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CLL Global Research Foundation

Date: March 18, 2005

by Chaya Venkat

New Resources and Initiatives

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Some of you may be aware that a new research foundation has been established. The CLL Global Research Foundation (CGRF) is under the leadership of Dr. Michael Keating of M. D. Anderson. This is welcome news, several of our best known experts are also participating in this foundation. You can learn more about it by clicking on the link www.cllglobal.org.  We can use all the help we get, as we search for new therapy options and cutting edge clinical trials that will improve survival odds for our patients.

We have recently reached our target for fund-raising for Project Alpha (High Fives!) and it is time for us to look around and see what is next on the horizon. It is a sad fact of life that drugs that do not have a huge profit potential often do not get serious attention - these are the orphans that no one wants to adopt. Expensive monoclonal antibodies and the latest chemotherapy drugs that may become block-buster brand names have strong support from their pharmaceutical company owners. But what about the off-patent and inexpensive drugs such as valproic acid (Epigenetics) and over the counter drugs such as EGCG from green tea (Do You Like Drinking Green Tea?)? No one stands to make a killing selling this type of drug - no one owns the intellectual property rights to them and therefore no one has incentive in pushing for well designed clinical trials to examine the potential value of these drugs. No one, except us patients. We stand to gain the most of all, if low toxicity and inexpensive drugs that may have good potential are tested in rigorous clinical trials. The clinical trials are a must: there is way too much hype and misinformation for us to be able to judge the true value of such drugs in the absence of rigorous testing.

Not long ago we published a review on the potential health benefits of Omega-3 Poly Unsaturated Fatty Acids such as EPA (eicosapentanoic acid) and DHA (docosahexanoic acid) found in cold water fish oils. There is gathering evidence that our modern eating and farming habits have seriously skewed the ratio of Omega-3 to Omega-6 polyunsaturated fatty acids in our diet, and this may be contributing to some of the rapid increase in autoimmune disease, inflammation, heart disease, cancer, etc. I will not go into the details here. You can read the earlier article if you wish to refresh your memory.

The interesting thing is that fish oil components such as EPA and DHA profoundly influence the lipid membranes surrounding each cell in your body. They have a huge impact on the ease with which signals are transmitted in and out of the cell, including the all important "suicide" signals to cancer cells. One of the mechanisms involved is the ROS (Reactive Oxygen Species) pathway. The role of the ROS pathway in CLL has been explored extensively by researchers at M. D. Anderson (Mitochondrial Approaches). It may also interest you to know that everyone's favorite monoclonal, Rituxan, also depends on favorable functioning of the lipid membrane, with so called "raft-formation" that segregates the CD20 markers and corrals them into close proximity (Sons of Rituxan and Campath).

Hoping to put together the pieces of this puzzle, I contacted Dr. Keating on February 15, 2005. The idea is to see if we can get Dr. Keating's interest in ROS-mediated cell kill combined with a Rituxan based-therapy that includes EPA/DHA to make the cell membrane sensitive to ROS signals as well as Rituxan signals. A quote from my letter, "Combination of the CD20 lineage specific targeting capabilities of Rituxan with immune system adjuvants (GM-CSF or G-CSF), plus long chain omega-3 PUFA would make for a very interesting clinical trial, from our perspective". You can read below the full text of my letter to Dr. Keating, as well as the references I sent him in support of this combination.

Dr. Keating's response to my letter was encouraging, we will wait to see if this concept develops into a full fledged clinical trial at the CGRF. As always, your comments and input are most welcome.

Correspondence

February 15, 2005

Dear Dr. Keating:

I have been following with interest the launch of the CLL Global Research Foundation (CGRF) under your able leadership. I am particularly pleased that your website lists the EGCG clinical trial among the first recipients of funding from the CGRF. As you may know, this trial is of great interest to CLL Topics, we are one of the sponsors of this trial at the Mayo Clinic. Your support for this potentially valuable low toxicity and patient friendly approach is much appreciated by our patient community.

Along the similar lines, we would like to suggest another clinical trial for funding by the CGRF, one that may be of particular interest to you and M. D. Anderson since much of the pivotal work defining the scientific underpinnings and relevance to CLL was done by your group. I am referring to your several recent publications on the subject of using mitochondrial respiratory defects as a novel target for CLL therapy. We reviewed some of your papers in a recent article on our website, here is the link in case you wish to glance through our article: Target Mitochondrion.

In the interest of brevity, I would like to bullet point the logic of the clinical trial we would like to suggest for your consideration. I have attached some of the relevant abstracts below for convenience.

