Suhasini Joshi

Lymphocyte reconstitution after allogeneic blood stem cell transplantation for hematologic malignancies

Forty-one patients were studied at set times after allogeneic blood stem cell transplantation (alloBSCT) for recovery of lymphocyte numbers and function. Cells were mobilized with G-CSF from HLA-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate; G-CSF was administered post-transplant. Median time to absolute lymphocyte count (ALC) ⩾500/μl was 17 days vs 41 and 49 days in historical alloBMT patients with G-CSF (n = 23) or no cytokine (n = 29) post-transplant, respectively (P < 0.0001). CD4/CD8+ ratio was 1.9 on day 28 after alloBSCT, then gradually declined to 0.8 at 1 year due to more rapid CD8+ cell recovery. Mean phytohemagglutinin-induced T cell responses were lower than normal on day +28 (P < 0.05), then tended to recover towards normal values. Natural-killer cytotoxicity remained low from day +28 to 1 year post-alloBSCT, but considerable lymphokine-activated killer cytotoxicity was induced from cells already obtained on day +28. Faster lymphocyte recovery correlated with better survival in alloBSCT patients (median follow-up 287 days, P = 0.002), ALC recovery was not affected by acute GVHD, CMV infections or doses of infused cells. ALC recovery did not correlate with survival in either historical alloBMT group. These data suggest that after alloBSCT lymphocyte reconstitution is faster than after alloBMT, and that quicker lymphocyte recovery predicts better survival in the alloBSCT setting.

Graviola: A novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism

Pancreatic tumors are resistant to conventional chemotherapies. The present study was aimed at evaluating the potential of a novel plant-derived product as a therapeutic agent for pancreatic cancer (PC). The effects of an extract from the tropical tree Annona Muricata, commonly known as Graviola, was evaluated for cytotoxicity, cell metabolism, cancer-associated protein/gene expression, tumorigenicity, and metastatic properties of PC cells. Our experiments revealed that Graviola induced necrosis of PC cells by inhibiting cellular metabolism. The expression of molecules related to hypoxia and glycolysis in PC cells (i.e. HIF-1α, NF-κB, GLUT1, GLUT4, HKII, and LDHA) were downregulated in the presence of the extract. In vitro functional assays further confirmed the inhibition of tumorigenic properties of PC cells. Overall, the compounds that are naturally present in a Graviola extract inhibited multiple signaling pathways that regulate metabolism, cell cycle, survival, and metastatic properties in PC cells. Collectively, alterations in these parameters led to a decrease in tumorigenicity and metastasis of orthotopically implanted pancreatic tumors, indicating promising characteristics of the natural product against this lethal disease.

Dysregulation in T helper 1/T helper 2 cytokine ratios in patients with multiple myeloma

We have assessed alterations of Th1 and Th2 derived cytokines in the progression of multiple myeloma (MM) by determining their circulatory concentrations in patients with MM and healthy controls and correlated observations with grade and severity of the disease. The study group consisted of 112 subjects: 62 patients with MM and 50 healthy controls. Regarding serum levels of Th1 cytokines, IFN-γ was significantly lower and IL-2 was non-significantly increased, while regarding levels of Th2 cytokines, IL-4 and IL-10 were significantly elevated. Results of our study indicate that a marked polarization exists toward Th2 cytokines in MM while Th1 cytokines remain suppressed.

Smoking accelerates pancreatic cancer progression by promoting differentiation of Mdscs and inducing Hb-egf expression in macrophages

