Priya Weerasinghe

From traditional Ayurvedic medicine to modern medicine: identification of therapeutic targets for suppression of inflammation and cancer

Cancer is a hyperproliferative disorder that involves transformation, dysregulation of apoptosis, proliferation, invasion, angiogenesis and metastasis. Extensive research during the last 30 years has revealed much about the biology of cancer. Drugs used to treat most cancers are those that can block cell signalling, including growth factor signalling (e.g., epidermal growth factor); prostaglandin production (e.g., COX-2); inflammation (e.g., inflammatory cytokines: NF-κB, TNF, IL-1, IL-6, chemokines); drug resistance gene products (e.g., multi-drug resistance); cell cycle proteins (e.g., cyclin D1 and cyclin E); angiogenesis (e.g., vascular endothelial growth factor); invasion (e.g., matrix metalloproteinases); antiapoptosis (e.g., bcl-2, bcl-XL, XIAP, survivin, FLIP); and cellular proliferation (e.g., c-myc, AP-1, growth factors). Numerous reports have suggested that Ayurvedic plants and their components mediate their effects by modulating several of these recently identified therapeutic targets. However, Ayurvedic medicine requires rediscovery in light of our current knowledge of allopathic (modern) medicine. The focus of this review is to elucidate the Ayurvedic concept of cancer, including its classification, causes, pathogenesis and prevention; surgical removal of tumours; herbal remedies; dietary modifications; and spiritual treatments.

The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death

Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 microM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by Western blot analysis. However, 8.48 and 16.96 microM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status.

Oncosis: An important non-apoptotic mode of cell death

It is now increasingly accepted that apoptosis may not be the only form of cell death seen in vitro and in vivo; hence there is a need to study novel forms of cell death. The explosion of cell death research that followed the recognition of apoptosis by Kerr and colleagues in the late 1960s completely obscured the fact that apoptosis is not the only form of cell death. Apoptosis manifests itself by cell shrinkage followed by breakup; another form (oncosis) is almost the opposite: it involves cell swelling and coagulation of the cytoplasm. The name oncosis was chosen over a century ago by von Recklinghausen, a top collaborator of Rudolph Virchow and thereby one of the founders of cellular pathology. Nevertheless, oncosis was forgotten, largely because a satisfactory technique for preparing tissue sections did not exist at the time. Also confusion developed regarding the distinction between oncosis as a mode of cell injury and cell death, and necrosis as a degradation process following cell death. In this review we have described the many characteristics of oncosis from a morphological and biochemical standpoint, and we briefly examine the application of oncosis in disease processes.

Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low level induced apoptosis or programmed cell death (PCD) in the Bcl-2 low-expressing K562 human erythroleukemia cells, and that a high level induced blister cell death (BCD); whereas Bcl-2 overexpressing, sanguinarine-treated JM1 pre-B lymphoblastic cells displayed neither apoptosis nor BCD morphologies. Here, we report that sanguinarine-treated K562 cells, when analyzed by western blot, showed significant increase in expression of the pro-apoptotic Bax protein in apoptosis, but not in BCD. cDNA expression array of PCD in K562 cells failed to reveal the presence of Bax at the gene transcript level, which suggests that this cell death process does not require de novo protein synthesis. Treated JM1 cells, on the other hand, showed an increase in the expression of Bcl-2 protein in both forms of cell death, but failed to show Bax expression. The role of other members of the Bcl-2 family remained negligible. Caspase-3 activation was observed in apoptosis of K562 cells but not in BCD or in sanguinarine-treated JM1 cells. These results suggest that sanguinarine in K562 cells induces apoptosis through increasing Bax and activating caspase-3, whereas sanguinarine-induced BCD involves neither. These results also suggest that in JM1 cells, Bcl-2 may play a role in susceptibility of cells to induction of apoptosis and BCD.

Unresolved issues in myocardial reperfusion injury.