  • CLL cells are under oxidative stress, this is particularly true of late stage patients and those that have been through prior therapy, since mitochondrial defects seem to accumulate over time and after chemotherapy. As you point out, the "Warburg effect" makes for an attractive target and promising therapeutic strategy.
  • Your papers have demonstrated the potential value of drugs such as arsenic trioxide, as a potent ROS generator. Others have suggested Adaphostin, Fenretinide, OSU03012 etc, all of which are also ROS generators. Interestingly, in keeping with the Warburg effect, both Adaphostin and Fenretinide are reported to work better with later stage patients, and those that have been through prior therapy.
  • More recently it has been suggested that one of the mechanisms by which Rituxan exerts cytotoxicity is through ROS mediated mechanisms. Formation of lipid rafts and segregation of CD20 into these lipid rafts, enhanced cross linking and signaling are thought to play a part in the ROS effects of Rituxan. This may explain why combination of Rituxan with Fenretinide, a known ROS generator, has synergistic effects in CLL cell kill. I understand the Hutch is planning a clinical trial (Phase I / II) for CLL patients using a combination of Rituxan and Fenretinide.
  • It has been reported in hundreds of papers that the long chain omega-3 fatty acids EPA and DHA found in fish oil have very substantial impact on the composition and fluidity of cellular membranes, as well as dramatic impact on the formation of lipid rafts. Long chain omega-3 PUFA are also thought to exert ROS mediated cytotoxic culling of cancer cells, with no deleterious effects on normal tissue. A higher consumption of dietary omega-3 acids (as opposed to the omega-6 fatty acids) is also thought to down regulate COX mediated inflammatory cytokines and prostaglandins.
  • A recent paper has demonstrated very substantial synergy between DHA and arsenic trioxide in the killing of leukemic cells. There have been similar reports describing the synergy between long chain omega-3 fatty acids and conventional chemotherapy agents such as doxorubicin, epirubicin and 5-fluorouracil. Fish oil was shown to reduce the cardiac toxicity of these potent ROS generators, while at the same time enhancing the cytotoxicity of these chemotherapy agents in solid cancers.
  • Omega-3 long chain fatty acids such as DHA and EPA have been reported to down regulate the anti-apoptotic protein Bcl-2, as well as facilitate TRAIL signaling. As you are aware, most CLL patients (especially the poor prognostic cases) seem to have elevated expression of Bcl-2, and show poor response to TRAIL mediated cell kill.

I understand M.D. Anderson and several other CRC centers are conducting a clinical trial using a combination of Rituxan with growth factors (GM-CSF or G-CSF) as a way of improving ADCC mechanism of cell kill. Based on the logic of the "Warburg effect" you have highlighted, we would like to suggest further addition of long chain omega-3 PUFA (fish oil) to the mix, to take advantage of mitochondrial pathways of apoptosis as well. Rituxan therapy continues to be very popular with patients, because of its low toxicity, lower immune suppression, and relatively mild infusion related side effects. But as you know response rates to single agent Rituxan therapy even in chemo naive CLL patients is distressingly low. Any improvement in the response rates of Rituxan by the addition of immune system modulating agents such as growth factors and dietary supplements such as fish oil capsules will be of great value.

CLL Topics has grown over the last two years. Our website www.clltopics.org attracted more than 19,000 visitors last month, and the traffic keeps growing month after month. This has become a labor of love for me and the many volunteers that contribute to make our website a dynamic resource for CLL patients all over the world. Part of our charter mission is to work with research groups that are doing cutting edge research, bringing new and innovative therapy options to the patient bedside. Combination of the CD20 lineage specific targeting capabilities of Rituxan with immune system adjuvants (GM-CSF or G-CSF), plus long chain omega-3 PUFA would make for a very interesting clinical trial, from our perspective. We would like to urge funding of such a trial by the CGRF, to be conducted at M. D. Anderson and any of the other CRC sites that may be interested. CLL Topics would be honored to support such an effort, help in any way we can with the patient recruitment as well as publicize the trial in the patient community.

Thank you for your time, I look forward to hearing from you. My contact information is given below.

Best regards,

Chaya Venkat
President, CLL Topics Inc.
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Abstracts:

CLL and ROS

Cancer Res. 2005 Jan 15;65(2):613-21.

Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia.

Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, Keating MJ, Huang P.

Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX

Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associated with resistance to therapeutic agents. Here, we report that inhibition of glycolysis severely depletes ATP in cancer cells, especially in clones of cancer cells with mitochondrial respiration defects, and leads to rapid dephosphorylation of the glycolysis-apoptosis integrating molecule BAD at Ser(112), relocalization of BAX to mitochondria, and massive cell death. Importantly, inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment in which the cancer cells exhibit high glycolytic activity and decreased sensitivity to common anticancer agents. Depletion of ATP by glycolytic inhibition also potently induced apoptosis in multidrug-resistant cells, suggesting that deprivation of cellular energy supply may be an effective way to overcome multidrug resistance. Our study shows a promising therapeutic strategy to effectively kill cancer cells and overcome drug resistance. Because the Warburg effect and hypoxia are frequently seen in human cancers, these findings may have broad clinical implications.

