Prabhu Ramamoorthy

Curcumin induces cell death in esophageal cancer cells through modulating Notch signaling.

Background Curcumin inhibits the growth of esophageal cancer cell lines; however, the mechanism of action is not well understood. It is becoming increasingly clear that aberrant activation of Notch signaling has been associated with the development of esophageal cancer. Here, we have determined that curcumin inhibits esophageal cancer growth via a mechanism mediated through the Notch signaling pathway. Methodology/Principal Findings In this study, we show that curcumin treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in esophageal cancer cell lines. Furthermore, curcumin treatment induced apoptosis through caspase 3 activation, confirmed by an increase in the ratio of Bax to Bcl2. Cell cycle analysis demonstrated that curcumin treatment induced cell death and down regulated cyclin D1 levels. Curcumin treatment also resulted in reduced number and size of esophagospheres. Furthermore, curcumin treatment led to reduced Notch-1 activation, expression of Jagged-1 and its downstream target Hes-1. This reduction in Notch-1 activation was determined to be due to the down-regulation of critical components of the γ-secretase complex proteins such as Presenilin 1 and Nicastrin. The combination of a known γ-secretase inhibitor DAPT and curcumin further decreased proliferation and induced apoptosis in esophageal cancer cells. Finally, curcumin treatment down-regulate the expressions of Notch-1 specific microRNAs miR-21 and miR-34a, and upregulated tumor suppressor let-7a miRNA. Conclusion/Significance Curcumin is a potent inhibitor of esophageal cancer growth that targets the Notch-1 activating γ-secretase complex proteins. These data suggest that Notch signaling inhibition is a novel mechanism of action for curcumin during therapeutic intervention in esophageal cancers.

Methanolic Extracts of Bitter Melon Inhibit Colon Cancer Stem Cells by Affecting Energy Homeostasis and Autophagy

Bitter melon fruit is recommended in ancient Indian and Chinese medicine for prevention/treatment of diabetes. However its effects on cancer progression are not well understood. Here, we have determined the efficacy of methanolic extracts of bitter melon on colon cancer stem and progenitor cells. Both, whole fruit (BMW) and skin (BMSk) extracts showed significant inhibition of cell proliferation and colony formation, with BMW showing greater efficacy. In addition, the cells were arrested at the S phase of cell cycle. Moreover, BMW induced the cleavage of LC3B but not caspase 3/7, suggesting that the cells were undergoing autophagy and not apoptosis. Further confirmation of autophagy was obtained when western blots showed reduced Bcl-2 and increased Beclin-1, Atg 7 and 12 upon BMW treatment. BMW reduced cellular ATP levels coupled with activation of AMP activated protein kinase; on the other hand, exogenous additions of ATP lead to revival of cell proliferation. Finally, BMW treatment results in a dose-dependent reduction in the number and size of colonospheres. The extracts also decreased the expression of DCLK1 and Lgr5, markers of quiescent, and activated stem cells. Taken together, these results suggest that the extracts of bitter melon can be an effective preventive/therapeutic agent for colon cancer.

Honokiol inhibits melanoma stem cells by targeting notch signaling

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self‐renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor‐α‐Converting Enzyme (TACE) and γ‐secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch‐2. In addition, there was a reduction in the expression of downstream target proteins, Hes‐1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ‐secretase complex proteins in melanoma cells. To confirm that suppression of Notch‐2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes‐1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch‐2 signaling.

Differential changes of glutathione levels in astrocytes and neurons in ischemic brains by two-photon imaging

Using two-photon imaging techniques with monochlorobimane as a glutathione (GSH) probe, we investigated GSH levels in both core and penumbra regions of an ischemic brain after middle cerebral artery occlusion. We found that the GSH level significantly decreased in the ischemic core, but increased significantly in the penumbra. Furthermore, we observed a differential change of the GSH levels in neurons and astrocytes in the penumbra. The GSH level in neurons increased significantly whereas it decreased slightly in astrocytes in the penumbra. These findings reveal critical region and cell type-dependent changes of the GSH level in an ischemic brain.

