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1.  A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA 
PLoS Medicine  2008;5(5):e94.
Background
Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.
Methods and Findings
Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.
Conclusions
These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Shigetada Teshima-Kondo and colleagues find that cancer cells have a survival system that is regulated by vegf mRNA and that vegf mRNA and its protein may synergistically promote the malignancy of tumor cells.
Editors' Summary
Background
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. When a cancer is small, it uses the body's existing blood supply to get the oxygen and nutrients it needs for its growth and survival. But, when it gets bigger, it has to develop its own blood supply. This process is called angiogenesis. It involves the release by the cancer cells of proteins called growth factors that bind to other proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels). The receptors then send signals into the endothelial cells that tell them to make new blood vessels. One important angiogenic growth factor is “vascular endothelial growth factor” (VEGF). Tumors that make large amounts of VEGF tend to be more abnormal and more aggressive than those that make less VEGF. In addition, high levels of VEGF in the blood are often associated with poor responses to chemotherapy, drug regimens designed to kill cancer cells.
Why Was This Study Done?
Because VEGF is a key regulator of tumor development, several anti-VEGF therapies—drugs that target VEGF and its receptors—have been developed. These therapies strongly suppress the growth of tumor cells in the laboratory and in animals but, when used alone, are no better at increasing the survival times of patients with cancer than standard chemotherapy. Scientists are now looking for an explanation for this disappointing result. Like all proteins, cells make VEGF by “transcribing” its DNA blueprint into an mRNA copy (vegf mRNA), the coding region of which is “translated” into the VEGF protein. Other, “noncoding” regions of vegf mRNA control when and where VEGF is made. Scientists have recently discovered that the noncoding regions of some mRNAs suppress tumor development. In this study, therefore, the researchers investigate whether vegf mRNA has an unrecognized function in tumor cells that could explain the disappointing clinical results of anti-VEGF therapeutics.
What Did the Researchers Do and Find?
The researchers first used a technique called small interfering (si) RNA knockdown to stop VEGF expression in human colon cancer cells growing in dishes. siRNAs are short RNAs that bind to and destroy specific mRNAs in cells, thereby preventing the translation of those mRNAs into proteins. The treatment of human colon cancer cells with vegf-targeting siRNAs made the cells more sensitive to chemotherapy-induced apoptosis (a type of cell death). This sensitivity was only partly reversed by adding VEGF to the cells. By contrast, cancer cells engineered to make more vegf mRNA had increased resistance to chemotherapy-induced apoptosis. Treatment of these cells with an antibody that inhibited VEGF function did not completely block this resistance. Together, these results suggest that both vegf mRNA and VEGF protein have anti-apoptotic effects. The researchers show that the anti-apoptotic activity of vegf mRNA requires a noncoding part of the mRNA called the 5′ UTR, and that whereas human colon cancer cells expressing this 5′ UTR form tumors in mice, cells expressing a mutated 5′ UTR do not. Finally, they report that the expression of several pro-apoptotic genes and of an anti-tumor pathway known as the interferon/STAT1 tumor suppression pathway is down-regulated in tumors that express the vegf 5′ UTR.
What Do These Findings Mean?
These findings suggest that some cancer cells have a survival system that is regulated by vegf mRNA and are the first to show that a 5′UTR of mRNA can promote tumor growth. They indicate that VEGF and its mRNA work together to promote their development and to increase their resistance to chemotherapy drugs. They suggest that combining therapies that prevent the production of vegf mRNA (for example, siRNA-based gene silencing) with therapies that block the function of VEGF might improve survival times for patients whose tumors overexpress VEGF.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050094.
This study is discussed further in a PLoS Medicine Perspective by Hughes and Jones
The US National Cancer Institute provides information about all aspects of cancer, including information on angiogenesis, and on bevacizumab, an anti-VEGF therapeutic (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer, including angiogenesis (in several languages)
Cancer Research UK also provides basic information about what causes cancers and how they develop, grow, and spread, including information about angiogenesis
Wikipedia has pages on VEGF and on siRNA (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050094
PMCID: PMC2386836  PMID: 18494554
2.  Astragalus saponins modulates colon cancer development by regulating calpain-mediated glucose-regulated protein expression 
Background
Glucose-regulated proteins (GRP) are induced in the cancer microenvironment to promote tumor survival, metastasis and drug resistance. AST was obtained from the medicinal plant Astragalus membranaceus, which possesses anti-tumor and pro-apoptotic properties in colon cancer cells and tumor xenograft. The present study aimed to investigate the involvement of GRP in endoplasmic reticulum (ER) stress-mediated apoptosis during colon cancer development, with focus on the correlation between AST-evoked regulation of GRP and calpain activation.
Methods
The effects of AST on GRP and apoptotic activity were assessed in HCT 116 human colon adenocarcinoma cells. Calpain activity was examined by using a fluorescence assay kit. Immunofluorescence staining and immunoprecipitation were employed to determine the localization and association between calpains and GRP. GRP78 gene silencing was performed to confirm the importance of GRP in anticancer drug activities. The modulation of GRP and calpains was also studied in nude mice xenograft.
