Potential Anticancer Treatment Research Articles

Proteotoxic stress targeted therapy (PSTT): induction of protein misfolding enhances the antitumor effect of the proteasome inhibitor bortezomib

Proteotoxic stress (PS) is generated in cells under a variety of conditions involving accumulation of misfolded proteins. To avoid the toxicity of unmitigated PS, cells activate the heat shock response (HSR). HSR involves upregulation of factors such as ubiquitin and the non-housekeeping chaperone Hsp70 which assist with metabolism of aberrant proteins. The PS-HSR axis is a potential anticancer treatment target since many tumor cells display constitutive PS and dependence on HSR due to their rapid rates of proliferation and translation. In fact, induction of PS via stimulation of protein misfolding (hyperthermia), inhibition of proteasomes (bortezomib) or inhibition of Hsp90 (geldanamycin) have all been considered or used for cancer treatment. We found that combination of bortezomib with an inducer of protein misfolding (hyperthermia or puromycin) resulted in enhanced PS. HSR was also induced, but could not mitigate the elevated PS and the cells died via largely p53-independent apoptosis. Thus, combination treatments were more cytotoxic in vitro than the component single treatments. Consistent with this, combination of non-toxic doses of puromycin with bortezomib significantly increased the antitumor activity of bortezomib in a mouse model of multiple myeloma. These results provide support for using combination treatments that disrupt the balance of PS and HSR to increase the therapeutic index of anticancer therapies.

Abstract 4882: Loss of p300 reduces miR-let-7c and enhances ER-positive breast cancer cell tumorigenicity

Abstract Histone deacetylase (HDAC) inhibitors have been extensively studied as potential anticancer treatments and several are currently in clinical trials. p300 is a nuclear histone acetyltransferase that is an important coordinator of multiple transcription-dependent cellular processes. HDAC inhibitors may directly and indirectly increase the activity of p300. The role of p300 is dependent on cell context but most or all of its biological activity stems from its ability to acetylate histones or non-histone nucleoproteins including p53, HSP) which regulate cell proliferation, differentiation and apoptosis. Whether p300 acts as a tumor suppressor or promoter is controversial, and its role in estrogen positive breast cancer remains to be defined, however, structural studies indicate that it has significant potential as a target for small molecule therapy. To address this issue, we have used a lentiviral approach to achieve 60-70% stable knockdown (KD) of p300 levels in estrogen receptor-positive breast cancer lines MCF-7 and BT 474 respectively. Growth curve and FACS analysis showed that loss of p300 shortened doubling times and increased G2 phase cells in both lines. Xenograft tumors formed from p300 KD cells were on average about three times larger at six weeks post implantation than control tumors (p<0.05). H&E and CD31 staining showed that p300 KD xenograft tumors have a more aggressive phenotype with increased vascularity. In searching for targets of p300 that might regulate its anti-tumor effect, we observed that the microRNA miR-let-7c was downregulated in p300-depleted MCF-7 and BT474 cells. Consistent with this, the miR-let-7c target N-Ras was up-regulated in both p300 KD cell lines and in p300 KD xenograft tumors, and adenovirus-mediated over-expression of p300 caused downregulation of N-RAS. Finally, gene expression analysis showed that loss of p300 resulted in upregulation of E-cadherin, laminin, collagen, IL-6, IFN, TGF-β and components of the Wnt signaling pathway, indicating a shift from epithelial to mesenchymal phenotype. Our data show that p300 loss or inactivation results in altered transcription patterns and suppression of anti-tumor microRNAs, including let-7, and may induce a more invasive and potentially metastatic phenotype. We conclude that p300 likely acts as a tumor suppressor in breast cancer and that activation of p300 may contribute to the anti-tumor effects of TSA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4882.

