Dhanya K. Nambiar

All Publications

• NFE2L2 mutations enhance radioresistance in head and neck cancer by modulating intratumoral myeloid cells. Cancer research Guan, L., Nambiar, D. K., Cao, H., Viswanathan, V., Kwok, S., Hui, A. B., Hou, Y., Hildebrand, R., von Eyben, R., Holmes, B. J., Zhao, J., Kong, C. S., Wamsley, N., Zhang, W., Major, M. B., Seol, S. W., Sunwoo, J. B., Hayes, D. N., Diehn, M., Le, Q. T. 2023

  Abstract

  Radiotherapy is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, while the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. Radiotherapy increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3 and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an anti-tumor phenotype, supporting clinical testing of CB-839 with radiation in HNSCC with NFE2L2 mutations.

  View details for DOI 10.1158/0008-5472.CAN-22-1903

  View details for PubMedID 36652552

• Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance. The Journal of clinical investigation Nambiar, D. K., Aguilera, T., Cao, H., Kwok, S., Kong, C., Bloomstein, J., Wang, Z., Rangan, V. S., Jiang, D., von Eyben, R., Liang, R., Agarwal, S., Colevas, A. D., Korman, A., Allen, C. T., Uppaluri, R., Koong, A. C., Giaccia, A., Le, Q. T. 2019

  Abstract

  Immune checkpoint inhibitors (ICIs), although promising, have variable benefit in head and neck cancer (HNC). We noted that tumor galectin-1 (Gal1) levels were inversely correlated with treatment response and survival in patients with HNC who were treated with ICIs. Using multiple HNC mouse models, we show that tumor-secreted Gal1 mediates immune evasion by preventing T cell migration into the tumor. Mechanistically, Gal1 reprograms the tumor endothelium to upregulate cell-surface programmed death ligand 1 (PD-L1) and galectin-9. Using genetic and pharmacological approaches, we show that Gal1 blockade increases intratumoral T cell infiltration, leading to a better response to anti-PD1 therapy with or without radiotherapy. Our study reveals the function of Gal1 in transforming the tumor endothelium into an immune-suppressive barrier and that its inhibition synergizes with ICIs.

  View details for DOI 10.1172/JCI129025

  View details for PubMedID 31710313

• The Immune Subtypes and Landscape of Squamous Cell Carcinoma CLINICAL CANCER RESEARCH Li, B., Cui, Y., Nambiar, D. K., Sunwoo, J. B., Li, R. 2019; 25 (12): 3528–37

  View details for DOI 10.1158/1078-0432.CCR-18-4085

  View details for Web of Science ID 000472077200008

• Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling. Molecular cancer therapeutics Nambiar, D. K., Rajamani, P., Deep, G., Jain, A. K., Agarwal, R., Singh, R. P. 2015

  Abstract

  Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer. Mol Cancer Ther; 14(12); 1-13. ©2015 AACR.

  View details for DOI 10.1158/1535-7163.MCT-15-0348

  View details for PubMedID 26516160

• Hypoxia induces triglycerides accumulation in prostate cancer cells and extracellular vesicles supporting growth and invasiveness following reoxygenation. Oncotarget Schlaepfer, I. R., Nambiar, D. K., Ramteke, A., Kumar, R., Dhar, D., Agarwal, C., Bergman, B., Graner, M., Maroni, P., Singh, R. P., Agarwal, R., Deep, G. 2015; 6 (26): 22836-22856

  Abstract

  Hypoxia is an independent prognostic indicator of poor outcome in several malignancies. However, precise mechanism through which hypoxia promotes disease aggressiveness is still unclear. Here, we report that under hypoxia (1% O2), human prostate cancer (PCA) cells, and extracellular vesicles (EVs) released by these cells, are significantly enriched in triglycerides due to the activation of lipogenesis-related enzymes and signaling molecules. This is likely a survival response to hypoxic stress as accumulated lipids could support growth following reoxygenation. Consistent with this, significantly higher proliferation was observed in hypoxic PCA cells following reoxygenation associated with rapid use of accumulated lipids. Importantly, lipid utilization inhibition by CPT1 inhibitor etomoxir and shRNA-mediated CPT1-knockdown significantly compromised hypoxic PCA cell proliferation following reoxygenation. Furthermore, COX2 inhibitor celecoxib strongly reduced growth and invasiveness following hypoxic PCA cells reoxygenation, and inhibited invasiveness induced by hypoxic PCA EVs. This establishes a role for COX2 enzymatic products in the enhanced PCA growth and invasiveness. Importantly, concentration and loading of EVs secreted by PCA cells were significantly compromised under delipidized serum condition and by lipogenesis inhibitors (fatostatin and silibinin). Overall, present study highlights the biological significance of lipid accumulation in hypoxic PCA cells and its therapeutic relevance in PCA.

