Parmeswaran Diagaradjane

Curcumin Potentiates Antitumor Activity of Gemcitabine in an Orthotopic Model of Pancreatic Cancer through Suppression of Proliferation, Angiogenesis, and Inhibition of Nuclear Factor-κB–Regulated Gene Products

Gemcitabine is currently the best treatment available for pancreatic cancer, but the disease develops resistance to the drug over time. Agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Curcumin, a component of turmeric (Curcuma longa), is one such agent that has been shown to suppress the transcription factor nuclear factor-kappaB (NF-kappaB), which is implicated in proliferation, survival, angiogenesis, and chemoresistance. In this study, we investigated whether curcumin can sensitize pancreatic cancer to gemcitabine in vitro and in vivo. In vitro, curcumin inhibited the proliferation of various pancreatic cancer cell lines, potentiated the apoptosis induced by gemcitabine, and inhibited constitutive NF-kappaB activation in the cells. In vivo, tumors from nude mice injected with pancreatic cancer cells and treated with a combination of curcumin and gemcitabine showed significant reductions in volume (P = 0.008 versus control; P = 0.036 versus gemcitabine alone), Ki-67 proliferation index (P = 0.030 versus control), NF-kappaB activation, and expression of NF-kappaB-regulated gene products (cyclin D1, c-myc, Bcl-2, Bcl-xL, cellular inhibitor of apoptosis protein-1, cyclooxygenase-2, matrix metalloproteinase, and vascular endothelial growth factor) compared with tumors from control mice treated with olive oil only. The combination treatment was also highly effective in suppressing angiogenesis as indicated by a decrease in CD31(+) microvessel density (P = 0.018 versus control). Overall, our results suggest that curcumin potentiates the antitumor effects of gemcitabine in pancreatic cancer by suppressing proliferation, angiogenesis, NF-kappaB, and NF-kappaB-regulated gene products.

Imaging epidermal growth factor receptor expression in vivo: Pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe

Purpose: To develop and validate an optical imaging nanoprobe for the discrimination of epidermal growth factor (EGF) receptor (EGFR)–overexpressing tumors from surrounding normal tissues that also expresses EGFR. Experimental Design: Near-infrared (NIR) quantum dots (QD) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. In vitro binding affinity of these nanoprobes and unconjugated QDs was evaluated in a panel of cell lines, with and without anti-EGFR antibody pretreatment. Serial optical imaging of HCT116 xenograft tumors was done after systemic injection of QD and EGF-QD. Results: EGF-QD showed EGFR-specific binding in vitro. In vivo imaging showed three distinct phases, tumor influx (∼3 min), clearance (∼60 min), and accumulation (1-6 h), of EGF-QD nanoprobes. Both QD and EGF-QD showed comparable nonspecific rapid tumor influx and clearance followed by attainment of an apparent dynamic equilibrium at ∼60 min. Subsequently (1-6 h), whereas QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors. On delayed imaging at 24 h, tumor fluorescence decreased to near-baseline levels for both QD and EGF-QD. Ex vivo whole-organ fluorescence, tissue homogenate fluorescence, and confocal microscopic analyses confirmed tumor-specific accumulation of EGF-QD at 4 h. Immunofluorescence images showed diffuse colocalization of EGF-QD fluorescence within EGFR-expressing tumor parenchyma compared with patchy perivascular sequestration of QD. Conclusion: These results represent the first pharmacokinetic characterization of a robust EGFR imaging nanoprobe. The measurable contrast enhancement of tumors 4 h after systemic administration of EGF-QD and its subsequent normalization at 24 h imply that this nanoprobe may permit quantifiable and repetitive imaging of EGFR expression.

Modulation of in Vivo Tumor Radiation Response via Gold Nanoshell-Mediated Vascular-Focused Hyperthermia: Characterizing an Integrated Antihypoxic and Localized Vascular Disrupting Targeting Strategy

We report noninvasive modulation of in vivo tumor radiation response using gold nanoshells. Mild-temperature hyperthermia generated by near-infrared illumination of gold nanoshell-laden tumors, noninvasively quantified by magnetic resonance temperature imaging, causes an early increase in tumor perfusion that reduces the hypoxic fraction of tumors. A subsequent radiation dose induces vascular disruption with extensive tumor necrosis. Gold nanoshells sequestered in the perivascular space mediate these two tumor vasculature-focused effects to improve radiation response of tumors. This novel integrated antihypoxic and localized vascular disrupting therapy can potentially be combined with other conventional antitumor therapies.

