Sharmila K Makhija

University of Alabama at Birmingham, Birmingham, Alabama, United States

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Publications (29)136.96 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Human ovarian cancer is a highly lethal malignant neoplasm in woman with no effective treatment if conventional chemotherapy fails. In this regard, conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution to the development of CRAds was the introduction of tumor-selective viral replication to restrict amplification to the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells, killing the cells by cytolysis, leaving normal cells unaffected. However, to date, there have been limitations to the clinical application of these CRAd agents i.e. poor viral infectivity, poor tumor specificity and high toxicity. Here, we report the in vitro and in vivo comparison of four CRAd agents developed for ovarian cancer application, specifically, Ad-Delta24.F5/3, CRAd-C.F5/3, CRAd-M.F5/3 and CRAd-S.F5/3. All CRAd agents contained fiber knob chimeras of adenovirus serotype 3, which enhanced the viral infectivity at the transductional level via a non-Coxsackie-Adenovirus Receptor alternative pathway. In addition, these CRAds embodied distinct mechanisms for the achievement of replication specificity. Tumor cell killing was assessed by using an oncolytic assay and a cell viability assay (MTS) in vitro, while tumor growth was examined in a xenograft model in vivo by using a bioluminescent imaging assay. In addition, the replication rates of the CRAd agents were determined in human liver slices. Both the Ad-Delta24.F5/3 and CRAd-S.F5/3 were demonstrated to have higher tumor killing effects in tumor cells and a lower viral replication rate in human liver. These agents are thus excellent candidates for clinical trials of CRAd agents against human ovarian cancer.
    International Journal of Oncology 07/2008; 32(6):1179-88. · 3.03 Impact Factor
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    ABSTRACT: Hec1 (Highly Expressed in Cancer gene 1) has recently been shown to play an important role in the proper segregation of chromosomes during mitosis. Recently, an adenovirus delivery system carrying RNA interference (RNAi) of Hec1 has been reported in a cervical adenocarcinoma model. Adenoviral delivery systems, however, have the main limitation of poor viral infectivity due to lack of the native receptor, Coxsackie-Adenovirus Receptor (CAR), on the surface of tumor cells. We hypothesize that the viral infectivity of the adenovirus vector would be enhanced via a CAR-independent pathway by altering the targeting tropism, thus increasing the knockdown effect of Hec1 expression in ovarian carcinoma cells. Two adenoviruses (Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3), along with a negative control (Ad-siRNA-GAPDH.F5/3), were created using homologous recombination. HEY and SKOV3.ip1 cell lines were used to perform experiments. The following assays were then used to determine RNAi knockdown efficiency: (1) quantitative PCR (QPCR), (2) Western blot, (3) MTS assay, (4) Annexin V-FITC FACS, (5) crystal violet staining. In all experiments, a negative control served as a baseline measure. QPCR demonstrated a 2-log viral infectivity enhancement with Ad-siRNA-Hec1.F5/3 over Ad-siRNA-Hec1. QPCR at 72 h revealed mRNA knockdown induced by Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cells, respectively (71%/60%, and 32%/78% mRNA knockdown compared to negative control). Western blot revealed translational inhibition induced by both Hec1 Ads with the least knockdown seen with Ad-siRNA-GAPDH.F5/3. FACS analysis revealed increased annexin V positivity in RNAi-infected cells, suggesting a higher rate of apoptosis. MTS assay indicated increased cell death 8 days post-infection with Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cell lines, respectively (75% vs. 35% and 43% vs. 12% viable cells). Crystal violet staining revealed increased cell death with Ad-siRNA-Hec1.F5/3 in all tested cell lines. RNAi against Hec1 results in gene expression knockdown and apoptosis in vitro. The infectivity-enhanced adenovirus as delivery mechanism shows potential application in future gene therapy models of RNAi in ovarian cancer.
