excised by laser capture microdissection (LCM) followed by analysis of UPR markers (Grp78, ATF4 and CHOP) using quantitative PCR. Grp78 expression was also determined by immunostaining. Acidic stress was induced in primary human dermal microvascular endothelial cells (HDMECs) by treatment with conditioned medium (CM) from tumor cells grown under hypoxic conditions or by adjusting medium pH to 6.4 or 7.0 using lactic acid or hydrochloric acid (HCl). HDMEC resistance to the anti-angiogenic drug Sunitinib was assessed with SRB assay.
Results
UPR markers, Grp78, ATF4 and CHOP were significantly upregulated in TECs from OSCC compared to HDMECs. HDMECs cultured in acidic CM (pH 6.0–6.4) showed increased expression of the UPR markers. However, severe acidosis led to marked cell death in HDMECs. Alternatively, HDMECs were able to adapt when exposed to chronic acidosis at pH 7.0 for 7 days, with concomittant increase in Grp78 expression. Chronic acidosis also confers drug resistance to HDMECs against Sunitinib. Knockdown of Grp78 using shRNA resensitizes HDMECs to drug treatment.
Conclusions
UPR induction in ECs under acidic pH conditions is related to chemoresistance and may contribute to therapeutic failures in response to chemotherapy. Targeting Grp78, the key component of the UPR pathway, may provide a promising approach to overcome ECs resistance in cancer therapy.
doi:10.1371/journal.pone.0101053
PMCID: PMC4071032  PMID: 24964091
4.  RAIN-Droplet: A Novel 3-D in vitro Angiogenesis Model 
Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3-D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed as both outgrowths from endothelial cells on the surface of the droplets but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof-of-principle for the utility of the model showing significant inhibition of sprout formation (p<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay we also demonstrate a novel dose dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3-D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (p<0.05) more endothelial sprouts compared to controls over a 48-hour period. Higher doses of sorafenib (5 μM) resulted in a significant (p<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3-D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib which may impact the clinical application and dosing regimen of that drug.
doi:10.1038/labinvest.2012.77
PMCID: PMC4043634  PMID: 22565576
Endothelial; microvascular; puramatrix; 3-D cell-culture; drug screen; capillary formation assay; sorafenib; bevacizumab
5.  Regenerative Endodontics in light of the stem cell paradigm 
International dental journal  2011;61(0 1):23-28.
Stem cells play a critical role in development and in tissue regeneration. The dental pulp contains a small sub-population of stem cells that are involved in the response of the pulp to caries progression. Specifically, stem cells replace odontoblasts that have undergone cell death as a consequence of the cariogenic challenge. Stem cells also secrete factors that have the potential to enhance pulp vascularization and provide the oxygen and nutrients required for the dentinogenic response that is typically observed in teeth with deep caries. However, the same angiogenic factors that are required for dentin regeneration may ultimately contribute to the demise of the pulp by enhancing vascular permeability and interstitial pressure. Recent studies focused on the biology of dental pulp stem cells revealed that the multipotency and angiogenic capacity of these cells could be exploited therapeutically in dental pulp tissue engineering. Collectively, these findings suggest new treatment paradigms in the field of Endodontics. The goal of this review is to discuss the potential impact of dental pulp stem cells to Regenerative Endodontics.
doi:10.1111/j.1875-595X.2011.00026.x
PMCID: PMC3721644  PMID: 21726222
Tissue Engineering; Dental pulp; Odontoblasts; Angiogenesis; Differentiation
6.  Endothelial derived factors inhibit anoikis of head and neck cancer stem cells 
Oral Oncology  2011;48(1):26-32.
Recent evidence demonstrated that cancer stem cells reside in close proximity to blood vessels in human head and neck squamous cell carcinomas (HNSCC). These findings suggest the existence of a supporting perivascular niche for cancer stem cells.
Objective
The purpose of this study was to evaluate the effect of endothelial cell-secreted factors on the behavior of head and neck cancer stem-like cells (HNCSC).