Results
ER stress-mediated apoptosis was induced by AST, as shown by elevation in both spliced XBP-1 and CHOP levels, with parallel up-regulation of GRP. The expression of XBP-1 and CHOP continued to increase after the peak level of GRP was attained at 24 h. Nevertheless, the initial increase in calpain activity as well as calpain I and II protein level was gradually declined at later stage of drug treatment. Besides, the induction of GRP was partly reversed by calpain inhibitors, with concurrent promotion of AST-mediated apoptosis. The knockdown of GRP78 by gene silencing resulted in higher sensitivity of colon cancer cells to AST-induced apoptosis and reduction of colony formation. The association between calpains and GRP78 had been confirmed by immunofluorescence staining and immunoprecipitation. Modulation of GRP and calpains by AST was similarly demonstrated in nude mice xenograft, leading to significant inhibition of tumor growth.
Conclusions
Our findings exemplify that calpains, in particular calpain II, play a permissive role in the modulation of GRP78 and consequent regulation of ER stress-induced apoptosis. Combination of calpain inhibitors and AST could exhibit a more pronounced pro-apoptotic effect. These results help to envisage a new therapeutic approach in colon cancer by targeting calpain and GRP.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6882-14-401) contains supplementary material, which is available to authorized users.
doi:10.1186/1472-6882-14-401
PMCID: PMC4210535  PMID: 25319833
GRP78; Calpain inhibitor; ER stress; Gene silencing; AST; Apoptosis; Metastasis; Tumor xenograft
3.  α-santalol inhibits the angiogenesis and growth of human prostate tumor growth by targeting vascular endothelial growth factor receptor 2-mediated AKT/mTOR/P70S6K signaling pathway 
Molecular Cancer  2013;12:147.
Background
VEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied in the cancer treatment. However, recently, most of these anticancer drugs have some adverse effects. Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed.
Methods
We used α-santalol and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVECs) and Prostate tumor cells (PC-3 or LNCaP) in vitro. Tumor xenografts in nude mice were used to examine the in vivo activity of α-santalol.
Results
α-santalol significantly inhibits HUVEC proliferation, migration, invasion, and tube formation. Western blot analysis indicated that α-santalol inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including AKT, ERK, FAK, Src, mTOR, and pS6K in HUVEC, PC-3 and LNCaP cells. α-santalol treatment inhibited ex vivo and in vivo angiogenesis as evident by rat aortic and sponge implant angiogenesis assay. α-santalol significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model. The antiangiogenic effect by CD31 immunohistochemical staining indicated that α-santalol inhibited tumorigenesis by targeting angiogenesis. Furthermore, α-santalol reduced the cell viability and induced apoptosis in PC-3 cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Molecular docking simulation indicated that α-santalol form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.
Conclusion
α-santalol inhibits angiogenesis by targeting VEGFR2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.
doi:10.1186/1476-4598-12-147
PMCID: PMC4221991  PMID: 24261856
α-santalol; Angiogenesis; VEGFR2; AKT/mTOR/P70S6K; Molecular docking
4.  The antihypertension drug doxazosin inhibits tumor growth and angiogenesis by decreasing VEGFR-2/Akt/mTOR signaling and VEGF and HIF-1α expression 
Oncotarget  2014;5(13):4935-4944.
Doxazosin is an α1 adrenergic receptor blocker that also exerts antitumor effects. However, the underlying mechanisms by which it modulates PI3K/Akt intracellular signaling are poorly understood. In this study, we reveal that doxazosin functions as a novel antiangiogenic agent by inhibiting vascular endothelial growth factor (VEGF)-induced cell migration and proliferation. It also inhibited VEGF-induced capillary-like structure tube formation in vitro. Doxazosin inhibited the phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream signaling, including PI3K, Akt, 3-phosphoinositide-dependent protein kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor 1 (HIF-1α). However, it had no effect on VEGF-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, doxazosin reduced tumor growth and suppressed tumor vascularization in a xenograft human ovarian cancer model. These results provide evidence that doxazosin functions in the endothelial cell system to modulate angiogenesis by inhibiting Akt and mTOR phosphorylation and interacting with VEGFR-2.
PMCID: PMC4148112  PMID: 24952732
doxazosin; anti-angiogenic activity; VEGFR-2; Akt/mTOR phosphorylation; endothelial cell
5.  Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1 
PLoS Medicine  2007;4(6):e186.
Background
While vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial.
Methods and Findings
We investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope.
Conclusions
This study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.
Shalom Avraham and colleagues' study provides evidence of a survival system in breast cancer cells by which VEGF acts as an internal autocrine survival factor through its binding to VEGFR1.
Editors' Summary
Background.
One woman in eight will develop breast cancer during her lifetime. Most of these women live for many years after their diagnosis and many are cured of their cancer. However, sometimes the cancer grows inexorably and spreads (metastasizes) around the body despite the efforts of oncologists. Characteristics of the tumor known as prognostic factors can indicate whether this spreading is likely to happen. Large tumors that have metastasized have a poorer prognosis than small tumors that are confined to the breast. The expression of specific proteins within the tumor also provides prognostic information. One protein whose expression is associated with a poor prognosis is vascular endothelial growth factor (VEGF). VEGF stimulates angiogenesis—the growth of new blood vessels. Small tumors get the nutrients needed for their growth from existing blood vessels but large tumors need to organize their own blood supply. They do this, in part, by secreting VEGF. This compound binds to proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels), which then send a signal into the cell instructing it to make new blood vessels. Angiogenesis inhibitors, including molecules that block the activity of VEGF receptors, are being developed for the treatment of cancer.
Why Was This Study Done?