  View details for PubMedID 26087400

• The Combination of Radiotherapy and Complement C3a Inhibition Potentiates Natural Killer cell Functions Against Pancreatic Cancer. Cancer research communications Sodji, Q. H., Nambiar, D. K., Viswanathan, V., von Eyben, R., Colburg, D., Binkley, M. S., Li, C. G., Olcina, M. M., Chang, D. T., Le, Q., Giaccia, A. J. 2022; 2 (7): 725-738

  Abstract

  Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer, its combination with radiation therapy hold greater therapeutic benefit.

  View details for DOI 10.1158/2767-9764.crc-22-0069

  View details for PubMedID 35937458

• Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype. PNAS nexus Viswanathan, V., Cao, H., Saiki, J., Jiang, D., Mattingly, A., Nambiar, D., Bloomstein, J., Li, Y., Jiang, S., Chamoli, M., Sirjani, D., Kaplan, M., Holsinger, F. C., Liang, R., Von Eyben, R., Jiang, H., Guan, L., Lagory, E., Feng, Z., Nolan, G., Ye, J., Denko, N., Knox, S., Rosen, D., Le, Q. 2022; 1 (2): pgac056

  Abstract

  Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1 -/- mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

  View details for DOI 10.1093/pnasnexus/pgac056

  View details for PubMedID 35707206

• Relationship between KEAP1/NFE2L2 pathway activation and radiation resistance in oral cavity cancer. Guan, L., Cao, H., Hui, A., Kwok, S., Viswanathan, V., Nambiar, D., Eyben, R. V., Holmes, B., Kong, C., Diehn, M., Quynh-Thu Le AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000641160600094

• VISTA immune-checkpoint blunts radiotherapy induced anti-tumor immune response. Nambiar, D. K., Mehta, N., Maddineni, S., Cao, H., Viswanathan, V., Cheunkarndee, T., Cochran, J. R., Quynh Thu Le AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000641160600078

• Y box binding protein 1 inhibition as a targeted therapy for ovarian cancer. Cell chemical biology Tailor, D., Resendez, A., Garcia-Marques, F. J., Pandrala, M., Going, C. C., Bermudez, A., Kumar, V., Rafat, M., Nambiar, D. K., Honkala, A., Le, Q., Sledge, G. W., Graves, E., Pitteri, S. J., Malhotra, S. V. 2021

  Abstract

  Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. Invivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, invitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

  View details for DOI 10.1016/j.chembiol.2021.02.014

  View details for PubMedID 33713600

• The role of Glial cell derived neurotrophic factor in head and neck cancer. PloS one Cao, H., He, Q., Eyben, R. v., Bloomstein, J., Nambiar, D. K., Viswanathan, V., Aggarwal, S., Kwok, S., Liang, R., Koong, A. J., Lewis, J. S., Kong, C., Xiao, N., Le, Q. 2020; 15 (2): e0229311

