Shantaram S. Joshi

Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinsons disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells.

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL‐60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0–100 μM concentrations of the two cations for 0, 24, or 48 hours at 37°C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 μM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2‐ and 3.0‐fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 μM Cr(VI), respectively, and 1.2‐ and 1.7‐fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7‐ and 4.9‐fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 μM Cr(VI), respectively, and 1.6‐ and 3.3‐fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6‐fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 μM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL‐60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL‐60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL‐60 cells as compared with normal human peripheral blood mononuclear cells.

Hedgehog-induced survival of B-cell chronic lymphocytic leukemia cells in a stromal cell microenvironment: a potential new therapeutic target.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell–induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell–induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target. (Mol Cancer Res 2008;6(12):1928–36)

Targeting of sonic hedgehog-GLI signaling: a potential strategy to improve therapy for mantle cell lymphoma

Mantle cell lymphoma (MCL) has one of the worst clinical outcomes among the B-cell lymphomas, with a median survival of only 3 to 4 years. Therefore, a better understanding of the underlying mechanisms that regulate MCL proliferation/survival is needed to develop an effective therapy. Because sonic hedgehog (Shh)-GLI signaling has been shown to be important in the proliferation and survival of several cancers, and no such information is available for MCL, this study was undertaken. Our results show that the molecules associated with Shh-GLI signaling, such as PTCH and SMO receptors, and GLI1 and GLI2 target transcription factors were expressed in the human MCL cell lines and primary MCL cells from patients. Perturbation of this signaling in the presence of exogenous Shh/cyclopamine significantly (P < 0.001) influenced the proliferation of JVM2 MCL cells. Furthermore, down-regulation of GLI transcription factors using antisense oligonucleotides not only resulted in significantly (P < 0.001) decreased proliferation of the MCL cells but also significantly (P < 0.05) increased their susceptibility to chemotherapeutic drug, doxorubicin. Also, down-regulation of GLI decreased cyclin D1 and BCL2 transcript levels, which suggests that these key molecules might be regulated by GLI in MCL. Thus, our results indicate a significant role for Shh-GLI signaling in the proliferation of MCL, and molecular targeting of GLI is a potential therapeutic approach to improve the treatment for MCL. [Mol Cancer Ther 2008;7(6):1450–60]

Chemopreventive effects of grape seed proanthocyanidin extract on Chang liver cells

In an attempt to ameliorate the chemotherapy associated normal cell toxicity, in this study a known antioxidant, grape seed proanthocyanidin extract (GSPE) using Chang liver cells has been used. Chang liver cells were treated in vitro with idarubicin (Ida) (30 nM) and 4-hydroxyperoxycyclophosphamide (4-HC) (1 microg/ml) with or without proanthocyanidin (25 microg/ml). The cells were grown in vitro and the growth rate of the cells were determined using MTT assay. The results showed that the GSPE decreased growth inhibitory effects of Ida and 4-HC on Chang liver cells in vitro. Since these chemotherapeutic agents are known to induce apoptosis in the target cells, these cells were also analyzed for presence of apoptotic cells using flow cytometry. The GSPE decreased the number of apoptotic cell population induced by either chemotherapy. In an attempt to determine the mechanisms of ameliorating effects of proanthocyanidin, the expression of apoptosis/cell cycle/growth related genes, Bcl-2, p53 and c-myc was determined in the treated and control cells using Western blotting or reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. There was an increased expression of Bcl-2 in the cells treated with GSPE. However, there was a significant decrease in the expression of other cell cycle related genes such as p53 and c-myc in these cells following treatment with GSPE. Thus, these results indicate that proanthocyanidin can be a potential candidate to ameliorate the toxic effects associated with chemotherapeutic agents used in treatment of cancer.

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b-/- lymphomas, but not in Dnmt3b-/- pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b-/- lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.

ATM, CTLA4, MNDA, and HEM1 in High versus Low CD38–Expressing B-Cell Chronic Lymphocytic Leukemia

