Sivapriya Ponnurangam

Curcumin induces cell death in esophageal cancer cells through modulating Notch signaling.

Background Curcumin inhibits the growth of esophageal cancer cell lines; however, the mechanism of action is not well understood. It is becoming increasingly clear that aberrant activation of Notch signaling has been associated with the development of esophageal cancer. Here, we have determined that curcumin inhibits esophageal cancer growth via a mechanism mediated through the Notch signaling pathway. Methodology/Principal Findings In this study, we show that curcumin treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in esophageal cancer cell lines. Furthermore, curcumin treatment induced apoptosis through caspase 3 activation, confirmed by an increase in the ratio of Bax to Bcl2. Cell cycle analysis demonstrated that curcumin treatment induced cell death and down regulated cyclin D1 levels. Curcumin treatment also resulted in reduced number and size of esophagospheres. Furthermore, curcumin treatment led to reduced Notch-1 activation, expression of Jagged-1 and its downstream target Hes-1. This reduction in Notch-1 activation was determined to be due to the down-regulation of critical components of the γ-secretase complex proteins such as Presenilin 1 and Nicastrin. The combination of a known γ-secretase inhibitor DAPT and curcumin further decreased proliferation and induced apoptosis in esophageal cancer cells. Finally, curcumin treatment down-regulate the expressions of Notch-1 specific microRNAs miR-21 and miR-34a, and upregulated tumor suppressor let-7a miRNA. Conclusion/Significance Curcumin is a potent inhibitor of esophageal cancer growth that targets the Notch-1 activating γ-secretase complex proteins. These data suggest that Notch signaling inhibition is a novel mechanism of action for curcumin during therapeutic intervention in esophageal cancers.

Honokiol in Combination with Radiation Targets Notch Signaling to Inhibit Colon Cancer Stem Cells

Cancer stem cells are implicated in resistance to ionizing radiation (IR) and chemotherapy. Honokiol, a biphenolic compound has been used in traditional Chinese medicine for treating various ailments. In this study, we determined the ability of honokiol to enhance the sensitivity of colon cancer stem cells to IR. The combination of honokiol and IR suppressed proliferation and colony formation while inducing apoptosis of colon cancer cells in culture. There were also reduced numbers and size of spheroids, which was coupled with reduced expression of cancer stem cell marker protein DCLK1. Flow cytometry studies confirmed that the honokiol–IR combination reduced the number of DCLK1+ cells. In addition, there were reduced levels of activated Notch-1, its ligand Jagged-1, and the downstream target gene Hes-1. Furthermore, expression of components of the Notch-1 activating γ-secretase complex, presenilin 1, nicastrin, Pen2, and APH-1 was also suppressed. On the other hand, the honokiol effects were mitigated when the Notch intracellular domain was expressed. To determine the effect of honokiol–IR combination on tumor growth in vivo, nude mice tumor xenografts were administered honokiol intraperitoneally and exposed to IR. The honokiol–IR combination significantly inhibited tumor xenograft growth. In addition, there were reduced levels of DCLK1 and the Notch signaling–related proteins in the xenograft tissues. Together, these data suggest that honokiol is a potent inhibitor of colon cancer growth that targets the stem cells by inhibiting the γ-secretase complex and the Notch signaling pathway. These studies warrant further clinical evaluation for the combination of honokiol and IR for treating colon cancers. Mol Cancer Ther; 11(4); 963–72. ©2012 AACR.

Tandutinib Inhibits the Akt/mTOR Signaling Pathway to Inhibit Colon Cancer Growth

The c-Kit receptor can activate distinct signaling pathways including phosphoinositide 3-kinase (PI3K)/Akt and mTOR. Aberrant c-Kit activation protects cells from apoptosis and enhances invasion of colon carcinoma cells. Tandutinib is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases including c-Kit. We determined the effect of tandutinib on colon cancer growth and identified a mechanism of action. Tandutinib inhibited phosphorylation of c-Kit, Akt, mTOR, and p70S6 kinase. In addition, tandutinib significantly inhibited the proliferation and colony formation ability of colon cancer cell lines but did not affect normal colonic epithelial cells. There were increased levels of activated caspase-3 and Bax/Bcl2 ratio, coupled with a reduction in cyclin D1, suggesting apoptosis. There was also a downregulation of COX-2, VEGF, and interleukin-8 expression, suggesting effects on cancer-promoting genes. In addition, overexpressing constitutively active Akt partially suppressed tandutinib-mediated colon cancer cell growth. In vivo, intraperitoneal administration of tandutinib significantly suppressed growth of colon cancer tumor xenografts. There was a reduction in CD31-positive blood vessels, suggesting that there was an effect on angiogenesis. Tandutinib treatment also inhibited the expression of cancer-promoting genes COX-2 and VEGF and suppressed the activation of Akt/mTOR signaling proteins in the xenograft tissues. Together, these data suggest that tandutinib is a novel potent therapeutic agent that can target the Akt/mTOR/p70S6K signaling pathway to inhibit tumor growth and angiogenesis. Mol Cancer Ther; 12(5); 598–609. ©2013 AACR.

