Lubna Qamar

Involvement of the electrophile responsive element and p53 in the activation of hepatic stellate cells as a response to electrophile menadione

The cytotoxic effects of menadione and hydrogen peroxide were examined in two hepatic stellate cell lines derived from normal or cirrhotic rat liver. The cirrhotic fat-storing cells (CFSC) were found more resistant than the normal fat-storing cells (NFSC) to menadione cytotoxicity. No significant differences were observed in hydrogen peroxide toxicity in these two cell lines. Although protein levels and enzymatic activities of catalase, Cu,Zn-SOD, Mn-SOD, and NADPH cytochrome c reductase were similar in these cell lines, 20-fold increases of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzymatic activity and protein levels were detected in CFSC compared to those of NFSC. Gel mobility shift assays and functional analysis using transient transfection experiments indicated the involvement of the electrophile responsive element (EPRE) in the up-regulation of the NQO1 expression. Antibody supershift analysis revealed that, although Nrf2 is a member of the EPRE-binding complex in both NFSC and CFSC, Nrf1 was identified as a part of the protein/DNA complex only in CFSC. Expression of p53 tumor suppressor gene was found in higher levels in CFSC than in NFSC. We conclude that activation of the EPRE-signaling pathway, which up-regulates several phase II genes and affects p53 stabilization, may offer resistance to hepatic stellate cells against oxidative damage during hepatic injury. This resistance may be a part of the activation process of the hepatic stellate cells and could contribute to their increased proliferation and production of extracellular matrix.

Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer

OBJECTIVE The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. METHODS A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. RESULTS The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischers Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). CONCLUSIONS The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.

TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

Background Signaling by Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL) has been proposed to contribute to the chemosensitivity of tumor cells treated with various other anti-cancer agents. However, the importance of these effects and whether there are differences in vitro and in vivo is unclear. Methodology/Principal Findings To assess the relative contribution of death receptor pathways to this sensitivity and to determine whether these effects are intrinsic to the tumor cells, we compared the chemosensitivity of isogenic BJAB human lymphoma cells where Fas and TRAIL receptors or just TRAIL receptors were inhibited using mutants of the adaptor protein FADD or by altering the expression of the homeobox transcription factor Six1. Inhibition of TRAIL receptors did not affect in vitro tumor cell killing by various anti-cancer agents indicating that chemosensitivity is not significantly affected by the tumor cell-intrinsic activation of death receptor signaling. However, selective inhibition of TRAIL receptor signaling caused reduced tumor regression and clearance in vivo when tested in a NOD/SCID mouse model. Conclusions These data show that TRAIL receptor signaling in tumor cells can determine chemosensitivity in vivo but not in vitro and thus imply that TRAIL resistance makes tumors less susceptible to conventional cytotoxic anti-cancer drugs as well as drugs that directly target the TRAIL receptors.

Protein kinase C inhibitor Gö6976 augments caffeine-induced reversal of chemoresistance to cis-diamminedichloroplatinum-II (CDDP) in a human ovarian cancer model

OBJECTIVES Novel strategies for the treatment of chemoresistant ovarian cancer are needed. Caffeine and related compounds have been shown to over-ride G2/M arrest in ovarian cancer cells, increasing toxicity to chemotherapy. Newer compounds have been developed which may have the same effect as and exhibit synergism with caffeine, allowing the use of lower doses. METHODS We investigated the effects of caffeine and Gö6976 in the presence of CDDP in the SKOV3 and A2780 cell lines using proliferation, cell-cycle analysis, apoptosis, and AKT expression. RESULTS Proliferation of cancer cells was decreased in a dose-dependent manner with caffeine and CDDP, but doses of caffeine required for significant inhibition were higher than that achievable in patients. Gö6976, a global PKC inhibitor with G2/M over-ride capability similar to caffeine, when combined with caffeine and CDDP at doses below that required for cell-cycle over-ride produced the growth inhibitory effects of a ten-fold higher caffeine concentration in both cell lines. CDDP induced G2/M arrest was significantly abrogated by caffeine but not by Gö6976 alone and no additional effect was seen on G2/M over-ride by the addition of Gö6976 to caffeine. Addition of Gö6976 to caffeine and CDDP did increase apoptosis but without altering phospho-AKT. CONCLUSIONS Gö6976, when added to caffeine at doses below that required for cell-cycle over-ride, augments caffeine in overcoming CDDP resistance in this experimental system. G2/M over-ride is not the mechanism underlying the inhibition of proliferation. An AKT-independent apoptotic mechanism may be responsible.

Photoprotective Effects of Bucillamine Against UV-induced Damage in an SKH-1 Hairless Mouse Model†

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non‐DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N‐(2‐mercapto‐2‐methylpropionyl)‐l‐cysteine] is a cysteine‐derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB‐induced photodamage using the SKH‐1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm−2) on the dorsal skin of SKH‐1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg−1 of body weight, administered subcutaneously) markedly attenuated UVB‐mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser‐15 and Ser‐20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.

Progestin treatment decreases cd133 + cancer stem cell populations in endometrial cancer

OBJECTIVES Endometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells. METHODS The Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4μM medroxyprogesterone 17-acetate (MPA) for 6days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression was scored and evaluated for change over time on serial biopsies. RESULTS CD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment. CONCLUSION CD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells.

Cytokeratin 5-Positive Cells Represent a Therapy Resistant subpopulation in Epithelial Ovarian Cancer.

Objective Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)+ breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5+ cell populations to cisplatin therapy. Materials and Methods Cytokeratin 5 expression was evaluated in 2 ovarian tissue microarrays, representing 137 neoplasms, and 6 ovarian cancer cell lines. Cell lines were treated with IC50 (half-maximal inhibitory concentration) cisplatin, and the prevalence of CK5+ cells pretreatment and posttreatment was determined. Proliferation of CK5+ versus CK5− cell populations was determined using 5-bromo-2′-deoxyuridine incorporation. Chemotherapy-induced apoptosis in CK5+ versus CK5− cells was measured using immunohistochemical staining for cleaved caspase-3. Results Cytokeratin 5 was expressed in 39.3% (42 of 107) of epithelial ovarian cancers with a range of 1% to 80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5+ specimens. Cytokeratin 5 expression correlated with ER positivity (38 of 42 CK5+ tumors were also ER+). Cytokeratin 5 was expressed in 5 of 6 overall and 4 of 4 ER+ epithelial ovarian cancer cell lines ranging from 2.4% to 52.7% positive cells. Cytokeratin 5+ compared with CK5− cells were slower proliferating. The prevalence of CK5+ cells increased after 48-hour cisplatin treatment in 4 of 5 cell lines tested. Cytokeratin 5+ ovarian cancer cells compared with CK5− ovarian cancer cells were more resistant to cisplatin-induced apoptosis. Conclusions Cytokeratin 5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating chemoresistant subpopulation that may warrant cotargeting in combination therapy.

Statistical performance of image cytometry for DNA, lipids, cytokeratin, & CD45 in a model system for circulation tumor cell detection

Detection of circulating tumor cells (CTCs) in a blood sample is limited by the sensitivity and specificity of the biomarker panel used to identify CTCs over other blood cells. In this work, we present Bayesian theory that shows how test sensitivity and specificity set the rarity of cell that a test can detect. We perform our calculation of sensitivity and specificity on our image cytometry biomarker panel by testing on pure disease positive (D+) populations (MCF7 cells) and pure disease negative populations (D−) (leukocytes). In this system, we performed multi‐channel confocal fluorescence microscopy to image biomarkers of DNA, lipids, CD45, and Cytokeratin. Using custom software, we segmented our confocal images into regions of interest consisting of individual cells and computed the image metrics of total signal, second spatial moment, spatial frequency second moment, and the product of the spatial‐spatial frequency moments. We present our analysis of these 16 features. The best performing of the 16 features produced an average separation of three standard deviations between D+ and D– and an average detectable rarity of ∼1 in 200. We performed multivariable regression and feature selection to combine multiple features for increased performance and showed an average separation of seven standard deviations between the D+ and D– populations making our average detectable rarity of ∼1 in 480. Histograms and receiver operating characteristics (ROC) curves for these features and regressions are presented. We conclude that simple regression analysis holds promise to further improve the separation of rare cells in cytometry applications.

Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.