PMID: 15695406
____________

Cancer Chemother Pharmacol. 2004 Mar;53(3):209-19. Epub 2003 Nov 11.

Intrinsic oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity.

Hileman EO, Liu J, Albitar M, Keating MJ, Huang P.

Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX

PURPOSE: Therapeutic selectivity is one of the most important considerations in cancer chemotherapy. The design of therapeutic strategies to preferentially kill malignant cells while minimizing harmful effects to normal cells depends on our understanding of the biological differences between cancer and normal cells. We have previously demonstrated that certain agents generating reactive oxygen species (ROS) such as 2-methoxyestradiol (2-ME) preferentially kill human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The purpose of the current study was to investigate the biochemical basis for such selective anticancer activity.
METHODS: Flow cytometric analyses were utilized to measure intracellular O(2)(-) levels and apoptosis. MTT assays were used as indicators of cellular viability. Western blot analysis was used to measure the expression of antioxidant enzymes in cancer and normal cells.
RESULTS: Malignant cells in general are more active than normal cells in the production of O(2)(-), are under intrinsic oxidative stress, and thus are more vulnerable to damage by ROS-generating agents. The intrinsic oxidative stress in cancer cells was associated with the upregulation of SOD and catalase protein expression, likely as a mechanism to tolerate increased ROS stress. The increase in SOD and catalase expression was observed both in primary human leukemia cells and in primary ovarian cancer cells. Both malignant cell types were more sensitive to 2-ME than their normal counterparts, as demonstrated by the significant accumulation of O(2)(-) and subsequent apoptosis. The administration of ROS scavengers in combination with 2-ME prevented the accumulation of O(2)(-) and abrogated apoptosis induction.
CONCLUSIONS: O(2)(-) is an important mediator of 2-ME-induced apoptosis. The increased oxidative stress in cancer cells forces these cells to rely more on antioxidant enzymes such as SOD for O(2)(-) elimination, thus making the malignant cells more vulnerable to SOD inhibition than normal cells.

PMID: 14610616
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Blood. 2003 May 15;101(10):4098-104. Epub 2003 Jan 16

Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents.

Zhou Y, Hileman EO, Plunkett W, Keating MJ, Huang P.

Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX

2-Methoxyestradiol (2-ME), a new anticancer agent currently in clinical trials, has been demonstrated to inhibit superoxide dismutase (SOD) and to induce apoptosis in leukemia cells through a free radical-mediated mechanism. Because the accumulation of superoxide (O(2)-) by inhibition of SOD depends on the cellular generation of O(2)-, we hypothesized that the endogenous production of superoxide may be a critical factor that affects the antileukemia activity of 2-ME. In the present study, we investigated the relationship between cellular O(2)- contents and the cytotoxic activity of 2-ME in primary leukemia cells from 50 patients with chronic lymphocytic leukemia (CLL). Quantitation of O(2)- revealed that the basal cellular O(2)- contents are heterogeneous among patients with CLL. The O(2)- levels were significantly higher in CLL cells from patients with prior chemotherapy. CLL cells with higher basal O(2)- contents were more sensitive to 2-ME in vitro than those with lower O(2)- contents. There was a significant correlation between the 2-ME-induced O(2)- increase and the loss of cell viability. Importantly, addition of arsenic trioxide, a compound capable of causing reactive oxygen species (ROS) generation, significantly enhanced the activity of 2-ME, even in the CLL cells that were resistant to 2-ME alone. These results suggest that the cellular generation of O(2)- plays an important role in the cytotoxic action of 2-ME and that it is possible to use exogenous ROS-producing agents such as arsenic trioxide in combination with 2-ME to enhance the antileukemia activity and to overcome drug resistance. Such a combination strategy may have potential clinical applications.

PMID: 12531810
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J Biol Chem. 2003 Sep 26;278(39):37832-9. Epub 2003 Jul 9.

Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism.

Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W, Keating MJ, Huang P.

Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX

Cancer cells are under intrinsic increased oxidative stress and vulnerable to free radical-induced apoptosis. Here, we report a strategy to hinder mitochondrial electron transport and increase superoxide O2. radical generation in human leukemia cells as a novel mechanism to enhance apoptosis induced by anticancer agents. This strategy was first tested in a proof-of-principle study using rotenone, a specific inhibitor of mitochondrial electron transport complex I. Partial inhibition of mitochondrial respiration enhances electron leakage from the transport chain, leading to an increase in O2. generation and sensitization of the leukemia cells to anticancer agents whose action involve free radical generation. Using leukemia cells with genetic alterations in mitochondrial DNA and biochemical approaches, we further demonstrated that As2O3, a clinically active anti-leukemia agent, inhibits mitochondrial respiratory function, increases free radical generation, and enhances the activity of another O2.-generating agent against cultured leukemia cells and primary leukemia cells isolated from patients. Our study shows that interfering mitochondrial respiration is a novel mechanism by which As2O3 increases generation of free radicals. This novel mechanism of action provides a biochemical basis for developing new drug combination strategies using As2O3 to enhance the activity of anticancer agents by promoting generation of free radicals.