Isatis indigotica induces hepatoma cell apoptosis via caspase‐independent pathway in vitro and in vivo

We explored the chemotherapeutic potential of an Asian biennial herbaceous Cruciferae Isatis indigotica and investigated the mechanism by which it inhibits tumor growth in vitro and in vivo. Chloroform extract from dried leafs (CEDLI) had the strongest anti‐tumor activity among all fractions examined. CEDLI supplementation (0.26% for 2 mo) inhibited pre‐developed xenografted hepatoma growth by 43 % without inducing toxicity. Tumor immunohistochemical analysis showed a 33% decrease in proliferation and a 3.6‐fold increase in cell apoptosis in vivo. CEDLI induced sub‐G1 cell cycle arrest and apoptosis by activating p53, inducing Bax and reducing Bcl‐2, which in turn caused mitochondrial stress and led to the release of mitochondrial apoptosis inducing factor into the cytosol, followed by its translocation into the nuclear, resulting apoptosis in hepatoma cell‐lines. We provide novel in vivo evidence that Isatis indigotica has antitumor activity against liver tumorigenesis, and the chemotherapeutic activity of Isatis indigotica on hepatoma was associated with a distinguished caspase‐independent apoptotic pathway. Results from this study provide the molecular basis for Isatis indigotica as a potential anticancer treatment against human hepatoma.

Oncolytic herpes simplex virus 1 (HSV-1) vectors: Increasing treatment efficacy and range through strategic virus design

Viruses have long been considered potential anticancer treatments. Wild-type viruses have been tested as anticancer agents in clinical trials since the 1960s. The possibility of viral oncolysis as an alternate cancer therapy was transformed by the emergence of modern genetic engineering. The herpes simplex virus (HSV) family offers particular advantages for use as a viral oncolytic. The engineered vectors that make up oncolytic HSVs (oHSVs) have demonstrated remarkable safety in clinical trials, with some evidence of efficacy. The past decade has seen a focus on increasing the efficacy of oncolytic vectors by adding exogenous transgenes to enhance tumor destruction. The current paper describes the various strategies for engineering HSV for increased cancer tissue specificity and efficacy. Presented are the rationale, preclinical data and clinical data where available. This is meant to illustrate a basic framework for the development of a novel therapy meant to exploit the viral life cycle for the killing of cancer.

Abstract A67: Regulation of Eag1 expression by steroid hormones in cervical cancer cells

Abstract Introduction: Ether à go-go 1 (Eag1) channels have oncogenic properties. This channel is widely expressed in human tumors. It has been shown that inhibition of its expression and/or activity reduces cellular proliferation. Some cancer-associated factors up-regulate Eag1 expression; including estradiol and the E6 and E7 human papilloma virus (HPV) oncogenes. On the other hand, calcitriol (the most active metabolite of vitamin D) has shown antiproliferative properties and it has been considered as a potential anticancer treatment. In this work, we studied Eag1 regulation by estradiol, tamoxifen, ICI 182 780, genistein and calcitriol in the cervical cancer cell line SiHa, that express E6 an E7 HPV oncogenes. Methods: SiHa cells were cultivated with DMEM without phenol red and were treated with estradiol, tamoxifen, ICI 182 780, genistein or calcitriol during 48 hours. Total RNA was obtained with Trizol reagent, and cDNA was synthesized. Eag1 expression was evaluated by real time PCR. Eag1 expression was normalized against the housekeeping gene GAPDH. Using XTT colorimetric assay we evaluated the effect of calcitriol on SiHa cells proliferation. Results: Calcitriol decreased cell proliferation after 5 days of culture. We also identified estrogen receptors α and β and VDR (vitamin D receptor) proteins by Western blot. Estradiol, antiestrogens, genistein and calcitriol down-regulated Eag1 mRNA expression. Concomitant treatment of SiHa cells with estradiol and calcitriol seems to potentiate such Eag1 down-regulation. On the other hand, SiHa cells expressed higher levels of ERβ than ERα. Conclusions: Our data suggest that down regulation of Eag1 by calcitriol could be a new mechanism by which calcitriol reduce tumor cells proliferation. Together, estradiol, antiestrogens, genistein and calcitriol could be used as treatment for cancers that present ER pattern expression silimar to SiHa cells. Citation Information: Cancer Res 2009;69(23 Suppl):A67.