    Gynecologic Oncology 02/2008; 108(1):34-41. DOI:10.1016/j.ygyno.2007.08.096 · 3.69 Impact Factor
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    ABSTRACT: Conventional cancer treatments are not adequate for the majority of most patients stricken with squamous cell carcinomas of the head and neck (SCCHN). Conditionally replicating adenoviruses (CRAds) represent a promising new modality for treating of neoplastic diseases, including SCCHN. Specifically, CRAd agents infect tumor cells and selectively replicate within them, thus causing their death while sparing surrounding normal cells in the host. Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells, sparing normal host cells. However, to date there have been two main limitations to successful clinical application of these CRAd agents: poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.F5/3, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter, and the viral infectivity is enhanced by a fiber modification, F5/3, containing an Ad3 knob chimeric fiber protein. As expected, this agent improved both of the viral infectivity and tumor specificity as evaluated in established SCCHN tumor cell lines and in primary tumor tissues from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as described previously. Based on these data, the CRAd-CXCR4.F5/3 is a promising novel CRAd agent for SCCHN targeting with low host toxicity.
    International Journal of Oncology 12/2007; 31(5):1213-22. DOI:10.3892/ijo.31.5.1213 · 3.03 Impact Factor
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    ABSTRACT: Current virotherapy strategies for ovarian cancer have been hampered by limitations in target cell infectivity and nonspecific tissue replication. In an effort to circumvent these limitations, we evaluated various CRAds modified to incorporate novel capsid targeting motifs (RGD and chimeric Ad5/3) with a novel tissue-specific promoter (CXCR4). Two novel CRAds (Ad5-CXCR4-F5/3 and Ad5-CXCR4-RGD) were constructed via homologous recombination and verified by PCR and DNA sequencing. The infectivity and viral replication rates of these two CRAds were analyzed via quantitative real-time PCR (QRT-PCR) in cell line experiments using three ovarian cancer cell lines (SKOV3.ip1, Hey, and OV4) and compared to that achieved with a clinical grade CRAd (delta24-RGD) to be evaluated in a Phase I trial. Cytocidal effects were determined by crystal violet staining in these same cell lines infected with different concentrations of viral particles per cell (0, 0.1, 1, 10, 100, and 500). Additionally, viral replication was evaluated by QRT-PCR in primary ovarian cancer tissue slices from multiple patients with ovarian cancer as well as in primary human normal liver tissue slices in order to establish CRAd selectivity. All experiments incorporated appropriate controls and repeated in triplicate. Compared to RGD-capsid CRAds (delta24-RGD and CXCR4-RGD), the F5/3-capsid CRAd (CXCR4-F5/3) demonstrated significant improvements in infection rates (p=0.025, 0.006, and 0.006) in all ovarian cancer cell lines tested (SKOV3.ip1, Hey, and OV4, respectively). In addition to improved transduction of virus into the cells, the TSP CXCR4-based CRAds demonstrated improved viral replication. Specifically, CXCR4-F5/3 further enhanced viral replication 89-fold (p=0.009, 0.010, 0.003) in the same cancer cell lines. Furthermore, CXCR4-F5/3 showed a 4-log improvement in oncolytic potential over delta24-RGD. In the ex vivo primary ovarian tissue slices, CXCR4-F5/3 showed a 58-fold improvement in viral replication (p=0.005) compared to the clinical grade delta24-RGD. Both CXCR4-F5/3 and CXCR4-RGD demonstrated significant reduction of viral replication in normal liver slices (p=0.001). These data suggest that a dual targeted approach is feasible for the combined enhancement of infectivity and replication in ovarian cancer with a specificity that was attenuated in normal liver tissues. In fact, CXCR4-F5/3 outperformed our best CRAd agent to date nearly 60-fold in our most stringent ex vivo model of primary ovarian cancer tissue slices and suggests that this novel agent could be useful for the treatment of ovarian cancer.
    Gynecologic Oncology 05/2007; 105(1):113-21. DOI:10.1016/j.ygyno.2006.10.057 · 3.69 Impact Factor
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    ABSTRACT: The purpose of this study was to evaluate the biodistribution and toxicity of the tropism-modified infectivity-enhanced conditionally replicative adenovirus, Ad5-delta24-arginine-glycine-aspartate (RGD). Cohorts of cotton rats were treated intravenously or intraperitoneally for 3 consecutive days with 5 x 10(8) to 5 x 10(11) particles/kg of Ad5-delta24-RGD or controls and killed on day 8, 17, or 56. For biodistribution studies, tissue samples from 14 organ sites and serum samples were evaluated for the presence of virus with the use of quantitative polymerase chain reaction analysis. For toxicity experiments, tissue samples from more than 30 organ sites and serum samples were obtained for the assessment of vector-related tissue or laboratory effects. Ad5-delta24-RGD was noted in tested samples at days 8 and 17 in animals that were treated intravenously and intraperitoneally with clearance by day 56. There were lower copies of vector noted in the blood and liver specimens of intraperitoneally treated animals. Mild peritonitis histopathologic findings were noted in rats that were treated intraperitoneally with Ad5-delta24-RGD; pathologic findings did not vary significantly with dose, over time, or in comparison to that noted in animals that were treated with Ad5-delta24. These studies provide critical insights regarding Ad5-delta24-RGD dosing and anticipated toxicity for a planned clinical trial for ovarian cancer.