Hypoxia-Inducible Factor 1 Protects Hypoxic Astrocytes against Glutamate Toxicity

Stroke is a major neurological disorder characterized by an increase in the Glu (glutamate) concentration resulting in excitotoxicity and eventually cellular damage and death in the brain. HIF-1 (hypoxia-inducible factor-1), a transcription factor, plays an important protective role in promoting cellular adaptation to hypoxic conditions. It is known that HIF-1α, the regulatable subunit of HIF-1, is expressed by astrocytes under severe ischaemia. However, the effect of HIF-1 on astrocytes following Glu toxicity during ischaemia has not been well studied. We investigated the role of HIF-1 in protecting ischaemic astrocytes against Glu toxicity. Immunostaining with GFAP (glial fibrillary acidic protein) confirmed the morphological modification of astrocytes in the presence of 1 mM Glu under normoxia. Interestingly, when the astrocytes were exposed to severe hypoxia (0.1% O2), the altered cell morphology was ameliorated with up-regulation of HIF-1α. To ascertain HIF-1s protective role, effects of two HIF-1α inhibitors, YC-1 [3-(50-hydroxymethyl-20-furyl)-1-benzylindazole] and 2Me2 (2-methoxyoestradiol), were tested. Both the inhibitors decreased the recovery in astrocyte morphology and increased cell death. Given that ischaemia increases ROS (reactive oxygen species), we examined the role of GSH (reduced glutathione) in the mechanism for this protection. GSH was increased under hypoxia, and this correlated with an increase in HIF-1α stabilization in the astrocytes. Furthermore, inhibition of GSH with BSO (l-butathione sulfoximine) decreased HIF-1α expression, suggesting its role in the stabilization of HIF-1α. Overall, our results indicate that the expression of HIF-1α under hypoxia has a protective effect on astrocytes in maintaining cell morphology and viability in response to Glu toxicity.

Abstract 1893: Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function

Background: Despite advances in early detection, colon cancer remains the second leading cause of death in the United States. We are focused on developing dietary prevention strategies. HNK (HNK) is a biphenolic compound that is used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether HNK affected colon cancer stem cells and to identify a mechanism. Method: Colon cancer (CRC) 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 used the AOM/DSS-induced colonic tumorigenesis model. Immunohistochemistry was determined for stem cell markers and Hippo signaling proteins. Results: HNK induced a significant dose-dependent inhibition of proliferation and colony formation of the two CRC lines, but induced apoptosis. HNK did not affect the normal cells. To demonstrate HNK effects on stem cells, we performed colosphere assays. HNK 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 HNK reduced the number of DCLK1+ cells. We next determined whether stem cell signaling is affected. For this, we looked at the Hippo signaling pathway, which is active in intestinal stem cells. The key effector protein of this pathway, YAP1 is also oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm where it is degraded, thereby inhibiting downstream gene expression. HNK significantly reduced YAP1 levels. Furthermore, HNK inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from HNK-mediated growth suppression. To determine the in vivo effect of HNK on AOM/DSS induced colonic tumorigenesis, HNK were oral gavaged at a dose of 5mg/kg bw for 24 weeks. HNK treatment significantly reduced the colonic tumor numbers and size. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker proteins, oncogenic YAP1 phosphorylation and TEAD1 in the HNK-treated AOM/DSS colonic tumor tissues. Conclusion: Together, these data suggest that HNK prevents colonic tumorigenesis that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Deep Kwatra, Gaurav Kaushik, Prabhu Ramamoorthy, Satish Ramalingam, Ossama Tawfik, Scott J. Weir, Subhash Padhye, Dan A. Dixon, Shahid Umar, Roy A. Jensen, Shrikant Anant. Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2015-1893

Expression of Hypoxia Inducible Factor 1alpha Is Protein Kinase A-dependent in Primary Cortical Astrocytes Exposed to Severe Hypoxia

The hypoxia inducible factor 1 (HIF-1) and the cyclic AMP-responsive element binding protein (CREB) are two transcription factors that have been studied in the context of neuronal survival and neurodegeneration. HIF-1 upregulation and CREB activation have been observed not only in neurons but also in astrocytes under conditions of hypoxia. We hypothesized that activation of CREB regulate HIF-1α expression in the nucleus of cortical astrocytes under in vitro ischemic condition. To test the hypothesis, we determined the effects of inhibiting the CREB activation pathway on the expression of HIF-1α protein in astrocytes exposed to CoCl2 and severe hypoxia (near anoxia, 0.1% O2). The results demonstrated that inhibition of CaMKII and CaMKIV had no effect on both HIF-1α and pCREB expression in cortical astrocytes exposed to CoCl2 and anoxia. In contrast, PKA inhibition lowered the expression of HIF-1α and pCREB expression. Furthermore, the inhibition of PKA but not CaMKII or CaMKIV increased cell death of astrocytes exposed to near anoxia. The results suggest that PKA plays an important role in the cell survival signaling pathways in astrocytes.