Materials and methods
HNCSC were identified by sorting UM-SCC-22A (cell line derived from a primary squamous cell carcinoma of the oropharynx) and UM-SCC-22B (derived from the metastatic lymph node of the same patient) for CD44 expression and ALDH (aldehyde dehydrogenase) activity. HNCSC (ALDH+CD44+) and control (ALDH−CD44−) cells were cultured in ultra-low attachment plates in presence of conditioned medium from primary human endothelial cells.
Results
ALDH+CD44+ generated more orospheres than control cells when cultured in suspension. The growth factor milieu secreted by endothelial cells protected HNCSC against anoikis. Mechanistic studies revealed that endothelial cell-secreted vascular endothelial growth factor (VEGF) induces proliferation of HNCSC derived from primary UM-SCC-22A, but not from the metastatic UM-SCC-22B. Likewise, blockade of VEGF abrogated endothelial cell-induced Akt phosphorylation in HNCSC derived from UM-SCC-22A while it had a modest effect in Akt phosphorylation in HNCSC from UM-SCC-22B.
Conclusion
This study revealed that endothelial cells initiate a crosstalk that protect head and neck cancer stem cells against anoikis, and suggest that therapeutic interference with this crosstalk might be beneficial for patients with head and neck cancer.
doi:10.1016/j.oraloncology.2011.09.010
PMCID: PMC3261237  PMID: 22014666
Head and neck squamous cell carcinoma; Perivascular niche; Angiogenesis; Tumor microenvironment; Metastasis
7.  The biology of head and neck cancer stem cells 
Oral Oncology  2011;48(1):1-9.
Emerging evidence indicates that a small population of cancer cells is highly tumorigenic, endowed with self-renewal, and has the ability to differentiate into cells that constitute the bulk of tumors. These cells are considered the “drivers” of the tumorigenic process in some tumor types, and have been named cancer stem cells. Epithelial-mesenchymal transition (EMT) appears to be involved in the process leading to the acquisition of stemness by epithelial tumor cells. Through this process, cells acquire an invasive phenotype that may contribute to tumor recurrence and metastasis. Cancer stem cells have been identified in human head and neck squamous cell carcinomas (HNSCC) using markers such as CD133 and CD44 expression, and aldehyde dehydrogenase (ALDH) activity. The head and neck cancer stem cells reside primarily in perivascular niches in the invasive front where endothelial-cell initiated events contribute to their survival and function. In this review, we discuss the state-of-the-knowledge on the pathobiology of cancer stem cells, with a focus on the impact of these cells to head and neck tumor progression.
doi:10.1016/j.oraloncology.2011.10.004
PMCID: PMC3261238  PMID: 22070916
Oral cancer; Tumorigenesis; Epithelial-mesenchymal transition; EMT; Self-renewal; Stemness; Perivascular niche; Squamous cell carcinoma; Angiogenesis
8.  Endothelial cell-derived interleukin-6 regulates tumor growth 
BMC Cancer  2014;14:99.
Background
Endothelial cells play a complex role in the pathobiology of cancer. This role is not limited to the making of blood vessels to allow for influx of oxygen and nutrients required for the high metabolic demands of tumor cells. Indeed, it has been recently shown that tumor-associated endothelial cells secrete molecules that enhance tumor cell survival and cancer stem cell self-renewal. The hypothesis underlying this work is that specific disruption of endothelial cell-initiated signaling inhibits tumor growth.
Methods
Conditioned medium from primary human dermal microvascular endothelial cells (HDMEC) stably transduced with silencing RNA for IL-6 (or controls) was used to evaluate the role of endothelial-derived IL-6 on the activation of key signaling pathways in tumor cells. In addition, these endothelial cells were co-transplanted with tumor cells into immunodefficient mice to determine the impact of endothelial cell-derived IL-6 on tumor growth and angiogenesis.
Results
We observed that tumor cells adjacent to blood vessels show strong phosphorylation of STAT3, a key mediator of tumor progression. In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells. Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells.
Conclusions
Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells.
doi:10.1186/1471-2407-14-99
PMCID: PMC4016552  PMID: 24533454
Cervical Cancer; Signaling pathways; Molecular targeted therapy; STAT3
9.  Cisplatin Induces Bmi-1 and Enhances the Stem Cell Fraction in Head and Neck Cancer12 
Neoplasia (New York, N.Y.)  2014;16(2):137-146.