The PI3K Inhibitor ON 01910.Na Inhibits Critical Survival Pathways and Induces Apoptosis in CLL Cells through Induction of NOXA and BIM.

Abstract 412Chronic Lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a clinically heterogeneous disease characterized by the accumulation of mature B lymphocytes. Disease progression is driven by cell survival and proliferation signals provided by the tumor microenvironment through different receptors including the B-cell receptor, TNF, chemokine and cytokine receptors, and through other cell-cell interactions. ON 01910.Na (Onconova, Therapeutics) belongs to the family of styryl benzyl sulfones, a novel family of non-ATP competitive compounds that are currently under development as potential anticancer treatments. These compounds have shown activity in a variety of xenograft mouse models. Interestingly, it has recently been described that ON 01910.Na exerts potent antitumor activity against Mantle Cell Lymphoma (MCL) cells by inhibition of PI-3K/Akt/mTOR pathway and downregulation of Cyclin D1 translation. Here, we evaluated the cytotoxic effect of ON 01910.Na after 48 hours of in vitro exposure on PBMCs from CLL and normal donors by flow cytometry using AnnexinV-PE. We co-stained with CD3-APC and CD19-FITC to assess the effect on B and T cells separately. ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested (n=24), without affecting T-cell viability. Notably, ON 01910.Na did not affect B and T cells from normal donors at concentrations that induced apoptosis of CLL cells. Thirteen CLL samples were highly sensitive towards ON 01910.Na (mean B-cell IC50 0.71 μM) a concentration readily achieved in phase I clinical trials, eight samples were sensitive (mean B-cell IC50 1.38 μM) and three showed moderate sensitivity (mean B-cell IC50 4.26 μM). In contrast, the IC50 of T-cells was >8 μM in all samples. No significant differences were found in ON 01910.Na cytotoxicity against CLL expressing mutated (n=9, mean B-cell IC50 1.45 μM) or unmutated (n=15, mean B-cell IC50 1.34μM) IgVH sequences. ON 01910.Na was also effective against tumor cells having 17p (n=4, mean B-cell IC50 1.45 μM) or 11q deletions (n=3, mean B-cell IC50 0.96 μM). ON 01910.Na activated the mitochondrial apoptotic pathway, as shown by flow cytometry using conformation specific antibodies to detect activation of Bax and Bak, leading to mitochondrial depolarization (measured by mitotracker) and caspase-3 activation. ON 01910.Na also induced the upregulation of the proapoptotic BH3-only proteins Noxa and Bim and decreased expression of Mcl-1 at 4 hours, well before the onset of apoptosis. Inhibition of Noxa expression by retroviral transfection reduced the sensitivity to drug by 60%. ON 01910.Na induced reactive oxygen species (ROS) that may contribute to Noxa induction. Consistently ROS blockade using N-acetyl-cysteine reduced ON 01910.Na cytotoxic activity. To further delineate the biological processes underlying ON 01910.Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro for 4 and 10 hours with ON 01910.Na. GEP revealed the induction of Noxa and ATF3, consistent with an oxidative stress response. Noteworthy, Gene Set Enrichment Analysis (GSEA) of microarray data showed an induction of an AP-1 gene signature, (False Discovery Rate (FDR) = 0.10), validated by the nuclear accumulation of c-jun at 4 hours and a FOXO gene signature (FDR=0.13) that correlated with the upregulation of the proapoptotic BH3-only protein Bim. Strikingly, ON 01910.Na repressed B-cell receptor (FDR=0.023), NF-kB (FDR=0.084) and PI3K gene signatures. Consistent with inhibition of the BCR/PI3K/AKT axis, ON 01910.Na inhibited AKT phosphorylation by 65% after in vitro BCR activation of CLL cells. Our results identify ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. These data support the development of ON 01910.Na in CLL and a clinical trial has been initiated at our institution. Disclosures:No relevant conflicts of interest to declare.

