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1.  EWS-FLI1 induces developmental abnormalities and accelerates sarcoma formation in a transgenic mouse model 
Cancer research  2008;68(21):8968-8975.
Ewing’s sarcoma is characterized by the t(11;22)(q24:q12) reciprocal translocation. To study the effects of the fusion gene EWS-FLI1 on development and tumor formation, a transgenic mouse model was created. A strategy of conditional expression was used to limit the potentially deleterious effects of EWS-FLI1 to certain tissues. In the absence of Cre recombinase, EWS-FLI1 was not expressed in the EWS-FLI1 transgenic mice, and they had a normal phenotype. When crossed to the Prx1-Cre transgenic mouse, which expresses Cre recombinase in the primitive mesenchymal cells of the embryonic limb bud, the EF mice were noted to have a number of developmental defects of the limbs. These included shortening of the limbs, muscle atrophy, cartilage dysplasia, and immature bone. By itself, EWS-FLI1 did not induce the formation of tumors in the EF transgenic mice. However, in the setting of p53 deletion, EWS-FLI1 accelerated the formation of sarcomas from a median time of 50 weeks to 21 weeks. Furthermore, EWS-FLI1 altered the type of tumor that formed. Conditional deletion of p53 in mesenchymal cells (Prx1-Cre p53lox/lox) produced osteosarcomas as the predominant tumor. The presence of EWS-FLI1 shifted the tumor phenotype to a poorly differentiated sarcoma. The results taken together suggest that EWS-FLI1 inhibits normal limb development and accelerates the formation of poorly differentiated sarcomas.
PMCID: PMC4167779  PMID: 18974141
EWS-FLI1; p53; Ewing’s sarcoma; Prx1; development
2.  Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells 
PLoS ONE  2013;8(11):e78570.
Background and purpose
Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.
Experimental approach
For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.
Key results
The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC50 of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.
Conclusions and implications
Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.
PMCID: PMC3817219  PMID: 24223823
3.  Phenylalkyl isoselenocyanates vs phenylalkyl isothiocyanates: thiol reactivity and its implications 
Chemico-biological interactions  2012;200(1):28-37.
Phenylalkyl isoselenocyanate (ISC) compounds were recently designed in our laboratory by incorporating the anticancer element selenium into a panel of phenylalkyl isothiocyanates (ITCs), known to have anticancer properties. A structural activity investigation was carried out to compare the ISC and ITC panels. Cell viability assay and Annexin V staining for apoptosis showed ISC compounds to be more potent in killing A549 lung adenocarcinoma cells. Both ITCs and ISCs were able to deplete reduced glutathione (GSH) in cells, ISCs more rapidly, but ITCs to a greater extent. ISC compounds had a higher rate of reaction to thiol (-SH) groups as determined by pseudo first order kinetics than the corresponding carbon chain length ITC. The equilibrium concentrations of the GSH and protein thiol conjugates did not differ significantly when comparing sulfur to selenium compounds of the same carbon chain length, and did follow the same trend of displaying decreasing reactivity with increasing carbon chain length for both ITCs and ISCs. Furthermore, only ITCs were able to induce cell cycle arrest, suggesting that protein targets inside the cell may differ for the S and Se panels. Finally, the panels were tested for their ability to redox cycle when reacted with GSH to form superoxide and other reactive oxygen species (ROS). ISC compounds showed a much greater ability to redox cycle than corresponding ITCs, and were able to induce higher levels of ROS in A549 cells. Also, the direct proapoptotic effects of ISCs and ITCs were inhibited by GSH and potentiated by depletion of intracellular GSH by buthionine sulfoximine. In conclusion, our studies suggest that the redox-cycling capabilities of ISCs and thus generation of higher levels of ROS may be contributing to the increased cytotoxicity of ISC compounds in A549 cells, compared to that of the corresponding ITCs.
PMCID: PMC3480549  PMID: 22982772
Isoselenocyanates; Isothiocyanates; Thiol reactivity; Chemiluminesence; Redox cycling; ROS
4.  Galactosylated IgG1 links FcγRIIB and Dectin-1 to block complement-mediated inflammation 
Nature medicine  2012;18(9):1401-1406.
