Pochi R. Subbarayan

Tissue invasion and metastasis: Molecular, biological and clinical perspectives

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), β-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-β), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.

Modulation of thymidilate synthase and p53 expression by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with 5FU or Raltitrexed

Despite the introduction of several novel anticancer agents almost 50% of colorectal cancer (CRC) patients die for cancer suggesting the necessity of new therapeutical approaches. In this study we demonstrated that the HDAC inhibitor vorinostat exerted potent antiproliferative effect in a panel of mut- and wt-p53 human CRC cell lines. Moreover, in combination with 5-fluorouracil modulated by folinic acid (5FU-FA) or with Raltitrexed (RTX), both commonly used in the treatment of this disease, it showed a clear schedule-dependent synergistic antiproliferative interaction as demonstrated by calculating combination indexes. Only simultaneous, or 24 hrs pretreatment with vorinostat followed by either agent, produced synergistic effect paralleled by evident cell cycle perturbations with major S-phase arrest. Moreover, we provided for the first time evidences that vorinostat can overcome resistance to both 5FU and RTX. Down-modulation of TS protein induced by vorinostat within 24 hrs, represented a key factor in enhancing the effects of both drugs in sensitive as well as resistant tumor cells. Furthermore, p53, whose wild-type expression is critical for sensitivity to 5FU and RTX, was up-regulated by vorinostat in wt- and down-regulated in mut-p53 cells, suggesting an additional mechanism of the antiproliferative synergistic interactions observed. Overall these data add new insights in the mechanism of vorinostat antitumor effect and suggested that the association of vorinostat plus 5FU-FA and/or RTX should be clinically explored.

A Phase I Study of 5-Fluorouracil/Leucovorin and Arsenic Trioxide for Patients with Refractory/Relapsed Colorectal Carcinoma

Purpose: This Phase I study was designed to determine a safe combination dose of 5-fluorouracil (5-FU) and arsenic trioxide (ATO) to treat 5-FU–resistant relapsed/refractory colorectal cancer patients. We studied the effect of ATO in the downregulation of thymidylate synthase (TS) in peripheral blood mononuclear cells and in tumor biopsies. Experimental Design: ATO was administered for 5 consecutive days during the first week and twice during weeks 2 to 3 and once on week 4. 5-FU/leucovorin (LV) was administered on days 8, 15, and 22. A modified accelerated titration design was used. 5-FU was dose escalated first followed by a planned dose increase for ATO. Results: No dose-limiting toxicities were seen in seven patients who received 0.15 mg/kg ATO; grade 3 toxicities were as follows: neutropenia 1, diarrhea 1, and bowel obstruction 1. In patients receiving 0.20 mg/kg ATO, grade 3 toxicities were QTc prolongation 1, fatigue 4, alkaline phosphatase elevation 2, diarrhea 2, and peripheral edema 1. TS gene expression in peripheral blood mononuclear cell decreased in all patients. Eight tumors were biopsied, four showed TS downregulation, three showed upregulations, and one did not change. Estimated median progression-free survival and overall survival were 3.1 and 13.9 months, respectively. In patients who showed TS increase or no change versus TS reduction, estimated median progression-free survival was 2.6 versus 7.9 months (P = 0.188) and overall survival was 8.6 versus 11.7 months (P = 0.44), respectively. Conclusions: Thus, we determined 0.20 mg/kg ATO, 2,600 mg/m2 5-FU, and 500 mg/m2 leucovorin (LV) to be the recommended phase II dose. Clin Cancer Res; 16(11); 3019–27. ©2010 AACR.

Arsenic trioxide/ascorbic acid therapy in patients with refractory metastatic colorectal carcinoma: A clinical experience

Arsenic trioxide (As2O3) has demonstrated effectiveness in treating acute promyelocytic leukemia (APL). Therefore the FDA has approved it to treat APL. In patients with refractory metastatic colorectal carcinoma (CRC), we assessed the efficacy and toxicity of As2O3/AA (ascorbic acid) as the outcome of this trial. Five patients with refractory metastatic CRC who failed all previous standard chemotherapy were enrolled in this study. They were treated with 0.25 mg/kg body weight/day As2O3 and 1000 mg/day of ascorbic acid for 5 days a week for 5 weeks. Each treatment cycle extended for 7 weeks with 5 weeks of treatment and 2 weeks of rest. All the patients developed moderate to severe toxic side effects to arsenic trioxide/AA therapy and therefore the study was discontinued. No CR (complete remission) or PR (partial remission) was observed. CT scans demonstrated stable or progressive disease. Three of the five patients died within 2 to 5 months after cessation of the therapy. None of the deaths could be related to this clinical trial. Two years of follow-up study showed that two patients were alive with stable disease. Under the current treatment regimen all patients developed moderate to severe side effects with no clinically measurable activity. As an alternate, efforts may be made to reduce the dose and arsenic trioxide may be combined with other standard regimen in reversing the chemo resistance.