    American journal of obstetrics and gynecology 04/2007; 196(4):389.e1-9; discussion 389.e9-10. DOI:10.1016/j.ajog.2006.12.016 · 3.97 Impact Factor
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    ABSTRACT: Conventional treatments are not adequate for the majority of lung cancer patients. Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of neoplastic diseases, including non-small cell lung cancer. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect a new population of surrounding target cells, replicate again and eradicate the infected tumor cells while leaving normal cells unaffected. However, to date, there have been two main limitations to successful clinical application of these CRAd agents; i.e. poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.RGD, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter and the viral infectivity is enhanced by a capsid modification, RGD4C. This agent CRAd-CXCR4.RGD, as expected, improved both of the viral infectivity and tumor specificity as evaluated in an established lung tumor cell line and in primary tumor tissue from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as compared to three other promoters regularly used for targeting tumors. In addition, this agent has the potential of targeting multiple other tumor cell types. From these data, the CRAd-CXCR4.RGD appears to be a promising novel CRAd agent for lung cancer targeting with low host toxicity.
    Lung Cancer 03/2007; 55(2):145-56. DOI:10.1016/j.lungcan.2006.10.012 · 3.74 Impact Factor
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    ABSTRACT: Icodextrin, a novel glucose polymer solution utilized for peritoneal dialysis, has been demonstrated to have prolonged intraperitoneal (IP) instillation volumes in comparison to standard PBS solutions. In an animal model of ovarian cancer, we explored whether a survival advantage exists utilizing icodextrin rather than PBS as a delivery solution for an infectivity enhanced virotherapy approach. Initial experiments evaluated whether icodextrin would adversely affect replication of a clinical grade infectivity enhanced conditionally replicative adenovirus (Delta24-RGD). Virus was added to prepared blinded solutions of PBS or icodextrin (20%) and then evaluated in vitro in various human ovarian cancer cell lines (SKOV3.ip1, PA-1, and Hey) and in vivo in a SKOV3.ip1 human ovarian cancer IP murine model. Viral replication was measured by detecting adenovirus E4 gene levels utilizing QRT-PCR. Survival was subsequently evaluated in a separate SKOV3.ip1 ovarian cancer IP murine model. Cohorts of mice were treated in blinded fashion with PBS alone, icodextrin alone, PBS+Delta24-RGD, or icodextrin+Delta24-RGD. Survival data were plotted on Kaplan-Meier curve and statistical calculations performed using the log-rank test. There was no adverse affect of icodextrin on vector replication in the ovarian cancer cell lines nor murine model tumor samples evaluated. Median survival in the IP treated animal cohorts was 23 days for the PBS group, 40 days for the icodextrin group, 65 days for the PBS+Delta24-RGD group, and 105 days for icodextrin+Delta24-RGD (p=0.023). Of note, 5 of the 10 mice in the icodextrin+Delta24-RGD group were alive at the end of the study period, all without evidence of tumor (120 days). These experiments suggest that the use of dialysates such as icodextrin may further enhance the therapeutic effects of novel IP virotherapy and other gene therapy strategies for ovarian cancer. Phase I studies utilizing icodextrin-based virotherapy for ovarian cancer are currently in development.