Quinomycin A targets Notch signaling pathway in pancreatic cancer stem cells

Cancer stem cells (CSCs) appear to explain many aspects of the neoplastic evolution of tumors and likely account for enhanced therapeutic resistance following treatment. Dysregulated Notch signaling, which affects CSCs plays an important role in pancreatic cancer progression. We have determined the ability of Quinomycin to inhibit CSCs and the Notch signaling pathway. Quinomycin treatment resulted in significant inhibition of proliferation and colony formation in pancreatic cancer cell lines, but not in normal pancreatic epithelial cells. Moreover, Quinomycin affected pancreatosphere formation. The compound also decreased the expression of CSC marker proteins DCLK1, CD44, CD24 and EPCAM. In addition, flow cytometry studies demonstrated that Quinomycin reduced the number of DCLK1+ cells. Furthermore, levels of Notch 1–4 receptors, their ligands Jagged1, Jagged2, DLL1, DLL3, DLL4 and the downstream target protein Hes-1 were reduced. The γ-secretase complex proteins, Presenilin 1, Nicastrin, Pen2, and APH-1, required for Notch activation also exhibited decreased expression. Ectopic expression of the Notch Intracellular Domain (NICD) partially rescued the cells from Quinomycin mediated growth suppression. To determine the effect of Quinomycin on tumor growth in vivo, nude mice carrying tumor xenografts were administered Quinomycin intraperitoneally every day for 21 days. Treatment with the compound significantly inhibited tumor xenograft growth, coupled with significant reduction in the expression of CSC markers and Notch signaling proteins. Together, these data suggest that Quinomycin is a potent inhibitor of pancreatic cancer that targets the stem cells by inhibiting Notch signaling proteins.

Bitter melon extracts enhance the activity of chemotherapeutic agents through the modulation of multiple drug resistance.

Recently, we demonstrated that extracts of bitter melon (BME) can be used as a preventive/therapeutic agent in colon cancers. Here, we determined BME effects on anticancer activity and bioavailability of doxorubicin (DOX) in colon cancer cells. BME enhanced the effect of DOX on cell proliferation and sensitized the cells toward DOX upon pretreatment. Furthermore, there was both increased drug uptake and reduced drug efflux. We also observed a reduction in the expression of multidrug resistance conferring proteins (MDRCP) P-glycoprotein, MRP-2, and BCRP. Further BME suppressed DOX efflux in MDCK cells overexpressing the three efflux proteins individually, suggesting that BME is a potent inhibitor of MDR function. Next, we determined the effect of BME on PXR, a xenobiotic sensing nuclear receptor and a transcription factor that controls the expression of the three MDR genes. BME suppressed PXR promoter activity thereby suppressing its expression. Finally, we determined the effect of AMPK pathway on drug efflux because we have previously demonstrated that BME affects the pathway. However, inhibiting AMPK did not affect drug resistance, suggesting that BME may use different pathways for the anticancer and MDR modulating activities. Together, these results suggest that BME can enhance the bioavailability and efficacy of conventional chemotherapy.

CDK-4 Inhibitor P276 Sensitizes Pancreatic Cancer Cells to Gemcitabine-Induced Apoptosis

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/Akt and mTOR signaling pathways are frequently dysregulated in pancreatic cancer. Gemcitabine 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 gemcitabine-induced apoptosis, a mechanism-mediated through inhibition of Akt-mTOR signaling. In vitro, the combination of P276 and gemcitabine 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 showed that this combination downregulated Akt-mTOR signaling pathway, which was confirmed by Western blot analyses. There was also a downregulation of 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. Mol Cancer Ther; 11(7); 1598–608. ©2012 AACR.

Inhibition of angiogenesis- and inflammation-inducing factors in human colon cancer cells in vitro and in ovo by free and nanoparticle-encapsulated redox dye, DCPIP