Abstract 5882: Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer

Ciclopirox (CPX) is contained in a number of FDA-approved topical antifungal drug products as the free acid and olamine salt. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models. Its clinical utility is limited as an oral anticancer agent, however. The oral bioavailability of CPX is quite low due to extensive first pass effect. The poor water solubility of CPX and its olamine salt prevent formulation as an injectable drug product. Thirdly, dose-limiting gastrointestinal toxicities were observed following four times daily oral dosing of CPX in patients with advanced hematologic malignancies. Ciclopirox Prodrug (CPX-POM), in contrast, has demonstrated excellent bioavailability via injectable routes of administration. Here we describe the preclinical characterization of CPX-POM, a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Following IV, SQ and IP administration to mice, CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases. CPX and its major, inactive glucuronide metabolite are extensively eliminated in urine. At well-tolerated doses, steady-state urine concentrations of CPX exceed in vitro IC50 values in mice by 15-30 fold. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro in T24 (NMIBC) and 253JBV (MIBC) human cell lines in both concentration- and time-dependent manners with IC50 values of 2-4 µM. CPX exposure increased the percentage of NMIBC and MIBC cells arrested at the S and G0/G1 phases, and induced cell death. CPX exposure significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. CPX was shown to inhibit bladder cancer cell growth in vitro by inhibiting the Notch 1 signaling pathway. The validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen mouse model of bladder cancer was employed to establish in vivo preclinical proof of principle for CPX-POM. Over the once-daily IP dose range of 25-200 mg/kg, CPX-POM treatment resulted in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, as well as a reduction in PCNA-expressing cells. All CPX-POM doses were well tolerated with no evidence of toxicity to the urinary tract based on blinded pathologic evaluation. There were also dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 in bladder cancer tissues obtained from CPX-POM treated animals. Tumor response was similar, in vivo, following once-daily and three-times weekly CPX-POM administration. CPX-POM has received FDA clearance to proceed to Phase I, and is currently being evaluated in a first-in-human trial in patients with advanced solid tumors. Citation Format: Scott J. Weir, Partha Ranjarajan, Robyn Wood, Karl Schorno, Prabhu Ramamoorthy, Lian Rajweski, Kathy Heppert, Michael J. McKenna, William McCulloch, Greg A. Reed, Amanda Brinker, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5882.

Abstract B36: Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC

Background: Triple-negative breast cancers (TNBCs) are highly associated with an aggressive clinical course, resistance to chemotherapy, and poor prognosis compared to other breast cancer subtypes. However, breast cancers arising in BRCA1 mutation carriers appear to be particularly sensitive to platinum-based chemotherapy agents. The tumor suppressor BRCA1 (breast cancer type 1 susceptibility protein) is part of a protein complex that repairs DNA damage by homologous recombination. Recent studies have shown that tumor cells expressing high levels of BRCA1 are resistant to both ionizing radiation (IR) and platinum-based chemotherapy agents, and that ablation of BRCA1 expression can restore sensitivity to these agents. We have demonstrated that the chaperone protein heat shock protein 90 (HSP90) is required for BRCA1 stability. However, established HSP90 inhibitors exhibit a high level of toxicity unrelated to HSP90. Recently, we observed that the natural compounds gedunin and celastrol modulate HSP90 activity without inducing side effects. We believe that combinatory use of these compounds can further reduce BRCA1 expression beyond their individual effect to overcome BRCA1-mediated resistance to platinum-based chemotherapies. Study Design: The goal of these experiments is to demonstrate that combination treatment with the HSP90 inhibitors gedunin and celastrol will maximize BRCA1 degradation (via inhibition of HSP90), impair the DNA repair system, and allow carboplatin to effectively eradicate TNBC cells. We will confirm that this is a HSP90 mediated event that results in the impairment of DNA repair. Using in vivo models of breast cancer, we will demonstrate that natural compounds could sensitize BRCA1-expressing TNBC cells to carboplatin, inhibiting breast cancer progression and tumor growth. Results: Using Western blot analysis, we observed that the combination of gedunin and celastrol attenuates HSP90 activity, leading to a dose-dependent reduction in BRCA1 degradation in HCC1937BRCA1 TNBC cells. Additionally, we the combination facilitated carboplatin-mediated inhibition of proliferation in both BT-20 and HCC1937BRCA1 cells. Immunofluorescence staining for g-H2AX and RAD51 demonstrated that the two HSP90 inhibitors prevent BRCA1-mediated recruitment of DNA repair complexes following ionizing radiation-induced DNA damage. To test the effects of celastrol and gedunin in vivo, we generated mouse xenografts by performing fat pad injections of HCC1937BRCA1 cells. Mice were dosed I.P. every 5 days with celastrol (5 mg/kg), gedunin (5 mg/kg), and carboplatin (25 mg/kg) for 20 days. Treatment with the HSP90 inhibitors along with carboplatin resulted in significant reduction in both tumor size and weight. In contrast treatment with carboplatin alone resulted in tumors of size similar to vehicle-treated animals. Additionally, phospho-H3 and cleaved caspase 3 immunofluorescence staining indicated a corresponding decrease in cell proliferation and increased apoptosis in tumors from celastrol+gedunin+carboplatin -treated animals compared to either vehicle or carboplatin alone-treated. Future studies will focus on examining the effects of this combination treatment in TNBC xenografts created with patient specimens, and in defining the interaction between HSP90 and BRCA1. Significance: In order to increase survival rates in TNBC patients, it is essential that resistance to chemotherapy be overcome. Successful completion of these studies demonstrating that natural compounds can act as HSP90 inhibitors to reverse platinum resistance without added toxicity would facilitate their rapid incorporation into clinical trials and provide a critically needed therapy for women with TNBC. Citation Format: Kelli Valdez, Prabhu Ramamoorthy, Shrikant Anant, Roy Jensen. Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B36.

