Morphoproteomic-Guided Treatment of Chemotherapy Resistant Colon Cancer using Natural Based Therapies: A Case Study.

Colorectal cancer was estimated to have the fourth newest incident rate and to have the second highest death rate in 2015 according to the American Cancer Society. There were an estimated 132,700 new diagnoses of colorectal cancer made and approximately 69,000 deaths in 2015 attributing to this form of cancer. 5-fluorouracil (5-FU) or 5-FU with oxaliplatin (FOLFOX) continues to be the standard treatment protocol for patients presenting with colorectal cancer. However, treatment with FOLFOX and conventional chemotherapy has shown to lead to cancer recurrence and a number of toxic side effects. Alternative therapies based on natural ingredients show promise in combating cancer by inhibiting tumorigenic pathways, such as mTORC (mammalian target of rapamycin complex), c-MYC oncogene, and the COX-2 pathway. Using natural based agents and morphoproteomic-guided targeted therapy, the goal of this study was to evaluate the effectiveness of such an approach in a chemotherapy resistant, colon cancer patient. This case report describes a 46-year old woman with a non-metastasizing colorectal cancer that showed "minimal treatment effect" with chemotherapy despite being compliant. As of May 2016, CT scans and colonoscopy confirmed that the cancer was in remission after that patient had been compliant with morphoproteomic-guided, natural therapies for nine months.

Morphoproteomics Identifies SIRT1 and EZH2 Pathways as Commonalities in B-cell Acute Lymphoblastic Leukemia: Pathogenetic Implications and Opportunities for Therapeutic Intervention.

B-cell acute lymphoblastic leukemia (ALL) represents a malignant process in which bone marrow-derived lymphoblasts retain their undifferentiated state. Genetic testing has revealed either no identifiable cytogenetic and genomic abnormalities in such patients or a wide range of aberrations that may or may not contribute to the block in differentiation and the associated proliferation of the malignant lymphoblasts in cases of B-cell ALL. In this study, we applied morphoproteomics to a representative spectrum of cases of newly diagnosed B-cell ALL in order to identify pathways that are known to be associated with the maintenance of the undifferentiated state while promoting proliferation. Our results showed nuclear expression in a majority of the lymphoblasts from bone marrow clot preparations of each of the study cases for both silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+ histone deacetylase and enhancer of Zeste homolog 2 (EZH2), a histone methyltransferase. These represent pathogenetic pathways capable of blocking differentiation and promoting proliferation of the B-cell ALL lymphoblasts. Data mining of the National Library of Medicine's MEDLINE Database and Ingenuity Pathway analysis revealed agents of relatively low toxicity-melatonin, metformin, curcumin and sulforaphane-that are capable of inhibiting directly or pharmacogenomically one or both of the SIRT1 and EZH2 pathways and should, in a combinatorial fashion, remove the block in differentiation and decrease the proliferation of the B-cell ALL lymphoblasts.

A model for cardiomyocyte cell death: insights into mechanisms of oncosis.

It is now known that there are at least two basic patterns of cell injury progressing to cell death: cell injury with swelling, known as oncosis, and cell injury with shrinkage, known as apoptosis. Both types of cell death are "programmed" in the sense that the genetic information and many of the enzymes and other factors pre-exist in the cell. Previous investigation has pointed to cardiomyocyte ischemic injury evolving as the oncotic pattern of injury, although apoptosis has also been implicated. This study was designed, using a unique cell model system, to gain insight into the molecular events of anticancer agent-induced cardiomyocyte injury. Cardiomyocytes exposed for 2 h to 1.5 µg/ml sanguinarine consistently displayed the morphology of apoptosis in over 80% of cells, whereas a higher dose of 25 µg/ml at 2 h yielded the pattern of oncosis in over 90% of cells. Microarray analysis revealed altered expression of 2514 probes in sanguinarine-induced oncosis and 1643 probes in apoptosis at a level of significance of p<0.001. Some of the inductions such as perforin were found to be higher than 11-fold in oncosis. When perforin was blocked by perforin-specific siRNA we found a reduction in oncotic cell death. These results strengthen the notion that oncosis is not representative of nonspecific necrosis, but constitutes a genetically controlled form of "programmed cell death"; and also that oncosis might represent a pathogenetic mechanism of cardiomyocyte injury. This is also the first demonstration of the involvement of perforin in cardiomyocyte oncosis.

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.

Sorafenib downregulates ERK/Akt and STAT3 survival pathways and induces apoptosis in a human neuroblastoma cell line.