Smoking is an established risk factor for pancreatic cancer (PC), but late diagnosis limits the evaluation of its mechanistic role in the progression of PC. We used a well-established genetically engineered mouse model (LSL-K-rasG12D) of PC to elucidate the role of smoking during initiation and development of pancreatic intraepithelial neoplasia (PanIN). The 10-week-old floxed mice (K-rasG12D; Pdx-1cre) and their control unfloxed (LSL-K-rasG12D) littermates were exposed to cigarette smoke (total suspended particles: 150 mg/m3) for 20 weeks. Smoke exposure significantly accelerated the development of PanIN lesions in the floxed mice, which correlated with tenfold increase in the expression of cytokeratin19. The systemic accumulation of myeloid-derived suppressor cells (MDSCs) decreased significantly in floxed mice compared with unfloxed controls (P<0.01) after the smoke exposure with the concurrent increase in the macrophage (P<0.05) and dendritic cell (DCs) (P<0.01) population. Further, smoking-induced inflammation (IFN-γ, CXCL2; P<0.05) was accompanied by enhanced activation of pancreatic stellate cells and elevated levels of serum retinoic acid-binding protein 4, indicating increased bioavailability of retinoic acid which contributes to differentiation of MDSCs to tumor-associated macrophages (TAMs) and DCs. TAMs predominantly contribute to the increased expression of heparin-binding epidermal growth factor-like growth factor (EGFR ligand) in pre-neoplastic lesions in smoke-exposed floxed mice that facilitate acinar-to-ductal metaplasia (ADM). Further, smoke exposure also resulted in partial suppression of the immune system early during PC progression. Overall, the present study provides a novel mechanism of smoking-induced increase in ADM in the presence of constitutively active K-ras mutation.

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells.

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.

Effects of BCR-ABL Antisense Oligonucleotides (AS-ODN) on Human Chronic Myeloid Leukemic Cells: AS-ODN as Effective Purging Agents

We examined phosphorothioate oligodeoxyribonucleotides (ODNs) directed against bcr in exon 3 or exon 2, which are rearranged with exon 2 of abl (B3A2 and B2A2) at t(9;22) of chronic myelogenous leukemia (CML). Since these ODNs are designed to be CML cell specific, we studied their effects on the human CML cell line K562, which is known to have B3A2 rearrangement, and leukemic cells from patients, as well as normal hematopoietic stem cells in vitro. In vitro experiments were performed to determine a potential role of these two ODNs as ex vivo purging agents. Incubation of B3A2 antisense at 40, 80, and 120 micrograms/ml with K562 CML cells for 72 hours at 37 degrees C resulted in 44%, 56%, and 63% reduction of CFU-L as compared to controls. In contrast, B3A2 sense and B2A2 antisense had no significant growth inhibitory effect on K562 cells. Incubation of B3A2 and B2A2 antisense ODNs at concentration of 80 micrograms/ml at 37 degrees C for 36 hours with normal peripheral blood stem/progenitor cells (PBSC) resulted in 124% and 98% CFU-GM formation as compared to untreated controls, respectively. However, incubation of PBSC with B3A2 and B2A2 sense-ODNs resulted in a 22% and 44% reduction in CFU-GM, respectively. In order to determine the ex vivo purging effects of bcr-abl ODNs, the K562 cells were mixed with PBSC from normal donors at a ratio of 1:20 (CML:PBSC). The mixture of cells was then incubated with B3A2 antisense at 80 micrograms/ml for 36 hrs at 37 degrees C. After incubation, no CML cells were detected by fluorescence in situ hybridization (FISH) as compared to untreated controls. These results were confirmed by RT-PCR using bcr-abl primers and mRNA isolated from the mixture of cells. Further, these results support the hypothesis that bcr-abl antisense ODNs are potentially effective agents for ex vivo purging of autologous stem cells before transplantation to eliminate/reduce the burden of leukemic cells. No significant toxicity to normal hematopoietic stem/progenitor cell population by the bcr-abl antisense ODNs was observed. Although unanticipated reductions in normal hematopoietic progenitor cells (CFU-GM) were observed with sense ODNs, no reduction in CFU-GM was observed with unrelated phosphorothioate ODN controls.

Angiopoietin-2: A potential novel diagnostic marker in multiple myeloma

BACKGROUND Multiple myeloma (MM) was the first hematological malignancy in which a prognostic relevance of bone marrow (BM) angiogenesis was reported. Microenvironment formed by reactive oxygen species, growth factors and chemokines initiates the process of BM angiogenesis. Among the angiopoietins, angiopoietin-1 is involved in vessel stability, whereas angiopoietin-2 is involved in vessel sprouting. DESIGN AND METHODS Circulatory levels of angiopoietin-1, angiopoietin-2 and VEGF in 62 MM patients and 50 healthy controls were determined using ELISA. RESULTS Significant increase in VEGF and angiopoietin-2 level was observed in patients which correlated positively with the severity of the disease, whereas no alteration was observed in angiopoietin-1 levels. CONCLUSION Significant elevation in angiopoietin-2 and VEGF levels and their correlation with severity of the disease indicate their utility as potential tumor markers along with β(2)- microglobulin and might also suggest new therapeutic target for anticancer treatment.

Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival

Pancreatic cancer (PC) and associated pre-neoplastic lesions have been reported to be hypoxic, primarily due to hypovascular nature of PC. Though the presence of hypoxia under cancerous condition has been associated with the overexpression of oncogenic proteins (MUC1), multiple emerging reports have also indicated the growth inhibitory effects of hypoxia. In spite of being recognized as the top-most differentially expressed and established oncogenic protein in PC, MUC4 regulation in terms of micro-environmental stress has not been determined. Herein, for the first time, we are reporting that MUC4 protein stability is drastically affected in PC, under hypoxic condition in a hypoxia inducible factor 1α (HIF-1α)-independent manner. Mechanistically, we have demonstrated that hypoxia-mediated induction of reactive oxygen species (ROS) promotes autophagy by inhibiting pAkt/mTORC1 pathway, one of the central regulators of autophagy. Immunohistofluorescence analyses revealed significant negative correlation (P-value=0.017) between 8-hydroxy guanosine (8-OHG) and MUC4 in primary pancreatic tumors (n=25). Moreover, we found pronounced colocalization between MUC4 and LAMP1/LC3 (microtubule-associated protein 1A/1B-light chain 3) in PC tissues and also observed their negative relationship in their expression pattern, suggesting that areas with high autophagy rate had less MUC4 expression. We also found that hypoxia and ROS have negative impact on overall cell growth and viability, which was partially, though significantly (P<0.05), rescued in the presence of MUC4. Altogether, hypoxia-mediated oxidative stress induces autophagy in PC, leading to the MUC4 degradation to enhance survival, possibly by offering required metabolites to stressed cells.

Immunological properties of mononuclear cells from blood stem cell harvests following mobilization with erythropoietin + G-CSF in cancer patients.

BACKGROUND Although the recombinant granulocyte colony-stimulating factor (G-CSF) is a good CD34 cell mobilizer, the effects of G-CSF mobilization on the immune effector function of the individual is not always optimal. We studied the functional and phenotypic properties of peripheral blood stem cells (PBSC) collected from 15 cancer patients mobilized with G-CSF plus recombinant erythropoietin (EPO). METHODS The patients received EPO (300 U/kg) and (G-CSF 1 microg/kg) per day as mobilizing cytokines and an autologous graft product was collected with at least daily apheresis procedures until a target number of CD34 cells and mononuclear cells were obtained. Mononuclear cells from the first four PBSC collections were tested for their natural killer (NK), activated NK and lymphokine activated killer (LAK) cytotoxicity in vitro against K562 and Raji tumor target cells. RESULTS There was a significant increase in NK, activated NK and LAK cytotoxicity in EPO + G-CSF mobilized cells when compared to mononuclear cells from premobilization blood baseline values. Although there was no increase in CD3+ T cells, there was a significant increase in myeloid cells (CD14+), B-cells (CD20+) and NK cells (CD56+) following mobilization. There was no difference in T cell response to the mitogens PHA and Con-A, but there was an increase in B-cell response to PWM following mobilization. Thus, the combination of EPO + G-CSF not only mobilized hematopoietic precursor cells but also increased the number of immune effector cells in the PBSC collections.

NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer

Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P<0.01). Immunohistochemistry analysis in PC tissues and metastatic lesions established an association between NCOA3 and mucin (MUC1 and MUC4) expression. Spontaneous mouse model of PC (K-rasG12D; Pdx-1cre) showed early expression of Ncoa3 during pre-neoplastic lesions. Mechanistically, NCOA3 knockdown abrogated retinoic acid-mediated MUC4 upregulation by restricting MUC4 promoter accessibility as demonstrated by micrococcus nuclease digestion (P<0.05) and chromatin immuno-precipitation analysis. NCOA3 also created pro-inflammatory conditions by upregulating chemokines like CXCL1, 2, 5 and CCL20 (P<0.001). AKT, ubiquitin C, ERK1/2 and NF-κB occupied dominant nodes in the networks significantly modulated after NCOA3 silencing. In addition, NCOA3 stabilized mucins post translationally through fucosylation by FUT8, as the knockdown of FUT8 resulted in the downregulation of MUC4 and MUC1 at protein levels.