Some breast cancer cell lines (cells isolated from breast cancers and grown in the laboratory) make VEGF and VEGF receptors (VEGFR1, VEGFR2, and neuropilin 1 [NRP1]). But, although some studies have reported an association between VEGFR1 expression in breast tumors and a poor prognosis, other studies have found no expression of VEGFR1 in breast tumors. Consequently, the role of VEGF receptors in breast cancer is unclear. In this study, the researchers analyzed the expression and function of VEGF and its receptors in breast cancer cells to investigate whether and how VEGF helps these cells to survive.
What Did the Researchers Do and Find?
The researchers first examined the expression of VEGF receptors in several human breast cancer cell lines. All of them expressed VEGFR1, some expressed NRP1, but VEGFR2 expression was universally low. They then investigated the function of VEGF and its receptors in two human breast cancer cell lines (MDA-MB-231 and MCF-7). In both cell lines, blocking the expression of VEGF or of VEGFR1 (but not of the other two receptors) reduced cell survival by stimulating a specific process of cell death called apoptosis. Unexpectedly, adding VEGF to the cultures did not reverse the effect of blocking VEGF expression, a result that suggests that VEGF and VEGFR1 do not affect breast cancer cell survival by acting at the cell surface. Accordingly, when the researchers examined where VEGFR1 occurs in the cell, they found it on the membranes around the nucleus of the breast cancer cell lines and not on the cell surface; several primary breast tumors and normal breast tissue had the same localization pattern. Finally, the researchers showed that inhibitors of VEGF action that act at the cell surface did not affect the survival of the breast cancer cell lines.
What Do These Findings Mean?
These findings suggest that VEGF helps breast cancer cells to survive in a unique way: by binding to VEGFR1 inside the cell. In other words, whereas VEGF normally acts as a paracrine growth factor (it is released by one cell and affects another cell), in breast cancer cells it might act as an internal autocrine (intracrine) survival factor, a factor that affects the cells in which it is produced. These findings need confirming in more cell lines and in primary breast cancers but could have important implications for the treatment of breast cancer. Inhibitors of VEGF and VEGFR1 that act inside the cell (small molecule drugs) might block breast cancer growth more effectively than inhibitors that act at the cell surface (for example, proteins that bind to the receptor), because internally acting inhibitors might both kill the tumor directly and have antiangiogenic effects, whereas externally acting inhibitors could only have the second effect.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040186.
US National Cancer Institute information for patients and professionals on breast cancer (in English and Spanish) and on angiogenesis (in English and Spanish)
MedlinePlus Encyclopedia information for patients on breast cancer (in English and Spanish)
CancerQuest, information from Emory University on cancer biology and on angiogenesis and angiogenesis inhibitors (in several languages)
Wikipedia pages on VEGF (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0040186
PMCID: PMC1885450  PMID: 17550303
6.  CDK-4 inhibitor P276 sensitizes Pancreatic Cancer cells to Gemcitabine induced Apoptosis 
Molecular Cancer Therapeutics  2012;11(7):1598-1608.
Despite advances in molecular pathogenesis, pancreatic cancer remains a major unsolved health problem. It is a rapidly invasive, metastatic tumor that is resistant to standard therapies. The phosphatidylinositol-3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in pancreatic cancer. Gemcitabine (Gem) is the mainstay treatment for metastatic pancreatic cancer. P276 is a novel CDK inhibitor that induces G2/M arrest and inhibits tumor growth in vivo models. Here, we determined that P276 sensitizes pancreatic cancer cells to Gem induced apoptosis, a mechanism mediated through inhibition of Akt-mTOR signaling. In vitro, the combination of P276 and Gem resulted in a dose- and time-dependent inhibition of proliferation and colony formation of pancreatic cancer cells but not with normal pancreatic ductal cells. This combination also induced apoptosis, as seen by activated caspase 3 and increased Bax/Bcl2 ratio. Gene profiling studies demonstrated that this combination downregulated Akt-mTOR signaling pathway, which was confirmed by western blot analyses. There was also a downregulation of vascular endothelial growth factor (VEGF) and interleukin-8 expression suggesting effects on angiogenesis pathway. In vivo, intraperitoneal administration of the P276-Gem combination significantly suppressed the growth of pancreatic cancer tumor xenografts. There was a reduction in CD31 positive blood vessels, and reduced VEGF expression, again suggesting an effect on angiogenesis. Taken together, these data suggest that P276-Gem combination is a novel potent therapeutic agent that can target the Akt-mTOR signaling pathway to inhibit both tumor growth and angiogenesis.
doi:10.1158/1535-7163.MCT-12-0102
PMCID: PMC3392497  PMID: 22532602
apoptosis; tumor xenograft; microarray; cell cycle; Akt; mTOR
7.  Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways 
PLoS ONE  2012;7(10):e47516.
Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.
doi:10.1371/journal.pone.0047516
PMCID: PMC3475699  PMID: 23094058
8.  Methylnaltrexone Potentiates the Anti-Angiogenic Effects of mTOR Inhibitors 
Background
Recent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose.
Methods
Methylnaltrexone (MNTX), a peripheral mu opioid receptor (MOR) antagonist, in combination with the mTOR inhibitors temsirolimus and/or rapamycin, was evaluated for inhibition of VEGF-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration as well as in vivo angiogenesis (mouse Matrigel plug assay).
Results
MNTX inhibited VEGF-induced EC proliferation and migration with an IC50 of ~100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation and migration from ~10 nM to ~1 nM and from ~50 to ~10 nM respectively. We observed similar effects with rapamycin. On a mechanistic level, we observed that MNTX increased EC plasma membrane-associated tyrosine phosphate activity. Inhibition of tyrosine phosphatase activity (3,4-dephostatin) blocked the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR Complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR Complex 1 (rapamycin and temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events.