Abstract 3832: Combination use of natural Hsp90 inhibitors to reverse BRCA1-mediated platinum resistance in triple negative breast cancer

Background: Triple-negative breast cancers (TNBCs) are highly associated with an aggressive clinical course and poor prognosis, largely due to resistance to chemotherapy. Platinum-based therapies act by causing DNA damage, which in the absence of a functional DNA repair system, leads to cancer cell death. BRCA1, a well-studied tumor suppressor, is critical for repairing DNA damage by homologous recombination. This ability makes BRCA1 an attractive therapeutic target for the re-sensitization of cancer cells to platinum chemotherapies. Recently, we have demonstrated that the chaperone protein heat shock protein 90 (HSP90) is required for BRCA1 stability. While targeted elimination of HSP90s results in a loss of BRCA1, current pharmacological inhibitors also exhibit toxicity unrelated to HSP90. We have found that the natural compounds gedunin, triptolide, and celastrol can modulate HSP90 expression without negative side effects. We believe that combinatory use of these non-toxic compounds can further reduce BRCA1 expression beyond their individual effect to overcome BRCA1-mediated resistance to platinum-based chemotherapies. Study Design: The goal of these experiments is to identify a combination of gedunin, celastrol, and triptolide to maximize BRCA1 degradation (via inhibition of HSP90), impairing the DNA repair system, and allowing carboplatin to effectively eradicate TNBC cells. Using in vitro techniques we will identify the most potent combination of gedunin, celastrol and triptolide to maximize BRCA1 degradation. We will then confirm that this is a HSP90 mediate event that results in the impairment of DNA repair. Using in vivo models of breast cancer we will demonstrate that natural compounds will sensitize BRCA-expressing TNBC cells to carboplatin, inhibiting breast cancer progression and tumor growth. Results: Using Western analysis, we have found that gedunin, celastrol and triptolide each can attenuate HSP90 activity, leading to BRCA1 degradation in BT-20 and MDA-MB-231 TNBC cells. Additionally, we have observed that gedunin, celastrol, and triptolide can each prevent formation of radiation-induced DNA repair complex, as evident by γ-H2AX staining, in HCC1937BRCA1 TNBC cells. Once we identify the most potent combination of our natural compounds, TNBC xenografts with be created and treated with the combination and carboplatin to demonstrate sensitivity to platinum treatment and inhibition of breast cancer progression. Significance: In order to increase survival rates in TNBC patients, it is essential that resistance to chemotherapy be overcome. Successful completion of these studies demonstrating that natural compounds can act as HSP90 inhibitors to reverse platinum resistance without added toxicity would facilitate their rapid incorporation into clinical trials and provide a critically needed therapy for women with TNBC. Citation Format: Kelli E. Valdez, Prabhu Ramamoorthy, Shrikant Anant, Roy Jensen. Combination use of natural Hsp90 inhibitors to reverse BRCA1-mediated platinum resistance in triple negative breast cancer. [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 3832.