The Radiolabeling of a Gly-Sar Dipeptide Derivative with Flourine-18 and Its Use as a Potential Peptide Transporter PET Imaging Agent.

: We have developed a novel fluorine-18 radiotracer, dipeptide 1, radiolabeled in two steps from mesylate 3. The initial radiolabeling is achieved in a short reaction time (10 min) and purified through solid-phase extraction (SPE) with modest radiochemical yields (rcy = 10 ± 2%, n = 5) in excellent radiochemical purity (rcp > 99%, n = 5). The de-protection of the tert-butyloxycarbonyl (Boc) and trityl group was achieved with mild heating under acidic conditions to provide 18F-tagged dipeptide 1. Preliminary analysis of 18F-dipeptide 1 was performed to confirm uptake by peptide transporters (PepTs) in human pancreatic carcinoma cell lines Panc1, BxPC3, and ASpc1, which are reported to express the peptide transporter 1 (PepT1) . Furthermore, we confirmed in vivo uptake of 18F-dipeptide tracer 1 using microPET/CT in mice harboring subcutaneous flank Panc1, BxPC3, and Aspc1 tumors. In conclusion, we have established the radiolabeling of dipeptide 1 with fluoride-18, and demonstrated its potential as an imaging agent which may have clinical applications for the diagnosis of pancreatic carcinomas.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-Troglitazone-induced Apoptosis in Prostate Cancer Cells Involve AMP-activated Protein Kinase.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers in men with limited treatment options for the hormone-resistant forms. Development of novel therapeutic options is critically needed to target advanced forms. Here we demonstrate that combinatorial treatment with the thiazolidinedione troglitazone (TZD) and TNF-related apoptosis-inducing ligand (TRAIL) can induce significant apoptosis in various PCa cells independent of androgen receptor status. Because TZD is known to activate AMP-activated protein kinase (AMPK), we determined whether AMPK is a molecular target mediating this apoptotic cascade by utilizing PCa cell lines stably overexpressing AMPKα1 dominant negative (C4-2-DN) or empty vector (C4-2-EV). Our results indicated a significantly higher degree of apoptosis with TRAIL-TZD combination in C4-2-EV cells compared with C4-2-DN cells. Similarly, results from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a larger reduction of viability of C4-2-EV cells compared with C4-2-DN cells when treated with TRAIL-TZD, thus suggesting that C4-2-DN cells were more apoptosis-resistant. Additionally, siRNA-mediated knockdown of endogenous AMPKα1 expression showed a reduction of TRAIL-TZD-induced apoptosis, further confirming the participation of AMPK in mediating this apoptosis. Apoptosis induction by this combinatorial treatment was also associated with a cleavage of ß-catenin that was inhibited in both C4-2-DN cells and those cells in which AMPKα1 was knocked down. In addition, time course studies showed an increase in pACC(S79) (AMPK target) levels coinciding with the time of apoptosis. These studies indicate the involvement of AMPK in TRAIL-TZD-mediated apoptosis and ß-catenin cleavage and suggest the possibility of utilizing AMPK as a therapeutic target in apoptosis-resistant prostate cancer.

Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses.

More than 120 human papillomaviruses (HPVs) have now been identified and have been associated with a variety of clinical lesions. To understand the molecular differences among these viruses that result in lesions with distinct pathologies, we have begun a MS-based proteomic analysis of HPV-host cellular protein interactions and have created the plasmid and cell line libraries required for these studies. To validate our system, we have characterized the host cellular proteins that bind to the E7 proteins expressed from 17 different HPV types. These studies reveal a number of interactions, some of which are conserved across HPV types and others that are unique to a single HPV species or HPV genus. Binding of E7 to UBR4/p600 is conserved across all virus types, whereas the cellular protein ENC1 binds specifically to the E7s from HPV18 and HPV45, both members of genus alpha, species 7. We identify a specific interaction of HPV16 E7 with ZER1, a substrate specificity factor for a cullin 2 (CUL2)-RING ubiquitin ligase, and show that ZER1 is required for the binding of HPV16 E7 to CUL2. We further show that ZER1 is required for the destabilization of the retinoblastoma tumor suppressor RB1 in HPV16 E7-expressing cells and propose that a CUL2-ZER1 complex functions to target RB1 for degradation in HPV16 E7-expressing cells. These studies refine the current understanding of HPV E7 functions and establish a platform for the rapid identification of virus-host interactions.