Curcumin Sensitizes Human Colorectal Cancer Xenografts in Nude Mice to γ-Radiation by Targeting Nuclear Factor-κB–Regulated Gene Products

Purpose: How colorectal cancer develops resistance to γ-radiation is not fully understood, but the transcription factor nuclear factor-κB (NF-κB) and NF-κB–regulated gene products have been proposed as mediators. Because curcumin, a component of turmeric (Curcuma longa), has been shown to suppress NF-κB activation, whether it can sensitize the colorectal cancer to γ-radiation was investigated in colorectal cancer xenografts in nude mice. Experimental Design: We established HCT 116 xenograft in nude mice, randomized into four groups, and treated with vehicle (corn oil), curcumin, γ-radiation, and curcumin in combination with γ-radiation. NF-κB modulation was ascertained using electrophoretic mobility shift assay and immunohistochemistry. Markers of proliferation, angiogenesis, and invasion were monitored by immunohistochemistry and Western blot analysis. Results: Curcumin significantly enhanced the efficacy of fractionated radiation therapy by prolonging the time to tumor regrowth (P = 0.02) and by reducing the Ki-67 proliferation index (P < 0. 001). Moreover, curcumin suppressed NF-κB activity and the expression of NF-κB–regulated gene products (cyclin D1, c-myc, Bcl-2, Bcl-xL, cellular inhibitor of apoptosis protein-1, cyclooxygenase-2, matrix metalloproteinase-9, and vascular endothelial growth factor), many of which were induced by radiation therapy and mediate radioresistance. The combination of curcumin and radiation therapy also suppressed angiogenesis, as indicated by a decrease in vascular endothelial growth factor and microvessel density (P = 0.002 versus radiation alone). Conclusion: Collectively, our results suggest that curcumin potentiates the antitumor effects of radiation therapy in colorectal cancer by suppressing NF-κB and NF-κB–regulated gene products, leading to inhibition of proliferation and angiogenesis.

Resveratrol, a multitargeted agent, can enhance antitumor activity of gemcitabine in vitro and in orthotopic mouse model of human pancreatic cancer

Gemcitabine, while a standard treatment of advanced pancreatic cancer (PaCa), alone is not very effective. New agents that are safe and effective are highly needed. Resveratrol is one such agent which is safe and multitargeted; and has been linked with suppression of survival, proliferation, invasion and angiogenesis of cancer. Whether resveratrol can sensitize PaCa to gemcitabine in vitro and in vivo was investigated. We established PaCa xenografts in nude mice, randomized into 4 groups, and treated with vehicle, gemcitabine, resveratrol and with combination. Modulation of NF‐κB and markers of proliferation, angiogenesis and invasion were ascertained using electrophoretic mobility shift assay (EMSA), immunohistochemistry and western blot analysis. Resveratrol inhibited the proliferation of 4 different human PaCa cell lines, synergized the apoptotic effects of gemcitabine, inhibited the constitutive activation of NF‐κB and expression of bcl‐2, bcl‐xL, COX‐2, cyclin D1 MMP‐9 and VEGF. In an orthotopic model of human PaCa, we found that resveratrol significantly suppressed the growth of the tumor (p < 0.001) and this effect was further enhanced by gemcitabine (p < 0.001). Both the markers of proliferation index Ki‐67 and the micro vessel density CD31 were significantly downregulated in tumor tissue by the combination of gemcitabine and resveratrol (p < 0.001 vs. control; p < 0.01 vs. gemcitabine). As compared to vehicle control, resveratrol also suppressed the NF‐κB activation and expression of cyclin D1, COX‐2, ICAM‐1, MMP‐9 and survivin. Overall our results demonstrate that resveratrol can potentiate the effects of gemcitabine through suppression of markers of proliferation, invasion, angiogenesis and metastasis.

Thermal Enhancement with Optically Activated Gold Nanoshells Sensitizes Breast Cancer Stem Cells to Radiation Therapy