    Gynecologic Oncology 01/2007; 103(3):985-9. DOI:10.1016/j.ygyno.2006.06.005 · 3.69 Impact Factor
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    ABSTRACT: Cholangiocarcinoma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to the clinical application of these CRAd agents, i.e. poor viral infectivity and tumor specificity. Here we report the construction of three new CRAd agents, CRAd-S.RGD, CRAd-S.F5/3 and CRAd-S.pk7, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD, F5/3 or pk7) in the adenovirus fiber region. These CRAd agents effectively target cholangiocarcinoma cells, induce strong cytoxicity in these cells in vitro and inhibit tumor growth in a murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting cholangiocarcinoma with low host toxicity. Such results should provide important insights into the identification of novel therapeutic strategies for cholangiocarcinoma.
    International Journal of Oncology 12/2006; 29(5):1319-29. DOI:10.3892/ijo.29.5.1319 · 3.03 Impact Factor
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    ABSTRACT: Mesothelioma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard is the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; viral infectivity and tumor specificity have been poor. Herein we report on two CRAd agents, CRAd-S.RGD and CRAd-S.F5/3, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD or F5/3) in the adenovirus fiber region. These CRAd agents effectively target human mesothelioma cell lines, induce strong cytoxicity in these cells in vitro, and viral replication in a H226 murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting mesothelioma with low host toxicity. These agents should provide important insights into the identification of novel therapeutic strategies for mesothelioma.
    Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 10/2006; 1(7):701-11. DOI:10.1097/01243894-200609000-00017 · 5.80 Impact Factor
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    ABSTRACT: Induction of tumor cell resistance to therapeutics has been a major obstacle in cancer therapy. Targeting of the death receptors by a natural ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or agonistic monoclonal antibodies against TRAIL receptor 1 (TRAIL-R1) or TRAIL receptor 2 (TRAIL-R2) has been thought to be a promising cancer therapy. To determine whether tumor cells are able to generate a resistance to apoptosis induced by an anti-TRAIL-R2 antibody, TRA-8, we examined the apoptotic response of human breast and ovarian cancer cell lines after treatment with TRA-8. Our results show that tumor cell resistance to TRA-8 can be induced by repeated treatment of tumor cells with low, non-apoptosis-inducing doses of TRA-8. Interestingly, the induced resistance to apoptosis was not due to a global apoptotic defect in tumor cells but rather a selective defect in the TRAIL-R2 signaling pathway. Whereas TRA-8-treated tumor cells developed a selective resistance to TRAIL-R2-mediated apoptosis, the apoptotic responses induced by TRAIL, an anti-TRAIL-R1 antibody (2E12), and other apoptotic stimuli were not impaired. The expression levels of cell surface TRAIL-R2 were not altered and mutations of TRAIL-R2 were not found in the resistant cells. The induced TRA-8 resistance was due to a selective blockade at the level of the death domain and could be reversed by a wide array of chemotherapeutic agents. Proteomic analysis of death-inducing signaling complex formation during TRA-8 treatment shows that the translocation of TRAIL-R2-associated apoptotic proteins was significantly altered. Our results suggest that the prevention of tumor cell resistance to therapeutic agents that target the death receptors must be taken into consideration.
    Cancer Research 10/2006; 66(17):8520-8. DOI:10.1158/0008-5472.CAN-05-4364 · 9.28 Impact Factor
  • Lung Cancer 10/2006; 54. DOI:10.1016/S0169-5002(07)70303-3 · 3.74 Impact Factor
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    ABSTRACT: Molecular Therapy (2006) 13, S425|[ndash]|S425; doi: 10.1016/j.ymthe.2006.08.1210 1105. A Phase I Clinical Trial of an IL-12 Gene Plasmid Formulated with a Novel Lipopolymeric Gene Delivery System for Intraperitoneal Treatment of Recurrent Ovarian Cancer Ronald D. Alvarez1, Mack N. Barnes1, Connie McCombs1, Sharmila Makhija1, Jason Fewell2, Danny H. Lewis2 and Kursheed Anwer21Ob/Gyn, The University of Alabama at Birmingham, Birmingham, AL2Expression Genetics, Inc., Huntsville, AL
    Molecular Therapy 04/2006; 13:S425-S425. DOI:10.1016/j.ymthe.2006.08.1210 · 6.43 Impact Factor
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    ABSTRACT: Despite advances in therapy, advanced ovarian cancer maintains a dismal overall survival of 15-30%. Thus, the need for novel therapeutic modalities exists. Gene therapy represents one such approach and the purpose of this review is to present a logical rationale for the investigation of gene therapy for the treatment of ovarian cancer. The different strategies of gene therapy (molecular chemotherapy (prodrugs), mutation compensation, immunotherapy approaches, altered drug sensitivity, and virotherapy) for cancer treatment are discussed separately with attention to investigations with clinical applicability. Furthermore, the different viral vectors utilized for improvements in targeted therapy are presented. The advancements, discovery, and shortcomings are reviewed which lend itself to future directions. These future directions involve coxsackie-adenovirus receptor (CAR) independent pathways to improve infectivity and specificity to ovarian tumor cells, the potential of utilizing gene therapy as an imaging modality in detecting cancer, and incorporating the recently described technique of RNA interference. Due to the advancements in detection and targeting of ovarian cancer, coupled with the containment to the intraperitoneal cavity, gene therapy remains a promising treatment modality for ovarian cancer.