  Abstract

  Glial cell-derived neurotrophic factor (GDNF) is reported to promote the survival of neurons and salivary gland regeneration after radiation damage. This study investigated the effect of GDNF on cell migration, growth, and response to radiation in preclinical models of head and neck squamous cell carcinoma (HNSCC) and correlated GDNF expression to treatment outcomes in HNSCC patients. Our ultimate goal is to determine whether systemic administration of GDNF at high dose is safe for the management of hyposalivation or xerostomia in HNSCC patients. Three HPV-positive and three HPV-negative cell lines were examined for cell migration, growth, and clonogenic survival in vitro and tumor growth assay in vivo. Immunohistochemical staining of GDNF, its receptors GFRalpha1 and its co-receptor RET was performed on two independent HNSCC tissue microarrays (TMA) and correlated to treatment outcomes. Results showed that GDNF only enhanced cell migration in two HPV-positive cells at supra-physiologic doses, but not in HPV-negative cells. GDNF did not increase cell survival in the tested cell lines post-irradiation. Likewise, GDNF treatment affected neither tumor growth in vitro nor response to radiation in xenografts in two HPV-positive and two HPV-negative HNSCC models. High stromal expression of GDNF protein was associated with worse overall survival in HPV-negative HNSCC on multivariate analysis in a combined cohort of patients from Stanford University (n = 82) and Washington University (n = 189); however, the association between GDNF gene expression and worse survival was not confirmed in a separate group of HPV-negative HNSCC patients identified from the Cancer Genome Atlas (TCGA) database. Based on these data, we do not believe that GNDF is a safe systemic treatment to prevent or treat xerostomia in HNSCC and a local delivery approach such as intraglandular injection needs to be explored.

  View details for DOI 10.1371/journal.pone.0229311

  View details for PubMedID 32084217

• Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer. British journal of cancer Tailor, D. n., Going, C. C., Resendez, A. n., Kumar, V. n., Nambiar, D. K., Li, Y. n., Dheeraj, A. n., LaGory, E. L., Ghoochani, A. n., Birk, A. M., Stoyanova, T. n., Ye, J. n., Giaccia, A. J., Le, Q. T., Singh, R. P., Sledge, G. W., Pitteri, S. J., Malhotra, S. V. 2020

  Abstract

  To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment.Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models.We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis.SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

  View details for DOI 10.1038/s41416-020-01137-4

  View details for PubMedID 33139797

• Fisetin sensitizes the hypoxia induced chemotherapy resistance in head and neck cancer Singh, R., Tailor, D., Nambiar, D., Malhotra, S. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000525061502269

• Galectin-1 intensifies immunosuppression in head and neck cancer by boosting myeloid-derived suppressive cell (MDSC) expansion Nambiar, D. K., Aguilera, T. A., Bloomstein, J., Jiang, D., Cao, H., Koong, A., Quynh Thu Le AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-4068

  View details for Web of Science ID 000468819502295

• Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes. Proceedings of the National Academy of Sciences of the United States of America Saiki, J. P., Cao, H., Van Wassenhove, L. D., Viswanathan, V., Bloomstein, J., Nambiar, D. K., Mattingly, A. J., Jiang, D., Chen, C., Stevens, M. C., Simmons, A. L., Park, H. S., von Eyben, R., Kool, E. T., Sirjani, D., Knox, S. M., Le, Q. T., Mochly-Rosen, D. 2018

  Abstract

  Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1-/- adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy.

  View details for PubMedID 29794221

• Chemical Space Mimicry for Drug Discovery JOURNAL OF CHEMICAL INFORMATION AND MODELING Yuan, W., Jiang, D., Nambiar, D. K., Liew, L. P., Hay, M. P., Bloomstein, J., Lu, P., Turner, B., Le, Q., Tibshirani, R., Khatri, P., Moloney, M. G., Koong, A. C. 2017; 57 (4): 875-882

  Abstract

  We describe a new library generation method, Machine-based Identification of Molecules Inside Characterized Space (MIMICS), that generates sets of molecules inspired by a text-based input. MIMICS-generated libraries were found to preserve distributions of properties while simultaneously increasing structural diversity. Newly identified MIMICS-generated compounds were found to be bioactive as inhibitors of specific components of the unfolded protein response (UPR) and the VEGFR2 pathway in cell-based assays, thus confirming the applicability of this methodology toward drug design applications. Wider application of MIMICS could facilitate the efficient utilization of chemical space.

  View details for DOI 10.1021/acs.jcim.6b00754

  View details for Web of Science ID 000400204900023

  View details for PubMedID 28257191

• Targeting galectin-1 to improve therapeutic efficacy in head and neck cancers Nambiar, D. K., Cao, H., Quynh Thu Le AMER ASSOC CANCER RESEARCH. 2017

  View details for DOI 10.1158/2326-6074.TUMIMM16-B51

  View details for Web of Science ID 000419245100103

• Silibinin attenuates ionizing radiation-induced pro-angiogenic response and EMT in prostate cancer cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Nambiar, D. K., Rajamani, P., Singh, R. P. 2015; 456 (1): 262-268

  Abstract

  Radiotherapy of is well established and frequently utilized in prostate cancer (PCa) patients. However, recurrence following therapy and distant metastases are commonly encountered problems. Previous studies underline that, in addition to its therapeutic effects, ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of IR-induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. Here, we demonstrate that a plant flavonoid, silibinin can radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only strongly down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it strongly (p<0.001) reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. Most of the pro-angiogenic (VEGF, iNOS), migratory (MMP-2) and EMT promoting proteins (uPA, vimentin, N-cadherin) were up-regulated by IR in PCa cells. Interestingly, all of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR-activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3, by silibinin.

