David Standing

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.

Methanolic Extracts of Bitter Melon Inhibit Colon Cancer Stem Cells by Affecting Energy Homeostasis and Autophagy

Bitter melon fruit is recommended in ancient Indian and Chinese medicine for prevention/treatment of diabetes. However its effects on cancer progression are not well understood. Here, we have determined the efficacy of methanolic extracts of bitter melon on colon cancer stem and progenitor cells. Both, whole fruit (BMW) and skin (BMSk) extracts showed significant inhibition of cell proliferation and colony formation, with BMW showing greater efficacy. In addition, the cells were arrested at the S phase of cell cycle. Moreover, BMW induced the cleavage of LC3B but not caspase 3/7, suggesting that the cells were undergoing autophagy and not apoptosis. Further confirmation of autophagy was obtained when western blots showed reduced Bcl-2 and increased Beclin-1, Atg 7 and 12 upon BMW treatment. BMW reduced cellular ATP levels coupled with activation of AMP activated protein kinase; on the other hand, exogenous additions of ATP lead to revival of cell proliferation. Finally, BMW treatment results in a dose-dependent reduction in the number and size of colonospheres. The extracts also decreased the expression of DCLK1 and Lgr5, markers of quiescent, and activated stem cells. Taken together, these results suggest that the extracts of bitter melon can be an effective preventive/therapeutic agent for colon cancer.

Honokiol inhibits melanoma stem cells by targeting notch signaling

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self‐renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor‐α‐Converting Enzyme (TACE) and γ‐secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch‐2. In addition, there was a reduction in the expression of downstream target proteins, Hes‐1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ‐secretase complex proteins in melanoma cells. To confirm that suppression of Notch‐2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes‐1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch‐2 signaling.

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.

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.

Abstract 2953: Notch signaling is a key pathway involved in drug resistance in melanoma cells

Background: Melanoma expresses a plastic and aggressive phenotype, lacking the majority of regulatory mechanisms due to the aberrant activation of various signaling pathways including the Notch pathway. Oncogenic BRAF mutation has the target for therapeutic interventions such as the drug vemurafinib but recent studies have indicated development of resistance. Unfortunately, however, the mechanisms of vemurafenib-induced resistance in melanoma are still poorly understood. We explored the role of Notch signaling in development of vemurafenib drug resistance in melanoma cells. Methods: We have utilized melanoma cell lines (especially B16/F10, SKMEL-28, A2058, UACC275 SKMEL103 and M14) with and without the common hot spot BRAFV600E mutation for the studies. We performed hexoseaminidase and clonogenicity assays to determine the cell growth rate and IC50 values in the cell lines. To generate drug resistant cells, UACC275 and SKMEL-28 cells were repetitively grown in the presence of vemurafenib. For stem cells, we did melanosphere formation assay. For protein expression, we performed western blots. Results: In the initial screening with vemurafinib, we observed a pattern of increased IC50 values in drug resistant cell lines, with UACC275 and SKMEL-28 being the more sensitive cells. Following sequential exposure, we developed vemurafinib-resistant cell lines, and observed that the IC50 values for proliferation inhibition to be ∼8-10 fold higher than the parental cells. Colony forming potential of the drug resistant cells was also not affected by increasing concentrations of vemurafenib, confirming acquisition of resistance. Furthermore, the drug resistant UACC275 cells presented a smaller and round morphology compared to the usual elongated and stretched appearance of the parent lines. Additionally, we also observed a significant increase in size of melanospheres for the drug resistance cells suggesting enrichment of stem cells. We further studied the expression and activation of various notch signaling cascade proteins and observed a significant increase in the levels of cleaved Notch-2, and -4 in the drug resistant cells. Interestingly, early passages of cell culture for drug resistant cells showed decrease in cleaved Notch-2 levels in cells with significant increase in basal levels of cleaved Notch-2 levels at Conclusion: As Notch-2 and -4 levels were higher in most of the resistant cells, therefore, notch signaling may play critical role in the development of vemurafenib drug resistance in melanoma cells. Targeting specific notch in patients on chemotherapy especially on BRAF inhibitors will be a key event for better progression and treatment of melanoma. Citation Format: Gaurav Kaushik, Jonathan Sheldon, Prasad Dandawate, Dharmalingam Subramaniam, David Standing, Shrikant Anant, Joshua M.V. Mammen. Notch signaling is a key pathway involved in drug resistance in melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2953.