Proceedings of the First International Conference on PEP005

The sap of Euphorbia peplus, commonly know as 'petty spurge', 'radium weed' or 'milkweed' has been used for centuries as a traditional treatment for skin conditions, including warts, corns and cancers of the skin. Documentation of its use by medical professionals to treat basal cell carcinoma (BCC) dates from the early 19 century. Individuals who participated in a 1988 survey of home treatments for cancer indicated the sap of E. peplus was an effective cure for actinic lesions leading the investigators to suggest that this potential utility should be further explored in controlled clinical trials. The fractionation of the sap E. peplus using solvents of varying polarity yielded several macrocyclic diterpenes, many of which were found to have cytotoxic activity or the ability to influence cellular differentiation. Ultimately, ingenol 3-angelate (I3A) of PEP005, emerged as a promising potential new anti-cancer treatment. Here we report the proceedings from the First International Conference on PEP005, covering the exciting potential of PEP005 as the therapeutic agent for the treatment of skin cancer, leukemia and bladder cancer.

VEGF Inhibitors in Cancer Therapy

Vascular endothelial growth factor (VEGF)-mediated angiogenesis is thought to play a critical role in tumor growth and metastasis. Consequently, anti-VEGF therapies are being actively investigated as potential anti-cancer treatments, either as alternatives or adjuncts to conventional chemo or radiation therapy. Among the techniques used to block the VEGF pathway are: 1) neutralizing monoclonal antibodies against VEGF or its receptor, 2) small molecule tyrosine kinase inhibitors of VEGF receptors, 3) soluble VEGF receptors which act as decoy receptors for VEGF, and 4) ribozymes which specifically target VEGF mRNA. Recent evidence from phase III clinical trials led to the approval of bevacizumab, an anti-VEGF monoclonal antibody, by the FDA as first line therapy in metastatic colorectal carcinoma in combination with other chemotherapeutic agents. However, may challenges still remain, and the role of anti-VEGF therapy in the treatment of other solid tumors remains to be elucidated. The aim of this article is to review the progress of clinical investigations involving VEGF inhibitors in the treatment of different types of solid tumors.

322. Syngeneic Cotton Rat Cancer Model for Replicating Adenoviral Vectors

Background: Oncolytic adenoviruses are under study as a potential anticancer treatment. Their preclinical antitumor activity; however, has not been effectively translated to the clinic. A contributing factor may be the clinical relevance of immunodeficient mouse tumor xenograft models used to study these vectors. While these demonstrate viral replication in the xenograft, they are limited by the lack of natural host immune responses to the virus, associated toxicity, dissemination and systemic viral replication. Cotton rats (Sigmodon hispidus) are semi-permissive for the replication of human adenovirus and have been used to study these vectors. Until recently this model lacked a transplantable tumor to assess oncolytic activity. We characterized three syngeneic transplantable cotton rat tumor cells lines that support adenovirus replication and oncolysis in this immunocompetent rodent model. Methods: Three tumor cell lines: CCRT, LCRT and VCRT were derived from a spontaneously arising osteosarcoma, and fibrosarcomas of the breast and jaw in animals from an inbred cotton rat colony. The ability of the cells to be infected with adenovirus was examined using an adenovirus expressing green fluorescent protein (Ad.GFP). Viral burst assays were used to determine titers of infective adenovirus produced by each cell line at 4 and 72 hours following infection with wild type adenovirus type-5 (Ad5). Transmission electron microscopy (TEM) was used to visualize the relative number of virions at each time point. We also evaluated replication and oncolysis of the cell lines in vivo. Results: Using Ad.GFP, the cell lines were readily infected and demonstrated GFP expression comparable to human A549 cells. All cell lines showed significant higher (P<0.05) intracellular titers of adenovirus and in supernatants 72 hours after infection compared to 4 hours after infection with Ad5 indicating viral replication and increased number of functional virions. In addition, all the cell lines exhibited greater expression of adenoviral hexon protein at 72 hours than at 4 hours after infection, indicating late transcriptional unit gene activation. The cotton rat cell lines formed tumors in cotton rats when injected subcutaneously. On days 5 and 8, PCR demonstrated 2 to 3-log greater number of Ad5 genome copies in tumors injected with Ad5 compared to those injected with a non- replicating adenovirus (Ad.null). This was consistent for CCRT, LCRT and VCRT. Ad5 slowed tumor growth in cotton rats subcutaneously injected with CCRT and VCRT cells. Despite in vitro oncolysis, no anti-tumor effect was seen in the LCRT tumors with the virus rapidly cleared from the tumor. Conclusions: CCRT, LCRT and VCRT, three novel transplantable cotton rat tumor-derived cell lines demonstrate replication of Ad5 both in vitro and in vivo. In addition, CCRT and VCRT demonstrated in vivo oncolysis. The varied antitumor effect in this model mirrors the results of human clinical trials highlighting the potential relevance of these cotton rat tumor models for assessing replication competent adenovirus vectors.