  View details for DOI 10.1016/j.bbrc.2014.11.069

  View details for Web of Science ID 000348483600040

  View details for PubMedID 25446081

• Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1 ONCOTARGET Nambiar, D. K., Deep, G., Singh, R. P., Agarwal, C., Agarwal, R. 2014; 5 (20): 10017-10033

  Abstract

  Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.

  View details for Web of Science ID 000348036500039

  View details for PubMedID 25294820

• Acacetin Inhibits In Vitro and In Vivo Angiogenesis and Downregulates Stat Signaling and VEGF Expression CANCER PREVENTION RESEARCH Bhat, T. A., Nambiar, D., Tailor, D., Pal, A., Agarwal, R., Singh, R. P. 2013; 6 (10): 1128-1139

  Abstract

  Angiogenesis is an effective target in cancer control. The antiangiogenic efficacy and associated mechanisms of acacetin, a plant flavone, are poorly known. In the present study, acacetin inhibited growth and survival (up to 92%; P < 0.001), and capillary-like tube formation on Matrigel (up to 98%; P < 0.001) by human umbilical vein endothelial cells (HUVEC) in regular condition, as well as VEGF-induced and tumor cells conditioned medium-stimulated growth conditions. It caused retraction and disintegration of preformed capillary networks (up to 91%; P < 0.001). HUVEC migration and invasion were suppressed by 68% to 100% (P < 0.001). Acacetin inhibited Stat-1 (Tyr701) and Stat-3 (Tyr705) phosphorylation, and downregulated proangiogenic factors including VEGF, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP-2), and basic fibroblast growth factor (bFGF) in HUVEC. It also suppressed nuclear localization of pStat-3 (Tyr705). Acacetin strongly inhibited capillary sprouting and networking from rat aortic rings and fertilized chicken egg chorioallantoic membrane (CAM; ∼71%; P < 0.001). Furthermore, it suppressed angiogenesis in Matrigel plugs implanted in Swiss albino mice. Acacetin also inhibited tyrosine phosphorylation of Stat-1 and -3, and expression of VEGF in cancer cells. Overall, acacetin inhibits Stat signaling and suppresses angiogenesis in vitro, ex vivo, and in vivo, and therefore, it could be a potential agent to inhibit tumor angiogenesis and growth.

  View details for DOI 10.1158/1940-6207.CAPR-13-0209

  View details for Web of Science ID 000325272400015

  View details for PubMedID 23943785

  View details for PubMedCentralID PMC3808880

• In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells CANCER LETTERS Nambiar, D., Prajapati, V., Agarwal, R., Singh, R. P. 2013; 334 (1): 109-117

  Abstract

  Silibinin suppresses the growth of many cancers; however, its efficacy against pancreatic cancer has not been evaluated in established preclinical models. Here, we investigated in vitro and in vivo effects of silibinin against lower and advanced stages of human pancreatic carcinoma cells. Silibinin (25-100μM) treatment for 24-72h caused a dose- and time-dependent cell growth inhibition of 27-77% (P<0.05-0.001) in BxPC-3 cells, and 22-45% (P<0.01-0.001) in PANC-1 cells. Silibinin showed a strong dose-dependent G1 arrest in BxPC-3 cells (upto 72% versus 45% in control; P<0.001), but a moderate response in advanced PANC-1 cells. Cell death observed in cell growth assay, was accompanied by up to 3-fold increase (P<0.001) in apoptosis in BxPC-3 cells, and showed only slight effect on PANC-1 cells. Dietary feeding of silibinin (0.5%, w/w in AIN-93M diet for 7weeks) inhibited BxPC-3 and PANC-1 tumor xenografts growth in nude mice without any apparent change in body weight gain and diet consumption. Tumor volume and weight were decreased by 47% and 34% (P⩽0.001) in BxPC-3 xenograft, respectively. PANC-1 xenograft showed slower growth kinetics and silibinin decreased tumor volume by 34% (P<0.001) by 7weeks. Another 4weeks of silibinin treatment to PANC-1 xenograft showed 28% and 33% decrease in tumor volume and weight, respectively. Silibinin-fed group of BxPC-3 tumors showed decreased cell proliferation and angiogenesis and an increased apoptosis, however, considerable inhibitory effect was observed only for angiogenesis in PANC-1 tumors. Overall, these findings show both in vitro as well as in vivo anticancer efficacy of silibinin against pancreatic cancer that could involve inhibition of cell proliferation, cell cycle arrest, apoptosis induction and/or decrease in tumor angiogenesis.