While the basic pathobiology of myocardial ischemic injury and reperfusion has been determined over the last 50 years, there are important, unresolved, or at least not completely elucidated, issues in the field. These include the relative contributions of different modes of cell injury and death to evolving myocardial infarcts; interactions of phenomena produced by reperfusion, including stunning and preconditioning; and potential new approaches for successfully combining adjuvant therapy with coronary artery opening. A model of myocardial ischemic and reperfusion injury is proposed involving the potential expression of apoptotic and oncotic pathways in the same perturbed cardiomyocytes. Promising new cardioprotective strategies for reducing lethal reperfusion injury are discussed, including ischemic postconditioning, activators of the reperfusion injury salvage kinase (RISK) pathway, inhibitors of protein kinase c-delta, and inhibitors of the mitochondrial membrane permeability transition pore (PTP). Major outstanding clinical challenges are also discussed, including the development of clinical care systems that can routinely deliver very timely coronary opening and reperfusion, perhaps combined with adjuvant therapy, and the development of strategies to retard adverse remodeling and congestive heart failure in patients with significant myocardial infarction and scarring, perhaps by refinements in stem cell therapy.

Sanguinarine induces bimodal cell death in K562 but not in high Bcl-2-expressing JM1 cells

Our previous studies with low Bcl-2-expressing K562 cells have shown that, when treated with the putative anti-cancer drug sanguinarine, concentrations of 1.5 microg/ml induced the morphology of apoptosis or programmed cell death (PCD), while concentrations of 12.5 microg/ml induced a morphology of blister formation or blister cell death (BCD). To elucidate the possible role of Bcl-2 in this dual cell death modality induced by sanguinarine, K562 and the high Bcl-2-expressing JM1 cells were treated with sanguinarine concentrations of 1.5 microg/ml and 12.5 microg/ml respectively, and multiple parameters of their effects were studied using light and electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling, 51Cr release, trypan blue exclusion, propidium iodide exclusion, and annexin-V binding. In general, we found that, while K562 cells underwent PCD and BCD when treated with sanguinarine, JM1 cells failed to undergo either PCD or BCD under the same experimental conditions. Thus, the over-expression of anti-apoptotic Bcl-2 may have prevented sanguinarine from inducing PCD and BCD in JM1 cells. These results indicate that the resistance of JM1 cells to the alkaloid sanguinarine may have been due to an anti-BCD role played by Bcl-2, in addition to its widely reported anti-apoptotic role.

T40214/PEI complex: A potent therapeutics for prostate cancer that targets STAT3 signaling

Prostate cancer (PC) is the most common cancer among men in American and the second leading cause of cancer death. The treatment options employed for patients with advanced and metastatic PC are limited. As a critical mediator of oncogenic signaling, STAT3 is active in 82% of patients with PC. STAT3 has become a very important molecular target for PC therapy since it upregulates the oncogenes encoding apoptosis inhibitors, cell cycle regulators, and inducers of angiogenesis. However, no anti‐tumor drug whose primary mode of action is to target STAT3 has yet reached the clinic. To this end, we have laid the initial groundwork to develop the STAT3‐inhibiting G‐quartet oligodeoxynucleotide (GQ‐ODN), T40214, for treatment of PCs.

Aurintricarboxylic Acid Inhibits Protein Synthesis Independent, Sanguinarine-Induced Apoptosis and Oncosis

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low concentration (1.5 μg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 μg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 μg/ml) induced diffuse swelling or oncosis-diffuse cell swelling (DCS). To assess the underlying mechanism of sanguinarine-induced apoptosis and oncosis-BCD in K562 cells, we studied their response to pre-treatment with two chemical compounds: aurintricarboxylic acid (ATA) and cycloheximide (CHX). The pretreatment effects of both chemical compounds on apoptosis and oncosis-BCD were evaluated by measuring multiple parameters using quantitative morphology, electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling and annexin-V-binding. ATA, a DNA endonuclease inhibitor, efficiently prevented DNA nicking and inhibited apoptosis almost completely and oncosis-BCD by about 40%, while CHX, a protein synthesis inhibitor, failed to inhibit both apoptosis and oncosis-BCD. These results demonstrate, first, the importance of endonuclease in sanguinarine-induced apoptosis and to some extent in oncosis-BCD and, second, that this inhibition does not require de novo protein synthesis.