Recent evidence has unveiled a subpopulation of highly tumorigenic, multipotent cells capable of self-renewal in head and neck squamous cell carcinomas (HNSCCs). These unique cells, named here cancer stem cells (CSCs), proliferate slowly and might be involved in resistance to conventional chemotherapy. We have shown that CSCs are found in perivascular niches and rely on endothelial cell-secreted factors [particularly interleukin-6 (IL-6)] for their survival and self-renewal in HNSCC. Here, we hypothesized that cisplatin enhances the stem cell fraction in HNSCC. To address this hypothesis, we generated xenograft HNSCC tumors with University of Michigan-squamous cell carcinoma 22B (UM-SCC-22B) cells and observed that cisplatin treatment increased (P = .0013) the fraction of CSCs [i.e., aldehyde dehydrogenase activity high and cluster of differentiation 44 high (ALDHhighCD44high)]. Cisplatin promoted self-renewal and survival of CSCs in vitro, as seen by an increase in the number of orospheres in ultralow attachment plates and induction in B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and octamer-binding transcription factor 4 expression. Cisplatin-resistant cells expressed more Bmi-1 than cisplatinsensitive cells. IL-6 potentiated cisplatin-induced orosphere formation generated when primary human HNSCC cells were sorted for ALDHhighCD44high immediately after surgery and plated onto ultralow attachment plates. IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (indicative of stemness) was unaffected by treatment with cisplatin in UM-SCC-22B cells, whereas IL-6-induced extracellular signal-regulated kinase (ERK) phosphorylation (indicative of differentiation processes) was partially inhibited by cisplatin. Notably, cisplatin-induced Bmi-1 was inhibited by interleukin-6 receptor blockade in parental and cisplatin-resistant cells. Taken together, these results demonstrate that cisplatin enhances the fraction of CSCs and suggest a mechanism for resistance to cisplatin therapy in head and neck cancer.
PMCID: PMC3978394  PMID: 24709421
10.  A Hydrogel Scaffold That Maintains Viability and Supports Differentiation of Dental Pulp Stem Cells 
Objectives
The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation.
Methods
DPSC cells were grown in 0.05 to 0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3-D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1.
Results
DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation.
Significance
Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics.
doi:10.1016/j.dental.2012.08.002
PMCID: PMC3515741  PMID: 22901827
Tissue engineering; Hydrogel; Dental pulp; Regenerative Endodontics; Odontoblast; Stem cells
11.  Xenograft Tumors Vascularized with Murine Blood Vessels May Overestimate the Effect of Anti-Tumor Drugs: A Pilot Study 
PLoS ONE  2013;8(12):e84236.
Recent evidence demonstrated that endothelial cells initiate signaling events that enhance tumor cell survival, proliferation, invasion, and tumor recurrence. Under this new paradigm for cellular crosstalk within the tumor microenvironment, the origin of endothelial cells and tumor cells may have a direct impact on the pathobiology of cancer. The purpose of this pilot study was to evaluate the effect of endothelial cell species (i.e. murine or human) on xenograft tumor growth and response to therapy. Tumor xenografts vascularized either with human or with murine microvascular endothelial cells were engineered, side-by-side, subcutaneously in the dorsum of immunodefficient mice. When tumors reached 200 mm3, mice were treated for 30 days with either 4 mg/kg cisplatin (i.p.) every 5 days or with 40 mg/kg sunitinib (p.o.) daily. Xenograft human tumors vascularized with human endothelial cells grow faster than xenograft tumors vascularized with mouse endothelial cells (P<0.05). Notably, human tumors vascularized with human endothelial cells exhibited nuclear translocation of p65 (indicative of high NF-kB activity), and were more resistant to treatment with cisplatin or sunitinib than the contralateral tumors vascularized with murine endothelial cells (P<0.05). Collectively, these studies suggest that the species of endothelial cells has a direct impact on xenograft tumor growth and response to treatment with the chemotherapeutic drug cisplatin or with the anti-angiogenic drug sunitinib.
doi:10.1371/journal.pone.0084236
PMCID: PMC3877272  PMID: 24391922
13.  The Unfolded Protein Response Induces the Angiogenic Switch in Human Tumor Cells through the PERK/ATF4 Pathway 
Cancer research  2012;72(20):5396-5406.