    Current Gene Therapy 01/2006; 5(6):643-53. DOI:10.2174/156652305774964668 · 4.91 Impact Factor
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    ABSTRACT: Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect surrounding target cells, replicate and eradicate the infected tumor cells, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; i.e., both infectivity and tumor specificity are poor. Survivin protein is a novel member of the inhibitor of apoptosis (IAP) protein family, which plays an important role in the survival of cancer cells and progression of malignancies. Previous data have shown the survivin promoter has high activities in multiple cancer cells with a low activity in mouse liver. In this study, we propose an improved CRAd agent to circumvent the obstacles. We constructed a novel CRAd agent, CRAd-Survivin-RGD, which contains both the survivin promoter (either the short version, S-S, or the long version, S-L) to selectively drive E1 gene expression in tumor cells and a capsid modification and RGD4C to specifically enhance the tumor infectivity of CRAd agents. Both CRAd agents (S-S and S-L) showed high replication rates in the breast cancer cell line, MDA-MB-361, and low promoter activity in both normal mouse and human liver, thus signifying the CRAd agents have the phenotype of 'tumor on/liver off'. In cytocidal experiments, the CRAd agents demonstrated a high cytocidal effect on multiple cancer cell lines, including the breast cancer cell line, MDA-MB-231; the glioma cell line, D65, the melanoma cell line, MEL-28; and mesothelioma, Meso2374. The results also showed the tumor growth was dramatically inhibited by intertumoral administration of the CRAd agents in a breast cancer (MDA-MB-361) xenograft animal model. These data clearly demonstrate that CRAd-Survivin-RGD is a potential novel therapeutic agent for treatment in many, but not all, human cancers.
    International Journal of Oncology 08/2005; 27(1):237-46. DOI:10.3892/ijo.27.1.237 · 3.03 Impact Factor
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    ABSTRACT: Gene therapy is a novel therapy for melanoma. To date, however, there is still no powerful tumor specific promoter (TSP) to restrict the transgene expression in melanoma cells. In order to define a useful TSP for targeting in the context of melanoma gene therapy, four promoters, the cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (CXCR4), epithelial glycoprotein 2 (EGP-2), and survivin, were tested in both established melanoma cell lines and primary melanoma cells. We employed recombinant adenoviral vectors (reAds) each with a candidate TSP (the Cox-2, CXCR4, EGP-2, or survivin), a reporter luciferase gene, and a poly-A signal, all of which were inserted into the E1-deleted region. A reAdGL3Bcytomegalovirus (CMV), containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity. Luciferase activity was measured in multiple tumor cell lines and two primary melanoma cell cultures after infection with reAds. Human epithelial melanocytes, HEM, were used as normal control. In contrast to three other promoters, the survivin promoter exhibited the highest activities within both melanoma cell lines and primary melanoma cells, but not in HEMs. Additionally, the survivin promoter exhibited very low activities in major mouse organs including the liver, in vivo. EGP-2 is not active in melanoma; messenger RNA expressions were correlated to promoter activities both in melanoma cell lines and primary cell cultures. Thus, these data suggest that the survivin promoter achieved a 'tumor-on/liver-off' profile, and thus represents a potentially useful tumor-specific promoter with applications for transcriptional targeting of Ad vector-based cancer gene therapy or oncolysis to melanoma.