FL118, acting as a 'molecular glue degrader', binds to dephosphorylates and degrades the oncoprotein DDX5 (p68) to control c-Myc, survivin and mutant Kras against colorectal and pancreatic cancer with high efficacy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), a difficult-to-treat cancer, is expected to become the second-largest cause of cancer-related deaths by 2030, while colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths. Currently, there is no effective treatment for PDAC patients. The development of novel agents to effectively treat these cancers remains an unmet clinical need. FL118, a novel anticancer small molecule, exhibits high efficacy against cancers; however, the direct biochemical target of FL118 is unknown. METHODS: FL118 affinity purification, mass spectrometry, Nanosep centrifugal device and isothermal titration calorimetry were used for identifying and confirming FL118 binding to DDX5/p68 and its binding affinity. Immunoprecipitation (IP), western blots, real-time reverse transcription PCR, gene silencing, overexpression (OE) and knockout (KO) were used for analysing gene/protein function and expression. Chromatin IP was used for analysing protein-DNA interactions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid assay and human PDAC/CRC cell/tumour models were used for determining PDAC/CRC cell/tumour in vitro and in vivo growth. RESULTS: We discovered that FL118 strongly binds to dephosphorylates and degrades the DDX5 oncoprotein via the proteasome degradation pathway without decreasing DDX5 mRNA. Silencing and OE of DDX5 indicated that DDX5 is a master regulator for controlling the expression of multiple oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras. Genetic manipulation of DDX5 in PDAC cells affects tumour growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Our human tumour animal model studies further indicated that FL118 exhibits high efficacy to eliminate human PDAC and CRC tumours that have a high expression of DDX5, while FL118 exhibits less effectiveness in PDAC and CRC tumours with low DDX5 expression. CONCLUSION: DDX5 is a bona fide FL118 direct target and can act as a biomarker for predicting PDAC and CRC tumour sensitivity to FL118. This would greatly impact FL118 precision medicine for patients with advanced PDAC or advanced CRC in the clinic. FL118 may act as a 'molecular glue degrader' to directly glue DDX5 and ubiquitination regulators together to degrade DDX5.

Mutant Kras as a Biomarker Plays a Favorable Role in FL118-Induced Apoptosis, Reactive Oxygen Species (ROS) Production and Modulation of Survivin, Mcl-1 and XIAP in Human Bladder Cancer.

Tumor heterogeneity in key gene mutations in bladder cancer (BC) is a major hurdle for the development of effective treatments. Using molecular, cellular, proteomics and animal models, we demonstrated that FL118, an innovative small molecule, is highly effective at killing T24 and UMUC3 high-grade BC cells, which have Hras and Kras mutations, respectively. In contrast, HT1376 BC cells with wild-type Ras are insensitive to FL118. This concept was further demonstrated in additional BC and colorectal cancer cells with mutant Kras versus those with wild-type Kras. FL118 strongly induced PARP cleavage (apoptosis hallmark) and inhibited survivin, XIAP and/or Mcl-1 in both T24 and UMUC3 cells, but not in the HT1376 cells. Silencing mutant Kras reduced both FL118-induced PARP cleavage and downregulation of survivin, XIAP and Mcl-1 in UMUC3 cells, suggesting mutant Kras is required for FL118 to exhibit higher anticancer efficacy. FL118 increased reactive oxygen species (ROS) production in T24 and UMUC3 cells, but not in HT1376 cells. Silencing mutant Kras in UMUC3 cells reduced FL118-mediated ROS generation. Proteomics analysis revealed that a profound and opposing Kras-relevant signaling protein is changed in UMUC3 cells and not in HT1376 cells. Consistently, in vivo studies indicated that UMUC3 tumors are highly sensitive to FL118 treatment, while HT1376 tumors are highly resistant to this agent. Silencing mutant Kras in UMUC3 cell-derived tumors decreases UMUC3 tumor sensitivity to FL118 treatment. Together, our studies revealed that mutant Kras is a favorable biomarker for FL118 targeted treatment.