Anti-proliferative and anti-cancer properties of Achyranthes aspera: specific inhibitory activity against pancreatic cancer cells.

AIMS OF THE STUDY Achyranthes aspera (Family: Amaranthacea) is a medicinal plant used as an anti-cancer agent in ayurveda, a traditional system of medicine practiced in subcontinental India. The aim of the study was to systematically investigate the anti-proliferative properties of Achyranthes aspera leaves extracted in methanol (LE) on human cancer cells in vitro. MATERIALS AND METHODS We tested time, dose dependent and specific anti-proliferative activity of LE by clonogenic cell survival assay on human cancer and normal epithelial cell lines in vitro. We further investigated its effect on the expression of metastatic and angiogenic genes by real time polymerase chain reaction. On silica gel column, we carried out initial fractionation analysis. RESULTS LE exhibited time and dose dependent cytotoxicity on several tumor cells. Compared to cancer cells of colon, breast, lung and prostate origin, pancreatic cancer cells were significantly more sensitive to LE. Preliminary mechanistic studies suggested that LE selectively suppressed the transcription of metalloproteases (MMP-1 and -2), inhibitors of MMPs (TIMP-2) and angiogenic factors (VEGF-A and VEGF-B). Fractionation of LE on methanol equilibrated silica gel column resolved into three fractions of which fraction (F 3) was found to be enriched with anti-proliferative activity. CONCLUSION Methanolic extract of Achyranthes aspera contains potent anti-proliferative compound with specific activity against pancreatic cancer. Further studies are needed to confirm the in vivo anti-tumorigenicity and subsequent chemical characterization of the active molecule(s).

In the War Against Solid Tumors Arsenic Trioxide Need Partners

In the past decade, the therapeutic potential of arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL) was recognized. This encouraged other investigators to test the efficacy of ATO in the management of other hematological and solid tumor malignancies. Notably, as a single agent, arsenic trioxide did not benefit patients diagnosed with solid tumors. However, when it was combined with other agents, treatment benefit emerged. In this article, we have summarized the outcome of clinical trials that used arsenic trioxide as a single agent as well as in combination settings in patients diagnosed with solid tumors. We have also reviewed possible additional mechanisms by which ATO may be useful as a chemosensitizer in combination therapy. We hope that our review will encourage clinical investigators to rationally combine ATO with additional chemotherapeutic agents in treating patients diagnosed with solid tumors.

Achyranthes aspera (Apamarg) leaf extract inhibits human pancreatic tumor growth in athymic mice by apoptosis

ETHNOPHARMACOLOGICAL RELEVANCE Achyranthes aspera (Family Amaranthacea) is used for cancer therapy by ayurvedic medical practitioners in India. However, due to the non formal nature of its use, there are no systematic studies validating its medicinal properties. Thus, its utility as an anti cancer agent remains anecdotal. Earlier, we demonstrated A. aspera to exhibit time and dose-dependent preferential cytotoxicity to cultured human pancreatic cancer cells. In this report we validate in vivo anti tumor properties of A. aspera. MATERIALS AND METHODS The in vivo anti tumor activity of leaf extract (LE) was tested by intraperitoneal (IP) injections into athymic mice harboring human pancreatic tumor subcutaneous xenograft. Toxicity was monitored by recording changes in behavioral, histological, hematological and body weight parameters. RESULTS Dosing LE to athymic mice by I.P. injection for 32 days showed no adverse reactions in treated mice. Compared to the control set, IP administration of LE to tumor bearing mice significantly reduced both tumor weight and volume. Gene expression analysis using Real time PCR methods revealed that LE significantly induced caspase-3 mRNA (p<0.001) and suppressed expression of the pro survival kinase Akt-1 (p<0.05). TUNEL assay and immunohistochemistry confirmed apoptosis induction by activation of caspase-3 and inhibiting Akt phosphorylation in treated sets. These results are in agreement with RT PCR data. CONCLUSION Taken together, these data suggest A. aspera to have potent anti cancer property.