Complement is an ancient danger sensing system playing critical roles in host defense, immune surveillance and homeostasis1. C5a and its G-Protein-coupled receptor mediate many of the pro-inflammatory properties of complement2. Despite its critical role in allergic asthma3, autoimmune arthritis4, sepsis5 and cancer6, our knowledge about C5a regulation is limited. Here we demonstrate an unexpected link through which IgG1 immune complexes (IC), the inhibitory IgG receptor FcγRIIB and the C-type lectin-like receptor Dectin-1 suppress C5a receptor (C5aR) functions. Specifically, we found that IgG1 IC associate FcγRIIB with Dectin-1, resulting in phosphorylation of spleen tyrosine kinase (Syk) downstream of Dectin-1 and Src homology 2 domain containing inositol phosphatase (SHIP) downstream of FcγRIIB. This pathway blocks C5a receptor-mediated ERK1/2 phosphorylation and C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo including the development of skin blisters in experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin disorder. Notably, high galactosylation of IgG N-glycan is critical for this inhibitory property of IgG1 IC as it promotes the association between FcγRIIB and Dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert immunoregulatory properties beyond their impact on activating FcγRs that may control allergy, autoimmunity and cancer.
PMCID: PMC3492054  PMID: 22922409
5.  Knockdown of c-Fos suppresses the growth of human colon carcinoma cells in athymic mice 
Here we have investigated whether inhibition of c-Fos expression in RKO human colon carcinoma cells (HCCCs) would result in reduced TGFβ1 expression and suppression of tumor growth in athymic mice. We stably transfected RKO cells with c-Fos small interfering RNA (siRNA) or with the corresponding control siRNA. Using these stable cell lines, we demonstrated that siRNA-c-Fos significantly suppressed both AP-1 binding, promoter reporter activity at the proximal AP-1 site in the TGFβ1 promoter, and TGFβ1 production. Further, we established colon cancer xenografts with each of RKO-siRNA-EV, RKO-siRNA-Ctrl, and RKO-siRNA-c-Fos cells. By 24 days, the tumor size of RKO-siRNA-c-Fos xenografts was 40% that of either RKO-EV or RKO-siRNA-Ctrl. Immunohistochemistry (IHC) of tumor xenografts demonstrated that siRNA-c-Fos significantly blocked c-Fos expression, and consequently expression of TGFβ1. However, expression of TGFβ2 and TGFβ3 were unaffected. Overall, our results demonstrate that blockade of TGFβ1 production by siRNA-c-Fos effectively suppressed tumor growth in vivo.
PMCID: PMC3163111  PMID: 21344377
TGFβ; AP-1; c-Fos; colon carcinoma; xenograft
6.  Overexpression of the dynein light chain km23-1 in human ovarian carcinoma cells inhibits tumor formation in vivo and causes mitotic delay at prometaphase/metaphase 
km23-1 is a dynein light chain that was identified as a TGFß receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells. More importantly, induction of km23-1 expression substantially reduced the tumorigenicity of SKOV-3 cells in a xenograft model in vivo. Fluorescence-activated cell sorting analysis of SKOV-3 and IGROV-1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho-MEK, phospho-ERK, and cyclin B1 were elevated, as was the mitotic index, suggesting that km23-1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, while the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/− cells, it was inappropriately retained at the metaphase kinetochore in Dox− cells. Thus, the mechanism by which high levels of km23-1 suppresses ovarian carcinoma growth in vitro and inhibits ovary tumor formation in vivo appears to involve a BubR1-related mitotic delay.
PMCID: PMC3103626  PMID: 21469138
dynein; km23-1; ovarian cancer; mitosis
7.  Gambogic Acid Inhibits STAT3 Phosphorylation Through Activation of Protein Tyrosine Phosphatase SHP-1: Potential Role in Proliferation and Apoptosis 
The transcription factor, signal transducer and activator of transcription 3 (STAT3), is associated with proliferation, survival, and metastasis of cancer cells. We investigated whether gambogic acid (GA), a xanthone derived from the resin of traditional Chinese medicine, Gamboge hanburyi (mangosteen), can regulate the STAT3 pathway, leading to suppression of growth and sensitization of cancer cells. We found that GA induced apoptosis in human multiple myeloma cells that correlated with the inhibition of both constitutive and inducible STAT3 activation. STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 was inhibited by GA. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase (JAK) 1, and JAK2. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the GA-induced down-regulation of STAT3, suggesting the involvement of a PTP. We also found that GA induced the expression of the PTP SHP-1. Deletion of the SHP-1 gene by small interfering RNA suppressed the ability of GA to inhibit STAT3 activation and to induce apoptosis, suggesting the critical role of SHP-1 in its action. Moreover, GA down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) proteins, and this correlated with suppression of proliferation and induction of apoptosis. Overall, these results suggest that GA blocks STAT3 activation, leading to suppression of tumor cell proliferation and induction of apoptosis.