PMID: 12853461
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Curr Pharm Des. 2004;10(8):841-53.

Cytotoxic effects of B lymphocytes mediated by reactive oxygen species.

Villamor N, Montserrat E, Colomer D.

Hematopathology Unit and Department of Hematology, Institut Clinic de Malaties Hematologiques i Oncologiques (ICMHO), Hospital Clinic, Institut d'Investigacions Biomediques Ausgust Pi iSunyer (IDIBAPS), Barcelona, Spain.

Reactive oxygen species (ROS) are produced in all mammalian cells as a result of norman cellular metabolism and due to the activation of oxidant-producing enzymes in response to exogenous stimuli. The balance between ROS production and antioxidant defenses determines the degree of oxidative stress. Generation of ROS has been associated with cell signaling, stress responses, cell proliferation, aging and cancer development. The ability of ROS to induce cellular damage and to cause cell death opens the possibility to exploit this property in the treatment of cancer through a fee radical-mediated mechanism.

PMID: 15040354
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ROS Pathways, DHA & Synergy with Chemotherapy Drugs

Int J Cancer. 2004 Nov 20;112(4):707-12.

Enhancement of arsenic trioxide-mediated apoptosis using docosahexaenoic acid in arsenic trioxide-resistant solid tumor cells.

Baumgartner M, Sturlan S, Roth E, Wessner B, Bachleitner-Hofmann T.

Surgical Research Laboratories, Department of Surgery, Medical University of Vienna, Vienna, Austria.

It has been shown that the polyunsaturated fatty acid docosahexaenoic acid (DHA) can sensitize various tumor cells to reactive oxygen species (ROS)-inducing anticancer agents. Recently, we demonstrated that DHA also enhances the apoptotic effect of clinically achievable concentrations (1-2 microM) of arsenic trioxide (As2O3) in several As2O3-resistant human leukemic cell lines via a ROS-dependent mechanism. The aim of the present study was to evaluate whether this combined effect of As2O3 and DHA is also applicable to As2O3-resistant solid tumor cells. We have tested 12 different tumor cell lines, including MDA-MB-468, SK-BR-3, MCF-7 (breast cancer), ES-2, SKOV-3 (ovarian cancer), HT-29, SW-620, LS-174T (colon cancer), PC-3 (prostate cancer), HeLa (cervical cancer), PANC-1 (pancreatic cancer) and one primary melanoma cell line. With the exception of MDA-MB-468 and ES-2, all cells were resistant to treatment with either As2O3 or DHA alone. However, combined treatment with As2O3 and DHA significantly reduced viability in 7 of the 10 As2O3-resistant solid tumors tested. The cytotoxic effect of As2O3 and DHA was associated with the induction of apoptosis and a concomitant increase of intracellular lipid peroxidation products. Importantly, the combined effect of As2O3 and DHA was selectively toxic for malignant cells since no cytotoxic effect was observed in normal skin fibroblasts, human microvascular endothelial cells and peripheral blood mononuclear cells derived from healthy donors. Our data indicate that DHA may help to extend the therapeutic spectrum of As2O3 in the treatment of solid tumors since it may overcome de novo or acquired resistance to As2O3.

PMID: 15382055
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Clin Cancer Res. 2001 Jul;7(7):2041-9.

Three percent dietary fish oil concentrate increased efficacy of doxorubicin against MDA-MB 231 breast cancer xenografts.

Hardman WE, Avula CP, Fernandes G, Cameron IL.

Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX.

Omega 3 polyunsaturated fatty acids (the type of fat found in fish oil) have been used to kill or slow the growth of cancer cells in culture and in animal models and to increase the effectiveness of cancer chemotherapeutic drugs. An AIN-76 diet containing 5% corn oil (CO) was modified to contain 3% w/w fish oil concentrate (FOC) and 2% CO to test whether a clinically applicable amount of FOC is beneficial during doxorubicin (DOX) treatment of cancer xenografts in mice. Compared with the diet containing 5% CO, consumption of FOC increased omega 3 polyunsaturated fatty acids and lipid peroxidation in tumor and liver, significantly decreased the ratio of glutathione peroxidase activity to superoxide dismutase activity (a putative indicator of increased oxidative stress) in tumor but not in the liver, and significantly decreased the tumor-growth rate. The decreased glutathione peroxidase:superoxide dismutase ratio, indicating an altered redox state, in the tumor of FOC-fed mice was significantly correlated with decreased tumor-growth rate. Assay of the body weight change, blood cell counts, and number of micronuclei in peripheral erythrocytes indicated that the toxicity of DOX to the host mouse was not increased in mice fed FOC. Thus, a small amount of FOC increased the effectiveness of DOX but did not increase the toxicity of DOX to the host mouse. These positive results justify clinical testing of FOC in conjunction with cancer chemotherapy.