Conclusions
Our data indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. Therefore, addition of MNTX could potentially lower the dose of mTOR inhibitors which could improve therapeutic index.
doi:10.1186/2040-2384-2-5
PMCID: PMC2831839  PMID: 20298531
9.  Stathmin Regulates Hypoxia-Inducible Factor-1α Expression through the Mammalian Target of Rapamycin Pathway in Ovarian Clear Cell Adenocarcinoma 
ISRN Pharmacology  2013;2013:279593.
Stathmin, a microtubule-destabilizing phosphoprotein, is highly expressed in ovarian cancer, but the pathophysiological significance of this protein in ovarian carcinoma cells remains poorly understood. This study reports the involvement of stathmin in the mTOR/HIF-1α/VEGF pathway in ovarian clear cell adenocarcinoma (CCA) during hypoxia. HIF-1α protein and VEGF mRNA levels were markedly elevated in RMG-1 cells, a CCA cell line, cultured under hypoxic conditions. Rapamycin, an inhibitor of mTOR complex 1, reduced the level of HIF-1α and blocked phosphorylation of ribosomal protein S6 kinase 1 (S6K), a transcriptional regulator of mTOR, demonstrating that hypoxia activates mTOR/S6K/HIF-1α signaling in CCA. Furthermore, stathmin knockdown inhibited hypoxia-induced HIF-1α and VEGF expression and S6K phosphorylation. The silencing of stathmin expression also reduced Akt phosphorylation, a critical event in the mTOR/HIF-1α/VEGF signaling pathway. By contrast, stathmin overexpression upregulated hypoxia-induced HIF-1α and VEGF expression in OVCAR-3 cells, another CCA cell line. In addition, suppression of Akt activation by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, decreased HIF-1α and VEGF expression. These results illustrate that regulation of HIF-1α through the PI3K/Akt/mTOR pathway is controlled by stathmin in CCA. Our findings point to a new mechanism of stathmin regulation during ovarian cancer.
doi:10.1155/2013/279593
PMCID: PMC3683482  PMID: 23819061
10.  Luteolin Inhibits Human Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis 
PLoS ONE  2012;7(12):e52279.
Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.
doi:10.1371/journal.pone.0052279
PMCID: PMC3534088  PMID: 23300633
11.  A Retro-inhibition Approach Reveals a Tumor Cell-Autonomous Response to Rapamycin in Head and Neck Squamous Carcinoma 
Cancer research  2008;68(4):1144-1153.
Emerging evidence supporting the activation of the Akt-mTOR signaling network in head and neck squamous cell carcinoma (HNSCC) progression has provided the rationale for exploring the therapeutic potential of inhibiting this pathway for HNSCC treatment. Indeed, rapamycin, a clinically relevant mTOR inhibitor, promotes the rapid regression of HNSCC-tumor xenografts in mice. However, rapamycin does not affect the growth of HNSCC cells in vitro, thus raising the possibility that, as for other cancer types, rapamycin may not target cancer cells directly but may instead act on a component of the tumor microenvioronment, such as tumor-associated vasculature. Here, we utilized a retro-inhibition approach to assess the contribution of cancer cell-autonomous actions of rapamycin to its antitumor activity in HNSCC. A rapamycin-resistant form of mTOR (mTOR-RR) was expressed in HNSCC cells, while retaining the wild-type (rapamycin-sensitive) mTOR alleles in host-derived endothelial and stromal cells. Expression of mTOR-RR prevented the decrease in phospho-S6 levels caused by rapamycin through mTOR in HNSCC cells but not in stromal cells, and rendered HNSCC xenografts completely resistant to the antitumoral activity of rapamycin. This reverse-pharmacology strategy also enabled monitoring the direct consequences of inhibiting mTOR in cancer cells within the complex tumor micro-environment, which revealed that mTOR controls the accumulation of HIF-1α and the consequent expression of VEGF and a glucose transporter, Glut-1, in HNSCC cells. These findings indicate that HNSCC cells are the primary target of rapamycin in vivo, and provide evidence that its anti-angiogenic effects may represent a downstream consequence of mTOR inhibition in HNSCC cells.
doi:10.1158/0008-5472.CAN-07-1756
PMCID: PMC3443567  PMID: 18281490
mTOR; xenograft; signal transduction; human squamous cell carcinoma; drug discovery; rapamycin; lentivirus
12.  Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer 
Strahlentherapie Und Onkologie  2014;190(12):1154-1162.
Background
The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells.
Materials and methods
Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase–polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay.
Results
Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells.
Conclusions
Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.
doi:10.1007/s00066-014-0708-z
PMCID: PMC4240909  PMID: 24989178
Radiation; Vascular endothelial growth factor C; Lung cancer cells; Endothelial cells; PI3K/Akt/mTOR signaling pathway; Strahlung; Vaskulärer endothelialer Wachstumsfaktor C; Lungenkrebszellen; Endothelzellen; PI3K/Akt/mTOR-Signalweg
13.  Potent Anti-Cancer Effect of 3′-Hydroxypterostilbene in Human Colon Xenograft Tumors 
PLoS ONE  2014;9(11):e111814.