    Gene Therapy 03/2005; 12(4):330-8. DOI:10.1038/sj.gt.3302385 · 4.20 Impact Factor
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    ABSTRACT: Chlorhexidine has been proposed as a potent chemotherapeutic agent against oral bacteria. However, there are some inconsistent results regarding the usefulness of chlorhexidine mouthrinse as an antimicrobial for Streptococcus mutans. The purpose of this study was to investigate the effectiveness of combining oral rinses to reduce S. mutans levels in human saliva. Sixteen healthy adult subjects were randomly assigned to one of four rinse groups using a 4-cell crossover design. The groups rinsed twice a day for 7 days with one of the following: 0.12% chlorhexidine (PerioGard), 1.5% hydrogen peroxide (Peroxyl), a combined chlorhexidine+hydrogen peroxide, or water (control). Every 5 weeks, each group initiated a different rinse. Saline wash samples were collected on days 7 and 21 for assessment of S. mutans and total streptococci. No significant differences were seen in S. mutans levels among the groups; however, the levels of total streptococci on day 7 samples were significantly lower in the chlorhexidine and chlorhexidine+hydrogen peroxide groups than in the hydrogen peroxide and control groups. There was no additional decrease seen in S. mutans or total streptococci levels in the group receiving chlorhexidine+hydrogen peroxide compared to chlorhexidine alone. Sample variation was high throughout the study, with a significant trend toward lower counts as the study progressed. Adding hydrogen peroxide to the chlorhexidine mouthrinse did not result in a further decrease in S. mutans levels.
    Oral Microbiology and Immunology 03/2005; 20(1):31-4. DOI:10.1111/j.1399-302X.2004.00189.x · 2.81 Impact Factor
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    V Sahasrabuddhe, S Makhija
    International Journal of Gynecological Cancer 01/2005; 15(1):1-3. DOI:10.1111/j.1048-891X.2005.15001.x · 1.95 Impact Factor
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    ABSTRACT: Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 +/- 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood-brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery.
    Virology 08/2004; 325(1):116-28. DOI:10.1016/j.virol.2004.04.021 · 3.28 Impact Factor
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    ABSTRACT: Adenoviral vectors are considered to be good gene delivery vectors for cancer gene therapy due to their wide host tissue range and cell cycle-independent infectivity. However, the disadvantages include the lack of specificity for cancer cells and the high liver accumulation in vivo. The human CXCR4 gene is expressed at high levels in many types of cancers, but is repressed in the liver. We explored the CXCR4 promoter as a candidate to restrict adenoviral transgene expression to tumor cells with a low expression in host tissues. The luciferase activities in multiple cancer cell lines infected with recombinant adenovirus reAdGL3BCXCR4 or the control vector reAdGL3BCMV revealed that the CXCR4 promoter exhibited relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and two ovarian cancer cell lines, OVCAR-3 and SKOV3. ip1, 65% (P=0.0087), 16.7% (P=0.1) and 20% (P=0.0079) compared to that of the CMV promoter, respectively, and low expression, 4.9 and 0.1%, respectively, in both normal cell lines HFBC and HMEC. In addition, CXCR4 had a low expression of luciferase (0.32%) compared to that of the CMV promoter in mouse liver in vivo. The data also revealed that the CXCR4 promoter was a stronger tumor-specific promoter (TSP) than the Cox-2M promoter in primary melanomas obtained from two patients. The CXCR4 promoter is shown to have a 'tumor-on' and 'liver-off' status in vitro and in vivo, and CXCR4 may prove to be a good candidate TSP for cancer gene therapy approaches for melanoma and breast cancers.
    Gene Therapy 05/2004; 11(7):645-8. DOI:10.1038/sj.gt.3302089 · 4.20 Impact Factor
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    ABSTRACT: It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a "tumor on" and "liver off" profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.
    Cancer Gene Therapy 05/2004; 11(4):256-62. DOI:10.1038/sj.cgt.7700679 · 2.55 Impact Factor

Publication Stats

724 Citations
136.96 Total Impact Points

Institutions

  • 2002–2008
    • University of Alabama at Birmingham
      • • Department of Medicine
      • • Division of Gynecologic Oncology
      Birmingham, Alabama, United States
  • 1999–2001
    • Memorial Sloan-Kettering Cancer Center
      • • Department of Medicine
      • • Department of Surgery
      New York City, New York, United States