PMCID: PMC3131433  PMID: 21490133
Gambogic acid; STAT3; Apoptosis; Proliferation; Cancer
8.  Functional basis for complement evasion by staphylococcal superantigen-like 7 
Cellular microbiology  2010;12(10):1506-1516.
The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen-like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA-FcαRI binding and serum killing of E. coli. As C5a generation, in contrast to C5b-9-mediated lysis, is crucial for immune defense against staphylococci, we investigated the impact of SSL7 on staphylococcal-induced C5a-mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA-binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a-induced phagocytosis of S. aureus and oxidative burst in an in vitro whole blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a-driven influx of neutrophils in mouse peritoneum.
PMCID: PMC2941559  PMID: 20545943
Staphylococcus aureus; SSL7; complement evasion; C5a; IgA
9.  Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells 
STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents.
PMCID: PMC2877157  PMID: 19937797
Betulinic acid; STAT3; JAK1; JAK2; SHP-1; apoptosis
10.  Sesamin Manifests Chemopreventive Effects through Suppression of NF-κB-regulated Cell Survival, Proliferation, Invasion and Angiogenic Gene products 
Molecular cancer research : MCR  2010;8(5):751-761.
Agents that are safe, affordable and efficacious are urgently needed for prevention of chronic diseases such as cancer. Sesamin, a lipid-soluble lignan is one such agent that belongs to a class of phytoestrogens, isolated from sesame (Sesamum indicum), and has been linked with prevention of hyperlipidemia, hypertension, and carcinogenesis through an unknown mechanism. Because the transcription factor NF-κB has been associated with inflammation, carcinogenesis, tumor cell survival, proliferation, invasion and angiogenesis of cancer, we postulated that sesamin might mediate its effect through the modulation of NF-κB pathway. We found that sesamin inhibited the proliferation of wide variety of tumor cells including leukemia, multiple myeloma, and cancers of the colon, prostate, breast, pancreas and lung. Sesamin also potentiated TNF-α induced apoptosis and this correlated with suppression of gene products linked to cell survival (e.g.; Bcl-2 and survivin), proliferation (e.g.; cyclin D1), inflammation (e.g.; COX-2), invasion (e.g.; MMP-9, ICAM-1) and angiogenesis (e.g.; VEGF). Sesamin downregulated constitutive and inducible NF-κB activation induced by various inflammatory stimuli and carcinogens; and inhibited degradation of IκBα, the inhibitor of NF-κB through the suppression of phosphorylation of IκBα and inhibition of activation of IκBα protein kinase (IKK); thus resulting in the suppression of p65 phosphorylation and nuclear translocation, and NF-κB-mediated reporter gene transcription. The inhibition of IKK activation was found to be mediated through the inhibition of TAK1 kinase. Overall our results demonstrated that sesamin may have potential against cancer and other chronic diseases through the suppression of pathway linked to the NF-κB signaling.
PMCID: PMC2895997  PMID: 20460401
11.  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.
PMCID: PMC3090706  PMID: 19908231
apoptosis; chemoresistance; chemotherapeutic agents; NF-κB; pancreatic cancer
12.  Curcumin Modulates the Radiosensitivity of Colorectal Cancer Cells by Suppressing Constitutive and Inducible NF-κB Activity 
Radiation therapy is an integral part of the preoperative treatment of rectal cancers. However, only a minority of patients achieve a complete pathological response to therapy due to resistance of these tumors to radiation therapy. This resistance may be mediated by constitutively active pro-survival signaling pathways or by inducible/acquired mechanisms in response to radiation therapy. Simultaneous inhibition of these pathways can sensitize these tumors to radiation therapy.
Methods and Materials
Human colorectal cancer cells were exposed to clinically relevant doses of gamma rays and the mechanism of their radioresistance was investigated. We characterize the transcription factor nuclear factor-κB (NF-κB) activation as a mechanism of inducible radioresistance in colorectal cancer and use curcumin, the active ingredient in the yellow spice turmeric to overcome this resistance.
Curcumin inhibited the proliferation and the post-irradiation clonogenic survival of multiple colorectal cancer cell lines. Radiation stimulated NF-κB activity in a dose- and time-dependent manner while curcumin suppressed this radiation-induced NF-κB activation via inhibition of radiation-induced phosphorylation and degradation of IκBα, inhibition of IKK activity, and inhibition of Akt phosphorylation. Curcumin also suppressed NF-κB regulated gene products (Bcl-2, Bcl-xL, inhibitor of apoptosis protein-2, cyclooxygenase-2, and cyclin D1).