G-rich Oligonucleotides Inhibit HIF-1α and HIF-2α and Block Tumor Growth

Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in tumor promotion by upregulating its target genes, which are involved in energy metabolism, angiogenesis, cell survival, invasion, metastasis, and drug resistance. The HIF-1alpha subunit, which is regulated by O2-dependent hydroxylation, ubiquitination, and degradation, has been identified as an important molecular target for cancer therapy. We have rationally designed G-rich oligodeoxynucleotides (ODNs) as inhibitors of HIF-1alpha for human cancer therapy. The lead compounds, JG243 and JG244, which form an intramolecular parallel G-quartet structure, selectively target HIF-1alpha and decreased levels of both HIF-1alpha and HIF-2alpha (IC50 < 2 micromol/l) and also inhibited the expression of HIF-1-regulated proteins [vascular endothelial growth factor (VEGF), Bcl-2, and Bcl-XL], but did not disrupt the expression of p300, Stat3, or p53. JG-ODNs induced proteasomal degradation of HIF-1alpha and HIF-2alpha that was dependent on the hydroxylase activity of prolyl-4-hydroxylase-2. JG243 and JG244 dramatically suppressed the growth of prostate, breast, and pancreatic tumor xenografts. Western blots from tumor tissues showed that JG-ODNs significantly decreased HIF-1alpha and HIF-2alpha levels and blocked the expression of VEGF. The JG-ODNs are novel anticancer agents that suppress tumor growth by inhibiting HIF-1.

A Morphoproteomic Study of Metformin and Vorinostat, Alone and in Combination, Induced Apoptosis in MOLT-4 Acute Leukemia Cells

Emerging evidence suggests that human leukocyte antigen (HLA) antibody-mediated rejection (AMR) through donorspecific antibodies (DSAs) contributes to lower graft and patient survival in liver transplant patients. Therapeutic plasma exchange (TPE) is currently a category I indication for AMR in ABO-compatible renal transplant patients and a category III indication for AMR in lung allograft rejection. Current guidelines do not include AMR in liver allografts as an indication for TPE; however, criteria for chronic antibodymediated rejection in liver allograft patients, including the presence of DSA, were recently proposed. Over the past 2 years, the transfusion medicine service at Houston Methodist Hospital has been consulted for TPE in 8 liver transplant patients with potential AMR and positive DSAs. Herein, we describe our relevant observations from these patients, which included five women and three men, ranging in age from 3271 years. All had received an orthotopic liver transplant ranging from 1 month to 2 years prior to TPE. All patients had DSAs. All but one patient had class II DSAs, including six patients with antibodies directed against the DQ antigens. Four patients had class I DSAs. The number of DSAs ranged from one to eight, with two patients having eight DSAs prior to TPE. For certain DSAs, TPE led to rapid reduction in DSA mean fluorescence intensity to undetectable levels, as determined by the single antigen bead assay. Class I antibodies and antibodies with lower mean fluorescence intensity were more likely to decrease or disappear after two to three sessions of TPE performed approximately every other day. Despite this, some DSAs, especially Class II DSAs directed against DQ antigens with high pre-TPE mean fluorescence intensity, persisted after repeated, regular TPE sessions. In two patients, TPE led to complete reduction in DSAs to undetectable levels. Patients tolerated TPE with 5% albumin as a replacement fluid without major bleeding or thrombotic complications. No adverse events occurred during the TPE procedures; however, one patient experienced major hematoma/ hemothorax during central line placement for TPE. Fresh frozen plasma was used as a replacement fluid in only rare circumstances to replace fibrinogen or prior to an invasive procedure. The overall data suggest that liver transplant patients tolerate TPE without major complications. However, TPE appears effective for removal of DSAs as assessed by the single antigen bead assay for only certain antibodies. TPE practitioners should become familiar with the potential risks and benefits of TPE in liver transplant patients being treated for AMR as the definition of AMR and the role of DSAs becomes more established. AJCP / MEETING ABSTRACTS