Here we report that 3′-hydroxypterostilbene (HPSB), a natural pterostilbene analogue, was more potent than pterostilbene against the growth of human cancer cells (COLO 205, HCT-116, and HT-29) with measured IC50 values of 9.0, 40.2, and 70.9 µM, respectively. We found that HPSB effectively inhibited the growth of human colon cancer cells by inducing apoptosis and autophagy. Autophagy occurred at an early stage and was observed through the formation of acidic vesicular organelles and microtubule-associated protein 1 light chain 3-II production. At the molecular levels, the results from western blot analysis showed that HPSB significantly down-regulated phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) signalings including decreased the phosphorylation of mammalian target of rapamycin (mTOR). Significant therapeutic effects were demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with HPSB (10 mg/kg i.p.). These inhibitory effects were accompanied by mechanistic down-regulation of the protein levels of cyclooxygenase-2 (COX-2), matrix metallopeptidase-9 (MMP-9), vascular endothelial growth factor (VEGF), and cyclin D1, as well as by the induction of apoptosis in colon tumors. Our findings suggest that HPSB could serve as a novel promising agent for colon cancer treatment.
doi:10.1371/journal.pone.0111814
PMCID: PMC4229093  PMID: 25389774
14.  Predictive value of phosphorylated mammalian target of rapamycin for disease-free survival in breast cancer patients receiving neoadjuvant chemotherapy 
Oncology Letters  2014;8(6):2642-2648.
The mammalian target of rapamycin (mTOR)/eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) pathway plays a critical role in cell growth, survival and angiogenesis, and has been demonstrated to correlate with human epidermal growth factor receptor 2 (HER2) status. Neoadjuvant chemotherapy (NAC), also known as preoperative therapy, is now well established in the treatment of inoperable locally advanced and inflammatory breast cancer. In vitro study has shown that mTOR inhibitors, together with cytotoxic agents, exhibit tumor cell killing activity. A number of non-randomized studies in HER2-positive trastuzumab-resistant metastatic breast cancer have revealed the antitumor activity of mTOR inhibitors when used together with standard chemotherapy plus trastuzumab. In the present study, the expression levels of phosphorylated (p)-mTOR and p-4E-BP1 were analyzed in breast cancer patients prior to and following NAC, to determine whether p-mTOR and p-4E-BP1 affect the response to NAC and the subsequent survival. Formalin-fixed, paraffin-embedded tissues representing matched pairs of core biopsy (pre-NAC) and surgical specimen (post-NAC) from 83 patients with invasive ductal carcinomas were collected. Immunohistochemistry was performed to evaluate the expression of p-mTOR and p-4E-BP1 using a semi-quantitative scoring system by two pathologists. It was found that the expression of p-mTOR and p-4E-BP1 was downregulated following NAC. The decrease in mTOR expression following NAC was found to positively correlate with HER2 expression and the reduction of tumor sizes. The high expression of p-mTOR and p-4E-BP1 in pre-NAC specimens was associated with poor disease-free survival (DFS). Furthermore, the high expression of p-mTOR in post-NAC specimens was associated with poor DFS, regardless of whether the expression was high or low in the pre-NAC specimens. In conclusion, NAC was found to decrease the expression levels of p-mTOR and p-4E-BP1. The p-mTOR expression post-NAC may potentially serve as a predictor for DFS. However, further study is required to clarify the mechanism and to evaluate the predictive value of the phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway in NAC.
doi:10.3892/ol.2014.2551
PMCID: PMC4214504  PMID: 25364442
phosphorylated-mammalian target of rapamycin; phosphorylated-eukaryotic translation initiation factor 4E-binding protein; neoadjuvant chemotherapy; predictive value
15.  PI3K/AKT/mTOR Pathway in Angiogenesis 
The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is activated in the majority of human cancers. This pathway is known to play a key role in numerous cellular functions including proliferation, adhesion, migration, invasion, metabolism, and survival, but in the current review we focus on its role in angiogenesis. PI3K activation may occur via RAS mutation, loss of phosphatase and tensin homolog (PTEN), or by increased expression of growth factor receptors such as epidermal growth factor receptor. There is a connection between the PI3K pathway and angiogenesis. Hypoxia leads to HIF-1α stabilization and is a major stimulus for increased vascular endothelial growth factor (VEGF) production by tumor cells. However, activation of the PI3K/AKT pathway in tumor cells can also increase VEGF secretion, both by hypoxia-inducible factor 1 (HIF-1) dependent and independent mechanisms. The PI3K/AKT pathway also modulates the expression of other angiogenic factors such as nitric oxide and angiopoietins. Numerous inhibitors targeting the PI3K/AKT/mTOR pathway have been developed, and these agents have been shown to decrease VEGF secretion and angiogenesis. The effect of these inhibitors on tumor vasculature can be difficult to predict. The vasculature of tumors is aberrant, leading to sluggish bloodflow and elevated interstitial blood pressure, which can be perpetuated by the high levels of VEGF. Hence, decreasing VEGF expression can paradoxically lead to vascular normalization and improved bloodflow in some tumors. In addition to its importance in cancer, the PI3K pathway also plays an essential role in the formation of normal blood vessels during development. Embryos with kinase-dead p110α catalytic subunit of PI3K develop vascular defects. Stimulation of endothelial cells by VEGF leads to activation of the PI3K pathway within these cells, which is important for cell migration. Sustained endothelial activation of AKT1 has been shown to induce the formation of structurally abnormal blood vessels that recapitulate the aberrations of tumor vessels. Hence, the PI3K pathway plays an important role in regulating angiogenesis both in normal tissues and in cancers.
doi:10.3389/fnmol.2011.00051
PMCID: PMC3228996  PMID: 22144946
angiogenesis; PI3K/AKT/mTOR; VEGF; nitric oxide; angiopoietins
16.  Phosphorylation of mTOR and S6RP predicts the efficacy of everolimus in patients with metastatic renal cell carcinoma 
BMC Cancer  2014;14:376.