Abstract 972: Bitter melon extracts enhance the activity of chemotherapeutic agents through the modulation of multiple drug resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnPurpose: Bitter melon (BM) is a tropical and subtropical vine, widely grown in Asia, Africa, and the Caribbean for its edible fruit. The fruit is recommended in ancient Indian and Chinese medicine for prevention/treatment of diabetes. Recent publications have demonstrated that BM extracts inhibit the growth of breast and prostate cancers. Our previous studies also suggest that it can be used as a preventive/therapeutic agent in colon cancers. Phytochemical usage and dietary supplementation can often lead to improved efficacy of conventional therapies but they may also lead to drug-drug interactions. Thus, we studied the effects of bitter melon on the anticancer activity as well as bioavailability of doxorubicin (DOX) in colon cancer cells (SW480 and HT29)nnMethods: Cell proliferation studies using DOX were carried out in colon cancer cells either pretreated or co-treated with BM extract. To identify the effects of BM on active permeation of DOX drug uptake and efflux studies were carried out in colon cancer cells. PCR and western blots were performed to determine the changes in major efflux protein levels involved in limiting the uptake of DOX in cancer cells. Further, luciferase based activity assays for PXR, a xenobiotic sensing nuclear receptor were performed to identify the mechanism with which BM altered the efflux transporter expression.nnResults: BM was found to enhance the effect of DOX as well as sensitize the cells towards DOX upon pretreatment. Combinatorial index was calculated to identify synergism between the therapeutics. This effect may be result of altered drug permeation into the cells as both drug uptake and efflux were found to reduce in the presence of BM pre- and co- treatment. The reduced efflux and uptake maybe due to inhibition as well as alteration in expression of expression of Multidrug resistance genes (P-glycoprotein, MRP-2 and BCRP) as was observed with PCR and western blots. The BM extracts were seen to effect PXR activation as well as expression in colon cancer cells which might be responsible for the altered efflux protein expression. Since AMPK pathway is the main target of BM we also studied the effect of other AMPK activators to see whether there is a correlation between AMPK pathway and efflux. Our results were interesting in showing potential correlation between AMPK pathway and drug efflux.nnConclusion: Taken together these results suggest that BM can enhance the bioavailability and efficacy of conventional chemotherapy. This can lead to reduction in minimum effective doses of chemotherapeutics thereby reducing toxicity/side effects.nnCitation Format: Deep Kwatra, Dharmalingam Subramaniam, David Standing, Ashim K. Mitra, Shrikant Anant. Bitter melon extracts enhance the activity of chemotherapeutic agents through the modulation of multiple drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 972. doi:10.1158/1538-7445.AM2013-972

Abstract 2584: Tandutinib inhibits the PI3 Kinase/Akt/mTOR signaling pathway to inhibit colon cancer growth.

Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. The c-Kit receptor can activate distinct signaling pathways including phosphatidylinositol-3- kinase (PI3K)/Akt and mammalian target of rapamycin (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 and PDGFR. The current study is designed to determine the effect of tandutinib on colon cancer growth and identified a mechanism of action. Method: Colon cancer cell lines HCT116, HT-29 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. Cell cycle analysis was measured by Flow cytometry. Signaling proteins were quantified by western blot analysis. For in vivo effects, HCT116 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for CD31 and Akt/mTOR signaling proteins. Results: Tandutinib treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in all three cell lines but did not affect normal colonic epithelial cells. Treatment also induced colon cancer cells to undergo apoptosis and G0/G1 arrest. Apoptosis was further confirmed by increased levels of activated caspase 3 and Bax/Bcl2 ratio, coupled with a reduction in cyclin D1. There was also a downregulation of cyclooxygenase 2 (COX-2), vascular endothelial growth factor (VEGF) and interleukin-8 expression suggesting effects on cancer promoting genes. Moreover, tandutinib inhibited phosphorylation of c-Kit, Akt, mTOR, p70S6 Kinase and 4EBP1. Overexpression of constitutively active Akt1 overcame this inhibitory effect suggesting a critical role for this pathway. To determine the effect of tandutinib on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Tandutinib treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following tandutinib treatment when compared to controls suggesting inhibition of angiogenesis. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression cancer promoting genes COX-2 and VEGF, and suppressed the activation of Akt/mTOR signaling proteins in the tandutinib-treated xenograft tissues. Conclusion: 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. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, David Standing, Parthasarathy Rangarajan. Tandutinib inhibits the PI3 Kinase/Akt/mTOR signaling pathway to inhibit colon cancer growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2584. doi:10.1158/1538-7445.AM2013-2584