  View details for DOI 10.1016/j.canlet.2012.09.004

  View details for Web of Science ID 000320413500017

  View details for PubMedID 23022268

• Advances in Prostate Cancer Chemoprevention: A Translational Perspective NUTRITION AND CANCER-AN INTERNATIONAL JOURNAL Nambiar, D., Singh, R. P. 2013; 65: 12-25

  Abstract

  Chemopreventive interventions are steadily emerging as an important aspect of cancer management and control. Herein, we have discussed the major epidemiological and clinical studies advocating the role of androgen inhibitors, flavonoids and antioxidants in preventing prostate cancer (PCa). Androgen inhibitors have lately been discussed not only in treatment of PCa, but also as preventive agents especially after trials with Finasteride and Dutasteride. Flavonoids such as silibinin, green tea polyphenols, genistein, curcumin have shown great promise, but avenues to improve their bioavailability are requisite. Agents with antioxidant potentials like lycopene, selenium, and vitamin E have also been explored. Antioxidant trials have yielded mixed results or benefitted only a subgroup of population, although further studies are needed to establish them as preventive agent. Although a majority of the trials resulted in positive outcomes supporting their role as preventive agents; one should be cautious of neutral or negative results as well. For clinical applicability of these agents, we need to identify the ideal target population, time of intervention, appropriate dosage, and extent of intervention required. Incoherency of data with these agents urges for a stringent study design and thorough interpretation to accurately judge the necessity and feasibility of the preventive measures.

  View details for DOI 10.1080/01635581.2013.785006

  View details for Web of Science ID 000319511100003

  View details for PubMedID 23682779

• Usnic Acid Inhibits Growth and Induces Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma A549 Cells NUTRITION AND CANCER-AN INTERNATIONAL JOURNAL Singh, N., Nambiar, D., Kale, R. K., Singh, R. P. 2013; 65: 36-43

  Abstract

  Usnic acid (UA) is a secondary metabolite abundantly found in lichens. Some studies have shown the anticancer potential of UA; however, its efficacy and associated mechanisms are yet to be fully explored. Herein, we assessed the anticancer potency and associated molecular alterations by UA in human lung carcinoma A549 cells. UA treatment (25-100 μM) for 24 and 48 h decreased total cell number by 39-67% (P < 0.01) and 68-89% (P < 0.001), respectively, and enhanced cell death by up to twofold and eightfold (P < 0.001), respectively. UA (1-10 μM) also significantly (P < 0.001) suppressed colony formation of A549 cells. The cell growth inhibition was associated with cell cycle arrest at G0/ G1 phase. UA decreased the expression of cyclin-dependent kinase (CDK)4, CDK6, and cyclin D1 and increased the expression of CDK inhibitor (CDKI) p21/cip1 protein. While examining the cell death associated molecular changes, we observed that UA induces mitochondrial membrane depolarization and led to more than twofold increase (P < 0.01) in apoptotic cells. The apoptotic effect of UA was accompanied by enhanced poly(ADP-ribose) polymerase cleavage. This study shows that UA inhibits cell growth involving G0/G1 phase cell cycle arrest and induces cell death via mitochondrial membrane depolarization and induction of apoptosis in human lung carcinoma cells.