Neovascularization is a limiting factor in tumor growth and progression. It is well known that changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can induce VEGF production. However, the mechanism linking GD to tumor growth and angiogenesis is unclear. We hypothesize that GD induces the angiogenic switch in tumors through activation of the unfolded protein response (UPR). We report that UPR activation in human tumors results in elevated expression of proangiogenic mediators and a concomitant decrease in angiogenesis inhibitors. cDNA microarray results showed that GD-induced UPR activation promoted upregulation of a number of proangiogenic mediators (VEGF, FGF2, IL6, etc.) and downregulation of several angiogenic inhibitors (THBS1, CXCL14 and CXCL10). In vitro studies revealed that partially blocking UPR signaling by silencing PERK or ATF4 significantly reduced the production of angiogenesis mediators induced by GD. However, suppressing the alpha subunit of hypoxia-inducible factors had no effect on this process. Chromatin immunoprecipitation confirmed binding of ATF4 to a regulatory site in the VEGF gene. In vivo results confirmed that knockdown of PERK in tumor cells slows down tumor growth and decreases tumor blood vessel density. Collectively, these results demonstrate that the PERK/ATF4 arm of UPR mediates the angiogenic switch and is a potential target for antiangiogenic cancer therapy.
doi:10.1158/0008-5472.CAN-12-0474
PMCID: PMC3743425  PMID: 22915762
Glucose deprivation; UPR; angiogenic switch; VEGF; THBS1
14.  Transcriptional targeting of tumor endothelial cells for gene therapy 
Advanced drug delivery reviews  2009;61(7-8):542-553.
It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy.
doi:10.1016/j.addr.2009.02.006
PMCID: PMC2727054  PMID: 19393703
Cancer; angiogenesis; promoter; tumor microenvironment; review
15.  Expanding Circle of Inhibition: Small-Molecule Inhibitors of Bcl-2 as Anticancer Cell and Antiangiogenic Agents 
Journal of Clinical Oncology  2008;26(25):4180-4188.
The specific targeting of diseases, particularly cancer, is a primary aim in drug development, as specificity reduces unwelcome effects on healthy tissue and increases drug efficacy at the target site. Drug specificity can be increased by improving the delivery system or by selecting drugs with affinity for a molecular ligand specific to the disease state. The role of the prosurvival Bcl-2 protein in maintaining the normal balance between apoptosis and cellular survival has been recognized for more than a decade. Bcl-2 is vital during development, much less so in adults. It has also been noted that some cancers evade apoptosis and obtain a survival advantage through aberrant expression of Bcl-2. The new and remarkably diverse class of drugs, small-molecule inhibitors of Bcl-2 (molecular weight approximately 400 to 800 Daltons), is examined herein. We present the activities of these compounds along with clinical observations, where available. The effects of Bcl-2 inhibition on attenuation of tumor cell growth are discussed, as are studies revealing the potential for Bcl-2 inhibitors as antiangiogenic agents. Despite an enormous body of work published for the Bcl-2 family of proteins, we are still learning exactly how this group of molecules interacts and indeed what they do. The small-molecule inhibitors of Bcl-2, in addition to their therapeutic potential, are proving to be an important investigative tool for understanding the function of Bcl-2.
doi:10.1200/JCO.2007.15.7693
PMCID: PMC2654265  PMID: 18757333
16.  An expanding circle of inhibition: Small molecule inhibitors of Bcl-2 as anti-cancer cell and anti-angiogenic agents 
The specific targeting of diseases, particularly cancer, is a primary aim in drug development, as specificity reduces unwelcome effects on healthy tissue and increases drug efficacy at the target site. Drug specificity can be increased by improving the delivery system or by selecting drugs with affinity for a molecular ligand specific to the disease state. The role of the pro-survival Bcl-2 protein in maintaining the normal balance between apoptosis and cellular survival has been recognized for over a decade. Bcl-2 is vital during development, much less so in adults. It has also been noted that some cancers evade apoptosis and obtain a survival advantage through aberrant expression of Bcl-2. The new and remarkably diverse class of drugs, small molecule inhibitors of Bcl-2 (mw ~400–800), is examined herein. We present the activities of these compounds along with clinical observations, where available. The effects of Bcl-2 inhibition on attenuation of tumor cell growth are discussed, as are studies revealing the potential for Bcl-2 inhibitors as anti-angiogenic agents. Despite an enormous body of work published for the Bcl-2 family of proteins, we are still learning exactly how this group of molecules interacts and indeed what they do. The small molecule inhibitors of Bcl-2, in addition to their therapeutic potential, are proving to be an important investigative tool for understanding the function of Bcl-2.