Background
The incidence of renal cell cancer (RCC) has been increasing for the past decade, and the 5-year survival for patients with metastatic RCC (mRCC) is rather low. Everolimus (RAD001), a new inhibitor for mammalian target of rapamycin (mTOR), is generally well tolerated, and demonstrates clinical benefit to patients with anti-VEGF-refractory mRCC. However, factors for selection of patients who may benefit from everolimus remain largely unknown. Here we aimed to explore potential molecular indicators for mRCC patients who may benefit from everolimus treatment.
Methods
Paraffin-embedded tumor tissue specimens derived from 18 mRCC patients before everolimus treatment, who participated the phase 1b trial of everolimus in VEGF receptor (VEGFR)-tyrosine kinase inhibitor (TKI)-refractory Chinese patients with mRCC (clinicaltrials.gov, NCT01152801), were examined for the expression levels of phosphorylated AKT, mTOR, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4EBP1) and 40S ribosomal protein S6 (S6RP) by immunohistochemistry. Clinical benefit rate (complete response [CR], partial response [PR], plus stable disease [SD] ≥ 6 months) and progression-free survival time (PFS) were correlated with expression levels of these mTOR-associated molecules.
Results
In these 18 patients, there were 1 PR, 15 SDs (including 9 SDs ≥ 6 months), and 2 progressive diseases (PD). The clinical benefit rate (CBR) was 55.6% (10/18), and the median PFS time was 8.4 months. Patients with positive expression of phospho-mTOR showed a better CBR (71.4% versus 0%, P = 0.023) and PFS time (11.3 versus 3.7 months, P = 0.001) than those patients with negative expression. The median PFS of patients with positive phospho-S6RP expression was longer (11.3 versus 3.7 months, P = 0.002) than that of patients negative for phospho-S6RP expression. However, expression levels of phospho-4EBP1 and phospho-AKT were unassociated to efficacy of everolimus treatment with respect to CBR and PFS. Co-expression of phosphorylated mTOR, S6RP and/or 4EBP1 may improve the predictive value of the biomarkers for patients treated with everolimus.
Conclusions
The expression levels of phospho-mTOR and phospho-S6RP may be potential predictive biomarkers for efficacy of everolimus in patients with mRCC. Combining examinations of phosphorylated mTOR, S6RP and/or 4EBP1 may be a potential strategy to select mRCC patients sensitive to mTOR inhibitor treatment.
doi:10.1186/1471-2407-14-376
PMCID: PMC4041340  PMID: 24886512
Metastatic renal cell carcinoma; Targeted therapy; Mammalian target of rapamycin; Clinical response; Predictive biomarker
17.  Role of mTOR in anticancer drug resistance 
The mammalian target of rapamycin (mTOR) pathway plays a central role in regulating protein synthesis, ribosomal protein translation, and cap-dependent translation. Deregulations in mTOR signaling are frequently associated with tumorigenesis, angiogenesis, tumor growth and metastasis. This review highlights the role of the mTOR in anticancer drug resistance. We discuss the network of signaling pathways in which the mTOR kinase is involved, including the structure and activation of the mTOR complex and the pathways upstream and downstream of mTOR as well as other molecular interactions of mTOR. Major upstream signaling components in control of mTOR activity are PI3K/PTEN/AKT and Ras/Raf/MEK/ERK pathways. We discuss the central role of mTOR in mediating the translation of mRNAs of proteins related to cell cycle progression, those involved in cell survival such as c-myc, hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), cyclin A, cyclin dependent kinases (cdk1/2), cdk inhibitors (p21Cip1 and p27Kip1), retinoblastoma (Rb) protein, and RNA polymerases I and III. We then discuss the potential therapeutic opportunities for using mTOR inhibitors rapamycin, CCI-779, RAD001, and AP-23573 in cancer therapy as single agents or in combinations to reverse drug resistance.
doi:10.1016/j.drup.2008.03.001
PMCID: PMC2519122  PMID: 18440854
mTOR; drug resistance; p70S6K1; PI3K; AKT; MAP kinase; VEGF; CCI-779; RAD001 (everolimus); AP-23573; neurofibromatosis 1
18.  Antitumor effect of Kanglaite® injection in human pancreatic cancer xenografts 
Background
Kanglaite® injection (KLT), with a main ingredient of Coix seed oil (a traditional Chinese medicine), has been widely used for cancer treatment in China. KLT has an inhibitory effect on many kinds of tumors and PI3K/Akt/mTOR signaling promotes cell survival, proliferation, and progression in cancer cells. Therefore, targeting this pathway may lead to the development of novel therapeutic approaches for human cancers.
Methods
Here, we examined the effects of KLT on the PI3K/Akt/mTOR pathway in pancreatic cancer xenografts in mice, and assessed its therapeutic potential. Growth and apoptosis of tumor xenografts were examined, and the expression levels of genes and proteins involved in the PI3K/Akt/mTOR pathway were measured by RT-PCR and western blotting, respectively.
Results
Our results revealed that KLT dramatically inhibited the growth of pancreatic cancer xenografts and induced apoptosis simultaneously. Furthermore, it downregulated the expression of phospho-Akt and phospho-mTOR.