Local hyperthermia with gold nanoshells sensitizes cancer stem cells to radiation treatment in mouse and human breast cancer preclinical models. A Midas Touch for Breast Cancer Treatment One of the biggest hurdles to beating breast cancer is that a small population of stem cell–like cells (CSCs) within the tumor are stubbornly resistant to radiation therapy and chemotherapy. It is this CSC subpopulation that is responsible for relapse after successful treatment with radiation and drugs. Atkinson and colleagues now take a nanotech approach to solving this problem. Working in two different mouse models of breast cancer, they use gold nanoshells to turn up the heat on CSCs, making them more sensitive to radiation therapy. To sensitize breast tumor CSCs to radiation treatment, Atkinson and colleagues engineered gold nanoshells (nanoparticles comprising a silica core with an ultrathin gold layer) that accumulate preferentially in solid tumors and can be activated by near-infrared light (which is able to penetrate tissues). When activated by a laser, the nanoshells cause local heating of the tumors in which they have accumulated. The investigators first tested their gold nanoshells in mice bearing breast tumors that were particularly aggressive and radioresistant. Using surface markers and flow cytometry, the authors found that these breast tumors contained a cell population similar to the CSCs of human breast tumors. They injected their gold nanoshells intravenously into the tumor-bearing mice and then exposed the animals to both near-infrared laser light and 6 gray of ionizing radiation. This dual treatment not only shrank the tumors but also decreased the number of CSCs. Atkinson and colleagues then transplanted the treated tumors into syngeneic recipient mice and found that the tumors had become less aggressive and more differentiated in response to the dual laser radiation treatment. The researchers then went a step further, repeating these experiments with human breast tumor biopsy samples propagated in mice. Once again, they saw that the nanoshell-induced heating effect rendered the human breast tumors and their CSCs much more sensitive to ionizing radiation. But how does the combined treatment work? The investigators demonstrated that nanoshell-induced heating prevented breast tumor cells from repairing DNA double-strand breaks induced by ionizing radiation, resulting in an increase in their radiation sensitivity. Although the gold nanoshells still require further testing, hyperthermia treatments are already in clinical trials, and ionizing radiation is a staple of cancer therapy. This suggests that the dual hyperthermia-radiation cancer therapy of Atkinson et al. should be amenable to translation to a clinical setting. Breast cancer metastasis and disease recurrence are hypothesized to result from residual cancer stem cells, also referred to as tumor-initiating cells, which evade initial treatment. Using both syngeneic mouse and human xenograft models of triple-negative breast cancer, we have demonstrated that a subpopulation enriched in cancer stem cells was more resistant to treatment with 6 gray of ionizing radiation than the bulk of the tumor cells, and accordingly their relative proportion increased 48 to 72 hours after ionizing radiation treatment. In contrast, we achieved a larger reduction in tumor size without a concomitant increase in the percentage of cancer stem cells by treating with local hyperthermia for 20 minutes at 42°C after ionizing radiation using intravenously administered, optically activated gold nanoshells. Forty-eight hours after treatment, cells derived from the tumors treated with ionizing radiation plus hyperthermia exhibited both a marked decrease in tumorigenicity and a more differentiated phenotype than mock- and ionizing radiation–treated tumors. Thus, we have confirmed that these cancer stem cells are responsible for accelerated repopulation in vivo and demonstrated that hyperthermia sensitizes this cell population to radiation treatment. These findings suggest that local hyperthermia delivered by gold nanoshells plus radiation can eliminate radioresistant breast cancer stem cells.

Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model

Because of the poor prognosis and the development of resistance against chemotherapeutic drugs, the current treatment for advanced metastatic colorectal cancer (CRC) is ineffective. Whether curcumin (a component of turmeric) can potentiate the effect of capecitabine against growth and metastasis of CRC was investigated. The effect of curcumin on proliferation of CRC cell lines was examined by mitochondrial dye‐uptake assay, apoptosis by esterase staining, nuclear factor‐kappaB (NF‐κB) by electrophoretic mobility shift assay and gene expression by Western blot analysis. The effect of curcumin on the growth and metastasis of CRC was also examined in orthotopically implanted tumors in nude mice. In vitro, curcumin inhibited the proliferation of human CRC cell lines, potentiated capecitabine‐induced apoptosis, inhibited NF‐κB activation and suppressed NF‐κB‐regulated gene products. In nude mice, the combination of curcumin and capecitabine was found to be more effective than either agent alone in reducing tumor volume (p = 0.001 vs. control; p = 0.031 vs. capecitabine alone), Ki‐67 proliferation index (p = 0.001 vs. control) and microvessel density marker CD31. The combination treatment was also highly effective in suppressing ascites and distant metastasis to the liver, intestines, lungs, rectum and spleen. This effect was accompanied by suppressed expression of activated NF‐κB and NF‐κB‐regulated gene products (cyclin D1,c‐myc, bcl‐2, bcl‐xL, cIAP‐1, COX‐2, ICAM‐1, MMP‐9, CXCR4 and VEGF). Overall, our results suggest that curcumin sensitizes CRC to the antitumor and antimetastatic effects of capecitabine by suppressing NF‐κB cell signaling pathway.