BackgroundThe redox dye, DCPIP, has recently shown to exhibit anti-melanoma activity in vitro and in vivo. On the other hand, there is increasing evidence that synthetic nanoparticles can serve as highly efficient carriers of drugs and vaccines for treatment of various diseases. These nanoparticles have shown to serve as potent tools that can increase the bioavailability of the drug/vaccine by facilitating absorption or conferring sustained and improved release. Here, we describe results on the effects of free- and nanoparticle-enclosed DCPIP as anti-angiogenesis and anti-inflammation agents in a human colon cancer HCT116 cell line in vitro, and in induced angiogenesis in ovo.ResultsThe studies described in this report indicate that (a) DCPIP inhibits proliferation of HCT116 cells in vitro; (b) DCPIP can selectively downregulate expression of the pro-angiogenesis growth factor, VEGF; (c) DCPIP inhibits activation of the transcriptional nuclear factor, NF-κB; (d) DCPIP can attenuate or completely inhibit VEGF-induced angiogenesis in the chick chorioallantoic membrane; (e) DCPIP at concentrations higher than 6 μg/ml induces apoptosis in HCT116 cells as confirmed by detection of caspase-3 and PARP degradation; and (f) DCPIP encapsulated in nanoparticles is equally or more effective than free DCPIP in exhibiting the aforementioned properties (a-e) in addition to reducing the expression of COX-2, and pro-inflammatory proteins IL-6 and IL-8.ConclusionsWe propose that, DCPIP may serve as a potent tool to prevent or disrupt the processes of cell proliferation, tissue angiogenesis and inflammation by directly or indirectly targeting expression of specific cellular factors. We also propose that the activities of DCPIP may be long-lasting and/or enhanced if it is delivered enclosed in specific nanoparticles.

Abstract 1893: Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function

Background: Despite advances in early detection, colon cancer remains the second leading cause of death in the United States. We are focused on developing dietary prevention strategies. HNK (HNK) is a biphenolic compound that is used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether HNK affected colon cancer stem cells and to identify a mechanism. Method: Colon cancer (CRC) cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we used the AOM/DSS-induced colonic tumorigenesis model. Immunohistochemistry was determined for stem cell markers and Hippo signaling proteins. Results: HNK induced a significant dose-dependent inhibition of proliferation and colony formation of the two CRC lines, but induced apoptosis. HNK did not affect the normal cells. To demonstrate HNK effects on stem cells, we performed colosphere assays. HNK significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where HNK reduced the number of DCLK1+ cells. We next determined whether stem cell signaling is affected. For this, we looked at the Hippo signaling pathway, which is active in intestinal stem cells. The key effector protein of this pathway, YAP1 is also oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm where it is degraded, thereby inhibiting downstream gene expression. HNK significantly reduced YAP1 levels. Furthermore, HNK inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from HNK-mediated growth suppression. To determine the in vivo effect of HNK on AOM/DSS induced colonic tumorigenesis, HNK were oral gavaged at a dose of 5mg/kg bw for 24 weeks. HNK treatment significantly reduced the colonic tumor numbers and size. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker proteins, oncogenic YAP1 phosphorylation and TEAD1 in the HNK-treated AOM/DSS colonic tumor tissues. Conclusion: Together, these data suggest that HNK prevents colonic tumorigenesis that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Deep Kwatra, Gaurav Kaushik, Prabhu Ramamoorthy, Satish Ramalingam, Ossama Tawfik, Scott J. Weir, Subhash Padhye, Dan A. Dixon, Shahid Umar, Roy A. Jensen, Shrikant Anant. Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2015-1893

Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769-82. ©2018 AACR.

Abstract 3227: Novel Marmelin analog DBQ targets Notch signaling pathway in colon cancer stem cells

Background: Colon cancer is the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound Marmelin from Aegle marmelos and potent anti-colon cancer activity. We have developed novel Marmelin analogue THB from this structure showed potent anti-cancer activity and its inhibitory constant value was 10 µM. From this, we developed a second series of analogs, of which DBQ is even more potent than THB. The current study is designed to determine whether DBQ affects colon cancer stem cells and identify a mechanism. Method: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we performed studies in HCT116 tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins. Results: DBQ treatment induced significant dose-dependent inhibition of proliferation and colony formation of HCT116 and SW480 cells, but not that of the normal FHC colon epithelial cells. DBQ also significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, DBQ reduced the levels of colon stem cell marker proteins DCLK1, LGR5, and CD44. We obtained further confirmation by flow cytometry, where DBQ treatment reduced the number of DCLK1+ cells. We next determined whether DBQ affects the Notch signaling, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. DBQ treatment significantly downregulated the expression of all four Notch isoforms, its ligands Jagged 1, 2 and DLL1, 3, 4 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. DBQ treatment inhibits the expression of γ-secretase complex proteins. To confirm that DBQ effect is thorough downregulating Notch activation, we ectopically expressed the Notch Intracellular domain. DBQ effect was significantly mitigated in this condition. To determine the effect of DBQ on tumor growth in vivo, we administered DBQ intraperitoneally (5mg/kg bw) every day for 21 days in mice carrying HCT116 tumor xenografts. DBQ treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the DBQ-treated xenograft tissues. Conclusion: Together, these data suggest that DBQ treatment suppresses colon cancer growth that targets stem cells in part by inhibiting Notch signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Prasad R. Dandawate, Gaurav Kaushik, Ossama W. Tawfik, Roy A. Jensen, Santimukul Santra, Subhash B. Padhye, Scott J. Weir, Shrikant Anant. Novel Marmelin analog DBQ targets Notch signaling pathway in colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3227. doi:10.1158/1538-7445.AM2017-3227