Neuroblastoma is a common solid tumor in children and its tumorigenicity is enhanced by the expression of survival pathways such as Akt and signal transducer and activator of transcription 3 (STAT3). Sorafenib is a multikinase inhibitor that also inhibits STAT3 signaling and induces apoptosis. In this study, we will examine the efficacy of sorafenib on a human neuroblastoma cell line (SK-N-AS) and also investigate its possible mechanisms. After cells reached 50-60% confluence, they were treated with various concentrations of sorafenib (0, 0.1, 1, 5, 10 and 20 microM) for different periods of time. The cell viability and apoptosis were determined by MTS colorimetric assay and TUNEL, respectively. Phosphorylation of Akt1/2/3 (p-Akt1/2/3), extracellular signal-regulated kinase 1/2 (p-ERK1/2), STAT3 (p-STAT3), and AMP-activated protein kinase alpha subunit (p-AMPKalpha) were determined with Western blot. The results indicate that as early as 2 hours post-treatment, cell viability was significantly decreased at 10 microM concentration. In 24 hours or longer treatment groups, sorafenib at 5 microM and above significantly decreased cell viability. TUNEL assay showed a significant increased of apoptosis in 5 and 20 microM treatment groups 24 hours after treatment. Western blots showed a decrease of p-ERK1/2, p-Akt1/2/3, p-STAT3, and p-AMPKalpha expression levels in various sorafenib treatment groups. Our results indicate that sorafenib significantly decreased cell viability and increased apoptosis in human neuroblastoma cell line in association with down-regulation of p-ERK1/2, p-Akt, p-STAT3 survival pathways. These data suggested potential clinical application of sorafenib in the treatment of neuroblastoma.

G-rich oligonucleotides inhibit HIF-1alpha and HIF-2alpha 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.

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.

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

BACKGROUND: 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. METHODS: We employed in vitro and in vivo assays, including Western blots, EMSA, cell cycle analysis, TUNEL and xenograft models, to determine the drug efficacy and mechanism of T40214/PEI complex. RESULTS: The results demonstrated that (i) T40214 significantly inhibited STAT3 activation and induced apoptosis in both androgen-dependent and androgen-independent PC cells; (ii) T40214 delivered by ployethylenimine (PEI) significantly suppressed prostate tumor growth in tumor-bearing nude mice due to that T40214 inhibited STAT3 activation and then greatly promoted apoptosis, reduced angiogenesis and cell proliferation in prostate tumors. CONCLUSION: Our studies suggested that STAT3 is a critical oncogenic signal, which strongly influences the progression of PCs and that T40214/PEI complex is a promising candidate for treatment of patients with prostate tumors and represents a novel strategy for PC therapy.

Inhibition of Stat3 activation and tumor growth suppression of non-small cell lung cancer by G-quartet oligonucleotides.

Lung cancer is the leading cause of cancer mortality in the United States. Despite advances made over the past decades, the overall survival of patients with lung cancer remains dismal. Here we report novel G-quartet oligodeoxynucleotides (GQ-ODN) that were designed to selectively target signal transducer and activator of transcription 3 (Stat3), in the treatment of human non-small cell lung cancer (NSCLC). The objective of this study was to evaluate the effects of two novel GQ-ODN STAT3 inhibitors, T40214 and T40231, on NSCLC bearing nude mice. NSCLC bearing nude mice were assigned to 5 groups, which were treated by vehicle, control ODN, T40214, T40231, and Paclitaxel, respectively. Tumors were measured, isolated and analyzed using Western blotting, immuno-histochemistry, RPA and TUNEL. Results show that GQ-ODN T40214 and T40231 significantly suppress the growth of NSCLC tumors in nude mice by selectively inhibiting the activation of Stat3 and its downstream proteins Bcl-2, Bcl-xL, Mcl-1, survivin, VEGF, Cyclin D1 and c-myc; thereby, promoting apoptosis and reducing angiogenesis and cell proliferation. These findings validate Stat3 as an important molecular target for NSCLC therapy and demonstrate the efficacy of GQ-ODN in inhibiting Stat3 phosphorylation.

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 microg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 microg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 microg/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.

Sanguinarine overcomes P-glycoprotein-mediated multidrug-resistance via induction of apoptosis and oncosis in CEM-VLB 1000 cells.

Permeability-glycoprotein (Pgp) positive cells are known to be encoded by the multidrug-resistance gene (MDR1), and characterized by a reduced ability to accumulate drugs. The vinblastin-resistant, Pgp positive CEM-VLB 1000 and its wild type (Pgp-negative and vinblastin-sensitive) counterpart CEM-T4 human leukemia cells, when treated with the alkaloid sanguinarine, were both found to undergo apoptosis at concentrations of 1.5 microg/ml and oncosis/blister cell death (BCD) at concentrations of 12.5 microg/ml. The aim of this study was to assess the ability of sanguinarine to overcome Pgp-mediated multidrug-resistance (MDR), and also to characterize the cell death processes of apoptosis and oncosis (or bimodal cell death) induced by sanguinarine in MDR cells. The cell death processes of apoptosis and oncosis in CEM-VLB 1000 and CEM-T4 cell lines were found to be qualitatively similar when assessed by light microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling, annexin-V-binding, trypan blue exclusion and western blot analysis. Western blotting revealed an increase in the Bax/Bcl-2 ratio and activation of caspase-3 in apoptosis but not oncosis in both cell lines. The Pgp-positive CEM-VLB 1000 cells and their wild type CEM-T4 cells were both equally sensitive to sanguinarine. Thus, sanguinarine may overcome the phenomenon of Pgp-mediated MDR by inducing apoptosis through increasing the Bax/Bcl-2 ratio and activating caspase-3, and oncosis, which involved neither.

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-kappaB, 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-X(L), 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.