Editor's note: DOX, doxorubicin, is a well established chemotherapy drug that works by potent ROS generation.

PMID: 11448922
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Cancer Chemother Pharmacol. 2005 Jan;55(1):12-20. Epub 2004 Sep 10.

Docosahexaenoic acid enhances the susceptibility of human colorectal cancer cells to 5-fluorouracil.

Calviello G, Di Nicuolo F, Serini S, Piccioni E, Boninsegna A, Maggiano N, Ranelletti FO, Palozza P.

Institute of General Pathology, Catholic University, L.go F. Vito, 1, 00168, Rome, Italy.

PURPOSE: Powerful growth-inhibitory action has been shown for n-3 polyunsaturated fatty acids against colon cancer cells. We have previously described their ability to inhibit proliferation of colon epithelial cells in patients at high risk of colon cancer. In the work reported here we investigated the ability of docosahexaenoic acid (DHA) to potentiate the antineoplastic activity of 5-fluorouracil (5-FU) in p53-wildtype (LS-174 and Colo 320) and p53-mutant (HT-29 and Colo 205) human colon cancer cells.
METHODS: When in combination with DHA, 5-FU was used at concentrations ranging from 0.1 to 1.0 microM, much lower than those currently found in plasma patients after infusion of this drug. Similarly, the DHA concentrations (< or =10 microM) used in combination with 5-FU were lower than those widely used in vitro and known to cause peroxidative effects in vivo.
RESULTS: Whereas the cells showed different sensitivity to the growth-inhibitory action of 5-FU, DHA reduced cell growth independently of p53 cellular status. DHA synergized with 5-FU in reducing colon cancer cell growth. The potentiating effect of DHA was attributable to the enhancement of the proapoptotic effect of 5-FU. DHA markedly increased the inhibitory effect of 5-FU on the expression of the antiapoptotic proteins BCL-2 and BCL-XL, and induced overexpression of c-MYC which has recently been shown to drive apoptosis and, when overexpressed, to sensitize cancer cells to the action of proapoptotic agents, including 5-FU.
CONCLUSION: Our results indicate that DHA strongly increases the antineoplastic effects of low concentrations of 5-FU. Overall, the results suggest that combinations of low doses of the two compounds could represent a chemotherapeutic approach with low toxicity.

Editor's note: 5-FU, 5-fluorouracil, is yet another well established chemotherapy drug that seems to have strong synergy with DHA from fish oil.

PMID: 15365767
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Med Hypotheses. 2003 May;60(5):615-23.

A low toxicity maintenance regime, using eicosapentaenoic acid and readily available drugs, for mantle cell lymphoma and other malignancies with excess cyclin D1 levels.

Horrobin DF.

Laxdale Ltd., Stirling, Scotland, UK.

Mantle cell lymphoma is a difficult to treat non-Hodgkin's lymphoma (NHL) whose biochemistry is unusually well characterised. Almost all and perhaps all patients overexpress the cyclin D1 protein which is crucial in driving cells from the G1 to the S phase. This overexpression may be responsible for the refractoriness. Despite this understanding, treatments for mantle cell lymphoma are based on standard NHL regimes of cyclophosphamide, doxorubicin, vincristine and prednisone, perhaps supplemented with the monoclonal antibody rituximab. There has never been any attempt to direct treatment to the cyclin D1 mechanism or to angiogenesis which is now known to be important in all lymphomas. Both these targets lend themselves to long-term maintenance regimes of relatively low toxicity which can be used as adjuvants to standard therapy. Agents which have recently been shown to block cyclin D1 translation by regulating calcium levels are the unsaturated essential fatty acid, eicosapentaenoic acid (EPA), the antidiabetic thiazolidinediones, and the antifungal agent, clotrimazole. Two types of agent which have been shown to inhibit angiogenesis are the teratogen, thalidomide, and the selective inhibitors of cyclo-oxygenase 2 (COX-2). Retinoids exert synergistic effects with EPA and have been shown to inhibit both tumour growth and angiogenesis. The mechanisms of action of these various agents are discussed, and specific suggestions are made for low toxicity maintenance therapy of mantle cell lymphoma and of other tumours which overexpress cyclin D1.

PMID: 12710892
____________

Mini Rev Med Chem. 2004 Oct;4(8):859-71.

Omega 3-fatty acids: health benefits and cellular mechanisms of action.

Siddiqui RA, Shaikh SR, Sech LA, Yount HR, Stillwell W, Zaloga GP.

Methodist Research Institute, Indianapolis, IN.