Hypoxia signature of splice forms of tryptophanyl-tRNA synthetase marks pancreatic cancer cells with distinct metastatic abilities.

Objectives: Pancreatic cancer is one of most deadly because of its aggressive growth and high metastatic ability that correlates with intratumoral hypoxia. Earlier diagnosis and prognosis marker of pancreatic cancer is not yet available. In colorectal cancer, protein biosynthesis enzyme, tryptophanyl-tRNA synthetase (TrpRS), is up-regulated in good-prognosis tumors and down-regulated in metastatic poor-prognosis tumors. Tryptophanyl-tRNA synthetase status in pancreatic cancer is unknown. To correlate metastatic ability with hypoxia and TrpRS as a possible prognostic marker, we examined mRNA and protein expression in 2 human pancreatic cancer cell lines with different metastatic abilities and TrpRS levels using our site-specific monoclonal antibodies directed to conformation-dependent epitopes on pancreatic TrpRS. Methods: Pancreatic MIAPaCa-2, Panc-1, cervical HeLa, and prostate cancer PC-3 cells were cultivated under normoxia or in hypoxic chamber. Expression of full-length TrpRS, antiangiogenic TrpRS, cyclin B1, hypoxia-inducible factor 1&agr;, and Glut-1 was determined with reverse transcriptase-polymerase chain reaction, immunoblotting, and immunocytochemistry. Results: We demonstrate that hypoxia regulates differentially TrpRS splice forms. Pronounced down-regulation of full-length TrpRS by hypoxia is concomitant with higher metastatic ability. Conclusions: Tryptophanyl-tRNA synthetase down-regulation by hypoxia may be a factor responsible for low TrpRS in tumors with high metastatic ability. Tryptophanyl-tRNA synthetase recognizability is important for pancreatic cancer prognosis and as a new target for metastasis treatment.

5-Fluorouracil/Leucovorin and arsenic trioxide for patients with refractory/relapsed colorectal carcinoma: A clinical experience

5-Fluorouracil (5-FU) has been the most active che-motherapeutic agent for metastatic colorectal cancer (CRC), and the mainstay of treatment for the last 40 years. The reported response rate to single-agent 5-FU ranges from 10 – 30%, yet the median survival remains approximately 12 months [1]. Over the past decade, new agents oxaliplatin, irinotecan, Bevacizumab, Pani-tumumab, and Cetuximab, have been introduced into clinical practice. However, it is only when these agents are combined with 5-FU that signifi cant improvements in response rates and median survival are seen [1 – 5]. Consequently, development of 5-FU resistance in CRC patients presents a major challenge. Strategies to reverse cellular resistance to 5-FU may create a treatment choice for approximately 16,000 patients annually who otherwise have no alternative options. We recently dem-onstrated

Arsenic Attenuates GLI Signaling, Increasing or Decreasing its Transcriptional Program in a Context Dependent Manner

The metalloid arsenic is a worldwide environmental toxicant, exposure to which is associated with many adverse outcomes. Arsenic is also an effective therapeutic agent in certain disease settings. Arsenic was recently shown to regulate the activity of the Hedgehog (HH) signal transduction pathway, and this regulation of HH signaling was proposed to be responsible for a subset of arsenic’s biologic effects. Surprisingly, these separate reports proposed contradictory activities for arsenic, as either an agonist or antagonist of HH signaling. Here we provide in vitro and in vivo evidence that arsenic acts as a modulator of the activity of the HH effector protein glioma-associated oncogene family zinc finger (GLI), activating or inhibiting GLI activity in a context-dependent manner. This arsenic-induced modulation of HH signaling is observed in cultured cells, patients with colorectal cancer who have received arsenic-based therapy, and a mouse colorectal cancer xenograft model. Our results show that arsenic activates GLI signaling when the intrinsic GLI activity is low but inhibits signaling in the presence of high-level GLI activity. Furthermore, we show that this modulation occurs downstream of primary cilia, evidenced by experiments in suppressor of fused homolog (SUFU) deficient cells. Combining our findings with previous reports, we present an inclusive model in which arsenic plays dual roles in GLI signaling modulation: when GLIs are primarily in their repressor form, arsenic antagonizes their repression capacity, leading to low-level GLI activation, but when GLIs are primarily in their activator form, arsenic attenuates their activity.