Role of Intracellular Glutathione in Cell Sensitivity to the Apoptosis Induced by Tumor Necrosis Factor α–Related Apoptosis-Inducing Ligand/Anticancer Drug Combinations

We have recently shown that combination of tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) with anticancer drugs induced an apoptotic cell death pathway involving both caspases and mitochondria. The present work further explores the role of intracellular reduced glutathione (GSH) level in cell sensitivity to this cell death pathway. Intracellular GSH level was measured by high-performance liquid chromatography. Cell death was detected by immunofluorescence after Hoechst 33342/propidium iodide staining. Reactive oxygen species production was evaluated by flow cytometry after dihydroethidium probe labeling. Western blot analysis was done to study stress-activated protein kinase/c-jun NH(2)-terminal kinase (SAPK/JNK) phosphorylation. The Student's t test was used to determine significance of the results. Three to six experiments were done. GSH depletion enhanced apoptosis induced by TRAIL/cisplatin (CDDP) or TRAIL/5-fluorouracil (5-FU) combinations in both human HT29 colon carcinoma and HepG2 hepatocarcinoma cells, whereas it enhanced cytotoxicity induced only by TRAIL/CDDP in human primary hepatocytes. Our results further suggested that GSH depletion enhanced SAPK/JNK phosphorylation upon TRAIL/5-FU exposure and likely reduced the detoxification mechanisms of CDDP in HT29 cells. Resistance of Bcl-2-expressing HT29 and HepG2 cells to combined treatment was not overcome by GSH depletion, thus indicating that Bcl-2-mediated antiapoptotic effect occurs independently of intracellular GSH level. GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment by TRAIL/anticancer drug combinations. Furthermore, TRAIL/5-FU combination might be a potential anticancer treatment of human tumors, being ineffective on human primary hepatocytes and thus could be of interest in clinical cancer treatment. Nevertheless, Bcl-2 expression remains an important resistance factor.

Uncovering the Hidden Potential of Intravenous Immunoglobulin as an Anticancer Therapy

Intravenous immunoglobulin (IVIg) is a safe preparation made from human plasma. The original concept of IVIg as an anticancer drug was built up over the years, after numeral reports were accumulated indicating cancer regressions after IVIg administration. Because IVIg is basically an established remedy for immunodeficiencies and several autoimmune diseases, the association between beneficial effects in cancer patients after IVIg was first seen in patients who had both cancer and autoimmune or immunodeficiency diseases. Interestingly, cancer and autoimmunity share several common features, which together enhance the notion of using IVIg to treat cancer. Several studies tested the broad range of the antimetastatic effects of IVIg. IVIg was found to operate in many different and complex ways, among them (a) induction of interleukin-12 secretion, leading to natural-killer-cell activation; (b) inhibition of matrix metalloproteinase-9 mRNA expression; (c) suppression of tumor cell growth; (d) hindrance of nuclear factor kappaB activation and IkappaB degradation; and (e) G1 cell-cycle arrest. In conclusion, IVIg is a potential anticancer treatment for several reasons: (a) the bidirectional relationship between cancer and autoimmunity; (b) the apparent association between cancer regression and IVIg administration; (c) a variety of anticancer effects of IVIg observed; and (d) IVIg is considered to be a safe preparation with minimal side effects. Obviously, prospective controlled studies that will establish the antitumor effects of IVIg are needed.