Epidemiological evidence has established that ingestion of long-chain polyunsaturated omega-3 fatty acids (omega-3 PUFAs), abundant in fish oils, have profound effects on many human disorders and diseases, including cardiovascular disease and cancer. Here we briefly review the dietary recommendations and the food sources that are naturally enriched by these fatty acids. There are also a number of products including eggs, bread, and cereals available to supplement omega-3 fatty acid dietary intake. Some of these supplements are proposed to aid different pathological conditions. While the beneficial effects of omega-3 fatty acids can no longer be doubted, their molecular mechanism of action remains elusive. Without question, the action of omega-3 fatty acids is complex and involves a number of integrated signaling pathways. This review focuses on one of the possible cellular mechanisms by which the omega-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), may function. Studies with cancer cells suggest that DHA induces cell cycle arrest and apoptosis by activating protein phosphatases, leading to dephosphorylation of retinoblastoma protein (pRB). Protein phosphatases are also involved with the protein Bcl2, which regulates the release of cytochrome c from mitochondria, and eventually, activation of the apoptotic enzyme caspase 3.

Editor's note: Write to us if you wish to locate the full text of this very thorough literature review article.

PMID: 15544547
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Int J Oncol. 2004 Sep;25(3):737-44.

Docosahexaenoic acid modulates different genes in cell cycle and apoptosis to control growth of human leukemia HL-60 cells.

Chiu LC, Wong EY, Ooi VE.

TCM Pharmacology Research Laboratory, Department of Biology, Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, P.R. China.

Although previous studies have shown that docosahexaenoic acid (DHA; 22:6 omega 3) from fish oils inhibits growth of different cancers, safety issues have been raised repeatedly about contaminations of toxins in these oils. Cultured microalgae are suggested recently as an alternative cleaner and safer source of the fatty acid. We investigated in this study the function of DHA from the enriched microalga Crypthecodinium cohnii (ADHA) in cell-growth control and its mechanism in human leukemia HL-60 cells. ADHA retarded proliferation of the leukemia cells dose-dependently by 4-93% of the control level, after 72-h incubations with 10-160 micro M of the fatty acid; and the 50% inhibitory concentration (IC50) was estimated as 74 micro M. DNA-flow cytometry study showed that ADHA arrested G0/G1 cells by 12-22% and induced apoptotic cells by 569-906% of their controls, after incubation with the IC50 of ADHA for 24, 48 and 72 h. The modes of cell-cycle arrest and pro-apoptotic actions of ADHA were further elucidated. Gene-array analysis illustrated that ADHA modulated a number of cell-cycle and apoptosis genes to control the cell growth; in particular, the fatty acid up-regulated the transcriptional repressor E2F-6 and pro-apoptotic Bax by 1435 and 4172% respectively, after 24 h of incubation. Semi-quantitative RT-PCR study further showed that ADHA induced elevation of the Bax mRNA transcript time-dependently. In meanwhile, ADHA also induced phosphorylation and thus inactivation of Rb protein in the leukemia cells. All these results suggest that ADHA up-regulates Bax and inactivates Rb protein to induce the cell-growth control and apoptosis in human leukemia HL-60 cells.

PMID: 15289877
____________

Int Oncol. 2004 Mar;24(3):591-608

Novel signaling molecules implicated in tumor-associated fatty acid synthase-dependent breast cancer cell proliferation and survival: Role of exogenous dietary fatty acids, p53-p21WAF1/CIP1, ERK1/2 MAPK, p27KIP1, BRCA1, and NF-kappaB.

Menendez JA, Mehmi I, Atlas E, Colomer R, Lupu R.

Department of Medicine, Evanston Northwestern Research Institute, Evanston, IL.

PMID: 14767544
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"Lipid Raft" Formation & Impact on Rituxan Efficacy

Biochemistry. 2003 Oct 21;42(41):12028-37.

Interaction of cholesterol with a docosahexaenoic acid-containing phosphatidylethanolamine: trigger for microdomain/raft formation?

Shaikh SR, Cherezov V, Caffrey M, Stillwell W, Wassall SR.

Department of Biology, Indiana University, IN.

Docosahexaenoic acid (DHA, 22:6) containing phospholipids have been postulated to be involved in promoting lateral segregation within membranes into cholesterol- (CHOL-) rich and CHOL-poor lipid microdomains. Here we investigated the specific molecular interactions of phospholipid bilayers composed of 1-[(2)H(31)]palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE-d(31)) or 1-[(2)H(31)]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE-d(31)) with equimolar CHOL using solid-state (2)H NMR spectroscopy and low- and wide-angle X-ray diffraction (XRD). Moment analysis of (2)H NMR spectra obtained as a function of temperature reveals that the main chain melting transition and the lamellar-to-inverted hexagonal (H(II)) phase transition of 16:0-22:6PE-d(31) remain in the presence of equimolar CHOL, whereas addition of equimolar CHOL essentially obliterates the gel-to-liquid crystalline transition of 16:0-18:1PE-d(31). (2)H NMR order parameter measurements show that the addition of equimolar CHOL in the lamellar liquid crystalline phase causes a smaller increase in order for the perdeuterated sn-1 chain by 22% for 16:0-22:6PE-d(31) as opposed to 33% for 16:0-18:1PE-d(31). XRD experiments determined markedly lower solubility of 32 +/- 3 mol % for CHOL in 16:0-22:6PE bilayers in contrast to the value of approximately 51 mol % for 16:0-18:1PE. Our findings provide further evidence that cholesterol has a low affinity for DHA-containing phospholipids and that this reduced affinity may serve as a mechanism for triggering the formation of lipid microdomains such as rafts.