Our results suggest that transient inducible NF-κB activation provides a pro-survival response to radiation that may account for development of radioresistance. Curcumin blocks this signaling pathway and potentiates the anti-tumor effects of radiation therapy.
PMCID: PMC3090721  PMID: 19735878
Curcumin; Radiation; NF-κB; Radiosensitization; Colorectal Cancer
13.  Garcinol Potentiates TRAIL-Induced Apoptosis through Modulation of Death Receptors and Antiapoptotic Proteins 
Molecular cancer therapeutics  2010;9(4):856-868.
Whether garcinol, the active component from Garcinia indica, can modulate the sensitivity of cancer cells to TRAIL, a cytokine currently in phase II clinical trial, was investigated. We found that garcinol potentiated TRAIL-induced apoptosis of cancer cells as indicated by intracellular esterase activity, DNA strand breaks, accumulation of the membrane phospholipid phosphatidylserine, mitochondrial activity, and activation of caspase-8, -9, and -3. We found that garcinol, independent of the cell type, induced both of the TRAIL receptors, death receptors (DR)-4 and DR5. Garcinol neither induced the receptors on normal cells, nor sensitized them to TRAIL. Deletion of DR5 or DR4 by small interfering RNA significantly reduced the apoptosis induced by TRAIL and garcinol. In addition, garcinol downregulated various cell survival proteins including survivin, bcl-2, XIAP and cFLIP; and induced bid cleavage, bax and cytochrome c release. Induction of DRs by garcinol was found to be independent of modulation of CHOP, p53, bax, ERK or JNK. The effect of garcinol was mediated through the generation of reactive oxygen species, in as much as both induction of DRs, modulation of antiapoptotic and proapoptotic proteins and potentiation of TRAIL-induced apoptosis were abolished by N-acetyl cysteine and glutathione. Interestingly, garcinol also converted TRAIL-resistant cells to TRAIL-sensitive. Overall, our results indicate that garcinol can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of antiapoptotic proteins.
PMCID: PMC2852472  PMID: 20371723
TRAIL; garcinol; apoptosis; death receptors; potentiation
14.  C5a receptor-deficient dendritic cells promote induction of Treg and Th17 
European journal of immunology  2010;40(3):710-721.
C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in dendritic cells (DC) affects the development of regulatory T cell (Treg) and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen-derived DC results in increased production of TGF-β leading to de novo differentiation of Foxp3+ Treg within 12h after co-incubation with CD4+ T cells from DO11.10/RAG2-/- mice. Stimulation of C5aR-/- DC with ovalbumin and TLR2 ligand Pam3CSK4 increased TGF-β production and induced high levels of IL-6 and IL-23 but only minor amounts of IL-12 leading to differentiation of Th cells producing IL-17A and IL-21. Th17 differentiation was also found in vivo after adoptive transfer of CD4+ Th cell into C5aR-/- mice immunized with OVA and Pam3CSK4. The altered cytokine production of C5aR-/- DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17 cell differentiation through regulation of DC function.
PMCID: PMC3040298  PMID: 20017191
C5a; complement; dendritic cell; T cells; IL-17A
15.  Plumbagin, Vitamin K3 Analogue, Suppresses STAT3 Activation Pathway through Induction of Protein Tyrosine Phosphatase, SHP-1: Potential Role in Chemosensitization 
Molecular cancer research : MCR  2010;8(1):107-118.
The activation of STAT3 has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), an analogue of Vitamin K and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and IL-6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, JAK1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1; and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and VEGF, activated caspase-3, induced PARP cleavage, and increased the sub-G1 population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through induction of SHP-1 and this may mediate sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.
PMCID: PMC2808447  PMID: 20068065
Plumbagin; STAT3; JAK1; JAK2; SHP-1; Apoptosis
16.  Targeted mutation of p53 and Rb in mesenchymal cells of the limb bud produces sarcomas in mice 
Carcinogenesis  2009;30(10):1789-1795.