Conclusions
These results suggest that KLT can suppress growth and induce apoptosis of pancreatic cancer xenografts. Moreover, KLT can downregulate the expression of phospho-Akt and phospho-mTOR to modulate the PI3K/Akt/mTOR signaling pathway.
doi:10.1186/1472-6882-14-228
PMCID: PMC4105135  PMID: 25005526
Kanglaite® injection; Pancreatic cancer; PI3K/Akt/mTOR signaling; Traditional Chinese medicine
19.  p16INK4A Represses Breast Stromal Fibroblasts Migration/Invasion and Their VEGF-A-dependent Promotion of Angiogenesis through Akt Inhibition1 
Neoplasia (New York, N.Y.)  2012;14(12):1269-1277.
Stromal fibroblasts, the most abundant and probably the most active cellular component of breast cancer-associated stroma, become active and promote angiogenesis through paracrine effects. However, it still unclear how these processes are regulated. Here, we have shown that down-regulation of the tumor suppressor p16INK4A protein enhances the migration/invasion abilities of breast stromal fibroblasts, which form dendritic network of extensions into matrigel. Furthermore, we present clear evidence that p16INK4A represses the expression/secretion of the proangiogenesis protein vascular endothelial growth factor A (VEGF-A). Consequently, p16INK4A-deficient breast stromal fibroblasts and mouse embryonic fibroblasts enhanced endothelial cell differentiation into capillary-like structures in a paracrine manner. This effect was suppressed by adding bevacizumab, a specific VEGF-A inhibitor. Additionally, p16INK4A-defective mouse embryonic fibroblasts enhanced angiogenesis in breast cancer xenografts in mice. Furthermore, we have shown that p16INK4A suppresses the Akt/mammalian target of rapamycin (mTOR) signaling pathway and its downstream effector hypoxia-inducible factor 1-alpha (HIF-1α), which transactivates VEGF-A. Consequently, Akt inactivation suppressed both the p16INK4A-dependent autocrine effect on fibroblast migration/invasion and the paracrine effect on angiogenesis, showing the important role of this protein kinase in mediating the various effects related to p16INK4A deficiency. These results indicate that p16INK4A is an efficient inhibitor of the migration/invasion abilities of breast stromal fibroblasts and also their paracrine proangiogenic effects, through inhibition of Akt. Therefore, pharmacologic restoration of p16INK4A level in stromal fibroblasts may be exploited as therapeutic strategy to help eradicate tumor cells and/or prevent their recurrence, through suppressing cell non-autonomous procarcinogenic mediators.
PMCID: PMC3540951  PMID: 23308058
20.  Celastrol Suppresses Angiogenesis-Mediated Tumor Growth through Inhibition of AKT/Mammalian Target of Rapamycin Pathway 
Cancer research  2010;70(5):1951-1959.
Understanding the molecular basis and target of traditional medicine is critical for drug development. Celastrol, derived from Trypterygium wilfordii Hook F. (“Thunder of God Vine”), a traditional Chinese medicine plant, has been assigned anticancer activities but its mechanism is not well understood. Here, we investigated whether Celestrol could inhibit angiogenesis-mediated tumor growth and if so through what mechanism. When administered subcutaneously to mice bearing human prostate cancer (PC-3 cell) xenografts, Celastrol (2 mg/kg/d) significantly reduced the volume and the weight of solid tumors and decreased tumor angiogenesis. We found that this agent inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, and capillary-like structure formation by primary cultured human umbilical endothelial cells (HUVECs) in a dose-dependent manner. Further, Celastrol abrogated VEGF-induced sprouting of the vessels from aortic rings and inhibited vascular formation in the Matrigel plug assay in vivo. To understand the molecular mechanism of these activities, we next examined the signaling pathways in treated HUVECs and PC-3 tumor cells. Celastrol suppressed the VEGF-induced activation of AKT, mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (P70S6K). Additionally, we found that Celastrol inhibited the proliferation of prostate cancer cells and induced apoptosis, and these effects correlated with the extent of inhibition of AKT/mTOR/P70S6 kinase signaling. Taken together, our results suggest that Celastrol targets the AKT/mTOR/P70S6K pathway, which leads to suppression of tumor growth and angiogenesis.
doi:10.1158/0008-5472.CAN-09-3201
PMCID: PMC2854134  PMID: 20160026
Celastrol; tumor angiogenesis; mTOR; Akt; p70S6 kinase; prostate tumor
21.  Targeting mTOR pathway: A new concept in cancer therapy 
This article highlights the current knowledge of mTOR biology and provides new insights into the role of mTOR in different cancers. An active mTOR coordinates a response in cell growth directly through its effects on cell cycle regulators and indirectly by sustaining nutrient supply into the cell through the production of nutrient transporters and also through the promotion of angiogenesis. A primary way that mTOR exerts its regulatory effects on cell proliferation is by controlling the production of cyclin D1. mTOR increases the translation of hypoxia-inducible factor 1 (HIF-1)/HIF-2. The HIF transcription factors drive the expression of hypoxic stress response genes, including angiogenic growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor β (PDGF-β), and transforming growth factor a (TGF-α). mTOR also increases the surface expression of nutrient transporters proteins. An increase in these proteins results in greater uptake of amino acids and other nutrients by the cell leading to adequate nutrient support to abnormal cell growth and survival. There is also emerging evidence that mTOR activation may play a role in promoting cell survival through the activation of antiapoptotic proteins that contribute to tumor progression. Given that the mTOR pathway is deregulated in a number of cancers, it is anticipated that mTOR inhibitors will have broad therapeutic application across many tumor types. Until now, no treatment demonstrated Phase III evidence after disease progression on an initial VEGF-targeted therapy in advanced renal cell carcinoma. Everolimus is the first and only therapy with Phase III evidence after failure of VEGF-targeted therapy. Everolimus is a once-daily, oral inhibitor of mTOR (mammalian target of rapamycin) indicated for the treatment of advanced renal cell carcinoma in patients, whose disease has progressed on or after treatment with VEGF-targeted therapy.
doi:10.4103/0971-5851.76197
PMCID: PMC3089921  PMID: 21584218
Angiogenesis; bioenergetics; everolimus
22.  E2F1 Inhibits c-Myc-driven Apoptosis via PIK3CA/Akt/mTOR and COX-2 in a Mouse Model of Human Liver Cancer 
Gastroenterology  2008;135(4):1322-1332.