Purpose: In B-cell chronic lymphocytic leukemia (CLL), high CD38 expression has been associated with unfavorable clinical course, advanced disease, resistance to therapy, shorter time to first treatment, and shorter survival. However, the genes associated with CLL patient subgroups with high and low CD38 expression and their potential role in disease progression is not known. Experimental Design: To identify the genes associated with the clinical disparity in CLL patients with high versus low CD38 expression, transcriptional profiles were obtained from CLL cells from 39 different patients using oligonucleotide microarray. Gene expression was also compared between CLL cells and B cells from healthy individuals. Results: Gene expression analysis identified 76 differentially expressed genes in CD38 high versus low groups. Out of these genes, HEM1, CTLA4, and MNDA were selected for further studies and their differential expression was confirmed by real-time PCR. HEM1 overexpression was associated with poor outcome, whereas the overexpression of CTLA4 and MNDA was associated with good outcome. Down-regulation of HEM1 expression in patient CLL cells resulted in a significant increase in their susceptibility to fludarabine-mediated killing. In addition, when gene expression patterns in CD38 high and low CLL cells were compared with normal B-cell profiles, ATM expression was found to be significantly lower in CD38 high compared with CD38 low CLL as confirmed by real-time reverse transcription-PCR. Conclusions: These results identify the possible genes that may be involved in cell proliferation and survival and, thus, determining the clinical behavior of CLL patients expressing high or low CD38.

Protective effects of antioxidants against smokeless tobacco-induced oxidative stress and modulation of Bcl-2 and p53 genes in human oral keratinocytes.

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and β-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992–1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0–200 μg/ml) for 24h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 μg/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher concentration of STE as reported earlier. GSPE significantly modulated STE-induced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased with STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was observed following incubation with STE and/or GSPE. Thus, c-myc may not be involved in STE-induced cytotoxicity towards NHOK cells. These results suggest that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.

Chronic lymphocytic leukemia cells in a lymph node microenvironment depict molecular signature associated with an aggressive disease.

Chronic lymphocytic leukemia (CLL) cells survive longer in vivo than in vitro, suggesting that the tissue microenvironment provides prosurvival signals to tumor cells. Primary and secondary lymphoid tissues are involved in the pathogenesis of CLL, and the role of these tissue microenvironments has not been explored completely. To elucidate host-tumor interactions, we performed gene expression profiling (GEP) of purified CLL cells from peripheral blood (PB; n = 20), bone marrow (BM; n = 18), and lymph node (LN; n = 15) and validated key pathway genes by real-time polymerase chain reaction, immunohistochemistry and/or TCL1 trans-genic mice. Gene signatures representing several pathways critical for survival and activation of B cells were altered in CLL cells from different tissue compartments. Molecules associated with the B-cell receptor (BCR), B cell-activating factor/a proliferation-inducing ligand (BAFF/APRIL), nuclear factor (NF)-κB pathway and immune suppression signature were enriched in LN-CLL, suggesting LNs as the primary site for tumor growth. Immune suppression genes may help LN-CLL cells to modulate antigen-presenting and T-cell behavior to suppress antitumor activity. PB CLL cells overexpressed chemokine receptors, and their cognate ligands were enriched in LN and BM, suggesting that a chemokine gradient instructs B cells to migrate toward LN or BM. Of several chemokine ligands, the expression of CCL3 was associated with poor prognostic factors. The BM gene signature was enriched with antiapoptotic, cytoskeleton and adhesion molecules. Interestingly, PB cells from lymphadenopathy patients shared GEP with LN cells. In Eμ-TCL1 transgenic mice (the mouse model of the disease), a high percentage of leukemic cells from the lymphoid compartment express key BCR and NF-κB molecules. Together, our findings demonstrate that the lymphoid microenvironment promotes survival, proliferation and progression of CLL cells via chronic activation of BCR, BAFF/APRIL and NF-κB activation while suppressing the immune response.

IRF4 is a suppressor of c-Myc induced B cell leukemia.

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator in B cell development and function. We have previously shown that IRF4, together with IRF8, orchestrates pre-B cell development by limiting pre-B cell expansion and by promoting pre-B cell differentiation. Here, we report that IRF4 suppresses c-Myc induced leukemia in EμMyc mice. Our results show that c-Myc induced leukemia was greatly accelerated in the IRF4 heterozygous mice (IRF4+/−Myc); the average age of mortality in the IRF4+/−Myc mice was only 7 to 8 weeks but was 20 weeks in the control mice. Our results show that IRF4+/−Myc leukemic cells were derived from large pre-B cells and were hyperproliferative and resistant to apoptosis. Further analysis revealed that the majority of IRF4+/−Myc leukemic cells inactivated the wild-type IRF4 allele and contained defects in Arf-p53 tumor suppressor pathway. p27kip is part of the molecular circuitry that controls pre-B cell expansion. Our results show that expression of p27kip was lost in the IRF4+/−Myc leukemic cells and reconstitution of IRF4 expression in those cells induced p27kip and inhibited their expansion. Thus, IRF4 functions as a classical tumor suppressor to inhibit c-Myc induced B cell leukemia in EμMyc mice.