Mice bearing germ line mutations of p53 develop sarcomas at a significant rate. Since they are susceptible to a variety of other malignancies, they are not ideally suited to the study of sarcomas. To test the possibility that targeted mutation of tumor suppressor genes in early mesenchymal cells would induce formation of sarcomas, the Prx1-cre transgenic mouse was crossed to mice-bearing floxed alleles of p53 and Rb. Mice with homozygous deletion of p53 (Prx1-cre p53lox/lox) developed sarcomas in the extremities at a mean time of 50 weeks. Osteosarcomas (OS) were the most common type of sarcoma (61%) followed by poorly differentiated soft tissue sarcomas (PDSTS) (32%). Homozygous deletion of p53 produced sarcomas significantly more rapidly than heterozygous deletion, which resulted in sarcoma formation after a mean of 96 weeks. Mice with homozygous Rb mutation (Prx1-cre Rblox/lox) developed normally and had no ostensible defects in the limbs. In contrast to p53, targeted deletion of Rb did not produce sarcomas in the limbs. However, simultaneous deletion of Rb and p53 accelerated the time to sarcoma formation, and a greater percentage of PDSTS were found. Deletion of p53 in committed osteoblasts by the Col1a1-cre transgenic mouse bearing an osteoblast-specific enhancer resulted in a high percentage of OS. These findings suggest that deletion of p53 in mesenchymal cells that give rise to osteoblasts is a powerful initiator of OS. Deletion of Rb does not initiate sarcoma formation in mice, but it accelerates formation of both soft tissue sarcomas and OS.
PMCID: PMC4141195  PMID: 19635748
17.  Boswellic Acid Blocks STAT3 Signaling, Proliferation, and Survival of Multiple Myeloma via the Protein Tyrosine Phosphatase SHP-1 
Molecular cancer research : MCR  2009;7(1):118-128.
Activation of signal transducers and activators of transcription (STAT)-3 factors has been linked with survival, proliferation, chemoresistance and angiogenesis of tumor cells, including human multiple myeloma (MM). Thus agents that can suppress STAT3 activation have potential as cancer therapeutics. In our search for such agents, we identified acetyl-11-keto-β-boswellic acid (AKBA), originally isolated from Boswellia serrata. Our results show that AKBA inhibited constitutive STAT3 activation in human MM cells. AKBA suppressed IL-6-induced STAT3 activation, and the inhibition was reversible. The phosphorylation of both Jak 2 and Src, constituents of the STAT3 pathway, was inhibited by AKBA. Interestingly, treatment of cells with pervanadate suppressed AKBA’s effect to inhibit the phosphorylation of STAT3, thus suggesting the involvement of a protein tyrosine phosphatase. We found that AKBA induced Src homology region 2 domain-containing phosphatase 1 (SHP-1), which may account for its role in dephosphorylation of STAT3. Moreover, deletion of SHP-1 gene by SiRNA abolished the ability of AKBA to inhibit STAT3 activation. The inhibition of STAT3 activation by AKBA led to the suppression of gene products involved in proliferation (cyclin D1), survival (Bcl-2, Bcl-xL and Mcl-1), and angiogenesis (VEGF). This affect correlated with the inhibition of proliferation and apoptosis in MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the AKBA induced apoptosis. Overall, our results suggest that AKBA is a novel inhibitor of STAT3 activation and has potential in the treatment of cancer.
PMCID: PMC2677182  PMID: 19147543
Acetyl-11-Keto-{beta}-Boswellic Acid; STAT3; c-Src; JAK2; SHP-1; Apoptosis
18.  Role of Prooxidants and Antioxidants in the Anti-Inflammatory and Apoptotic Effects of Curcumin (Diferuloylmethane) 
Free radical biology & medicine  2007;43(4):568-580.
Extensive research within last half a century has indicated that curcumin (diferuloylmethane), a yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory and proapoptotic activities. Whether anti-inflammatory and proapoptotic activities assigned to curcumin, are mediated through its antioxidant mechanism was investigated. We found that TNF-mediated NF-κB activation was inhibited by curcumin; and glutathione reversed the inhibition. Similarly, suppression of TNF-induced AKT activation by curcumin, was also abrogated by glutathione. The reducing agent also counteracted the inhibitory effect of curcumin on TNF-induced NF-κB regulated antiapoptotic (Bcl-2, Bcl-xL, IAP1), proliferative (cyclin D1) and proinflammatory (COX-2, iNOS and MMP-9) gene products. The suppression of TNF-induced AP-1 activation by curcumin was also reversed by glutathione. Also, the direct proapoptotic effects of curcumin were inhibited by glutathione and potentiated by depletion of intracellular glutathione by buthionine sulfoximine. Moreover, curcumin induced the production of reactive oxygen species (ROS) and modulated the intracellular GSH levels. Quenchers of hydroxyl radicals, however, were ineffective in inhibiting curcumin mediated NF-κB suppression. Further, N-acetylcysteine partially reversed the effect of curcumin. Based on these results we conclude that curcumin mediate its apoptotic and anti-inflammatory activities through modulation of the redox status of the cell.