Background & Aims
Resistance to apoptosis is essential for cancer growth. We previously reported that hepatic co-expression of c-Myc and E2F1, two key regulators of proliferation and apoptosis, enhanced HCC development in transgenic mice. Here, we investigated the molecular mechanisms underlying oncogenic cooperation between c-Myc and E2F1 in relationship to human liver cancer.
Methods
Activation of pro- and anti-apoptotic cascades was assessed by immunoblotting in in vivo and in vitro HCC models, and in primary human HCC. Effect of antisense oligodeoxy nucleotides against c-Myc and E2F1 was studied in human HCC cell lines. Suppression of PIK3CA/AKT, mTOR, and COX-2 pathways was achieved by pharmacological inhibitors and specific siRNAs in human and mouse HCC cell lines.
Results
Co-expression with E2F1 did not increase proliferation triggered by c-Myc overexpression but conferred a strong resistance to c-Myc-initiated apoptosis via concomitant induction of PIK3CA/Akt/mTOR and c-Myb/COX-2 survival pathways. COX-2 was not induced in c-Myc and rarely in E2F1 tumors. In human HCC, PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways were similarly activated, with levels of PIK3CA/Akt, mTOR, and c-Myb being inversely associated with patients’ survival length. Knocking down c-Myc and E2F1 oncoproteins reduced PIK3CA/Akt and mTOR and completely abolished c-Myb and COX-2 expression in human HCC cell lines. Finally, simultaneous inhibition of PIK3CA/Akt/mTOR and COX-2 activity in in vitro models caused massive apoptosis of neoplastic hepatocytes.
Conclusion
E2F1 may function as a critical anti-apoptotic factor both in human and rodent liver cancer through its ability to counteract c-Myc-driven apoptosis via activation of PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways.
doi:10.1053/j.gastro.2008.07.012
PMCID: PMC2614075  PMID: 18722373
23.  Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2 
Cancer research  2014;74(4):1067-1078.
Melanoma is one of the cancers of fastest-rising incidence in the world. iNOS is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K/AKT/mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p-P70S6K, p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of TSC2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Rheb, a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of mTOR pathway members. Exogenously-supplied NO was also sufficient to reverse mTOR pathway inhibition by the B-Raf inhibitor Vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers.
doi:10.1158/0008-5472.CAN-13-0588
PMCID: PMC3960077  PMID: 24398473
nitric oxide; mTOR; melanoma; nitrosylation; inducible nitric oxide synthase
24.  Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells 
BMC Cancer  2013;13:229.
Background
Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells.
Methods
To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system.
Results
Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines.
Conclusions
Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.
doi:10.1186/1471-2407-13-229
PMCID: PMC3658959  PMID: 23651517
25.  Acetyl-11-Keto-β-Boswellic Acid Inhibits Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis 
Cancer research  2009;69(14):5893-5900.
The role of angiogenesis in tumor growth and metastasis is well established. Identification of small molecule that blocks tumor angiogenesis and is safe and affordable has been a challenge in drug development. In this study, we demonstrated that acetyl-11-keto-β-boswellic acid (AKBA), an active component from an Ayurvedic medicinal plant (Boswellia serrata), could strongly inhibit tumor angiogenesis. AKBA suppressed tumor growth in the human prostate tumor xenograft mice treated daily (10 mg/kg of AKBA) after solid tumors reached about 100 mm3 (n=5). The inhibitory effect of AKBA on tumor growth was well correlated with suppression of angiogenesis. When examined for the molecular mechanism, we found that AKBA significantly inhibited blood vessel formation in the Matrigel plug assay in mice and effectively and suppressed vascular endothelial growth factor (VEGF)-induced microvessel sprouting in rat aortic ring assay ex vivo. Furthermore, AKBA inhibited VEGF-induced cell proliferation, chemotactic motility, and the formation of capillary-like structures from primary cultured human umbilical vascular endothelial cells (HUVECs) in a dose-dependent manner. Western blot analysis and in vitro kinase assay revealed that AKBA suppressed VEGF-induced phosphorylation of VEGF receptor 2 kinase (KDR/Flk-1) with IC50 of 1.68 μmol/L. Specifically, AKBA suppressed the downstream protein kinases of VEGFR2, including Src family kinase, focal adhesion kinase, extracellular signal-related kinase, AKT, mTOR, and ribosomal protein S6 kinase. Our findings suggest that AKBA potently inhibits human prostate tumor growth through inhibition of angiogenesis induced by VEGFR2 signaling pathways.
doi:10.1158/0008-5472.CAN-09-0755
PMCID: PMC2724674  PMID: 19567671
AKBA; KDR/Flk-1; tumor angiogenesis; mTOR; prostate tumor

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