PMID: 14556634
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Cancer Res. 2003 Jan 15;63(2):534-40.

Anti-CD20 therapeutic antibody rituximab modifies the functional organization of rafts/microdomains of B lymphoma cells.

Semac I, Palomba C, Kulangara K, Klages N, van Echten-Deckert G, Borisch B, Hoessli DC.

Department of Pathology, Faculty of Medicine, Centre medical universitaire, 1211 Geneva 4, Switzerland.

Incubation of Burkitt lymphoma-derived Raji cells at physiological temperature with submicromolar concentrations of humanized anti-CD20 antibody rituximab (RTX) redistributes CD20 to liquid-ordered, plasma membrane rafts. This accumulation of the CD20 tetraspan protein in rafts does not change the existing lipid and phosphoprotein composition but makes sphingolipids and the Src regulator Cbp/PAG (Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain) transmembrane phosphoprotein more resistant to n-octyl-beta-pyranoside, a detergent that dissociates sphingolipid clusters. On the contrary, sphingolipids and Cbp/PAG are not protected by the presence of CD20 against the disruptive effects of methyl-beta-cyclodextrin, a cyclic carbohydrate that removes membrane cholesterol. After accumulation of CD20, the activity of the raft-associated Lyn kinase is down-regulated without apparent alteration of its relationship to substrates. Moreover, in rafts of lymphoblastoid cells that express lower amounts of Cbp/PAG, RTX redistributes CD20 to rafts but does not modulate the raft-associated protein tyrosine kinase activity, suggesting that the presence of Cbp/PAG protein in rafts is necessary for RTX to exert its transmembrane "signaling effects." Lastly, redistribution of CD20 in rafts renders the glycosylphosphatidyl inositol (GPI)-linked CD55 C'-defense protein hypersensitive to glycosylphosphatidyl inositol-specific phospholipases. By redistributing CD20 to rafts, RTX modifies their stability and organization and modulates the associated signaling pathways and C' defense capacity.

PMID: 12543813
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J Immunol. 2002 Sep 15;169(6):2886-91.

Colocalization of the B cell receptor and CD20 followed by activation-dependent dissociation in distinct lipid rafts.

Petrie RJ, Deans JP.

Department of Biochemistry and Molecular Biology, Immunology Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.

The B cell Ag receptor (BCR) and CD20, a putative calcium channel, inducibly associate with cholesterol-dependent membrane microdomains known as lipid rafts. A functional association between the BCR and CD20 is suggested by the effects of CD20-specific mAbs, which can modulate cell cycle transitions elicited by BCR signaling. Using immunofluorescence microscopy we show here that the BCR and CD20 colocalize after receptor ligation and then rapidly dissociate at the cell surface before endocytosis of the BCR. After separation, surface BCR and CD20 were detected in distinct lipid rafts isolated as low density, detergent-resistant membrane fragments. Pretreatment with methyl-beta-cyclodextrin, which we have previously shown to enhance receptor-mediated calcium mobilization, did not prevent colocalization of the BCR and CD20, but slowed their dissociation. The data demonstrate rapid dynamics of the BCR in relation to CD20 at the cell surface. Activation-dependent dissociation of the BCR from CD20 occurs before receptor endocytosis and appears to require in part the integrity of lipid rafts.

PMID: 12218101
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Rituxan Cell Kill May Be Mediated by ROS Pathways

Blood. 2001 Nov 1;98(9):2771-7.

Complement-mediated cell death induced by rituximab in B-cell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species.

Bellosillo B, Villamor N, Lopez-Guillermo A, Marce S, Esteve J, Campo E, Colomer D, Montserrat E.

Hematopathology Unit, Department of Hematology, Institute of Hematology and Oncology, Postgraduate School of Hematology Farreras-Valenti, Barcelona, Spain.