Involvement of AMP-activated protein kinase and Death Receptor 5 in TRAIL-Berberine-induced apoptosis of cancer cells.

Our previous studies indicated that combination of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PPARγ ligand Troglitazone (TZD), can induce significant apoptosis in various TRAIL-resistant prostate and hepatocellular carcinoma (HCC) cells. These also suggested serine/threonine kinase AMP-activated protein kinase (AMPK) to be a mediator of TRAIL-TZD-induced apoptosis. To further validate AMPK's role in TRAIL sensitization, we determined the apoptotic potential of TRAIL in combination with the natural compound Berberine (BBR), the latter being a potent activator of AMPK. These demonstrated a significant reduction of cell viability and induction of apoptosis (increased cleavage of caspase 3, 8, 9) when treated with TRAIL-BBR combination. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN) or following AMPKα knockdown, confirming involvement of AMPK. To identify potential downstream mediators involved, an apoptosis RT2 PCR array analysis was performed. These showed induction of several genes including TNFRSF10B (expresses DR5) and Harakiri following BBR treatment, which were further validated by qPCR analysis. Furthermore, knocking down DR5 expression significantly attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a mediator of this apoptosis. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of ameliorating TRAIL-resistance involving DR5 in advanced cancer.

Antineoplastic Effects of Honokiol on Melanoma.

Honokiol, a plant lignan has been shown to have antineoplastic effects against nonmelanoma skin cancer developments in mice. In this study, antineoplastic effects of honokiol were investigated in malignant melanoma models. In vitro effects of honokiol treatment on SKMEL-2 and UACC-62 melanoma cells were evaluated by measuring the cell viability, proliferation, apoptosis, cell cycle analysis, and expressions of various proteins associated with cell cycle progression and apoptosis. For the in vivo study, male nude mice inoculated with SKMEL-2 or UACC-62 cells received injections of sesame oil or honokiol for two to seven weeks. In vitro honokiol treatment caused significant decrease in cell viability, proliferation, cell cycle arrest, increased apoptosis, and modulation of apoptotic and cell cycle regulatory proteins. Honokiol caused an accumulation of cells in the G2/M phase of the cell cycle in SKMEL-2 and G0/G1 phase in UACC-62 cells. An elevated level of caspases and PARP were observed in both cell lines treated with honokiol. A decrease in the expression of various cell cycle regulatory proteins was also observed in honokiol treated cells. Honokiol caused a significant reduction of tumor growth in SKMEL-2 and UACC-62 melanoma xenografts. These findings suggest that honokiol is a good candidate for further studies as a possible treatment for malignant melanoma.

Modulation of glycogen synthase kinase-3ß following TRAIL combinatorial treatment in cancer cells.

Glycogen Synthase Kinase-3ß (GSK3ß) is a serine/threonine kinase, known to regulate various cellular processes including proliferation, differentiation, survival, apoptosis as well as TRAIL-resistance. Thus pathways that can modulate GSK3ß axis are important targets for cancer drug development. Our earlier studies have shown that combinatorial treatment with Troglitazone (TZD) and TRAIL can induce apoptosis in TRAIL-resistant cancer cells. The current studies were undertaken to investigate whether GSK3ß pathway was modulated during this apoptosis. Our results indicated an increase in inhibitory GSK3ßSer9 phosphorylation during apoptosis, mediated via AKT. At a later time, however, TZD alone and TRAIL-TZD combination produced a dramatic reduction of GSK3ß expression, which was abolished by cycloheximide. Luciferase assays with GSK3ß-luc promoter reporter showed that TZD can effectively antagonize GSK3ß promoter activity. Since TZD is a ligand for transcription factor PPARγ and can activate AMPK, we determined their roles on antagonism of GSK3ß. Knockdown of PPARγ was unable to restore GSK3ß expression or antagonize GSK3ßSer9 phosphorylation. Although pretreatment with Compound C (pharmacological inhibitor of AMPK) partially rescued GSK3ß expression, knockdown of AMPKα1 or α2 alone or in combination were ineffective. These studies suggested a novel PPARγ-AMPK-independent mechanism of targeting GSK3ß by TZD, elucidation of which might provide newer insights to improve our understanding of TRAIL-resistance.

