Prasad Dandawate

Bitter melon: a panacea for inflammation and cancer

Nature is a rich source of medicinal plants and their products that are useful for treatment of various diseases and disorders. Momordica charantia, commonly known as bitter melon or bitter gourd, is one of such plants known for its biological activities used in traditional system of medicines. This plant is cultivated in all over the world, including tropical areas of Asia, Amazon, east Africa, and the Caribbean and used as a vegetable as well as folk medicine. All parts of the plant, including the fruit, are commonly consumed and cooked with different vegetables, stir-fried, stuffed or used in small quantities in soups or beans to give a slightly bitter flavor and taste. The plant is reported to possess anti-oxidant, anti-inflammatory, anti-cancer, anti-diabetic, anti-bacterial, anti-obesity, and immunomodulatory activities. The plant extract inhibits cancer cell growth by inducing apoptosis, cell cycle arrest, autophagy and inhibiting cancer stem cells. The plant is rich in bioactive chemical constituents like cucurbitane type triterpenoids, triterpene glycosides, phenolic acids, flavonoids, essential oils, saponins, fatty acids, and proteins. Some of the isolated compounds (Kuguacin J, Karaviloside XI, Kuguaglycoside C, Momordicoside Q-U, Charantin, α-eleostearic acid) and proteins (α-Momorcharin, RNase MC2, MAP30) possess potent biological activity. In the present review, we are summarizing the anti-oxidant, anti-inflammatory, and anti-cancer activities of Momordica charantia along with a short account of important chemical constituents, providing a basis for establishing detail biological activities of the plant and developing novel drug molecules based on the active chemical constituents.

Natural compounds targeting major cell signaling pathways: a novel paradigm for osteosarcoma therapy

Osteosarcoma is the most common primary bone cancer affecting children and adolescents worldwide. Despite an incidence of three cases per million annually, it accounts for an inordinate amount of morbidity and mortality. While the use of chemotherapy (cisplatin, doxorubicin, and methotrexate) in the last century initially resulted in marginal improvement in survival over surgery alone, survival has not improved further in the past four decades. Patients with metastatic osteosarcoma have an especially poor prognosis, with only 30% overall survival. Hence, there is a substantial need for new therapies. The inability to control the metastatic progression of this localized cancer stems from a lack of complete knowledge of the biology of osteosarcoma. Consequently, there has been an aggressive undertaking of scientific investigation of various signaling pathways that could be instrumental in understanding the pathogenesis of osteosarcoma. Here, we review these cancer signaling pathways, including Notch, Wnt, Hedgehog, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT, and JAK/STAT, and their specific role in osteosarcoma. In addition, we highlight numerous natural compounds that have been documented to target these pathways effectively, including curcumin, diallyl trisulfide, resveratrol, apigenin, cyclopamine, and sulforaphane. We elucidate through references that these natural compounds can induce cancer signaling pathway manipulation and possibly facilitate new treatment modalities for osteosarcoma.

New targets for the treatment of follicular lymphoma

The last two decades have witnessed striking advances in our understanding of the biological factors underlying the development of Follicular lymphoma (FL). Development of newer treatment approaches have improved the outlook for many individuals with these disorders; however, with these advances come new questions. Given the long-term survival of patients with FL, drugs with favourable side-effect profile and minimal long-term risks are desired. FL is incurable with current treatment modalities. It often runs an indolent course with multiple relapses and progressively shorter intervals of remission. The identification of new targets and development of novel targeted therapies is imperative to exploit the biology of FL while inherently preventing relapse and prolonging survival. This review summarizes the growing body of knowledge regarding novel therapeutic targets, enabling the concept of individualized targeted therapy for the treatment of FL.

Targeting HSF1 disrupts HSP90 chaperone function in chronic lymphocytic leukemia

CLL is a disease characterized by chromosomal deletions, acquired copy number changes and aneuploidy. Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. Interestingly, several independent studies have demonstrated that HSF1 expression and activity also affects the chaperoning of HSP90 kinase clients, although the mechanism underlying this observation is unclear. Here, we determined how HSF1 regulates HSP90 function using CLL as a model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. We demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Brutons Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). Treatment with triptolide or HSF1 knockdown disrupts the cytosolic complex between HSF1, p97, HSP90 and the HSP90 deacetylase- Histone deacetylase 6 (HDAC6). Consequently, HSF1 inhibition results in HSP90 acetylation and abrogation of its chaperone function. Finally, tail vein injection of Mec-1 cells into Rag2−/−IL2Rγc−/− mice followed by treatment with minnelide (a pro-drug of triptolide), reduced leukemia, increased survival and attenuated HSP90-dependent survival signaling in vivo. In conclusion, our study provides a strong rationale to target HSF1 and test the activity of minnelide against human CLL.

Bitter Melon as a Therapy for Diabetes, Inflammation, and Cancer: a Panacea?

Natural products have been used for centuries for cures prevention, treatment, and cure of multiple diseases. Some dietary agents are present in multiple systems of medicines as proposed treatments for chronic and difficult to treat diseases. Once such natural product is Momordica charantia or bitter melon. Bitter melon is cultivated in multiple regions across the world, and various parts of the plant, such as fruit, leaves seed, etc. have been shown to possess medicinal properties in ancient literature. Over the last few decades, multiple well-structured scientific studies have been performed to study the effects of bitter melon in various diseases. Some of the properties for which bitter melon has been studied include: antioxidant, antidiabetic, anticancer, anti-inflammatory, antibacterial, antifungal, antiviral, anti-HIV, anthelmintic, hypotensive, anti-obesity, immuno-modulatory, antihyperlipidemic, hepato-protective, and neuro-protective activities. This review attempts to summarize the various literature findings regarding medicinal properties of bitter melon. With such strong scientific support on so many medicinal claims, bitter melon comes close to being considered a panacea.

Abstract 1310: Targetingprolactin signaling to suppress pancreatic cancer stem cells

Background: Pancreatic cancer (PCa) is a major cause of cancer related mortality in United States with Methods: PCa cells (MiaPaCa-2 and PanC-1) were grown to 70-80% of confluency and treated with and without prolactin (PRL) and JAK2, STAT3 and ERK phosphorylation in presence and absence of antipsychotic compound were evaluated by western blot. Growth of PCa lines (MiaPaCa-2, PanC-1, BxPC-3, AsPC-1) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, respectively. Pancosphere formation was used to identify effects on stem cells. Results: We have recently identified that the receptor for the pituitary hormone prolactin is overexpressed in pancreatic cancers, and in pancreatic cancer cell lines. When prolactin (PRL) binds its cognate receptor (PRLR), it induces various downstream events including the JAK-STAT and ERK MAPK pathways. In pancreatic cancer cell lines, we observe that PRL treatment induced dose- and time-dependent JAK2, STAT3, and ERK1/2 phosphorylation. Furthermore, there was an increase in the expression of cancer stem cell (CSC) markers DCLK1 (doublecortin calmodulin like kinase 1) and CD44. In addition, PRL-induced pancosphere formation further suggesting that PRL affects stem cells. Based on these data, we conclude that PRL signaling enhances stemness in pancreatic cancers, and therefore we decided to target it for therapeutic intervention. For this, we developed a homology model for the C-terminal intracellular region of the receptor and performed a virtual screening in silico with FDA approved drugs. One compound, a first generation antipsychotic drug diphenylbutylpiperidine, also called Penfluridol was found to interact with the region of the receptor that also binds site for JAK2. The compound has a long half-life, and is used in the treatment of chronic schizophrenia and similar psychotic disorders. We have further determined that Penfluridol inhibits PRL-induced STAT3 and ERK phosphorylation. In addition, the compound reduced proliferation, colony formation, and spheroid formation. Moreover, it induced cells to undergo autophagy by activating LC3B and increasing expression of autophagy-related genes ATG5, 7 and 12. Conclusions: PRL signaling through its cognate PRLR receptor is critical for aggressive pancreatic cancer behavior, and therefore may be an effective therapeutic strategy. Citation Format: Prasad Dandawate, Gaurav Kaushik, Dharmalingam Subramaniam, Prabhu Ramamoorthy, Scott J. Weir, Roy A. Jensen, Shrikant Anant. Targetingprolactin signaling to suppress pancreatic cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1310.

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

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

Abstract 4748: Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling

Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound, HDNC or Marmelin from Aegle marmelos with potent anti-colon cancer activity. We have now developed a novel marmelin analogue THB and made it water soluble THB using β-cyclodextrin (THBCD). The current study is designed to determine whether THB affects stem cells and to identify a mechanism. Method: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we have performed HCT116 cells induced tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins. Results: THB treatment induced a significant dose-dependent inhibition of proliferation and colony formation of the two colon cancer cell lines, but not that of the normal cells. To demonstrate THB effects on stem cells, we performed colosphere assays. THB treatment significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where THB reduced the number of DCLK1+ cells. We next determined whether THB affects the Notch signaling pathway, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. THB treatment significantly downregulated the expression of Notch1, its ligand Jagged1 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. THB treatment inhibits the expression of γ-secretase complex proteins Presenilin1, Nicastrin, APH1 and PEN2. Moreover, ectopic expression of the Notch Intracellular domain (NICD) rescued the cells from THB mediated growth suppression. These data demonstrate that THB mediated effects of colon cancer stem cells is in part through downregulating Notch1 activation. To determine the effect of THB on tumor growth in vivo, mice carrying HCT116 tumor xenografts were administered the compound intraperitoneally (5mg/kg bw) every day for 21 days. THB treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the THB-treated xenograft tissues. Conclusion: Together, these data suggest that THB treatment suppresses colon cancer growth that targets stem cells by inhibiting Notch1 signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Prasad R. Dandawate, Ossama W. Tawfik, Roy A. Jensen, Scott J. Weir, Subhash B. Padhye, Shrikant Anant. Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4748.