Mechanisms involving the in vitro effect of rituximab in cells from 55 patients with B-cell lymphoproliferative disorders were investigated. No cytotoxic effect was observed when cells were incubated with rituximab alone, but in the presence of human AB serum rituximab induced complement-dependent cell death (R-CDC). A cytotoxic effect was observed in cells from 9 of 33 patients with B-cell chronic lymphocytic leukemia, 16 of 16 patients with mantle-cell lymphoma, 4 of 4 patients with follicular lymphoma, and 2 of 2 patients with hairy-cell leukemia. R-CDC was observed in cells from patients expressing more than 50 x 10(3) CD20 molecules per cell, and directly correlated with the number of CD20 molecules per cell. Preincubation with anti-CD59 increased the cytotoxic effect of rituximab and sensitized cells from nonsensitive cases. Neither cleavage of poly-ADP ribose polymerase (PARP) nor activation of caspase-3 was observed in R-CDC. In addition, no cells with a hypodiploid DNA content were detected and R-CDC was not prevented by a broad-spectrum caspase inhibitor, suggesting a caspase-independent mechanism. Incubation with rituximab in the presence of AB serum induced a rapid and intense production of reactive oxygen species (ROS). R-CDC was blocked by the incubation of cells with N-acetyl-L-cysteine (NAC) or Tiron, 2 ROS scavengers, indicating that the cytotoxic effect was due to the generation of superoxide (O) radicals. In conclusion, the results of the present study suggest that CD20, CD59, and complement have a role in the in vitro cytotoxic effect of rituximab, which is mediated by a caspase-independent process that involves ROS generation.

PMID: 11675350
____________

Blood. 2004 Sep 23;

Adaphostin-induced apoptosis in CLL B-cells is associated with induction of oxidative stress and exhibits synergy with fludarabine.

Shanafelt TD, Lee YK, Bone ND, Strege AK, Narayanan VL, Sausville EA, Geyer SM, Kaufmann SH, Kay NE.

Department of Medicine, Division of Hematology, Division of Oncology Research, and Division of Biostatistics, Mayo Clinic, Rochester, MN, USA.

B-cell chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. We evaluated the ability of the investigational antileukemic agent adaphostin to induce apoptosis in CLL B-cells and synergize with fludarabine in vitro. Analysis by Annexin V/propidium iodide (PI) staining revealed that the mean IC50 for adaphostin at 24 hours was 4.2 micro M (range 1.10 micro M - 11.25 M; median = 4.25; n = 29) for CLL isolates and >10 micro M for B and T-cells from normal donors. Immunoblots demonstrated adaphostin-induced PARP cleavage and cleavage of caspase 3 substrates, suggesting that adaphostin induces apoptosis. Adaphostin increased the level of reactive oxygen species (ROS) within CLL B-cells; and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis. Adaphostin also caused a decrease in the level of the anti-apoptotic protein Bcl-2. When adaphostin was combined with fludarabine (F-ARA-AMP), a synergistic effect on cell death was observed in all 10 CLL samples. These findings not only indicate that adaphostin induces apoptosis selectively in CLL B-cells through a mechanism that involves ROS generation, but also demonstrate its ability to augment the effects of fludarabine. Further preclinical development of adaphostin as a novel agent for the treatment of CLL appears warranted.

PMID: 15388586
____________

Blood. 2004 Sep 28;

Blood. 2004 May 1;103(9):3516-20. Epub 2003 Dec 24.

Fenretinide enhances rituximab-induced cytotoxicity against B-cell lymphoma xenografts through a caspase-dependent mechanism.

Gopal AK, Pagel JM, Hedin N, Press OW.

Seattle Cancer Care Alliance, Mailstop G6-800, Rm 6802, University of Washington, 825 Eastlake Ave E, Seattle, WA.

The anti-CD20 monoclonal antibody rituximab induces remission in 40% to 60% of patients with indolent B-cell lymphoma, but virtually all patients have relapses. We evaluated the efficacy of concurrent administration of another biologic agent, N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) with rituximab against a variety of human B-cell lymphoma cell lines (Ramos, DHL-4, and FL-18) in vivo. Concurrent 4HPR and rituximab administration prevented tumor progression of lymphoma-bearing mice in a minimal disease model (rituximab + 4HPR, 100% progression free; rituximab alone, 37.5% progression free, P =.01; 4HPR alone, 12.5% progression free, P <.01; controls, 0% progression free, P <.01). Combinations of 4HPR + rituximab exceeded the predicted 50% additive rate of disease control from each agent alone (P =.038). Administering 4HPR and rituximab to mice with established tumors induced complete responses (CRs) in 80% of animals compared with 20% to 40% CRs using either agent alone (P =.07), resulting in significantly improved survival. Tumors harvested from 4HPR + rituximab-treated mice displayed elevated caspase activation compared with untreated controls (P =.02). Adding a broad-spectrum caspase inhibitor in vivo fully abrogated the antitumor effects of 4HPR + rituximab (P =.05). These results establish the efficacy of 4HPR/rituximab combinations, confirm their caspase-mediated mechanism of action, and offer the potential for disease control with minimal toxicity for patients with B-cell malignancies.

Editor's note: Fenretinide, a vitamin A analogue, has been tested extensively in many different cancers. The ROS pathway is one of its known mechanisms, and here the authors report its synergy with Rituxan.

PMID: 14695237
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