doi:10.1200/JCO.2007.15.7693
PMCID: PMC2654265  PMID: 18757333
Developmental therapeutics; Tumor; Neovascularization; Pharmacology
17.  Anti-tumor and anti-angiogenic effects of metronomic dosing of BH3-mimetics 
Cancer research  2011;72(3):716-725.
Recent studies have shown that Bcl-2 functions as a pro-angiogenic signaling molecule in addition to its well-known effect as an inhibitor of apoptosis. The discovery of AT101, a BH3-mimetic drug that is effective and well tolerated when administered orally, suggested the possibility of using a molecularly targeted drug in a metronomic regimen. Here, we generated xenograft squamous cell carcinomas (SCC) with humanized vasculature in immunodeficient mice. Mice received taxotere in combination with either daily 10 mg/kg AT101 (metronomic regimen) or weekly 70 mg/kg AT101 (bolus regimen). The effect of single drug AT101 on angiogenesis, and combination AT101/taxotere on the survival of endothelial cells and SCC cells, were also evaluated in vitro. Metronomic AT101 increased mouse survival (p=0.02), decreased tumor mitotic index (p=0.0009), and decreased tumor microvessel density (p=0.0052), as compared to bolus delivery of AT101. Notably, the substantial potentiation of the anti-tumor effect observed in the metronomic AT101 group was achieved using the same amount of drug and without significant changes in systemic toxicities. In vitro, combination of AT101 and taxotere showed additive toxicity for endothelial cells and synergistic or additive toxicity for tumor cells (SCC). Interestingly, low-dose (sub-apoptotic) concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, these data unveiled the benefit of metronomic delivery of a molecularly targeted drug, and suggested that patients with squamous cell carcinomas might benefit from continuous administration of low dose BH3-mimetic drugs.
doi:10.1158/0008-5472.CAN-10-2873
PMCID: PMC3748951  PMID: 22158856
Developmental therapeutics; targeted therapy; angiogenesis; Bcl-2; squamous cell carcinoma
18.  Amino Acid Deprivation Promotes Tumor Angiogenesis through the GCN2/ATF4 Pathway1 
Neoplasia (New York, N.Y.)  2013;15(8):989-997.
As tumors continue to grow and exceed their blood supply, nutrients become limited leading to deficiencies in amino acids (AAD), glucose (GD), and oxygen (hypoxia). These alterations result in significant changes in gene expression. While tumors have been shown to overcome the stress associated with GD or hypoxia by stimulating vascular endothelial growth factor (VEGF)-mediated angiogenesis, the role of AAD in tumor angiogenesis remains to be elucidated. We found that in human tumors, the expression of the general control non-derepressible 2 (GCN2, an AAD sensor) kinase is elevated at both protein and mRNA levels. In vitro studies revealed that VEGF expression is universally induced by AAD treatment in all five cell lines tested (five of five). This is in contrast to two other angiogenesis mediators interleukin-6 (two of five) and fibroblast growth factor 2 (two of five) that have a more restricted expression. Suppressing GCN2 expression significantly decreased AAD-induced VEGF expression. Silencing activating transcription factor 4 (ATF4), a downstream transcription factor of the GCN2 signaling pathway, is also associated with strong inhibition of AAD-induced VEGF expression. PKR-like kinase, the key player in GD-induced unfolded protein response is not involved in this process. In vivo xenograft tumor studies in nonobese diabetic/severe combined immunodeficient mice confirmed that knockdown of GCN2 in tumor cells retards tumor growth and decreases tumor blood vessel density. Our results reveal that the GCN2/ATF4 pathway promotes tumor growth and angiogenesis through AAD-mediated VEGF expression and, thus, is a potential target in cancer therapy.