325 CELF2 Induced Splicing Results in Loss of the Bir Domain in Survivin: Critical Role of XIAP in Radiation Induced Mitotic Catastrophe

ized wild-type(CVN), MyD88f/f (WT), MyD88-/and MyD88IEC-/mice (4-8/group) were subjected to ileal ischemia by obstructing the distal branches of the superior mesenteric artery for 60 minutes (ischemia) followed by 90 minutes of reperfusion. Sections of the ileum inside the ischemic region (damage) and outside (healthy) were collected and fixed in 10% formalin. Tissue damage was assessed using a histological necrosis scoring system. Expression of ileal pro-inflammatory mediators (Cxcl1, Il1b, Tnf) was evaluated using ABIPrism sequence detection system. Neutrophil infiltration wasmeasured by stainingmyeloperoxidase (MPO) using IHC. Results: Intestinal RNA showed the expected WT MyD88 product of 405 bp (MyD88f/f), whereas a fragment length of 226 bp was found in MyD88IEC-/mice. I/R-induced ileal injury was greater in GF mice vs. CVN mice (6+/-0.9vs2.8+/0.8,p<0.05). This finding suggests that bacteria play a protective role during I/R-induced injury. To link this effect to host innate signaling, we performed I/R in MyD88 deficient mice. No difference in histological damage was observed between genotypes after 60 min ischemia, but I/R-induced intestinal damage diminished in MyD88IEC-/compared to MyD88f/f control mice after 90min reperfusion (4.6+/-1.3vs10+/-1.7,p<0.05). This improvement was similar to the one observed in MyD88-/mice. Neutrophil infiltration per crypt/ villus significantly decreased in I/R-exposed MyD88IEC-/mice compared to MyD88f/f control mice (1.7+/-0.3vs4+/-0.3,p<0.05). In addition, Cxcl1 mRNA accumulation (foldincrease over healthy WT control) significantly decreased in I/R-exposed MyD88IEC-/mice compared to MyD88f/f control mice (205+/-130vs1372+/-538,p<0.05). In contrast Tnf mRNA expression was significantly upregulated in I/R-exposed MyD88IEC-/mice compared to MyD88f/f control mice (8.37+/-1.9-fold over WT,p<0.05) whereas the Il1b mRNA expression remained unchanged. Conclusion: Our data suggests that while bacteria protect against I/R-induced intestinal injury, this effect is independent of IEC-derived MyD88 signaling. In contrast, epithelial-derived MyD88 signaling contributes to intestinal damage and is associated with enhanced Cxcl1 expression and crypt neutrophil infiltration.

Abstract 5287: RNA binding protein RBM3 promotes metastasis through induction of hemangiogenesis and lymphangiogenesis

Introduction: Tumor metastasis, rather than primary tumors is the main cause of cancer-related deaths. A critical aspect of tumor metastasis, driven by vascular endothelial growth factors (VEGF) is the development of new vessels through hemangiogenesis and lymphangiogenesis. RNA binding protein RBM3 is a novel protooncogene that transforms normal cells when overexpressed. Here, we report that RBM3 mediates both hemangiogenesis and lymphangiogenesis. Results: Immunohistochemistry analysis of a human tissue microarray demonstrated increased RBM3 expression in tumors when compared to normal tissue, with even higher levels in metastatic tissues. Furthermore, RBM3-transformed NIH3T3 cells were injected into flanks of athymic nude mice metastasized to the liver and lungs. Immunohistochemistry analysis of the primary NIH3T3-RBM3 xenograft demonstrated increased number of microvessels that were lined by either CD31 or LYVE1 positive cells suggesting the increase in hem- and lymphangiogenesis. To demonstrate that RBM3 induces hemangiogenesis, tube formation assay using human umbilical vein endothelial (HUVEC) cells was performed. RBM3 overexpression increased, while RBM3 knockdown inhibited tube formation. Furthermore, NIH3T3-RBM3 cells significantly induced de novo capillary formation in the Chorio-Allantoic Membrane assay. Similarly, RBM3 overexpression enhanced, while knockdown suppressed tube formation by human lymphatic endothelial (HLEC) cells suggesting that it also affects lymphangiogenesis. To confirm the RBM3 role in hem- and lymphangiogenesis, HCT116 conditioned media was used in the tube formation assays. Tube formation by both HUVEC and HLEC cells was significantly increased in media from HCT116-RBM3 overexpressing cells. Moreover, both HUVEC and HLEC cells showed significant migration through collagen in transwell invasion assay. We further determined that this was due to the increased expression of VEGF A, VEGF C and VEGF D because siRNA mediated suppression of either one of these factors significantly reduced RBM3-mediated enhanced HLEC tube formation and migration through collagen. We next determined the mechanism of RBM3 mediated upregulation of the three VEGFs. HCT116 cells overexpressing RBM3 showed increased steady state levels of VEGF A, C and D mRNAs when compared to control. In addition, actinomycin D-stability assays demonstrated that there was increased stability of the three VEGF mRNAs in HCT116-RBM3 cells when compared to HCT116 control cells. Conclusion: Taken together these data suggests that RBM3 is a key regulator of hemangiogenesis and lymphangiogenesis, thereby inducing a highly metastatic phenotype through upregulation of VEGF A, C and D at the posttranscriptional levels of mRNA stability and translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5287. doi:1538-7445.AM2012-5287