Activation of hERG3 channel stimulates autophagy and promotes cellular senescence in melanoma.

Ion channels play a major factor in maintaining cellular homeostasis but very little is known about the role of these proteins in cancer biology. In this work we have discovered that, the Kv11.3 (hERG3) a plasma-membrane potassium channel plays a critical role in the regulation of autophagy in a cancer cell model. We have found that pharmacologic stimulation of the Kv11.3 channel with a small molecule activator, NS1643 induced autophagy via activation of an AMPK-dependent signaling pathway in melanoma cell line. In addition, we have found that NS1643 produced a strong inhibition of cell proliferation by activating a cellular senescence program. Furthermore, inhibition of autophagy via siRNA targeting AMPK or treatment with hydroxychloroquine an autophagy inhibitor activates apoptosis in NS1643-treated cells. Thus, we propose that, Kv11.3 is a novel mediator of autophagy, autophagy can be a survival mechanism contributing to cellular senescence, and that use of a combinatorial pharmacologic approach of Kv11.3 activator with inhibitors of autophagy represents a novel therapeutic approach against melanoma.

Anticancer Effects of Sandalwood (Santalum album).

Effective management of tumorigenesis requires development of better anticancer agents with greater efficacy and fewer side-effects. Natural products are important sources for the development of chemotherapeutic agents and almost 60% of anticancer drugs are of natural origin. α-Santlol, a sesquiterpene isolated from Sandalwood, is known for a variety of therapeutic properties including anti-inflammatory, anti-oxidant, anti-viral and anti-bacterial activities. Cell line and animal studies reported chemopreventive effects of sandalwood oil and α-santalol without causing toxic side-effects. Our laboratory identified its anticancer effects in chemically-induced skin carcinogenesis in CD-1 and SENCAR mice, ultraviolet-B-induced skin carcinogenesis in SKH-1 mice and in vitro models of melanoma, non-melanoma, breast and prostate cancer. Its ability to induce cell-cycle arrest and apoptosis in cancer cells is its most reported anticancer mechanism of action. The present review discusses studies that support the anticancer effect and the mode of action of sandalwood oil and α-santalol in carcinogenesis.

α-Santalol, a skin cancer chemopreventive agent with potential to target various pathways involved in photocarcinogenesis.

This study is designed to investigate the chemopreventive effect and molecular mechanisms of α-santalol on UVB-induced skin tumor development in SKH-1 hairless mouse, a widely used model for human photocarcinogenesis. A dose of UVB radiation (30 mJ cm(-2) day(-1)) that is in the range of human sunlight exposure was used for the initiation and promotion of tumor. Topical treatment of mice with α-santalol (10%, wt/vol in acetone) caused reduction in tumor incidence, multiplicity and volume. In our study, the anticarcinogenic action of α-santalol against UVB-induced photocarcinogenesis was found to be associated with inhibition of inflammation and epidermal cell proliferation, cell cycle arrest and induction of apoptosis. α-Santalol pretreatment strongly inhibited UVB-induced epidermal hyperplasia and thickness of the epidermis, expression of proliferation and inflammation markers proliferating cell nuclear antigen (PCNA), Ki-67 and cyclooxygenase 2 (Cox-2). Significant decrease in the expression of cyclins A, B1, D1 and D2 and cyclin-dependent kinases (Cdk)s Cdk1 (Cdc2), Cdk2, Cdk4 and Cdk6 and an upregulated expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21 were found in α-santalol pretreated group. Furthermore, an elevated level of cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) were observed in α-santalol-treated group. Our data suggested that α-santalol is a safer and promising skin cancer chemopreventive agent with potential to target various pathways involved in photocarcinogenesis.

Antineoplastic effects of α-santalol on estrogen receptor-positive and estrogen receptor-negative breast cancer cells through cell cycle arrest at G2/M phase and induction of apoptosis.

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor (ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.

α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation.

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 µM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells.