Publications by authors named "Awtar Krishan"

40 Publications

Novel GHRH antagonists suppress the growth of human malignant melanoma by restoring nuclear p27 function.

Cell Cycle 2014 ;13(17):2790-7

a Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Research and Education ; Miami , FL USA.

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitro using the MTS assay. The effect of MIA-690 (5 μg/day 28 d) was further evaluated in vivo in nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 μM, and 19.2% at 5 μM, P < 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P < 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P < 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitro and in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.
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http://dx.doi.org/10.4161/15384101.2015.945879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615138PMC
July 2015

Chameleon dyes which change their color on excitation.

Authors:
Awtar Krishan

Cytometry A 2013 May;83(5):422-3

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http://dx.doi.org/10.1002/cyto.a.22279DOI Listing
May 2013

Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist.

Proc Natl Acad Sci U S A 2013 Feb 28;110(7):2617-22. Epub 2013 Jan 28.

Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Research and Education, Miami, FL 33125, USA.

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 μg/d; and a 18.4% reduction with 50 μg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κβ/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.
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http://dx.doi.org/10.1073/pnas.1222355110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574942PMC
February 2013

Mechanisms of synergism between antagonists of growth hormone-releasing hormone and antagonists of luteinizing hormone-releasing hormone in shrinking experimental benign prostatic hyperplasia.

Prostate 2013 Jun 31;73(8):873-83. Epub 2012 Dec 31.

Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Research and Education, Miami, Florida 33125, USA.

Background: Benign prostatic hyperplasia (BPH) affects aging men. Combined therapy with antagonists of growth hormone-releasing hormone (GHRH) and of luteinizing hormone-releasing hormone (LHRH or GnRH) induces prostate shrinkage in rat models. We investigated the mechanisms of action of this combination on cell cycle traverse and expression of prostatic genes.

Methods: Effects of GHRH antagonist, JMR-132 (40 µg/day), the LHRH antagonist, cetrorelix (0.625 mg/kg), and their combination were evaluated on testosterone-induced benign prostatic hyperplasia in male Wistar rats. Influence of JMR-132, cetrorelix, and their combinations on cell viability was assessed by MTS assay in BPH-1 human prostate epithelial cells and WPMY-1 normal prostate stromal cells. Cell cycle was analyzed by laser flow cytometry. Real-time PCR arrays were performed.

Results: The combination of antagonists caused marked shrinkage of rat prostate (29.5%). In vitro, JMR-132 plus cetrorelix (both 5µM) produced synergistic (57.4%) inhibition of growth of BPH-1 cells, but a lesser inhibition (46%) of WPMY-1 cells. Co-treatment of with JMR-132 plus cetrorelix induced a significant increase of BPH-1 cells blocked in S-phase plus cells with lower G0 /G1 and G2 /M DNA content. Significant changes in expression of >40 gene transcripts related to growth factors, inflammatory cytokines, and signal transduction were identified.

Conclusions: GHRH antagonist and LHRH antagonist combination potentiates rat prostate weight reduction and synergistically inhibits of growth of BPH-1 leading to cell cycle arrest in S-phase. These effects were lesser in normal stromal prostate cell line, WPMY-1. Our findings suggest that GHRH antagonists could be useful for BPH therapy, possibly in combination with LHRH antagonists.
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http://dx.doi.org/10.1002/pros.22633DOI Listing
June 2013

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer.

Cell Cycle 2012 Nov 24;11(22):4203-10. Epub 2012 Oct 24.

Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center and South Florida Veterans Affairs Foundation for Research and Education, Miami, FL, USA.

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G 1 fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40-55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56-85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.
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http://dx.doi.org/10.4161/cc.22498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524216PMC
November 2012

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers.

Cell Cycle 2012 Jul 1;11(13):2518-25. Epub 2012 Jul 1.

Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, USA.

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G 2/M and cells with lower G(0)/G(1) DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14-27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.
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http://dx.doi.org/10.4161/cc.20900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404878PMC
July 2012

Click-iT proliferation assay with improved DNA histograms.

Curr Protoc Cytom 2010 Apr;Chapter 7:Unit7.36

University of Miami Miller School of Medicine, Miami, Florida, USA.

The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.
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http://dx.doi.org/10.1002/0471142956.cy0736s52DOI Listing
April 2010

Targeted cytotoxic somatostatin analog AN-162 inhibits growth of human colon carcinomas and increases sensitivity of doxorubicin resistant murine leukemia cells.

Cancer Lett 2010 Aug 13;294(1):35-42. Epub 2010 Feb 13.

Department of Internal Medicine, General Hospital Oberndorf, Teaching Hospital of the Paracelsus Private Medical University of Salzburg, Paracelsusstrasse 37, Oberndorf, Austria.

The effect of the targeted cytotoxic somatostatin (SST) analog AN-162, consisting of doxorubicin (DOX) conjugated to SST carrier RC-121, was investigated on the growth of human colorectal cancer (CRC) cell lines HT-29, HCT-15, and HCT-116 and a DOX-resistant mouse leukemia cell line P388/R84. mRNA for SST-receptors and high affinity binding sites for SST were detected in all CRC cell lines and in P388/R84 cells. In contrast to DOX alone, AN-162 blocked HCT-116 cells and P388/R84 cells in S/G2 phase and increased the number of apoptotic cells. In vivo, AN-162 reduced the volume of CRC xenografts more effectively than its unconjugated components. Our results suggest that AN-162 inhibits growth of experimental CRC more effectively than DOX and increases sensitivity of DOX resistant human leukemia cells.
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http://dx.doi.org/10.1016/j.canlet.2010.01.018DOI Listing
August 2010

Electronic volume, aldehyde dehydrogenase, and stem cell marker expression in cells from human peripheral blood apheresis samples.

Cytometry B Clin Cytom 2010 Mar;78(2):123-9

Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida, USA.

Background: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples.

Methods: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34, CD90, CD117, and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression.

Results: In ALDH1(bright)/SSC(low) cells, 0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)).

Conclusions: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression.
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http://dx.doi.org/10.1002/cyto.b.20505DOI Listing
March 2010

ALDH(+)/CD44(+)/CD24(-) expression in cells from body cavity fluids.

Cytometry B Clin Cytom 2010 May;78(3):176-82

Department of Pathology (R-71), Miller School of Medicine, University of Miami, Miami, Florida 33101, USA.

Background: Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44(+)/CD24(-)) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity fluids of female patients suspected to have a malignancy.

Methods: Cells from peritoneal and pleural fluids of 100 female patients were examined by diagnostic cytology and analyzed by laser flow cytometry for enhanced ALDH1 expression. Cells from 36 body cavity fluids with ALDH1(bright) fluorescence were then analyzed for the expression of CD44 and CD24 markers.

Results: In samples positive for malignancy, ALDH1(bright) cells with both SSC(low) and SSC(high) were seen. In 15 body cavity fluids positive for malignancy, the percentage of ALDH1(bright) cells ranged from 0.26 to 6.34% of the total cells. The percentage of ALDH1(bright) cells with CD44(+)/CD24(-) expression in these samples ranged from 0.02 to 3.66%. ALDH1(bright) cells with CD44(+)/CD24(-) expression were also present in body cavity fluids of patients in whom diagnostic cytology could not detect any malignancy. However, the percentage of ALDH1(bright) and CD44(+)/CD24(-) cells amongst the 21 body cavity fluids with negative cytology was lower than that of samples with malignancy.

Conclusions: Expression of ALDH1(bright) and the CD44(+)/CD24(-) phenotype in body cavity fluids in which diagnostic cytology could not find any malignant cells suggests that this phenotype may not be restricted to the putative breast tumor stem cells. It is possible that only subsets of cells with this phenotype are the putative breast tumor stem cells.
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http://dx.doi.org/10.1002/cyto.b.20509DOI Listing
May 2010

Flow immunocytochemistry of marker expression in cells from body cavity fluids.

Cytometry A 2010 Feb;77(2):132-43

Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida, USA.

Diagnostic cytology based on the examination of cells from body cavity fluids misses approximately 50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non-malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1(pos)/CD44(pos)/CD24(neg) expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.
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http://dx.doi.org/10.1002/cyto.a.20824DOI Listing
February 2010

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells.

Cell Cycle 2009 Oct 3;8(19):3149-56. Epub 2009 Oct 3.

Department of Internal Medicine, General Hospital Oberndorf, Oberndorf, Austria.

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10 muM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (Delta Psi m). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21(Waf1/Cip1) was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p < 0.05) and extended tumor doubling time (p < 0.001). Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21(Waf1/Cip1) mediated S-phase arrest along with apoptosis involving the intrinsic pathway.
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http://dx.doi.org/10.4161/cc.8.19.9698DOI Listing
October 2009

Triple-negative breast cancers express receptors for luteinizing hormone-releasing hormone (LHRH) and respond to LHRH antagonist cetrorelix with growth inhibition.

Int J Oncol 2009 Oct;35(4):789-96

The Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center and South Florida VA Foundation for Research and Education, Miami, FL 33125, USA.

The aim of the present study was to evaluate the expression of receptors for luteinizing hormone-releasing hormone (LHRH) in human specimens of triple-negative breast cancers (TNBC). In addition, we used in vitro and in vivo models of TNBC to investigate if these receptors are suitable targets for the treatment with the LHRH antagonist cetrorelix. Receptors for LHRH were expressed in all tumor samples and in the TNBC cell lines HCC1806 and HCC1937. The proliferation of both TNBC cell lines was significantly inhibited in vitro by 1 microM cetrorelix. Injections of 3 mg cetrorelix on day 1 and 21 resulted in a significant growth inhibition of HCC1806 tumors xenografted into nude mice. Tumors of mice treated with cetrorelix expressed less mRNA for EGFR and HER3 receptors than untreated tumors. After treatment of cells with Cetrorelix a flow cytometric analysis of the cell cycle revealed a decrease in S-phase. Given the low toxicity and clinical availability of cetrorelix, this peptide antagonist should be considered for phase II studies in patients with advanced TNBC.
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http://dx.doi.org/10.3892/ijo_00000391DOI Listing
October 2009

Click-iT assay with improved DNA distribution histograms.

Cytometry A 2009 Oct;75(10):862-5

Department of Pathology, University of Miami Miller School of Medicine, Miami 33101, Florida, USA.

The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.
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http://dx.doi.org/10.1002/cyto.a.20780DOI Listing
October 2009

Hormone receptor-related gene polymorphisms and prostate cancer risk in North Indian population.

Mol Cell Biochem 2008 Jul 16;314(1-2):25-35. Epub 2008 May 16.

Department of Biotechnology, Panjab University, Chandigarh, India.

The purpose of this study was to analyse the frequency and type of mutations in the coding region of androgen receptor (AR) and to determine the role of polymorphisms in the intron 1 of ERalpha, exon 5 of ERbeta, intron 7 of progesterone, exon 7 of the aromatase (CYP19) and exon 9 of VDR genes in the risk of prostate cancer. PCR-RFLP analysis of all above the genes was on 100 prostate cancer patients and an equal number of matching controls. The study also included PCR-SSCP analyses of exons 2-8 of AR gene. The genotype containing -/- allele of ERalpha gene was statistically significant for the risk of prostate cancer pose (OR, 2.70; 95% CI, 1.08-6.70, P = 0.032) Rr genotype of ERbeta gene also have a higher risk (OR, 1.65; 95% CI, 0.52-5.23) for prostate cancer. The Cys allele of CYP19 gene was also associated with statistically significant increased risk of prostate cancer (OR; 2.28, 95% CI, 1.20-4.35, P = 0.012). tt genotype of codon 352 of VDR gene showed an OR of 0.43 for (95% CI, 0.13-1.39) and an OR for Tt genotype was 0.65 (95% CI, 0.36-1.16). Taken together, the results showed that in North Indian population, ERalpha and CYP19 genes may be playing a role in the risk of prostate cancer.
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http://dx.doi.org/10.1007/s11010-008-9761-1DOI Listing
July 2008

Electronic volume of CD34 positive cells from peripheral blood apheresis samples.

Cytometry B Clin Cytom 2008 May;74(3):182-8

Department of Pathology, Jackson Memorial Medical Center and Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami 33101, Florida, USA.

Background: The authors have used a flow analyzer to measure electronic cellular volume of peripheral blood hematopoietic stem/progenitor cells obtained by granulocyte-colony stimulating factor (G-CSF) mobilization and apheresis (HPC-A) of patients with hematological malignancies.

Methods: Fifty three apheresis samples stained with CD45-fluorescein isothiocyanate (FITC) and CD34-R-phycoerythrin (PE)-labeled antibodies after erythrocyte lysis with BD FACS Lysing Solution were analyzed for electronic cell volume and two-color FITC and PE fluorescence.

Results: Lymphocytes, monocytes and granulocytes in the HPC-A samples had a mean electronic volume of 414, 797, and 670 microm(3), respectively corresponding to cell diameter of 9.25, 11.5, and 10.85 microm. In 53 HPC-A samples analyzed, the mean electronic volume of the CD34 positive mononuclear cells was 407 microm(3) while the CD45 positive cells had mean volume of 453 microm(3).

Conclusions: CD34 positive stem/progenitor cells have a smaller volume and diameter than CD45 positive mononuclear cells in HPC-A samples. In the present study the CD34 stem/progenitor cells had a considerably larger diameter than that of stem cells previously reported in the literature. With the availability of electronic cell volume as a parameter in flow cytometric analysis, further studies can be carried out to correlate stem cell volume with specific phenotypic marker expression.
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http://dx.doi.org/10.1002/cyto.b.20399DOI Listing
May 2008

Cellular volume and marker expression in human peripheral blood apheresis stem cells.

Cytometry A 2008 Feb;73(2):160-7

Department of Pathology, University of Miami Miller School of Medicine, Sylvester Cancer Center, Miami, Florida 33101, USA.

Coulter volume is far more accurate measure of cell volume than forward angle light scatter. In this report, we have used Coulter volume to determine the mean cell volume and diameter of normal human peripheral blood cells and hematopoietic progenitor cells obtained by apheresis (HPC-A) from patients with hematological malignancies. Fresh peripheral blood samples (treated with Beckman Coulter IMMUNOPrep erythrocyte lysis solution), HPC-A samples (treated with BD Biosciences FACSLysing solution), or processed by Ficoll Hypaque sedimentation method were stained with CD45-FITC and PE-labeled CD34, CD90, CD117, and CD133 antibodies and analyzed for electronic volume and two color fluorescence. The mean electronic volume and diameter of mononuclear cells from fresh peripheral blood samples prepared with IMMUNOPrep were lymphocytes (191 microm(3), 7.16 microm), monocytes (370 microm(3), 9.91 microm), and granulocytes (328 microm(3), 8.56 microm). In mononuclear cells of HPC-A samples prepared by Histopaque-1077 sedimentation, the lymphocytes had volume and diameter of 311 microm(3), 8.4 microm, monocytes were 486 microm(3), 9.76 microm, and granulocytes were 515 microm(3), 9.95 microm. In contrast, HPC-A samples prepared after lysis with FACSLysing solution had mean electronic volume and diameter of lymphocytes (414 microm(3), 9.25 microm), monocytes (797 microm(3), 11.5 microm), and granulocytes (670 microm(3), 10.85 microm). Cell volume of mononuclear cells in the HPC-A samples prepared by Histopaque-1077 sedimentation method was correlated with the expression of stem cell markers CD34, CD90, CD117, and CD133. CD90 positive cells had the smallest mean electronic volume of 299.93 microm(3) when compared with cells with positive expression of CD133 (322 microm(3)), CD117 (349 microm(3)), CD34 (407 microm(3)), and CD45 (453 microm(3)). Correlation of cell volume with stem cell marker expression may allow for the identification of small stem cells, which may not express the conventional markers used for the identification of stem cells in HPC-A samples.
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http://dx.doi.org/10.1002/cyto.a.20524DOI Listing
February 2008

What links cod liver oil, spinach, and flow cytometry? Flow cytometric detection and quantitation of folic acid receptors in tumor cells.

Authors:
Awtar Krishan

Cytometry A 2007 Nov;71(11):897-8

Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.

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http://dx.doi.org/10.1002/cyto.a.20467DOI Listing
November 2007

Are increased tumor aneuploidy and heightened cell proliferation along with heterogeneity associated with patient outcome for carcinomas of the uterine cervix? A combined analysis of subjects treated in RTOG 9001 and a single-institution trial.

Int J Radiat Oncol Biol Phys 2008 Jan 24;70(1):111-7. Epub 2007 Oct 24.

Department of Radiation Oncology, University of Miami Miller School of Medicine, 1475 NW 12th Avenue, D-31, Miami, FL 33136, USA.

Purpose: To look for possible associations between measurements of DNA index (DI), S-phase fraction (SPF), and tumor heterogeneity (TH) using flow cytometry and overall survival for patients with invasive cervical carcinoma treated with definitive irradiation.

Methods And Materials: A total of 57 patients with International Federation of Obstetrics and Gynecology Stages IB(2) through IVB cervical carcinomas treated with definitive radiotherapy with or without concurrent chemotherapy were enrolled into this registry study that involved flow cytometric analysis of fresh tissue from each cervical cancer obtained by pretreatment biopsy. These specimens were evaluated for DNA aneuploidy (DI 1.5), SPF (15%), and TH (uniploid vs. multiploid).

Results: In these analyses 27 of the patients were treated in Radiation Therapy Oncology Group protocol 9001, and an additional 30 were offered chemoradiation at a single institution. Forty-one patients had DI 1.5. Twenty-nine patients had SPF 15%, and 2 had no determinable SPF. Forty-three patients had uniploid and 14 multiploid tumors. The 4-year estimated overall survival rate for the entire study cohort was 62% (95% confidence interval 48%-74%). With a median follow-up of 3.7 years, there were no observable associations by univariate analysis for DI, SPF, or TH concerning patient survival.

Conclusions: There were no statistically significant associations among DI, SPF, or TH and patient outcome. Additional studies are indicated to identify tumor biomarkers that could predict patients at risk for disseminated disease.
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http://dx.doi.org/10.1016/j.ijrobp.2007.05.069DOI Listing
January 2008

The minimal instrumentation requirements for Hoechst side population analysis: stem cell analysis on low-cost flow cytometry platforms.

Stem Cells 2006 Nov 3;24(11):2573-81. Epub 2006 Aug 3.

NPE Systems, Pembroke Pines, Florida, USA.

The Hoechst side population (SP) technique is a critical method of identifying stem cells and early progenitors in rodent, nonhuman primate, and human hematopoietic and nonhematopoietic tissues. In this technique, the cell-permeable DNA-binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells and early progenitors subsequently pump this dye out via an ATP-binding cassette membrane pump-dependent mechanism, resulting in a low-fluorescence "tail" (the SP) when the cells are analyzed by flow cytometry. This population contains stem cells and early progenitors. One significant drawback of this method is the requirement of an UV laser to excite the Hoechst 33342. Unfortunately, flow cytometers equipped with UV sources are expensive to own and operate and are not readily available to many laboratories or institutions. In the interests of designing a less expensive flow cytometric system for stem cell analysis, we determined the minimum UV excitation and instrumentation requirements for measuring Hoechst SP. Less than 3 mW of UV laser output was required for adequate resolution of Hoechst SP on two cuvette-based flow cytometers, one of which was a simple, inexpensive benchtop analyzer (the Quanta Analyzer; NPE Systems). Furthermore, Hoechst SP could also be adequately resolved on this epifluorescence-based cytometer platform using two nonlaser UV sources, a mercury arc lamp with a UV bandpass filter and a UV-emitting light-emitting diode. These results suggest that an economical flow cytometric system can be designed that is capable of resolving Hoechst SP, with a cost far lower than most UV laser-equipped commercial systems. An inexpensive system of this type would make Hoechst SP analysis available to a much broader group of stem cell investigators.
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http://dx.doi.org/10.1634/stemcells.2006-0266DOI Listing
November 2006

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis.

Diagn Cytopathol 2006 Aug;34(8):528-41

Department of Pathology, University of Miami Miller School of Medicine, Miami, Florida 33101, USA.

Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of malignant cells in body cavity fluids.
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http://dx.doi.org/10.1002/dc.20496DOI Listing
August 2006

Androgen & vitamin D nuclear receptor expression in archival breast tumour samples.

Indian J Med Res 2006 Jan;123(1):73-82

Department of Radiobiology, Life Sciences Center, Kasturba Medical College, Manipal, India.

Background & Objective: Breast tumour cells have receptors for androgen and vitamin D and their clinical significance is not completely understood. Therefore, the present study was undertaken to analyze androgen and vitamin D receptor levels in human primary infiltrating ductal breast carcinomas (IDC) and benign breast tumour archival samples and to find out their correlation, if any, with the clinical findings.

Methods: Paraffin blocks of benign and malignant breast tumours were sectioned, deparaffinized, and nuclei released by pepsin digestion. After antigen retrieval, nuclei were stained with primary antibodies for androgen or vitamin D receptors and secondary fluorescein isothiocyanate (FITC) labeled antibodies and propidium iodide respectively, to quantitative receptor expression and DNA content by flow cytometry.

Results: Androgen receptor positive nuclei ranged from 16-66 per cent in the IDC tumours as compared to 36-67 per cent in the benign tumours. Based on flow cytometric comparison of AR expression in AR positive and negative cell lines established earlier, 24 of 28 tumours from postmenopausal women were AR positive compared to all benign tumours and 32 of 33 tumours from pre-menopausal patients. Vitamin D receptor positive nuclei ranged from 14-89 and 2-75 per cent in IDC and benign tumours, respectively. All pre- or post-menopausal tumours were VDR positive as compared to 10 of 15 benign tumours that were VDR positive. No correlation was seen between nuclear androgen and vitamin D receptor expression of the IDC or benign tumours. There was a positive correlation between per cent of receptor positive nuclei and antigen density as measured by ratio of the mean log fluorescence channel value (MFC). No statistically significant correlation was found between nuclear receptor expression (per cent positive nuclei or antigen density) with that of tumour stage, lymph node status, tumour grade, patient age or menopausal status.

Interpretation & Conclusion: There was no significant correlation between androgen or vitamin D receptor expression and clinical findings. The expression of AR and VDR and the antigen density in the nuclei of the archival breast tumour samples were highly variable because of the tumour heterogeneity. Future studies with fresh biopsy samples of tumour on AR and VDR levels and their up- or down-regulation may be useful while stratifying the patients for hormonal therapy.
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January 2006

Efficacy of 2-halogen substituted D-glucose analogs in blocking glycolysis and killing "hypoxic tumor cells".

Cancer Chemother Pharmacol 2006 Dec 23;58(6):725-34. Epub 2006 Mar 23.

School of Medicine and Sylvester Cancer Center, The University of Miami, Miami, FL, USA.

Purpose: Since 2-deoxy-D-glucose (2-DG) is currently in phase I clinical trials to selectively target slow-growing hypoxic tumor cells, 2-halogenated D-glucose analogs were synthesized for improved activity. Given the fact that 2-DG competes with D-glucose for binding to hexokinase, in silico modeling of molecular interactions between hexokinase I and these new analogs was used to determine whether binding energies correlate with biological effects, i.e. inhibition of glycolysis and subsequent toxicity in hypoxic tumor cells.

Methods And Results: Using a QSAR-like approach along with a flexible docking strategy, it was determined that the binding affinities of the analogs to hexokinase I decrease as a function of increasing halogen size as follows: 2-fluoro-2-deoxy-D-glucose (2-FG) > 2-chloro-2-deoxy-D-glucose (2-CG) > 2-bromo-2-deoxy-D-glucose (2-BG). Furthermore, D-glucose was found to have the highest affinity followed by 2-FG and 2-DG, respectively. Similarly, flow cytometry and trypan blue exclusion assays showed that the efficacy of the halogenated analogs in preferentially inhibiting growth and killing hypoxic vs. aerobic cells increases as a function of their relative binding affinities. These results correlate with the inhibition of glycolysis as measured by lactate inhibition, i.e. ID50 1 mM for 2-FG, 6 mM for 2-CG and > 6 mM for 2-BG. Moreover, 2-FG was found to be more potent than 2-DG for both glycolytic inhibition and cytotoxicity.

Conclusions: Overall, our in vitro results suggest that 2-FG is more potent than 2-DG in killing hypoxic tumor cells, and therefore may be more clinically effective when combined with standard chemotherapeutic protocols.
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http://dx.doi.org/10.1007/s00280-006-0207-8DOI Listing
December 2006

Influence of number of CAG repeats on local control in the RTOG 86-10 protocol.

Am J Clin Oncol 2006 Feb;29(1):14-20

Department of Radiation Oncology, University of Miami School of Medicine, Miami, FL 33136, USA.

Objectives: The number of CAG repeats on the androgen receptor (AR) gene is inversely proportional to transcriptional activity. The purpose of this study was to determine if short-term androgen deprivation therapy (RT + HT) can improve outcome in patients with tumors with short CAG repeats (<19).

Materials And Methods: Prostate cancer patients were randomized to receive either radiotherapy (RT) alone or (RT + HT) in the RTOG 86-10 study. CAG repeats were measured in 94 tumor specimens (21%; test cohort) of the 456 (parent cohort) analyzable cases. AR flow cytometry measurements were done on 13 patients. The effect on local failure (LF), distant metastases (DM), prostate cancer survival (PSS), and overall survival (OS) was studied.

Results: Pretreatment characteristics and assigned treatment arm were not significantly different between the parent and test groups except for a significantly higher risk of death (P = 0.049) in the test group. The median CAG repeat was 19. There were no significant differences in stage, or Gleason score between high (19 or greater) and low CAG (<19) patients within each treatment group. Number of CAG repeats alone did not significantly influence LF, DM, PSS, and OS. However, when the CAG repeat outcome was studied in conjunction with androgen deprivation therapy, patients with CAG <19 who received H + RT had improved local control as compared with patients who received RT alone (P = 0.026, 5-year rates 4.6% versus 36.4%) and improved local control over patients with CAG > or =19 that received H + RT (P = 0.028).

Conclusions: Patients with short CAG repeats show a local control benefit with short-term androgen deprivation therapy, but no improvement in survival.
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http://dx.doi.org/10.1097/01.coc.0000195085.34162.88DOI Listing
February 2006

DAPI fluorescence in nuclei isolated from tumors.

J Histochem Cytochem 2005 Aug;53(8):1033-6

Division of Experimental Therapeutics, Radiation Oncology Department, Sylvester Cancer Center, University of Miami, School of Medicine, Miami, FL 33136, USA.

In DNA histograms of some human solid tumors stained with nuclear isolation medium--4,6-diamidino-2-phenylindole dihydrochloride (NIM-DAPI), the coefficient of variation (CV) of the G0/G1 peak was broad, and in nuclear volume vs DNA scattergrams, a prominent slope was seen. To determine the cause for this, nuclei from frozen breast tumors were stained with NIM-DAPI and analyzed after dilution or resuspension in PBS. In two-color (blue vs red) analysis, most of the slope and broad CV was due to red fluorescence of nuclei stained with NIM-DAPI, which was reduced on dilution or resuspension in PBS, resulting in elimination of the slope and tightening of the CV.
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http://dx.doi.org/10.1369/jhc.4B6563.2005DOI Listing
August 2005

Flow cytometric monitoring of fluorescent drug retention and efflux.

Methods Mol Med 2005 ;111:149-66

Division of Experimental Therapeutics, University of Miami, FL, USA.

Laser flow cytometry has been used for monitoring cellular retention of fluorescent drugs such as fluorescent anticancer antibiotics (e.g., doxorubicin) and fluorochromes used for the detection of cellular drug efflux and resistance (e.g., rhodamine 123, Hoechst 33342). Multiparametric flow cytometry can be used for identification of tumor cell subpopulations based on their drug retention profiles with or without the presence of an efflux blocker. This rapid procedure can be used for identification of tumor cells with the drug-resistance phenotype based on drug efflux as well as for efflux blockers that may block efflux of a chemotherapeutic agent and thus increase cellular retention and sensitivity. It has been reported recently that some of the bone marrow stem cells (SP cells) efflux the Hoechst 33342 fluorochrome and thus can be rapidly identified by comparing red vs blue fluorescence in the presence or absence of an efflux blocker such as verapamil. The present chapter discusses some of the flow cytometric methods used for the study of cellular drug retention and the artifacts that may arise in such analysis.
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http://dx.doi.org/10.1385/1-59259-889-7:149DOI Listing
June 2005

DNA index, genome size, and electronic nuclear volume of vertebrates from the Miami Metro Zoo.

Cytometry A 2005 May;65(1):26-34

Division of Experimental Therapeutics (R-71), Department of Radiation Oncology, University of Miami School of Medicine, Miami, Florida 33101, USA.

Background: Flow cytometry is a rapid and reliable method for measuring nuclear DNA content and genome size. Fluorochrome binding characteristics, sample preparation and differences in DNA condensation, and availability of binding sites can cause variations in results obtained.

Methods: Blood samples from 82 vertebrate species were collected in 10% dimethyl sulfoxide and stained with propidium iodide/hypotonic citrate or 4,6-diamidino-2-phenylindole dihydrochloride for analysis of DNA content and electronic nuclear volume (ENV). Trout red blood cells (TRBCs), human peripheral blood lymphocytes, and human buccal cavity cells were used as internal standards.

Results: Mean fluorescence channel (MFC) values of TRBC and buccal cavity cells used as internal standards were stable at 15 to 120 min of propidium iodide staining. TRBCs mixed with other cells especially human peripheral blood cells showed an increase in MFC. ENV and MCF values were less variable in different species of birds than in reptiles or mammals. Genome size based on use of buccal cavity cells as the internal standard showed a high degree of correlation with previous reports.

Conclusions: Proper selection and use of internal standards and sample preparation are essential for reliable determination of DNA content and genome size in vertebrates by flow cytometry.
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http://dx.doi.org/10.1002/cyto.a.20130DOI Listing
May 2005

Androgen and vitamin D receptor expression in archival human breast tumors.

Cytometry B Clin Cytom 2004 Mar;58(1):53-60

Department of Radiation Oncology, University of Miami, School of Medicine, Miami, Florida 33101, USA.

Background: The present study was undertaken for quantitation of androgen (AR) and vitamin D (VDR) receptor expression in human male and female breast tumors by flow cytometry.

Methods: Nuclei isolated from sections of paraffin-embedded tumors by pepsin digestion were treated for antigen unmasking and incubated with antibodies to AR and VDR. Flow cytometric analysis was used to determine the percentage of receptor-positive nuclei with fluorescence greater than 95% of the isotype nuclei. Mean log fluorescence channel values were used for comparing antigen density of the isotype and the antibody-treated nuclei.

Results: Six of 23 female breast tumors had aneuploid DNA content. Nineteen of 20 estrogen receptor-positive female tumors by immunohistochemical analysis (IHC) were also AR positive by flow analysis. Aneuploid subpopulations had higher percentages of AR-positive nuclei than did diploid populations. Eight of 33 male breast tumors had aneuploid DNA content. Twenty-three of 33 male breast tumors were AR positive by flow analysis compared with six that were AR positive by IHC. Six AR-positive (IHC) male tumors were also AR positive by flow analysis. VDR expression was higher in diploid female tumors than in aneuploid tumors.

Conclusions: Lack of a strong correlation between IHC and flow analysis may be due to differences in criteria used for identification of receptor-positive and -negative tumors by the two methods.
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http://dx.doi.org/10.1002/cyto.b.10060DOI Listing
March 2004

Synergistic cytotoxicity of pyrazoloacridine with doxorubicin, etoposide, and topotecan in drug-resistant tumor cells.

Clin Cancer Res 2004 Feb;10(3):1160-9

Department of Advanced Therapeutics, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3.

Pyrazoloacridine (NSC 366140, PD115934, PZA) is a new class of acridine anticancer agents under investigation in Phase II clinical trials in patients with advanced cancers. Although poor responses in patients to the treatment with PZA alone have been observed, this class of agents remains of interest because of its distinct mechanism of action from other topoisomerase poisons. Therefore, the combination of PZA with conventional anticancer agents presents an attractive approach to treat drug-resistant human tumors. In the present study, the cytotoxic effects of PZA combined with doxorubicin, topotecan, and etoposide were determined using paired parental and doxorubicin-resistant human colon carcinoma (SW-620 and SW620/AD-300) and breast cancer cell lines (MCF-7 and MCF-7/TH). Cytotoxicity was measured by soft agar clonogenic assays. Dose effect and combination effects were analyzed by the method of Chou and Talalay. The combination of PZA with doxorubicin, topotecan, and etoposide in fixed ratios demonstrated synergistic cytotoxicity on both SW-620 and SW620/AD-300 cell lines. The combination of PZA with doxorubicin also exhibited synergistic cytotoxicity against both MCF-7 and MCF-7/TH cell lines. The mechanism of synergism appeared independent of topoisomerase I and II inhibition, and interference with protein-DNA complexes. Strategies to define optimal drug combinations are proving to be of significant value when considering potential clinical applications of new and established agents.
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http://dx.doi.org/10.1158/1078-0432.ccr-1044-3DOI Listing
February 2004
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