Dhanya K. Nambiar

Effects of phytochemicals on ionization radiation-mediated carcinogenesis and cancer therapy

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.

Fisetin inhibits various attributes of angiogenesis in vitro and in vivo—implications for angioprevention

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.

Usnic Acid Inhibits Growth and Induces Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma A549 Cells

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.

Acacetin Inhibits In Vitro and In Vivo Angiogenesis and Downregulates Stat Signaling and VEGF Expression

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. Cancer Prev Res; 6(10); 1128–39. ©2013 AACR.

In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells.

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.

Silibinin attenuates ionizing radiation-induced pro-angiogenic response and EMT in prostate cancer cells

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.

Silibinin preferentially radiosensitizes prostate cancer by inhibiting DNA repair signaling

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); 2722–34. ©2015 AACR.

Advances in Prostate Cancer Chemoprevention: A Translational Perspective

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.

Chemical Space Mimicry for Drug Discovery

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.

Abstract LB-180: Targeting galectin-1 in combination with radiation and immune checkpoint therapy in head and neck cancers

Head and neck cancers (HNC) development shows significant changes in the immune profile, with lower systemic lymphocyte counts and reduced tumor-infiltrating lymphocytes, which inversely correlate with survival. Galectin-1 (Gal-1) is secreted at high level by cancer cells, including HNC, and contributes to tumor-immune escape and disease progression. Previously, we have shown that both hypoxia and radiotherapy (RT) can stimulate Gal-1 secretion. Immune checkpoint therapies, specifically targeting the PD1 pathway, allows T cells to be effective in shrinking tumors and lengthen survival in patients with metastatic HNC. However, these therapies work only in a small number of patients with tumors showing significant T cell infiltration. Targeting Gal-1 in HNC therefore, could be a useful therapeutic approach, as it may synergize with radiation and immunotherapy. Using the CRISPR/Cas9 deletion approach, we developed an orthotopic model of HNC with/without Gal-1 expression. We found that in both subcutaneous and orthotopic model of oral cancer, Gal-1 KO (knock-out) tumors showed marked decrease in tumor growth and nodal metastases. These differences were attributed to enhanced infiltration of CD4+ and CD8+ T cells, in the absence of Gal-1. Using a series of transendothelial migration assays in vitro and tracking adoptively transferred T cells in vivo, we show that presence of Gal-1 in the tumor microenvironment strongly suppresses T cell infiltration into tumors. We found that Gal-1 secretion led to a systemic immune suppressive response not just intra-tumorally but also in the circulating blood compartment, resulting in reduction of both CD4+ and CD8+ T cells. Concurrently, we detected higher levels of IFN-γ secretion in mice with Gal-1 KO tumors. We also observed a higher expression of PD-L1 and Gal-9 on the endothelium of WT-Gal1 compared to Gal-1 KO tumors, leading to T cell exhaustion and ineffective systemic immune response. In addition, there was noteworthy increase in the PD1+ and Tim3+ T cells in Gal-1 WT tumors compared to Gal-1 KO tumors, suggesting a role of Gal-1 in T cell anergy. Finally, we show that knocking out Gal-1 either genetically or by a blocking antibody, we could convert an anti-PD1 Ab non-responsive tumor into a responsive tumor with a significant decrease in tumor burden. In summary, our study shows Gal-1 in the tumor microenvironment could hamper immune response by impeding transendothelial T cell migration into the tumor, blocking pro-inflammatory cytokine secretions and up-regulating inhibitory immune checkpoints ligands in both tumor endothelial and T cells. Our data suggests that Gal-1 may mediate poor-response or resistance to anti-PD1 therapy and combinatorial approaches of galectin-1 inhibition with PD-1 checkpoint inhibitors may enhance therapeutic efficacy in HNC. (Acknowledgements: BMS for Anti-PD1 and Anti-Gal1 Abs. Dr R. Uppaluri, for MOC-2 oral cancer cells). Citation Format: Dhanya K. Nambiar, Todd Aguilera, Joshua Daniel Bloomstein, Hongbin Cao, Albert Koong, Quynh Thu Le. Targeting galectin-1 in combination with radiation and immune checkpoint therapy in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2017-LB-180