Neutrophil Gelatinase–Associated Lipocalin: A Novel Suppressor of Invasion and Angiogenesis in Pancreatic Cancer

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa secreted acute phase protein, which is also up-regulated in multiple cancers, including breast, lung, and pancreas. Recently, NGAL has been proposed as an early biomarker in pancreatic cancer (PaCa). However, its biological role in PaCa is unknown. In this study, we examined in vitro and in vivo the functional role of NGAL in PaCa. Well- to moderately differentiated PaCa cells (AsPC-1, BxPC-3, and Capan-2) expressed high levels of NGAL but moderately to poorly differentiated PaCa cells (PANC-1 and MIAPaCa-2) expressed undetectable NGAL levels. Immunohistochemistry of untreated tissue microarray showed specific NGAL staining in resected PaCa specimens (P = 0.0167). Stable NGAL overexpression (MIAPaCa-2 and PANC-1) significantly blocked PaCa cell adhesion and invasion in vitro and vice versa with stable PaCa clones (BxPC-3 and AsPC-1). Moreover, NGAL overexpression reduced focal adhesion kinase (FAK) tyrosine-397 phosphorylation in PaCa cells. Furthermore, NGAL overexpression potently decreased angiogenesis in vitro partly through reduced vascular endothelial growth factor (VEGF) production and vice versa. Stable NGAL overexpression or underexpression had no effect on PaCa cell survival, viability, and response to chemotherapeutic drugs. Finally, MIAPaCa-2 cells overexpressing NGAL reduced tumor volume (P = 0.012), local and distant metastasis (P = 0.002), and angiogenesis (P = 0.05) with no effect on K-67 proliferation index (P > 0.1) in an orthotopic nude mouse PaCa model. Collectively, our results suggest that NGAL reduces adhesion/invasion partly by suppressing FAK activation and inhibits angiogenesis partly by blocking VEGF production in PaCa cells. Thus, NGAL is a potential suppressor of invasion and angiogenesis in advanced PaCa.

Nanoparticle-mediated thermal therapy: Evolving strategies for prostate cancer therapy

Purpose: Recent advances in nanotechnology have resulted in the manufacture of a plethora of nanoparticles of different sizes, shapes, core physicochemical properties and surface modifications that are being investigated for potential medical applications, particularly for the treatment of cancer. This review focuses on the therapeutic use of customised gold nanoparticles, magnetic nanoparticles and carbon nanotubes that efficiently generate heat upon electromagnetic (light and magnetic fields) stimulation after direct injection into tumours or preferential accumulation in tumours following systemic administration. This review will also focus on the evolving strategies to improve the therapeutic index of prostate cancer treatment using nanoparticle-mediated hyperthermia. Conclusions: Nanoparticle-mediated thermal therapy is a new and minimally invasive tool in the armamentarium for the treatment of cancers. Unique challenges posed by this form of hyperthermia include the non-target biodistribution of nanoparticles in the reticuloendothelial system when administered systemically, the inability to visualise or quantify the global concentration and spatial distribution of these particles within tumours, the lack of standardised thermal modelling and dosimetry algorithms, and the concerns regarding their biocompatibility. Nevertheless, novel particle compositions, geometries, activation strategies, targeting techniques, payload delivery strategies, and radiation dose enhancement concepts are unique attributes of this form of hyperthermia that warrant further exploration. Capitalising on these opportunities and overcoming these challenges offers the possibility of seamless and logical translation of this nanoparticle-mediated hyperthermia paradigm from the bench to the bedside.

γ-Tocotrienol Inhibits Pancreatic Tumors and Sensitizes Them to Gemcitabine Treatment by Modulating the Inflammatory Microenvironment

Pancreatic cancers generally respond poorly to chemotherapy, prompting a need to identify agents that could sensitize tumors to treatment. In this study, we investigated the response of human pancreatic cells to γ-tocotrienol (γ-T3), a novel, unsaturated form of vitamin E found in palm oil and rice bran oil, to determine whether it could potentiate the effects of gemcitabine, a standard of care in clinical treatment of pancreatic cancer. γ-T3 inhibited the in vitro proliferation of pancreatic cancer cell lines with variable p53 status and potentiated gemcitabine-induced apoptosis. These effects correlated with an inhibition of NF-κB activation by γ-T3 and a suppression of key cellular regulators including cyclin D1, c-Myc, cyclooxygenase-2 (COX-2), Bcl-2, cellular inhibitor of apoptosis protein, survivin, vascular endothelial growth factor (VEGF), ICAM-1, and CXCR4. In an orthotopic nude mouse model of human pancreatic cancer, p.o. administration of γ-T3 inhibited tumor growth and enhanced the antitumor properties of gemcitabine. Immunohistochemical analysis indicated a correlation between tumor growth inhibition and reduced expression of Ki-67, COX-2, matrix metalloproteinase-9 (MMP-9), NF-κB p65, and VEGF in the tissue. Combination treatment also downregulated NF-κB activity along with the NF-κB-regulated gene products, such as cyclin D1, c-Myc, VEGF, MMP-9, and CXCR4. Consistent with an enhancement of tumor apoptosis, caspase activation was observed in tumor tissues. Overall, our findings suggest that γ-T3 can inhibit the growth of human pancreatic tumors and sensitize them to gemcitabine by suppressing NF-κB-mediated inflammatory pathways linked to tumorigenesis.