PMCID: PMC2754304  PMID: 17640567
Curcumin; Glutathione; Tumor necrosis factor; Nuclear factor-κB; Reactive oxygen species
19.  Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing VEGFR2 signaling 
Cancer research  2008;68(6):1843-1850.
Gambogic acid (GA), the main active compound of Gamboge Hanburyi, has been previously reported to activate apoptosis in many types of cancer cell lines by targeting transferrin receptor and modulating NF-kappa B signaling pathway. Whether GA inhibits angiogenesis, which is crucial for cancer and other human diseases, remains unknown. Here we found that GA significantly inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, invasion, tube formation, and micro-vessel growth at nM concentration. In a xenograft prostate tumor model, we found that GA effectively inhibited tumor angiogenesis and suppressed tumor growth with low side effects using metronomic chemotherapy with GA. GA was more effective in activating apoptosis and inhibiting proliferation and migration in HUVECs than in human prostate cancer cells (PC3), suggesting GA might be a potential drug candidate in cancer therapy through angioprevention with low chemotoxicity. Furthermore, we demonstrated that GA inhibited the activations of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream protein kinases, c-Src, FAK and AKT. Together, these data suggest that GA inhibits angiogenesis and may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.
PMCID: PMC2587446  PMID: 18339865
Gambogic acid; anti-angiogenesis; tumor angiogenesis; VEGF receptor 2 inhibitor; prostate cancer
20.  Staphylococcal complement evasion by various convertase-blocking molecules 
The Journal of Experimental Medicine  2007;204(10):2461-2471.
To combat the human immune response, bacteria should be able to divert the effectiveness of the complement system. We identify four potent complement inhibitors in Staphylococcus aureus that are part of a new immune evasion cluster. Two are homologues of the C3 convertase modulator staphylococcal complement inhibitor (SCIN) and function in a similar way as SCIN. Extracellular fibrinogen-binding protein (Efb) and its homologue extracellular complement-binding protein (Ecb) are identified as potent complement evasion molecules, and their inhibitory mechanism was pinpointed to blocking C3b-containing convertases: the alternative pathway C3 convertase C3bBb and the C5 convertases C4b2aC3b and C3b2Bb. The potency of Efb and Ecb to block C5 convertase activity was demonstrated by their ability to block C5a generation and C5a-mediated neutrophil activation in vitro. Further, Ecb blocks C5a-dependent neutrophil recruitment into the peritoneal cavity in a mouse model of immune complex peritonitis. The strong antiinflammatory properties of these novel S. aureus–derived convertase inhibitors make these compounds interesting drug candidates for complement-mediated diseases.
PMCID: PMC2118443  PMID: 17893203
21.  A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma 
Journal of Clinical Investigation  2006;116(3):783-796.
Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.
PMCID: PMC1386108  PMID: 16511606
22.  Humoral immune response to an allogenic foetus in normal fertile women and recurrent aborters 
Several humoral immune factors are responsible for a successful pregnancy. There are a few studies, which demonstrate the role of antipaternal cytotoxic antibodies (APCA) and mixed lymphocyte reaction blocking factor (MLR-Bf) in the maintenance of pregnancy. However, these factors have not been studied in women with history of recurrent spontaneous abortion (RSA). We designed this study to review the role of APCA and MLR-Bf in normal pregnancy as well as in women with a history of RSA.
One hundred and five women with unexplained recurrent spontaneous abortion were included in the present study. These women were screened for all other known causes of recurrent abortion. We also included 60 normal fertile women, fifteen from each trimester and fifteen women during the post partum period (up to six months). RSA and controls (normal pregnancy) were matched for age, caste, and socio economic background and also for parity. APCA and MLR-Bf were evaluated in all the groups. All women with RSA who conceived during the study period were on follow up.
We have analyzed the status of APCA and MLR-Bf in normal pregnancy (different gestational periods and during post partum), and in women with history of RSA. Our results show that APCA was significantly higher in controls as compared to RSA women. MLR-Bf was directed against the husbands' cells in normal pregnancy and was virtually absent in RSA women.
Our results indicate that there is a significantly low titer of APCA and MLR-Bf in women with recurrent spontaneous abortion. This highlights the role of these factors in the maintenance of successful pregnancy.
PMCID: PMC122080  PMID: 12162792

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