PMCID: PMC3730049  PMID: 23908598
19.  TGF-β1 regulates the invasive and metastatic potential of mucoepidermoid carcinoma cells 
Background
Patients with mucoepidermoid carcinoma exhibit poor long-term prognosis due to the lack of therapeutic strategies that effectively block tumor progression. We have previously characterized the Ms cells as a highly metastatic mucoepidermoid carcinoma cell line that expresses high levels of transforming growth factor β1 (TGF-β1). Here, we studied the effect of suppressing TGF-β1 by RNA silencing on the invasive and metastatic potential of mucoepidermoid carcinoma.
Methods
Cell motility, substratum adhesion and transmembrane invasion were estimated by migration, matrigel adhesion and metrigel invasion assay. Matrix metalloproteinase (MMP)-2 and MMP-9 activity were determined using gelatin gel zymography. Mice lung metastatic model was used to test the matastatic ability. Lung metastatic tumors were experimentally induced by mice tail vein inoculation of cancer cells.
Results
TGF-β1 silencing inhibits cell motility, substratum adhesion and transmembrane invasion. In vivo, a significant decrease in lung metastasis was observed when mice received tail vein injections of TGF-β1-silenced mucoepidermoid carcinoma cells, as compared to controls.
Conclusion
These results unveil a critical role for TGF-β1 in the progression of mucoepidermoid carcinomas, and suggest that patients with this malignancy may benefit from therapeutic inhibition of the effectors of the TGF-β1 pathway.
doi:10.1111/j.1600-0714.2011.01051.x
PMCID: PMC3723136  PMID: 21689159
Tumor progression; Metastasis; Salivary gland cancer; Extracellular matrix; Matrix metalloproteinase
20.  The stimulation of adipose-derived stem cell differentiation and mineralization by ordered rod-like fluorapatite coatings 
Biomaterials  2012;33(20):5036-5046.
In this study, the effect of ordered rod-like FA coatings of metal discs on adipose-derived stem cell (ASC)’s growth, differentiation and mineralization was studied in vitro; and their mineral inductive effects in vivo. After 3 and 7 days, the cell number on the metal surfaces was significantly higher than those on the ordered and disordered FA surfaces. However, after 4 weeks much greater amounts of mineral formation was induced on the two FA surfaces with and even without osteogenesis induction. The osteogenic profiles showed the up regulation of a set of pro-osteogenic transcripts and bone mineralization phenotypic markers when the ASCs were grown on FA surfaces compared to metal surfaces at 7 and 21 days. In addition to BMP and TGFβ signaling pathways, EGF and FGF pathways also appeared to be involved in ASC differentiation and mineralization. In vivo studies showed accelerated and enhanced mineralized tissue formation integrated within ordered FA coatings. After 5 weeks, over 80 % of the ordered FA coating was integrated with a mineralized tissue layer covering the implants. Both the intrinsic properties of the FA crystals and the topography of the FA coating appeared to dominate the cell differentiation and mineralization process.
doi:10.1016/j.biomaterials.2012.03.050
PMCID: PMC3341557  PMID: 22483243
Fluorapatite; differentiation; mineralization; gene profile; stem cells
21.  Endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma 
Background
Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells.
Methods
Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection (LCM). RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells.
Results
We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells.
Conclusions
Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells, and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.
doi:10.1111/j.1600-0714.2011.01081.x
PMCID: PMC3246528  PMID: 21936874
Oral cancer; Angiogenesis; Lymphangiogenesis; Biomarkers; VEGF; Head and Neck Cancer
22.  Efficient in vivo Vascularization of Tissue Engineering Scaffolds 
The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and peri-vascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial- and vascular smooth muscle cells were seeded at different ratios in poly-L lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue engineering context was strongly enhanced in the implants seeded with a complete complement of blood vessel components: Human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3-D image reconstruction analysis of hPASMC distribution within vascularized implants was non-random and displayed a preferential peri-vascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.
doi:10.1002/term.336
PMCID: PMC3010488  PMID: 20865694
angiogenesis; scaffold; endothelial; mural cell; microcirculation; multiphoton
23.  Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells 
Cancer research  2010;70(23):9969-9978.
Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used Aldehyde Dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin−) led to tumors in 13 (out of 15) mice, while 10,000 non-cancer stem cells (NCSC; ALDH−CD44−Lin−) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a sub-population of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin− cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-µm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared to controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck cancer stem cells.
doi:10.1158/0008-5472.CAN-10-1712
PMCID: PMC3058885  PMID: 21098716
Tumor microenvironment; perivascular niche; anti-angiogenic therapy; squamous cell carcinoma; stemness
24.  Metronomic small molecule inhibitor of Bcl-2 (TW-37) is anti-angiogenic and potentiates the anti-tumor effect of ionizing radiation 
Purpose
To investigate the effect of a metronomic (low dose, high frequency) small molecule inhibitor of Bcl-2 (TW-37) in combination with radiotherapy on microvascular endothelial cells in vitro and in tumor angiogenesis in vivo.
Methods and materials
Primary human dermal microvascular endothelial cells (HDMEC) were exposed to ionizing radiation and/or TW-37, and colony formation as well as capillary sprouting in 3-D collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by co-transplantation of human squamous cell carcinoma cells (OSCC3) and HDMEC seeded in highly porous biodegradable scaffolds into the subcutaneous space of immunodeficient mice. Mice were treated with metronomic TW-37 and/or radiation, and tumor growth was evaluated.
Results
Low dose TW-37 sensitized primary endothelial cells to radiation-induced inhibition of colony formation. Low dose TW-37 or radiation partially inhibited endothelial cell sprout formation, while in combination these therapies abrogated new sprouting. Combination of metronomic TW-37 and low dose radiation inhibited tumor growth and resulted in significant increase in time to failure as compared to controls, whereas single agents did not. Notably, histopathological analysis revealed that tumors treated with TW-37 (with or without radiation) are more differentiated and showed more cohesive invasive fronts, which is consistent with less aggressive phenotype.
Conclusions
These results demonstrate that metronomic TW-37 potentiates the anti-tumor effects of radiotherapy, and suggest that patients with head and neck cancer might benefit from the combination of small molecule inhibitor of Bcl-2 and radiation therapy.
doi:10.1016/j.ijrobp.2010.04.024
PMCID: PMC2946486  PMID: 20675079
Developmental therapeutics; Radiotherapy; Head and Neck Cancer; Apoptosis; Neovascularization
25.  The Effect of Novel Fluorapatite Surfaces on Osteoblast-Like Cell Adhesion, Growth, and Mineralization 
Tissue Engineering. Part A  2010;16(9):2977-2986.
There is increasing demand for biomedical implants to correct skeletal defects caused by trauma, disease, or genetic disorder. In this study, the MG-63 cells were grown on metals coated with ordered and disordered fluorapatite (FA) crystal surfaces to study the biocompatibility, initial cellular response, and the underlying mechanisms during this process. The long-term growth and mineralization of the cells were also investigated. After 3 days, the cell numbers on etched metal surface are significantly higher than those on the ordered and disordered FA surfaces, but the initial adherence of a greater number of cells did not lead to earlier mineral formation at the cell–implant interface. Of the 84 cell adhesion and matrix-focused pathway genes, an up- or down-regulation of a total of 15 genes such as integrin molecules, integrin alpha M and integrin alpha 7 and 8 was noted, suggesting a modulating effect on these adhesion molecules by the ordered FA surface compared with the disordered. Osteocalcin expression and the mineral nodule formation are most evident on the FA surfaces after osteogenic induction (OI) for 7 weeks. The binding of the ordered FA surfaces to the metal, with and without OI, was significantly higher than that of the disordered FA surfaces with OI. Most significantly, even without the OI supplement, the MG-63 cells grown on FA crystal surfaces start to differentiate and mineralize, suggesting that the FA crystal could be a simple and bioactive implant coating material.
doi:10.1089/ten.tea.2009.0632
PMCID: PMC2928044  PMID: 20412028

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