  View details for DOI 10.1080/01635581.2013.785007

  View details for Web of Science ID 000319511100005

  View details for PubMedID 23682781

• Fisetin inhibits various attributes of angiogenesis in vitro and in vivo-implications for angioprevention CARCINOGENESIS Bhat, T. A., Nambiar, D., Pal, A., Agarwal, R., Singh, R. P. 2012; 33 (2): 385-393

  Abstract

  Studies have shown that fisetin, a small phytochemical molecule, has antitumor activity; however, its antiangiogenic activity has not yet been examined. Accordingly, herein, we investigated the antiangiogenic efficacy and associated mechanisms of fisetin in human umbilical vein endothelial cells (HUVECs). Fisetin (10-50 μM) strongly inhibited the regular serum plus growth supplement- and vascular endothelial growth factor (VEGF)-induced growth (up to 92%, P < 0.001) and survival (up to 16%, P < 0.001) of HUVEC in a dose- and time-dependent manner. Fisetin also caused cell cycle arrest at G(1) (strong) and G(2)/M (moderate) phases together with a decrease in cyclin D1 and an increase in p53 levels. Fisetin-caused cell death was accompanied by decreased expression of survivin and an increase in cleaved levels of caspases-3 and -7 and poly-(ADP-ribose) polymerase along with an increased ratio of Bax to Bcl-2. Furthermore, fisetin inhibited capillary-like tube formation on Matrigel (up to 85%, P < 0.001) as well as migration (up to 66%, P < 0.001), which were associated with decreased expression of endothelial nitric oxide synthase (eNOS) and VEGF in HUVEC. It also decreased the expression of eNOS, VEGF, inducible nitric oxide synthase, matrix metalloproteinase-2 and -9 in A549 and DU145 human cancer cells. In vivo matrigel plug assay in mice showed significant decrease in size (up to 43%, P < 0.001), vascularization and hemoglobin content (up to 94%, P < 0.001) in the plugs from fisetin-treated, compared with control mice. Overall, these results suggest that fisetin inhibits various attributes of angiogenesis, which might contribute to its reported antitumor effects, and therefore, fisetin warrants further investigation for its angiopreventive potential toward cancer control.

  View details for DOI 10.1093/carcin/bgr282

  View details for Web of Science ID 000300039800020

  View details for PubMedID 22139440

• Effects of phytochemicals on ionization radiation-mediated carcinogenesis and cancer therapy MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH Nambiar, D., Rajamani, P., Singh, R. P. 2011; 728 (3): 139-157

  Abstract

  Ionizing radiation (IR)-induced cellular damage is implicated in carcinogenesis as well as therapy of cancer. Advances in radiation therapy have led to the decrease in dosage and localizing the effects to the tumor; however, the development of radioresistance in cancer cells and radiation toxicity to normal tissues are still the major concerns. The development of radioresistance involves several mechanisms, including the activation of mitogenic and survival signaling, induction of DNA repair, and changes in redox signaling and epigenetic regulation. The current strategy of combining radiation with standard cytotoxic chemotherapeutic agents can potentially lead to unwanted side effects due to both agents. Thus agents are needed that could improve the efficacy of radiation killing of cancer cells and prevent the damage to normal cells and tissues caused by the direct and bystander effects of radiation, without have its own systemic toxicity. Chemopreventive phytochemicals, usually non-toxic agents with both cancer preventive and therapeutic activities, could rightly fit in this approach. In this regard, naturally occurring compounds, including curcumin, parthenolide, genistein, gossypol, ellagic acid, withaferin, plumbagin and resveratrol, have shown considerable potential. These agents suppress the radiation-induced activation of receptor tyrosine kinases and nuclear factor-κB signaling, can modify cell survival and DNA repair efficacy, and may potentiate ceramide signaling. These radiosensitizing and counter radioresistance mechanisms of phytochemicals in cancer cells are also associated with changes in epigenetic gene regulation. Because radioresistance involves multiple mechanisms, more studies are needed to discover novel phytochemicals having multiple mechanisms of radiosensitization and to overcome radioresistance of cancer cells. Pre-clinical studies are needed to address the appropriate dosage, timing, and duration of the application of phytochemicals with radiation to justify clinical trials. Nonetheless, some phytochemicals in combination with IR may play a significant role in enhancing the therapeutic index of cancer treatment.

  View details for DOI 10.1016/j.mrrev.2011.07.005

  View details for Web of Science ID 000296933400006

  View details for PubMedID 22030216